Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

SECTION 8 Clinical Microbiology: Bacteria

181 

Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli HILMAR WISPLINGHOFF

KEY CONCEPTS • Nonfermenting gram-negative bacilli (NFGNB), such as Pseu­ domonas aeruginosa, Acinetobacter baumannii, and Steno­ trophomonas maltophilia, are important nosocomial pathogens contributing significantly to morbidity and mortality, while others are being increasingly recognized as clinical pathogens. • NFGNBs such as A. baumannii or P. aeruginosa have been implicated in nearly all kinds of infections, including bloodstream infections (BSI), pneumonia and meningitis. • Clinical presentation of BSI due to A. baumannii or P. aerugi­ nosa varies. It may range from benign transient bacteremia to fulminant septic shock with high mortality and is not different from other gram-negative bacteria.

branes (such as the presence of mucus, lactoferrin and lactoperoxidase) and to environmental conditions, these organisms may pose an important problem in the susceptible host and in healthcare facilities.

Pathogenic Role Aerobic NFGNB can be part of the transient physiologic flora. Although most species are considered low-virulent, some like P. aeruginosa and A. baumannii are recognized as important human pathogens (Table 181-1).1,2

Antibiotic Therapy Most aerobic NFGNB have a high intrinsic resistance to the major classes of antimicrobial agents, often leaving few therapeutic options.1–4 Various mechanisms of both intrinsic and acquired

• Most NFGNB have a high intrinsic resistance which makes them frequently resistant to the major classes of antimicrobial agents, often leaving few therapeutic options. • Several NFGNB are highly resistant to environmental conditions favoring epidemic spread.

Introduction Strictly aerobic gram-negative bacilli have become increasingly important as human pathogens over the past decades.1–3 Molecular methods for identification have led to a number of changes in taxonomy, contributing insights into the clinical significance and epidemiology. While Pseudomonas aeruginosa1 remains the clinically most prevalent species among the nonfermenting gram-negative bacilli (NFGNB), other species, mainly Acinetobacter baumannii, have emerged as important nosocomial pathogens.2 Stenotrophomonas maltophilia and Burkholderia cepacia are commonly isolated from intensive care unit (ICU) patients and patients with cystic fibrosis (CF).3,4 Species of Acidovorax, Alcaligenes, Brevundimonas, Comamonas, Chryseobacte­ rium (Flavobacterium), Pandoraea and Ralstonia groups have been recognized as potential pathogens since the late 1980s.4

Epidemiology Most of the NFGNB can survive or even replicate under adverse environmental conditions. Pseudomonas spp. and other NFGNBs, in particular Acinetobacter spp., may survive for extended periods of time in dry, cold or warm environments, and have been isolated from a variety of surfaces, medical products and foods such as dairy products, poultry and frozen foods. Most species are saprophytic organisms and can be recovered from water, soil, plants, vegetables, insects and various other sources due to their ability to use a wide variety of substrates as sole carbon and energy sources and to grow in environments providing only limited nutrients.1–4 Combining intrinsic resistance to antimicrobial agents with the protective conditions of the skin (such as dryness, low pH, the resident normal flora and toxic lipids), the mucous mem-



TABLE

181-1 

Predominant Sites and Incidences of Nosocomial Infections due to Acinetobacter baumannii, Burkholderia cepacia, Pseudomonas aeruginosa and Stenotrophomonas maltophilia Incidence (% of Patients)

Organism

Clinical Presentation

Pseudomonas aeruginosa

Bloodstream infection Pneumonia (VAP) Urinary tract infection Wound infection Cystic fibrosis respiratory infection Burn wound infection

3–5 21–24 11–15 8–10 70–90

Bloodstream infection Pneumonia (VAP) Urinary tract infection Wound infection Cystic fibrosis respiratory infection Burn wound infection

<1 to 6 2 3–9 8–17 7

Burkholderia cepacia

Bloodstream infection Pneumonia (VAP) Urinary tract infection Wound infection Cystic fibrosis respiratory infection Burn wound infection

<1 <1 4 – 80

Acinetobacter baumannii*

Bloodstream infection Pneumonia (VAP) Urinary tract infection Wound infection Cystic fibrosis respiratory infection Burn wound infection

<1 to 14 5–10 10–30 2–32 –

Stenotrophomonas maltophilia

1–10

–

–

–

*Includes Acinetobacter genomic species 2, 3 and 13TU. –, no data available; VAP, ventilator-associated pneumonia.

1579

1580

SECTION 8  Clinical Microbiology: Bacteria

resistance have been identified, their type and frequency varying widely among different species. Antimicrobial therapy should be guided by in vitro susceptibility testing results; when possible, minimal inhibitory concentrations (MICs) of the respective antimicrobials should be determined.

Pseudomonas aeruginosa and Other Pseudomonas spp. Nature and Taxonomy The taxonomic classification of Pseudomonas spp. (historically, based on the utilization of various organic compounds as sole energy sources) has significantly changed since the proposition of the genus in 1894. Using rRNA–DNA hybridization, five different groups have been identified:1 I: Pseudomonas spp. II: Burkholderia spp. and Ralstonia spp. III: Comamonas spp., Acidovorax spp., Delftia spp. and Hydroge­ nophaga spp. IV: Brevundimonas spp. V: Xanthomonas spp. and Stenotrophomonas spp. The taxonomic status of Pseudomonas spp. (see Table 181-2) continues to evolve with the advances of polyphasic taxonomy. Some species display a high genetic diversity, such as P. stutzeri (at least 18 genovars, additional three proposed),5 P. fluorescens (biotype A (I), B (II, reclassified as P. marginalis), C (III), D and E (IV, V, reclassified together as P. chlororaphis), F and G), and P. putida (at least biovars A and B). However, most have so far not been isolated from clinical specimens.1

Epidemiology Currently, there are 160 Pseudomonas spp., most of which are ubiquitous organisms and are widely distributed in nature.1 To date, at least 12 Pseudomonas spp., including P. aeruginosa, P. alcaligines, P. fluore­ scens, P. luteola, P. mendocina, P. oryzihabitans, P. putida and P. stutzeri,1,6,7 have been implicated in human infections. P. aeruginosa remains the most prevalent aerobic NFGNB identified as the causative pathogen in a large variety of nosocomial infections. Studies have shown the ability of P. aeruginosa strains to acquire or discard genomic segments, giving rise to strains that can survive in a wide range of environmental habitats and serve as reservoirs for the infecting organisms.8 For example, P. aeruginosa has been isolated from surgical instruments, disinfectants (quaternary ammonium compounds) and contact lens cleaning solutions, ventilatory equipment in ICUs and surgical equipment such as ultrasonic aspirators used in neurosurgical procedures.7,9 Surveys using molecular typing methods (exotoxin A typing) also linked P. aeruginosa isolates from sinks, wash basins and toilets to those isolated from the hands of staff and the urinary tracts of paraplegic patients. Transmission over a 6-month period in an ICU for newborns has also been related to a source implicating air valves in the ventilator tubes.10 Pseudomonas spp. are not part of the normal flora and colonization of healthy individuals with Pseudomonas spp., particularly P. aeruginosa, is rare.11 Recovery of these bacteria, especially mucoid variants, should prompt the search for infections.1 In hospitalized patients intestinal colonization reaches 18% (up to 73% in patients recovering from gastrointestinal surgery). In neutropenic patients intestinal colonization has been identified as the source of subsequent P. aeruginosa bloodstream infection (BSI). Pseudomonas spp., such as P. fluorescens, P. stutzeri and Sphingomo­ nas paucimobilis (P. paucimobilis), are more frequently isolated from clinical specimens than other P. non-aeruginosa species and have been implicated in a variety of infections in adult and pediatric patients. There have also been individual reports of common source outbreaks due to these organisms.

Figure 181-1  Pseudomonas aeruginosa monotrichous polar flagellum seen on electron microscopy. (Courtesy of Professor A. Marty.)

EPIDEMIOLOGIC MARKERS A variety of typing methods (phenotypic and molecular) have been assessed for epidemiologic and clinical purposes.12,13 A selection of methods is summarized in Table 181-3.

Diagnostic Microbiology BACTERIOLOGY OF PSEUDOMONAS SPP. Pseudomonas spp. are thin, rod-shaped, non spore-forming gramnegative bacilli with a guanine/cytosine content of 57–68 mol%. Pseu­ domonas spp. are motile due to one (e.g. P. aeruginosa, P. stutzeri, P. oryzihabitans; Figure 181-1) or more polar flagella (e.g. P. fluorescens, P. putida, P. luteola). Pseudomonas spp. are strictly aerobic, although in some cases anaerobic growth is possible if a nitrate source can be utilized. P. aeruginosa and other clinically relevant Pseudomonas spp. are oxidase-positive with the exception of P. luteola and P. oryzi­ habitans.1,6 Pseudomonas spp. can be recovered from clinical and environmental specimens using standard collection, transport and storage techniques. It can also be cultured using standard broth or solid media including all nonselective (e.g. Columbia or tryptic-soy agar) as well as a number of selective media including MacConkey agar. While selective media containing inhibitors such as acetamide, nitrofurantoin or cetrimide may be helpful for the isolation of Pseu­ domonas spp., inhibition of some strains of P. aeruginosa (isolated from specimens from CF patients) by cetrimide and nalidixic acid has been reported. Some Pseudomonas spp. (P. fluorescens, P. putida) have the ability to grow at temperatures as low as 4 °C (39 °F), but most grow between 28 and 42 °C (82 and 108 °F), achieving visible growth within 24–48 hours. P. aeruginosa colonies on solid media are usually flat, but small colony variants (SCV)14 and mucoid forms have been described (Figure 181-2). Mucoid forms are particularly observed in patients suffering from CF and can pose problems for identification and susceptibility testing. Pyoverdin (fluorescein, yellow) is produced by all fluorescent Pseudomonas spp.; P. aeruginosa may also produce pyocyanin (blue), pyorubin (red) or pyomelanin (black). The distinct green color of P. aeruginosa colonies (Figure 181-2) usually results from a combination of pyoverdin and pyocyanin. P. aeruginosa can be separated from the other fluorescent Pseudomonas spp. (e.g. P. fluorescens, P. putida) by its ability to grow at 42 °C (108 °F). Automated systems can usually identify P. aeruginosa; however, additional testing is often required for identification of Pseudomonas spp., including mucoid or other forms of P. aeruginosa from cystic fibrosis (CF) patients. Other identification methods include conventional and real-time polymerase chain reaction (PCR), probes directed at or sequencing of species-specific 16S rRNA,15 fluorescence in situ hybridization (FISH) and matrix-assisted laser desorption/ionization-time of flight (MALDITOF) mass spectrometry.16



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

TABLE

181-2 

1581

Current Nomenclature of Nonfermenting Gram-Negative Bacilli*

Genus

Current Name

Achromobacter

Achromobacter Achromobacter Achromobacter Achromobacter

Acidovorax

Acidovorax delafieldii Acidovorax facilis Acidovorax temperans

Pseudomonas delafieldii Pseudomonas facilis Pseudomonas temperans

Acinetobacter

Acinetobacter baumannii

Acinetobacter anitratus, A. calcoaceticus, Diplococcus mucosus, Micrococcus calcoaceticus, Alcaligenes haemolysans, Mima polymorpha, Herellea vaginicola, Bacterium anitratum, Moraxella lwoffi var. glucidolytica, Neisseria winogradskyi, Achromobacter anitratus, Achromobacter mucosus

Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter Acinetobacter

Previous Name(s) denitrificans piechaudii ruhlandii xylosoxidans

baylyi bouvetii calcoaceticus gerneri grimontii haemolyticus johnsonii junii lwoffii nosocomialis parvus pittii radioresistens schindleri tandoii tjernbergiae towneri ursingii venetianus

Alcaligenes xylosoxidans subsp. denitrificans, Alcaligenes denitrificans CDC group VD-3 Alcaligenes piechaudii Alcaligenes ruhlandii Alcaligenes xylosoxidans subsp. xylosoxidans, Alcaligenes denitrificans subsp. xylosoxidans, Achromobacter xylosoxidans CDC groups IIIa, IIIb

Acinetobacter anitratus, Acinetobacter calcoaceticus subsp. anitratus Acinetobacter anitratus Acinetobacter anitratus, Moraxella lwoffi, Acinetobacter calcoaceticus subsp. lwoffi Acinetobacter genospecies 13 Acinetobacter genospecies 3

Agrobacterium

Agrobacterium tumefaciens

Alcaligenes

Alcaligenes faecalis

Alcaligenes odorans, Pseudomonas odorans

Bergeyella

Bergeyella zoohelcum

Weeksella zoohelcum

Brevundimonas

Brevundimonas Brevundimonas Brevundimonas Brevundimonas Brevundimonas Brevundimonas

Caulobacter henricii subsp. aurantiacus Pseudomonas diminuta Caulobacter intermedius Caulobacter subvibrioides Caulobacter variabilis Pseudomonas vesicularis

Burkholderia

Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia

aurantiaca diminuta intermedia subvibrioides variabilis vesicularis

ambifaria anthina arboris cenocepacia cepacia contaminans diffusa dolosa gladioli lata latens mallei metallidurans multivorans pseudomallei pyrocinia seminalis stabilis vietnamensis

Burkholderia cepacia complex genomovar III Pseudomonas cepacia, Pseudomonas multivorans, Pseudomonas kingae CDC group EO-1

Pseudomonas gladioli, Pseudomonas marginata Pseudomonas mallei, Actinobacillus mallei Pseudomonas pseudomallei

Chryseobacterium

Chryseobacterium gleum Chryseobacterium indologenes Chryseomonas luteola

Flavobacterium gleum CDC group IIb Flavobacterium indologenes CDC group IIb Pseudomonas luteola CDC group Ve-1

Comamonas

Comamonas Comamonas Comamonas Comamonas

Pseudomonas terrigena Pseudomonas testosteroni CDC group EF-19

aquatica kerstersii terrigena testosteroni

Cupriavidus

Cupriavidus necator

Wautersia eutropha, Ralstonia eutropha, Pseudomonas/Alcaligenes eutrophus

Delftia

Delftia acidovorans

Comamonas acidovorans, Pseudomonas acidovorans

Continued on following page

1582 TABLE

181-2 

SECTION 8  Clinical Microbiology: Bacteria

Current Nomenclature of Nonfermenting Gram-Negative Bacilli* (Continued)

Genus

Current Name

Previous Name(s)

Elizabethkingia

Elizabethkingia meningoseptica

Chryseobacterium meningosepticum, Flavobacterium meningosepticum CDC group IIa, Flavobacterium meningosepticum

Kerstersia

Kerstersia gyiorum

Alacligines faecalis (some strains)

Myroides

Myroides odoratus

Chryseobacterium odoratum, Flavobacterium odoratum CDC group M-4f

Ochrobactrum

Ochrobactrum anthropi Ochrobactrum intermedium

Achromobacter Vd Ochrobactrum sp. nov.

Oligella

Oligella ureolytica Oligella urethralis

CDC group IVe Moraxella urethralis, CDC group M-4

Pandoraea

Pandoraea spp.

Burkholderia spp.

Pedobacter

Pedobacter heparinum Pedobacter piscium

Sphingobacterium heparinum Sphingobacterium piscium

Pseudomonas

Pseudomonas aeruginosa Pseudomonas alcaligenes Pseudomonas chlororaphis

Pseudomonas fluorescens Biotype D Pseudomonas fluorescens Biotype E Pseudomonas aureofaciens

Pseudomonas delafieldii Pseudomonas fluorescens Pseudomonas kingii Pseudomonas mendocina Pseudomonas oryzihabitans Pseudomonas pertucinogena Pseudomonas pseudoalcaligenes Pseudomonas putida Pseudomonas sp. group 1 Pseudomonas stutzeri Pseudomonas stutzeri-like Pseudomonas-like group 2 Psyrobacter

CDC group Vb-2 Bordetella pertussis rough phase IV Pseudomonas alcaligenes biotype B Pseudomonas denitrificans CDC group Vb-1 CDC group Vb-3 CDC group IV-d

Psyrobacter faecali Psyrobacter pulmonis

Psyrobacter immobilis

Ralstonia

Ralstonia pickettii

Burkholderia pickettii, Pseudomonas pickettii, CDC groups Va-1, Va-2, Pseudomonas thomasii

Rhizobium

Rhizobium radiobacter Rhizobium rhizogenes Rhizobium rubi Rhizobium vitis

Agrobacterium group Vd-3 Agrobacterium Agrobacterium Agrobacterium

Shewanella

Shewanella algae Shewanella hanedai Shewanella putrefaciens

Alteromonas hanedai Pseudomonas/Alteromonas putrefaciens, CDC group Ib, Achromobacter putrefaciens

Sphingobacterium

Sphingobacterium Sphingobacterium Sphingobacterium Sphingobacterium Sphingobacterium Sphingobacterium Sphingobacterium

Flavobacterium Flavobacterium Flavobacterium Flavobacterium Flavobacterium

antarcticum faecium mizutaii multivorum spiritivorum thalpophilum yabuuchiae

radiobacter, Agrobacterium tumefaciens, Agrobacterium radiobacter CDC rhizogenes rubi vitis

mizutaii multivorum CDC group IIk-2 spiritivorum, Sphingobacterium versatilis CDC group IIk-3 thalpophilum yabuuchiae

Sphingomonas

Sphingomonas paucimobilis

Pseudomonas paucimobilis CDC group IIk-11

Stenotrophomonas

Stenotrophomonas maltophilia

Xanthomonas maltophilia, Pseudomonas maltophilia

Weeksella

Weeksella virosa

Flavobacterium genitale CDC group II-f 1,6,7

*The list is limited to those potentially involved in human infections.

Pathogenicity and Pathogenesis P. aeruginosa expresses a variety of virulence factors some of which have been characterized in recent years (Table 181-4).

HOST-RELATED FACTORS Host-related factors such as anatomic and physiologic barriers play an important role in the pathogenesis of P. aeruginosa infections. Several important P. aeruginosa infections such as burn wound infections or the colonization of the upper respiratory or gastrointestinal tract, largely depend on the impairment of the natural barriers provided by

intact skin or mucous membranes. Animal studies showed that P. aeruginosa can cause lethal infections originating from as little as 10 colony forming units (cfu) inoculated into burned skin, while similar infections of the intact skin require five to six logs more organisms. Disruption of the integrity of the corneal mucosa has been identified as the major factor in severe P. aeruginosa infections of the eye. Mucosal surfaces provide protection against P. aeruginosa colonization and infection by a variety of mechanisms such as mucus-containing antimicrobial factors including lysozyme, lactoferrin and defensins, the presence of physiologic flora, and mucociliary clearance. However, if impaired by underlying diseases such as CF or therapeutic



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

TABLE

181-3 

1583

Epidemiologic Markers for Acinetobacter baumannii and Pseudomonas aeruginosa Typing

Epidemiologic Markers

Species

Principles and Characteristics

Advantages

Drawbacks

Phenotypic

A. baumannii

Utilization of substrates, production of enzymes, biotyping schemes for identification, commonly carbon-source utilization test using levulinate, citraconate, L-phenylalanine, phenylacetate, 4-hydroxybenzoate and L-tartrate

Rapid, easy to perform, inexpensive, also useful for identification to species level

Unstable, variability of metabolic characters, limited discriminatory power

P. aeruginosa

Utilization of substrates, production of enzymes, biotyping schemes for identification

Rapid, easy to perform, API 20NE panel or automated identification systems (Vitek, MicroScan, Phoenix), inexpensive

Unstable, variability of metabolic characters, poorly discriminating

Resistance phenotype

A. baumannii, P. aeruginosa

Antimicrobial susceptibility pattern always obtained, multiple resistance markers

Rapid, easy to perform, standardized (national/ international guidelines), automated systems (Vitek, MicroScan, Phoenix) can be used, early and often useful during outbreak

Unstable profiles, plasmid acquisition or loss during an outbreak, derepression of inducible enzymes, mutations, poorly discriminating, unreliable

Serotyping

A. baumannii

O-antigenic polysaccharide of the lipopolysaccharide, 34 O-antisera

No advantages for A. baumannii

Not all strains typeable, low discriminatory power

P. aeruginosa

Based on somatic O-specific antigen (LPS), polyclonal/monoclonal antibodies, 20 serotypes, 17 antisera (IATS)

Rapid, early results, easy to perform, inexpensive

50–70% of CF strains nontypeable, polyagglutination of some CF strains, reproducibility of anti-LPS monoclonal antibodies is 75%, only available in specialized laboratories

A. baumannii

Two systems (21 and 14 phages, respectively)

Limited requirements, inexpensive

Lack of reproducibility, low discriminatory power, approx. 20% of strains nontypeable, only available in specialized laboratories

P. aeruginosa

Colindale set of 21 phages, cell surface receptors (OM, LPS, slime)

Limited requirements, inexpensive

Lack of reproducibility, low discrimination, insensitivity of CF- and LPS-defective strains, only available in specialized laboratories

Pyocin typing

P. aeruginosa

R, F, S pyocins, specific lytic activity, 105 types, 26 subtypes

Limited requirements, inexpensive

Poor discrimination, complexity of the system, timeconsuming technique, only available in specialized laboratories

Plasmidotyping

A. baumannii

Plasmids present in many A. baumannii strains

No advantage for A. baumannii typing

Plasmids are easily transferable and may be gained or lost, cumbersome technique

P. aeruginosa

Relatively rare plasmid in P. aeruginosa, plasmids of 1.2–60 MDa in 15% of strains

No advantage for P. aeruginosa typing

Low frequency, acquisition or loss during epidemics

Genomic DNA, total DNA

P. aeruginosa

Polymorphism of DNA, REA endonucleases (EcoR1, HindII, SmaI), conventional agarose gel electrophoresis

Good discriminatory power

Large number of fragments making resolution of bands difficult to interpret

DNA RFLP

P. aeruginosa

Detection of genes coding for exotoxin A (exoA), elastase (lasB), alginate (algD), two probes necessary

Good discriminatory power, good correlation with ribotyping

Laborious techniques, small numbers of isolates can be compared

Ribotyping (ribosomal DNA)

A. baumannii

HindIII, HincII, EcoR1, ClaI

Results comparable to AFLP, automated systems available

Limited discriminatory power, labor intensive, PFGE and others provide more accurate results for A. baumannii

P. aeruginosa

Three genes coding for rRNA, probes for 16S and 23S RNA, restriction enzymes (EcoR1, ClaI, SalI)

Universal, excellent reproducibility, stable ribotype patterns within outbreaks

Laborious techniques, sensitivity and specificity not established for P. aeruginosa

Biotyping

Phage typing

Genotypic

Continued on following page

1584 TABLE

181-3 

SECTION 8  Clinical Microbiology: Bacteria

Epidemiologic Markers for Acinetobacter baumannii and Pseudomonas aeruginosa Typing (Continued)

Epidemiologic Markers

Species

Principles and Characteristics

Advantages

Drawbacks

Genotypic

A. baumannii

ApaI and/or SmaI

Gold standard for A. baumannii, highest discriminatory power, interlaboratory reproducibility possible with standardized protocols

Labor intensive

P. aeruginosa

DNA fingerprinting, restriction enzymes DraI, SpeI

The most specific discriminatory technique

Interpretation somewhat delicate, heavy workload

S. maltophilia

DNA fingerprinting

The most specific discriminatory technique

Interpretation somewhat delicate, heavy workload

ERIC-PCR

S. maltophilia

Amplification of DNA sequences using ERIC2 primer

Good discriminatory power

Inter- and intralaboratory reproducibility difficult to achieve

RAPD-PCR

A. baumannii

Amplification of random DNA sequences

Fast, easy and low cost, relatively high discriminatory power

Inter- and intralaboratory reproducibility difficult to achieve, not suited for large-scale epidemiologic studies, discriminatory power inferior to PFGE

P. aeruginosa

Amplification of random DNA sequences

Fast, easy and low cost, relatively high discriminatory power

Inter- and intralaboratory reproducibility difficult to achieve

S. maltophilia

Amplification of random DNA sequences

Fast, easy and low cost, relatively high discriminatory power

Inter- and intralaboratory reproducibility difficult to achieve

A. baumannii

PCR-based typing method, amplification of highly conserved REP sequences using specific primers

Simple, rapid

Inter- and intralaboratory reproducibility remains to be determined, expensive

S. maltophilia

PCR-based typing method, amplification of highly conserved REP sequences using specific primers

Simple, rapid

Inter- and intralaboratory reproducibility remains to be determined, expensive

AFLP

A. baumannii

PCR-based typing method, amplification of restriction fragments (HindIII and TaqI) using specific primers

Can be used for typing and species identification, relatively robust method, high discriminatory power

Acinetobacter only, requires a high level of standardization and extensive experience, cumbersome and expensive, restricted to reference laboratories, not suited for routine epidemiologic analyses, data are not readily transportable

MLST

A. baumannii, Acinetobacter genomic species 13TU, P. aeruginosa

Amplification and sequencing of several specific loci in different housekeeping genes: A. baumannii and Acinetobacter genomic species 13TU: gltA, gyrB, gdhB, recA, cpn60, gpi, rpoD; P. aeruginosa: acsA, aroE, guaA, mutL, nuoD, ppsA, trpE

Highly portable, highly reproducible

Time-consuming, very expensive

PCR/ESI-MS

A. baumannii

Amplification of specific sequences of six housekeeping genes (efp, trpE, adk, mutY, fumC, ppa), detection by ESI-MS

Results similar to MLST, easy, high throughput, fastest method to date

Novel method, not suited for routine laboratories, expensive equipment, detailed evaluation warranted

Pulsotyping (PFGE)

REP-PCR

AFLP, amplified fragment length polymorphism; CF, cystic fibrosis; ERIC-PCR, enterobacterial repetitive intergenic consensus polymerase chain reaction; ESI-MS, electrospray ionization mass spectrometry; IATS, International Antigenic Typing Scheme; LPS, lipopolysaccharide; MLST, multilocus sequence typing; OM, outer membrane; PCR, polymerase chain reaction; PFGE, pulsed-field gel electrophoresis; RAPD, random amplified polymorphic DNA; REA, restriction endonuclease analysis; REP-PCR, repetitive extragenic palindromic polymerase chain reaction; RFLP, restriction fragment length polymorphism.

interventions, such as antimicrobial or anticancer chemotherapy, P. aeruginosa has a high propensity for colonization and infection of the respective site. A variety of soluble factors is involved in the pathogenesis of P. aeruginosa infections, such as complement, collectins, cytokines, chemokines and immunoglobulins, as well as cellular mediators. A detailed summary of their function can be found elsewhere.1,17

BACTERIAL FACTORS Virtually all major classes of bacterial virulence systems (see Table 181-4) have been reported in P. aeruginosa, including endo- and exotoxins, pili, flagella, proteases, lipases, iron-binding proteins and exopolysaccharides. Adherence of P. aeruginosa to host tissue is in part promoted by pili (pilA gene, regulated by rpoN, pilS and pilR), flagella (fliC [flagella] and fliD [protein caps]) and other appendage-like



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

TABLE

181-4 

Acinetobacter baumannii and Pseudomonas aeruginosa Virulence-Associated Factors

Category

Pathogen

Classes of Virulence Factors (examples)

Effects in Humans

Enzyme

A. baumannii

Antibiotic-inactivating enzymes (β-lactamases) Proteases Protein S

Inactivation of antimicrobial agents Tissue damage Interference with phagocytosis

P. aeruginosa

Adhesins (exoenzyme S) Antibiotic-inactivating enzymes (β-lactamases) Elastolytic activity (two enzymes Las A, Las B) Glycolipid-rhamnolipid (heat-stable) Hemolysins (phospholipase C)

Binding specificity for glycolipids (glycosphingolipid) Inactivation of antimicrobial agents Break down elastin of blood vessels, hemorrhages

Neuraminidase Cytoplasmic lectins, P. aeruginosa lectin I (PA I), PA II Proteases (alkaline and neutral metalloproteinase) Structural

Disruption of phospholipids of cell membranes, hydrolysis of lung surfactant and ciliostatic action Enhances pilin-mediated adherence PA I specific for D-galactose, PA II specific for D-mannose Tissue damage (active on elastin, collagen, fibrin), digestion of protecting host defense proteins

A. baumannii

Aerobactin Fimbriae Iron-repressible outer membrane receptor proteins Iron-uptake components Lipopolysaccharide Lipopolysaccharide (hydrophobic sugars in the O side chain) Outer membrane proteins Pili Polysaccharide capsule

Increased virulence (mechanism to be determined) Adhesion to epithelial cells Increased virulence (mechanism to be determined) Survival in the bloodstream Proinflammatory response Adhesion to host cells Interference with cell permeability Adhesion to epithelial cells Interference with phagocytosis, survival in dry environment

P. aeruginosa

Efflux pumps Lipopolysaccharide Mucopolysaccharide capsule (alginate)

Quorum sensing Type III secretion factors

Increase resistance to antimicrobial agents Cascade of inflammatory events Adhesion to epithelial cells, barrier to antibiotics, increased viscosity of bronchial secretions (cystic fibrosis) Adhesion to epithelial cells Help growth in iron-limited condition, generation of toxic oxygen-free radicals Biofilm formation, regulation of virulence factors Facilitate injection of type III effectors

A. baumannii

Lipid A Outer membrane protein A (Omp 38)

Toxicity, pyrogenicity Cytotoxicity, apoptosis, cell death

P. aeruginosa

Cytotoxins (leukocidin)

Cytopathic effects on leukocytes and alteration of phospholipids of cell membrane Septic shock Tissue damage, inhibition of phagocytes, inhibition of protein synthesis Cytotoxicity, cell death Ciliary disruption

Pili (fimbriae) Siderophores (pyochelin and pyoverdin)

Toxin

1585

Endotoxins Exotoxin A Type III secretion (ExoS, ExoT, ExoU, ExoY) Phenazine pigment (pyocyanin)

Figure 181-2  Pseudomonas aeruginosa mucoid (right side of plate) and nonmucoid colonies (left side of plate) on MH-agar. (Courtesy of H. Wisplinghoff.)

structures that have recently been identified in multidrug-resistant strains of P. aeruginosa. In addition, lipopolysaccharide may bind to the cystic fibrosis transmembrane conductance regulator (CFTR). Defective expression of the CFTR contributes to the hypersusceptibility of CF patients to P. aeruginosa infection. Recent studies have identified numerous CFTR-dependent factors that are recruited to the epithelial plasma membrane in response to infection and are needed for bacterial clearance. Several factors mediating resistance to host defenses have been identified in P. aeruginosa – including lipopolysaccharide O (LPS O) which prevents lysis by complement and proteases (e.g. protease IV, elastolytic proteases and alkaline protease) – that are involved in the evasion of innate immunity, degradation of cells and host proteins, and syndecan 1. Manifestation of infection largely depends on the expression of a variety of substances that damage host cells, such as ferripyochelin (destroying endothelial cells) alone or in combination with hydrogen peroxide. P. aeruginosa toxins include exotoxin A (inhibition of protein biosynthesis by inactivating elongation factor 2), leukocidin, plcHR (a hemolytic phospholipase C), pyocyanin and rhamnolipids. Most toxins and hydrolytic enzymes involved in virulence are regulated by a complex system, components of which are influenced by environmental factors. Intoxication of the host cells via type III secretion systems is another important factor in P. aeruginosa infections and the production of type III secretor proteins (PcrG, PcrV, PcrH, PopB, and PopD, encoded by the pcrGVH-popBD operon) has repeatedly been identified as an independent risk factor for poor outcome.18

1586

SECTION 8  Clinical Microbiology: Bacteria

In P. aeruginosa three major quorum-sensing systems have been identified to date, termed las, rhl and PQS. These interacting systems seem to play an important role in the regulation of biofilm formation and expression of virulence factors. Degradation of acyl-homoserine lactones, the signaling molecules of the quorum-sensing system, has been discussed as a treatment option. In addition, the GacS/GacA twocomponent system positively controls the transcription of two sRNAs (RsmY, RsmZ) that are crucial for the expression of virulence genes and regulate the response to oxidative stress in P. fluorescens. The spread of P. aeruginosa and invasion of the bloodstream requires a smooth LPS substituted with O side chains and is likely enhanced by bacterial factors stimulating the release of tumor necrosis factor (TNF) and quorum-sensing molecules.

MUCOID PSEUDOMONAS AERUGINOSA In the early 1960s the importance of mucoid variants of P. aeruginosa was recognized. Mucoid strains are morphologic and functional variants characterized by the ability to produce copious amounts of alginate, an acetylated polymer of D-mannuronic and L-glucuronic acids regulated by ClpXP proteases, AlgB, Alg8 and Alg44 (Figures 181-3 and 181-4). Expression is regulated by the extracytoplasmatic sigma factor (σ(22), AlgU/T) and negatively impacted by the cognate antisigma factor MucA. Alginate has been implicated in the pathogenesis of respiratory tract infections due to P. aeruginosa as well as in chronic oropharyngeal colonization.19,20 In vivo, in areas with impaired local defenses, such as in the airways of CF patients, the organism grows in microcolonies surrounded by a

Figure 181-3  Anatomic pathology of Pseudomonas aeruginosa pneumonia showing acute inflammatory exudate, necrosis of alveolar membranes and fibrinous thrombosis in a venula. Hematoxylin–eosin stain. (Courtesy of Professor Groussard.)

thick polysaccharide matrix adherent to the walls of larger airways and in the alveoli. Mucoid P. aeruginosa is present in foci of active inflammation in small bronchioles but not in destroyed parenchymal areas. This is consistent with the simultaneous role of bacterial growth in the active inflammatory process and of toxins produced by P. aeruginosa diffusing away from microcolonies. Several studies have addressed the role of antibodies against alginate and their failure to confer protection against infection in CF patients, showing that the biofilm-like growth may interfere with the opsonizing capabilities of the alginate antibodies. An exopolysaccharide–alginate conjugate vaccine has been discussed as a therapeutic approach.

Clinical Manifestations BACTEREMIA/BLOODSTREAM INFECTION Assessment of the incidence of P. aeruginosa BSI is difficult. P. aerugi­ nosa accounted for 20–35% of all BSI isolates in older series, but only for 4.4% in recent series. Among 24 179 nosocomial BSIs from 52 hospitals in the USA over a 7-year period, P. aeruginosa (incidence 2.1 per 10 000 admissions) accounted for 3.8% of all BSI isolates from non-ICU wards and 4.7% from ICUs, making it the seventh and fifth most commonly isolated pathogen, respectively.21 Historically, crude mortality rates of 50–70% have been reported in patients with P. aeruginosa BSI, while more recent studies found crude mortality rates of 28% in non-ICU and 48% in ICU patients, respectively,21 as well as an attributable mortality ranging from 15% to 44%. Secondary P. aeruginosa BSI most often originates from the respiratory tract (Table 181-5) followed by the urinary tract or burn wounds. Colonization of the gastrointestinal tract, which may develop in the presence of risk factors such as hospitalization in an ICU, neutropenia or treatment with cytotoxic chemotherapy, has also been discussed as a source of P. aeruginosa BSI, particularly in neutropenic patients receiving chemotherapy. Approximately 50% of these patients develop intestinal carriage, compared to only 5–15% of the general population, and translocation has been implicated as the potential mechanism for invasion. In addition, contaminated medical equipment used for endoscopic retrograde cholangiopancreatography (ERCP), placement of left ventricular assist devices and other invasive procedures have been reported as sources of P. aerugi­ nosa BSI.22 The clinical presentation of P. aeruginosa BSI does not differ from sepsis due to other gram-negative pathogens and may range from benign transient bacteremia to fulminant septic shock with high mortality. In neutropenic patients, typical skin lesions (e.g. ecthyma gangrenosum; see Chapter 13) may present. Even though these lesions may on rare occasions present in non-neutropenic patients or be associated with other pathogens, such as Aspergillus spp. or Mucor spp., ecthyma gangrenosum in a septic patient should prompt antimicrobial chemotherapy active against P. aeruginosa.1

ENDOCARDITIS (see also Chapter 51)

Figure 181-4  Burned leg superinfected with Pseudomonas aeruginosa. (Courtesy of Professor H. Carsin.)

P. aeruginosa is the second most common cause of endocarditis due to non-HACEK GNB (after Escherichia coli), accounting for 22% (n=11) of all cases caused by non-HACEK GNB in a recent observational study from the International Collaboration on EndocarditisProspective Cohort Study (ICE-PCS) database, including 2761 cases of definite endocarditis from 61 hospitals in 28 countries.23 P. aerugi­ nosa is also the second most common cause of cardiac device-related endocarditis (CDE) in a retrospective survey of CDE-cases occurring between 1980 and 2011. In contrast to older studies that reported endocarditis due to P. aeruginosa predominantly in intravenous drug users,22 the ICE-PCS study found that 57% of patients with nonHACEK GNB endocarditis had a healthcare-associated infection (IVDA in only 4%), and implanted endovascular devices and prosthetic heart valves were frequent risk factors.23 The in-hospital mortality of patients with endocarditis due to P. aeruginosa reached 21% in recent studies despite high rates of cardiac surgery (55%).23



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

TABLE

181-5 

1587

Infections Due to Acinetobacter baumannii and Pseudomonas aeruginosa

Infection Respiratory tract infections

Associated Factors A. baumannii pneumonia, nosocomial, 60% mortality with BSI P. aeruginosa pneumonia (30–60% mortality rate) P. aeruginosa pneumonia, nosocomial, with BSI (80–100% mortality rate)

A. baumannii pneumonia (community acquired, 60% mortality in bacteremic pneumonia) P. aeruginosa respiratory tract infection in people with cystic fibrosis (ultimately fatal unless a pulmonary transplant is carried out)

Mechanical ventilation Endotracheal or tracheostomy tube Neurologic disease Nasogastric tube Prolonged stay in intensive care unit Broad-spectrum antibiotics Neutropenia Underlying malignant neoplasm Cytotoxic chemotherapy Chronic bronchiectasis (terminal state) Diabetes mellitus Severe immunosuppression Severe burns Chronic obstructive pulmonary disease Smoking Alcoholism Presence of the lethal genetic disease cystic fibrosis Chronic colonization with P. aeruginosa Progressive lung deterioration Altered immune response to P. aeruginosa

A. baumannii

Prolonged stay in intensive care unit, mechanical ventilation, underlying immunosuppression, intravenous devices Leukemia, lymphoma Colonization with A. baumannii Various endoscopic instrumentation procedures

P. aeruginosa

Prolonged stay in intensive care unit, broad-spectrum antibiotics, invasive procedures, underlying immunosuppression, intravenous devices Leukemia, lymphoma Mechanical ventilation Intravenous drug use Trauma Prematurity Ulceration of the gastrointestinal tract Solid organ or bone marrow transplant Various endoscopic instrumentation procedures

Skin and soft tissue infections

P. aeruginosa

Burn wound sepsis Wound infection Ecthyma gangrenosum Invasive procedures, surgery Dermatitis, pyoderma

UTI

A. baumannii P. aeruginosa

Invasive procedures, urinary catheters Acute (rare) Chronic (obstruction)

Endocarditis

P. aeruginosa

Intravenous drug use Prosthetic heart valves

Miscellaneous

A. baumannii P. aeruginosa

Meningitis (secondary) Brain abscesses, meningitis (secondary, following neurosurgical procedures) Bone and joint infections (chronic osteomyelitis) Ear infections (otitis externa, malignant external otitis) Eye infections (keratitis, endophthalmitis, contact lens keratitis)

Bloodstream infection (BSI)

LOWER RESPIRATORY TRACT INFECTIONS (see also Chapter 29) P. aeruginosa is the most common gram-negative pathogen causing ventilator-associated pneumonia (VAP) and is consistently associated with a measurable attributable mortality.24,25 In a recent prospective multicenter study from Spain, P. aeruginosa (together with L. pneumophila) was the second most common bacterial pathogen (after S. pneumoniae) in patients with healthcare associated pneumonia accounting for 4.8% of cases.26 Since P. aeruginosa is also a frequent colonizer of endotracheal tubes and the nasopharynx of patients with mechanical ventilation receiving antimicrobial chemotherapy, the significance of a culture from the upper respiratory tract yielding P. aeruginosa in the absence of clinical and/or radiologic signs of pneumonia is less clear. The delay in eradication of P. aeruginosa in patients with VAP may be explained by an increased apoptosis in neutrophils.24

Typical pathologic features include necrosis of alveolar septa (see Figure 181-3) and arterial walls, with areas of focal hemorrhage and, in intact areas, infiltration with macrophages, mononuclear cells and polymorphonuclear leukocytes. A different lung pathology has been described in bacteremic pneumonia, which is rarely seen and mainly affects patients with severe underlying conditions. Risk factors are listed in Table 181-6. The clinical course is characterized by a rapid progression with a diffuse and often bilateral infiltration, usually in combination with pleural effusion. On cross-section, the lesions are nodular, hemorrhagic with necrotic foci or umbilicated nodules surrounded by dark hemorrhage. Intra-alveolar hemorrhage with patchy alveolar septal necrosis is seen on microscopy. Lesions contain many bacteria but lack infiltration with macrophages, mononuclear cells and polymorphonuclear leukocytes. Pulmonary edema and necrotizing bronchopneumonia are associated with a poor prognosis. The case fatality rate in patients with fulminant bacteremic pneumonia due to P. aeruginosa is extremely high (80–100%).

1588

TABLE

181-6 

SECTION 8  Clinical Microbiology: Bacteria

Sources, Means of Transmission and Risk Factors for Nosocomial Infections Due to Aerobic Gram-Negative Bacilli

Organisms

Settings*

Sources/Means of Transmission

Risk Factor/Comments

Achromobacter (Alcaligenes) xylosoxidans

ICU, hemodialysis units

Contaminated chlorhexidine solution, dialysis fluid, aerosols, respirators

Aqueous source, hemodialysis, severe underlying disease

Acinetobacter baumannii

ICU

Contaminated ventilators, intravascular catheters

Severely ill patients, cross-contamination, outbreaks

Burkholderia cepacia

ICU

Airborne transmission, contaminated skin preparations, ventilator, thermometer, antiseptic solutions

Cystic fibrosis patients, hand carriage, immunocompromised patients

Elizabethkingia (Chryseobacterium) meningosepticum

NICU

Contaminated water, ice, disinfectants, humidifiers

Wounds, mechanical ventilation

Pseudomonas aeruginosa

ICU, burns units

Contaminated equipment, solutions, antiseptics, endogenous

Cross-contamination, exposure to broad-spectrum antibiotics, severely ill patients, outbreaks in burn patients

Pseudomonas putida, Pseudomonas fluorescens

ICU

Contaminated blood and blood by-products, antiseptics

Large (burn) wounds, intravascular devices

Stenotrophomonas maltophilia

ICU, hematology/ oncology units

Contaminated devices, disinfectants, catheters

Dialysis fluids, exploratory procedures, neutropenia, respiratory devices, tracheostomized patients, backflow from nonsterile tubes

*Most frequently reported occurrence. ICU, intensive care unit; NICU, neonatal intensive care unit.

considerably from 10% in older reports to about 1% in current studies.28 Colonization of the burned skin may result from the patient’s own flora or from environmental sources. The bacteria penetrate into the subcutaneous tissues via hair follicles and break in the burned skin and may enter the bloodstream with the help of proteolytic enzymes. Other virulence factors (see Table 181-4) contribute significantly to the severity of the infection. Gr-1+/CD11b+ cells have been identified as an accelerator of sepsis originating from wound infection in thermally injured mice. Sepsis requires specific management in burn centers (Figures 181-4 and 181-5). Recent studies comparing BSI in burn patients due to P. aeruginosa, A. baumannii, Klebsiella pneumoniae and S. aureus showed that K. pneumoniae had the greatest impact on mortality relative to all other pathogens. Figure 181-5  Burned abdominal wall superinfected with Pseudomonas aeruginosa. (Courtesy of Professor H. Carsin.)

Historically, community-acquired pneumonia due to P. aeruginosa was mainly seen in immunocompromised patients, such as human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) patients with low CD4 counts or patients suffering from CF, but has also been reported in previously healthy nonimmunocompromised patients, especially in patients with structural lung defects (e.g. COPD). Inappropriate antibiotic therapy has been identified as a significant risk factor for adverse outcome.24

SKIN AND SOFT TISSUE INFECTIONS Skin and soft tissue infections are usually dermatitis or superinfections of predisposing skin lesions. Folliculitis due to P. aeruginosa has been described and outbreaks have been linked to swimming pools, whirlpools, hot tubs and, recently, contamination of an industrial closed loop water recycling system.27 See also Chapters 10, 11 and 13.

Burn Wound Sepsis P. aeruginosa is the most common cause of burn wound sepsis, the predominant form of skin and soft tissue infection complicating thermal injury (see Figures 181-4 and 181-5). The mortality rate is high (50–78%) despite improvements in management and antibiotic therapy.22 The incidence of P. aeruginosa BSI, however, has declined

EYE INFECTIONS (see also Chapters 16 to 18) P. aeruginosa is the most common gram-negative pathogen causing infections of the eye. Infections most commonly originate from an exogenous source and are related to (superficial) injuries. Ocular infections vary from mild (conjunctivitis) to extremely severe (orbital cellulitis).

Keratitis Keratitis is the most frequent eye infection. Keratitis due to P. aerugi­ nosa has been reported only secondary to an injury of the corneal surface. The predominant predisposing factors are contact lenses, congenital abnormalities, burns or trauma, altered host defenses (in particular HIV/AIDS) and prematurity. Contact lens-associated keratitis due to P. aeruginosa has mainly been observed in association with extended-wear contact lenses, inappropriate disinfecting regimens and poor hygiene. The bacteria may adhere to the lens, resulting in a coat of mucopolysaccharides forming a biofilm similar to other prosthetic devices. Viral keratitis may also be associated with secondary bacterial infection. Exocellular products of P. aeruginosa and strong adhesion to the exposed basement membrane of the epithelium increase the corneal damage; exotoxins, proteases and phospholipases degrade the corneal stroma, resulting in extensive loss of collagen fibers from the stroma. Keratitis due to P. aeruginosa may progress rapidly, leading to endoph­ thalmitis and loss of eyesight.



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

Endophthalmitis Endophthalmitis most often results from an endogenous origin, occurring by hematogenous spread from other infected sites or after intraocular inoculation of P. aeruginosa by trauma, burns or ocular surgery. Endophthalmitis may present as an acute fulminant disease with severe pain, chemosis and decreased acuity, and can progress rapidly to panophthalmitis. The prognosis is poor without appropriate local and systemic management.

URINARY TRACT INFECTIONS (see also Chapter 57–60) Urinary tract infections (UTI) due to P. aeruginosa are usually healthcare associated. Risk factors include the presence of a foreign body (e.g. long-term catheter or stent), surgery, obstruction of the urinary flow or persistent infection (e.g. chronic prostatitis). While P. aeruginosa UTI usually affect patients with prolonged hospitalization, antimicrobial therapy and/or other risk factors, infections in otherwise healthy children with no known risk factors have also been reported. P. aeruginosa UTIs have no specific clinical presentation but tend to evolve with frequent recurrences, treatment failures and chronic evolution. A characteristic picture of ulcerative or necrotic lesions and multiple renal abscesses is seen in patients who have metastatic bacteremia with urinary tract invasion.22

EAR INFECTIONS P. aeruginosa is frequently isolated from the external auditory canal, particularly in infants. Infections may range from benign transient colonization to severe infections with a prolonged clinical course that may be associated with permanent neurologic sequelae and adverse outcome. P. aeruginosa has been identified as the cause of a mild superficial infection of the external ear canal (e.g. swimmer’s ear). Although this benign infection usually resolves without sequelae, it may proceed to invasion of the epithelium between cartilage and bone in the lateral portion of the auditory canal, penetrating soft tissue, cartilage and bone.

Malignant (Necrotizing) Otitis Externa Malignant or necrotizing otitis externa is a severe invasive ear infection, clinically characterized by decreased hearing, otalgia, otorrhea, early facial nerve paralysis and a swollen erythematous external auditory canal. Adjacent soft tissue is often involved and the infection may progress to the middle ear, mastoid, temporal bone and cranial nerves. Clinical presentation may include visible extension with cellulitis, bone erosions and purulent discharge from the inner ear if the tympanic membrane is perforated.22 P. aeruginosa may be isolated from superficial swabs of the external auditory canal and from surgical specimens. Most cases occur in elderly people with diabetes mellitus, but cases have also been reported from HIV/AIDS patients, elderly patients without immunosuppression or infants with severe underlying diseases. Management requires prolonged antibiotic therapy, surgical debridement and drainage. The fatality rate is high (about 15–20%). Relapses are frequent and malignant external otitis requires prolonged follow-up.

MISCELLANEOUS Central Nervous System Infections (see also Chapters 19, 21 and 24) Infections of the central nervous system are rarely due to P. aeruginosa. Meningitis, epidural or subdural empyema and brain abscesses have been reported in adults and children, usually as a result of either direct inoculation (head trauma, surgery), a contiguous infection (sinus, mastoid) or following BSI.

Bone and Joint Infections (see also Chapters 43 to 45) Bone and joint infections are infrequently caused by P. aeruginosa. Direct inoculation (trauma, surgery), contiguous infection from

1589

surrounding tissue or hematogenous seeding following BSI are the most common routes of infection. Studies in war casualties found that P. aeruginosa was more frequently seen in primary osteomyelitis, whereas gram-positive pathogens were more likely to be isolated from recurrent episodes. Infections predominantly occur in patients with predisposing factors such as diabetes mellitus, intravenous drug use and chronic debilitation. More frequently reported associations include: • vertebral osteomyelitis and arthritis involving the sternoclavicular or sternochondral joints in intravenous drug users • vertebral osteomyelitis in elderly patients following genitourinary instrumentation or surgery • osteochondritis of the foot in children following puncture wounds or in patients with diabetic foot ulcers. While P. aeruginosa seems to have a particular affinity for cartilaginous joints of the axial skeleton, infections at a distant site, such as pneumonia, rarely spread to the vertebral column or the axial skeleton. Following pelvic surgery or femoral catheterization, osteomyelitis of the pubic symphysis has been reported as well as rare cases of pelvic osteomyelitis in children. Clinical presentation may be discrete; pain, swelling, fever and other systemic signs are variable.22 The diagnosis may be difficult as blood cultures are frequently negative and imaging studies may also be normal in the earlier stages.

Pathogenicity and Clinical Manifestations of Pseudomonas spp. Other than P. aeruginosa Some of the non-aeruginosa Pseudomonas spp. have been isolated from human clinical specimens (blood, urine, stools) and occasional cases of opportunistic infection as a result of transfusions, contamination of indwelling catheters, antiseptics or dialysis fluid and various other mechanisms of transmission have been reported. For example, P. fluo­ rescens may grow at 4 °C (39 °F), which favors its presence in blood products and infusates such as heparin. Outbreaks of bacteremia, respiratory infections, wound infections and rare cases of communityacquired pneumonia have been reported. Other Pseudomonas spp., particularly P. fluorescens, P. stutzeri and Sphingomonas paucimobilis (P. paucimobilis), have also been implicated in rare cases of brain abscess, arthritis, endophthalmitis and keratitis while P. alcaligenes and P. mendocina have been isolated from patients with endocarditis. Most cases occur in patients with severely impaired host defenses such as immunocompromised patients in the ICU setting (Table 181-7).22

Antimicrobial Resistance and Therapy Although P. aeruginosa is intrinsically resistant to many antimicrobials, several agents from different classes remain potentially active (Tables 181-8 and 181-9). However, the percentage of multi- or pandrugresistant clinical P. aeruginosa strains has increased considerably in the past decade. Antimicrobial agents with potential antipseudomonal activity include semisynthetic penicillins such as carboxypenicillins (ticarcillin), ureidopenicillins (piperacillin), some third- (group IIIb), fourth-, and fifth generation cephalosporins (ceftazidime, cefpirome, cefepime, ceftaroline, ceftobiprole), carbapenems (imipenem, meropenem, doripenem), monobactams (aztreonam), aminoglycosides (gentamicin, tobramycin, amikacin), fluoroquinolones (ciprofloxacin, levofloxacin) and polymyxins (polymyxin B, polymyxin E). A summary of anti-pseudomonal treatment options in various indications is given in Table 181-10. P. aeruginosa displays a wide array of resistance determinants, most of which are chromosomally located. The incidence of plasmids is relatively low. Mechanisms of resistance include altered outer membrane permeability (altered protein porins or lack of protein porin OprD), production of β-lactamases, aminoglycoside-inactivating enzymes and efflux pump systems actively removing different antibiotic classes from the bacterial cell (Table 181-11).

1590 TABLE

181-7 

SECTION 8  Clinical Microbiology: Bacteria

Epidemiology and Pathogenicity of Acinetobacter spp. and Pseudomonas spp.

Species

Habitat and Epidemiology

Clinical Significance

Acinetobacter non-baumannii, i.e. A. johnsonii, A. junii, A. lwoffii, A. radioresistens, A. ursingii

Ubiquitous, soil, water, sewage water, hospital environment, antiseptics, injectable solutions, more susceptible to antibiotics than other species

Outbreaks of pseudobacteremia involved in catheter-related bloodstream infection, isolation from wounds, urine, blood, cerebrospinal fluid

A. baumannii, A. pittii, A. nosocomialis (Acinetobacter genomic species 2, 3, 13TU)

Hospital environment, colonized patients, healthcare personnel

Opportunistic pathogen, severe infections mostly in immunocompromised patients

Pseudomonas alcaligenes, P. pseudoalcaligenes

Environment, water, plants, hospital environment, rare opportunistic pathogens

Occasional bacteremia (contaminated blood products, solutions)

P. putida, P. fluorescens, P. aeruginosa

Soil, water, plants, hospital sinks, floor, food spoilage (eggs, meat, fish, milk), opportunistic pathogens

Rarely isolated from clinical specimens, rare cases of colonization in patients with cystic fibrosis, bacteremia, urinary tract infection, wounds

P. stutzeri

Ubiquitous, soil, water, sewage water, hospital environment, antiseptics, injectable solutions, relatively more frequent than other non-aeruginosa Pseudomonas spp., opportunist, more susceptible to antibiotics than other species

Outbreaks of pseudobacteremia, isolation from pus, urine, blood, cerebrospinal fluid, contamination of bone marrow transplant

P. aeruginosa, P. fluorescens

Hospital environment, patients, healthcare personnel

Opportunistic pathogen, severe infections mostly in immunocompromised patients

TABLE

181-8 

Antibiotic Susceptibility of Acinetobacter baumannii and Pseudomonas aeruginosa A. baumannii

Antibiotic Class

MIC50 (mg/L)

β-Lactams

Ampicillin Ampicillin–sulbactam Amoxicillin Amoxicillin–clavulanic acid Mezlocillin Piperacillin Piperacillin–tazobactam Ticarcillin Ticarcillin–clavulanic acid Cefazolin Cefuroxime Ceftriaxone Cefotaxime Ceftazidime Cefepime Imipenem Meropenem Aztreonam

Aminoglycosides

P. aeruginosa

MIC90 (mg/L)

MIC50 (mg/L)

64 1 32 2 16 8 4 64 32 256 32 8 16 4 4 0.06 0.25 16

>256 16 256 256 512 512 128 512 256 512 128 128 128 128 16 0.25 1 64

Amikacin Gentamicin Tobramycin

2 1 0.5

8 32 8

0.5 0.5 0.125

Quinolones

Ofloxacin Ciprofloxacin Levofloxacin Moxifloxacin

0.5 0.5 0.25 0.12

16 64 8 16

2 0.06 1 4

Miscellaneous

Trimethoprim–sulfamethoxazole

2

256

64 32 32 32 16 8 4 32 16 128 32 8 16 4 4 1 0.5 4

128

MIC90 (mg/L) >256 256 >256 256 128 512 128 512 512 512 256 128 64 32 16 2 2 16 2 8 4 4 0.25 32 32 >128

From Zhanel et al. Diagn Microbiol Infect Dis 2008; 62:67–80 and Seifert et al. J Antimicrob Chemother 2006; 58:1099-1100.

β-Lactamases as well as IMP, GES and VIM metallocarbapenem­ ases may be augmented by a number of efflux systems and decreased OprD expression which together confer multidrug resistance to β-lactam antibiotics. In clinical settings, these are often encountered in combination with other mechanisms conferring resistance to fluoroquinolones and aminoglycosides, thus considerably limiting the remaining therapeutic options. Recent multicenter studies report rates of multidrug-resistant (MDR, i.e. resistance to three or more antimicrobial classes) P. aerugi­ nosa ranging from 3% to 50%, with considerable variation between

countries. In the USA, the prevalence of MDR-P. aeruginosa was approximately 15-fold higher than the prevalence of carbapemenresistant Enterobacteriaceae in nationwide data from 2000 to 2009.29,30 The therapy of serious Pseudomonas infections should be based on the results of adequately performed antimicrobial susceptibility testing, including determination of MICs. Of note, in mucoid strains of P. aeruginosa, susceptibility testing with commercial systems may not be accurate, and the addition of agar dilution or gradient diffusion techniques (such as E-test) may be warranted. Data shown in Table 181-9



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

TABLE

181-9 

1591

Antibiotic Susceptibility of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia Isolated from Patients Hospitalized with Pneumonia in US and European Hospitals Acinetobacter spp.

P. aeruginosa

S. maltophilia

USA (n = 251)

Europe (n = 449)

USA (n = 1439)

Europe (n = 1250)

TZP

29.5

20.3

72.9

63.9

n.r.

n.r.

Ceftazidime

31.5

18.3

79.6

68.7

36.8

30.5

Cefepime

27.1

18.7

80.4

72.1

n.r.

n.r.

Meropenem

33.9

28.4

76.3

65.8

n.r.

n.r.

Amikacin

48.6

34.3

96.2

88.8

n.r.

n.r.

Gentamicin

29.5

27.6

87.0

75.2

n.r.

n.r.

Tobramycin

41.0

46.5

91.7

76.9

n.r.

n.r.

Levofloxacin

27.9

16.9

70.5

63.4

75.1

83.8

Minocycline

64.8

63.3

n.r.

n.r.

99.5

99.2

Tigecycline

91.2

98.1

n.r.

n-r-

n.r.

n.r.

Colistin

95.2

97.3

98.9

99.0

47.9

38.7

TMP–SMX

n.r.

n.r.

n.r.

n.r.

96.3

97.7

Antibiotic

USA (n = 302)

Europe (n = 192)

TZP, piperacillin–tazobactam; TMP–SMX, trimethoprim–sulfamethoxazole. n.r., not reported. From Sader et al. Int J Antimicrob Agents 2014; 43: 328-334.

TABLE

181-10 

Empiric Antibiotic Therapy for Acinetobacter baumannii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia Infections

Choices

Organism

Drugs

Indications

Monotherapy

P. aeruginosa

Antipseudomonal penicillins: ticarcillin, piperacillin, azlocillin Cephalosporins: ceftazidime, cefoperazone, cefpirome Carbapenems: imipenem, meropenem Fluoroquinolones: ciprofloxacin Carbapenems: imipenem, meropenem Fluoroquinolones: ciprofloxacin, levofloxacin Polymyxins: colistin (in carbapenem-resistant strains) Glycylcycline: tigecycline (in multidrug-resistant strains) TMP–SMX

Limited to nongranulocytopenic patients, non-lifethreatening infections

P. aeruginosa

Aztreonam, ticarcillin or ceftazidime plus tobramycin, imipenem plus amikacin, ciprofloxacin plus ceftazidime, ciprofloxacin plus fosfomycin

A. baumannii

Combination of two in vivo active compounds such as imipenem plus ciprofloxacin or amikacin, combination of any monotherapy drug with sulbactam or polymyxin TMP–SMX plus ceftazidime Ticarcillin–clavulanate plus ciprofloxacin Ticarcillin–clavulanate plus TMP–SMX Tigecycline plus colistin

Severe P. aeruginosa infections – pneumonia, bacteremia, burns (plus topical), malignant external otitis media (plus surgery), central nervous system infection (plus intrathecal), cystic fibrosis (plus topical, colistin or tobramycin inhalative) Critically ill patients with severe infections; data on sulbactam and polymyxin therapy are from in vitro and animal models and from rare case studies in humans Benefit not determined

A. baumannii

S. maltophilia Conventional combinations

S. maltophilia

Alternatives

P. aeruginosa A. baumannii S. maltophilia

Antipseudomonal penicillin plus fluoroquinolones, aztreonam plus aminoglycoside, aminoglycoside plus fluoroquinolone Various combinations of β-lactams and amikacin, imipenem plus rifampin (rifampicin) Ticarcillin–clavulanate Fluoroquinolones Tigecycline Colistin

indicate current susceptibility rates, but empiric therapy should be based on local susceptibility data owing to considerable geographic variation. Pseudomonas spp. other than P. aeruginosa are generally more susceptible to currently used antimicrobial agents including trimethoprim–sulfamethoxazole (TMP–SMX). Based on in vitro

Due to high and increasing resistance therapy must be based on susceptibility testing results

Salvage therapy, combinations of antibacterial agents that are resistant by in vitro testing should be used with great caution or avoided Situations when alternative treatments are not suitable

susceptibilities, carbapenems and fluoroquinolones remain the drugs of choice, while aminoglycosides have considerably less activity, especially in P. putida and P. fluorescens, which are also resistant to TMP– SMX. While P. stutzeri is usually susceptible to all antipseudomonal agents, higher rates of resistance to aztreonam and β-lactam antibiotics have been reported in P. putida, P. fluorescens and P. oryzihabitans.

1592 TABLE

181-11 

SECTION 8  Clinical Microbiology: Bacteria

Resistance Mechanisms in Acinetobacter baumannii and Pseudomonas aeruginosa

Antibiotic Class

Antibiotics

Organism

Mechanisms

Genetic Basis (examples)

Aminoglycosides

Aminoglycosides

A. baumannii

aph(3’)-VIa and aac(6’)-I

Aminoglycosides Aminoglycosides Aminoglycosides Aminoglycosides Aminoglycosides Gentamicin

A. baumannii A. baumannii P. aeruginosa P. aeruginosa S. maltophilia S. maltophilia

Aminoglycoside-modifying enzymes Ribosomal (16s rRNA) methylation Efflux Enzymatic inactivation Reduced permeability

Chloramphenicol

Chloramphenicol

S. maltophilia

Efflux

SmeDEF, SmeABC, SmrA

Fosfomycin

Fosfomycin

P. aeruginosa

Altered transport system

Chromosomal mutation (GlpT), plasmidmediated (fosA, fosB)

Quinolones

Quinolones Quinolones Quinolones Quinolones Quinolones Quinolones Quinolones

A. baumannii A. baumannii P. aeruginosa P. aeruginosa S. maltophilia S. maltophilia S. maltophilia

Modification of target binding site Efflux Altered DNA gyrase target Efflux Efflux Intrinsic resistance Modification of target binding site

gyrA, parC AdeABC, AdeM gyrA, parC mexCD-oprJ, mexAB-oprM SmeDEF, SmeABC, SmrA qnr Mutation in topoisomerase and gyrase genes

Rifampin (rifampicin)

Rifampin

P. aeruginosa

Altered DNA polymerase target

arr-2

Tetracyclines and glycylcyclines

Tetracyclines Tetracyclines Tetracyclines Tetracyclines

glycylcyclines glycylcyclines glycylcyclines glycylcyclines

A. baumannii A. baumannii A. baumannii S. maltophilia

Tetracycline-specific efflux Ribosomal protection Efflux Efflux

Tet(A), tet(B) tet(M) AdeABC SmeDEF, SmeABC, SmrA

Trimethoprim– sulfamethoxazole

Trimethoprim–sulfamethoxazole

S. maltophilia

β-Lactams

Carbapenems Carbapenems Carbapenems Carbapenems Carbapenems Cephalosporins Cephalosporins

A. baumannii A. baumannii P. aeruginosa P. aeruginosa S. maltophilia P. aeruginosa P. aeruginosa

Multidrug efflux Carbapenemases Altered protein porin D2 Imipenemase Efflux Reduced permeability β-Lactamase inactivation (83%)

Monobactams

P. aeruginosa

β-Lactamase inactivation

Penicillins

A. baumannii

Penicillins β-Lactams

P. aeruginosa A. baumannii

Altered penicillin binding proteins (PBPs) Altered PBP targets β-Lactamases

β-Lactams

A. baumannii

Outer membrane proteins

β-Lactams β-Lactams

A. baumannii S. maltophilia

Efflux β-Lactamase inactivation

and and and and

Treatment of infections due to P. aeruginosa with a single agent is possible using third-generation cephalosporins, such as ceftazidime or cefpirome, carbapenems, such as imipenem or meropenem, or fluoroquinolones, such as ciprofloxacin, if the strain is susceptible.31 A meta-analysis of controlled clinical trials has also shown that monotherapy (β-lactam) provided similar survival compared to combination therapy (β-lactam and aminoglycoside) in patients with cancer and neutropenic fever.32 However, outcome results in individual studies vary considerably, therefore these data may not be entirely applicable to any individual neutropenic patient with BSI.33 In addition, especially with fluoroquinolones and carbapenems, resistance may develop quickly during therapy and close microbiologic monitoring is advised, including repeated determination of susceptibility. In addition to systemic antimicrobial therapy, other interventions, such as surgical debridement or local antimicrobial therapy, have been shown to be useful in specific infections due to P. aeruginosa. Central nervous system infections require bactericidal antibiotics that reach high concentrations in cerebrospinal fluid (CSF). Patients with meningitis require a combination of ceftazidime, which is highly concentrated in CSF when the meninges are inflamed, and an

AdeABC, AdeM aph(3’)-IIb PprA, PprB

Class1 integrons, ISCR elements (sul1,sul2) AdeABC OXA, IMP Chromosomal, orpD VIM, IMP SmeDEF, SmeABC, SmrA Chromosomal AmpC, PSE-1, PSE-3, PSE-4, OXA-10 (= PSE-2), IMP-1, TEM-1, TEM-2, OXA-1 AmpC, PSE-1, PSE-3, PSE-4, OXA-10 (= PSE-2), IMP-1, TEM-1, TEM-2, OXA-1 Chromosomal (altered PBP-4) Chromosomal (altered PBP-4) TEM, SHV, ADCs, VEB, PER, CTX-M, OXA, IMP, VIM, SIM CarO (29-kDa), 22-, 33-, 36-, 43-, 44-, 47-kDa, HMP-AB, OmpW AdeABC Chromosomal (L1, L2)

aminoglycoside. If there is obstruction of the subarachnoid space the aminoglycoside may be instilled directly into the ventricular system. P. aeruginosa brain abscesses may require surgical intervention in addition to prolonged antibiotic therapy.22 Malignant external otitis, which may present as an extremely severe infection, usually requires a combination of local surgical debridement and drainage together with antimicrobial therapy consisting of a (local) aminoglycoside or quinolone preparation and a parenteral antipseudomonal β-lactam. In burn wound sepsis frequent emergence of resistance due to high bacterial counts and limited access of antibiotic to burn sites has been reported. Local procedures with topical agents and surgical debridement of necrotic tissue are always applied in addition to systemic antibiotics.22 Strategies for the treatment of P. aeruginosa pneumonia have emphasized the importance of early initiation of appropriate empiric therapy. Combination therapy is generally recommended (antipseudomonal β-lactam and an aminoglycoside), but there is a poor correlation between clinical response and the in vitro synergistic effects of antibiotics.22 In patients with ventilator-associated pneumonia due



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

to colistin-only susceptible P. aeruginosa, aerosolized colistin may be a beneficial adjunct to intravenous colistin.34 In CF patients, eradication of Pseudomonas spp. as well as other pathogens, such as Burkholderia cepacia or Stenotrophomonas maltophilia, from the airways occurs temporarily only, whatever strategy is used. A recent review did not find any conclusive evidence that oral anti-pseudomonal antibiotics are more or less effective than alternative treatments for either pulmonary exacerbations or long-term treatment.35 Acute exacerbations of Pseu­ domonas lung infection in these patients usually require combination therapy and higher dosing. Fluoroquinolones have been used successfully both in adult and pediatric CF patients, and a combination of ciprofloxacin with fosfomycin has demonstrated in vitro synergy. Repeated courses of aggressive antibiotic therapy every 3 months in combination with other measures such as mucolytics, antiproteases, topical (inhaled) antibiotic therapy and physiotherapy have increased the long-term survival of patients with CF. Treatment options have been extensively reviewed elsewhere. Inhalation therapy utilizing aminoglycosides and colistin is generally well tolerated, although some reduction of the maximum expired volume per second has occasionally been observed. Currently, tobramycin, polymyxin B and aztreonam are being used successfully in prevention and therapy of respiratory tract infections due to P. aeruginosa as well as B. cepacia in patients with CF.

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Figure 181-6  Example of A. non-baumannii spp. Colonies of Acinetobacter lwoffii on TSA-blood agar. (Courtesy of H. Wisplinghoff.)

Prevention Prevention plays a major role in controlling Pseudomonas infections. Preventive measures can be based on the identification of sources and interruption of ways of transmission (see Table 181-3). Guidelines have been established in the USA and Europe, implementing isolation policies, administrative and regulatory measures and hospital epidemiology surveillance. Attempts to reduce the risk of colonization in high-risk patients have included: • elimination of endogenous nosocomial P. aeruginosa and reduction of oropharyngeal, intestinal and skin colonization in ICU patients • prevention of cross-contamination and monitoring of various sources of P. aeruginosa • prevention of contamination in burn patients, in surgical wounds and in the oropharyngeal area in ventilated patients. Active vaccination has been discussed and a variety of antigenic determinants are being evaluated as potential vaccine targets including outer membrane proteins, flagella and pili. In addition, vaccination with live attenuated strains has been shown to be efficacious in animal models. Current approaches to vaccines and immunotherapy against P. aeruginosa have recently been reviewed in detail.36

Acinetobacter spp. Nature and Taxonomy The genus Acinetobacter currently consists of 41 different species, nine of which have not been assigned names (see Table 181-2). While most Acinetobacter spp. (Figure 181-6) are considered to be of minor clinical importance, A. baumannii (see Figure 181-7), A. pittii and A. nosoco­ mialis (together also referred to the A. baumannii group, ABG) have emerged as important clinical pathogens. Due to their phenotypic similarity, these three species have been grouped together with the environmental organism A. calcoaceticus in the so-called A. calcoaceticus–A. baumannii (Acb) complex. Overall, A. baumannii has emerged as one of the most significant nosocomial pathogens, especially in patients with impaired host defenses in the ICU, and has been implicated in a variety of infections including BSI, pneumonia and meningitis, with mortality rates as high as 64%. Major epidemiologic features of these organisms include their propensity for clonal spread and their involvement in hospital outbreaks as well as the ability to express or acquire resistance determinants, making it one of the most resistant organisms known to date.

Figure 181-7  Acinetobacter baumannii on TSA-medium. (Courtesy of H. Wisplinghoff.)

Epidemiology Acinetobacter spp. are widely distributed in nature, but not all Acineto­ bacter spp. are found in the environment (some, such as A. schindleri and A. ursingii, have until now been recovered only from human specimens) and the clinically important species (e.g. A. baumannii) are in fact not ubiquitous even though reservoirs outside the hospital have been described.3,37 A variety of Acinetobacter spp. recovered from the axilla, groin and toe webs, the oral cavity, the respiratory tract and normal intestine have been identified as part of the commensal human flora. In contrast, some Acinetobacter spp., such as A. baylyi, A. bou­ vetii, A. grimontii, A. tandoii, A. tjernbergiae and A. towneri, have as yet never been observed in human specimens. Studies investigating the colonization of human skin and mucous membranes found Acineto­ bacter spp. in up to 44% of nonhospitalized and up to 75% of hospitalized individuals. The most frequently isolated species were A. lwoffii (58–61%), A. johnsonii (20%), Acinetobacter genomic species 15BJ (12%), A. junii (10%), A. radioresistens (8%) and A. pittii (5%), whereas A. baumannii was found only rarely on human skin (0.5–3%) and in human feces (0.8%).38 Recent studies investigating potential skin contamination in healthy US soldiers did not report any Acineto­ bacter spp., but the lack of enrichment techniques and long transport time may have contributed to this finding. In addition, seasonal and geographic variations in skin colonization with Acinetobacter spp. have been reported from different geographic locations where A. pittii (36%), A. nosocomialis (15%), Acinetobacter genomic species 15TU (6%) and A. baumannii (4%) were the most frequently recovered species, whereas A. lwoffii, A. johnsonii and A. junii were only rarely found.39,40

MOLECULAR EPIDEMIOLOGY After 1986, the taxonomy of the genus Acinetobacter was revised when molecular methods enabled identification of Acinetobacter at the species level, and studies of the epidemiology of the different members of this genus became possible. Methods include ribotyping, pulsed-field gel electrophoresis, random amplified polymorphic

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SECTION 8  Clinical Microbiology: Bacteria

DNA analysis, amplified fragment length polymorphism, multilocus sequence typing41,42 and polymerase chain reaction/electrospray ionization mass spectrometry.43 Acinetobacter outbreaks published between 1977 and 2004 have been extensively reviewed.44,45 Recent studies have increasingly reported outbreaks involving MDR A. baumannii. The persistence of endemic strains of A. baumannii over an extended period of time and the spread of single clones within a medical center have been documented in several institutions worldwide. Some outbreaks have been linked to a common source, such as computer keyboards, blood pressure cuffs, enteral nutrition or dust inside of mechanical ventilators or continuous venovenous hemofiltration dialysis machines, others do not seem to have a common source despite extensive environmental surveillance. Several large studies failed to detect interinstitutional spread of A. baumannii while studies from New York, London and Johannesburg reported involvement of several different medical centers within a city, or even of healthcare facilities in several cities within one country. Following reports of the European clones I, II and III,46 some authors suggested that few epidemic strains may be involved in outbreaks across countries; however, no epidemiologic link in time or space could be established. Studies investigating the population structure using MLST and REP-PCR show evidence of the distribution of several A. baumannii-clones designated as worldwide or international clones.47,48

Diagnostic Microbiology Acinetobacter spp. are nonfermenting, nonmotile, oxidase-negative, aerobic gram-negative coccobacilli (Figure 181-8) and may be mistaken for gram-negative (or even gram-positive) cocci. Strictly aerobic, Acinetobacter spp. grow on most routinely used media at temperatures of 20–44 °C (68–111 °F). Acinetobacter spp. isolated from human specimens grow readily at 37 °C (99 °F). In contrast to the species of the Acb complex, other Acinetobacter spp. do not grow on MacConkey agar or may show hemolysis on sheep blood agar (A. haemolyticus, Acineto­ bacter genomic species 6, 13BJ, 14BK, 15BJ, 16 and 17). Presumptive identification at genus level is possible using the above-mentioned criteria; unambiguous identification of Acineto­ bacter spp. is possible by transformation of the naturally transformable tryptophan auxotroph A. baylyi ADP1 by crude DNA of any Acineto­ bacter to wild-type phenotype.49 Phenotypic identification of 11 of the 12 initially described (but not the novel) species is possible using a scheme proposed by Bouvet and Grimont.50 A variety of molecular methods may be used for identification to species level.41 While most

of these methods are not suitable for the routine laboratory, MALDITOF mass spectrometry can be used to identify at least the members of the A. baumannii group and may be an interesting option in the future for identification of all Acinetobacter spp.51 Species identification using current commercial systems such as API 20NE, Vitek 2, Phoenix and MicroScan WalkAway remains problematic, especially since A. baumannii, A. pittii and A. nosocomialis are uniformly identified as A. baumannii by the most widely used identification systems.2

Pathogenicity and Pathogenesis Several factors may be responsible for the virulence of Acinetobacter spp., including a polysaccharide capsule formed of L-rhamnose, Lglucose, D-glucuronic acid and D-mannose, factors facilitating adhesion to human epithelial cells in the presence of fimbriae and/or mediated by the capsular polysaccharide; enzymes, such as butyrate esterase, caprylate esterase and leucine arylamidase, which are potentially involved in damaging tissue lipids; and the LPS component of the cell wall and the presence of lipid A, which are likely to participate in the pathogenicity of Acinetobacter spp. Recent studies using whole genome sequencing identified a large number of antibiotic drug resistance determinants as well as several pathogenicity islands,52 some of which likely originated in other species including Pseudomonas spp., Salmonella spp. and E. coli.52 Relevant genes included those encoding the cell envelope, pilus biogenesis, iron uptake and metabolism, as well as sensor kinases. Several studies in A. baumannii have described siderophore-mediated iron acquisition systems, biofilm formation, adherence and outer membrane protein function and a specific lipopolysaccharide. The LPS of Acinetobacter seems to be equally potent to E. coli LPS at similar concentrations. Humoral immune responses include antibodies targeted toward iron-repressible outer membrane proteins and the O-polysaccharide component of LPS that have bactericidal and opsonizing in vitro activity. While several potential host response mechanisms have been described in recent studies, the role of the host responses in the pathogenesis of A. baumannii infections remains to be determined.

CLINICAL MANIFESTATIONS Acinetobacter spp. (mainly A. baumannii, A. pittii and A. nosocomialis) have been implicated as the causative pathogen in nearly all types of nosocomial infection, including BSI, pneumonia, urinary tract infection, wound infection and meningitis (see Table 181-5). Overall, clinical significance remains controversial. Some studies report high mortality in patients with pneumonia and BSI, others argue that these rates are associated rather with the underlying conditions. Crude mortality in patients with A. baumannii BSI ranges from 32% to 52%,53 but mortality rates as high as 73% have been reported in patients with meningitis due to A. baumannii. Acinetobacter spp. other than the ABG, such as A. johnsonii, A. junii, A. lwoffii, A. parvus, A. radioresistens, A. schindleri or A. ursingii, have been isolated from clinical specimens, representing transient colonizers of the human skin rather than true pathogens.54 Community-acquired infections – with the exception of A. baumannii pneumonia – are less common and usually less severe. In contrast to other pathogens, A. baumannii infections are frequently reported in association with natural disasters (Marmara earthquake, 1999; Indian Ocean tsunami, 2004) or military operations (Operation Enduring Freedom, from 2001).

Pneumonia

Figure 181-8  Morphology of Acinetobacter baumannii on Gram stain. (Courtesy of H. Wisplinghoff.)

A. baumannii accounts for 5–10% of cases of ICU-acquired pneumonia in the USA and is usually observed in patients with a prolonged ICU stay. In a recent series analyzing almost 13 000 patients hospitalized with pneumonia in the USA and Europe, Acinetobacter spp. were the eighth (USA) and fifth (Europe) most common pathogens, accounting for 3.7 and 7.5% of isolates, respectively.55 Predisposing factors include endotracheal intubation, surgery, prior antibiotic therapy and underlying pulmonary disease. The clinical presentation



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

may include multilobular involvement, pleural effusion and formation of a bronchopulmonary fistula. Mortality rates of up to 70% have been reported, but this may reflect the patients’ underlying condition rather than the virulence of the organism. Community-acquired pneumonia due to A. baumannii, which is characterized by a fulminant clinical course, secondary BSI and high mortality of 40–60%, has mainly been reported from tropical regions of Australia and Asia, affecting patients with impaired host defenses (diabetes, renal failure, chronic alcohol abuse) or underlying pulmonary disease.

Bloodstream Infection In a recent series analyzing almost 25 000 cases of nosocomial BSI, A. baumannii was the tenth most common pathogen, accounting for 1.3% of all monomicrobial BSIs (0.6 BSIs per 10 000 admissions), occurring late during hospitalization (mean, 26 days after admission).21 Sources include intravascular catheters, pneumonia, urinary tract infection and wound infection. Crude mortality rates in patients with A. baumannii BSI ranged from 16.3% to 43.4% and was exceeded only by P. aeruginosa and Candida spp. Even though studies reported a significantly higher mortality in A. baumannii BSI, none of the studies formally adjusted for severity of illness or co-morbidities. Aci­ netobacter spp. other than the members of the ABG – in particular A. johnsonii, A. lwoffii, as well as A. haemolyticus, A. junii, A. radioresistens and Acinetobacter genomic species,10 A. ursingii and A. schindleri – have been mainly associated with catheter-related BSIs.

Wound Infection A. baumannii has been implicated in 2.1% of ICU-acquired skin/soft tissue infections, but has been isolated from up to 32.5% of wound infections in combat casualties sustained in Iraq or Afghanistan. Even though colonization is one of the major risk factors for BSI in these patients, the impact of A. baumannii infection on the outcome of burn patients or combat casualties remains to be determined. While a combination of early microbiologic diagnosis, adequate antimicrobial therapy, surgical debridement and early wound closure may be effective, there are no data on the impact of A. baumannii colonization on the wound healing process.56

Miscellaneous A. baumannii is responsible for 1.6% of ICU-acquired UTI. Most cases of pyelonephritis and other UTIs have been associated with a urinary catheter or nephrolithiasis. Nosocomial, post-neurosurgical A. bau­ mannii meningitis has increasingly been reported. Risk factors include neurosurgery and external ventricular drainage. Crude mortality rates of up to 73% have been reported. Other infections include endocarditis (commonly associated with prosthetic valves), endophthalmitis or keratitis, as well as osteomyelitis and arthritis.

Management and Resistance ANTIBIOTIC RESISTANCE Since 1980 successive surveys have shown increasing resistance in clinical isolates of A. baumannii. High proportions of strains are now resistant to the most commonly used antibacterial drugs, including aminopenicillins, ureidopenicillins, cephalosporins of the first (cephalothin) and second generation (cefamandole), cephamycins, such as cefoxitin, chloramphenicol and tetracyclines, and resistance to all known antibiotics (i.e. pandrug resistance) has been reported. In addition, with reports of resistance to polymyxins and tigecycline there is currently no antimicrobial agent to which A. baumannii can be considered uniformly susceptible. Therapy of serious A. baumannii infections should be based on the results of adequately performed antimicrobial susceptibility testing, and empiric therapy should consider recent institutional level susceptibility data.2 To date, carbapenems remain the agents of choice for serious A. baumannii infections, but increasing resistance (up to 70%) has been reported from several countries, including Portugal, Spain, France and the USA.48 The increasing prevalence of carbapenemresistant A. baumannii isolates (CRAB) is highly problematic since

1595

there are few therapeutic options and studies have indicated that patients with CRAB infections may have a worse prognosis. In the case of multi- or pandrug-resistant strains, combination therapy or the use of agents such as colistin may be considered. Even though polymyxins remain highly active in recent in vitro studies, there are increasing reports of resistant strains and clinical data suggest that combination therapy may be benficial.57 Tigecycline remains controversial, despite being active in vitro against most strains including CRAB, because several studies reported development of resistance or the emergence of A. baumannii infections despite tigecycline therapy as well as higher mortality associated with a tigecycline MIC of ≥2 mg/L.58,59 Several recent studies summarize the currently available antimicrobial agents and their potential use in the therapy of A. baumannii infections, even though there are no data from prospective trials and most recommendations are based on in vitro data and small case series.

Burkholderia spp. Nature and Taxonomy Burkholderia spp. (as well as Ralstonia spp.) were transferred from rRNA group II of the former genus Pseudomonas. The genus Burkhold­ eria currently consists of more than 60 species, most of which have been assigned species names. Some of the novel Burkholderia spp. have recently been reclassified as Pandoraea species. Burkholderia spp. are ubiquitous organisms, being widespread in water, soil and plants, and are present in the human environment. The B. cepacia complex currently harbors nine genovars: I: B. cepacia II: B. multivorans, B. gladioli III: genovar III, B. cenocepacia IV: B. stabilis V: B. vietnamensis VI: genovar VI VII: B. ambifaria VIII: B. anthina IX: B. pyrrocinia and represents the most frequently isolated clinical pathogen among the Burkholderia spp., followed by B. mallei and B. pseudomallei. It has recently been proposed that five other species be added to the novel species within the B. cepacia complex: Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkhold­ eria seminalis sp. nov. and Burkholderia metallica sp. nov.60

Epidemiology Burkholderia spp. can be isolated from a variety of environmental sources. B. cepacia has no specific nutritional requirements and may survive for months in water, sinks, antiseptic solutions (chlorhexidine, quaternary ammoniums, povidone–iodine) and pharmaceutical products. It may also survive on environmental surfaces4 and has been found in nebulizers and other medical devices (see Table 181-6). Person-to-person transmission has been reported for strains of the B. cepacia complex and B. pseudomallei, but so far there have been no reports for B. mallei and B. gladioli. Overall, about 3% of pediatric and 7% of adult CF patients are colonized or infected with strains of the B. cepacia complex, with a considerable geographic variation of species. Ralstonia pickettii (B. pickettii) and B. gladioli are ubiquitous organisms that can be found in water and soil and may play a role as nosocomial pathogens. Rare outbreaks of infection have been described and emergence of multidrug resistance is a potential problem.4 B. mallei and B. pseudomallei are predominantly found in Asia, Africa and South America. B. pseudomallei, the causative agent of melioidosis, can be isolated from environmental samples such as soil and water. Transmission to humans usually occurs by percutaneous inoculation, ingestion or inhalation from the environment; however,

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SECTION 8  Clinical Microbiology: Bacteria

person-to-person transmission, zoonotic disease and laboratoryacquired infections have been reported. B. mallei is the causative agent of glanders, a disease primarily affecting horses and donkeys. In contrast to B. pseudomallei, B. mallei does not survive in the environment, although laboratory infections have been reported.61

commonly associated with BSI and sepsis (see Chapter 125). Clinical presentation of BSI may range from transient bacteremia to fulminant septic shock with mortality rates of up to 87%.

Diagnostic Microbiology

In vitro efficacy against B. cepacia has been demonstrated for ureidopenicillins, third-generation cephalosporins, carbapenems, fluoroquinolones, TMP–SMX, chloramphenicol and minocycline.65 Among the novel antimicrobials, tigecycline has shown less activity compared to minocycline. Resistance to several classes of antimicrobial agents is often observed, especially in patients receiving multiple courses of antibiotics over a prolonged period of time. Therapy of B. cepacia infections should be based on the results of antimicrobial susceptibility testing. In cases of multi- or pandrug-resistant strains, combination therapy is recommended and the use of agents such as polymyxins may be considered. In addition, inhalation therapy in combination with intravenously administered antimicrobials can control pulmonary exacerbation by B. cepacia infection.66 Management of B. mallei and B. pseudomallei infections is discussed elsewhere in this book (see Chapter 125) and has been recently reviewed.64

Commercial test systems such as API 20NE, Phoenix, MicroScan and Vitek 2 can identify bacteria of the B. cepacia complex. However, misidentification (B. gladioli as B. cepacia) is frequent and differentiation of species within the complex, as well as differentiation from other NFGNB such as Achromobacter spp. or Ralstonia spp., may require additional testing. B. pseudomallei and B. mallei cannot be distinguished by morphology or serologic tests and may be falsely identified as B. cepacia, P. stutzeri or other Pseudomonas spp., and molecular methods such as 16S rRNA gene sequencing or MALDI-TOF MS may be required for species identification.62

Pathogenicity and Pathogenesis Although only weakly virulent with a limited invasive capacity in the normal host, B. cepacia has become an important nosocomial pathogen that has been isolated from a variety of nosocomial infections including BSI, UTI, arthritis, peritonitis, endophthalmitis and pneumonia,22 most commonly in patients with impaired host defenses such as CF patients or patients in the ICU. The predominant site of infection is the respiratory tract (Table 181-1). Virulence factors of B. cepacia include exoproducts (proteases, lipases, exopolysaccharides) that act in addition to the LPS-forming part of O-antigen and which are responsible for severe pneumonia and sepsis in CF patients. Cellular virulence factors have recently been reviewed in detail.63 Attachment to epithelial cells is mediated by pili, followed by penetration, biofilm formation and invasion, which is in part aided by flagella and LPS. In addition, intracellular survival of clinical B. cepacia has been demonstrated. As in Pseudomonas spp., quorum-sensing system(s) may be responsible for the regulation of virulence factors. In B. pseudomallei, host-related factors such as diabetes, chronic renal disease or alcoholism seem to play a major role in the acquisition and clinical course of disease. Production of LPS and the ability of B. pseudomallei to survive intracellularly have been identified as important factors in the pathogenesis of melioidosis.

Clinical Manifestations While several Burkholderia spp. such as B. gladioli, B. thailandiensis or B. oklahomensis have been isolated only occasionally from clinical specimens, the B. cepacia complex (including B. cepacia, B. multi­ vorans, B. cenocepacia), B. mallei and B. pseudomallei are classified as human pathogens. B. gladioli has been reported to cause disease in patients with CF or chronic granulomatous disease and other immunocompromising conditions. Respiratory tract infection and pneumonia are the most frequent manifestations of the B. cepacia complex, mainly affecting patients with CF. Most commonly isolated species are B. cenocepacia, B. mul­ tivorans and in some series B. cepacia, even though with the exception of B. ubonensis all species of the B. cepacia complex have been recovered from clinical specimens (see Table 181-1). In patients with CF, increased mortality and a higher rate of fatal complications following lung transplantation has been associated with B. cepacia colonization. While chronic colonization of the respiratory tract with little or no clinical symptoms is frequent, cases of fulminant necrotizing pneumonia and sepsis with a rapidly fatal outcome have been reported. In addition, B. cepacia has been associated with catheter-related BSI, ventilator-associated pneumonia, and skin and soft tissue infections following burns, surgery or invasive diagnostic procedures. B. mallei and B. pseudomallei can cause glanders and melioidosis in humans. Clinical manifestations of melioidosis (B. pseudomallei, recently reviewed in detail64) in humans include pneumonia, genitourinary manifestations, osteomyelitis and skin or soft tissue abscesses,

Management and Resistance

Stenotrophomonas maltophilia Nature and Taxonomy Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has also emerged as an important nosocomial pathogen contributing substantially to morbidity and mortality of immunocompromised patients, particularly in the ICU setting.3

Epidemiology S. maltophilia is a water-borne organism that can be readily isolated from soil, plants, water and raw milk. It can also be recovered from the hospital environment where it has been isolated from a variety of sources such as ventilatory equipment, nebulizers, endoscopes, prosthetic devices, as well as dialysis fluids and antiseptic solutions (see Table 181-6). There is a high incidence of infections due to S. malto­ philia in immunocompromised patients such as those with solid malignancies, leukemia or lymphoma.67 In addition, S. maltophilia is increasingly implicated in pulmonary infections in CF patients (see Table 181-1). Recent surveys report a point prevalence of 11% of CF patients with transient colonization with S. maltophilia, even though the importance of S. maltophilia in patients with CF remains to be determined.3

Diagnostic Microbiology S. maltophilia grows readily on most routinely used media and is characterized by the presence of a single or a small number of polar flagella (motile bacteria), frequently pigmented colonies (yellow or yellowishorange) and a negative oxidase reaction, even though some isolates may be oxidase-positive.3 S. maltophilia acidifies sugars (except for rhamnose and mannitol) and is generally proteolytic. Co-isolation of S. maltophilia with other NFGNB, such as A. baumannii, Burkholderia spp. and P. aeruginosa, from respiratory specimens can be challenging. Current automated biochemical panels usually identify S. maltophilia with high certainty as well as molecular methods or MALDI-TOF.3

Pathogenicity and Pathogenesis S. maltophilia produces proteolytic enzymes and other pathogenic extracellular enzymes such as DNAse, RNAse, elastase, lipase, hyaluronidase, mucinase and hemolysin, which may contribute to the severity of S. maltophilia infection. In addition, cytotoxic activity has been reported from clinical isolates. Other pathogenicity factors include LPS, flagella and a diffusible signal factor system which may play a role in adherence to and invasion of bronchial epithelial cells, biofilm formation, chronic colonization and antimicrobial resistance.



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

Pathogenicity, including the role of S. maltophilia in the CF lung environment, has been recently reviewed in detail.3

Clinical Manifestations S. maltophilia has been implicated in respiratory tract infections, endocarditis, bacteremia, meningitis and UTI. In addition, severe cutaneous infections (ecthyma gangrenosum similar to that due to P. aeruginosa), cellulitis and abscesses (wounds resulting from agricultural machinery) have been reported. Several studies listing S. malto­ philia among the top 15 pathogens with recovery rates around 3% from hospitalized patients with pneumonia have been summarized in a recent review.3 In a series analyzing almost 13 000 patients hospitalized with pneumonia in the USA and Europe, S. maltophilia was the sixth (USA) and eighth (Europe) most common pathogen, accounting for 4.4% and 3.2% of isolates, respectively. Crude mortality in patients with S. maltophilia BSI ranges from 14% to 69%, and attributable mortality as high as 37.5% has been reported.3,55

Management and Resistance S. maltophilia displays intrinsic resistance to most classes of antimicrobial agents (see Table 181-8). Mechanisms of resistance include production of several β-lactamases, rendering it susceptible only to latamoxef and combinations of ticarcillin plus clavulanic acid or piperacillin plus tazobactam, as well as carbapenemase production conferring resistance to carbapenems.3,68 Few strains are susceptible to gentamicin, neomycin and kanamycin, and susceptibility to doxycycline is reported in less than 50% of strains. Currently, TMP–SMX remains the drug of choice for treatment of infections due to S. maltophilia, although in vitro studies and retrospective case series indicate that ticarcillin–clavulanic acid, minocycline, some of the new fluoroquinolones, colistin and tigecycline may be alternative agents in case of resistance or allergy.68,69 Due to increasing rates of resistance, therapy should always be guided by susceptibility testing. Of note, susceptibility testing results may not correctly predict clinical treatment response. Severe infections may require combination therapy.

Miscellaneous Aerobic Nonfermenting Gram-Negative Bacteria Many other NFGNB have been identified from clinical specimens, some of which are increasingly involved in human infection.3,70 These genera and species have undergone many taxonomic changes; some have been identified recently and the wide use of analysis of ribosomal 16S RNA gene sequences has allowed a clearer taxonomic position to be established for most of these organisms. The following section includes a short description of the pathogenic role of NFGNB involved in human infections and of the management of these infections. For easy reading, the generic groups are described in alphabetical order. Details regarding conventional identification have been summarized elsewhere.3,70

Alcaligenes spp. (Including Achromobacter spp., Ochrobactrum spp., Kerstersia spp. and Advenella spp.) The genera Alcaligenes, Achromobacter and Ochrobactrum have undergone confusing taxonomic changes in the past decade (see Table 1812). Currently Alcaligenes faecalis remains the only Alcaligenes species of clinical importance, after several other Alcaligenes spp. have been transferred to the genus Achromobacter, including A. denitrificans, A. xylosoxidans, A. ruhlandii and A. piechaudii. The genus Kertersia harbors Alcaligines faecalis strains that have been reclassified as K. gyiorum, while Advenella consists of A. incenata and several other currently unnamed species.

1597

EPIDEMIOLOGY The natural habitat of Alcaligenes spp. is the same as that of Pseu­ domonas spp. In the hospital environment, A. faecalis and A. xylosoxi­ dans have been isolated from various environmental sources such as respirators, hemodialysis systems, intravenous solutions and disinfectants.1

DIAGNOSTIC MICROBIOLOGY Achromobacter spp. and Alcaligines spp. are gram-negative, oxidaseand catalase-positive, indole-negative nonfermenting rods, strictly aerobic and motile, with one to eight peritrichous flagella. In contrast to A. xylosoxidans, A. faecalis, A. piechaudii and A. denitrificans are not saccharolytic. Biochemical identification to species level is not possible for all Alcaligenes spp. or Achromobacter spp.

PATHOGENESIS AND CLINICAL MANIFESTATIONS Alcaligenes spp. and Achromobacter spp. have been isolated from various clinical sources such as blood, feces, sputum, urine, CSF, wounds, burns and swabs taken from throat, eyes and ear discharges. Alcaligenes strains do not seem to possess any specific virulence determinants. They are infrequent causes of hospital-acquired infections in patients who often have severe underlying disease. Rare cases of peritonitis, pneumonia, bacteremia, meningitis and UTI have been reported. In many instances the organism is considered to be a colonizer.71 Nosocomial outbreaks are usually associated with an aqueous source. Alcaligenes spp. are predominantly isolated from respiratory tract specimens and recovery of these organisms from the sputum of CF patients has been associated with an exacerbation of pulmonary symptoms. Alcaligenes xylosoxidans has been implicated in BSI (mostly catheter-related), pneumonia, endocarditis, meningitis, osteomyelitis, peritonitis and urinary tract infection, often in patients with underlying malignancy, HIV and CF.

MANAGEMENT As with other NFGNB, available susceptibility data for Alcaligenes spp. and Achromobacter spp. are based on a limited number of isolates, and antimicrobial therapy should be guided by appropriate susceptibility testing. Alcaligenes faecalis is generally resistant to aminoglycosides, chloramphenicol and tetracyclines and usually susceptible to TMP– SMX and β-lactam antibiotics such as ureidopenicillins, ticarcillin– clavulanic acid, cephalosporins and carbapenems. A. xylosoxidans is usually susceptible to ureidopenicillins, imipenem and polymyxins and variably resistant to fluoroquinolones. In contrast to A. faecalis, Achromobacter spp. are often resistant to cephalosporins. There have been several reports of resistance to broad-spectrum penicillins in A. xylosoxidans due to constitutive β-lactamase production. Kerstersia spp. are usually susceptible to ciprofloxacin and cefotaxime.

Bergeyella spp. and Weeksella spp. Bergeyella zoohelcum and Weeksella virosa (see Table 181-2) have been implicated in infections in humans. W. virosa has been recovered from urine and vaginal swabs, B. zoohelcum has been isolated from wound infections following animal bites, but individual cases of BSI and meningitis have also been reported.72 Both grow as pigmented colonies (brown or yellow) and can be distinguished by urease (positive in B. zoohelcum) and susceptibility to polymyxin B (B. zoohelcum is resistant). Both organisms are usually susceptible to most antimicrobial agents, but susceptibility testing is nevertheless warranted in all cases.

Chryseobacterium spp., Elizabethkingia spp., Flavobacterium spp. and Myroides spp. The clinically important Chryseobacterium spp. (C. meningosepticum and C. indologenes) have been reclassified from the genus Flavobacte­ rium (see Table 181-2), while other Flavobacterium spp. such as F.

1598

SECTION 8  Clinical Microbiology: Bacteria

multivorum and F. spiritivorum have been moved to the genus Sphin­ gobacterium. In addition, F. odoratum has been reclassified as Myroides odoratus and M. odoratimimus. C. meningosepticum has been reclassified as Elizabethkingia menin­ goseptica.73 Chryseobacterium spp. and Elizabethkingia spp. are ubiquitous organisms that can be found in soil and water and have also been recovered from foods and the hospital environment. Epidemiologic studies have traced the bacterial source to contaminated water, ice machines and humidifiers. Phenotypic markers used for the delineation of outbreaks of E. meningoseptica infections were serology based on the O-antigenic type; nine O-serovars have been identified (A–H and K).

MICROBIOLOGY Chryseobacterium spp. usually grow between 5° and 30° C (41° and 86 °F), but strains isolated from human specimens including E. menin­ goseptica readily grow at 37 °C (99 °F). On nutrient agar, colonies are 1–2 mm in diameter, and are frequently pigmented light yellow or yellowish-orange. The metabolism is strictly aerobic, except for M. odoratus and Sphingobacterium multivorum which do not acidify glucose. Indole-positive species (i.e. E. meningoseptica, C. gleum) are usually strongly proteolytic; esculin, citrate and urease tests are variably positive.

CLINICAL MANIFESTATIONS E. meningoseptica and C. indologenes have been isolated from patients with sepsis, osteomyelitis, meningitis and endocarditis.74 Meningitis due to E. meningoseptica has often been observed in neonates but has been reported infrequently in immunocompromised adult patients. BSIs due to E. meningoseptica, E. miricola and C. indologenes have been associated with intravascular catheters or contaminated infusates and often present as benign transient bacteremia. In otherwise healthy individuals E. meningiseptica has been implicated in cellulitis, arthritis and community-acquired pneumonia.74

MANAGEMENT E. meningoseptica and Chryseobacterium spp. are intrinsically resistant to many commonly used antimicrobial agents, including aminoglycosides, third-generation cephalosporins, penicillins (mezlocillin, piperacillin, ticarcillin), aztreonam, imipenem and tetracycline. However, most of these species, including E. meningoseptica, are generally susceptible to agents that are usually active against gram-positive bacteria such as rifampin (rifampicin), clindamycin, erythromycin and vancomycin. Cases of neonatal sepsis have been treated with clindamycin combined with piperacillin. Recent studies have reported the highest in vitro activities in minocycline, rifampin, TMP–SMX and levofloxacin. One reported case of E. miricola BSI has been successfully treated with tigecycline and levofloxacin. C. indologenes is uniformly resistant to aztreonam, third-generation cephalosporins, aminoglycosides, erythromycin, clindamycin, vancomycin and teicoplanin. Therapy should be guided by antimicrobial susceptibility testing using MICs, since disk-diffusion results are unreliable in predicting susceptibility of Chryseobacterium spp.70

Comamonas spp., Delftia spp. and Acidovorax spp. Previously designated as Pseudomonas rRNA homology group III, the family Comamonadaceae now includes the genera Comamonas, Delftia and Acidovorax. The genus Comamonas consists of four named species – C. aquatica, C. kerstersii, C. terrigena and C. testosteronei – that have been isolated from human specimens, as well as several other species that so far have been recovered from environmental samples only. The genus Delftia consists of D. acidovorans, formerly designated Comamo­ nas acidovorans. Three clinically relevant species – Acidovorax facilis, A. delafieldii and A. temperans – currently belong to the genus Acidovo­

rax in addition to several environmental species that have recently been identified or reclassified from the genus Pseudomonas. Members of these genera are aerobic, gram-negative, oxidasepositive rods that are commonly found in soil, water and on plants but are seldom implicated in human infections. Rare cases of catheterrelated bacteremia (C. testosteroni, D. acidovorans, Acidovorax spp.), meningitis (C. testosteroni), endocarditis (C. testosteroni, D. acido­ vorans), conjunctivitis (C. testosteroni) and otitis media (D. acidov­ orans) have been reported.75

Ochrobactrum spp. Derived from the genus Achromobacter, two species have been recognized as clinical pathogens: Ochrobactrum anthropi and O. interme­ dium. These nonfastidious bacteria grow readily on most conventional media and can be identified to genus level by classic biochemical tests; however, no biochemical reaction can separate the two species. Ochrobactrum spp. are environmental organisms and are considered opportunistic pathogens of low virulence in humans. O. anthropi has been associated with catheter-related BSIs, and individual cases of meningitis, endocarditis and other infections have been published. In contrast, O. intermedium has been implicated only in one case of pyogenic liver infection; however, due to the biochemical indistinguishability, these data should be interpreted with caution. Ochrobactrum spp. are usually resistant to most β-lactam antibiotics except carbapenems. Aminoglycosides (except tobramycin in O. intermedium), fluoroquinolones, tetracycline and TMP–SMX are usually active. In addition, O. intermedium is resistant to polymyxins.70

Oligella spp. This genus was created in 1987 and includes O. urethralis (derived from Moraxella urethralis and CDC group M-4) and O. ureolytica (derived from CDC group IVe). These small rods, often occurring in pairs, grow slowly on blood agar and exhibit only limited metabolic activity. They are oxidase- and catalase-positive. O. ureolytica is motile, while O. urethralis is not. Both species have been implicated in bacteremia, arthritis and genitourinary infections including urosepsis, although the causative role could not be established in all cases.76 Both species are usually susceptible to most antimicrobial agents, with O. ureolytica being the more resistant species. Therapy should be guided by the results of antimicrobial susceptibility testing.

Pandoraea spp. Pandoraea spp. are aerobic, gram-negative, nonspore-forming rods that are usually isolated from soil, water, plants, fruits and vegetables. Pandoraea spp. have been recovered from blood cultures and other specimens in patients with CF or other predisposing pulmonary conditions.4

Psyrobacter spp. Most of the more than 30 species of the genus Psyrobacter have so far not been isolated from human specimens. Recent studies using 16S rRNA data suggest that P. faecalis and P. pulmonis (both coccoid gramnegative rods) are the only species isolated from clinical material, and infections attributed to P. immobilis in earlier reports may have been also due to one of the other Psyrobacter spp.70

Ralstonia spp. and Cupriavidus spp. Ralstonia spp. were reclassified from Pseudomonas rRNA group II and originally consisted of R. pickettii (formerly Burkholderia pickettii or Pseudomonas pickettii) and R. eutropha (formerly Alcaligenes eutro­ phus). More recently, R. eutropha has been reclassified as Cupriavidus necator, after intermittently being named Wautersia eutropha.77 R. mannitolilytica (formerly R. pickettii biovar 3) has been reported to account for the majority of infections due to Ralstonia spp. and has been implicated in nosocomial outbreaks. C. pauculus has been associated with BSI and peritonitis. While other species such as R. pickettii,



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli

R.insidiosa, C. respiraculi and C. metallidurans have been recovered from CF patients, their role has not been entirely clarified.2,77,78

Rhizobium spp. (Formerly Agrobacterium spp.) The genus Rhizobium currently consists of four species – R. radio­ bacter, R. rhizogenes, R. rubi and R. vitis – all of which were transferred from the genus Agrobacterium (see Table 181-2). Rhizobium spp. are phytopathogenic organisms, present in water, soil and environmental plants; they are strictly aerobic coccobacilli, motile with peritrichous flagella (one to six). They grow easily on conventional media, produce oxidase and catalase and can be identified by most commercially available systems. Thus far, only R. radiobacter has been implicated in infections in humans, mostly device-related. Individual cases of endocarditis, catheter-related BSI, peritonitis and UTI have been published.79 Rhizobium spp. are generally susceptible to cephalosporins (secondand third-generation), ticarcillin, imipenem, tetracyclines, colistin, TMP–SMX and fluoroquinolones. In device-related infections, removal of the device may be necessary.79

Shewanella spp. Shewanella putrefaciens, formerly CDC group Ib, Alteromonas, Pseudo­ monas and Achromobacter putrefaciens, currently belongs to the genus Shewanella, which also includes Shewanella algae. Shewanella spp. grow in media used for Enterobacteriaceae and produce H2S, which may result in misidentification as Salmonella spp. or Proteus spp., even though Shewanella spp. are nonfermenting. S. putrefaciens is present

1599

in the environment and has occasionally been isolated from meningitis, otitis media, keratitis, intra-abdominal infections and bacteremia, most cases occurring in immunocompromised patients.80 In contrast, S. algae accounts for the majority of clinical isolates and has been associated with a broad range of diseases, including BSI, peritonitis, osteomyelitis, skin and soft tissue infections and otitis media.80 Shewanella spp. are generally resistant to penicillins but susceptible to third-generation cephalosporins, imipenem, ciprofloxacin, aminoglycosides, TMP–SMX and tetracyclines.80

Sphingobacterium spp. Two species of the genus Sphingobacterium, S. multivorum and S. spiri­ tivorum, are derived from several Flavobacterium spp. and CDC groups IIk-2 and -3 (see Table 181-2). In addition, the genus also harbors S. antarcticum, S. faecium, S. thalpophilum and Sphingobacterium genospecies 1 and 2. S. mizutaii has been transferred to the genus Flavobac­ terium. Other species formerly included in the genus, S. heparinum and S. piscium, have been reclassified as Pedobacter spp., none of which has been implicated in clinical manifestations in humans. Sphingobacte­ rium spp. are characterized by colonies that develop a yellow pigment after a few days at room temperature. S. multivorum, S. spiritivorum and S. thalpophilum have been isolated from a variety of infections, including BSIs, peritonitis, wound infections, UTI and abscesses.81 Sphingobacterium spp. in vitro are usually resistant to aminoglycosides and polymyxin B and susceptible to fluoroquinolones and TMP–SMX. References available online at expertconsult.com.

KEY REFERENCES Antunes L.C., Visca P., Towner K.J.: Acinetobacter bauman­ nii: evolution of a global pathogen. Pathog Dis 2013; 71(3):292-301. Brooke J.S.: Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 2012; 25(1):2-41. Eveillard M., Kempf M., Belmonte O., et al.: Reservoirs of Acinetobacter baumannii outside the hospital and potential involvement in emerging human communityacquired infections. Int J Infect Dis 2013; 17:e802-e805. Higgins P.G., Dammhayn C., Hackel M., et al.: Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother 2010; 65:233-238.

Mathee K., Narasimhan G., Valdes C., et al.: Dynamics of Pseudomonas aeruginosa genome evolution. Proc Natl Acad Sci USA 2008; 105:3100-3105. Peleg A.Y., Seifert H., Paterson D.L.: Acinetobacter bauman­ nii: the emergence of a successful pathogen. Clin Micro­ biol Rev 2008; 21(3):538-582. Polverino E., Torres A., Menendez R., et al.: Microbial aetiology of healthcare associated pneumonia in Spain: a prospective, multicentre, case–control study. Thorax 2013; 68:1007-1014. Samonis G., Karageorgopoulos D.E., Maraki S., et al.: Ste­ notrophomonas maltophilia infections in a general hospi-

tal: patient characteristics, antimicrobial susceptibility, and treatment outcome. PLoS ONE 2012; 7:e37375. Wiersinga W.J., Currie B.J., Peacock S.J.: Melioidosis. N Engl J Med 2012; 367:1035-1044. Zilberberg M.D., Shorr A.F.: Prevalence of multidrugresistant Pseudomonas aeruginosa and carbapenemresistant Enterobacteriaceae among specimens from hospitalized patients with pneumonia and bloodstream infections in the United States from 2000 to 2009. J Hosp Med 2013; 8:559-563.



Chapter 181  Pseudomonas spp., Acinetobacter spp. and Miscellaneous Gram-Negative Bacilli 1599.e1

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42. Diancourt L., Passet V., Nemec A., et al.: The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS ONE 2010; 5:e10034. 43. Ecker J.A., Massire C., Hall T.A., et al.: Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry. J Clin Microbiol 2006; 44:2921-2932. 44. Villegas M.V., Hartstein A.I.: Acinetobacter outbreaks, 1977–2000. Infect Control Hosp Epidemiol 2003; 24:284295. 45. Fournier P.E., Richet H.: The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis 2006; 42:692-699. 46. Nemec A., Dolzani L., Brisse S., et al.: Diversity of aminoglycoside-resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones. J Med Microbiol 2004; 53:1233-1240. 47. Higgins P.G., Dammhayn C., Hackel M., et al.: Global spread of carbapenem-resistant Acinetobacter bauman­ nii. J Antimicrob Chemother 2010; 65:233-238. 48. Antunes L.C., Visca P., Towner K.J.: Acinetobacter bau­ mannii: evolution of a global pathogen. Pathog Dis 2013; 71(3):292-301. 49. Juni E.: Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J Bacteriol 1972; 112:917-931. 50. Bouvet P.J., Grimont P.A.: Taxonomy of the genus Aci­ netobacter with the recognition of Acinetobacter bau­ mannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., and Acinetobacter junii sp. nov., and emended description of Acinetobacter cal­ coaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 1986; 36:228-240. 51. Espinal P., Seifert H., Dijkshoorn L., et al.: Rapid and accurate identification of genomic species from the Aci­ netobacter baumannii (Ab) group by MALDI-TOF MS. Clin Microbiol Infect 2012; 18:1097-1103. 52. Fournier P.E., Vallenet D., Barbe V., et al.: Comparative genomics of multidrug resistance in Acinetobacter bau­ mannii. PLoS Genet 2006; 2:7. 53. Wisplinghoff H., Edmond M.B., Pfaller M.A., et al.: Nosocomial bloodstream infections caused by Acineto­ bacter species in United States hospitals: clinical features, molecular epidemiology, and antimicrobial susceptibility. Clin Infect Dis 2000; 31:690-697. 54. Wisplinghoff H., Seifert H.: Infektionen mit Acineto­ bacter baumannii – Klinische Bedeutung und Therapieoptionen. Hyg Med 2012; 37:8-15. 55. Sader H.S., Farrell D.J., Flamm R.K., et al.: Antimicrobial susceptibility of Gram-negative organisms isolated from patients hospitalised with pneumonia in US and European hospitals: results from the SENTRY Antimicrobial Surveillance Program, 2009–2012. Int J Antimi­ crob Agents 2014; 43:328-334. 56. Tekin R., Dal T., Bozkurt F., et al.: Risk factors for nosocomial burn wound infection caused by multidrug resistant Acinetobacter baumannii. J Burn Care Res 2014; 35:e73-e80. 57. Batirel A., Balkan I.I., Karabay O., et al.: Comparison of colistin–carbapenem, colistin–sulbactam, and colistin plus other antibacterial agents for the treatment of extremely drug-resistant Acinetobacter baumannii bloodstream infections. Eur J Clin Microbiol Infect Dis 2014; 33(8):1311-1322. 58. Seifert H., Stefanik D., Wisplinghoff H.: Comparative in vitro activities of tigecycline and 11 other antimicrobial agents against 215 epidemiologically defined multidrug-resistant Acinetobacter baumannii isolates. J Antimicrob Chemother 2006; 58:1099-1100. 59. Chuang Y.C., Cheng C.Y., Sheng W.H., et al.: Effectiveness of tigecycline-based versus colistin-based therapy for treatment of pneumonia caused by multidrugresistant Acinetobacter baumannii in a critical setting: a matched cohort analysis. BMC Infect Dis 2014; 14:102. 60. Vanlaere E., Lipuma J.J., Baldwin A., et al.: Burkholde­ ria latens sp. nov., Burkholderia diffusa sp. nov., Burk­ holderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex. Int J Syst Evol Microbiol 2008; 58:1580-1590.

1599.e2

SECTION 8  Clinical Microbiology: Bacteria

61. Peacock S.J., Schweizer H.P., Dance D.A., et al.: Online report: Management of accidental laboratory exposure to Burkholderia pseudomallei and B. mallei. Emerg Infect Dis 2008; 14:2. 62. Karger A., Stock R., Ziller M., et al.: Rapid identification of Burkholderia mallei and Burkholderia pseudom­ allei by intact cell Matrix-assisted Laser Desorption/ Ionisation mass spectrometric typing. BMC Microbiol 2012; 12:229. 63. Sousa S.A., Ulrich M., Bragonzi A., et al.: Virulence of Burkholderia cepacia complex strains in gp91phox–/– mice. Cell Microbiol 2007; 9:2817-2825. 64. Wiersinga W.J., Currie B.J., Peacock S.J.: Melioidosis. N Engl J Med 2012; 367:1035-1044. 65. Milatovic D., Schmitz F.J., Verhoef J., et al.: Activities of the glycylcycline tigecycline (GAR-936) against 1,924 recent European clinical bacterial isolates. Antimicrob Agents Chemother 2003; 47:400-404. 66. Moss R.B.: Long-term benefits of inhaled tobramycin in adolescent patients with cystic fibrosis. Chest 2002; 121:55-63. 67. Safdar A., Rolston K.V.: Stenotrophomonas maltophilia: changing spectrum of a serious bacterial pathogen in patients with cancer. Clin Infect Dis 2007; 45:1602-1609. 68. Muder R.R.: Optimizing therapy for Stenotrophomonas maltophilia. Semin Respir Crit Care Med 2007; 28:672677.

69. Samonis G., Karageorgopoulos D.E., Maraki S., et al.: Stenotrophomonas maltophilia infections in a general hospital: patient characteristics, antimicrobial susceptibility, and treatment outcome. PLoS ONE 2012; 7:e37375. 70. Vaneechoutte M., Dijkshoorn L., Nemec A., et al.: Aci­ netobacter, Chryseobacterium, Moraxella, and other nonfermentative gram-negative rods. In: Versalovic J., Carroll K.C., Funke G., et al., eds. Manual of clinical microbiology. 10th ed. Washington, DC: ASM Press; 2011. 71. Ledger S.G., Cordy P.: Successful treatment of Alcalig­ enes xylosoxidans in automated peritoneal dialysisrelated peritonitis. Perit Dial Int 2007; 27:596-598. 72. Lin W.R., Chen Y.S., Liu Y.C.: Cellulitis and bacteremia caused by Bergeyella zoohelcum. J Formos Med Assoc 2007; 106:573-576. 73. Kim K.K., Kim M.K., Lim J.H., et al.: Transfer of Chry­ seobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen. nov. as Elizabethkingia meningoseptica comb. nov. and Elizabethkingia miricola comb. nov. Int J Syst Evol Microbiol 2005; 55:12871293. 74. Lee C.H., Lin W.C., Chia J.H., et al.: Communityacquired osteomyelitis caused by Chryseobacterium meningosepticum: case report and literature review. Diagn Microbiol Infect Dis 2008; 60:89-93.

75. Cooper G.R., Staples E.D., Iczkowski K.A., et al.: Coma­ monas (Pseudomonas) testosteroni endocarditis. Cardio­ vasc Pathol 2005; 14:145-149. 76. Escobar Mora S., Marne Trapero C., Gascón Val M., et al.: Urinary infection caused by Oligella urethralis. Aten Primaria 2001; 28:622-623. 77. Vaneechoutte M., Kampfer P., De Baere T.: Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia europha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov. Int J Syst Evol Microbiol 2004; 54:317-327. 78. Forgie S., Kirkland T., Rennie R., et al.: Ralstonia picket­ tii bacteremia associated with pediatric extracorporeal membrane oxygenation therapy in a Canadian hospital. Infect Control Hosp Epidemiol 2007; 28:1016-1018. 79. Chen C.Y., Hansen K.S., Hansen L.K.: Rhizobium radio­ bacter as an opportunistic pathogen in central venous catheter-associated bloodstream infection: case report and review. J Hosp Infect 2008; 68:203-207. 80. Holt H.M., Gahrn-Hansen B., Bruun B.: Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics. Clin Microbiol Infect 2005; 11:347-352. 81. Tronel H., Plesiat P., Ageron E., et al.: Bacteremia caused by a novel species of Sphingobacterium. Clin Microbiol Infect 2003; 9:1242-1244.