Non-fermentative Gram-negative bacteria

International Journal of Antimicrobial Agents 29 Suppl. 3 (2007) S33–S41

www.ischemo.org

Non-fermentative Gram-negative bacteria D.A. Enoch*, C.I. Birkett, H.A. Ludlam Clinical Microbiology & Public Health Laboratory, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QW, UK

Abstract Over the past decade, non-fermenting Gram-negative bacteria have emerged as important opportunistic pathogens in the increasing population of patients who are immunocompromised by their disease or medical treatment. These bacteria are assisted by their ubiquitous distribution in the environment and have a propensity for multiple, intrinsic or acquired drug resistance. The infections that they cause now pose significant problems in terms of treatment and infection control, whilst the commonly observed rapid emergence of bacterial resistance to new antimicrobial compounds raises concerns regarding the clinical lifespan of these agents. Studies are urgently required to assess whether combination therapy can improve the long-term utility of new drugs in the treatment of patients infected with non-fermenters. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Non-fermenters; Pseudomonas aeruginosa; Stenotrophomonas maltophilia; Burkholderia cepacia; Acinetobacter baumannii

1. Introduction Non-fermenting Gram-negative bacteria (“non-fermenters”) are widespread in the environment and are an increasing cause of serious infections in hospital practice, primarily affecting the expanding population of patients immunocompromised by disease or by medical and surgical treatments. Many species are notable for their resistance to multiple antibiotics and the facility with which they may acquire further resistances. Acquisition of resistance may also promote their proliferation in hospitals, leading to significant antibiotic treatment and infection control challenges. There is a pressing need for novel antimicrobials to address the problem caused by these organisms. Non-fermenters comprise numerous species belonging to many genera. This review will focus on the four species most often causing significant problems in hospital practice, namely Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia and members of the Burkholderia cepacia complex, discussing the in vitro activity of the drugs available to treat infections caused by these organisms. Surveillance programmes demonstrate the increasing problem of antimicrobial resistance globally. The SENTRY Program was established in 1997 to measure the predominant pathogens and their antimicrobial resistance patterns over a broad network of sentinel hospitals in the USA, Canada, South America and Europe [1]. * Corresponding author. D.A. Enoch. Tel.: +44 1223 257 035; fax: +44 1223 242 775. E-mail: [email protected] (D.A. Enoch).

The European Antimicrobial Resistance Surveillance System (EARSS) provides data on the prevalence and spread of resistance for seven major invasive bacteria, including P. aeruginosa (but not A. baumannii, B. cepacia complex and S. maltophilia), in Europe (http://www.earss.rivm.nl/), illustrating the wide geographical variation in antimicrobial resistance. The British Society for Antimicrobial Chemotherapy (BSAC) Resistance Surveillance Programme (http://www.bsacsurv.org) provides local data for the UK and Irish Republic. In this paper, we review recent data from these programmes and other literature for P. aeruginosa, A. baumannii, S. maltophilia and B. cepacia complex. 2. The organisms 2.1. Pseudomonas aeruginosa Pseudomonas aeruginosa is widely distributed in the environment. It is a leading cause of nosocomial infections, being responsible for 10% of all hospital-acquired infections (HAIs) [2]. Infection is usually opportunistic and associated with invasive devices, mechanical ventilation, burn wounds or surgery. Cross-infection from other colonised patients occurs and is encouraged by suppression of hospital patients’ normal flora with broad-spectrum antibiotics to which this organism is widely resistant. The organism also has a propensity to colonise and infect the lungs of cystic fibrosis (CF) patients and is virtually ineradicable in this setting. Pseudomonas aeruginosa has an impressive armamentarium of virulence factors, allowing it to combat host

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D.A. Enoch et al. / International Journal of Antimicrobial Agents 29 Suppl. 3 (2007) S33–S41

defences. These include the flagellum, pili, alginate production, mucoid phenotypes in CF, lipopolysaccharide, type III secretion factors, secreted proteases, oxidative factors and toxins [3]. Quorum sensing may regulate expression of virulence factors [4]. The emergence and gradual dissemination of metallo-b-lactamases, with their ability to hydrolyze all b-lactams except aztreonam, has further compromised the activity of many agents traditionally used for the treatment of P. aeruginosa infections [5], although it remains more common for pan-resistance to arise by accumulation of successive mutations affecting efflux, impermeability and AmpC b-lactamase expression. Susceptibility testing gives variable results for isolates recovered from samples in patients with CF, and its role in antibiotic choice has been questioned as patients commonly improve despite receiving apparently inappropriate therapy [6]. Outside this setting, laboratory-detected resistance is believed to be significant. 2.1.1. Trends in isolation and resistance In the SENTRY Program, the numbers of isolates of P. aeruginosa reported are increasing, although distinct differences exist by geographic region and site of infection. SENTRY data reported that multidrug-resistant (resistant to piperacillin, ceftazidime, imipenem and gentamicin) P. aeruginosa bloodstream isolates were most prevalent in Latin America (12.0−17.6%; average 15.0%), followed by Europe (5.1−14.2%; average 9.3%) and North America (1.6−2.5%; average 2.1%) between 1997 and 2001 [1] (Fig. 1). Multidrug resistance in bloodstream infections saw a steady increase between 1997 and 2001, with resistance even to the carbapenems widespread in Europe (Fig. 2). The UK situation is better: the BSAC bacteraemia survey reported relatively stable or declining rates of resistance among P. aeruginosa isolates between 2002 and 2005, except possibly to ciprofloxacin (Fig. 3). Europe (1152)

Latin America (608)

North America (1550)

% MDR Resistance

20.0

17.6 14.3

15.0

16.0 14.2

14.5

12.0 10.1

10.4

9.5

10.0 5.1

5.0

2.5

1.6

2.1

2.0

2.0

0.0 1997

1998

1999

2000

2001

Fig. 1. SENTRY Program data for multidrug-resistant (MDR) Pseudomonas aeruginosa from bloodstream isolates (1997–2001). Adapted from ref. [1]. Whilst multidrug resistance remained relatively stable between 1997 and 2001 in North America, it increased from 12.0 to 17.6% of P. aeruginosa isolates in Latin America over the same time period and more than doubled in Europe.

EARSS 2005 Report: http://www.rivm.nl/earss/Images/EARSS%202005_tcm61-34899.pdf

Fig. 2. Proportion of invasive Pseudomonas aeruginosa isolates resistant to carbapenems in 2005, from the European Antimicrobial Resistance Surveillance System (EARSS) (http://www.earss.rivm.nl). Carbapenem resistance is high all over Europe, with only Norway and The Netherlands reporting proportions of <5%. Reproduced with permission from EARSS. 2002 (n=171)

2003 (n=185)

2004 (n=207)

2005 (n=197)

20.0 18.0 16.0 14.0 % resistance

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12.0 10.0 8.0 6.0 4.0 2.0 0.0 Ceftazidime

Gentamicin

Imipenem

Ciprofloxacin

Fig. 3. British Society for Antimicrobial Chemotherapy bacteraemia surveillance data for Pseudomonas aeruginosa from 2002–2005 [7] for the UK and Republic of Ireland.

North American surveillance studies report a rise in aminoglycoside [8], carbapenem [9] and fluoroquinolone [8] resistance. By 2005, US studies reported ceftazidime and imipenem MIC50 values (minimum inhibitory concentration for 50% of the organisms) ranging from <1 mg/L to >32 mg/L [10,11] and from 0.06 mg/L to 16 mg/L [10], respectively, with this great diversity perhaps reflecting different patient groups or the effects of local outbreak strains with more or less resistance. In CF, MIC50 values for ceftazidime and imipenem ranged from 0.25 mg/L to >512 mg/L and 0.5 mg/L to >512 mg/L, respectively [12]. Reported rates of multidrug-resistant P. aeruginosa from the SENTRY Program varied from 1.1% to 31.2% according to geographic location [13]. 2.2. Acinetobacter baumannii Acinetobacter baumannii is also widely distributed in the environment. It is generally non-pathogenic in healthy

2.2.1. Trends in isolation and resistance BSAC surveillance data illustrate resistance trends in A. baumannii for bacteraemias only since 2001 [7]. In 2005, >30% of bacteraemia isolates were resistant to gentamicin and piperacillin/tazobactam [7] (Fig. 4). Many isolates from non-bacteraemic sources are even more resistant [19,20]. Similar trends have been reported in other countries [21]. A recent study from Greece reported >94% resistance to ceftazidime, amikacin, piperacillin/tazobactam and imipenem, whilst colistin MICs ranged from 0.25 mg/L to >1024 mg/L and those of tigecycline from 0.12 mg/L to 4 mg/L [22]. In the USA, ceftazidime and amikacin MICs range from <0.06 mg/L to 128 mg/L and <0.5 mg/L to >64 mg/L, respectively. Several mechanisms for carbapenem resistance have been described, namely class D 2002 (n=28)

2003 (n=38)

2004 (n=41)

2005 (n=43)

45.0 40.0 35.0

% resistance

30.0 25.0 20.0 15.0 10.0 5.0

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80 70 60 50 40 30 20 10

S.E. clone

OXA-23 clone 1

Q2 Q3 Q4

Q2 Q3 Q4 200 4

Q2 Q3 Q4 200 1 Q2 Q3 Q4 200 2 Q2 Q3 Q4 200 3

0

200 0

individuals, except possibly as an agent of wound infection in military casualties from the Middle East [14,15]. Over the last decade it has become an increasing cause of serious opportunistic infection, particularly ventilator-associated pneumonia and invasion of burns wounds, in susceptible patients in Intensive Care Units (ICUs). Virulence factors are limited, consistent with the organism’s limited invasive potential. It produces no cytotoxins and its lipopolysaccharide is of uncertain endotoxigenic potential. Its enhanced survival is due to a combination of bacteriocin production, the presence of a capsule and prolonged viability under dry conditions [16]. The population structure is highly clonal, with single strains commonly affecting multiple patients in a unit or hospital. Indeed, a few lineages of A. baumannii have achieved “epidemic” status as nosocomial pathogens, spreading to cause outbreaks of infections in many hospitals and countries [17]. Many of these outbreaks have involved strains that were multidrug resistant, including to carbapenems and amikacin, with colistin and tigecycline sometimes representing the only remaining therapeutic options [17,18].

Cumulative no. hospitals affected

D.A. Enoch et al. / International Journal of Antimicrobial Agents 29 Suppl. 3 (2007) S33–S41

OXA-23 clone 2

Fig. 5. Carbapenem-resistant Acinetobacter clones in the UK (2000–2004). Reproduced from ref. [23]. These data illustrate the rise of carbapenemresistant clones in the UK over a 5-year period between 2000 and 2005. There was no evidence of widely disseminated clones at the beginning in 2000, whereas by 2004 the SE clone and the OXA-23 clone affected ca. 40 hospitals in the UK.

OXA-type carbapenemases and class B metalloenzymes (IMP or VIM family), whilst PER extended-spectrum enzymes cause resistance to third-generation cephalosporins in many isolates in Turkey [24]. Multidrug-resistant isolates of A. baumannii have increased significantly in the UK since 2002, becoming most prevalent in hospitals in London and southeast England [19, 20]. Pulsed-field gel electrophoresis has shown that many of these isolates belong to three clones that possess the nonmetallo OXA-23 or -51 carbapenemases as the predominant mechanism for their carbapenem resistance. All three exhibit resistance to b-lactams (including carbapenems), fluoroquinolones and many aminoglycosides, although they vary in susceptibility to amikacin. The most prevalent clones, each recorded from ca. 40 hospitals, are the SE clone and the OXA-23 clone l; their spread since 2000 is illustrated in Fig. 5 [23]. Risk factors for acquisition of multidrug-resistant A. baumannii include presence on an ICU or burns unit, large hospital size (>500 beds) [25] and previous exposure to antibiotics [25,26], notably carbapenems [27]. Immunosuppression, emergency admission, respiratory failure or mechanical ventilation, invasive procedures, urinary catheterisation and recent surgery are further risk factors [25–27]. Attributable mortality is hard to define, since A. baumannii tends only to cause infection in very vulnerable patients and its properties may be clone-specific; nevertheless, rates up to 20% [28] and >61% [29] have been claimed in bacteraemia. Its capacity to acquire resistance to multiple antimicrobial agents has made this organism a significant challenge in many healthcare institutions.

0.0 Gentamicin

Pip/tazo

Imipenem

Ciprofloxacin

Fig. 4. British Society for Antimicrobial Chemotherapy bacteraemia surveillance data for Acinetobacter baumannii from 2002–2005 [7]. The data show increasing rates of resistance, with levels reaching over 30% for gentamicin, piperacillin/tazobactam (Pip/tazo) and ciprofloxacin and over 5% for imipenem by 2005.

2.3. Burkholderia cepacia complex Members of B. cepacia complex are well recognised, if uncommon, nosocomial pathogens. They are more important in CF, where the spread of epidemic strains has presented significant infection control problems, both

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in hospital units and among the CF community as a whole [30]. A number of potential virulence factors have been identified, although not all of these have been proven to affect the pathogenesis of human disease [31]. They include intrinsic antimicrobial resistances (e.g. to polymyxins and aminoglycosides), the ability to form biofilms, pili, the cenocepacia (pathogenicity) island [32], production of exopolysaccharide, haemolysin, melanin and extracellular proteases, flagella, lipopolysaccharide, siderophore production, type III secretion factors and the ability to invade and survive inside respiratory cells and macrophages. Burkholderia multivorans (genomovar II) and Burkholderia cenocepacia (genomovar III) account for the majority of infecting strains in Europe and the USA, and isolation of B. cepacia complex isolates from respiratory CF samples is accepted as a poor prognostic marker [33]. A fulminant pneumonic and septicaemic process called “cepacia syndrome” may occur in 10% of colonised CF patients, more commonly with B. cenocepacia than B. multivorans [32]. Patients with B. cenocepacia had a shorter survival than patients with P. aeruginosa (P = 0.01) in a recent study but there was no difference in survival between P. aeruginosa and B. multivorans [32]. Some centres decline lung transplantation to patients colonised with B. cepacia complex [34]. Burkholderia pseudomallei is prevalent in Southeast Asia and the Northern Territory of Australia both as a soil organism and as a primary pathogen, often in patients with underlying diabetes mellitus. It is associated with 30−40% mortality in septicaemic disease, even when treated with appropriate antibiotics, although with much lower mortality rates for pulmonary disease. Tigecycline has an MIC90 of 2 mg/L [35] and may be an alternative therapy to ceftazidime for melioidosis, although clinical trials are needed to support this hypothesis. 2.3.1. Trends in isolation and resistance A recent review of HAIs related to contaminated substances reported B. cepacia to be a significant contaminant of substances other than blood products [36]. SENTRY data reported B. cepacia to comprise 7.7% of “uncommonly isolated” non-enteric Gram-negative bacilli between 1997 and 2003 [11]. In the USA, resistance ranges from 9.3% for co-trimoxazole to 48% for imipenem [11]. Among patients with CF, resistance to imipenem was >84% in Europe and the USA [12,37]. MIC50 values for piperacillin ranged from 4 mg/L [38] to 256 mg/L [12] for multidrug-resistant CF isolates. 2.4. Stenotrophomonas maltophilia Independent risk factors for infection with S. maltophilia include chronic obstructive pulmonary disease, neutropenia, the presence of a central venous catheter, prolonged hospitalisation and previous therapy with broad-spectrum

antibiotics [39]. Nosocomial transmission of S. maltophilia is rare and most infections are with unique strains. Stenotrophomonas maltophilia is associated with significant morbidity and mortality in immunocompromised patients [39]. Putative, but poorly understood, virulence factors include a range of exoenzymes such as DNase, RNase, fibrinolysin, lipases, hyaluronidase, protease and elastase, the ability to survive and then multiply in medical solutions and the ability to adhere to prosthetic materials. Bacteraemias related to central venous catheters and nosocomial pneumonia are the two most common manifestations of true infection due to S. maltophilia, although most isolates originating from the respiratory tract represent colonisation rather than infection. Recovery of the organism from this site, or from urinary catheters, should not ordinarily prompt antibiotic treatment. In rare cases, S. maltophilia is associated with skin and soft-tissue infections, endocarditis, meningitis, urinary tract infections, intra-abdominal infections and ophthalmological syndromes related to applied or implanted foreign materials. Mortality rates of 10−60% in patients with bacteraemia due to S. maltophilia have been reported [40,41], although attributable mortality is again hard to define in the types of patient sufficiently ill to be affected by this species. The majority of isolates remain sensitive to co-trimoxazole, although toxicity or intolerance remains the major problem in some patients, for whom ticarcillin/clavulanic acid may be considered as an alternative. Fluoroquinolones may be useful as adjuncts to co-trimoxazole or ticarcillin/clavulanic acid therapy [42]. 2.4.1. Trends in isolation and resistance The number of isolates of S. maltophilia reported are increasing worldwide, although reports tend to be from individual institutions and associated with the introduction of carbapenem use [43]. This increase may reflect a change in patient types rather than a change in epidemiology. Among ‘uncommonly isolated’ non-enteric Gram-negative bacilli, recent SENTRY data report S. maltophilia to comprise 59.2%, with a roughly even distribution between respiratory and blood isolates (49% and 41%, respectively) [11]. Distinct differences exist in the frequency by geographic region and site of infection [43]. Co-trimoxazole resistance varies widely. Betriu et al. [44, 45] reported that
2% of Spanish isolates of S. maltophilia were resistant, whereas an Italian study reported 80.9% resistance [46]. The highest levels of resistance are seen in CF isolates, with 84% resistance to co-trimoxazole, 50% to ticarcillin/clavulanic acid, 74% to colistin and 11% to doxycycline [47]. A recent surveillance study reported that >40% of pneumonia isolates from the USA are resistant to b-lactams and ciprofloxacin [48]. A further problem – and caution – when reading surveillance data is that the MICs of b-lactams vary with the medium [49]; those of aminoglycosides vary with the test temperature [49].

D.A. Enoch et al. / International Journal of Antimicrobial Agents 29 Suppl. 3 (2007) S33–S41

3. Antibiotics for treatment of infections due to non-fermentative Gram-negative bacteria 3.1. Aminoglycosides Aminoglycosides are a long-established component of therapy for infections due to non-fermenters, although they should be seen as adjuncts rather than as sole agents [50]. Against P. aeruginosa they are generally combined with b-lactams, although the evidence for synergy is weak except with penicillins. Bacterial resistance may occur owing to reduced drug uptake, increased efflux pump activity and enzymatic inactivation. This topic has been reviewed elsewhere [51]. They have no in vitro activity against S. maltophilia and B. cepacia. Tobramycin is inherently the most active of the group against P. aeruginosa, where most resistance to aminoglycosides in general is mutational due to impermeability or efflux [51]. Pseudomonas aeruginosa isolates with the highest MICs were most commonly from CF patients, with tobramycin MIC50 and MIC90 values of 8 mg/L and 64 mg/L for mucoid isolates and 16 mg/L and 256 mg/L for non-mucoid isolates, respectively [12]. 3.2. Co-trimoxazole The combination of trimethoprim and sulphamethoxazole is active in vitro against many isolates of B. cepacia and S. maltophilia, but not P. aeruginosa. Acquired resistance is common in A. baumannii. Co-trimoxazole is the treatment of choice for infections with S. maltophilia [52] and significant morbidity may occur in patients with co-trimoxazole-resistant S. maltophilia bacteraemia [53]. Despite the Committee for Safety of Medicines in the UK advising against its general use, co-trimoxazole remains the standard therapy for these infections, and trimethoprim therapy alone is not active. 3.3. Colistin The polymyxins are a group of five chemically different bactericidal antibiotics (polymyxins A to E). Only polymyxin B and polymyxin E (colistin) have been used in clinical practice. Resistance may develop through mutation and occurs of a result of alterations of the outer membrane. Colistin has a wide spectrum of activity against Gramnegative organisms, including A. baumannii, P. aeruginosa and S. maltophilia, but is inactive against B. cepacia owing to its unusual lipopolysaccharide structure. Although resistance to polymyxin B has recently been reported in P. aeruginosa isolates from the USA and elsewhere [54], it remains rare and is almost exclusive to CF isolates. Recent clinical reports and reviews of experience with colistin are encouraging, with side effects observed less often than expected from historical data [55–60].

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3.4. Penicillins with activity against non-fermenters A number of semisynthetic penicillins, including ticarcillin and piperacillin, have activity against non-fermenters. These agents can be used in combination with the b-lactamase inhibitors clavulanic acid, tazobactam or sulbactam, although these do not inhibit all b-lactamases of these species. Moreover, most resistance in P. aeruginosa is due to non-b-lactamase mechanisms, principally efflux, and b-lactamase inhibitors have no effect on this mechanism. Resistance rates are typically highest in isolates from CF patients [12] and in outbreaks of carbapenem-resistant P. aeruginosa and A. baumannii [61]. Temocillin is used for the treatment of B. cepacia in CF, but otherwise has little activity against non-fermenters [62]. 3.5. Cephalosporins The cephalosporin with most potent activity against nonfermenters is ceftazidime [63]. Its spectrum of activity includes P. aeruginosa, S. maltophilia, A. baumannii, B. cepacia and B. pseudomallei, for which it is standard therapy. Resistance occurs via a variety of mechanisms, including hydrolysis by b-lactamases and efflux (e.g. by upregulation of MexAB–OprM in P. aeruginosa) [64]. 3.6. Carbapenems Carbapenems are b-lactams with exceptionally broad antibacterial spectra. Meropenem and imipenem/cilastatin are active against non-fermenters. Ertapenem has no activity against these organisms and will not be discussed here [65]. Resistance in Gram-negative organisms usually results from a combination of impaired drug entry, efflux and the presence of a b-lactamase. Diminished permeability is often a result of loss of the outer membrane proteins through which carbapenems enter the periplasmic space and is a well known mechanism for P. aeruginosa where loss of OprD is associated with resistance [66]. Multidrug efflux systems (e.g. MexAB–OprM) are present in P. aeruginosa and can excrete meropenem as well as penicillins, fluoroquinolones and cephalosporins [66], although it is important to note that imipenem escapes this type of mechanism [67]. Stenotrophomonas maltophilia is intrinsically resistant to carbapenems owing to production of inducible zinc-metalloenzyme (L-1 enzyme) with carbapenemase activity [68]. Meropenem is one of the most active agents versus B. cepacia and some would regard it as the agent of choice. Doripenem is currently undergoing six phase III trials for a variety of conditions including complicated urinary tract infection, pneumonia and complicated intra-abdominal infections. It has similar activity to meropenem and imipenem against carbapenemase-negative A. baumannii strains, but no carbapenem retains activity against strains with expression of OXA or metallo-carbapenemases [69].

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D.A. Enoch et al. / International Journal of Antimicrobial Agents 29 Suppl. 3 (2007) S33–S41 Table 1 Potency of tigecycline against non-fermentative Gram-negative bacteria (adapted from refs. [38,77]) Organism

MIC a (mg/L)

% Susceptible b

% Resistant b

Range

MIC50

MIC90 16

5.1

2

94.5

0.9

Pseudomonas aeruginosa (n = 1121) [77]

0.12 to >32

8

Acinetobacter spp. (n = 326) [77]

0.06−8

0.5

Burkholderia cepacia (n = 21) [38]

0.25−32

1

16

67.0

29.0

Stenotrophomonas maltophilia (n = 203) [77]

0.12−8

1

2

93.1

3.0

a b

77.2

MIC, minimum inhibitory concentration (MIC50/ 90 , MIC for 50% and 90% of the organisms, respectively). The tigecycline susceptible breakpoint was defined as
2 mg/L and the resistance breakpoint as 8 mg/L for comparison purposes only [78]; these values are now accepted by the US Food and Drug Administration (FDA) but are one doubling dilution higher than the values adopted for Enterobacteriaceae by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)/European Medicines Agency (EMEA), who presently have no values specific for non-fermenters.

It is unlikely that doripenem will prove to be superior to meropenem, but this requires additional clinical studies. 3.7. Fluoroquinolones In general, ciprofloxacin remains the most potent agent in this class against non-fermenters and is particularly valued and widely used. It is the only oral agent with activity against P. aeruginosa. Fluoroquinolones have been widely used for the treatment of a wide variety of infections. Perhaps predictably, their use is increasingly compromised by the widespread emergence of bacterial resistance [70]. For the treatment of S. maltophilia, there is some clinical evidence that addition of moxifloxacin (more active than ciprofloxacin against this species) to co-trimoxazole may be therapeutically advantageous [42]. Resistance mechanisms have recently been reviewed elsewhere [70] and mostly entail reduction in permeability, upregulation of efflux (also affecting b-lactams) and mutation of DNA gyrase and topoisomerase IV genes. 3.8. Tigecycline Tigecycline is a glycylcycline, a semisynthetic derivative of minocycline with a 9-t-butylglycylamido substitution. Glycylcyclines act by binding to the bacterial 30S ribosomal subunit, blocking entry of the aminoacyl-tRNA molecules into the acceptor site of the ribosome, and are mostly considered to be bacteriostatic. The main determinants of acquired resistance to tetracyclines, namely active efflux and ribosomal protection, are overcome by steric hindrance from the large R-group at position 9. Tigecycline has in vitro activity against a number of Gram-negative species, including A. baumannii (including many carbapenemresistant isolates) and S. maltophilia. It lacks useful activity against P. aeruginosa. Tigecycline is able to evade Tet-type efflux pumps (responsible for plasmidmediated tetracycline resistance) but is susceptible to the chromosomal resistance–nodulation–cell division (RND) family of multidrug efflux pumps, including MexXY–OprM of P. aeruginosa [71], which explains the intrinsic resistance

of this species, and the AcrAB pump found in Proteus mirabilis [72]. Published resistance rates for tigecycline range from 58.5% to 97% for P. aeruginosa [38,73], 0% to 6% A. baumannii [74,75], 0% to 29% for B. cepacia complex [38,76] and 0% to 3.5% for S. maltophilia [45,48]. Table 1 [38,77] lists the MICs of tigecycline. 3.9. Combination therapy The potential benefits of combination antibiotic therapy are well recognised. They include a broadened and more reliable spectrum of activity, enhanced antibacterial activity due to any synergistic or additive effect allowing lower doses of toxic agents to be given, and prevention of the emergence of resistance. Enhanced in vitro activity has been noted for a number of antimicrobial combinations tested against A. baumannii. These include polymyxin B, imipenem and rifampicin [79], polymyxin B, rifampicin and sulbactam [80] and colistin and rifampicin [81]. There have been reports of some centres using tigecycline and nebulised colistin against Acinetobacter pneumonia [82], although studies are needed to evaluate this combination formally. Enhanced in vitro activity has also been noted for a number of antimicrobial combinations used against P. aeruginosa, and b-lactam/aminoglycoside combinations remain the standard of care for severe infections. In vitro synergy was demonstrated with the combination of cephalosporins and fluoroquinolones [83], colistin and ceftazidime [84] and a macrolide plus tobramycin [85]. Synergy was also demonstrated in vitro with the combination of macrolides with ceftazidime (for B. cepacia) and cotrimoxazole (for S. maltophilia) for infecting organisms from patients with CF [85]. Conversely, no enhanced in vitro antibacterial activity against P. aeruginosa was found with combinations of ciprofloxacin and colistin [84]. There are few clinical studies of combinations of antimicrobial agents for the treatment of multidrugresistant non-fermenters. Colistin was combined with a carbapenem in 19 of 25 patients, amikacin in 8 of 25 patients, tobramycin (3), cefepime (3), quinolone (2), ampicillin/sulbactam (3) and aztreonam (1 patient) in a

D.A. Enoch et al. / International Journal of Antimicrobial Agents 29 Suppl. 3 (2007) S33–S41

retrospective non-controlled observational study of 25 critically ill patients with infection due to multidrug-resistant P. aeruginosa or A. baumannii [86]. End-of-treatment mortality was 21%. Ten patients with carbapenem-resistant A. baumannii were treated with a combination of imipenem and rifampicin in a recent prospective study [87]. Three patients died, two because of therapeutic failure, and seven responded. High-level rifampicin resistance developed in 7 of 10 patients. The authors suggested this combination should not be used for the treatment of severe infection with these organisms.

4. Conclusions Pseudomonas aeruginosa, A. baumannii, S. maltophilia and members of the B. cepacia complex are an increasingly recognised cause of infection and are increasingly difficult to treat. Glycylcyclines demonstrate useful in vitro activity against many non-fermenters except P. aeruginosa, but clinical trials are needed to determine their role in the treatment of infections due to these organisms. Of particular concern are early reports of the development of resistance and treatment failure in two patients with carbapenem-resistant A. baumannii bacteraemia being treated with tigecycline for other conditions [88]. The two patients had received 9 days and 16 days of treatment with tigecycline when bacteraemias were detected. The MICs were 4 mg/L and 16 mg/L, respectively, but these fell to 1 mg/L and 4 mg/L after addition of phenyl-arginine-b-naphthylamide, a pump inhibitor, suggesting efflux as the mechanism of resistance. Noting a mean peak serum concentration of only 0.63 mg/L, the authors suggest that tigecycline should not be used to treat bloodstream infections caused by organisms with MICs >1 mg/L [88], which corresponds with the European Medicines Agency (EMEA)/European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint. There are currently no other drugs, besides doripenem, in phase II or III trials for the treatment of multidrugresistant non-fermenters. Novel efflux pump inhibitors have not yet entered human clinical trials, although they appear promising [89]. Therefore, currently available antibiotics require judicious and prudent use, following evidencebased trial data whenever possible. The role of combining these antibiotics in combating the development of further antibiotic resistance and delivering superior clinical efficacy requires more rigorous evaluation than in the past.

Acknowledgements Editorial assistance during the preparation of this manuscript was provided by Magus Strategic Communications Ltd.

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