Clostridium

Chapter 20

Clostridium Chapter outline Properties of the genus Antigenic characteristics Toxins and virulence factors produced Toxins Virulence factors Transmission Diagnosis Laboratory examinations Isolation of C. difficile Glutamate dehydrogenase assay (GDH) Detection of toxin Molecular biology

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Characteristics of antimicrobial resistance Clindamycin and erythromycin (macrolidee lincosamideestreptogramin B) resistance Fluoroquinolone (moxifloxacin) resistance Rifamycin (rifampicin) resistance Metronidazole resistance Vancomycin resistance Fidaxomicin resistance Tetracycline resistance Chloramphenicol resistance Cephalosporin resistance References

256 257 258 258 258 258 258 258 259 259 259

The bacterium was first isolated from the newborn infant’s intestinal flora by Hall and O’Toole (1935). It was described as Bacillus difficilis because of the ‘difficulty’ they faced during isolation. In 1978, Clostridium difficile was identified as the primary cause of pseudomembranous colitis in human (Bartlett et al., 1978). C. difficile infection (CDI) was found associated with antibiotic-induced diarrhoea (25%e33%) along with pseudomembranous enteritis (90%). In the United States, CDI alone causes 29,000 deaths (400,000 cases) each year with more than $1 billion economic burden (Lessa et al., 2015). A systematic review revealed categorization of CDI-associated excess cost in the United States and estimated $6774e$10212 for CDI-associated hospital admission (Gabriel and Beriot-Mathiot, 2014). Higher incidence rate of CDI (w17.1 cases/10,000) was observed in Europe (Sweden), Canada and China (Huang et al., 2008; Dubberke and Olsen, 2012). CDI is also observed as the most frequent aetiology of healthcare-associated infection even exceeding Staphylococcus aureus infections (Magill et al., 2014). In the United States, rate of hospitalizations with C. difficile tripled in 2011 (12.7/1000 discharges) in comparison to 2001 (5.6/1000 discharges) (Steiner et al., 2012).CDI is characterized by mild to severe diarrhoea causing toxic megacolon, fulminant colitis, piercing of intestine, sepsis and death (Rupnik et al., 2009). Therapeutic use of certain antibiotics (clindamycin, erythromycin, aminopenicillins, third-generation cephalosporins and fluoroquinolones) promotes CDI because of disruption of intestinal commensal bacteria (‘dysbiosis’), which favours the growth of ingested or resident C. difficile strains (Owens et al., 2008). Multidrug-resistant strains of C. difficile further aggravate the situation because of their growth in presence of antibiotics against which they are resistant. Antibiotic resistance or reduced susceptibility against moxifloxacin, metronidazole and vancomycin was correlated with epidemic spread of hypervirulent C. difficile strains such as polymerase chain reaction (PCR) ribotype (RT) 027 in the United States and European countries (He et al., 2013; Ofosu, 2016).

Properties of the genus Morphology: C. difficile are Gram-positive rods, measuring 3e5 mm in length and 0.5 mm in width. They are capsulated, motile by peritrichous flagella and sporulating in nature. Some strains also contain S-layer. The spores are oval, subterminal or terminal in position, do not bulge the parent cells and are produced in artificial medium after 72 h of incubation during decline phase of the growth. Some strains may produce polar fimbriae.

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254 Antimicrobial Resistance in Agriculture

Classification: C. difficile belongs to class Clostridia, order Clostridiales, family Clostridiaceae and genus Clostridium. On the basis of 16S rRNA gene sequences, C. difficile are closely related with Clostridium sordellii and Clostridium bifermentans (Elsayed and Zhang, 2004). Susceptibility to disinfectants: C. difficile spores are resistant to the solvents such as alcohol, enzymes, detergents, disinfectants and environmental stress conditions such as heat, ultraviolet light and ionizing radiation. Vegetative bacteria rapidly die during oxygen exposure. Natural habitat: They are commonly found in the soil, sand, river, lake, swimming pools, human hospitals and veterinary clinics surroundings, marine sediments, river bank mud, raw vegetables and floor swabs of private houses. It is found as a part of the normal flora of the human intestine (2%e4% in young adults). The isolation rate increases with the advancement of age (10%e20% in elderly persons). It is isolated from the faeces of cattle, horses, donkey, domestic birds, ducks, geese, dog and cat (Hensgens et al., 2012). Genome: The GC content of C. difficile genome is 29 mol%. Approximately 11% of the genome consists of mobile genetic elements (MGEs) (Sebaihia et al., 2006). The plasmids are detected in the bacterium with molecular weights ranging from 2.7
106 to 100
106, and the plasmids contain the genetic information required for their replication and maintenance only. A few bacteria may contain introns (group I/II) associated with MGEs. The first intron found in C. difficile was a group II intron detected within orf14 of a conjugative transposon (Tn5397), and the intron-encoded proteins are capable of splicing like eukaryotes (Mullany et al., 1996; Roberts et al., 2001). A combination of a group I intron and an insertion sequence (‘IStrons’) are widely distributed in C. difficile genome which can use alternative splice site to generate variant proteins (Braun et al., 2000). The varieties of conjugative transposons (CTns) and mobilisable transposons (MTns) are also detected in C. difficile genome which is transferred from donor to the recipient by a conjugation like process. The CTns and MTns carry genes for antibiotic resistance and ABC transporters (Brouwer et al., 2011). Tn5397, Tn916 (originated from Bacillus subtilis), Tn4453a and Tn4453b of C. difficile are well-characterized CTns, which confer resistance to tetracycline (Tn5397, Tn916; Mullany et al., 1996) and chloramphenicol (Tn4453a and Tn4453b; Wren et al., 1988). C. difficile Tn5398 confers resistance against macrolide, lincomycin and streptogramin B (Hächler et al., 1987).

Antigenic characteristics S-layer protein (SLP) is the major antigen of C. difficile used in the typing. This organism unusually expresses two kinds of SLPs, i.e., high molecular weight and low molecular weight proteins. Both the proteins are encoded by slpA gene and are produced by posttransitional cleavage of a single precursor. Currently, C. difficile comprises of 14 serogroups based on S-layer protein. Serologically it can cross-react with other Clostridia such as C. sordellii, C. bifermentans and Clostridium glycolicum. The virulence of C. difficile isolates is expressed by PCR ribotype (RT), North American pulsed field gel electrophoresis type (NAP) and restriction endonuclease analysis groups depending on the typing method used. PCR ribotyping is based on the differences in the spacer regions of 16S and 23S ribosomal RNA. RT027 and RT078 are the hypervirulent ribotypes, associated with high mortality and recurrence of infection, and have recently spread throughout the world (Collins et al., 2013). The RT027 was further characterized as toxinotype III, restriction endonuclease analysis group BI, North American pulsed-field gel electrophoresis type NAP1 and was designated as BI/NAP1/027 (O’Connor et al., 2009). The BI/NAP1/027 strains of C. difficile showed higher rate of sporulation, toxin production and marked antibiotic resistance (fluoroquinolone) (McDonald et al., 2005). A pan-European longitudinal surveillance study indicated RT356 (Italy) as antibiotic resistant and RT005 and RT087 as susceptible isolates in European countries during 2011e14 (Freeman et al., 2015). Distribution of C. difficile PCR ribotypes in different countries is described in Table 20.1.

Toxins and virulence factors produced Toxins C. difficile produces two large glycosylating toxins known as TcdA (308 KDa) and TcdB (270 KDa). They are glucosyl transferases that inactivate Rho, Rac and Cdc42 within target cells. The TcdA acts as enterotoxin causing accumulation of fluid in the intestine and the TcdB is a potent cytotoxin. On the basis of sequence variations of the toxins, there are 22 toxinotypes of the bacterium. These types are designated by roman numericals (IeXXII). The genes encoding the toxins (tcdA, tcdB) are located in a pathogenicity island (19.6 Kb, PaLoc) within the chromosome (Rupnik, 2008). The genes are expressed during the late log and stationary phases of growth in response to a

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255

TABLE 20.1 Distribution of Clostridium difficile polymerase chain reaction (PCR) ribotypes. PCR ribotype

Distribution

001

China, Japan, Korea, Spain, Germany, Scotland

002

Japan, Hong Kong, Korea

014

United States, Spain, France, Japan, China, Korea

017

China, Japan, Korea, Netherlands, Scotland

018

Japan, Korea

027

United States, Canada, Netherlands, Ireland, Germany, Chile, Asia

078

Spain, Germany, France

244

Australia

356

Italy

variety of environmental stimuli. There are three other genes (tcdC, tcdD and tcdE) found within the same pathogenicity island which encode regulatory proteins. The TcdC and TcdD function as a negative and positive regulator of the toxin production, respectively. The TcdE helps in release of the toxins by increasing the cell wall permeability. The production of the toxins is correlated with the stress conditions such as antibiotic exposure and ‘catabolite repression’. The major toxins (TcdA, TcdB) enter the cell via receptor-mediated endocytosis. The receptor for TcdA is a disaccharide (Galb1-4GlcNac) which is present in a variety of cells. Within the cell, TcdB glucosylates RhoA, Rac and cdc42 protein via transfer of a sugar moiety to Thr-37 of the GTPase with UDP-glucose as a co-substrate. TcdA can also glucosylate RhoA protein in a similar way (less active than TcdB). Thus both of the toxins inactivate small GTPases within the cell. It results in cellular actin filament condensation, rounding of the cells and membrane blebbing followed by apoptosis. A binary toxin was described in a few strains of C. difficile associated with formation of microtubule protrusions from cell wall that facilitated bacterial adhesion (Schwan et al., 2009).

Virulence factors Pili, capsule and degradative enzymes act as additional virulence factors. Enzymes such as hyaluronidase, neuraminidase, chondroitin-4-sulphatase and heparinase are detected. The hyaluronidase plays major role in degradation of connective tissues.

Transmission C. difficile spores are major vehicle of transmission in human. After entry of the spores into the human host, bile acids containing a specific protease (CspC) take prime role in germination of bacteria from the spores (Sorg and Sonenshein, 2008). C. difficile spores are detected in the environment on inert surfaces of hospitals or households (shoe-swabs), animal faeces, soils, rivers, sea, lakes, inland drainage, swimming pools, wastewater treatment plants, tap water and puddle water (Båverud et al., 2003; Alam et al., 2014; Janezic et al., 2016). C. difficile ribotypes prevalent in human were also detected in the intestine of food animals such as cattle, pigs, sheep, poultry and companion animals such as dogs and cats (Hensgens et al., 2012; Koene et al., 2012). Virulent ribotypes of C. difficile (RT078, RT014/020, RT045) were isolated from dogs and exotic pet such as reptile (sequence type 347, negative for toxin genes) (Davies et al., 2014; Andrés-Lasheras et al., 2018). Transmission of C. difficile between farmed pigs and humans was documented in the Netherlands where high-density pig farms are located (Ziakas et al., 2015). Occurrence of C. difficile was also detected in several raw food items such as retail vegetables, ready-to-eat salads, lettuce, eggplant, green pepper, uncooked meats, ground beef, ground pork, chicken meat, ready-to-eat summer sausage and ready-to-eat Braunschweiger (Songer et al., 2009; Bakri et al., 2009; Weese et al., 2009, 2010; Metcalf et al., 2010; Rodriguez-Palacios et al., 2014). The concentration of C. difficile spores in studied meats or their products is although quite low, the spores can survive cooking temperature (70e72 C) and may germinate if the cooked food is maintained at permissive temperatures. Consumption of beef was reported as a risk factor for CDI while no confirmed foodborne CDI was reported (Søes et al., 2014).

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Prolonged antibiotic use (ampicillin, amoxicillin, cephalosporins, clindamycin, fluoroquinolones) causing reduction of commensal Bacteroides and Firmicutes, advanced age, antineoplastic chemotherapy, organ transplantation, malnutrition, female gender, underlying menaces such as inflammatory bowel disease and chronic renal insufficiency and exposure to a carrier are the major risk factors for human transmission of C. difficile (Leffler and Lamont, 2015). Owing to lack of commensal-associated ‘colonization resistance’, the infants act as healthy carriers of C. difficile during the first year of life and the serum immunoglobulin G and A antitoxins (IgG and IgA) bind with C. difficile toxins and prevent the progress of infection (Jangi and Lamont, 2010). Loss of toll-like receptor signalling, accumulation of proinflammatory Th-17 cells and increased epithelial permeability due to antibiotic-induced dysbiosis were correlated with CDI (Chakra et al., 2014).

Diagnosis Diarrhoeic faeces, rectal swabs and tissues may be collected for diagnosis of CDI. Stool testing for C. difficile toxins is recommended for the patients with diarrhoea only. Posttreatment clinical samples may show positive reaction for several months in absence of clinical symptoms (Sethi et al., 2010).

Laboratory examinations Isolation of C. difficile C. difficile is a strict anaerobe and prefers enriched media such as blood agar for its growth. Selective media are cycloserine cefoxitin fructose agar, cefoxitin mannitol agar and cefoxitin mannitol blood agar. The colonies in blood agar are 3e5 mm in diameter with an irregular, lobate edge, grey in colour and nonhaemolytic. Young colonies produce yellow-green colour under long-wavelength ultraviolet illumination (365 nm). After 48e72 h of incubation, grey or white coloured centre develops in the middle of the colonies. The centre formation is associated with the sporulation of the bacteria. Colonies in CCFA are 4 mm in diameter, yellow or ground glass-like with filamentous edges and typical ‘horse manure’ smell. Isolation of C. difficile cannot differentiate between toxigenic and nontoxigenic strains.

Glutamate dehydrogenase assay (GDH) The assay is sensitive but cannot differentiate between toxigenic and nontoxigenic strains of C. difficile.

Detection of toxin C. difficile toxin in the processed samples (tissue, rectal swabs) can be detected by cell culture (cell cytotoxicity assay) or enzyme immunoassays. Cell culture in Chinese hamster ovary cell line is suitable for detection of the toxin from faecal samples. The cytotoxic activity of the toxin was confirmed by rounding of the cells and neutralization of the toxin by C. difficile antitoxin. The test is considered as ‘gold standard’ for C. difficile diagnosis as it can correlate with the clinical cases. Among the enzyme immunoassays, counterimmunoelectrophoresis, latex agglutination test and ELISA can be performed for detection of C. difficile toxins.

Molecular biology Nucleic acid amplification test (NAAT) detects genes for C. difficile toxins (A and/or B). NAAT is a sensitive method compared with the cell cytotoxicity assay. Detection of toxin genes is clinically irrelevant although, as negative isolates were found to be associated with clinical infection, and sometimes, the strains positive for toxin genes did not produce any toxins (Elliott et al., 2014). Instead of relying on a single test, two-step algorithms for accurate diagnosis of CDI are currently recommended considering the cost, rapidity, sensitivity and specificity. NAAT/GDH followed by enzyme immunoassays (Tox A/B) is the most accurate method for diagnosis of CDI (Polage et al., 2015). Most of the laboratories in the Europe and other places use single test although to detect CDI.

Characteristics of antimicrobial resistance Metronidazole and vancomycin are the drugs of choice for treatment of CDI, but reports of high recurrence rates and reduced susceptibility are increasing. The macrocyclic antimicrobial, fidaxomicin, is promising for treatment of CDI because of lower rates of recurrence and minimal commensal flora disruption (Eyre et al., 2013). The susceptibility testing

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257

TABLE 20.2 Prevalence/Occurrence of antimicrobial-resistant Clostridium difficile isolates. Continent/ country

Clindamycin

Moxifloxacin

Rifamycin/ Rifampicin

Metronidazole

Vancomycin

Europe

49.6%

40%

13%

0.11%

e

Freeman et al. (2015)

United States

30%

36%

7.9%

e

e

Tenover et al., 2012; Tickler et al., (2014)

Canada

e

83%

7.9%

e

e

Karlowsky et al., (2012); Tenover et al., 2012

United Kingdom

e

e

e

24.4% (reduced susceptibility)

e

Baines et al. (2008)

Spain

74%

43%

40%

6.3%

e

Pela´ez et al., (2008); Rodrı´guez-Pardo et al., (2013)

Poland

e

100%

80%

e

e

Obuch-Woszczaty
nski et al. (2014)

Czech Republic

e

e

63.6%

e

e

Freeman et al. (2015)

Denmark

e

e

e

e

Freeman et al. (2015)

56.5%

Italy

e

e

62.3%

e

e

Freeman et al. (2015)

New Zealand

61%

e

e

e

e

Roberts et al. (2011)

Germany

e

68%

e

e

e

Reil et al. (2012)

Hungary

e

e

58.6%

e

e

Freeman et al. (2015)

China

73.5%

e

19.8%

23% (Heteroresistance)

e

Huang et al., (2010); Lee et al., (2014)

Japan

87.7%

e

e

e

e

Oka et al. (2011)

Korea

81%

e

19.8%

e

e

Kim et al., (2012); Lee et al., (2014)

Iran

89.3%

e

e

5.3%

e

Goudarzi et al. (2013)

Brazil

e

8%

e

e

58%

Fraga et al. (2016)

Israel

e

4.7%

e

20%

31.5%

Tkhawkho et al. (2017)

Australia

e

3.4%

e

e

e

Knight et al. (2015)

trial in Europe comprising 22 countries (C. difficile European resistance, ClosER) revealed the presence of the ribotypes 027, 014, 001/072 and 078 with multiple antimicrobial resistance against rifampicin, moxifloxacin and clindamycin (Table 20.2). Reduced susceptibility to metronidazole and vancomycin was scarce and no evidence of resistance against fidaxomicin was observed in most of the European C. difficile isolates (Freeman et al., 2015). Reduced susceptibility to metronidazole of clinical C. difficile isolates was although detected in Poland, Spain, Texas and China (Huang et al., 2008; Obuch-Woszczaty
nski et al., 2014; Norman et al., 2014; Reigadas et al., 2015). Resistance to clindamycin and moxifloxacin of clinical C. difficile isolates varied between different countries (Table 20.2).

Clindamycin and erythromycin (macrolideelincosamideestreptogramin B) resistance Resistance to macrolideelincosamideestreptogramin B group of antibiotics is mediated through ribosomal target modifications or active efflux. The bacterial enzyme ribosomal methylase (ermB encoded) can modify ribosomal (23S) target to prevent the drug binding. C. difficile possessed the ermB (carried by Tn5398) or erm(FS) genes which conferred the ribosomal methylation (Farrow et al., 2001; Schmidt et al., 2007). There are 17 different genetic organisations of erm(B) (E1eE17) observed in C. difficile (Spigaglia et al., 2011). C. difficile strains negative for ermB can also show

258 Antimicrobial Resistance in Agriculture

erythromycin/clindamycin resistance and the vice versa, i.e., possession of ermB without resistance against clindamycin was also revealed (Tang-Feldman et al., 2005; Schmidt et al., 2007). Mutation in 23S rDNA (C656T substitution) was found to be associated with high level of erythromycin and low level of clindamycin resistance in ermB-negative C. difficile isolates (Schmidt et al., 2007). Certain hypervirulent ribotypes such as 027, 078, 001 and 017 were detected to be associated with erythromycin resistance.

Fluoroquinolone (moxifloxacin) resistance Fluoroquinolones inhibit bacterial DNA synthesis by producing a cleavage of enzyme (DNA gyrase and/or topoisomerase IV) DNA complexes. Mutational changes of GyrA and/or GyrB subunit of DNA gyrase induced by C. difficile is the mechanism of quinolone resistance (Oh and Edlund, 2003). The common sites of mutation are T82I (GyrA) and D71V, T82V, D81N, A83V, A118V and A118T (GyrB).

Rifamycin (rifampicin) resistance Rifamycin and rifaximin are commonly used for the treatment of CDI. Point mutation in DNA-dependent RNA polymerase b subunit (RpoB) is associated with rifamycin resistance in C. difficile isolates. The common mutations identified in C. difficile RpoB are R505K (MIC  32 mg/mL), H502N and I548M (O’Connor et al., 2008).

Metronidazole resistance Metronidazole and vancomycin are the drugs of choice for the treatment of CDI. Most of the clinical C. difficile isolates are still susceptible to metronidazole, a few instances of treatment failure and in vitro resistance were reported (Table 20.2; Debast et al., 2014). According to EUCAST and CLSI guidelines, anaerobic bacteria were classified as resistant to metronidazole at MIC 32 mg/mL, susceptible at MIC 8 mg/mL and intermediate at MIC ¼ 16 mg/mL (CLSI, 2012). The metronidazole metabolites inhibit bacterial DNA synthesis and break the DNA strand to initiate bacterial death. The precise mechanism of metronidazole resistance or reduced susceptibility in C. difficile isolates is yet to be elucidated. Mutation in ferric uptake regulator (fur) and associated deficient iron storage in C. difficile isolates was correlated with metronidazole resistance (Lynch et al., 2013; Moura et al., 2014).

Vancomycin resistance Reduced susceptibly to vancomycin of C. difficile isolates is defined as an MIC of >2 mg/mL. Resistance or reduced susceptibility to vancomycin in C. difficile isolates was reported from Poland, Brazil and Israel (Table 20.2; Dworczy
nski et al., 1991). Vancomycin binds with D-alanyl-D-alanine and inhibits bacterial peptidoglycan synthesis. The precise mechanism of vancomycin resistance or reduced susceptibility is unknown. Substitution of amino acid Pro108Leu in the MurG (helps in conversion of lipid I to lipid II during peptidoglycan synthesis) and biofilm formation in C. difficile isolates was found to be associated with vancomycin resistance (Ðapa et al., 2012; Leeds et al., 2013).

Fidaxomicin resistance Fidaxomicin (macrocyclic group of antibiotic) is used as an alternative drug of choice for CDI in the United States and Europe since 2011 (Obuch-Woszczaty
nski et al., 2014). Binding at a site distinct from rifamycin and inhibition of bacterial RNA polymerase is the major mechanism of action. Exceptional reports of fidaxomicin-reduced susceptibility (MIC 2e4 mg/L) (Leeds et al., 2013) or resistance (MIC 16 mg/L) are available (Goldstein et al., 2011). Mutation in rpoC (Glu/Arg at position 1073) and CD22120 (marR homologue) and associated target site modifications in the RNA polymerase were correlated with resistance against fidaxomicin in C. difficile isolates (Leeds et al., 2013).

Tetracycline resistance C. difficile resistance to tetracyclines is uncommon in clinical isolates and few studies indicated the highly variable occurrence rate (2.4%e41.6%) between the countries (Spigaglia, 2016). Resistance to tetracyclines in C. difficile isolates is mostly mediated by ribosomal protective protein (TetM) present in Tn916-like element (Spigaglia et al., 2006).

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259

Other resistance genes such as tetW and conjugative transposon (Tn6164) possessing tet(44) and ant(6)-Ib genes were also detected in C. difficile isolates (Corver et al., 2012; Fry et al., 2012).

Chloramphenicol resistance Chloramphenicol resistance of C. difficile is also uncommon and only 3.7% of European clinical isolates showed the resistance (Freeman et al., 2015). The resistance is mediated by catD gene (Tn4453a and Tn4453b) encoding chloramphenicol acetyl transferase (Wren et al., 1988).

Cephalosporin resistance Most of the C. difficile strains show constitutive resistance against cephalosporins and a few coding sequences present in the genome were identified to be associated with the resistance (Spigaglia, 2016).

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