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Journal of Clinical Microbiology, May 2007, p. 1607-1610, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.00306-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Toxin Profiles and Resistances to Macrolides and Newer Fluoroquinolones as Epidemicity Determinants of Clinical Isolates of Clostridium difficile from Warsaw, Poland{triangledown}

Hanna Pituch,1* Willem van Leeuwen,2 Kees Maquelin,3 Dorota Wultanska,1 Piotr Obuch-Woszczatynski,1 Grazyna Nurzynska,4 Haru Kato,5 Martin Reijans,6 Felicja Meisel-Mikolajczyk,1 Miroslaw Luczak,1,4 and Alex van Belkum2

Department of Medical Microbiology, Medical University of Warsaw, Warsaw, Poland,1 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands,2 Center for Optical Diagnostics and Therapy, Department of General Surgery, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands,3 Central Public Hospital of Medical University of Warsaw, Warsaw, Poland,4 Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo, Japan,5 Pathofinder BV, Oxfordlaan 70, Maastrich, The Netherlands6

Received 8 February 2007/ Accepted 14 February 2007


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ABSTRACT
 
Amplified fragment length polymorphism genotypes, antibiotic resistance profiles, and toxin profiles of Clostridium difficile strains from Warsaw were determined. The isolates segregate in six major genotypes, coinciding with toxin profiles. Most of the toxin A-negative toxin B-positive toxin CDT-negative strains possess ermB, and several strains were resistant to fluoroquinolones. Resistograms and toxin types of C. difficile strains are epidemicity determinants.


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TEXT
 
Clostridium difficile can cause antibiotic-associated diarrhea, antibiotic-associated colitis, and pseudomembranous colitis, collectively known as C. difficile-associated diseases (CDADs) (11, 16). The main effectors of CDADs are toxins A (TcdA) and B (TcdB) (3), the genes of which are located in the pathogenicity locus PaLoc (6). In nontoxigenic C. difficile strains, the PaLoc is truncated (20), with its upstream- and downstream-located cdu-2/2' and cdd-2/3 open reading frames still being intact. Some C. difficile strains produce a third (binary) toxin (CDT) encoded by the cdtA and cdtB genes outside of the PaLoc (13, 18). Overall, genotypes are important for deciphering clostridial epidemiology and population structure (9). Amplified fragment length polymorphism (AFLP) analysis allows the generation of many polymorphic markers suited for detailed epidemiological investigation (8, 19). We here define the AFLP-based population structure of C. difficile strains with different toxin profiles derived from Polish hospitals.

The isolation of C. difficile from fecal samples was performed using CCCA medium (bioMérieux, Marcy-l'Etoile, France) (14). Among 175 C. difficile strains, 92 clinical isolates were derived from adults in hospital A1 (2004 to 2006). Sixteen clinical C. difficile strains were isolated between May and September 2005 from adult patients in hospital A2. Patients were in transplantation, orthopedic, internal medicine, general surgery, urology, nephrology, dermatology, hematology, gynecology, and neurology wards (Fig. 1). In four cases, C. difficile was isolated from adult CDAD outpatients. A further 58 isolates originated from pediatric CDAD patients (4 to 16 years of age) hospitalized in pediatric hospitals P1 (38 isolates) and P2 (20 isolates). Pediatric patients were nursed in hematology, gastroenterology, and nephrology wards. Included were nine historic Polish C. difficile toxin A-negative toxin B-positive (A– B+) strains (1995 to 1998). Two strains were Japanese (GAI 97660 and GAI 97482) and belonged to the smz ribotype, and one strain belonged to the gr ribotype (GAI 97480). Two Japanese strains producing only tcdB (A– B+ CDT–) were included (GAI 95601 and GAI 95600). Four reference strains (VPI 10463 [A+ B+ CDT–, United States] NIHBRIGGS 8050 [A– B– CDT–, United States], 1470 [A– B+ CDT–, Belgium], and CCUG 20309 [8864] [A– B+ CDT+, United Kingdom]) were coanalyzed. Strains were stored at –80°C.


Figure 1
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  		FIG. 1. Survey of AFLP analysis, toxin profiles, and antibiotic susceptibility profiles of 175 strains of C. difficile from Poland, Japan, the United States, the United Kingdom, and Belgium. On the left, the Matlab-generated phylogenetic tree based on the AFLP scores is visualized. The dissimilarities in the 78-digit AFLP codes are indicated at the bottom of the figure (40% to 0%). The four major toxin-correlated groups are color coded in blue, red, black, and green. Six different genogroups, identified being as A to F between horizontal red lines, can be discerned. The first column indicates strain identification codes, whereas the second column gives the toxin profile (positive [+]/negative [–] scores for toxins A, B, and CDT from left to right). The third column summarizes the result of the ermB-specific PCR test. Next follow seven columns summarizing the antibiotic susceptibility values for macrolides (clindamycin [CM] and erythromycin [EM]), fluoroquinolones (ciprofloxacin [CI], moxifloxacin [MX], and gatifloxacin [GA]), metronidazole (MZ), and vancomycin (VA). The antimicrobial resistances are highlighted by a color-coded shadowed letter. The subsequent columns identify the originating hospitals and departments therein. Note that two separate transplantology (A and B) and three orthopedic (A to C) departments were involved. The last column on the right states the year of isolation (–, not known; n, not done; A1, adult hospital 1; A2, adult hospital 2; P1, pediatric hospital 1; P2, pediatric hospital 2; REF, reference strain; Jap epid, Japanese epidemic; *, strains with erythromycin resistance and clindamycin susceptibility).

For toxin detection, a C. difficile colony was transferred into brain heart infusion broth (Difco, Detroit, MI) and grown anaerobically, and the supernatant was collected. TcdA was detected by immunochromatography (toxin A test; Oxoid, Basingstoke, United Kingdom). Additionally, an immunoenzymatic assay for both TcdA and TcdB was used (TOX A/B test, TechLab). TcdB was detected by a cytotoxicity assay. Tenfold serial dilutions of culture filtrate were added in duplicate to cells and incubated for 24 h. C. difficile strain VPI 10463 was used as the positive control. The cytopathic effect was observed by inverse microscopy. If the cytopathic effect was neutralized by polyclonal antiserum to TcdB (TOX-B test; TechLab), the test was positive.

Template DNA for PCR was prepared using PREP-PLUS genomic DNA (A&A Biotechnology, Warsaw, Poland). For PCR of the nonrepeating regions of tcdA and tcdB, primer pairs YT28-YT29 and YT17-YT18 were used (14). Deletions in the repeating regions of tcdA were detected with primer pair NK9-NKV011 (7). Amplification of cdu-2 and cdd-3 was done with primer pairs Tim5-Struppi5 and Tim6-Struppi6, respectively (4). Previously described primers and PCR protocols for the amplification of cdtA and cdtB were used (15, 18). A fragment of the ermB gene was amplified using primer pair 2980-2981 (1).

AFLP analysis was performed as described above, after an initial optimization procedure (12). Reliable AFLP fragment markers were scored in a binary format (with 1 being present and 0 being absent). Similarity between each sample pair was calculated using the Dice correlation coefficient. Hierarchical clustering into a dendrogram was performed using an unweighted-pair group method using average linkages in Matlab version 7.1 (Mathworks, Natick, MA).

MICs for clindamycin, erythromycin, ciprofloxacin, moxifloxacin, gatifloxacin, metronidazole, and vancomycin were determined by Etest (AB Biodisc, Solna, Sweden). Resistance was defined according to Clinical and Laboratory Standards Institute (CLSI) recommendations (4a). For metronidazole testing, Bacteroides thetaiotaomicron ATCC 29791 and Bacteroides fragilis NCTC11295 were included as reference strains, as were Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 during fluoroquinolone testing.

Overall, we identified four major toxin profiles for the C. difficile strains under investigation: A+ B+ CDT– (n = 80; 46%), A+ B+ CDT+ (n = 15; 9%), A– B+ CDT– (n = 53; 30%), and A– B– CDT– (n = 27; 15%) (see color codes in Fig. 1). PCR for the detection of the repeating sequences with primer pair NK9-NKV011 generated the 700-bp product, as also observed for other A– B+ CDT– strains. This demonstrates that all isolates had an identical deletion in the TcdA gene, except for a single strain that showed a more extensive deletion (not shown). Clinical isolates that were positive for the binary toxin genes were also A+ B+ (n = 15). PCR for the detection of the open reading frames cdu-2 and cdd-3 confirmed the presence of the remainder of the PaLoc in all A– B– CDT– strains.

AFLP generated 78 markers that were scored for all strains. Six major AFLP subgroups can be discerned visually on the basis of the (color-coded) toxin types (A to F) (Fig. 1). Group A shows a small number of A+ B+ CDT– and A– B+ CDT– strains derived from two of the hospitals. Group A is quite similar to group C, which consists primarily of A+ B+ CDT– strains. However, group C also harbors A– B– CDT– strains (n = 19). Among these latter strains, a single cluster of nine isolates (including the American reference isolate NIHBRIGGS 8050) was observed. Interestingly, A+ B+ CDT– strains were found in all Polish hospitals. It appears as if deletions in the PaLoc occur regularly in the C complex. In addition, five strains from Japan, the United Kingdom, and the United States are part of this group of Polish strains. AFLP clusters B and D are relatively small and involve strains with the A+ B+ CDT+ toxin type. Apparently, the CDT genes were introduced into the AFLP C group upon independent occasions but not frequently. Cluster E strains were involved in an apparent long-term outbreak occurring in adult hospital A1. Thirty-two strains within this cluster were identical by AFLP. This shows that a clone has been circulating in one hospital between 2002 and 2006. It found its way to hospital P2 and the open population (positive outpatients). Cluster F contains a small number of A+ B+ CDT+ isolates recently recovered from three different hospitals.

All isolates were susceptible to metronidazole and vancomycin. Three of the 156 strains were susceptible to ciprofloxacin. Clindamycin resistance coupled to erythromycin resistance occurred in three of six genogroups, and solitary erythromycin resistance was documented for only three strains (Fig. 1). There is good concordance between positive ermB PCRs and combined resistance. Resistance to moxifloxacin and gatifloxacin is coupled, although in two cases, solitary moxifloxacin resistance was observed. The extended fluoroquinolone resistance occurred in three of six genogroups. In cluster E, 33 out of the 51 strains were resistant to both moxifloxacin and gatifloxacin. Overall, combined macrolide and fluoroquinolone resistance occurred in more than 20% of the clinical isolates, although this is biased because of the outbreak-related isolates in genogroup E.

In the United States and Canada, nationwide outbreaks of antibiotic-associated diarrhea were documented. These were associated with specific strains of C. difficile of a specific toxin type and with reduced susceptibility to fluoroquinolones (10). Clearly, such strains can travel the world (17). We have compared strains of four different toxin profiles by AFLP to define their genomic relatedness. The obvious clustering of toxin types suggests that the toxin profile is an important determinant of epidemic potential. Both persistence in medical institutions and geographic spreading of toxin variants were observed, and despite a certain degree of genetic heterogeneity generated during dissemination, the toxin types remain fairly constant. We report the emergence of an epidemic A– B+ CDT– variant belonging to ribotype 017 (data not shown), which turned out to be highly resistant to moxifloxacin and gatifloxacin. In addition, the strain was carrying ermB, which predisposes to macrolide-lincosamide-streptogramin B-type resistance profiles. This macrolide-lincosamide-streptogramin B-type resistance has been found in C. difficile strains previously (1, 2, 5). The link between toxin profiles, antibiograms, and epidemicity is an important one given the emergence and epidemic spread of pathogenic strains of C. difficile.


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ACKNOWLEDGMENTS
 
We do not have commercial or other associations that might pose a conflict of interest.

H. Pituch was supported by the Polish Ministry of Education and Science, grant 2 P05D 074 27. AFLP analysis was performed at PathoFinder BV (Maastricht, The Netherlands).

Guus Simons is acknowledged for enduring support.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology, Medical University of Warsaw, Chalubinski Street, 02-004 Warsaw, Poland. Phone: 48-22-628-2739. Fax: 48-22-628-2739. E-mail: hanna.pituch{at}ib.amwaw.edu.pl Back

{triangledown} Published ahead of print on 21 February 2007. Back


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Journal of Clinical Microbiology, May 2007, p. 1607-1610, Vol. 45, No. 5
0095-1137/07/$08.00+0     doi:10.1128/JCM.00306-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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