Phylogenetic distribution of CTX-M and non-esbl producing Escherichia coli isolates:

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1 JCM Accepts, published online ahead of print on 3 July 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Phylogenetic distribution of CTX-M and non-esbl producing Escherichia coli isolates: group B2 isolates, except clone ST131, rarely produce CTX-M enzymes Sylvain Brisse 1 *, Laure Diancourt 1, Cédric Laouénan 2, 3, Marie Vigan 2, 3, Valérie Caro 1, Guillaume Arlet 4, Laurence Drieux 5, 6, Véronique Leflon-Guibout 7, France Mentré 2, 3, Vincent Jarlier 6, 8, Marie-Hélène Nicolas-Chanoine 7, 9, 10, and the Coli β study group. 1. Institut Pasteur, Genotyping of Pathogens and Public Health, 28 rue du Dr Roux, Paris, France, 2. Université Paris Diderot, Sorbonne Paris Cité, UMR 738, F Paris, France; 3. Hôpital Bichat, AP-HP, Service de Biostatistique, Paris, France 4. Service de Bactériologie, Hôpital Tenon, AP-HP, Paris, France 5. Equipe Opérationnelle en Hygiène Hospitalière, Hôpital Charles-Foix, AP-HP, Ivry-sur- Seine, France 6. EA 1541, Université Pierre et Marie Curie-Paris, Paris, France 7. Service de Microbiologie, Hôpital Beaujon, AP-HP, Clichy, France 8. Laboratoire de Bactériologie Hygiène Hospitalière, Hôpital Pitié Salpêtrière, AP-HP, Paris, France 9. Université Paris Diderot, Sorbonne Paris Cité ; INSERM, UMR 773, Centre de Recherche Biomédicale Bichat-Beaujon (CRB3), Paris, France The Coli β study group: Anani Akpabie (Hôpital Emile Roux, AP-HP, Limeil Brévannes, France), Catherine Doit (Hôpital Robert Debré, AP-HP, Paris, France), Salah Gallah (Hôpital Charles Foix, AP-HP, Ivry, France), Najiby Kassis-Chikhani (Hôpital Paul Brousse, AP-HP, Villejuif, France), Estelle Marcon (Hôpital Beaujon, AP-HP, Clichy, France), Didier 1

2 Moissenet (Hôpital Tousseau, AP-HP, Paris, France), Isabelle Podglajen (Hôpital Georges Pompidou, AP-HP, Paris, France), Charlotte Verdet (Hôpital Tenon, AP-HP, Paris, France), Corine Vincent (Université D Diderot, Paris, France), and Jean-Ralph Zahar (Hôpital Necker, AP-HP, Paris, France) * Corresponding author: S. Brisse. Genotyping of Pathogens and Public Health (PF8), Institut Pasteur, 28 rue du Dr Roux, F Paris, France. sylvain.brisse@pasteur.fr; Phone Downloaded from on August 26, 2018 by guest 2

3 Abstract Escherichia coli is the most frequent species associated with clinical infections by extendedspectrum β-lactamase (ESBL)-producing isolates, with the CTX-M ESBL enzymes being predominant and found in genetically diverse E. coli isolates. The main objective of this study was to compare, based on a case-control study, the phylogenetic diversity of 152 CTX-Mproducing and 152 non-esbl-producing clinical E. coli isolates. Multilocus sequence typing (MLST) revealed that even though CTX-M enzymes were largely disseminated across the diversity of E. coli, phylogenetic group B2 showed a particularly heterogeneous situation. First, clone ST131 of group B2 was strongly associated with CTX-M production (55 out of 70 isolates, 79%), with CTX-M-15 being predominant. Second, the remaining members of group B2 were significantly less associated with CTX-M production (9 out of 75, 12%) than E. coli phylogenetic groups A, B1 and D (88 out of 159, 55%). CTX-M-producing ST131 E. coli isolates were significantly more frequent in patients hospitalized in geriatric wards or long term care facilities. Besides, the non-esbl ST131 isolates showed a significantly more frequent resistance to penicillins than the non-esbl, non-st131 isolates. In conclusion, the present study emphasizes the particular antimicrobial resistance and epidemiological characteristics of clone ST131 within group B2, which could result from the higher antibiotic exposure of this clone, as it is the predominant clone of group B2 in human gut carriage. 3

4 Introduction Escherichia coli is the Enterobacteriaceae species causing the largest number of infections, but this species is also a widespread gut commensal of humans and animals. Strains of E. coli are therefore frequently exposed to antimicrobial agents, which has resulted in the emergence and rapid diffusion of antibiotic resistant E. coli clinical isolates. Extendedspectrum β-lactamases (ESBL), which confer resistance to extended-spectrum cephalosporins, were until the year 2000, essentially produced by Klebsiella pneumoniae and Enterobacter spp isolates responsible for nosocomial infections. However, E. coli has now become dominant among the ESBL-producing enterobacterial species. This is especially worrisome as the human gut carriage of E. coli favors dissemination of ESBLs in the community (31). Interestingly, the switch of dominant species among the ESBL-producing enterobacterial isolates occurred concomitantly with the emergence of novel ESBL enzymes that belong to the CTX-M family. The CTX-M enzymes have superseded the TEM- and SHV ESBLs (2) and are found in genetically diverse E. coli isolates. Among CTX-M enzymes, CTX-M-15 predominates and has been associated with the dissemination of a particular E. coli clone of sequence type (ST) 131 (23, 27). The E. coli species is genetically diverse and strains have been classified into a number of phylogenetic groups, the most frequent ones being called A, B1, B2 and D (12, 14, 29, 32). The ST131 clone belongs to phylogenetic group B2 and is associated with extraintestinal infections including urinary tract infection, bacteremia and meningitis (27). However, ESBL-producing E. coli are genetically diverse. In a previous study, entitled Coliβ, we have performed a case-double control study to determine the risk factors associated with acquisition of CTX-M-producing E. coli clinical isolates (24). We have shown that in addition to health-care related factors, patient origin and living in collective housing were independently associated with isolation of a CTX-Mproducing E. coli clinical isolate. In the present study, the main objective was to compare the phylogenetic and clonal diversity of the CTX-M-producing and the non-esbl-producing E. coli isolates collected during the Coliβ study. The second objective was to assess the association of particular CTX- M enzymes with the clonal background of strains. A third objective was to compare the distribution of the ST131 isolates with the non-st131 isolates with regard to medical wards, 4

5 85 86 clinical samples and antibiotic susceptibility, in order to better understand the epidemiology of this prevalent clone that often produces CTX-M-15 enzymes (1, 6, 7, 11, 15, 25, 26, 30). 87 5

6 88 Materials and methods Bacterial isolates The 304 E. coli isolates studied, which comprised 152 CTX-M producers and 152 non-esbl producers, were prospectively collected from the 17th of November, 2008 to the 30th of June, 2009 in 10 hospitals (7,554 beds) of the Paris area (Assistance Publique Hôpitaux de Paris, France). They were collected in the frame of the Coliβ study, a casedouble control study aimed at determining the factors associated with a CTX-M-producing E. coli clinical isolate (24). This study was approved by the Ethics Committee of the Groupe Hospitalier Hôpitaux Universitaires Paris Nord Val de Seine (Institutional review board N IRB ). Each of the 152 studied CTX-M-producing clinical isolates came from a case to which was attributed a control patient with a non-esbl-producing isolate detected on the day or within the 3 days following the case detection. Control isolates caused infection but not necessarily the same as that caused by the CTX-M-producing isolate. Clinical origins of the studied E. coli isolates and the ward where the patients were hospitalized were recorded (Tables 1 and 2). The CTX-M-producing E. coli clinical isolates detected within the first 48 h of hospitalization were deemed as imported. Antibiotic susceptibility Antibiotic susceptibility of the 304 E. coli isolates was tested by using the disk agar diffusion method and interpreted according to the French Antibiogram Committee recommendations ( for the following antibiotics: ampicillin or amoxicillin, amoxicillin+ clavulanic acid, ticarcillin, ticarcillin+ clavulanic acid, piperacillin, piperacillin+tazobactam, cephalothin, cefoxitin, cefotaxime or ceftriaxone, ceftazidime, cefepime, imipenem, gentamicin, amikacin, nalidixic acid, ciprofloxacin, cotrimoxazole, and fosfomycin. β-lactamase detection ESBL production was detected by the double disk synergy test as recommended by the French Antibiogram Committee (13). ESBL-encoding bla genes were searched for by PCR as previously described (22) from the E. coli strains screened as ESBL producers. The amplified 6

7 fragments were sequenced by using primers specific for bla CTX -M, bla TEM and bla SHV genes, as previously described (22) Multilocus Sequence Typing (MLST) MLST was carried out based on an international MLST scheme (32) using previously described primers and guidelines as specified at The allele and profile assignments were determined based on the central E. coli MLST database at the above website. Data analysis Sequence chromatograms were edited and stored using BioNumerics v6.6 (Applied- Maths, Sint Maartens-Latem, Belgium). To achieve high levels of confidence on each nucleotide substitution, all nucleotides within the internal gene portion chosen for MLST analysis were supported by at least two sequence chromatograms. Microevolutionary relationships among STs were investigated by comparing allelic profiles using the eburstlike minimum spanning tree (MStree) method in BioNumerics. To determine the deep phylogenetic relationships among strains and assign them to the main E. coli phylogenetic groups, we first constructed a neighbor-joining tree based on the concatenated sequences of the seven MLST genes for the 304 isolates as well as 230 isolates of known phylogenetic group, including the ECOR reference collection, previously analyzed with the same genes (32) ( Based on their clustering position relative to reference strains, the isolates were assigned to phylogenetic groups A, B1, B2 and D. The latter group was broadly defined as including strains previously labeled as F (14) or ABD (32). To confirm the phylogenetic group assignments using a recombination-aware method, ClonalFrame (8) analysis was performed. Two runs each with a total of 150,000 iterations were performed, the 50,000 first ones being discarded as burn-in; the two runs gave consistent results. Finally, program Structure was used to determine for each isolate, the proportion of ancestry of nucleotides from variable numbers of ancestral populations. These results were used to confirm the group assignments determined above. Finally, the triplex PCR pattern (4) was determined for some isolates to control the correspondence between our sequence-based 7

8 groupings and the triplex PCR method. Genotypic diversity was estimated by the Simpson index computed from To compare the distribution of categorical variables between the paired CTX-Mproducing E. coli isolates and non-esbl- producing E. coli isolates, the non-parametric Cochran-Mantel-Haenszel paired test was used. To compare the distribution of the categorical variables between non paired ST131 isolates and non-st131 isolates among the CTX-M producers or the non-esbl producers with regard to medical wards, clinical samples and antibiotic susceptibility, the non-parametric Fisher exact test was used. If the overall distribution of categorical variables differed significantly, an adjustment for these two tests was used to adjust p-values for multiple comparisons class by class (using SAS proc MULTTEST). All statistical analyses were performed with SAS software, version 9.1 (SAS Institute, Cary, NC, USA). All tests were two-sided at the 0.05 significance level. Downloaded from on August 26, 2018 by guest 8

9 161 Results Phylogenetic diversity To test whether CTX-M-producing E. coli isolates were distributed differently from non-esbl-producing isolates into the main E. coli groups, we determined the phylogenetic positioning of the 304 isolates based on the seven gene sequences. Internal portions of the seven MLST genes (3,423 nucleotides in total) were obtained for the 304 isolates. A total of 286 (8.4%) nucleotide positions were variable, among which 225 were parsimony informative, providing a high quantity of information for phylogenetic analysis. For selected isolates, we also determined the triplex PCR pattern. Overall, the number of isolates classified into groups A, B1, B2 and D based on gene sequences were 70 (23%), 42 (14%), 145 (48%), and 47 (15%), respectively. The two phylogenetic group assignment methods disagreed for six strains (2%) that were clearly assigned to group A or B1 based on gene sequences, but to B2 or D based on triplex PCR. As sequence-based assignment is more reliable, we used the assignments based on this method in subsequent analyses. The phylogenetic relationships of the 304 isolates (Fig. 1) clearly demarcated group B2 and isolates assigned to group D. However, the branching order between groups B1, A and the branch that included ST648, which was previously described as group F (14), was not resolved. This lack of resolution is consistent with previous work that demonstrated high frequency of recombination among E. coli groups, in particular between groups A and B1 (32). The CTX-M-producing E. coli were grouped as follows: 44 (29%) in group A, 19 (13%) in group B1, 64 (42%) in group B2, and 25 (16%) in group D (Fig. 1). The non-esblproducing isolates were grouped as follows: 26 (17%) in group A, 23 (15%) in group B1, 81 (53%) in group B2 and 22 (15%) in group D. The distribution into phylogenetic groups was not significantly different between the 2 populations of E. coli. Genotypic diversity To compare the genotypic diversity of CTX-M-producing E. coli with that of non- ESBL-producing E. coli, the MLST data were used to group isolates into sequence types (ST) and clonal complexes (CC). To group closely related STs into meaningful clonal groups, we 9

10 used the standard definition of clonal complexes (CC) as groups of STs differing by only one gene from at least one other member of the group (10). The 304 E. coli isolates were grouped into 116 distinct ST. Of these, 36 were novel STs, which were assigned ST numbers 1648 to 1682, and The most frequent STs were ST131 (group B2) with 70 isolates (23%), ST10 (group A) with 22 isolates (7%) and ST73 (group B2) with 16 isolates (5%). In contrast, 87 STs (including all novel STs) comprised a single isolate. There were 13 CCs, including four that were represented at high frequency: CC131 with 71 isolates (23%), CC10 with 46 (15%), CC73 with 18 (6%) and CC23 with 15 (5%) (Fig. 2). CC131 included only two STs, ST131 (70 isolates) and the newly defined ST1680 (1 isolate), which differed from ST131 at gene gyrb. In contrast, CC10 (11 STs) and CC23 (5 STs) were more heterogeneous (Fig. 2). Of note, CC10, CC23, CC73 and CC131 correspond to the frequent clonal complexes CC2, CC4, CC66 and CC43, respectively, found previously among bacteremia isolates using an alternative MLST scheme (14) ( The overall genotypic diversity of the 304 isolates was 93.5% and was homogeneous across the ten hospital centers, with frequent STs being recovered from many of them. Notably, the genotypic diversity of CTX-M-producing E. coli and non-esbl-producing E. coli was distinct, as Simpson's index of diversity was 85.7% for the first population of E. coli but was much higher, 96.9% for the second one. The lower diversity among CTX-Mproducing E. coli was in a large part explained by the high frequency of ST131 (55 CTX-Mproducing ST131 isolates, 36%) and ST10 (13 isolates, 8.6%). In addition, non-esblproducing E. coli corresponded more frequently to STs with a unique isolate (36%) than CTX-M-producing E. coli (21%, p = 0.005). The clonal composition of the populations of CTX-M-producing E. coli and non- ESBL-producing E. coli differed significantly both at the level of STs and at the level of CCs (p < 10-4 ). Four individual group B2 STs were distributed differently between CTX-Mproducing E. coli and non-esbl-producing E. coli: ST131 (36.2% versus 9.9%, p < 10-4 ), ST73 (0% versus 10.5%, p = ), ST95 (0% versus 5.3%, p = 0.01) and ST141 (0% versus 4.6%, p = 0.02). Similarly, when comparing the distribution of the 13 recognized CCs, two of them, belonging to group B2, had a significantly distinct distribution between CTX-Mproducing E. coli and non-esbl-producing E. coli: CC131 (36.8%versus 9.9%, p < 10-4 ) and CC73 (0% versus 11.9%, p = 0.005). CC10 (group A) was slightly more frequent among CTX-M-producers than non-esbl-producers (21.7% versus 8.6%, respectively, p = 0.08). In 10

11 contrast, CC23 (group A), the third most frequent CC among CTX-M producers (after CC131 and CC10), was also frequent in non-esbl-producers (p = 0.9) CTX-M enzymes distribution among phylogenetic groups and CCs Of the 152 CTX-M- producing isolates, 10 different variants of CTX-M enzymes were detected. There were 119 (78%) enzymes belonging to group CTX-M-1, including 78 (51%) CTX-M-15 and 36 (24%) CTX-M-1 enzymes. There were 30 (20%) enzymes of group CTX- M-9, including 18 (12%) CTX-M-14 enzymes. Finally, there were 3 (2%) group CTX-M-2 enzymes. CTX-M enzymes were distributed into all phylogenetic groups, with a frequency per group ranging from 44% (B1 and B2) to 53% in group D and 62.5% in group A. However, the distribution of the CTX-M variants showed striking differences among and within groups (Fig. 1). First, a sharp contrast was observed within group B2 between ST131, where CTX- M-15 and other CTX-M enzyme producers predominated, and the remaining isolates of group B2, which were mostly negative for CTX-M enzymes: 55 out of 70 ST131 isolates were CTX-M producers, whereas the other genotypes within group B2, 66 out of 75, were not ESBL producers (p < 10-4 ). Among the CTX-M enzymes, CTX-M-15 was clearly distributed differently (p = ) than the other enzymes. CTX-M-15 enzymes were predominantly found in group B2 (56%), whereas this group accounted for only 25.7% of other CTX-M enzymes (p = ). In particular, ST131 was significantly associated with CTX-M-15 production rather than other CTX-M enzymes (p <10-4 ). Second, when ST131 was put aside, the remaining genotypes of group B2 were less associated with CTX-M enzyme production than the other E. coli groups. When excluding ST131, the proportion of ESBL producers within B2 (9 out of 75, 12%) was strikingly lower than the proportion in groups A, B1 and D (89 out of 162, 55%) (p < 10-4 ). Notably, the frequent B2 group CC73 contained no CTX-M-producing isolate. These observations show that with the remarkable exception of ST131 (and in fact, CC131, as the only isolate of ST1680 was a CTX-M-15 producer), group B2 as a whole is significantly less associated with CTX-M production than the other E. coli groups. Some heterogeneity was also observed within other groups. For example, within group A, CC10 showed a high proportion of CTX- M producers (Fig. 1). 11

12 Antibiotic susceptibility In each participating hospital, the susceptibility of amoxicillin or ampicillin, amoxicillin + clavulanic acid, cephalothin, cefotaxime or ceftriaxone, imipenem and gentamicin was systematically tested (Table 3). For the other antibiotics, the susceptibility was not systematically tested. However, in each participating hospital, the susceptibility to the same antibiotics was tested for each pair of CTX-M-producer and non-esbl producer control. The percentage of antibiotic resistant isolates was significantly higher for CTX-M producers than for non-esbl producers for all the antibiotics tested (p = from to < 10-4 ) except for imipenem (no resistant isolate in both populations) and fosfomycin (0 % for CTX- M producers and 1% for non-esbl producers: p = 0.2). The percentage of resistant isolates ranged from 14% (cefoxitin) to 100 % (amoxicillin or ampicillin, ticarcillin, piperacillin, cephalothin, cefotaxime or ceftriaxone, ceftazidime and cefepime) for CTX-M producers and from 0% (cefepime) to 51% (penicillins) for non-esbl producers (Table 3). Epidemiological and antimicrobial resistance characteristics of ST131 Because ST131 accounts for a large fraction of CTX-M-producing E. coli strains, we investigated the distribution of this clone among the participating hospitals and among medical wards. CTX-M-producing ST131 isolates were identified in each of the ten participating hospitals and distributed, in each hospital, into various wards, as were the non- ST131 CTX-M producers (Table 1). Considering wards (Table 2), among the CTX-M producer population, ST131 was distributed significantly differently among the seven wards (p = ) compared with the other STs. This could be attributed to the strong association of ST131 with geriatric/long-term care hospitalization (27% for ST131 versus 6% for other STs [p = 0.01]). Among the non-esbl-producing E. coli, the distribution of ST131 was also globally distinct from the other STs (p = 0.02). The antibiotic susceptibility of ST131 E. coli was compared with that of non-st131 E. coli for the two E. coli populations: CTX-M producers and non-esbl producers (Table 3). Irrespective of the two E. coli populations, ST131 isolates were significantly more often resistant to quinolones (nalidixic acid and ciprofloxacin) than non-st131 isolates (p < 10-4 ). In contrast, the percentage of cotrimoxazole-resistant E. coli was significantly higher among 12

13 non-st131 E. coli than among ST131 E. coli when comparing the CTX-M producers (p <10-4 ). Concerning the aminoglycosides, when a significant difference was observed (amikacin for CTX-producers [p =0.01], and gentamicin for the non-esbl producers [p =0.03]), the highest percentage of resistance always involved ST131 E. coli. With regard to the β-lactams cefoxitin and imipenem, which are classically not hydrolyzed by class A β-lactamases such as ESBL enzymes, there was no significant difference between ST131 and non-st131 E. coli for the two E. coli populations. For the other β-lactam molecules, the analysis (which focused on the non-esbl producers because those producing CTX-M were consequently resistant to these molecules) showed that ST131 E. coli were significantly more often resistant to penicillins (aminopenicillin: p = 0.03, ticarcillin: p = 0.03 and piperacillin: p = 0.009) than non-st131 E. coli. The non-esbl ST131 were not significantly more hospital-acquired than the non-st131, non-esbl producers (p = 0.16). In contrast, with regard to clinical sources, the distribution of ST131 was not significantly different from that of the non-st131 isolates, within the CTX-M-producing E. coli and the non- ESBL-producing E. coli (p = 0.3 and 0.2, respectively). Finally, we did not find that ST131 was significantly more prevalent than non-st131 among the CTX producers isolated within the first 48 h of hospitalization (36% and 47%, respectively; p = 0.2). 13

14 Discussion In this study, we compared for the first time using a case-control study, the clonal composition of CTX-M-producing and non-esbl producing E. coli clinical isolates. In addition, we determined the association of the different CTX-M enzymes with clonal groups, and investigated the epidemiological features and antimicrobial susceptibility of ST131, the major genotype among isolates producing CTX-M enzymes. Both CTX-M and non-esbl-producing populations were polyclonal and were distributed across the major phylogenetic groups A, B1, B2 and D in proportions that are consistent with previous studies, e.g. (14, 20). The amount and nature of the clones found in this study is consistent with previous studies that showed the important contribution of ST131, CC10 and CC23 to the CTX-M-producing E. coli population (3, 11, 26). This study demonstrates for the first time using a case-control study design, that the population of CTX-M-producing E. coli clinical isolates differs from non-esbl-producing populations in their clonal composition, with a few prevalent clones being clearly distributed non-randomly among the two populations. In particular, within group B2, most CC131 were CTX-M producers, whereas most CC95 and all CC73 and ST141 were not, as was the case for most of the remaining STs of phylogenetic group B2. As ST131 producing CTX-M isolates were identified in each of the ten participating hospitals and as a great variety and number of wards within each hospital were concerned by ST131, it can be expected that the situation observed in these hospitals reflects, at a local level, the global ST131 clonal diversity rather than local cross-transmission. Although CTX-M-15 was predominant in ST131, there is clear evidence of several independent acquisitions of distinct CTX-M types by strains of ST131, as found previously (27). The presence of CTX-M enzymes in all major phylogenetic groups reflects the wide dissemination of these enzymes across the diversity of E. coli. However, a striking observation of our study is an apparent restriction against acquisition of CTX-M enzymes by members of group B2, since most group B2 genotypes were devoid of CTX-M enzymes, except for ST131 (Fig. 1). To our knowledge, this is the first demonstration that ST131 is more associated with CTX-M enzymes than the other STs of group B2. In turn, non-st131 B2 isolates were clearly less associated with CTX-M production than other E. coli groups. Three hypotheses might explain these observations. First, group B2 might be less exposed to antibiotics than groups A, B1 and D, which are predominant in the fecal E. coli population of 14

15 healthy subjects (9, 21). Second, among group B2 fecal E. coli, those belonging to ST131 might display features different from non-st131 group B2 fecal E. coli. It has to be noted that ST131 has been shown to be the most prevalent clone, among group B2, in the dominant fecal E. coli population of healthy subjects (21). Thus, ST131 E. coli, similar to E. coli of non-b2 groups, might be more exposed to antibiotics. Third, clone ST131 might differ in some important biological properties from other group B2 E. coli. Notably, ST131 was shown to belong, within phylogenetic group B2, to subgroup I (5). Interestingly, this subgroup was suggested to have a basal position within the diversity of group B2 (19). Although incompletely resolved, our phylogeny (Fig. 1) is consistent with this proposal, as ST131 and closely related STs 429, 555, 1680 and 1650 formed one of the most basal branches of group B2. This basal position could imply that common characteristics shared by non-st131 B2 strains could have evolved after the evolutionary separation of ST131 and related genotypes. This independent evolution could have resulted for example, in intrinsic differences in the ability of ST131 to acquire the mobile elements that carry CTX-M encoding genes. Thus, further studies are required to explore the higher propensity of ST131, compared to non- ST131 group B2 E.coli isolates, to harbor CTX-M enzymes. Another new feature of ST131, showed in this study by comparing the non-esbl ST131 isolates with the non-esbl, non-st131 isolates, is the significantly higher percentage of ST131 isolates resistant to penicillins. In addition, ST131 was commonly resistant to fluoroquinolones, as previously reported (16). These observations add to the phenotypic resemblance of ST131 with non-b2 isolates, rather than with other B2 members. Interestingly, ST131 has already been shown to differ from classical extra-pathogenic group B2 E. coli strains with regards to its potential virulence, as assessed by the absence in ST131 of several virulence factors that are common in other B2 strains (5, 18) and by its reduced capacity to induce death or infection in different animal models (17, 18). With regards to the clinical epidemiology of ST131, we noted that CTX-M-producing ST131 E. coli isolates were significantly associated to patients hospitalized in geriatric wards or long term care facilities. In contrast, this characteristic was not identified for all CTX-Mproducing E. coli isolates when compared with non-esbl-producing isolates (24). In this previous study, we showed that living in collective housing was a factor associated with a CTX-M producing clinical E. coli isolate. Consistently, nursing homes in Ireland were shown to be the reservoir of ST131 isolates that produce CTX-M-15 (28). A large proportion of the patients hospitalized in geriatric wards and long term care facilities may come from collective 15

16 housing such as nursing homes. Therefore, we hypothesize that French nursing homes, similarly to Irish nursing homes, might be a reservoir of CTX-M-producing ST131. The absence of significant dominance of ST131 in the others wards (ICU and medical wards), and the absence of difference between the proportion of ST131 among the hospital-acquired and the imported CTX-M producers, reinforce the hypothesis of reservoirs of CTX-M-producing ST131 E. coli outside the hospital. In conclusion, the present study emphasizes the particular characteristics of B2 clone ST131 with regards to its epidemiology, carriage of genes coding for CTX-M enzymes, and antimicrobial susceptibility. We hypothesize that the latter features are due to an ecological adaptation of ST131 to the gut. As clone ST131 is the predominant clone of group B2 in human gut (9, 21), it should as a consequence be more exposed to antimicrobial agents. In contrast, non-st131 members of group B2 carry CTX-M enzymes much less frequently than other E. coli groups, which may result from a lower exposure to antimicrobial agents due to their lower frequency in gut carriage. Acknowledgments This study was supported by a grant (PAS 7010) from the Programme Régional de Recherche Clinique AP-HP/Institut Pasteur, Direction de la Recherche Clinique AP-HP, Paris, France. 16

17 Fig. 1. Phylogenetic distribution of CTX-M enzymes in E. coli. The phylogeny was constructed based on the seven MLST gene sequences using software ClonalFrame. CTX-M- producing isolates are indicated by circles colored according to the CTX-M enzyme type as indicated. Phylogenetic groups A, B1, B2 and D are identified by grey background. Sequence types (ST) of particular interest (see main text) are indicated. ST117 belongs to group ABD (32), whereas ST648 belongs to group F (14). Fig. 2. Clonal composition of CTX-M- producing and non-esbl- producing populations. The minimum spanning tree was constructed based on MLST allelic profiles using BioNumerics v Each circle corresponds to a sequence type (ST), which number is indicated besides it. The size of circles is related to the number of isolates with the corresponding ST. Pie sections colors within circles correspond to CTX-M enzyme types as indicated in the legend and correspond to colors in Fig. 1 (white: non-esbl producing isolates). STs that belong to a single clonal complex are surrounded by grey shade. The clonal complex (CC) number is indicated, for the major CCs, with grey background. The links between circles depict the number of allelic mismatches between STs: plain bold, 1 mismatch; plain, 2 mismatches; plain grey, 3 mismatches; dashed grey, four mismatches; dotted grey, 5 or more mismatches. 17

18 Table 1. Distribution of ST131and non-st131 CTX-M producers among the participating hospitals and number of wards concerned in each hospital by the patients infected by these isolates. 408 Hospital No. of CTX producers (No. of STs) / No. of wards concerned 409 (type) ST131 Non ST I (STCT) 3 / 3 15 (11) / 9 II (LTCF*) 9 / 4 9 (8) / 5 III (LTCF*) 8 / 5 4 (4) / 3 IV (STCF) 2 / 2 5 (5) / 4 V (STCF) 11 / (14) / 15 VI (Pediatrics) 2 / 2 4 (4) / 4 VII (STCF) 12 / 8 16 (13) / 9 VIII (STCF) 1 / 1 12 (9) / 8 IX (Pediatrics) 1/ 1 1 / 1 X (STCF*) 6 / 5 11 (7) / 5 STCT Short term care facility; LTCF: long term care facility * Hospital with a rehabilitation department

19 Table 2. Distribution of the ST131 and non-st131 Escherichia coli among the CTX-M and the non-esbl E. coli producers according to medical ward. Ward Number (%) of isolates CTX-M producers Non-ESBL producers ST131 Non ST131 Adjusted P* ST131 Non ST131 Adjusted P* Medicine 15 (27) 47 (49) (40) 12 (9) 0.2 Geriatric/Long term care 15 (27) 6 (6) (27) 21 (15) 0.9 Rehabilitation 10 (18) 9 (9) (13) 59 (43) 0.5 Surgery 7 (13) 16 (17) (13) 29 (21) 1.0 Gynecologic / Obstetric 5 (9) 5 (5) (7) 7 (5) 1.0 Pediatrics 2 (4) 1 (1) (0) 6 (5) 1.0 Intensive care 1 (2) 13 (13) (0) 3 (2) * Fisher Exact Test (adjusted for multiple comparisons)

20 29 30 Table 3. Comparison of the antibiotic susceptibility of Escherichia coli between CTX-M producers and non-esbl producers and between ST131 and non-st131 among the CTX-M and the non-esbl E. coli producers. Antibiotic Percentage of E. coli isolates intermediate susceptible or resistant No. of pairs CTX producers Non-ESBL producers CTXproducers producers ST131 Non ST131 ST131 Non-ESBL tested P* P* Non ST131 P** Amoxicillin or Ampicillin < Amoxicillin + clavulanic acid < Ticarcillin < Ticarcillin + clavulanic acid < Piperacillin < Piperacillin + tazobactam < Cephalothin < Cefoxitin Cefotaxime or Ceftriaxone < Ceftazidime < Cefepime < Imipenem Gentamicin < Amikacin < Cotrimoxazole < < Nalidixic acid < < <10-4 Ciprofloxacin < < <10-4 Fosfomycin NS

21 31 * Cochran-Mantel-Haenszel paired test; ** Fisher Exact Test (adjusted for multiple comparisons) 21

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24 Le Gall, T., O. Clermont, S. Gouriou, B. Picard, X. Nassif, E. Denamur, and O. Tenaillon Extraintestinal virulence is a coincidental by-product of commensalism in B2 phylogenetic group Escherichia coli strains. Mol Biol Evol. 11: Lee, S., J. K. Yu, K. Park, E. J. Oh, S. Y. Kim, and Y. J. Park Phylogenetic groups and virulence factors in pathogenic and commensal strains of Escherichia coli and their association with blactx-m. Ann Clin Lab Sci 40: Leflon-Guibout, V., J. Blanco, K. Amaqdouf, A. Mora, L. Guize, and M. H. Nicolas- Chanoine Absence of CTX-M enzymes but high prevalence of clones, including clone ST131, among fecal Escherichia coli isolates from healthy subjects living in the area of Paris, France. J Clin Microbiol 46: Leflon-Guibout, V., C. Jurand, S. Bonacorsi, F. Espinasse, M. C. Guelfi, F. Duportail, B. Heym, E. Bingen, and M. H. Nicolas-Chanoine Emergence and spread of three clonally related virulent isolates of CTX-M-15-producing Escherichia coli with variable resistance to aminoglycosides and tetracycline in a French geriatric hospital. Antimicrob Agents Chemother 48: Nicolas-Chanoine, M. H., J. Blanco, V. Leflon-Guibout, R. Demarty, M. P. Alonso, M. M. Canica, Y. J. Park, J. P. Lavigne, J. Pitout, and J. R. Johnson Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX- M-15. J Antimicrob Chemother 61: Nicolas-Chanoine, M. H., V. Jarlier, J. Robert, G. Arlet, L. Drieux, V. Leflon- Guibout, C. Laouénan, B. Larroque, V. Caro, and F. Mentré Patient's Origin and Lifestyle Associated with CTX-M-Producing Escherichia coli: A Case-Control- Control Study. PLoS One 7:e Oteo, J., K. Diestra, C. Juan, V. Bautista, A. Novais, M. Perez-Vazquez, B. Moya, E. Miro, T. M. Coque, A. Oliver, R. Canton, F. Navarro, and J. Campos Extended-spectrum beta-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/a, ST23 complex/a and ST131/B2. Int J Antimicrob Agents 34: Peirano, G., A. K. van der Bij, D. B. Gregson, and J. D. Pitout Molecular epidemiology over an 11-year period (2000 to 2010) of extended-spectrum betalactamase-producing Escherichia coli causing bacteremia in a centralized Canadian region. J Clin Microbiol 50:

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26 Figure 1 B1 A CC23 D ST648 CTX-M-15 CTX-M-1 CTX-M-14 CTX-M-27 CTX-M-28 ST10 CTX-M-2 CTX-M-9 CTX-M-24 CTX-M % CTX-M-32 ST117 ST141 ST131 ST95 B2 ST1650 ST1680 ST555 ST73

27 Figure CTX-M-15 CTX-M-1 CTX-M-14 CTX-M-27 CTX-M-28 CTX-M-2 CTX-M CTX M 9 CTX-M-24 CTX-M-22 CTX-M-32 CC CC CC CC CC73 CC on August 26, 2018 by guest Downloaded from