Temocillin as an alternative treatment for acute bacterial cholangitis: a retrospective microbiology susceptibility-based study of 140 episodes.

With rising antibiotic resistance, alternatives to carbapenems are needed for acute cholangitis (AC). Temocillin reaches high biliary concentrations with limited impact on microbiota. We retrospectively included 140 AC episodes and assessed the efficacy of temocillin using microbiology susceptibility testing from blood cultures. Considering all bacteria collected by episode, resistance to temocillin, PIP/TAZ and 3GC occurred in 27/140 (26%), 32 (22.8%) and 31 (22%) episodes, respectively (p = 0.7). After documentation, temocillin could have spared PIP/TAZ or carbapenems in 14/26 and 4/11 episodes. Temocillin may constitute an alternative treatment after microbiological documentation by sparing carbapenems and/or PIP/TAZ, but not as an empirical therapeutic option.

Success of Escherichia coli O25b:H4 Sequence Type 131 Clade C Associated with a Decrease in Virulence.

Escherichia coli O25b:H4 sequence type 131 (ST131), which is resistant to fluoroquinolones and which is a producer of CTX-M-15, is globally one of the major extraintestinal pathogenic E. coli (ExPEC) lineages. Phylogenetic analyses showed that multidrug-resistant ST131 strains belong to clade C, which recently emerged from clade B by stepwise evolution. It has been hypothesized that features other than multidrug resistance could contribute to this dissemination since other major global ExPEC lineages (ST73 and ST95) are mostly antibiotic susceptible. To test this hypothesis, we compared early biofilm production, presence of ExPEC virulence factors (VFs), and in vivo virulence in a mouse sepsis model in 19 and 20 epidemiologically relevant strains of clades B and C, respectively. Clade B strains were significantly earlier biofilm producers (P < 0.001), carriers of more VFs (P = 4e-07), and faster killers of mice (P = 2e-10) than clade C strains. Gene inactivation experiments showed that the H30-fimB and ibeART genes were associated with in vivo virulence. Competition assays in sepsis, gut colonization, and urinary tract infection models between the most anciently diverged strain (B1 subclade), one C1 subclade strain, and a B4 subclade recombining strain harboring some clade C-specific genetic events showed that the B1 strain always outcompeted the C1 strain, whereas the B4 strain outcompeted the C1 strain, depending on the mouse niches. All these findings strongly suggest that clade C evolution includes a progressive loss of virulence involving multiple genes, possibly enhancing overall strain fitness by avoiding severe infections, even if it comes at the cost of a lower colonization ability.

Independent Host Factors and Bacterial Genetic Determinants of the Emergence and Dominance of Escherichia coli Sequence Type 131 CTX-M-27 in a Community Pediatric Cohort Study.

The recent emergence and diffusion in the community of Escherichia coli isolates belonging to the multidrug-resistant and CTX-M-27-producing sequence type 131 (ST131) C1-M27 cluster makes this cluster potentially as epidemic as the worldwide E. coli ST131 subclade C2 composed of multidrug-resistant isolates producing CTX-M-15. Thirty-five extended-spectrum beta-lactamase (ESBL)-producing ST131 isolates were identified in a cohort of 1,885 French children over a 5-year period. They were sequenced to characterize the ST131 E. coli isolates producing CTX-M-27 recently emerging in France. ST131 isolates producing CTX-M-27 (n = 17), and particularly those belonging to the C1-M27 cluster (n = 14), carried many resistance-encoding genes and predominantly an F1:A2:B20 plasmid type. In multivariate analysis, having been hospitalized since birth (odds ratio [OR], 10.9; 95% confidence interval [CI], 2.4 to 48.8; P = 0.002) and being cared for in a day care center (OR, 9.4; 95% CI, 1.5 to 59.0; P = 0.017) were independent risk factors for ST131 CTX-M-27 fecal carriage compared with ESBL-producing non-ST131 isolates. No independent risk factor was found when comparing CTX-M-15 (n = 11)- and CTX-M-1/14 (n = 7)-producing ST131 isolates with ESBL-producing non-ST131 isolates or with non-ESBL-producing isolates. Several factors may contribute to the increase in fecal carriage of CTX-M-27-producing E. coli isolates, namely, resistance to multiple antibiotics, capacity of the CTX-M-27 enzyme to hydrolyze both cefotaxime and ceftazidime, carriage of a peculiar F-type plasmid, and/or capacity to colonize children who have been hospitalized since birth or who attend day care centers.

Risk factors for carbapenem-resistant Enterobacteriaceae infections: a French case-control-control study.

This study aimed to assess characteristics associated with infections due to carbapenem-resistant Enterobacteriaceae (CRE), producing (CPE) or not producing (non-CPE) carbapenemase, among hospitalised patients in 2014-2016 in France. Case-patients with CRE were compared to two control populations. In multivariate analysis comparing 160 CRE cases to 160 controls C1 (patients with a clinical sample positive for carbapenem-susceptible Enterobacteriaceae), five characteristics were linked to CRE: male gender (OR = 1.9; 95% CI = 1.3-3.4), travel in Asia (OR = 10.0; 95% CI = 1.1-91.2) and hospitalisation in (OR = 2.4; 95% CI = 1.3-4.4) or out of (OR = 4.4; 95% CI = 0.8-24.1) France in the preceding 12 months, infection in the preceding 3 months (OR = 3.0; 95% CI = 1.5-5.9), and antibiotic receipt between admission and inclusion (OR = 1.9; 95% CI = 1.0-3.3). In multivariate analysis comparing 148 CRE cases to 148 controls C2 [patients with culture-negative sample(s)], four characteristics were identified: prior infection (OR = 3.3; 95% CI = 1.6-6.8), urine drainage (OR = 3.0; 95% CI = 1.5-6.1) and mechanical ventilation (OR = 3.7; 95% CI = 1.1-13.0) during the current hospitalisation, and antibiotic receipt between admission and inclusion (OR = 6.6; 95% CI = 2.8-15.5). Univariate analyses comparing separately CPE cases to controls (39 CPE vs C1 and 36 CPE vs C2) and non-CPE cases to controls (121 non-CPE vs C1 and 112 non-CPE vs C2), concomitantly with comparison of CPE to non-CPE cases showed that only CPE cases were at risk of previous travel and hospitalisation abroad. This study shows that, among CRE, risk factors are different for CPE and non-CPE infection, and suggests that question patients about their medical history and lifestyle should help for early identification of patients at risk of CPE among patients with CRE.

Hypervirulent Klebsiella pneumoniae in Cryptogenic Liver Abscesses, Paris, France.

Liver abscesses containing hypervirulent Klebsiella pneumoniae have emerged during the past 2 decades, originally in Southeast Asia and then worldwide. We hypothesized that hypervirulent K. pneumoniae might also be emerging in France. In a retrospective, monocentric, cohort study, we analyzed characteristics and outcomes for 199 consecutive patients in Paris, France, with liver abscesses during 2010-2015. We focused on 31 patients with abscesses containing K. pneumoniae. This bacterium was present in most (14/27, 52%) cryptogenic liver abscesses. Cryptogenic K. pneumoniae abscesses were more frequently community-acquired (p<0.00001) and monomicrobial (p = 0.008), less likely to involve cancer patients (p<0.01), and relapsed less often (p<0.01) than did noncryptogenic K. pneumoniae liver abscesses. K. pneumoniae isolates from cryptogenic abscesses belonged to either the K1 or K2 serotypes and had more virulence factors than noncryptogenic K. pneumoniae isolates. Hypervirulent K. pneumoniae are emerging as the main pathogen isolated from cryptogenic liver abscesses in the study area.

The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone.

BACKGROUND: In 2006, we found healthy subjects carrying ST131 Escherichia coli in their intestinal microbiota consisting of two populations: a subdominant population of fluoroquinolone-resistant E. coli belonging to subclone H30 (H30-R or subclade C1), the current worldwide dominant ST131 subclone, and a dominant E. coli population composed of antibiotic-susceptible E. coli belonging to subclone H22 (clade B), the precursor of subclone H30. We sequenced the whole genome of fecal H22 strain S250, compared it to the genomes of ExPEC ST131 H30-Rx strain JJ1886 and commensal ST131 H41 strain SE15, sought the H22-H30 genomic differences in our fecal strains and assessed their phenotypic consequences. RESULTS: We detected 173 genes found in the Virulence Factor Database, of which 148 were shared by the three ST131 genomes, whereas some were genome-specific, notably those allowing determination of virotype (D for S250 and C for JJ1886). We found three sequences of the FimH site involved in adhesion: two in S250 and SE15 close and identical, respectively, to that previously reported to confer strong intestinal adhesion, and one in JJ1886, corresponding to that commonly present in uropathogenic E. coli. Among the genes involved in sugar metabolism, one encoding a gluconate kinase lacked in S250 and JJ1886. Although this gene was also absent in both our fecal H22 and H30-R strains, H22 strains showed a higher capacity to grow in minimal medium with gluconate. Among the genes involved in gluconate metabolism, only the ghrB gene differed between S250/H22 and JJ1886/H30-R strains, resulting in different gluconate reductases. Of the genes involved in biofilm formation, two were absent in the three genomes and one, fimB, in the JJ1886 genome. Our fecal H30-R strains lacking intact fimB displayed delayed biofilm formation relative to our fecal H22 strains. The H22 strains differed by subclade B type and plasmid content, whereas the H30-R strains were identical. CONCLUSIONS: Phenotypic analysis of our fecal strains based on observed genomic differences between S250 and JJ1886 strains suggests the presence of traits related to bacterial commensalism in our H22 strains and traits commonly found in uropathogenic E. coli in our H30-R strains.

Development of an algorithm for phenotypic screening of carbapenemase-producing Enterobacteriaceae in the routine laboratory.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are difficult to identify among carbapenem non-susceptible Enterobacteriaceae (NSE). We designed phenotypic strategies giving priority to high sensitivity for screening putative CPE before further testing. METHODS: Presence of carbapenemase-encoding genes in ertapenem NSE (MIC > 0.5 mg/l) consecutively isolated in 80 French laboratories between November 2011 and April 2012 was determined by the Check-MDR-CT103 array method. Using the Mueller-Hinton (MH) disk diffusion method, clinical diameter breakpoints of carbapenems other than ertapenem, piperazicillin+tazobactam, ticarcillin+clavulanate and cefepime as well as diameter cut-offs for these antibiotics and temocillin were evaluated alone or combined to determine their performances (sensitivity, specificity, positive and negative likelihood ratios) for identifying putative CPE among these ertapenem-NSE isolates. To increase the screening specificity, these antibiotics were also tested on cloxacillin-containing MH when carbapenem NSE isolates belonged to species producing chromosomal cephalosporinase (AmpC) but Escherichia coli. RESULTS: Out of the 349 ertapenem NSE, 52 (14.9%) were CPE, including 39 producing OXA-48 group carbapenemase, eight KPC and five MBL. A screening strategy based on the following diameter cut offs, ticarcillin+clavulanate <15 mm, temocillin <15 mm, meropenem or imipenem <22 mm, and cefepime <26 mm, showed 100% sensitivity and 68.1% specificity with the better likelihood ratios combination. The specificity increased when a diameter cut-off <32 mm for imipenem (76.1%) or meropenem (78.8%) further tested on cloxacillin-containing MH was added to the previous strategy for AmpC-producing isolates. CONCLUSION: The proposed strategies that allowed for increasing the likelihood of CPE among ertapenem-NSE isolates should be considered as a surrogate for carbapenemase production before further CPE confirmatory testing.

The challenges of multi-drug-resistance in hepatology.

Antimicrobial resistance has become a major global public health security problem that needs coordinated approaches at regional, national and international levels. Antibiotic overuse and the failure of control measures to prevent the spread of resistant bacteria in the healthcare environment have led to an alarming increase in the number of infections caused by resistant bacteria, organisms that resist many (multi-drug and extensively drug-resistant strains), if not all (pan-drug-resistant bacteria) currently available antibiotics. While Gram-positive cocci resistance (methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci) shows a heterogeneous geographical distribution, extended-spectrum ß-lactamase-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae have become pandemic worldwide and endemic in some parts of the world, respectively. Moreover, currently available therapeutic options for resistant bacteria are very limited, with very few new agents in development. Antimicrobial resistance is especially relevant in decompensated cirrhosis. Firstly, cirrhotic patients are highly susceptible to develop infections caused by resistant bacteria as risk factors of multiresistance concentrate in this population (mainly repeated hospitalizations and antibiotic exposure). Secondly, inappropriate empirical antibiotic schedules easily translate into increased morbidity (acute kidney injury, acute-on-chronic liver failure, septic shock) and hospital mortality in advanced cirrhosis. Therefore, hepatologists must face nowadays a complex clinical scenario that requires new empirical antibiotic strategies that may further spread resistance. Global, regional and local preventive measures should therefore be implemented to combat antimicrobial resistance in cirrhosis including the restriction of antibiotic prophylaxis to high-risk populations, investigation on non-antibiotic prophylaxis, stewardship programs on adequate antibiotic prescription and on increasing awareness of the problem among health professionals, and well-defined early de-escalation policies based on rapid microbiological diagnostic tests. Other infection control practices such as hand hygiene and barrier precautions are also important. Clinical impact and cost-effectiveness of epidemiological surveillance programs (periodic rectal and nasal swabs) should also be explored.

Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model.

Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence.

A new mechanism to render clinical isolates of Escherichia coli non-susceptible to imipenem: substitutions in the PBP2 penicillin-binding domain.

OBJECTIVES: So far, two types of mechanism are known to be involved in carbapenem non-susceptibility of Escherichia coli clinical isolates: reduced outer membrane permeability associated with production of ESBLs and/or overproduction of class C ß-lactamases; and production of carbapenemases. Non-susceptibility to only imipenem observed in two clinical isolates suggested a new mechanism, described in the present study. METHODS: The ST was determined for the two isolates of E. coli (strains LSNy and VSBj), and their chromosomal region encoding the penicillin-binding domain of PBP2 was amplified, sequenced and then used for recombination experiments in E. coli K12 C600. Antibiotic MICs were determined using the Etest method. RESULTS: Strains LSNy and VSBj, which displayed ST23 and ST345, respectively, showed amino acid substitutions in their PBP2 penicillin-binding domain. Substitution Ala388Ser located in motif 2 (SXD) was common to the two strains. Two additional substitutions (Ala488Thr and Leu573Val) located outside the two other motifs were identified in strain LSNy, whereas another one (Thr331Pro) located in motif 1 was identified in strain VSBj. Recombination experiments to reproduce non-susceptibility to imipenem in E. coli K12 C600 were not successful when only the common substitution was transferred, whereas recombination with DNA fragments including either the three substitutions (strain LSNy) or the two substitutions (strain VSBj) were successful. CONCLUSIONS: Substitution of amino acids in the penicillin-binding domain of PBP2 is a new mechanism by which E. coli clinical isolates specifically resist imipenem.

Escherichia coli ST131, an intriguing clonal group.

In 2008, a previously unknown Escherichia coli clonal group, sequence type 131 (ST131), was identified on three continents. Today, ST131 is the predominant E. coli lineage among extraintestinal pathogenic E. coli (ExPEC) isolates worldwide. Retrospective studies have suggested that it may originally have risen to prominence as early as 2003. Unlike other classical group B2 ExPEC isolates, ST131 isolates are commonly reported to produce extended-spectrum ß-lactamases, such as CTX-M-15, and almost all are resistant to fluoroquinolones. Moreover, ST131 E. coli isolates are considered to be truly pathogenic, due to the spectrum of infections they cause in both community and hospital settings and the large number of virulence-associated genes they contain. ST131 isolates therefore seem to contradict the widely held view that high levels of antimicrobial resistance are necessarily associated with a fitness cost leading to a decrease in pathogenesis. Six years after the first description of E. coli ST131, this review outlines the principal traits of ST131 clonal group isolates, based on the growing body of published data, and highlights what is currently known and what we need to find out to provide public health authorities with better information to help combat ST131.

Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae.

OBJECTIVES: In Klebsiella pneumoniae, overexpression of the AcrAB efflux pump and the more recently described OqxAB efflux pump has been linked to an antibiotic cross-resistance phenotype, but the mechanisms of regulation are largely unknown. Moreover, while AcrAB has been shown to participate in K. pneumoniae virulence, the contribution of OqxAB has not yet been assessed. METHODS: In the present study we investigated a K. pneumoniae clinical isolate (KPBj1 E+), displaying cross-resistance to quinolones, chloramphenicol and cefoxitin, and its phenotypic revertant (KPBj1 Rev, susceptible to antibiotics) by using whole-genome sequencing, RT-PCR, complementation and a Caenorhabditis elegans virulence model. RESULTS: We detected a point mutation in the oqxR repressor gene of KPBj1 E+, which overexpressed genes rarA, encoding a transcriptional regulator, and oqxB, but not acrB. Complementation with wild-type oqxR restored antibiotic susceptibility and normalized rarA and oqxB expression levels. Whole-genome sequencing showed that KPBj1 Rev had lost the entire rarA-oqxABR locus, situated close to an integration hot spot of phage P4. This large deletion seemed responsible for the significantly lower virulence potential of strain KPBj1 Rev compared with KPBj1 E+. Moreover, we found that KPBj1 E+ ΔacrB was significantly less virulent than its parental strain. CONCLUSIONS: This work demonstrates the role of the overexpression of efflux pump OqxAB, due to a mutation in gene oqxR, in the antibiotic resistance phenotype of a clinical isolate, and suggests that the presence of AcrAB, associated with overexpression of OqxAB, is required for high virulence potential.

Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups.

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected.

Incidence rates of carbapenemase-producing Enterobacteriaceae clinical isolates in France: a prospective nationwide study in 2011-12.

OBJECTIVES: To determine proportions and incidence rates of Enterobacteriaceae producing carbapenemase among those non-susceptible (NS) to carbapenems in France. METHODS: From November 2011 to April 2012, 71 laboratories recorded non-duplicate Enterobacteriaceae clinical isolates NS to at least one carbapenem and the total number of isolates of the different species. Carbapenem MICs were determined by broth microdilution and the ß-lactamase content by DNA microarray. RESULTS: During the study period, the 71 laboratories identified 133 244 Enterobacteriaceae isolates, of which 846 (0.63%) were NS to at least one carbapenem. Carbapenem-NS isolates accounted for 0.07% (61/90 148) among Escherichia coli isolates, 1.1% (111/10 436) among Klebsiella pneumoniae, 8.2% (492/5971) among Enterobacter cloacae and 4.0% (84/2104) among Enterobacter aerogenes. Among the 541 available carbapenem-NS isolates, 222 (including 63 randomly selected E. cloacae) were further analysed after confirmation of carbapenem non-susceptibility. None of the Enterobacter spp. isolates produced carbapenemase. Among the other species, 28 isolates produced carbapenemases (22 OXA-48, 4 KPC and 2 NDM), accounting for an estimated proportion of carbapenemase-producing isolates of 0.08% for all species, 0.01% for E. coli and 0.27% for K. pneumoniae. The incidence-density rate in the participating hospitals was 0.0041 per 1000 hospital-days and the incidence rate was 0.0027 per 100 admissions. CONCLUSIONS: The incidence-density rate of carbapenemase-producing isolates per 1000 hospital-days was low and 30-fold lower than that of carbapenem-NS isolates (0.125) and almost 300-fold lower than that of ESBL-producing isolates (1.104) in these French hospitals.

Prevalence of day-care centre children (France) with faecal CTX-M-producing Escherichia coli comprising O25b:H4 and O16:H5 ST131 strains.

OBJECTIVES: Determining the prevalence of children in day-care centres (DCCs) carrying faecal extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and molecularly characterizing those belonging to the Escherichia coli species. METHODS: Stools were collected from children's diapers (January-April 2012) in randomly chosen DCCs and plated onto ChromID ESBL. Colonies growing on this medium were identified by the Vitek 2 system and tested for antibiotic susceptibility and for ESBL production by the double-disc synergy test. ESBL genotypes were determined as well as phylogenetic groups, ERIC-2 (enterobacterial repetitive intergenic consensus) PCR profiles and sequence types (STs) for the E. coli isolates. Serotypes, virotypes, fimH alleles, ESBL-carrying plasmids and PFGE patterns were determined for the ST131 E. coli isolates. RESULTS: Among 419 children from 25 participating DCCs, 1 was colonized by CTX-M-15-producing Klebsiella pneumoniae and 27 (6.4%) by E. coli, which all produced CTX-M enzymes [CTX-M-15 (37%), CTX-M-1 (26%), CTX-M-14 (22%), CTX-M-27 (11%) and CTX-M-22 (4%)]. The 27 E. coli isolates, 55.5% belonging to group B2, displayed 20 ERIC-2 PCR profiles and 16 STs. The ST131 E. coli isolates were dominant (44%), displayed serotypes O25b:H4 and O16:H5, fimH alleles 30 and 41 and virotypes A and C. According to the PFGE patterns, one strain of E. coli ST131 producing a CTX-M-15 enzyme carried by an IncF F2:A1:B- plasmid had spread within one DCC. CONCLUSIONS: This study shows a notable prevalence (6.4%) of DCC children with faecal CTX-M-producing E. coli isolates comprising a high proportion of E. coli ST131 isolates, suggesting that these children might be a reservoir of this clone.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry assigns Escherichia coli to the phylogroups A, B1, B2 and D.

Escherichia coli classification into phylogroups reflects the diversity of their pathogenicity and their ecological niche, B2 isolates being the most virulent among extra-intestinal strains. MALDI-TOF MS allows a quick, automated, simple and inexpensive bacterial identification. We evaluated the MALDI-TOF MS as a tool for E. coli phylogroup differentiation. We used 656 E. coli isolates, previously assigned to phylogroup A, B1, B2, and D by multiplex PCR, to constitute independent training and validation sets. We then defined two phylogrouping strategies, both validated on spectra obtained by the 'direct transfer method'. The first strategy used the MALDI Biotyper software (Bruker Daltonik) that identified a single peak shift between isolates of phylogroup B2 and those of groups A, B1 and D. It accurately classified 89% of the isolates. The second strategy used the ClinProTools software (Bruker Daltonik) and was based on three successive models. The model 1 adequately differentiated 92% of phylogroup B2-isolates from those belonging to phylogroups A, B1, D. The model 2 adequately discriminated 87% of phylogroup D-isolates from those of phylogroups A and B1. The model 3 correctly sorted 69% of A and B1-isolates. We concluded that clinical laboratories could routinely and very quickly assign E. coli isolates to phylogroups with MALDI-TOF MS. These methods could (i) expedite the detection of the most virulent strains belonging to phylogroup B2 and (ii) be a first-line tool to monitor the epidemiology of extra-intestinal pathogenic E. coli.

Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli.

BACKGROUND: Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS: Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS: Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS: Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Four main virotypes among extended-spectrum-ß-lactamase-producing isolates of Escherichia coli O25b:H4-B2-ST131: bacterial, epidemiological, and clinical characteristics.

A total of 1,021 extended-spectrum-ß-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.

10-Fold increase (2006-11) in the rate of healthy subjects with extended-spectrum ß-lactamase-producing Escherichia coli faecal carriage in a Parisian check-up centre.

OBJECTIVES: In 2006, 0.6% of healthy subjects living in the Paris area had extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in their gut. To assess the evolution of this rate, a study identical to that of 2006 was conducted in 2011. PARTICIPANTS AND METHODS: Healthy adults who visited the IPC check-up centre in February-March 2011 and agreed to participate, provided stools and answered a questionnaire on the visit day. Stools were analysed to detect ESBL producers and to isolate the dominant E. coli population. ESBLs were molecularly characterized. For the subjects harbouring ESBL-producing E. coli, the phylogenetic group and sequence type (ST) were determined for both ESBL-producing and dominant E. coli isolates. PFGE profiles were also determined when two types of isolates had the same ST. RESULTS: Among the 345 subjects included, 21 (6%) had ESBL-producing E. coli faecal carriage. None of the previously published risk factors was identified. CTX-M accounted for 86% and SHV-12 for 14%. Dominant and ESBL-producing E. coli were similarly distributed into phylogenetic groups (A, 52%-48%; B1, 5%; B2, 24%-14%; and D, 19%-33%). Dominant and ESBL-producing E. coli displayed a polyclonal structure (18 STs each). However, ST10 and ST131 were identified in dominant and ESBL-producing E. coli isolates from different subjects. Most (20/21) ESBL producers were subdominant and belonged (16/21) to STs different from that of the corresponding dominant E. coli. CONCLUSIONS: The 10-fold increase in the rate of healthy subjects with ESBL-producing E. coli faecal carriage over a 5 year period suggests wide dissemination of these isolates in the Parisian community.