Long-Term Colonization by blaCTX-M-Harboring Escherichia coli in Healthy Japanese People Engaged in Food Handling

The actual state of intestinal long-term colonization by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in healthy Japanese people remains unclear. Therefore, a total of 4,314 fecal samples were collected from 2,563 food handlers from January 2010 to December 2011. Approximately 0.1 g of each fecal sample was inoculated onto a MacConkey agar plate containing cefotaxime (1 μg/ml). The bacterial colonies that grew on each plate were checked for ESBL production by the double-disk synergy test, as recommended by the Clinical and Laboratory Standards Institute. The bacterial serotype, antimicrobial susceptibility, pulsotype, sequence type (ST), and ESBL genotype were checked, and the replicon types of plasmids harboring the ESBL gene were also determined after conjugation experiments. ESBL producers were recovered from 70 (3.1%) of 2,230 participants who were checked only once. On the other hand, ESBL producers were isolated at least once from 52 (15.6%) of 333 participants who were checked more than twice, and 13 of the 52 participants carried ESBL producers for from more than 3 months to up to 2 years. Fluoroquinolone (FQ)-resistant E. coli strains harboring bla_CTX-M were repeatedly recovered from 11 of the 13 carriers of bla_CTX-M-harboring E. coli. A genetically related FQ-resistant E. coli O25b:H4-ST131 isolate harboring bla_CTX-M-27 was recovered from 4 of the 13 carriers for more than 6 months. Three FQ-resistant E. coli O1:H6-ST648 isolates that harbored bla_CTX-M-15 or bla_CTX-M-14 were recovered from 3 carriers. Moreover, multiple CTX-M-14- or CTX-M-15-producing E. coli isolates with different serotypes were recovered from 2 respective carriers. These findings predict a provable further spread of ESBL producers in both community and clinical settings.     

Association of Veterinary Third-Generation Cephalosporin Use with the Risk of Emergence of Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Dairy Cattle in Japan

The use of extended-spectrum cephalosporins in food animals has been suggested to increase the risk of spread of Enterobacteriaceae carrying extended-spectrum β-lactamases to humans. However, evidence that selection of extended-spectrum cephalosporin–resistant bacteria owing to the actual veterinary use of these drugs according to criteria established in cattle has not been demonstrated. In this study, we investigated the natural occurrence of cephalosporin-resistant Escherichia coli in dairy cattle following clinical application of ceftiofur. E. coli isolates were obtained from rectal samples of treated and untreated cattle (n = 20/group) cultured on deoxycholate-hydrogen sulfide-lactose agar in the presence or absence of ceftiofur. Eleven cefazoline-resistant isolates were obtained from two of the ceftiofur-treated cattle; no cefazoline-resistant isolates were found in untreated cattle. The cefazoline-resistant isolates had mutations in the chromosomal ampC promoter region and remained susceptible to ceftiofur. Eighteen extended-spectrum cephalosporin–resistant isolates from two ceftiofur-treated cows were obtained on ceftiofur-supplemented agar; no extended-spectrum cephalosporin–resistant isolates were obtained from untreated cattle. These extended-spectrum cephalosporin–resistant isolates possessed plasmid-mediated β-lactamase genes, including bla _CTX-M-2 (9 isolates), bla _CTX-M-14 (8 isolates), or bla _CMY-2 (1 isolate); isolates possessing bla _CTX-M-2 and bla _CTX-M-14 were clonally related. These genes were located on self-transmissible plasmids. Our results suggest that appropriate veterinary use of ceftiofur did not trigger growth extended-spectrum cephalosporin–resistant E. coli in the bovine rectal flora; however, ceftiofur selection in vitro suggested that additional ceftiofur exposure enhanced selection for specific extended-spectrum cephalosporin–resistant β-lactamase-expressing E. coli clones     

Emergence and dissemination of Enterobacteriaceae with plasmid-mediated CMY-6 and CTX-M-15 beta-lactamases in community in North-India

A collection of 88 Escherichia coli and 22 Klebsiella pneumoniae strains previously studied for the carriage of bla _CTX-M-15 were screened for the presence of bla _ampC using multiplex PCR, and analysed for antibiotic resistance and co-carriage of bla _ampC and bla _CTX-M-15. Cefoxitin resistance was noticed in total of 41.8% isolates. Concomitant resistance to third and fourth generation cephalosporins, aztreonam, gentamicin, trimethoprim and ciprofloxacin was noted. A total of 20% (22/110) isolates (14.5% E. coli and 5.5% K. pneumoniae) carried plasmidic bla _ampC of the CIT-family. Sequencing of the representative isolates revealed CMY-6 AmpC-beta-lactamase. Simultaneous occurrence of bla _ampC and bla _CTX-M was noticed in 77.3% (17/22) isolates. Plasmid analysis of the selected isolates showed the presence of a single plasmid of ca. ~23 kb. Curing and transconjugation experiments provided evidence of carriage of bla _CMY and bla _CTX-M on same plasmid. RAPD typing revealed the presence of bla _CMY and bla _CTX-M in a wider range of strains of E. coli and K. pneumoniae.     

Molecular Characteristics of Extended-Spectrum Cephalosporin-Resistant Enterobacteriaceae from Humans in the Community

Objective

To investigate the molecular characteristics of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae collected during a cross-sectional study examining the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in humans living in areas with high or low broiler density.

Methods

ESC-resistant Enterobacteriaceae were identified by combination disc-diffusion test. ESBL/AmpC/carbapenemase genes were analysed using PCR and sequencing. For E. coli, phylogenetic groups and MLST were determined. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing.

Results

175 ESC-resistant Enterobacteriaceae were cultured from 165/1,033 individuals. The isolates were Escherichia coli(n=65), Citrobacter freundii (n=52), Enterobacter cloacae (n=38), Morganella morganii (n=5), Enterobacter aerogenes (n=4), Klebsiella pneumoniae (n=3), Hafnia alvei (n=2), Shigella spp. (n=2), Citrobacter amalonaticus (n=1), Escherichia hermannii (n=1), Kluyvera cryocrescens (n=1), and Pantoea agglomerans (n=1). The following ESBL genes were recovered in 55 isolates originating from 49 of 1,033 (4.7 %) persons: bla _CTX-M-1 (n=17), bla _CTX-M-15 (n=16), bla _CTX-M-14 (n=9), bla _CTX-M-2 (n=3), bla _CTX-M-3 (n=2), bla _CTX-M-24 (n=2), bla _CTX-M-27 (n=1), bla _CTX-M-32 (n=1), bla _SHV-12 (n=2), bla _SHV-65 (n=1) and bla _TEM-52 (n=1). Plasmidic AmpC (pAmpC) genes were discovered in 6 out of 1,033 (0.6 %) persons. One person carried two different E. coli isolates, one with bla _CTX-M-1 and the other with bla _CMY-2 and therefore the prevalence of persons carrying Enterobacteriaceae harboring ESBL and/or pAmpC genes was 5.2 %. In eight E. coli isolates the AmpC phenotype was caused by mutations in the AmpC promoter region. No carbapenemase genes were identified. A large variety of E. coli genotypes was found, ST131 and ST10 being most common.

Conclusions

ESBL/pAmpC genes resembled those from patients in Dutch hospitals, indicating that healthy humans form a reservoir for transmission of these determinants to vulnerable people. The role of poultry in the transmission to humans in the community remains to be elucidated.     

Plasmid-Mediated Resistance to Cephalosporins and Fluoroquinolones in Various Escherichia coli Sequence Types Isolated from Rooks Wintering in Europe

Extended-spectrum-beta-lactamase (ESBL)-producing, AmpC beta-lactamase-producing, and plasmid-mediated quinolone resistance (PMQR) gene-positive strains of Escherichia coli were investigated in wintering rooks (Corvus frugilegus) from eight European countries. Fecal samples (n = 1,073) from rooks wintering in the Czech Republic, France, Germany, Italy, Poland, Serbia, Spain, and Switzerland were examined. Resistant isolates obtained from selective cultivation were screened for ESBL, AmpC, and PMQR genes by PCR and sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were performed to reveal their clonal relatedness. In total, from the 1,073 samples, 152 (14%) cefotaxime-resistant E. coli isolates and 355 (33%) E. coli isolates with reduced susceptibility to ciprofloxacin were found. Eighty-two (54%) of these cefotaxime-resistant E. coli isolates carried the following ESBL genes: bla_CTX-M-1 (n = 39 isolates), bla_CTX-M-15 (n = 25), bla_CTX-M-24 (n = 4), bla_TEM-52 (n = 4), bla_CTX-M-14 (n = 2), bla_CTX-M-55 (n = 2), bla_SHV-12 (n = 2), bla_CTX-M-8 (n = 1), bla_CTX-M-25 (n = 1), bla_CTX-M-28 (n = 1), and an unspecified gene (n = 1). Forty-seven (31%) cefotaxime-resistant E. coli isolates carried the bla_CMY-2 AmpC beta-lactamase gene. Sixty-two (17%) of the E. coli isolates with reduced susceptibility to ciprofloxacin were positive for the PMQR genes qnrS1 (n = 54), qnrB19 (n = 4), qnrS1 and qnrB19 (n = 2), qnrS2 (n = 1), and aac(6′)-Ib-cr (n = 1). Eleven isolates from the Czech Republic (n = 8) and Serbia (n = 3) were identified to be CTX-M-15-producing E. coli clone B2-O25b-ST131 isolates. Ninety-one different sequence types (STs) among 191 ESBL-producing, AmpC-producing, and PMQR gene-positive E. coli isolates were determined, with ST58 (n = 15), ST10 (n = 14), and ST131 (n = 12) predominating. The widespread occurrence of highly diverse ESBL- and AmpC-producing and PMQR gene-positive E. coli isolates, including the clinically important multiresistant ST69, ST95, ST117, ST131, and ST405 clones, was demonstrated in rooks wintering in various European countries.     

A study on the effect of different rifle calibres in euthanisation of grey seals (Halichoerus grypus) in seal traps in the Baltic Sea

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla _CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla _CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla _CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla _CTX-M-1.     

Prevalence and Characteristics of Extended-Spectrum β-Lactamase and Plasmid-Mediated Fluoroquinolone Resistance Genes in Escherichia coli Isolated from Chickens in Anhui Province,China

The aim of this study was to characterize the prevalence of extended-spectrum β-lactamase (ESBL) genes and plasmid-mediated fluoroquinolone resistance (PMQR) determinants in 202 Escherichia coli isolates from chickens in Anhui Province, China, and to determine whether ESBL and PMQR genes co-localized in the isolates. Antimicrobial susceptibility for 12 antimicrobials was determined by broth microdilution. Polymerase chain reactions (PCRs), DNA sequencing, and pulsed field gel electrophoresis (PFGE) were employed to characterize the molecular basis for β-lactam and fluoroquinolone resistance. High rates of antimicrobial resistance were observed, 147 out of the 202 (72.8%) isolates were resistant to at least 6 antimicrobial agents and 28 (13.9%) of the isolates were resistant to at least 10 antimicrobials. The prevalence of bla _CTX-M, bla _TEM-1 and bla _TEM-206 genes was 19.8%, 24.3% and 11.9%, respectively. Seventy-five out of the 202 (37.1%) isolates possessed a plasmid-mediated quinolone resistance determinant in the form of qnrS (n = 21); this determinant occurred occasionally in combination with aac(6′)-1b-cr (n = 65). Coexistence of ESBL and/or PMQR genes was identified in 31 of the isolates. Two E. coli isolates carried bla _TEM-1, bla _CTX-M and qnrS, while two others carried bla _CTX-M, qnrS and aac(6′)-1b-cr. In addition, bla _TEM-1, qnrS and aac(6′)-1b-cr were co-located in two other E. coli isolates. PFGE analysis showed that these isolates were not clonally related and were genetically diverse. To the best of our knowledge, this study is the first to describe detection of TEM-206-producing E. coli in farmed chickens, and the presence of bla _TEM-206, qnrS and aac(6′)-1b-cr in one of the isolates.     

Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq

Background

Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq.

Methodology/Principal Findings

In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥3) plasmids compared to their non-MDR counterparts, which carried ≤2 plasmids (p<0.01). Various large plasmids (∼52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (bla _TEM1, bla _AMPC, bla _CTX-M-15, bla _OXA-1, bla _VIM-2 and bla _SHV), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates.

Conclusions/Significance

This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.     

Molecular Typing of CTX-M-Producing Escherichia coli Isolates from Environmental Water,Swine Feces,Specimens from Healthy Humans,and Human Patients

CTX-M-producing Escherichia coli is the predominant type of extended-spectrum β-lactamase (ESBL)-producing E. coli worldwide. In this study, molecular typing was conducted for 139 CTX-M-producing E. coli isolates, phenotypically positive for ESBLs, isolated from environmental water, swine, healthy humans, and hospitalized patients in Hangzhou, China. The antibiotic resistance profiles of the isolates for the cephalosporins and fluoroquinolones were determined. The isolates showed 100% resistance to cefotaxime and ceftriaxone while maintaining relatively high susceptibility to cefoxitin, cefepime, and ceftazidime. A total of 61.9% (86/139) of the isolates, regardless of origin, showed high resistance to fluoroquinolones. PCRs and DNA sequencing indicated that bla_CTX-M-14 was the most prevalent CTX-M-9 group gene and that bla_CTX-M-15 and bla_CTX-M-55 were the dominant CTX-M-1 group genes. Isolates from all sources with CTX-M types belonging to the CTX-M-1 or CTX-M-9 group were most frequently associated with epidemics. Molecular homology analysis of the isolates, conducted by phylogenetic grouping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), demonstrated that the dominant clones belonged to B2-ST131, D-ST648, D-ST38, or A-CC10. These four sequence types (STs) were discovered in E. coli isolates both from humans and from environmental water, suggesting frequent and continuous intercompartment transmission between humans and the aquatic environment. Seven novel sequence types were identified in the current study. In conclusion, this study is the first to report the molecular homology analysis of CTX-M-producing E. coli isolates collected from water, swine, and healthy and hospitalized humans, suggesting that pathogens in the environment might originate both from humans and from animals.     

Antimicrobial Resistance,Virulence Factors and Genetic Diversity of Escherichia coli Isolates from Household Water Supply in Dhaka,Bangladesh

Background

Unsafe water supplies continue to raise public health concerns, especially in urban areas in low resource countries. To understand the extent of public health risk attributed to supply water in Dhaka city, Bangladesh, Escherichia coli isolated from tap water samples collected from different locations of the city were characterized for their antibiotic resistance, pathogenic properties and genetic diversity.

Methodology/Principal Findings

A total of 233 E. coli isolates obtained from 175 tap water samples were analysed for susceptibility to 16 different antibiotics and for the presence of genes associated with virulence and antibiotic resistance. Nearly 36% (n = 84) of the isolates were multi-drug(≥3 classes of antibiotics) resistant (MDR) and 26% (n = 22) of these were positive for extended spectrum β-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for bla _CTX-M-15, 7 for bla _OXA-1-group (all had bla _OXA-47) and 2 for bla _CMY-2. Quinolone resistance genes, qnrS and qnrB were detected in 6 and 2 isolates, respectively. Around 7% (n = 16) of the isolates carried virulence gene(s) characteristic of pathogenic E. coli; 11 of these contained lt and/or st and thus belonged to enterotoxigenic E. coli and 5 contained bfp and eae and thus belonged to enteropathogenic E. coli. All MDR isolates carried multiple plasmids (2 to 8) of varying sizes ranging from 1.2 to >120 MDa. Ampicillin and ceftriaxone resistance were co-transferred in conjugative plasmids of 70 to 100 MDa in size, while ampicillin, trimethoprim-sulfamethoxazole and tetracycline resistance were co-transferred in conjugative plasmids of 50 to 90 MDa. Pulsed-field gel electrophoresis analysis revealed diverse genetic fingerprints of pathogenic isolates.

Significance

Multi-drug resistant E. coli are wide spread in public water supply in Dhaka city, Bangladesh. Transmission of resistant bacteria and plasmids through supply water pose serious threats to public health in urban areas.     

Multi-Locus Variable Number of Tandem Repeat Analysis for Rapid and Accurate Typing of Virulent Multidrug Resistant Escherichia coli Clones

One hundred E. coli isolates from Norway (n = 37), Sweden (n = 24), UK (n = 20) and Spain (n = 19), producing CTX-M-type - (n = 84), or SHV-12 (n = 4) extended spectrum β-lactamases, or the plasmid mediated AmpC, CMY-2 (n = 12), were typed using multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes (MTCs). A strong agreement between the MLST profile and MLVA typing results was observed, in which all ST131-isolates (n = 39) and most of the STC-648 (n = 10), STC-38 (n = 9), STC-10 (n = 9), STC-405 (n = 8) and STC-23 (n = 6) isolates were clustered distinctly into MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate tool for genotyping isolates of globally disseminated virulent multidrug resistant E. coli lineages, including ST131.     

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131

Background

The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.

Methods

A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.

Results

Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The bla _CTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The bla _CTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.

Conclusions

Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.     

Houseflies (Musca domestica) as Vectors for Extended-Spectrum β-Lactamase-Producing Escherichia coli on Spanish Broiler Farms

Flies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence of Escherichia coli strains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producing E. coli. Of these isolates, 23 contained bla_CTX-M-1, 18 contained bla_CTX-M-14, and 1 contained bla_CTX-M-9. ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harbored qnrS. Identical PFGE profiles were found for E. coli isolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenic E. coli (APEC) and 29% were considered extraintestinal pathogenic E. coli (ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistant E. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches.     

Phylogenetic Distribution and Prevalence of Genes Encoding Class I Integrons and CTX-M-15 Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Healthy Humans in Chandigarh,India

Escherichia coli is generally considered as a commensal inhabitant of gastrointestinal tract of humans and animals. The aim of this study was to gain insight on the distribution of phylotypes and presence of genes encoding integrons, extended β-lactamases and resistance to other antimicrobials in the commensal E. coli isolates from healthy adults in Chandigarh, India. PCR and DNA sequencing were used for phylogenetic classification, detections of integrase genes, gene cassettes within the integron and extended β-lactamases. The genetic structure of E. coli revealed a non-uniform distribution of isolates among the seven phylogenetic groups with significant representation of group A. Integron-encoded integrases were detected in 25 isolates with class 1 integron-encoded intI1 integrase being in the majority (22 isolates). The gene cassettes identified were those for trimethoprim, streptomycin, spectinomycin and streptothricin. The dfrA12-orfF-aadA2 was the most commonly found gene cassette in intI1 positive isolates. Phenotypic assay for screening the potential ESBL producers suggested 16 isolates to be ESBL producers. PCR detection using gene-specific primers showed that 15 out of these 16 ESBL-producing E. coli harboured the bla _CTX-M-15 gene. Furthermore, molecular studies helped in characterizing the genes responsible for tetracycline, chloramphenicol and sulphonamides resistance. Collectively, our study outlines the intra-species phylogenetic structure and highlights the prevalence of class 1 integron and bla _CTX-M-15 in commensal E. coli isolates of healthy adults in Chandigarh, India. Our findings further reinforce the relevance of commensal E. coli strains on the growing burden of antimicrobial resistance.     

Dissemination of Escherichia coli with CTX-M Type ESBL between Humans and Yellow-Legged Gulls in the South of France

Extended Spectrum β-Lactamase (ESBL) producing Enterobacteriaceae started to appear in the 1980s, and have since emerged as some of the most significant hospital-acquired infections with Escherichia coli and Klebsiella being main players. More than 100 different ESBL types have been described, the most widespread being the CTX-M β-lactamase enzymes (bla _CTX-M genes). This study focuses on the zoonotic dissemination of ESBL bacteria, mainly CTX-M type, in the southern coastal region of France. We found that the level of general antibiotic resistance in single randomly selected E. coli isolates from wild Yellow-legged Gulls in France was high. Nearly half the isolates (47,1%) carried resistance to one or more antibiotics (in a panel of six antibiotics), and resistance to tetracycline, ampicillin and streptomycin was most widespread. In an ESBL selective screen, 9,4% of the gulls carried ESBL producing bacteria and notably, 6% of the gulls carried bacteria harboring CTX-M-1 group of ESBL enzymes, a recently introduced and yet the most common clinical CTX-M group in France. Multi locus sequence type and phylogenetic group designations were established for the ESBL isolates, revealing that birds and humans share E. coli populations. Several ESBL producing E. coli isolated from birds were identical to or clustered with isolates with human origin. Hence, wild birds pick up E. coli of human origin, and with human resistance traits, and may accordingly also act as an environmental reservoir and melting pot of bacterial resistance with a potential to re-infect human populations.     

Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern India

Background

The study investigated the presence of CTX-M-15 type extended spectrum beta-lactamases (ESBL), compared their genetic arrangements and plasmid types in gram negative isolates of hospital and food origin in north-east India. From September 2013 to April 2014, a total of 252 consecutive, non-duplicate clinical isolates and 88 gram negative food isolates were selected. Phenotypic and molecular characterization of ESBL genes was performed. Presence of integrons and gene cassettes were analyzed by integrase and 59 base-element PCR respectively. The molecular environments surrounding bla _CTX-M and plasmid types were investigated by PCR and PCR-based replicon typing respectively. Transformation was carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla _CTX-M-15 genes. DNA fingerprinting was performed by ERIC-PCR.

Results

Prevalence of ESBL was found to be 40.8% (103/252) in clinical and 31.8% (28/88) in food-borne isolates. Molecular characterization revealed the presence of 56.3% (58/103) and 53.5% (15/28) bla _CTX-M-15 in clinical and food isolates respectively. Strains of clinical and food origin were non-clonal. Replicon typing revealed that IncI1 and IncFII plasmid were carrying bla _CTX-M-15 in clinical and food isolates and were horizontally transferable. The ISEcp1 element was associated with bla _CTX-M-15 in both clinical and food isolates.

Conclusions

The simultaneous presence of resistance determinants in non-clonal isolates of two different groups thus suggests that the microbiota of common food products consumed may serve as a reservoir for some of the drug resistance genes prevalent in human pathogens.     

Molecular Characterization of CTX-M β-Lactamase and Associated Addiction Systems in Escherichia coli Circulating among Cattle,Farm Workers,and the Farm Environment

A total of 84 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from cattle, farm workers, and the farm environment isolated from February to September 2008 in the Republic of Korea were investigated. All 84 ESBL-producing isolates carried bla_CTX-M genes that belonged to the CTX-M-1 (n = 35) or CTX-M-9 (n = 49) family. The most predominant CTX-M type identified was CTX-M-14 (n = 49), followed by CTX-M-32 (n = 26). The bla_CTX-M genes were identified most commonly in E. coli isolates from feces (n = 29), teats (n = 25), and milk (n = 14). A bla_CTX-M-14 gene was also detected in an E. coli isolate from a farmer''s hand. Transfer of the bla_CTX-M gene from 60 bla_CTX-M-positive E. coli isolates to the recipient E. coli J53 strain by conjugation was demonstrated. Plasmid isolation from bla_CTX-M-positive transconjugants revealed a large (95- to 140-kb) conjugative plasmid. Almost all (82/84) bla_CTX-M genes possessed an insertion sequence, ISEcp1, upstream of the bla_CTX-M gene. Only in the case of the CTX-M-14 genes was IS903 downstream of the gene. The bla_CTX-M genes were associated with seven kinds of addiction systems. Among them, pndAC, hok-sok, and srnBC were the most frequently identified addiction systems in both wild strains and transconjugants. The spread of bla_CTX-M genes was attributed to both clonal expansion and horizontal dissemination. Our data suggest that a combination of multiple addiction systems in plasmids carrying bla_CTX-M genes could contribute to their maintenance in the host cells. To our knowledge, the bla_CTX-M-32 gene has not previously been reported in animal isolates from the Republic of Korea.     

Analysis of Drug Resistance Determinants in Klebsiella pneumoniae Isolates from a Tertiary-Care Hospital in Beijing, China

Background

The rates of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates among Enterobacteriaceae isolates, particularly Klebsiella pneumoniae, have risen substantially worldwide.

Methodology/Principal Findings

To better understand the molecular mechanisms of drug resistance in K. pneumoniae, we analyzed the drug resistance determinants for K. pneumoniae isolates collected from the 306 Hospital, a tertiary-care hospital in Beijing, China, for the period of September 1, 2010-October 31, 2011. Drug susceptibility testing, PCR amplification and sequencing of the drug resistance determinants were performed. Conjugation experiments were conducted to examine the natural ability of drug resistance to disseminate among Enterobacteriaceae strains using a sodium azide-resistant Escherichia coli J53 strain as a recipient. Among the 223 consecutive non-repetitive K. pneumoniae isolates included in this study, 101 (45.3%) were extended-spectrum beta-lactamases (ESBLs) positive. The rates of MDR, XDR, and PDR isolates were 61.4% (n = 137), 22.0% (n = 49), and 1.8% (n = 4), respectively. Among the tested drug resistance-associated genes, the following ones were detected at relatively high rates bla _CTX-M-10 (80, 35.9%), aacC2 (73, 32.7%), dhfr (62, 27.8%), qnrS (58, 26.0%), aacA4 (57, 25.6%), aadA1 (56, 25.1%). Results from conjugation experiments indicate that many of the drug resistance genes were transmissible.

Conclusions/Significance

Our data give a “snapshot” of the complex genetic background responsible for drug resistance in K. pneumoniae in China and demonstrate that a high degree of awareness and monitoring of those drug resistance determinants are urgently needed in order to better control the emergence and transmission of drug-resistant K. pneumoniae isolates in hospital settings.     

A Five-Year Experience of Carbapenem Resistance in Enterobacteriaceae Causing Neonatal Septicaemia: Predominance of NDM-1

Treatment of neonatal sepsis has become a challenge with the emergence of carbapenemase-producing bacteria. This study documents the trend of carbapenem susceptibility in Enterobacteriaceae that caused septicaemia in neonates over a five year period (2007–2011) and the molecular characterisation of Enterobacteriaceae resistant to carbapenems and cephalosporins. Hundred and five Enterobacteriaceae including Escherichia coli (n = 27), Klebsiella pneumoniae (n = 68) and Enterobacter spp. (n = 10) were isolated from blood of septicaemic neonates followed by antibiotic susceptibility tests, determination of MIC values, phenotypic and genotypic detection of β-lactamases. Carbapenem was the most active antimicrobial tested after tigecycline. CTX-M type was the most prevalent ESBL throughout the period (82%). New Delhi Metallo-β-lactamase-1 (NDM-1), which is a recent addition to the carbapenemase list, was the only carbapenemase identified in our setting. Fourteen percent of the isolates possessed bla_NDM-1. Carbapenem non-susceptibility was first observed in 2007 and it was due to loss of Omp F/Ompk36 in combination with the presence of ESBLs/AmpCs. NDM-1 first emerged in E. coli during 2008; later in 2010, the resistance was detected in K. pneumoniae and E. cloacae isolates. NDM-1-producing isolates were resistant to other broad-spectrum antibiotics and possessed ESBLs, AmpCs, 16S-rRNA methylases, AAC(6′)-Ib-cr, bleomycin resistant gene and class 1 integron. Pulsed field gel electrophoresis of the NDM-1-producing isolates indicated that the isolates were clonally diverse. The study also showed that there was a significantly higher incidence of sepsis caused by NDM-1-harbouring isolates in the male sex, in neonates with low birth weight and neonates born at an extramural centre. However, sepsis with NDM-1-harbouring isolates did not result in a higher mortality rate. The study is the first to review the carbapenem resistance patterns in neonatal sepsis over an extended period of time. The study highlights the persistence of ESBLs (CTX-Ms) and the emergence of NDM-1 in Enterobacteriaceae in the unit.