Effects of Therapeutic Ceftiofur Administration to Dairy Cattle on Escherichia coli Dynamics in the Intestinal Tract

The goal of this study was to follow ceftiofur-treated and untreated cattle in a normally functioning dairy to examine enteric Escherichia coli for changes in antibiotic resistance profiles and genetic diversity. Prior to treatment, all of the bacteria cultured from the cows were susceptible to ceftiofur. Ceftiofur-resistant E. coli was only isolated from treated cows during and immediately following the cessation of treatment, and the 12 bla_CMY-2-positive isolates clustered into two genetic groups. E. coli bacterial counts dropped significantly in the treated animals (P < 0.027), reflecting a disappearance of the antibiotic-susceptible strains. The resistant bacterial population, however, did not increase in quantity within the treated cows; levels stayed low and were overtaken by a returning susceptible population. There was no difference in the genetic diversities of the E. coli between the treated and untreated cows prior to ceftiofur administration or after the susceptible population of E. coli returned in the treated cows. A cluster analysis of antibiotic susceptibility profiles resulted in six clusters, two of which were multidrug resistant and were comprised solely of isolates from the treated cows immediately following treatment. The antibiotic treatment provided a window to detect the presence of ceftiofur-resistant E. coli but did not appear to cause its emergence or result in its amplification. The finding of resistant isolates following antibiotic treatment is not sufficient to estimate the strength of selection pressure nor is it sufficient to demonstrate a causal link between antibiotic use and the emergence or amplification of resistance.     

Population Distribution of Beta-Lactamase Conferring Resistance to Third-Generation Cephalosporins in Human Clinical Enterobacteriaceae in The Netherlands

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne β-lactamases that confer resistance to third-generation cephalosporins. The epidemiology of these bacteria is not well understood, and was, therefore, investigated in a selection of 636 clinical Enterobacteriaceae with a minimal inhibitory concentration >1 mg/L for ceftazidime/ceftriaxone from a national survey (75% E. coli, 11% E. cloacae, 11% K. pneumoniae, 2% K. oxytoca, 2% P. mirabilis). Isolates were investigated for extended-spectrum β-lactamases (ESBLs) and ampC genes using microarray, PCR, gene sequencing and molecular straintyping (Diversilab and multi-locus sequence typing (MLST)). ESBL genes were demonstrated in 512 isolates (81%); of which 446 (87%) belonged to the CTX-M family. Among 314 randomly selected and sequenced isolates, bla _CTX-M-15 was most prevalent (n = 124, 39%), followed by bla _CTX-M-1 (n = 47, 15%), bla _CTX-M-14 (n = 15, 5%), bla _SHV-12 (n = 24, 8%) and bla _TEM-52 (n = 13, 4%). Among 181 isolates with MIC ≥16 mg/L for cefoxitin plasmid encoded AmpCs were detected in 32 and 27 were of the CMY-2 group. Among 102 E. coli isolates with MIC ≥16 mg/L for cefoxitin ampC promoter mutations were identified in 29 (28%). Based on Diversilab genotyping of 608 isolates (similarity cut-off >98%) discriminatory indices of bacteria with ESBL and/or ampC genes were 0.994, 0.985 and 0.994 for E. coli, K. pneumoniae and E. cloacae, respectively. Based on similarity cut-off >95% two large clusters of E. coli were apparent (of 43 and 30 isolates) and 21 of 21 that were typed by belonged to ST131 of which 13 contained bla _CTX-M-15. Our findings demonstrate that bla _CTX-M-15 is the most prevalent ESBL and we report a larger than previously reported prevalence of ampC genes among Enterobacteriaceae responsible for resistance to third-generation cephalosporins.     

Extended-Spectrum β-Lactamase CTX-M-1 in Escherichia coli Isolates from Healthy Poultry in France

Genes encoding extended-spectrum β-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla_CTX-M-1 gene located on transferable plasmids of different sizes and structures.     

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.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

Impact of the Use of β-Lactam Antimicrobials on the Emergence of Escherichia coli Isolates Resistant to Cephalosporins under Standard Pig-Rearing Conditions

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained bla_CTX-M-1, 10 contained bla_CTX-M-14, 4 contained bla_CTX-M-9, 2 contained bla_CTX-M-15, and 5 contained bla_SHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals.     

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.     

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.     

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.     

Role of Ceftiofur in Selection and Dissemination of blaCMY-2-Mediated Cephalosporin Resistance in Salmonella enterica and Commensal Escherichia coli Isolates from Cattle

Third-generation cephalosporin resistance of Salmonella and commensal Escherichia coli isolates from cattle in the United States is predominantly conferred by the cephamycinase CMY-2, which inactivates β-lactam antimicrobial drugs used to treat a wide variety of infections, including pediatric salmonellosis. The emergence and dissemination of bla_CMY-2--bearing plasmids followed and may in part be the result of selection pressure imposed by the widespread utilization of ceftiofur, a third-generation veterinary cephalosporin. This study assessed the potential effects of ceftiofur on bla_CMY-2 transfer and dissemination by (i) an in vivo experimental study in which calves were inoculated with competent bla_CMY-2-bearing plasmid donors and susceptible recipients and then subjected to ceftiofur selection and (ii) an observational study to determine whether ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed bla_CMY-2 plasmid transfer in both ceftiofur-treated and untreated calves but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P = 0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. However, herds with a history of salmonellosis (including both ceftiofur-resistant and ceftiofur-susceptible Salmonella isolates) used more ceftiofur than herds with no history of salmonellosis (P = 0.03) These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of bla_CMY-2-bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations.The major mechanism of third-generation cephalosporin resistance among U.S. human and veterinary clinical isolates of Salmonella enterica subsp. enterica is the beta-lactamase CMY-2 (12, 17, 43, 44, 46). bla_CMY-2, which likely originated from the chromosomal AmpC locus of Citrobacter freundii, is disseminated among a group of similar plasmids harbored by diverse Enterobacteriaceae species (1, 2, 20, 26, 30, 31, 42, 45). In Salmonella, bla_CMY-2-bearing plasmids have been observed in more than 30 serovars, notably including serovar Newport, which has gained specific attention from public health officials as a rapidly emerging threat (2, 6, 31).Commensal Escherichia coli frequently harbors bla_CMY-2-bearing plasmids (15, 33, 44), and these plasmids may be transferable to pathogens, since bla_CMY-2 plasmids isolated from E. coli and S. enterica share extensive sequence similarity in addition to the bla_CMY-2 open reading frame (5, 12, 42, 44). This transfer may occur in the gastrointestinal tracts of cattle, where these bacterial species periodically coexist and where transconjugants may be subjected to specific antimicrobial selection pressure. In fact, in vivo transfer of bla_CMY-2 in the gastrointestinal tract has been reported between a Klebsiella pneumoniae bla_CMY-2 plasmid donor and a Salmonella enterica serovar Typhimurium isolate in cattle and goats (29).Ceftiofur is the only third-generation cephalosporin antimicrobial drug that is used in cattle production systems and is labeled for the treatment of pneumonia, postpartum metritis, necrotizing pododermatitis, and mastitis. Two ceftiofur preparations, ceftiofur sodium (Naxcel) and ceftiofur hydrochloride (Excenel) (Pfizer Animal Health, New York, NY), are unique in the veterinary pharmacopeia because they require no withholding and discard of milk collected from treated cows, making them frequent therapeutic choices in lactating animals (19, 35). Ceftiofur was licensed in 1988 (41) and its resistance in Salmonella spp. isolated from U.S. cattle, presumably conferred by bla_CMY-2, was first documented in 1998 (6).The effects of ceftiofur use on selection of bla_CMY-2-bearing commensal E. coli has been examined for cattle both epidemiologically and experimentally. Tragesser et al. studied 18 Ohio dairy herds and determined that the 11 herds that used ceftiofur in any capacity (labeled indications and/or extralabel use) were 25 times more likely to have E. coli with reduced susceptibility to ceftriaxone (an expected bla_CMY-2 phenotype) than the seven herds that reported no ceftiofur use (40). Interestingly, however, within eight herds that had detailed treatment records, no association was detected between the prevalence of E. coli with reduced susceptibility to ceftriaxone and use of ceftiofur on an individual-animal basis (40). In an experimental study by Jiang et al., ceftiofur administered to dairy calves was correlated with a 14% increase in ceftriaxone-resistant fecal E. coli compared to untreated controls (21). Together, these studies show a correlation between selection pressure within the gastrointestinal tracts at the individual-animal level and show that ceftiofur use may promote the dissemination of resistance in commensal E. coli at the whole-herd level.Whether or not ceftiofur treatment directly affects in vivo horizontal transfer of bla_CMY-2-bearing elements among E. coli and Salmonella has yet to be addressed. The diversity of bla_CMY-2 plasmid-bearing bacterial hosts is consistent with wide dissemination of this genetic element. One hypothesis that could explain this wide dissemination is that ceftiofur may itself promote the in vivo horizontal transfer of bla_CMY-2-bearing plasmids. Specifically, due to the relatively slow bactericidal activity of aminothiazolyl cephalosporins such as ceftiofur, it has been suggested that exposure to these compounds promotes filament formation in gram-negative bacteria prior to cell death that may increase the surface area and increase receptiveness of the cells for resistance plasmids (11).Because bla_CMY-2 may be disseminated by horizontal transfer of R plasmids and/or clonal expansion of individual strains, we examined the effect of ceftiofur use on these processes with two approaches; the first approach specifically considered the issue of horizontal transfer in an experimental in vivo calf model, while the second approach, a field study, assessed the overall relationship between ceftiofur use and bla_CMY-2 prevalence in the primary agricultural animal niche where it is used.     

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.     

Prediction of Antibiotic Resistance Evolution by Growth Measurement of All Proximal Mutants of Beta-Lactamase

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum β-lactamase gene bla_CTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEAR^P and PEAR^R, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of bla_CTX-M-14 evolution, respectively. Single to quintuple mutations of bla_CTX-M-14 predicted to confer resistance by PEAR^P were significantly enriched among the clinical isolates harboring bla_CTX-M-14 variants, and the PEAR^R scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.     

Dominance of CTX-M-Type Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Isolated from Patients with Community-Onset and Hospital-Onset Infection in China

Objective

To investigate CTX-M genotypes among extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) isolated from patients with community-onset and hospital-onset infections in China, their clonality and the distribution of CTX-M variants in different specimens of community-onset and hospital-onset infections.

Methods

ESBL-EC isolates were collected from general hospitals from 2011 to 2012 in China. Broth microdilution method antimicrobial susceptibility testing of 16 antibiotics was performed. Clinical data from community-onset and hospital-onset infections due to ESBL-EC were analyzed. ESBL-encoding genes were amplified by PCR and sequenced, and multilocus sequence typing (MLST) was performed for a random selection of predominant CTX-M type strains identified.

Results

A total of 1,168 ESBL-EC isolates were obtained from various clinical specimens, 41.7% of which were responsible for causing community-onset infections. The presence of urinary calculi was higher in community-onset infections, whereas malignancy, cardiovascular and cerebrovascular diseases, dementia, chronic renal disease, diabetes mellitus and surgical treatment were found to have higher proportions in hospital-onset infections. There was no significant difference in trauma between community-onset and hospital-onset infections. 96.2% of the isolates were detected to harbor bla _CTX-M genes. bla _CTX-M-1 group and bla _CTX-M-9 group were detected at 40.7% and 48.7% respectively, and both positive group accounted for 10.6%. bla _CTX-M-55 (24.8%) and bla _CTX-M-15 (18.2%) were the major genotypes in bla _CTX-M-1 group while bla _CTX-M-14 (46.8%) was predominant in bla _CTX-M-9 group. A comparison of bla _CTX-M distribution in different specimens between ESBL-EC causing community-onset and hospital-onset infection showed no significant difference. A total of 229 isolates were tested for MLST. ST131 (14%) was the predominant type. ST648, ST405 and ST1193 were also detected.

Conclusions

Community-onset ESBL-EC has emerged as a common pathogen in China. CTX-M-14 is the most commonly encountered, CTX-M-55 and CTX-M-15 have spread rapidly. ST131 is the predominant clonal group, and the great diversity of CTX-M-producing isolates of E. coli has emerged in China.     

Molecular Characterization of Klebsiella pneumoniae Carbapenemase (KPC)-Producing Enterobacteriaceae in Ontario,Canada, 2008-2011

Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of bla _KPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as bla _KPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored bla _KPC-2 (n = 23) or bla _KPC-3 (n = 7). bla _TEM-1 (n = 27) was commonly detected and occasionally bla _OXA-1 (n = 3) and bla _CTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried bla _SHV-11. bla _KPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258.     

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.     

CTX-M-producing Escherichia coli in pigs from a Czech farm during production cycle

We evaluated the prevalence and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in pigs during production cycle on a Czech farm with the history of previous use of ceftiofur. ESBL-producing E. coli isolates were obtained from rectal swabs from pigs of different age groups (suckling piglets, weaned piglets, growers and sows). Collected samples were directly cultivated on MacConkey agar with cefotaxime (2 mg l⁻¹), whereas intestinal swabs of slaughtered pigs and surface swabs from pig carcasses were also pre-enriched in buffered peptone water without antimicrobials before the cultivation. Clonal relationship of selected isolates was determined by XbaI pulse-field gel electrophoresis and multi-locus sequence typing. The transferability of plasmids carrying bla_CTX-M genes was tested by conjugation experiments. From all examined samples, 141 (43·7%, n = 323) were positive for ESBL-producing E. coli. All ESBL-producing isolates showed resistance to multiple antimicrobials and were positive for bla_CTX-M genes. The bla_CTX-M-1 was carried by conjugative IncN/ST1 plasmids (c. 40–45 kb) while the bla_CTX-M-15 was located on conjugative F plasmids with F:18:A5:B1 formula (c. 165 kb). This study demonstrated the persistence of CTX-M-positive E. coli isolates 2 months after banner of ceftiofur usage and indicated possible risk of transmission of these isolates to humans via the food chain.     

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.     

Increase in Resistance to Extended-Spectrum Cephalosporins in Salmonella Isolated from Retail Chicken Products in Japan

Extended-spectrum β-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996–2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the bla_CMY, bla_CTX-M, bla_TEM, and bla_SHV resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (bla_CMY-2), and nine ESBL-producing isolates harboring bla_CTX-M (n = 4, consisting of two bla_CTX-M-2 and two bla_CTX-M-15 genes), bla_TEM (n = 4, consisting of one bla_TEM-20 and three bla_TEM-52 genes), and bla_SHV (n = 1, bla_SHV-12). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing bla_CMY-2 in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated bla_CMY-2 in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health.     

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.