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The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.
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BACKGROUND: As WGS comes of age, changes in EU legislation implemented in 2021 allow its usage for systematic monitoring of ESBL-producing Escherichia coli from livestock and meat, replacing phenotypic testing. Presently, phenotypic testing correlates well with antimicrobial resistance predicted from WGS data. WGS has added value in the wealth of additional information that is present in the data. OBJECTIVES: In this study we have detected the resistance phenotypes for a panel of antimicrobials while also analysing the molecular epidemiology of ESBL-producing E. coli. METHODS: Susceptibility testing was performed with broth microdilution of selectively isolated E. coli. Short-read WGS was performed in parallel and phenotypes predicted based on the sequence data, which was also used to determine the phylogeny of the isolates. RESULTS: The phenotypically determined resistance and the predicted resistance correlated 90%-100% for the different antimicrobial classes. Furthermore, clonal relationships were detected amongst ESBL-producing E. coli within livestock sectors and the meat produced by this sector. CONCLUSIONS: Further implementation of WGS analysis of ESBL/AmpC-producing E. coli within the AMR monitoring programme of EU member states and global surveillance programmes will contribute to determining the attribution of livestock in the prevalence of ESBL/AmpC-encoding E. coli in humans.
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Infecções por Escherichia coli , Escherichia coli , Animais , Humanos , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/veterinária , Gado , beta-Lactamases/genética , Antibacterianos/farmacologia , CarneRESUMO
Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A ß-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined.
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Proteínas de Bactérias/metabolismo , Enterobacter cloacae/enzimologia , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/metabolismo , Enterobacter/efeitos dos fármacos , Enterobacter cloacae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genéticaRESUMO
One of the factors that can affect conjugation of IncI1 plasmids, amongst others, is the genetic region known as the shufflon. This multiple inversion system modifies the pilus tip proteins used during conjugation, thus affecting the affinity for different recipient cells. Although recombination is known to occur in in vitro conditions, little is known about the regulation and the extent of recombination that occurs. To measure the recombination of the shufflon, we have amplified the entire shufflon region and sequenced the amplicons using nanopore long-read sequencing. This method was effective to determine the order of the segments of the shufflon and allow for the analysis of the shufflon variants that are present in a heterogeneous pool of templates. Analysis was performed over different growth phases and after addition of cefotaxime. Furthermore, analysis was performed in different E.â¯coli host cells to determine if recombination is likely to be influenced. Recombination of the shufflon was constantly ongoing in all conditions that were measured, although no differences in the amount of different shufflon variants or the rate at which novel variants were formed could be found. As previously reported, some variants were abundant in the population while others were scarce. This leads to the conclusion that the shufflon is continuously recombining at a constant rate, or that the method used here was not sensitive enough to detect differences in this rate. For one of the plasmids, the host cell appeared to have an effect on the specific shufflon variants that were formed which were not predominant in another host, indicating that host factors may be involved. As previously reported, the pilV-A and pilV-A' ORFs are formed at higher frequencies than other pilV ORFs. These results demonstrate that the recombination that occurs within the shufflon is not random. While any regulation of the shufflon affected by these in vitro conditions could not be revealed, the method of amplifying large regions for long-read sequencing for the analysis of multiple inversion systems proved effective.
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Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Rearranjo Gênico/genética , Plasmídeos/genética , Animais , Humanos , Plasmídeos/isolamento & purificaçãoRESUMO
Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains (JF6X01.0022/XbaI.0251, JF6X01.0326/XbaI.1966, JF6X01.0258/XbaI.1968, and JF6X01.0045/XbaI.1970) have been identified in the United States with pulsed-field gel electrophoresis. Our examination of isolates showed introduction of these strains in the Netherlands and highlight the need for active surveillance and intervention strategies by public health organizations.
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Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções por Salmonella/microbiologia , Salmonella enterica/efeitos dos fármacos , Animais , Microbiologia de Alimentos , Humanos , Testes de Sensibilidade Microbiana , Países Baixos , Infecções por Salmonella/epidemiologia , Salmonella enterica/classificaçãoRESUMO
OBJECTIVES: Several studies on faecal carriage of extended-spectrum ß-lactamase (ESBL)/AmpC-producing Escherichia coli have been performed in cattle, but little is known about faecal carriage in veal calves. This study describes the prevalence and molecular characteristics of ESBL/AmpC genes in E. coli isolated from faecal samples of veal calves from 1997 to 2010. METHODS: Pooled faecal samples were inoculated using selective enrichment broth and subsequently selective MacConkey agar. All isolates with reduced susceptibility to cefotaxime were screened by PCR and sequencing analysis for the presence of ESBL/AmpC genes. RESULTS: The prevalence of E. coli with reduced susceptibility to cefotaxime showed a discontinuous increasing trend, ranging from 4% in 1998 and 1999 to 39% in 2010. Promoter mutations of the chromosomal ampC gene were present in all years. In 2000, ESBL genes blaCTX-M-1, blaTEM-52 and blaTEM-20 were first observed. Before 2005 the majority of E. coli with reduced susceptibility to cefotaxime harboured ampC promoter mutations. From 2005 onwards the majority harboured blaCTX-M genes, of which blaCTX-M-1 was the most abundant, followed by blaCTX-M-14 and blaCTX-M-15. The diversity of blaCTX-M genes gradually increased from one variant in 2000 to six variants in 2010. The prevalence of blaTEM-52 was relatively low, but it was detected from 2000 onwards. blaCMY and blaSHV were found sporadically. CONCLUSIONS: The prevalence and molecular diversity of genes encoding cefotaxime resistance in E. coli isolated from veal calves over a 14 year period showed an increasing trend. From 2005 onwards, blaCTX-M genes were most abundant, especially blaCTX-M-1.
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Portador Sadio/veterinária , Infecções por Escherichia coli/veterinária , Escherichia coli/enzimologia , Variação Genética , beta-Lactamases/genética , Animais , Técnicas Bacteriológicas , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Bovinos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Reação em Cadeia da Polimerase , Prevalência , Análise de Sequência de DNA , beta-Lactamases/classificaçãoRESUMO
OBJECTIVES: To assess the within-farm dynamics of extended-spectrum ß-lactamase (ESBL)/AmpC-producing Escherichia coli in veal calves. METHODS: Three veal-calf fattening farms were screened. Faecal samples from all calves within a compartment (109-150 per farm) were taken upon arrival on the farm (T0) and after 3, 6, 8 and 10 weeks (T3-T10). ESBL/AmpC genes were characterized by PCR and sequencing. Plasmids were characterized by transformation, PCR-based replicon typing and plasmid multilocus sequence typing (MLST). E. coli genotypes were analysed by MLST. RESULTS: At T0 the prevalence of ESBL/AmpC-producing E. coli ranged from 18% to 26%. These were predominantly isolates carrying blaCTX-M-1 and blaCTX-M-15 genes, located on various plasmids and E. coli sequence types (STs). Farm 1 was negative for ESBL/AmpC-producing E. coli after T0. Farm 2 showed an increase up to 37% at T3, which subsequently decreased gradually to 0% at T10. The presence from T3 to T10 on farm 2 was mainly caused by the clonal spread of a multiresistant E. coli ST57 harbouring blaCTX-M-14 on an IncF F2:A-:B- plasmid. Farm 3 showed a gradual decrease in prevalence to 1.4% at T10, with a relative increase of the identical clonal variant as shown for farm 2. A second clonal variant found in farm 3 was a multiresistant E. coli ST10 harbouring blaCTX-M-14 on an IncK plasmid. CONCLUSIONS: The prevalence of ESBL/AmpC-producing E. coli decreased over time. A clonal spread was observed on farm 2 and farm 3, illustrative of the complex dynamics probably associated with the use of antimicrobials.
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Infecções por Escherichia coli/veterinária , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , beta-Lactamases/metabolismo , Animais , Animais Domésticos , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/classificação , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Genótipo , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Plasmídeos/análise , Reação em Cadeia da Polimerase , Prevalência , Transformação Bacteriana , beta-Lactamases/genéticaRESUMO
Cloacal swabs from carcasses of Dutch wild birds obtained in 2010 and 2011 were selectively cultured on media with cefotaxime to screen for the presence of extended-spectrum ß-lactamase (ESBL)/AmpC-producing Escherichia coli. Subsequently, all cefotaxime-resistant E. coli isolates were tested by broth microdilution and microarray. The presence of ESBL/AmpC and coexisting plasmid-mediated quinolone resistance (PMQR) genes was confirmed by PCR and sequencing. To determine the size of plasmids and the location of ESBL and PMQR genes, S1 pulsed-field gel electrophoresis (PFGE) was performed on transformants, followed by Southern blot hybridization. The study included 414 cloacal swabs originating from 55 different bird species. Cefotaxime-resistant E. coli isolates were identified in 65 birds (15.7%) from 21 different species. In all, 65 cefotaxime-resistant E. coli ESBL/AmpC genes were detected, mainly comprising variants of blaCTX-M and blaCMY-2. Furthermore, PMQR genes [aac(6')-lb-cr, qnrB1, and qnrS1] coincided in seven cefotaxime-resistant E. coli isolates. Overall, replicon typing of the ESBL/AmpC-carrying plasmids demonstrated the predominant presence of IncI1 (n = 31) and variants of IncF (n = 18). Our results indicate a wide dissemination of ESBL and AmpC genes in wild birds from The Netherlands, especially among aquatic-associated species (waterfowl, gulls, and waders). The identified genes and plasmids reflect the genes found predominantly in livestock animals as well as in humans.
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Antibacterianos/farmacologia , Aves/microbiologia , Cefotaxima/farmacologia , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Resistência beta-Lactâmica , Animais , Cloaca/microbiologia , Eletroforese em Gel de Campo Pulsado , Escherichia coli/classificação , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Análise em Microsséries , Testes de Sensibilidade Microbiana , Tipagem Molecular , Países Baixos , Plasmídeos/análise , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.
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Citoplasma/microbiologia , Mycobacterium/patogenicidade , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Técnicas de Introdução de Genes , Interações Hospedeiro-Patógeno , Humanos , Lisossomos/microbiologia , Lisossomos/ultraestrutura , Mycobacterium/genética , Mycobacterium/metabolismo , Fagossomos/microbiologia , Fagossomos/ultraestrutura , Estrutura Terciária de Proteína , Ubiquitina/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility.
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Panels of pre- and post-pandemic farm animals, wild boar and human sera, including human sera able to neutralize SARS-CoV-2 in vitro, were tested in serological tests to determine their cross-reactivity with ß- and α-CoV originating from farm animals. Sera were tested in neutralization assays with high ascending concentrations (up to 1 × 104 TCID50 units/well) of ß-CoV Bovine coronavirus (BCV), SARS-CoV-2, and porcine α-CoV-transmissible gastroenteritis virus (TGEV). In addition, sera were tested for immunostaining of cells infected with ß-CoV porcine hemagglutinating encephalomyelitis (PHEV). Testing revealed a significantly higher percentage of BCV neutralization (78%) for sera of humans that had experienced a SARS-CoV-2 infection (SARS-CoV-2 convalescent sera) than was observed for human pre-pandemic sera (37%). Also, 46% of these human SARS-CoV-2 convalescent sera neutralized the highest concentration of BCV (5 × 103 TCID50/well) tested, whereas only 9.6% of the pre-pandemic sera did. Largely similar percentages were observed for staining of PHEV-infected cells by these panels of human sera. Furthermore, post-pandemic sera collected from wild boars living near a densely populated area in The Netherlands also showed a higher percentage (43%) and stronger BCV neutralization than was observed for pre-pandemic sera from this area (21%) and for pre- (28%) and post-pandemic (20%) sera collected from wild boars living in a nature reserve park with limited access for the public. High percentages of BCV neutralization were observed for pre- and post-pandemic sera of cows (100%), pigs (up to 45%), sheep (36%) and rabbits (60%). However, this cross-neutralization was restricted to sera collected from specific herds or farms. TGEV was neutralized only by sera of pigs (68%) and a few wild boar sera (4.6%). None of the BCV and PHEV cross-reacting human pre-pandemic, wild boar and farm animal sera effectively neutralized SARS-CoV-2 in vitro. Preexisting antibodies in human sera effectively neutralized the animal ß-CoV BCV in vitro. This cross-neutralization was boosted after humans had experienced a SARS-CoV-2 infection, indicating that SARS-CoV-2 activated a "memory" antibody response against structurally related epitopes expressed on the surface of a broad range of heterologous CoV, including ß-CoV isolated from farm animals. Further research is needed to elucidate if a symptomless infection or environmental exposure to SARS-CoV-2 or another ß-CoV also triggers such a "memory" antibody response in wild boars and other free-living animals.
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COVID-19 , Vírus da Gastroenterite Transmissível , Humanos , Feminino , Animais , Bovinos , Coelhos , Ovinos , Suínos , Animais Domésticos , SARS-CoV-2 , Pandemias , COVID-19/epidemiologia , COVID-19/veterinária , Soroterapia para COVID-19 , Sus scrofaRESUMO
This study aimed to characterize the changes in fecal carriage of Extended-Spectrum ß-Lactamase (ESBL) producing Enterobacterales (ESBL-PE) in a single Dutch veal calves. During the rearing period at the Dutch veal farm, a decrease in fecal carriage of cefotaxime-resistant Escherichia coli isolates was observed after 2 weeks at the veal farm, while an increase of cefotaxime-resistant Klebsiella pneumoniae isolates was demonstrated. E. coli and K. pneumoniae were isolated from rectal swabs collected from 110 veal calves in week 2, 6, 10, 18, and 24 after their arrival at the farm. ESBL-PE isolates were selectively cultured and identified by MALDI-TOF. ESBL genes were characterized by RT-PCR, PCRs, and amplicon sequencing. A total of 80 E. coli and 174 K. pneumoniae strains were isolated from 104 out of 110 veal calves. The prevalence of ESBL-E. coli decreased from week 2 (61%) to week 6 (7%), while an unexpected increase in ESBL-K. pneumoniae colonization was detected in week 6 (80%). The predominant ESBL genes detected in E. coli isolates were bla CTX-M-15 and the non-ESBL gene bla TEM-1a, while in K. pneumoniae bla CTX-M-14 gene was detected in all isolates. Four cefotaxime-resistant K. pneumoniae isolates were randomly selected and characterized in deep by transformation, PCR-based replicon typing, and whole-genome sequencing (WGS). The clonal relatedness of a subgroup of nine animals carrying K. pneumoniae ESBL genes was investigated by Multi Locus sequence typing (MLST). In four ESBL-K. pneumoniae isolates, bla CTX-M-14 was located on IncFIIK and IncFIINK plasmid replicons and the isolates were multi-drug resistant (MDR). MLST demonstrated a clonal spread of ESBL-K. pneumoniae ST107. To the best of our knowledge, this is the first study to report a change in fecal carriage of ESBL-PE over time in the same veal calf during the rearing period.
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This study aimed to investigate the phylogenetic diversity and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae from chicken, chicken meat, and human clinical isolates in Sao Paolo, Brazil, and characterize their respective ESBL-encoding plasmids. Three hundred samples from chicken cloaca, chicken meat, and clinical isolates were phenotypically and genotypically assessed for ESBL resistance. Isolates were identified by MALDI TOF-MS and further characterized by MLST analysis and phylogenetic grouping. ESBL genes were characterized and their location was determined by I-Ceu-I-PFGE and Southern blot, conjugation, transformation, and PCR-based replicon typing experiments. Thirty-seven ESBL-producing isolates (28 E. coli and 9 K. pneumoniae) that were positive for the bla CTX-M-1 or bla CTX-M-2 gene groups were obtained. Two isolates were negative in the transformation assay, and the chromosomal location of the genes was deduced by Southern blot. The bla CTX-M genes identified were carried on plasmid replicon-types X1, HI2, N, FII-variants, I1 and R. The E. coli isolates belonged to nine sequence types, while the K. pneumoniae isolates belonged to four sequence types. The E. coli isolates belonged to phylotype classification groups A, B1, D, and F. This study demonstrated that isolates from cloacal swabs, chicken meat, and human feces had genetic diversity, with a high frequency of bla CTX-M-15 among chickens, chicken meat, and human feces. Thus, this reinforces the hypothesis that chickens, as well as their by-products, could be an important source of transmission for ESBL-producing pathogens to humans in South America.
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OBJECTIVES: Mobile colistin resistance (mcr) genes encoded on conjugative plasmids, although described only relatively recently, have been reported globally both in humans and livestock. The genes are often associated with the insertion sequence ISApl1 that can transpose the genes to novel genetic locations. Since its first report, multiple variants of mcr have been discovered in a variety of genetic locations in Escherichia coli, in plasmids and integrated into the chromosome. METHODS: Using hybrid assembly of short-read and long-read whole-genome sequencing data, the presence ofmcr-1 was confirmed on an IncI1 plasmid in E. coli. In vitro conjugation assays were performed to determine the potential to transfer between strains. Genetic comparison with previously reported IncI1 plasmids was performed. RESULTS: The genomic sequence identified thatmcr-1 is present on a complete IncI1 plasmid. Comparison with previously reported extended-spectrum ß-lactamase (ESBL)-encoding plasmids from E. coli in the Netherlands from the same time period indicated a distinct lineage for this plasmid. CONCLUSIONS: The observation ofmcr-1 on an IncI1 plasmid confirms that the genetic region of this gene is actively transposed between genetic locations. This active transposition has consequences for the study of the epidemiology of mcr in populations.
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Colistina , Proteínas de Escherichia coli , Colistina/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Carne , Países Baixos , Plasmídeos/genética , beta-Lactamases/genéticaRESUMO
Extended-spectrum ß-lactamase (ESBL) and plasmid-mediated AmpC ß-lactamase (pAmpC) genes confer resistance to extended spectrum cephalosporin's. The spread of these genes is mostly facilitated by plasmid-mediated horizontal transfer. National surveillance activities to detect ESBL/pAmpC-producers in commensal bacteria from livestock are in place in the Netherlands since several years. This study aimed at reporting gene and plasmid diversity of commensal ESBL/pAmpC-producing Escherichia coli isolated from healthy animals during surveillance activities between 2007 and 2017. A collection of 2304 extended-spectrum cephalosporin-resistant (ESC-R) E. coli isolated from feces of broilers, dairy cattle, slaughter pigs, turkeys, ducks, and veal calves was investigated and ESBL/pAmpC genes were determined. Gene location of a selection of 473 E. coli isolates was determined and typing of plasmids linked to the ESBL/pAmpC genes was performed. Twenty-two different ESBL/pAmpC genes were identified with bla CTX-M-1 being the most prevalent gene in livestock (43.7%), followed by bla CMY -2 and bla SHV -12, independent of the animal source. Prevalence of typically human associated bla CTX-M-15 was highest in cattle. Less than 10% E. coli isolates owed their ESC-R phenotype to promoter mutations of the chromosomal ampC gene. Majority (92%) of ESBL/pAmpC genes analyzed were plasmid located, with IncI1α being the most represented plasmid family in isolates from all animals, followed by IncF (veal calves, dairy cattle and slaughter pigs), IncK (broilers and laying hens), IncX1 in broilers, and emerging IncX3 in broilers and dairy cattle. Prevalence and molecular diversity of ESC-R E. coli isolated from livestock over an 11-year period revealed a composite scenario of gene-plasmid combinations.
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The blaSHV-12 ß-lactamase gene is one of the most prevalent genes conferring resistance to extended-spectrum ß-lactams in Enterobacteriaceae disseminating within and between reservoirs, mostly via plasmid-mediated horizontal gene transfer. Yet, studies regarding the biology of plasmids encoding blaSHV-12 are very limited. In this study, we revealed the emergence of IncX3 plasmids alongside IncI1α/γ in blaSHV-12 in animal-related Escherichia coli isolates. Four representative blaSHV-12-encoding IncX3 plasmids were selected for genome sequencing and further genetic and functional characterization. We report here the first complete sequences of IncX3 plasmids of animal origin and show that IncX3 plasmids exhibit remarkable synteny in their backbone, while the major differences lie in their blaSHV-12-flanking region. Our findings indicate that plasmids of this subgroup are conjugative and highly stable, while they exert no fitness cost on their bacterial host. These favourable features might have contributed to the emergence of IncX3 amongst SHV-12-producing E. coli in the Netherlands, highlighting the epidemic potential of these plasmids.
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Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Plasmídeos/genética , Doenças das Aves Domésticas/epidemiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/transmissão , Proteínas de Escherichia coli/metabolismo , Genômica , Humanos , Plasmídeos/metabolismo , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/transmissão , beta-Lactamases/metabolismo , beta-Lactamas/farmacologiaAssuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Quinolonas/farmacologia , beta-Lactamas/farmacologia , Animais , Animais Domésticos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Testes de Sensibilidade Microbiana , Países BaixosRESUMO
Since multidrug resistant bacteria are frequently reported from Southeast Asia, our study focused on the occurrence of ESBL-producing Enterobacteriaceae in fresh imported herbs from Thailand, Vietnam and Malaysia. Samples were collected from fresh culinary herbs imported from Southeast Asia in which ESBL-suspected isolates were obtained by selective culturing. Analysis included identification by MALDI-TOF mass spectrometry, susceptibility testing, XbaI-PFGE, microarray, PCR and sequencing of specific ESBL genes, PCR based replicon typing (PBRT) of plasmids and Southern blot hybridization. In addition, the quinolone resistance genotype was characterized by screening for plasmid mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance determining region (QRDR) of gyrA and parC. The study encompassed fifty samples of ten batches of culinary herbs (5 samples per batch) comprising nine different herb variants. The herbs originated from Thailand (Water morning glory, Acacia and Betel leaf), Vietnam (Parsley, Asian pennywort, Houttuynia leaf and Mint) and Malaysia (Holy basil and Parsley). By selective culturing 21 cefotaxime resistant Enterobacteriaceae were retrieved. Array analysis revealed 18 isolates with ESBL genes and one isolate with solely non-ESBL beta-lactamase genes. Mutations in the ampC promoter region were determined in two isolates with PCR and sequencing. The isolates were identified as Klebsiella pneumoniae (n=9), Escherichia coli (n=6), Enterobacter cloacae complex (n=5) and Enterobacter spp. (n=1). All isolates tested were multidrug resistant. Variants of CTX-M enzymes were predominantly found followed by SHV enzymes. PMQR genes (including aac(6')-1b-cr, qnrB and qnrS) were also frequently detected. In almost all cases ESBL and quinolone resistance genes were located on the same plasmid. Imported fresh culinary herbs from Southeast Asia are a potential source for contamination of food with multidrug resistant bacteria. Because these herbs are consumed without appropriate heating, transfer to human bacteria cannot be excluded.
Assuntos
Cefalosporinas/farmacologia , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Microbiologia de Alimentos , Plantas/microbiologia , Quinolonas/farmacologia , Antibacterianos/farmacologia , Sudeste Asiático , Resistência a Múltiplos Medicamentos/genética , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Genes Bacterianos/genética , Plasmídeos/genéticaRESUMO
Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one-/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0-24% to 96-100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms.
Assuntos
Proteínas de Bactérias , Galinhas , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli , Escherichia coli , Doenças das Aves Domésticas/microbiologia , beta-Lactamases , Animais , Infecções por Escherichia coli/epidemiologia , Humanos , Países Baixos/epidemiologia , Doenças das Aves Domésticas/epidemiologiaRESUMO
OBJECTIVES: The presence of ESBL/AmpC-producing E. coli in cattle has been reported previously, however information on veal calves is limited. This study describes the prevalence and molecular characteristics of E. coli with non-wild type susceptibility to cefotaxime in veal calves at slaughter. METHODS: Faecal samples from 100 herds, 10 individual animals per herd, were screened for E. coli with non-wild type susceptibility for cefotaxime. Molecular characterization of ESBL/AmpC genes and plasmids was performed on one isolate per herd by microarray, PCR and sequence analysis. RESULTS: 66% of the herds were positive for E. coli with non-wild type susceptibility for cefotaxime. Within-herd prevalence varied from zero to 90%. 83% of E. coli producing ESBL/AmpC carried bla(CTX-M) genes, of which bla(CTX-M-1), bla(CTX-M-14) and bla(CTX-M-15) were most prevalent. The dominant plasmids were IncI1 and IncF-type plasmids. CONCLUSIONS: A relatively high prevalence of various bla(CTX-M) producing E. coli was found in veal calves at slaughter. The genes were mainly located on IncI1 and IncF plasmids.