Relação ancestral e pan-resistoma plasmidial de Escherichia coli produtora de CTX-M-8 e MCR-1 na interface humana, animal e ambiental / Ancestral relationship and plasmid pan-resistome of CTX-M-8- and MCR-1-producing Escherichia coli in human-animal-environmental interface

Linhagens de Escherichia coli produtoras de ß-lactamase de espectro estendido (ESßL) do tipo CTX-M são endêmicas no Brasil, sendo prevalentes em casos de infecções hospitalares e ambulatoriais. Atualmente, cepas produtoras de CTX-M têm sido recuperadas de ambientes urbanos, animais de companhia ou de produção e de alimentos de origem animal, inclusive afetando o agronegócio, o que aponta uma possível rota de disseminação em diferentes ecossistemas. Recentemente, nesta espécie, foi descoberto um novo gene, chamado de mcr-1, que confere resistência transferível à colistina, um dos últimos antibióticos eficazes para o tratamento de infecções causadas por bactérias produtoras de ESBL e carbapenemases. Deste modo, o presente estudo tem como objetivo elucidar os aspectos sobre a caracterização e a relação de plasmídeos que carregam genes do tipo blaCTX-M-8 e mcr- 1 em cepas de E. coli isoladas de seres humanos, animais, ambiente aquático e alimentos, no Brasil. Neste estudo são apresentados os resultados da análise plasmidial de 25 cepas de E. coli, das quais nove apresentaram o genótipo blaCTX-M-8/IncI1, 11 apresentaram o genótipo mcr-1/IncX4 e cinco apresentaram ambos os genótipos blaCTX-M-8/IncI1 e mcr-1/IncX4. Dos resultados, podemos observar que plasmídeos IncI1 (blaCTX-M-8) e IncX4 (mcr-1) estão circulando no Brasil desde o ano de 2009 entre diferentes clones (STs) de E. coli e em diferentes ambientes e hospedeiros. Os plasmídeos IncI1 foram conjugativos e pertencentes ao ST113, exceto o plasmídeo recuperado de um isolado humano, que foi pertencente ao ST131. Os plasmídeos IncI1 apresentaram sua arquitetura conservada, com a presença de genes de replicação, transferência e estabilidade. A partir do alinhamento, os plasmídeos IncI1 apresentaram 94-99% de similaridade genética entre eles. Dentre os plasmídeos IncX4, independente da fonte de isolamento, todos permaneceram com sua arquitetura altamente conservada. Entretanto, apenas dois plasmídeos (um encontrado em uma cepa de animal e outro encontrado em uma cepa de ambiente aquático) apresentaram uma IS1294, truncando o gene de mobilização. Na análise comparativa, todos os plasmídeos IncX4 apresentaram similaridade genética de 95-99,9% entre eles. No alinhamento de plasmídeos IncX4 brasileiros contra plasmídeos de outras regiões geográficas, foi observada similaridade genética > 99,9%, o que confirma a estabilidade e conservação desses plasmídeos. Neste estudo foram reportados dados inéditos da primeira identificação do gene mcr-1 em diferentes ecossistemas no Brasil, assim como a nova variante mcr-5.3. A análise filogenética dos plasmídeos IncI1 e IncX4, destacam que ambos compartilham uma arquitetura conservada, e a evolução é atribuída à aquisição de genes de resistência. Adicionalmente, um novo vetor de disseminação do gene mcr-1 no Brasil foi identificado - o plasmídeo IncHI2. Os resultados desse estudo demonstram o grave problema da resistência bacteriana dentro do conceito One-health e que, com o avanço de ferramentas moleculares, a identificação e a resolução desse problema poderá estar cada vez mais próxima de ser elucidada

CTX-M-type extended-spectrum-ß-lactamase (ESßL)-producing-Escherichia coli are endemic in Brazil and are prevalent in cases of nosocomial and ambulatory infections. Currently, CTXM-producing strains have been recovered from urban environments, companion/production animals and animal source foods, which indicates a possible route of dissemination in different ecosystems. Recently, in this species, a new gene, called mcr-1, has been discovered, conferring transferable resistance to colistin, one of the last effective antibiotics for the treatment of infections caused by ESBL- and carbapenemases -producing bacteria. Thus, the present study aims to elucidate unknown aspects of the pan-resistome and ancestral relationship of plasmids carrying blaCTX-M-8 and mcr-1 genes in strains of E. coli isolated from humans, animals, aquatic environment and food, in Brazil. In this study, we present results from the plasmidial analysis of 25 E. coli strains, from which nine presented the blaCTX-M-8/IncI1 genotype, 11 presented the mcr-1/IncX4, and five presented both blaCTX-M-8/IncI1 and mcr-1/IncX4 genotypes. Among these results, we can observe that IncI1 (blaCTX-M-8) and IncX4 (mcr-1) plasmids are circulating in Brazil since 2009, between different E. coli clones (STs) and different hosts and environments. IncI1 plasmids were conjugative and assigned to ST113, with exception of a plasmid recovered from a human isolate, which was assigned to ST131. IncI1 plasmids presented conserved architecture, with the presence of genes of replication, transference, and stability. From the alignment analysis, IncI1 plasmids presented 94-99% genetic similarity among them. Among the IncX4 plasmids, regardless the isolation source, their architecture remained highly conserved. However, only two plasmids (one detected in an animal's strain and another detected in an aquatic environment's strain) presented an IS1294, truncating the mobilization gene. In the comparative analysis, all IncX4 plasmids presented 95-99,9% genetic similarity among them. In the alignment of Brazilian IncX4 plasmids against plasmids from other geographic regions, >99.9% genetic similarity was observed, confirming the stability and conservation of these plasmids. In this study, unprecedented data from the first identification of the mcr-1 gene in different ecosystems in Brazil, as well as the new variant, mcr-5.3. Additionally, it was identified a new dissemination vector of the mcr-1 gene in Brazil - the IncHI2 plasmid. Phylogenetic analysis of IncI1and IncX4 plasmids highlight that both share a conserved backbone, and evolution is attributed to the acquisition of clinically relevant antimicrobial resistance genes. The results from this study demonstrate the serious problem of the bacterial resistance within the "One-Health" concept and that, with the advance of molecular tools, identification and resolution of this problem may be increasingly closer to being elucidate

Hypervirulence and biofilm production in KPC-2-producing Klebsiella pneumoniae CG258 isolated in Brazil.

In this study, we describe the frequency of virulence genes in Klebsiella pneumoniae carbapenemase-2-producing Klebsiella pneumoniae (KPC-KP), including hypervirulent (hv) and hypermucoviscous (hm) strains by whole-genome sequencing. We also evaluate the capacity for biofilm formation by using phenotypic techniques. The occurrence of several virulence genes (fimABCDEFGHIK, mrkABCDFHJ, ecpA, wabG, entB, ugE, irp1, irp2, traT, iutA and ureADE) and a high frequency of hvhmKPC-KP isolates was found. Most hospital-associated lineages of KPC-KP belong to the international clonal group 258 (CG258). Biofilm formation was a constant feature among 90.9 % of KPC-KP strains. This report suggests a close relationship between ST437 and weak biofilm production, given that all weakly biofilm-producing strains belonged to this sequence type. This also supports the dissemination of KPC-KP containing numerous virulence determinants belonging to the biofilm-producing CG258 type in Brazil, including hv and hm strains. These factors allow this pathogen to cause infections, leading to its rapid expansion and persistence in hospital settings.

Genetic background of novel sequence types of CTX-M-8- and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae from public wastewater treatment plants in São Paulo, Brazil.

The release of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in São Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla ESBL-containing E. coli transformants. Strains harbored bla CTX-M-8, bla CTX-M-15, and/or bla SHV-28. Sequencing results showed that bla CTX-M-8 and bla CTX-M-15 genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in São Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children.

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Enterotoxigenic and non-enterotoxigenic Bacteroides fragilis from fecal microbiota of children

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

Presence of Shiga toxin 2e-producing Escherichia coli and atypical enteropathogenic E. coli in an asymptomatic child.

INTRODUCTION: Escherichia coli causes gastroenteritis in humans and animals. CASE PRESENTATION: In this study, both Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (EPEC) strains were identified in a stool sample from a healthy child, and they were serotyped as Shiga toxin-producing E. coli (STEC) ONT : H19 and atypical enteropathogenic E. coli (EPEC) O37 : H45. CONCLUSION: This is the first report, to our knowledge, of a concomitant presence of diarrhoeagenic E. coli (DEC) strains in an asymptomatic child. None of the microorganisms was able to produce diarrhoea, maybe because they were transient bacteria or because of the good immune status of the child. Attention should be paid to this result and it could be of interest in vaccine prospects.