RESUMO
Strains 97/79T and A121, recovered respectively from human faeces and well water, were compared to currently known species of the genus Citrobacter using genotypic and phenotypic approaches. Multilocus sequence analysis based on housekeeping genes fusA, leuS, pyrG, rpoB and recN, showed that the two strains formed a distinct phylogenetic lineage within the genus Citrobacter. Average nucleotide identity (ANI) between strains 97/79T and A121 was 99.2 %, whereas ANI values of strain 97/79T with the type strains of closely related species of the genus Citrobacter, C. werkmanii, C. braakii, C. freundii, C. youngae and C. pasteurii, were all below 93.0 %. The ability to metabolize different compounds also discriminated strains 97/79T and A121 from other species of the genus Citrobacter. Based on these results, strains 97/79T and A121 represent a novel species of the genus Citrobacter, for which the name Citrobacter europaeus sp. nov. is proposed, with strain 97/79T (=CIP 106467T=DSM 103031T) as the type strain. The DNA G+C content of strain 97/79T is 52.0 %.
Assuntos
Fezes/microbiologia , Filogenia , Microbiologia da Água , Técnicas de Tipagem Bacteriana , Composição de Bases , Citrobacter/classificação , DNA Bacteriano/genética , Europa (Continente) , Genes Bacterianos , Humanos , Tipagem de Sequências Multilocus , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
A Gram-stain-negative strain, A60T, isolated from a water well sample in Portugal, was characterized phenotypically, genotypically and phylogenetically. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A60T belonged to the genus Citrobacter, and recN gene phylogeny revealed one strongly supported clade encompassing strain A60T and 13 other strains from public databases, distinct from currently recognized species of the genus Citrobacter. Furthermore, multilocus sequence analysis (MLSA) based on concatenated partial fusA, leuS, pyrG and rpoB sequences confirmed the classification obtained with the recN sequence. In silico genomic comparisons, including average nucleotide identity (ANI) and the genome-to-genome distance calculator (GGDC), showed 94.6â% and 58.4â% identity to the closest relative Citrobacter freundii ATCC 8090T, respectively. The ability to metabolize different compounds further discriminated strain A60T from other species of the genus Citrobacter. The G+C content of strain A60T is 52.0â%. The results obtained support the description of a novel species within the genus Citrobacter, for which the name Citrobacter portucalensis sp. nov. is proposed, with the type strain A60T (=DSM 104542T=CECT 9236T).
Assuntos
Citrobacter/classificação , Filogenia , Microbiologia da Água , Poços de Água , Técnicas de Tipagem Bacteriana , Composição de Bases , Citrobacter/genética , Citrobacter/isolamento & purificação , DNA Bacteriano/genética , Genes Bacterianos , Tipagem de Sequências Multilocus , Portugal , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
To gain insights into the diversification trajectories of qnrB genes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose, Citrobacter sp. isolates (n = 21) and genome or plasmid sequences (n = 56) available in public databases harboring complete or truncated qnrB genes were analyzed. Citrobacter species identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location of qnrB genes, and the genetic surroundings of qnrB genes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification of Citrobacter isolates was achieved using leuS and recN gene sequences, and isolates characterized in this study were diverse and harbored chromosomal qnrB genes. Phylogenetic analysis of all known qnrB genes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprising pspF and sapA and varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of these qnrB clusters, and the reliable identification of all Citrobacter isolates revealed that each platform evolved in different recognizable (Citrobacter freundii, C. braakii, C. werkmanii, and C. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome of Citrobacter spp. and in different plasmids of Enterobacteriaceae. Our data corroborate Citrobacter as the origin of qnrB and further suggest divergent evolution of closely related qnrB genes/platforms in particular Citrobacter spp., which were delineated using particular genotypic markers.
Assuntos
Cromossomos Bacterianos/química , Citrobacter/genética , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Filogenia , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Sequência de Bases , Evolução Biológica , Mapeamento Cromossômico , Cromossomos Bacterianos/metabolismo , Citrobacter/classificação , Citrobacter/efeitos dos fármacos , Citrobacter/metabolismo , Eletroforese em Gel de Campo Pulsado , Infecções por Enterobacteriaceae/microbiologia , Fluoroquinolonas/farmacologia , Genótipo , Humanos , Dados de Sequência Molecular , Família Multigênica , Plasmídeos/química , Plasmídeos/metabolismo , Análise de Sequência de DNARESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.
Assuntos
Bacteriemia , Infecções por Escherichia coli/complicações , Infecções por Escherichia coli/epidemiologia , Escherichia coli , Doenças Inflamatórias Intestinais/complicações , Mycobacterium avium subsp. paratuberculosis , Paratuberculose/complicações , Paratuberculose/epidemiologia , Adulto , Idoso , Coinfecção , DNA Bacteriano , Escherichia coli/genética , Feminino , Genes Bacterianos , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium avium subsp. paratuberculosis/genética , Prevalência , Estudos Prospectivos , Adulto JovemRESUMO
The aim of this study was to characterize by a multi-level approach extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006-7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations. The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with the amplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12 and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species in Hospitals B and C in 2006-7. Particular plasmid types were linked to blaCTX-M-15 (IncR or non-typeable plasmids), blaSHV-12 (IncR or IncHI2), blaSHV-28/-55/-106 (IncFIIK1 or IncFIIK5), blaTEM-10 (IncL/M) or blaTEM-24 (IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-type ESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15, ST147, ST336) and plasmid types (IncR, IncFIIK). Furthermore, we highlight the potential of IncFIIK plasmids (here firstly associated with blaSHV-2/-28/-55/-106) to disseminate as antibiotic resistance plasmids.
Assuntos
Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , beta-Lactamases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Epidemias , Genótipo , Hospitais , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Tipagem Molecular , Plasmídeos/análise , Portugal/epidemiologia , Análise de Sequência de DNARESUMO
OBJECTIVES: The spread of ESBL-producing Enterobacteriaceae among food animals/products has raised concerns about their possible transmission through the food chain. We aimed to characterize piggeries (pigs, piggery environments) as reservoirs of TEM-52- and CTX-M-encoding plasmids and clones. METHODS: Forty-three samples from five Portuguese intensive production farms were studied (2006-07). Twenty-two ESBL-producing (13 TEM-52, 6 CTX-M-32, 3 CTX-M-1) Escherichia coli isolates from healthy pigs, feed and liquid manure were further characterized. Standard methods were used for clonal (PFGE, MLST) and plasmid (S1-PFGE, replicon typing, pMLST, RFLP) analysis. PCR and sequencing were used for analysis of blaCTX-M genetic context and plasmid-mediated quinolone resistance genes. RESULTS: TEM-52 (n = 13/22; 59%), CTX-M-32 (n = 6/22; 27%) and CTX-M-1 (n = 3/22; 14%) were identified in feed (36%), swine faeces (36%), swine hide (9%) and liquid manure (18%) at different farms. Diverse phylogenetic groups and clones were identified among TEM-52 (7 A, 3 B1, 2 B2, 1 D; 8 clones)-producing, CTX-M-1 (1 A, 1 B1, 1 D; 3 clones)-producing and CTX-M-32 (4 A, 2 B1; 4 clones)-producing isolates. However, the ST10 clonal complex was frequent among TEM-52 (n = 6) and CTX-M-32 (n = 3) producers. blaTEM-52 and blaCTX-M-1/-32 genes were identified within epidemic IncI1/ST3 and IncN/ST1 plasmid variants, respectively. CONCLUSIONS: We report for the first time a piggery reservoir for blaTEM-52. The spread of blaTEM-52 and blaCTX-M-1/-32 within and/or between different piggeries was mostly associated with epidemic plasmids and clones previously identified in humans and other animal hosts in different EU countries, highlighting possible distribution along the food chain.
Assuntos
Infecções por Escherichia coli/veterinária , Escherichia coli/genética , Transferência Genética Horizontal , Plasmídeos , beta-Lactamases/genética , Animais , Eletroforese em Gel de Campo Pulsado , Microbiologia Ambiental , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Genótipo , Tipagem de Sequências Multilocus , Polimorfismo de Fragmento de Restrição , Portugal , Suínos , Resistência beta-LactâmicaRESUMO
Pathobionts, particularly Mycobacterium avium subsp. paratuberculosis (MAP) and Escherichia coli isolates with adherence/invasive ability (AIEC) have been associated with inflammatory bowel disease (IBD), particularly Crohn's disease (CD). This study aimed to evaluate the frequency of viable MAP and AIEC in a cohort of IBD patients. As such, MAP and E. coli cultures were established from faecal and blood samples (with a total n = 62 for each) of patients with CD (n = 18), ulcerative colitis (UC, n = 15), or liver cirrhosis (n = 7), as well as from healthy controls (HC, n = 22). Presumptive positive cultures were tested by polymerase chain reaction (PCR), for a positive confirmation of MAP or E. coli identity. E. coli-confirmed isolates were then tested for AIEC identity using adherence and invasion assays in the epithelial cell line of Caco-2 and survival and replication assays in the macrophage cell line of J774. MAP sub-culture and genome sequencing were also performed. MAP was more frequently cultured from the blood and faecal samples of patients with CD and cirrhosis. E. coli presumptive colonies were isolated from the faecal samples of most individuals, in contrast to what was registered for the blood samples. Additionally, from the confirmed E. coli isolates, only three had an AIEC-like phenotype (i.e., one CD patient and two UC patients). This study confirmed the association between MAP and CD; however, it did not find a strong association between the presence of AIEC and CD. It may be hypothesized that the presence of viable MAP in the bloodstream of CD patients contributes to disease reactivation.
RESUMO
Extended-spectrum-ß-lactamase (ESBL)- and carbapenemase-producing bacteria are widespread in hospitals, but the extent of this problem in long-term care facilities (LTCFs) is poorly understood. We aimed to elucidate, in the Portuguese regional clinical context, the relevance of LTCFs as a reservoir of Escherichia coli and Klebsiella spp. producing ESBL- and/or carbapenemases (Ec/Kp-ESBL/CARB). Fourteen LTCFs from Portugal, corresponding to units of convalescence (UC/n = 3), medium-term internment and rehabilitation (UMDR/ n = 5), or long-term internment and maintenance (ULDM/n = 6), were analyzed (2016-2019). All patients with Ec/Kp-ESBL/CARB infections acquired during LTCF stay were included, and detailed information was collected. Prevalence of patients with healthcare-associated infections (HAIs) by Ec/Kp-ESBL/CARB did not vary significantly over time (1.48% in 2016-2017, 1.89% in 2017-2018, and 1.90% in 2018-2019), but a statistically significant association with the LTCF typology (ULDM, UMDR) was observed. HAIs were caused by K. pneumoniae (n = 51/54.3%), E. coli (n = 41/43.6%), or both (n = 2/2.1%), producing ESBL (96%) or carbapenemases (4%). Prior colonization (n = 14/16%) corresponded to seven Kp-CARB and seven Ec/Kp-ESBL. The worrying prevalence of patients acquiring HAIs by Ec/Kp-ESBL/CARB, associated with the estimated rates of those already colonized at admission, highlights a relevant role for LTCFs as a reservoir of Ec/Kp-ESBL/CARB. Epidemiological surveillance should be extended to the national level, and colonization screening at LTCF admission implemented systematically.
RESUMO
TEM-154, identified in Portugal in 2004, associated the substitutions observed in the extended-spectrum ß-lactamase (ESBL) TEM-12 and in the inhibitor-resistant penicillinase (IRT) TEM-33. This enzyme exhibited hydrolytic activity against ceftazidime and a low level of resistance to clavulanic acid. Surprisingly, the substitution Met69Leu enhanced the catalytic efficiency of oxyimino ß-lactams conferred by the substitution Arg164Ser. Its discovery confirms the dissemination of the complex mutant group of TEM enzymes in European countries.
Assuntos
beta-Lactamases/química , beta-Lactamases/metabolismo , Ceftazidima/metabolismo , Ácido Clavulânico/farmacologia , Inibidores Enzimáticos/farmacologia , Dados de Sequência Molecular , beta-Lactamases/genéticaAssuntos
Portador Sadio , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , beta-Lactamases/biossíntese , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Escherichia coli , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Voluntários Saudáveis , Humanos , Vigilância da População , Portugal/epidemiologia , beta-Lactamases/genéticaRESUMO
TEM-24 remains one of the most widespread TEM-type extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae. To analyze the reasons influencing its spread and persistence, a multilevel population genetics study was carried out on 28 representative TEM-24 producers from Belgium, France, Portugal, and Spain (13 Enterobacter aerogenes isolates, 6 Escherichia coli isolates, 6 Klebsiella pneumoniae isolates, 2 Proteus mirabilis isolates, and 1 Klebsiella oxytoca isolate, from 1998 to 2004). Clonal relatedness (XbaI pulsed-field gel electrophoresis [PFGE] and E. coli phylogroups) and antibiotic susceptibility were determined by standard procedures. Plasmid analysis included determination of the incompatibility group (by PCR, hybridization, and/or sequencing) and comparison of restriction fragment length polymorphism (RFLP) patterns. Characterization of genetic elements conferring antibiotic resistance included integrons (classes 1, 2, and 3) and transposons (Tn3, Tn21, and Tn402). Similar PFGE patterns were identified among E. aerogenes, K. pneumoniae, and P. mirabilis isolates, while E. coli strains were diverse (phylogenetic groups A, B2, and D). Highly related 180-kb IncA/C2 plasmids conferring resistance to kanamycin, tobramycin, chloramphenicol, trimethoprim, and sulfonamides were identified. Each plasmid contained defective In0-Tn402 (dfrA1-aadA1, aacA4, or aacA4-aacC1-orfE-aadA2-cmlA1) and In4-Tn402 (aacA4 or dfrA1-aadA1) variants. These integrons were located within Tn21, Tn1696, or hybrids of these transposons, with IS5075 interrupting their IRtnp and IRmer. In all cases, blaTEM-24 was part of an IS5075-DeltaTn1 transposon within tnp1696, mimicking other genetic elements containing blaTEM-2 and blaTEM-3 variants. The international dissemination of TEM-24 is fuelled by an IncA/C2 plasmid acquired by different enterobacterial clones which seem to evolve by gaining diverse genetic elements. This work highlights the risks of a confluence between highly penetrating clones and highly promiscuous plasmids in the spread of antibiotic resistance, and it contributes to the elucidation of the origin and evolution of TEM-2 ESBL derivatives.
Assuntos
Antibacterianos/farmacologia , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Plasmídeos/genética , Antibacterianos/uso terapêutico , Cloranfenicol/farmacologia , Cloranfenicol/uso terapêutico , Eletroforese em Gel de Campo Pulsado , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Europa (Continente)/epidemiologia , Canamicina/farmacologia , Canamicina/uso terapêutico , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Trimetoprima/farmacologia , Trimetoprima/uso terapêuticoRESUMO
We aimed to investigate the occurrence of acquired AmpC ß-lactamases (qAmpC), and characterize qAmpC-producing Enterobacteriaceae from different non-clinical environments in Portugal. We analysed 880 Enterobacteriaceae resistant to third-generation cephalosporins recovered from 632 non-clinical samples [healthy human and healthy animal (swine, chickens) faeces; uncooked chicken carcasses; aquatic and trout aquaculture samples]. Bacterial and qAmpC identification, antibiotic susceptibility, clonal (PFGE, MLST) and plasmid (S1-/I-CeuI-PFGE, replicon typing, hybridization) analysis were performed using standard methods. The occurrence of qAmpC among Enterobacteriaceae from non-clinical origins was low (0.6%; n = 4/628 samples), corresponding to CMY-2-producing Escherichia coli from three healthy humans (HH) and one uncooked chicken carcass (UCC). We highlight a slight increase in CMY-2 human faecal carriage in the two periods sampled [1.0% in 2013-2014 versus 0% in 2001-2004], which is in accordance with the trend observed in other European countries. CMY-2-producing E. coli belonged to B22-ST4953 (n = 2, HH), A0-ST665 (n = 1, HH) or A1-ST48 (n = 1, UCC) clones. blaCMY-2 was identified in non-typeable and IncA/C2 plasmids. This study is one of the few providing an integrated evaluation of the qAmpC-producing Enterobacteriaceae occurrence, which was low, from a very large collection of different non-clinical origins. Further surveillance in contemporary collections can provide an integrated epidemiological information of potential shifts in reservoirs, transmission routes and mechanisms of dissemination of blaqAmpC in non-clinical settings.
Assuntos
Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Genótipo , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Análise de Sequência de DNA , beta-Lactamases/metabolismo , beta-Lactamas/farmacologiaRESUMO
OBJECTIVES: To investigate the diversity of integrons and extended-spectrum beta-lactamases (ESBLs) among Enterobacteriaceae from chickens and swine in Portugal and analyse the clonal relationships between Portuguese ESBL-producing isolates of animal and human origin. METHODS: We analysed samples from faeces of healthy swine (HSF, n = 35), from uncooked chicken carcasses (CM, n = 20) and from faeces of healthy chickens (HCF, n = 20). Samples were plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). ESBLs were characterized by PCR and DNA sequencing. Bacterial identification, antibiotic susceptibility and conjugation assays were performed by standard procedures. Isolate clonal relatedness was established by PFGE and by RAPD for PFGE non-typeable isolates. Escherichia coli phylogenetic groups were identified by a multiplex PCR. Integron analysis was accomplished by PCR-RFLP and sequencing. RESULTS: ESBL-producing Enterobacteriaceae were identified in 60% of CM, 10% of HCF and 5.7% of HSF samples, respectively, mostly corresponding to E. coli (phylogroups A, D and B1). TEM-52, SHV-2 and CTX-M-1 were detected from chicken and SHV-12 from swine samples. High clonal diversity was observed and most bla(ESBL) genes were transferable (67%). Class 1 and/or class 2 integrons were identified in 80% of CM, 10% of HCF and 63% of HSF samples, with class 1 integrons more common than class 2 integrons (36% versus 12% of the isolates recovered, respectively). Ten class 1 integron types are described, aadA1 and dfrA1-aadA1 being the most frequently found. Two class 1 integron types (aadA13-estX and dfrA14-aadA1-catB2) and one class 2 integron (aadA1) are first reported here. CONCLUSIONS: This study is the first report of ESBLs and integrons from chickens and swine in Portugal and highlights the antibiotic-resistant bacteria and/or resistance genes that might be acquired by humans through the food chain.
Assuntos
Galinhas/microbiologia , Resistência Microbiana a Medicamentos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Integrons , Suínos/microbiologia , beta-Lactamases/genética , Animais , Análise por Conglomerados , Conjugação Genética , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Enterobacteriaceae/classificação , Enterobacteriaceae/isolamento & purificação , Fezes/microbiologia , Variação Genética , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Filogenia , Reação em Cadeia da Polimerase , Portugal , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise de Sequência de DNARESUMO
KPC-3-producing bacteria are endemic in many countries but only recently became apparent their wide distribution in different Portuguese hospitals. The aim of this study is to characterize genetic backgrounds associated with bla KPC-3 among Klebsiella pneumoniae isolates recently identified on non-hospitalized patients in Portugal. Twenty KPC-producing K. pneumoniae identified between October 2014 and November 2015 in three different community laboratories were characterized. Isolates were mainly from patients from long-term care facilities (n = 11) or nursing homes (n = 6), most of them (75%) previously hospitalized in different Portuguese hospitals. Standard methods were used for bacterial identification and antibiotic susceptibility testing. Carbapenemase production was assessed by the Blue-Carba test, and identification of bla genes was performed by PCR and sequencing. Epidemiological features of KPC-producing K. pneumoniae included population structure (XbaI-PFGE, MLST and wzi sequencing), genetic context (mapping of Tn4401), and plasmid (replicon typing, S1-PFGE, and hybridization) analysis. All K. pneumoniae isolates produced KPC-3, with two MDR K. pneumoniae epidemic clones representing 75% of the isolates, namely ST147 (wzi64/K14.64, February-November 2015) and ST15 (two lineages exhibiting capsular types wzi19/K19 or wzi93/K60, July-November 2015). Other sporadic clones were detected: ST231 (n = 3; wzi104), ST348 (n = 1; wzi94) and ST109 (n = 1, wzi22/K22.37). bla KPC-3 was identified within Tn4401d in all isolates, located in most cases (80%) on cointegrated plasmids (repA FIA+repA FII+ori ColE1;105-250 kb) or in 50 kb IncN plasmids. In conclusion, this study highlights a polyclonal structure of KPC-3-producing K. pneumoniae and the predominance of the ST147 clone among non-hospitalized patients in Portugal, linked to platforms still unnoticed in Europe (bla KPC-3-Tn4401d-IncFIA) or firstly reported (bla KPC-3-Tn4401d-IncN). This scenario underlines the recent penetration of successful mobile genetic elements in previously circulating MDR K. pneumoniae lineages (mainly ST147 and ST15) in Portugal, rather than the importation of the global lineages from clonal group 258.
RESUMO
AIM: To characterize temporal shifts in extended-spectrum ß-lactamases (ESBLs) and clones of clinical Escherichia coli isolates. MATERIALS & METHODS: All ESBL-producing E. coli isolates from a Portuguese hospital (n = 112; June 2006-June 2007 and January-December 2010) were characterized by identification of phylogenetic groups, ESBL-types and virulence genes, XbaI-PFGE and MLST. RESULTS: We observed a substantial increase in widespread E. coli clones from phylogroups A, B1 and D (e.g., ST10, ST23, ST117, ST155, ST648) producing mainly CTX-M-1, -14, -32 or SHV-12, along with a decrease in the proportion of the predominant CTX-M-15-producing B2-ST131 clone. CONCLUSION: The amplification of diverse CTX-M-producing A, B1 and D clonal complexes, which have been long identified in Portuguese nonclinical settings, unveils a role for these reservoirs in the landscape of ESBL-producing E. coli in the clinical setting.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , beta-Lactamases/biossíntese , Eletroforese em Gel de Campo Pulsado , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Genótipo , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Filogenia , Portugal/epidemiologia , Fatores de TempoRESUMO
Bacteria colonizing the human intestine have a relevant role in the spread of antimicrobial resistance. We investigated the faecal carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in healthy humans from Portugal and analyzed the distribution of sul genes and class 1 and 2 integrons. Faecal samples (n = 113) were recovered from healthy persons (North/Centre of Portugal, 2001-2004) and plated on MacConkey agar with and without ceftazidime (1 mg/L) or cefotaxime (1 mg/L). Isolates representing different morphotypes/plate and antibiotic susceptibility patterns (n = 201) were selected. Isolates resistant to sulfonamides and/or streptomycin, gentamicin, and trimethoprim were screened (PCR and sequencing) for sul genes (sul1, sul2, sul3) and class 1 and 2 integrons. Presence of ESBLs was inferred using the double disk synergy test (DDST) and further confirmed by PCR and sequencing. ESBL producers were selected for clonal analysis, plasmid characterization and conjugation assays by standard methods. ESBL-producing isolates were found in 1.8% (2/113) of samples, corresponding to Escherichia coli of phylogroups A (n = 1) and B1 (n = 1) carrying transferable bla CTX-M-14 and the new bla TEM-153, respectively. A 80kb IncK plasmid bearing bla CTX-M-14 was found, being highly related to that widely spread among CTX-M-14 producers of humans and animals from Portugal and other European countries. sul genes were found in 88% (22/25; sul2-60%, sul1-48%, sul3-4%) of the sulfonamide resistant isolates. Class 1 integrons were more frequently found than class 2 (7%, 14/201 vs. 3%, 6/201). Interestingly, gene cassette arrangements within these platforms were identical to those commonly observed among Enterobacteriaceae from Portuguese food-producing animals, although aadA13 is here firstly described in Morganella morganii. These results reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes including bla ESBLs, and highly transferable genetic platforms as IncK epidemic plasmids.
Assuntos
Infecção Hospitalar/epidemiologia , Farmacorresistência Bacteriana Múltipla , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Fezes/microbiologia , Feminino , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Assistência de Longa Duração , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , PortugalRESUMO
We analyse the diversity of extended-spectrum beta-lactamases (ESBLs), antibiotic resistance genes, and plasmid content among Escherichia coli from a community and a hospital setting in Rio de Janeiro, Brazil. ESBL producers (CTX-M-2, CTX-M-9, CTX-M-59) belonged to different phylogroups/sequence types, and bla(CTX-M) were identified in IncA/C plasmids, reinforcing the possible intraplasmid evolution of bla(CTX-M-59) from bla(CTX-M-2) and the implication of IncA/C on bla(CTX-M) spread.