RESUMO
Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.
Assuntos
Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Enterococcus faecalis/genética , Enterococcus faecium/genética , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos/genéticaRESUMO
BACKGROUND: ESBL- and carbapenemase-producing Pseudomonas aeruginosa are prevalent in, for example, the Middle East, Eastern Europe and Latin America, though rarer elsewhere. Because P. aeruginosa readily mutate to become carbapenem resistant via loss of OprD, isolates producing ESBLs are often as broadly resistant as those producing carbapenemases. We hypothesized that: (i) relebactam might overcome class A carbapenemases directly in P. aeruginosa; and (ii) relebactam's inhibition of AmpC, which gives a generalized potentiation of imipenem against the species, might restore imipenem susceptibility in OprD-deficient ESBL producers. METHODS: MICs were determined using CLSI agar dilution for P. aeruginosa isolates producing ESBLs, principally VEB types, and for those producing GES-5, KPC and other carbapenemases. RESULTS: Relebactam potentiated imipenem by around 4-8-fold for most P. aeruginosa isolates producing VEB and other ESBLs; however, MICs were typically only reduced to 4-16 mg/L, thus mostly remaining above EUCAST's susceptible range and only partly overlapping CLSI's intermediate range. Strong (approx. 64-fold) potentiation was seen for isolates producing KPC carbapenemases, but only 2-fold synergy for those with GES-5. Predictably, potentiation was not seen for isolates with class B or D carbapenemase activity. CONCLUSIONS: Relebactam did potentiate imipenem against ESBL-producing P. aeruginosa, which are mostly imipenem resistant via OprD loss, but this potentiation was generally insufficient to reduce imipenem MICs to the clinical range. Imipenem resistance owing to KPC carbapenemases was reversed by relebactam in P. aeruginosa, just as for Enterobacterales.
Assuntos
Compostos Azabicíclicos , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias , Europa Oriental , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Oriente Médio , beta-Lactamases/genéticaRESUMO
Carbapenem resistance in Gram-negative bacteria is a public health concern. Consequently, numerous government and agency reports discuss carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant organisms (CROs). Unfortunately, these terms are fuzzy. Do they include (1) Proteeae with inherent imipenem resistance; (2) porin-deficient Enterobacterales resistant to ertapenem but not other carbapenems; (3) Enterobacterales with OXA-48-like enzymes that remain "carbapenem susceptible" at breakpoint; and (4) Pseudomonas aeruginosa that merely lack porin OprD? Counting CPE or CPOs is better but still insufficient, because different carbapenemases have differing treatment implications, particularly for new ß-lactam/ß-lactamase inhibitor combinations. At the least, it is essential for authors, journals, and regulatory agencies to specify the carbapenemases meant. The future may demand even greater precision, for mutations can alter hydrolytic activity, and the ability to confer resistance, within carbapenemase families.
Assuntos
Antibacterianos , Carbapenêmicos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Humanos , beta-Lactamases/genéticaRESUMO
Objectives: To evaluate the AusDiagnostics MT CRE EU assay for the detection of carbapenemase and acquired colistin resistance genes in Gram-negative bacteria. Methods: The assay allows the detection of blaKPC, blaOXA-48-like, blaNDM, blaVIM, blaIMP, blaSIM, blaGIM, blaSPM, blaFRI, blaIMI, blaGES (differentiating ESBL and carbapenemase variants), blaSME and mcr-1/-2. It was evaluated against a panel of isolates including Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. retrospectively (n = 210) and prospectively (n = 182). Results: The CRE EU assay was able to detect 268/268 carbapenemase genes, with 239 belonging to the 'big five' families (KPC, OXA-48-like, NDM, VIM and IMP) and 29 carbapenemase genes of the SIM, GIM, SPM, FRI, IMI, SME and GES families. It could distinguish between ESBL and carbapenemase variants of GES. It also allowed detection of mcr-1/-2 colistin resistance genes on their own or in isolates co-producing a carbapenemase. Conclusions: The AusDiagnostics MT CRE EU assay offered wide coverage for detection of acquired carbapenemase genes. It required minimal hands-on time and delivered results in less than 4 h from bacterial culture.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Técnicas de Diagnóstico Molecular/normas , beta-Lactamases/genética , Proteínas de Bactérias/isolamento & purificação , Carbapenêmicos/farmacologia , Bactérias Gram-Negativas/enzimologia , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular/métodos , Estudos Retrospectivos , beta-Lactamases/isolamento & purificaçãoRESUMO
Background: Ceftazidime/avibactam combines an established oxyimino-cephalosporin with the first diazabicyclooctane ß-lactamase inhibitor to enter clinical use. We reviewed its activity against Gram-negative isolates, predominantly from the UK, referred for resistance investigation in the first year of routine testing, beginning in July 2015. Methods: Isolates were as received from referring laboratories; there is a bias to submit those with suspected carbapenem resistance. Identification was by MALDI-TOF mass spectroscopy, and susceptibility testing by BSAC agar dilution. Carbapenemase genes were sought by PCR; other resistance mechanisms were inferred using genetic data and interpretive reading. Results: Susceptibility rates to ceftazidime/avibactam exceeded 95% for: (i) Enterobacteriaceae with KPC, GES or other Class A carbapenemases; (ii) Enterobacteriaceae with OXA-48-like enzymes; and (iii) for ESBL or AmpC producers, even when these had impermeability-mediated ertapenem resistance. Almost all isolates with metallo-carbapenemases were resistant. Potentiation of ceftazidime by avibactam was seen for 87% of ceftazidime-resistant Enterobacteriaceae with 'unassigned' ceftazidime resistance mechanisms, including two widely referred groups of Klebsiella pneumoniae where no synergy was seen between cephalosporins and established ß-lactamase inhibitors. Potentiation here may be a diazabicyclooctane/cephalosporin enhancer effect. Activity was seen against Pseudomonas aeruginosa with derepressed AmpC, but not for those with efflux-mediated resistance. Conclusions: Of the available ß-lactams or inhibitor combinations, ceftazidime/avibactam has the widest activity spectrum against problem Enterobacteriaceae, covering all major types except metallo-carbapenemase producers; against P. aeruginosa it has a slightly narrower spectrum than ceftolozane/tazobactam, which also covers efflux-type resistance.
Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/epidemiologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Reino Unido/epidemiologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/biossíntese , beta-Lactamases/genéticaRESUMO
Objectives: Detection of rarer carbapenemases is challenging, as it requires molecular assays with comprehensive coverage or the use of phenotypic methods for the detection of carbapenemase activity. We describe a new class A carbapenemase, FRI-2, in an Enterobacter cloacae complex isolate following implementation of an in-house multiplex PCR for the detection of 'rare' class A carbapenemases. Methods: MICs were determined by agar dilution. A carbapenem-resistant E. cloacae complex isolate was tested by PCR for the class A carbapenemases blaKPC, blaFRI, blaIMI, blaGES and blaSME. Carbapenemase activity was assessed using Carba NP and the carbapenem inactivation method. Whole genome and plasmid analyses of the clinical isolate and the FRI-2 transformant were performed by WGS, respectively. Typing was carried out by PFGE. Results: The E. cloacae complex isolate showed resistance to imipenem (MIC = 16 mg/L), meropenem (MIC = 8 mg/L) and ertapenem (MIC = 8 mg/L), but remained susceptible to piperacillin/tazobactam (MIC = 8 mg/L). Carbapenemase activity was confirmed in the isolate by both phenotypic methods. A blaFRI-1-like gene was detected by PCR and analysis of WGS data of the clinical isolate identified an ORF of 885 bp, which showed 97% nucleotide identity with blaFRI-1 and was named blaFRI-2. WGS of the transformant indicated blaFRI-2 was located on a 108 kb IncF/IncR plasmid. The FRI-2-positive E. cloacae complex isolate belonged to a novel ST (ST829). Conclusions: The possible circulation of rarer carbapenemases in clinical settings highlights the role of phenotypic tests to detect carbapenemase activity when molecular assays are negative for the 'big 5' carbapenemase families.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Carbapenêmicos/metabolismo , Enterobacter cloacae/enzimologia , Enterobacter cloacae/genética , Infecções por Enterobacteriaceae/microbiologia , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Enterobacter cloacae/efeitos dos fármacos , Enterobacter cloacae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Multiplex , Plasmídeos , Reino Unido/epidemiologia , beta-Lactamases/genéticaRESUMO
Background: We assessed the activity of ceftolozane/tazobactam against consecutive isolates collected in the BSAC Bacteraemia Surveillance from 2011 to 2015 and against 'problem' isolates sent to the UK national reference laboratory from July 2015, when routine testing began. Methods: Susceptibility testing was by BSAC agar dilution with resistance mechanisms identified by PCR and interpretive reading. Results: Data were reviewed for 6080 BSAC surveillance isolates and 5473 referred organisms. Ceftolozane/tazobactam had good activity against unselected ESBL producers in the BSAC series, but activity was reduced against ertapenem-resistant ESBL producers, which were numerous among reference submissions. AmpC-derepressed Enterobacter spp. were widely resistant, but Escherichia coli with raised chromosomal AmpC frequently remained susceptible, as did Klebsiella pneumoniae with acquired DHA-1-type AmpC. Carbapenemase-producing Enterobacteriaceae were mostly resistant, except for ceftazidime-susceptible isolates with OXA-48-like enzymes. Ceftolozane/tazobactam was active against 99.8% of the BSAC Pseudomonas aeruginosa isolates; against referred P. aeruginosa it was active against 99.7% with moderately raised efflux, 94.7% with strongly raised efflux and 96.6% with derepressed AmpC. Resistance in P. aeruginosa was largely confined to isolates with metallo-ß-lactamases (MBLs) or ESBLs. MICs for referred Burkholderia spp. and Stenotrophomonas maltophilia were 2-4-fold lower than those of ceftazidime. Conclusions: Ceftolozane/tazobactam is active against ESBL-producing Enterobacteriaceae; gains against other problem Enterobacteriaceae groups were limited. Against P. aeruginosa it overcame the two most prevalent mechanisms (up-regulated efflux and derepressed AmpC) and was active against 51.9% of isolates non-susceptible to all other ß-lactams, rising to 80.9% if ESBL and MBL producers were excluded.
Assuntos
Anti-Infecciosos/farmacologia , Cefalosporinas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/microbiologia , Ácido Penicilânico/análogos & derivados , Inibidores de beta-Lactamases/farmacologia , Monitoramento Epidemiológico , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Ácido Penicilânico/farmacologia , Reação em Cadeia da Polimerase , Tazobactam , Reino UnidoRESUMO
Objectives: Carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for CPE confirmed by the national reference laboratory from laboratories in the West Midlands region from November 2007 to December 2014. Methods: MICs were determined by BSAC agar dilution methodology and isolates exhibiting resistance to one or more carbapenems were screened for carbapenemase genes by PCR. Plasmid analyses were performed after electro-transformation of carbapenemase-encoding plasmids. WGS was performed on both transformants and clinical isolates. Patient data provided by the sending laboratories were reviewed. Results: During the study period, CPE ( n = 139) were submitted from 13 laboratories in the West Midlands region, originating from 108 patients and including one environmental isolate. CPE submissions increased significantly from 2009 onwards. Isolates were predominantly Klebsiella pneumoniae (89/139) obtained from inpatients. WGS was performed on all clinical isolates and transformants. After deduplication 119 isolates and 96 transformants remained for analysis. Within these, four families of carbapenemase genes were identified: bla NDM (69/119), bla KPC (26/119), bla OXA-48-like (16/119) and bla VIM (7/119); one isolate carried both bla NDM and bla OXA-48-like . Isolates represented diverse STs and plasmid replicon types. Plasmid analyses identified plasmids of different replicon types encoding bla KPC , bla NDM and bla OXA-48-like genes, found across several species and STs. Conclusions: CPE have been reported increasingly in the West Midlands region over a 7â year period. bla NDM , bla KPC and bla OXA-48-like were the dominant carbapenemase genes and were found in a range of diverse genomic/plasmid environments, highlighting their ability to mobilize across different plasmids, often impeding the detection of outbreaks.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/classificação , Enterobacteriaceae/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/metabolismo , Inglaterra/epidemiologia , Enterobacteriaceae/enzimologia , Infecções por Enterobacteriaceae/epidemiologia , Feminino , Genoma Bacteriano , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Plasmídeos/análise , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transformação BacterianaRESUMO
Objectives: OXA-48-like carbapenemases have spread worldwide since 2001. We analysed patient and microbiological data for UK isolates with these enzymes as confirmed by the national reference laboratory from November 2007 to December 2014. Methods: MICs were determined using BSAC agar dilution. Isolates with reduced susceptibility or resistance to at least one carbapenem and high-level resistance to both piperacillin/tazobactam (MICs ≥64 mg/L) and temocillin (MICs ≥128 mg/L) were screened by PCR for bla OXA-48-like genes. The genomes of about half of the isolates were sequenced, with MLST types, resistance genes and plasmid replicon types inferred. Patient data provided by sending laboratories were reviewed. Results: Isolates ( n = 741) with OXA-48-like carbapenemases were submitted from 111 UK laboratories, representing 536 patients. Almost all (99%; 736 of 741) were Enterobacteriaceae, predominantly Klebsiella pneumoniae (55%; 408), and most (80%; 595) were from inpatients. WGS of 351 non-duplicate isolates identified bla OXA-48 as the most common variant, found in two-thirds (235 of 351) of isolates, followed by bla OXA-181 (68), bla OXA-232 (32), bla OXA-244 (10), bla OXA-484 (5) and bla OXA-245 (1). Among K. pneumoniae (163 of 351), Escherichia coli (114 of 351) and Enterobacter cloacae (42 of 351), 119 STs were identified. Mapping analyses revealed that 63% (222 of 351) of isolates harboured plasmids that shared >99% identity to one of four known plasmids [pOXA-48a (44%; 154 of 351), pOXA-232 (10%; 34 of 351), pOXA181 (9%; 30 of 351) and pKP3-A (1%; 4 of 351)]; the remaining 37% of isolates harboured bla OXA-48-like in unknown environments. Conclusions: OXA-48-like carbapenemases are an increasing problem in the UK. This study highlights both the role of successful plasmids and the polyclonal nature of their dissemination.
Assuntos
Proteínas de Bactérias/genética , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Plasmídeos , beta-Lactamases/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Análise de Sequência de DNA , Reino Unido/epidemiologiaRESUMO
OBJECTIVES: To estimate UK prevalence and incidence of clinically significant carbapenemase-producing Enterobacteriaceae (CPE), and to determine epidemiological characteristics, laboratory methods and infection prevention and control (IPC) measures in acute care facilities. METHODS: A 6 month survey was undertaken in November 2013-April 2014 in 21 sentinel UK laboratories as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) project. Up to 10 consecutive, non-duplicate, clinically significant and carbapenem-non-susceptible isolates of Escherichia coli or Klebsiella pneumoniae were submitted to a reference laboratory. Participants answered a questionnaire on relevant laboratory methods and IPC measures. RESULTS: Of 102 isolates submitted, 89 (87%) were non-susceptible to ≥1 carbapenem, and 32 (36%) were confirmed as CPE. CPE were resistant to most antibiotics, except colistin (94% susceptible), gentamicin (63%), tigecycline (56%) and amikacin (53%). The prevalence of CPE was 0.02% (95% CIâ=â0.01%-0.03%). The incidence of CPE was 0.007 per 1000 patient-days (95% CIâ=â0.005-0.010), with north-west England the most affected region at 0.033 per 1000 patient-days (95% CIâ=â0.012-0.072). Recommended IPC measures were not universally followed, notably screening high-risk patients on admission (applied by 86%), using a CPE 'flag' on patients' records (70%) and alerting neighbouring hospitals when transferring affected patients (only 30%). Most sites (86%) had a laboratory protocol for CPE screening, most frequently using chromogenic agar (52%) or MacConkey/CLED agars with carbapenem discs (38%). CONCLUSIONS: The UK prevalence and incidence of clinically significant CPE is currently low, but these MDR bacteria affect most UK regions. Improved IPC measures, vigilance and monitoring are required.
Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Carbapenêmicos/uso terapêutico , Infecções por Enterobacteriaceae/epidemiologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/genética , Técnicas de Tipagem Bacteriana , Farmacorresistência Bacteriana/genética , Infecções por Enterobacteriaceae/tratamento farmacológico , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Humanos , Incidência , Controle de Infecções , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Prevalência , Inquéritos e Questionários , Reino Unido/epidemiologiaRESUMO
OBJECTIVES: We evaluated the RAPIDEC(®) CARBA NP assay (bioMérieux SA, Marcy-l'Étoile, France), a colorimetric test for rapid detection of carbapenemases, at two sites: Karolinska University Laboratory and PHE's national reference laboratory. METHODS: A total of 138 bacterial isolates previously characterized as positive for class A, B and/or D carbapenemase genes and 138 supposed non-carbapenemase producers were tested with RAPIDEC(®) CARBA NP according to the manufacturer's protocol. Two carbapenemase-producing isolates carried both NDM and OXA-48-like genes. Molecular detection of the expected carbapenemase gene(s) was used as the gold standard, and was performed by conventional and real-time PCR in-house assays. RESULTS: The RAPIDEC(®) CARBA NP assay detected 135 of 138 carbapenemase producers; one OXA-48-producing Klebsiella pneumoniae and two Acinetobacter baumannii producing OXA-23 or OXA-24 were not detected. Among 'negative' controls, 135 of 138 isolates were negative by RAPIDEC(®) CARBA NP. The exceptions were one Klebsiella oxytoca, which was later found to produce GES-5 carbapenemase, one Pseudomonas aeruginosa with OprD loss and increased efflux, and one Enterobacter cloacae with impermeability. When numbers were adjusted for the GES-5 producer, the overall sensitivity of the RAPIDEC(®) CARBA NP test was 97.8% and its specificity was 98.5%. CONCLUSIONS: The assay took less than 2.5 h to carry out, was user-friendly and had a high overall performance, making it an attractive option for clinical laboratories.
Assuntos
Acinetobacter baumannii/enzimologia , Proteínas de Bactérias/análise , Técnicas Bacteriológicas/métodos , Colorimetria/métodos , Enterobacteriaceae/enzimologia , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/análise , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
OBJECTIVES: Klebsiella pneumoniae carbapenemases (KPCs) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for KPC-positive bacteria confirmed by the national reference laboratory from UK laboratories from August 2003 to August 2014, excluding North-West England, where the epidemiology has previously been studied. METHODS: MICs were determined by BSAC agar dilution. Carbapenem-resistant isolates lacking imipenem/EDTA synergy were tested by PCR for blaKPC. MLST and blaKPC sequencing were performed on a subset of isolates. Plasmid analysis was performed by transformation, PCR-based replicon typing and, in some cases, whole-plasmid sequencing. Patient data provided by the sending laboratories were reviewed. RESULTS: Two hundred and ten isolates with KPC enzymes were submitted from 71 UK laboratories outside North-West England, representing 160 patients. All were Enterobacteriaceae, predominantly K. pneumoniae (82%; 173/210), and most (91%; 191/210) were from hospitalized patients. Analysis of 100 isolates identified blaKPC-2 (62%), blaKPC-3 (30%) and blaKPC-4 (8%). Clonal group (CG) 258 was dominant among K. pneumoniae (64%; 54/84), but 21 unrelated STs were also identified. Plasmid analysis identified a diverse range of plasmids representing >11 different replicon types and found in multiple STs and species. Most (34/35) plasmids with IncFIB/FIIK replicons exhibited >99% sequence identity to pKpQIL. CONCLUSIONS: KPC enzymes are increasingly detected in Enterobacteriaceae in the UK, albeit without the major outbreaks seen in North-West England. K. pneumoniae CG258 are the dominant hosts, but plasmid spread plays a major role in KPC dissemination between other K. pneumoniae STs and enterobacterial species.
Assuntos
Proteínas de Bactérias/genética , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/análise , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Plasmídeos/análise , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Reino Unido , beta-Lactamases/análiseRESUMO
We report two cases of infantile diarrhea due to multidrug-resistant, NDM-1 metallo-ß-lactamase-producing Salmonella enterica serovar Agona from Pakistan. This study alerts toward possible risk of NDM-1 transmission to enteric fever pathogens and encourages microbiologists to consider active screening of carbapenem resistance in nontyphoidal Salmonella isolates.
Assuntos
Infecções por Salmonella/microbiologia , Salmonella enterica/enzimologia , beta-Lactamases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Fezes/microbiologia , Humanos , Lactente , Testes de Sensibilidade Microbiana , Paquistão , Infecções por Salmonella/tratamento farmacológico , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genéticaRESUMO
OBJECTIVES: To assess the performance of three commercial molecular assays for detecting major families of carbapenemases in pure bacterial isolates. METHODS: A panel of 450 isolates with previously defined carbapenem resistance mechanisms was tested using the Check-Direct CPE kit, the eazyplex(®) SuperBug complete A kit and the Xpert(®) Carba-R kit. Isolates included 438 Enterobacteriaceae and 12 Pseudomonas spp. comprising 100 isolates each with KPC, NDM, VIM or OXA-48-like enzymes, two isolates producing both an NDM and an OXA-48-like enzyme, 24 IMP producers and 24 isolates without a known carbapenemase gene. Discordant results (commercial versus in-house) were investigated using in-house PCR and amplicons were sequenced to define the carbapenemase allele present. RESULTS: All three commercial assays detected all isolates with KPC, VIM, NDM and classic OXA-48 carbapenemases (no false-negatives). Isolates producing the OXA-181 variant (nâ=â18) were not detected by the Xpert(®) Carba-R kit or the eazyplex(®) SuperBug complete A kit, but were subsequently detected with modified versions of these kits. Only the Xpert(®) Carba-R kit could detect IMP carbapenemases, although this was limited to the IMP-1 subgroup. Invalid or false-positive results were either not observed when following the manufacturer's protocols or were eliminated by making simple interpretative adjustments to allow use with bacterial isolates rather than clinical samples. CONCLUSIONS: Commercial assays offer a reliable means of detecting bacteria with clinically significant carbapenemases. Coverage of some assays required expansion to maximize the sensitivity for OXA-48-like carbapenemases. Choice will ultimately depend on preferred gene coverage, intended throughput, cost and ability to fit into local workflows.
Assuntos
Proteínas de Bactérias/análise , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Pseudomonas/enzimologia , beta-Lactamases/análise , Proteínas de Bactérias/genética , Enterobacteriaceae/genética , Humanos , Pseudomonas/genética , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , beta-Lactamases/genéticaAssuntos
Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae/efeitos dos fármacos , Meropeném/farmacologia , Penicilinas/farmacologia , beta-Lactamases/classificação , Antibacterianos/farmacologia , Biomarcadores/análise , Enterobacteriaceae/genética , Genes MDR , Testes de Sensibilidade Microbiana , Sequenciamento Completo do GenomaRESUMO
OBJECTIVES: To identify the mechanism(s) underlying cefotaxime resistance in 118 of 21,641 (0.55%) non-typhoidal Salmonella enterica isolates collected from humans throughout England and Wales from January 2010 to September 2012. METHODS: Non-duplicate isolates (n = 118) resistant to cefotaxime (MICs >1 mg/L) were screened by PCR for genes encoding CTX-M extended-spectrum ß-lactamases (ESBLs) and associated ISEcp1-like elements, and for genes encoding acquired AmpC, SHV, TEM, VEB, PER and GES ß-lactamases. Sequencing was used to identify specific alleles in selected isolates. Carbapenem resistance was sought by ertapenem disc screening. RESULTS: Seventy-nine isolates (0.37% of all referred S. enterica) produced ESBLs, 37 isolates (0.17%) produced CMY-type AmpC enzymes, and 1 isolate had both enzyme types; the mechanism of cefotaxime resistance in 3 isolates could not be identified. Group 1 CTX-M genes were identified in 57 isolates belonging to 22 serotypes, with CTX-M-1 (n = 11), -15 (n = 9) and -55/57 (n = 8) the most prevalent alleles among the 29 (51%) investigated. CTX-M-2 (n = 5), -14 (n = 5), -8 (n = 1) and -65 (n = 1) were also identified. TEM-52 was identified in two isolates and SHV-12 in seven isolates. There was no evidence of carbapenem resistance. ESBL and AmpC genes were detected in both domestically acquired and travel-associated salmonellae. Eighty-nine isolates (75%) were multidrug resistant (resistant to at least three antimicrobial classes) and 42 (36%) had decreased susceptibility to ciprofloxacin (MICs 0.25-1 mg/L), with a further 13 (11%) isolates resistant (MICs >1 mg/L). CONCLUSIONS: The prevalence of CTX-M and acquired AmpC genes in human non-typhoidal S. enterica from England and Wales is still low, but has increased from 0.03% in 2001-03 to 0.49% in 2010-12. Resistance to third-generation cephalosporins requires monitoring as it may reduce therapeutic options.
Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Salmonelose Animal/microbiologia , Infecções por Salmonella/microbiologia , Salmonella enterica/efeitos dos fármacos , Resistência beta-Lactâmica , Alelos , Animais , Proteínas de Bactérias/genética , Cefotaxima/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Inglaterra/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Prevalência , Infecções por Salmonella/epidemiologia , Salmonelose Animal/epidemiologia , Salmonella enterica/isolamento & purificação , Análise de Sequência de DNA , País de Gales/epidemiologia , beta-Lactamases/genéticaRESUMO
OBJECTIVES: Gram-negative bacteria with diverse carbapenemases, including New Delhi metallo-ß-lactamase (NDM) enzymes, have been increasingly recorded in the UK since 2007. We analysed patient data for NDM-positive isolates confirmed by the national reference laboratory from UK laboratories from February 2008 to July 2013. METHODS: Isolates resistant to carbapenems and with imipenem MICs reduced ≥8-fold by EDTA were tested by PCR for genes encoding acquired class B carbapenemases. MICs were determined by BSAC agar dilution methodology. When requested by the sender, or when they were members of apparent clusters, NDM-positive isolates were typed by variable number tandem repeat (VNTR) analysis or PFGE. Data provided by the sending laboratories were collated and reviewed. RESULTS: From February 2008 to July 2013 the reference laboratory confirmed 326 NDM-positive isolates from 250 patients, submitted by 83 laboratories. Most (85%, 213/250) patients were already hospitalized when the NDM-positive bacteria were detected, were male (61%, 152/250) and were aged >60 years (58%, 145/250). Travel history was available for only 40% of patients, but 52% (53/101) of these had documented healthcare contact within or travel to the Indian subcontinent. Most NDM-positive isolates (94%, 306/326) were Enterobacteriaceae with just 6% (20/326) non-fermenters; the predominant hosts were Klebsiella spp. (55%, 180/326) and Escherichia coli (25%, 80/326). Almost all NDM-positive isolates were resistant to multiple antibiotic classes, but 90% remained susceptible to colistin. CONCLUSIONS: Gram-negative bacteria with NDM carbapenemases are a growing challenge, especially for elderly hospitalized patients, including those with healthcare contact in the Indian subcontinent, and leave few therapeutic options. UK outbreaks remain rare and contained.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Reino UnidoRESUMO
We present a duplex, real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase (blaKPC) and New Delhi metallo-ß-lactamase (blaNDM) genes. Accuracy was assessed with 158 Gram-negative bacillary isolates, including 134 carbapenemase producers. The assay had 100% sensitivity and specificity compared with reference methods and a turnaround time of 90 min.