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1.
J Clin Microbiol ; 58(5)2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32102855

RESUMO

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-ß-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

2.
Nat Commun ; 11(1): 466, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980604

RESUMO

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Surtos de Doenças , Enterobacter/enzimologia , Enterobacter/genética , Infecções por Enterobacteriaceae/epidemiologia , beta-Lactamases/biossíntese , beta-Lactamases/genética , Queimaduras/microbiologia , Enterobacteriáceas Resistentes a Carbapenêmicos/patogenicidade , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Enterobacter/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Genoma Bacteriano , Humanos , Queensland/epidemiologia , Fatores R/genética , Engenharia Sanitária , Centros de Atenção Terciária , Sequenciamento Completo do Genoma/métodos , Resistência beta-Lactâmica/genética
3.
Microb Genom ; 6(1)2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31860437

RESUMO

Carbapenemase-producing Enterobacteriaceae (CPE) are an increasingly common cause of healthcare-associated infections and may occasionally be identified in patients without extensive healthcare exposure. bla IMP-4 is the most frequently detected carbapenemase gene in Enterobacteriaceae within Australia, but little is known about the mechanisms behind its persistence. Here we used whole genome sequencing (WGS) to investigate the molecular epidemiology of bla IMP-4 in Queensland, Australia. In total, 107 CPE were collected between 2014 and 2017 and sent for WGS on an Illumina NextSeq500. Resistance genes and plasmid types were detected using a combination of read mapping and nucleotide comparison of de novo assemblies. Six isolates were additionally sequenced using Oxford Nanopore MinION to generate long-reads and fully characterize the context of the bla IMP-4 gene. Of 107 CPE, 93 carried the bla IMP-4 gene; 74/107 also carried an IncHI2 plasmid, suggesting carriage of the bla IMP-4 gene on an IncHI2 plasmid. Comparison of these isolates to a previously characterized IncHI2 plasmid pMS7884A (isolated from an Enterobacter hormaechei strain in Brisbane) suggested that all isolates carried a similar plasmid. Five of six representative isolates sequenced using Nanopore long-read technology carried IncHI2 plasmids harbouring the bla IMP-4 gene. While the vast majority of isolates represented E. hormaechei, several other species were also found to carry the IncHI2 plasmid, including Klebsiella species, Escherichia coli and Citrobacter species. Several clonal groups of E. hormaechei were also identified, suggesting that persistence of bla IMP-4 is driven by both presence on a common plasmid and clonal spread of certain E. hormaechei lineages.

4.
Microb Genom ; 5(10)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31617838

RESUMO

Stenotrophomonas maltophilia is emerging as an important cause of disease in nosocomial and community-acquired settings, including bloodstream, wound and catheter-associated infections. Cystic fibrosis (CF) airways also provide optimal growth conditions for various opportunistic pathogens with high antibiotic tolerance, including S. maltophilia. Currently, there is no rapid, cost-effective and accurate molecular method for detecting this potentially life-threatening pathogen, particularly in polymicrobial specimens, suggesting that its true prevalence is underestimated. Here, we used large-scale comparative genomics to identify a specific genetic target for S. maltophilia, with subsequent development and validation of a real-time PCR assay for its detection. Analysis of 167 Stenotrophomonas spp. genomes identified a conserved 4 kb region in S. maltophilia, which was targeted for Black Hole Quencher assay design. Our assay yielded the positive detection of 89 of 89 (100%) clinical S. maltophilia strains, and no amplification of 23 non-S. maltophilia clinical isolates. S. maltophilia was detected in 10 of 16 CF sputa, demonstrating the assay's utility for direct detection in respiratory specimens. The assay demonstrated good sensitivity, with limits of detection and quantitation on pure culture of ~10 and ~100 genome equivalents, respectively. Our assay provides a highly specific, sensitive and cost-effective method for the accurate identification of S. maltophilia, and will improve the diagnosis and treatment of this under-recognized pathogen by enabling its accurate and rapid detection from polymicrobial clinical and environmental samples.


Assuntos
Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Stenotrophomonas maltophilia , Humanos , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/isolamento & purificação
5.
BMC Infect Dis ; 19(1): 571, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266450

RESUMO

BACKGROUND: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. METHODS: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. RESULTS: The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. CONCLUSIONS: The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay's suitability for direct screening of clinical specimens.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos , Bactérias/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Sensibilidade e Especificidade
6.
Emerg Infect Dis ; 25(1): 153-156, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30561297

RESUMO

We report 3 cases of koala bite wound infection with Lonepinella koalarum-like bacteria requiring antimicrobial and surgical management. The pathogens could not be identified by standard tests. Phylogenetic analysis of 16S rRNA and housekeeping genes identified the genus. Clinicians should isolate bacteria and determine antimicrobial susceptibilities when managing these infections.


Assuntos
Infecções por Pasteurellaceae/diagnóstico , Pasteurellaceae/isolamento & purificação , Phascolarctidae/microbiologia , Infecção dos Ferimentos/diagnóstico , Idoso , Animais , Mordeduras e Picadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Pasteurellaceae/microbiologia , Queensland , Infecção dos Ferimentos/microbiologia
7.
Am J Infect Control ; 45(9): 954-958, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28757084

RESUMO

BACKGROUND: We report an outbreak of Burkholderia cenocepacia bacteremia and infection in 11 patients predominately in intensive care units caused by contaminated ultrasound gel used in central line insertion and sterile procedures within 4 hospitals across Australia. METHODS: Burkholderia cenocepacia was first identified in the blood culture of a patient from the intensive care unit at the Gold Coast University Hospital on March 26, 2017, with 3 subsequent cases identified by April 7, 2017. The outbreak response team commenced investigative measures. RESULTS: The outbreak investigation identified the point source as contaminated gel packaged in sachets for use within the sterile ultrasound probe cover. In total, 11 patient isolates of B cenocepacia with the same multilocus sequence type were identified within 4 hospitals across Australia. This typing was the same as identified in the contaminated gel isolate with single nucleotide polymorphism-based typing, demonstrating that all linked isolates clustered together. CONCLUSION: Arresting the national point-source outbreak within multiple jurisdictions was critically reliant on a rapid, integrated, and coordinated response and the use of informal professional networks to first identify it. All institutions where the product is used should look back at Burkholderia sp blood culture isolates for speciation to ensure this outbreak is no larger than currently recognized given likely global distribution.


Assuntos
Bacteriemia/transmissão , Infecções por Burkholderia/transmissão , Burkholderia cenocepacia/isolamento & purificação , DNA Bacteriano/genética , Surtos de Doenças , Contaminação de Medicamentos , Adulto , Austrália/epidemiologia , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Bacteriemia/prevenção & controle , Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/microbiologia , Infecções por Burkholderia/prevenção & controle , Burkholderia cenocepacia/classificação , Burkholderia cenocepacia/genética , Cateterismo Periférico , Notificação de Doenças , Feminino , Géis , Hospitais Universitários , Humanos , Unidades de Terapia Intensiva , Masculino , Tipagem de Sequências Multilocus , Ultrassonografia/instrumentação
8.
Diagn Microbiol Infect Dis ; 85(1): 98-101, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26971634

RESUMO

Infection caused by Aeromonas spp. ranges from superficial wound infection to life-threatening septicemia. Carbapenem resistance due to metallo-beta-lactamase, CphA encoded by the cphA gene, is a significant problem. This study defines Aeromonas spp. causing clinical disease in Queensland, Australia. Phenotypic tests for carbapenemase detection were assessed. One hundred Aeromonas isolates from blood (22), wound (46), sterile sites (11), stool (18), eye (2), and sputum (1) were characterized by rpoB and gyrB sequencing. Meropenem susceptibility by VITEK2, disk diffusion, and E-test MIC were determined. Carbapenemase production was assessed by Carba NP test and cphA by PCR. Gene sequencing identified isolates as Aeromonas dhakensis (39), Aeromonas veronii (21), Aeromonas hydrophila (20), Aeromonas caviae (14), Aeromonas jandaei (4), Aeromonas bestiarum (1), and Aeromonas sanarellii (1). Disk diffusion and E-test failed to detect resistance in isolates with presence of cphA. Carba NP was performed with 97.4% sensitivity and 95.7% specificity. Carbapenem resistance gene cphA was detected in A. veronii (21; 100%), A. hydrophila (18; 90%), A. dhakensis (34; 87.2%), A. jandaei (3; 75%), and A. bestiarum (1; 100%) but not A. caviae. We found that A. dhakensis was the predominant species, a previously unrecognized pathogen in this region.


Assuntos
Aeromonas/efeitos dos fármacos , Aeromonas/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Genótipo , Testes de Sensibilidade Microbiana , Resistência beta-Lactâmica , beta-Lactamases/genética , Aeromonas/classificação , Aeromonas/isolamento & purificação , Austrália , Técnicas de Tipagem Bacteriana , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Sensibilidade e Especificidade
9.
J Infect ; 67(5): 439-47, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23872210

RESUMO

OBJECTIVE: To describe the changing prevalence of healthcare- and community-associated MRSA. METHODS: Susceptibility phenotypes of MRSA were observed from 2000 to 2012 using routine susceptibility data. Phenotypic definitions of major clones were validated by genotyping isolates from a nested period prevalence survey in 2011. RESULTS: The predominant healthcare-associated (AUS-2/3 like) MRSA phenotype decreased from 42 to 14 isolates per million occasions of service in outpatients (P < 0.0001) and from 650 to 75 isolates per million accrued patient days in inpatients (P 0.0005), while the respective rates of the healthcare-related EMRSA-15 like phenotype increased from 1 to 19 in outpatients (P < 0.0001) and from 11 to 83 in inpatients (P < 0.0001) and those of the community-associated MRSA phenotype increased from 17 to 296 in outpatients (P < 0.0001) and from 71 to 486 in inpatients (P < 0.0001). When compared with single nucleotide polymorphism genotyping the AUS-2/3 like phenotype had a sensitivity and positive predictive value (PPV) for CC239 of 1 and 0.791 respectively, while the EMRSA-15 like phenotype had a sensitivity and PPV for CC22 of 0.903 and 0.774. PVL-positive CA-MRSA, predominantly ST93 and CC30, accounted for 60.8% of MRSA, while PVL-negative CA-MRSA, mainly CC5 and CC1, accounted for 21.4%. CONCLUSIONS: The initially dominant healthcare-associated MRSA clone has been progressively replaced, mainly by four community-associated lineages.


Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Distribuição de Qui-Quadrado , Infecções Comunitárias Adquiridas/epidemiologia , Infecção Hospitalar/epidemiologia , Genótipo , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Tipagem Molecular , Estudos Prospectivos , Queensland/epidemiologia , Reprodutibilidade dos Testes , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação
10.
Pathology ; 45(5): 492-4, 2013 08.
Artigo em Inglês | MEDLINE | ID: mdl-23856840

RESUMO

AIMS: To investigate the evolutionary origins of Australian healthcare-associated (HCA) methicillin-resistant Staphylococcus aureus (MRSA) strains from a panel of historical isolates typed using current genotyping techniques. METHODS: Nineteen MRSA isolates from 1965 to 1981 were examined and antibiotic susceptibility profiles determined. Genetic characterisation included real-time (RT) polymerase chain reaction (PCR) assays to identify single nucleotide polymorhpism (SNP) clonal complexes (SNP CC) and sequence type (SNP ST), multi locus sequence typing (MLST) and staphylococcal chromosomal cassette mec typing. RESULTS: All SNP CC30 isolates belonged to a novel sequence type, ST2249. All SNP CC239 isolates were confirmed as ST239-MRSA-III, except for a new single locus variant of ST239, ST2275. A further new type, ST2276, was identified. CONCLUSIONS: The earliest MRSA examined from 1965 was confirmed as ST250-MRSA-I, consistent with archaic European types. Identification of ST1-MRSA-IV in 1981 is the earliest appearance of this clinically important lineage which manifested in Australia and the United States in the 1990s. A previously unknown multi-resistant clone, ST2249-MRSA-III, was identified from 1973. Gentamicin resistance first appeared in this novel strain from 1976 and not ST239 as previously suspected. Thus, ST2249 was present in the earliest phase of the HCA MRSA epidemic in eastern Australia and was perhaps related to the emergence of the globally epidemic strain ST239.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Genótipo , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/microbiologia , Antibacterianos/uso terapêutico , Austrália/epidemiologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Gentamicinas/uso terapêutico , Humanos , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia
12.
Res Microbiol ; 159(3): 194-9, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18356026

RESUMO

The Burkholderia cepacia complex (Bcc) is a group of significant opportunistic respiratory pathogens which affect people with cystic fibrosis. In this study, we sought to ascertain the epidemiology and geographic species distribution of 116 Bcc isolates collected from people with CF in Australia and New Zealand. We performed a combination of recA-based PCR, amplified rDNA restriction analysis (ARDRA), pulsed-field gel electrophoresis and repetitive extragenic palindromic PCR on each isolate. Each Burkholderia cenocepacia isolate was also screened by PCR for the presence of the B. cepacia epidemic strain marker. One hundred and fourteen isolates were assigned to a species using recA-based PCR and ARDRA. B. cenocepacia, B. multivorans and B. cepacia accounted for 45.7%, 29.3% and 11.2% of the isolates, respectively. Strain analysis of B. cenocepacia revealed that 85.3% of the isolates were unrelated. One related B. cenocepacia strain was identified amongst 15 people. Whilst full details of person-to-person contact was not available, all patients attended CF centres in Queensland (Qld) and New South Wales (NSW). Although person-to-person transmission of B. cenocepacia strains has occurred in Australia, the majority of CF-related Bcc infections in Australia and New Zealand are most likely acquired from the environment.


Assuntos
Infecções por Burkholderia/epidemiologia , Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/isolamento & purificação , Fibrose Cística/microbiologia , Reação em Cadeia da Polimerase/métodos , Austrália/epidemiologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Infecções por Burkholderia/transmissão , Complexo Burkholderia cepacia/classificação , Complexo Burkholderia cepacia/genética , DNA Bacteriano/genética , DNA Ribossômico/genética , Eletroforese em Gel de Campo Pulsado , Humanos , Nova Zelândia/epidemiologia , Polimorfismo de Fragmento de Restrição , Recombinases Rec A/genética
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