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1.
PLoS One ; 16(7): e0231119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34319973

RESUMO

Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene blaNDM, spread via plasmids among Enterobacterales in Vietnam. In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148-149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75-76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.


Assuntos
Enterobacteriaceae/enzimologia , Plasmídeos , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Testes de Sensibilidade Microbiana , Vietnã , beta-Lactamases/economia
2.
BMC Biotechnol ; 21(1): 26, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33757473

RESUMO

BACKGROUND: The production of agricultural wastes still growing as a consequence of the population growing. However, the majority of these residues are under-utilized due their chemical composition, which is mainly composed by cellulose. Actually, the search of cellulases with high efficiency to degrade this carbohydrate remains as the challenge. In the present experiment, two genes encoding an endoglucanase (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21) were overexpressed in Escherichia coli and their recombinant enzymes (egl-FZYE and cel-FZYE, respectively) characterized. Those genes were found in Trabulsiella odontermitis which was isolated from the gut of termite Heterotermes sp. Additionally, the capability to release sugars from agricultural wastes was evaluated in both enzymes, alone and in combination. RESULTS: The results have shown that optimal pH was 6.0 and 6.5, reaching an activity of 1051.65 ± 47.78 and 607.80 ± 10.19 U/mg at 39 °C, for egl-FZYE and cel-FZYE, respectively. The Km and Vmax for egl-FZYE using CMC as substrate were 11.25 mg/mL and 3921.57 U/mg, respectively, whereas using Avicel were 15.39 mg/mL and 2314.81 U/mg, respectively. The Km and Vmax for cel-FZYE using Avicel as substrate were 11.49 mg/mL and 2105.26 U/mg, respectively, whereas using CMC the enzyme did not had activity. Both enzymes had effect on agricultural wastes, and their effect was improved when they were combined reaching an activity of 955.1 ± 116.1, 4016.8 ± 332 and 1124.2 ± 241 U/mg on corn stover, sorghum stover and pine sawdust, respectively. CONCLUSIONS: Both enzymes were capable of degrading agricultural wastes, and their effectiveness was improved up to 60% of glucose released when combined. In summary, the results of the study demonstrate that the recombinant enzymes exhibit characteristics that indicate their value as potential feed additives and that the enzymes could be used to enhance the degradation of cellulose in the poor-quality forage generally used in ruminant feedstuffs.


Assuntos
Celulases/química , Enterobacteriaceae/enzimologia , Eliminação de Resíduos/métodos , Resíduos/análise , Agricultura , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Celulases/genética , Celulases/metabolismo , Celulose/metabolismo , Produtos Agrícolas/metabolismo , Produtos Agrícolas/microbiologia , Enterobacteriaceae/química , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Estabilidade Enzimática , Isópteros/microbiologia , Cinética
3.
J Med Microbiol ; 69(2): 228-232, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31922949

RESUMO

Introduction. Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) from surveillance cultures is critical in supporting a good infection control programme. We implemented a new algorithm for CPE detection incorporating the NG Test CARBA 5 in January 2019.Aim. Our goals were to compare turnaround time (TAT), costs and staff requirements between the old and new algorithm, and to evaluate the performance of the CARBA 5 test directly on colonies grown on CARBA Smart agar.Methodology. We analysed and compared the TAT of CPE surveillance cultures processed using the old and new CPE screening algorithm. The total actual reagent costs and staff requirements for the new CPE algorithm were compared with the estimated costs and staff requirements of the old CPE algorithm.Results. Of 197 isolates included in the evaluation of the new algorithm, 64 were positive for carbapenemases by both CARBA 5 and Xpert Carba-R assay. Of the 133 that were negative, two were found to harbour NDM and IMI genotypes. Significant improvements in TAT were achieved with 88.7 % of cultures with CPE, reported on the same day as growth was observed on CARBA Smart agar compared to none in the old algorithm. The new algorithm incurred lower costs and, based on our workload, the new algorithm is estimated to save 28.9 man-hours annually.Conclusion. CARBA 5 performs well on colonies growing on CARBA Smart agar and significant improvements in TAT can be achieved without incurring additional costs or staff requirements.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Laboratório Clínico/métodos , Contagem de Colônia Microbiana/métodos , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/crescimento & desenvolvimento , Enterobacteriaceae/isolamento & purificação , Ensaios Enzimáticos/métodos , Algoritmos , Proteínas de Bactérias/genética , Técnicas de Laboratório Clínico/economia , Contagem de Colônia Microbiana/economia , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/diagnóstico , Ensaios Enzimáticos/economia , Humanos , Sensibilidade e Especificidade , beta-Lactamases/genética , beta-Lactamases/metabolismo
4.
J Hosp Infect ; 104(1): 12-19, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634537

RESUMO

BACKGROUND: There has been an increase in the number of carbapenemase-producing organisms documented across the UK over the past 10 years. From these, the 'big five' carbapenemases (KPC, OXA-48, IMP, VIM, and NDM) are the most common types reported in the order Enterobacterales, identified from a variety of reactive screening, outbreak, inpatient surveillance, and diagnostic samples. AIM: To perform a point prevalence study to determine the inpatient carriage rate of carbapenemase-producing organisms at Barts Health NHS Trust, which encompasses 2.5 million patients across four London boroughs: Tower Hamlets, Newham, Redbridge, and Waltham Forest. METHODS: Rectal swabs were collected from consenting inpatients, alongside details of the ward's medical specialty, patient's country of birth, history of foreign travel, length of hospitalization, and history of prior hospitalization. Swabs were enriched and subcultured on to mSuperCARBA selective medium. All Enterobacterales, Acinetobacter, and Pseudomonas species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy and underwent antibiotic susceptibility testing by disc diffusion, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. All isolates were screened for the 'big five' carbapenemases using a modified version of a published reverse transcriptase-polymerase chain reaction assay. FINDINGS: Of the 977 inpatients tested, 35 CPOs were isolated from 30 patients. NDM was the most frequently detected carbapenemase, followed by OXA-48, with an overall prevalence of 3.1%. Organisms isolated included Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis, and Escherichia coli. Renal and elderly care patients had the highest prevalences of CPOs, whereas the intensive care unit prevalence was low. Statistical analysis found that hospitalization abroad, any previous hospitalization, foreign travel and, specifically, travel to India, Pakistan, and Bangladesh were associated with increased risk of CPO carriage. CONCLUSION: The overall prevalence of CPOs at Barts Health Trust was 3.1%, comprising NDM and OXA-48-type carbapenemases, which is in line with other London-based studies. Renal patients and the elderly had the highest burden of CPOs, whereas previous hospitalization and foreign travel were associated with an increased risk of CPO carriage.


Assuntos
Proteínas de Bactérias/genética , Pacientes Internados/estatística & dados numéricos , Testes de Sensibilidade Microbiana/métodos , beta-Lactamases/genética , Acinetobacter/enzimologia , Acinetobacter/genética , Idoso , Estudos de Casos e Controles , Enterobacter cloacae/isolamento & purificação , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Escherichia coli/isolamento & purificação , Humanos , Klebsiella pneumoniae/isolamento & purificação , Programas de Rastreamento/métodos , Prevalência , Proteus mirabilis/isolamento & purificação , Pseudomonas/enzimologia , Pseudomonas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Medicina Estatal/organização & administração , Reino Unido/epidemiologia
5.
J Clin Microbiol ; 57(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31462547

RESUMO

OXA-181 is the second most common global OXA-48-like carbapenemase and is endemic in the Indian subcontinent. Molecular studies have shown that Enterobacterales with OXA-181 are often introduced into regions of nonendemicity. Distinguishing OXA-181 from other OXA-48-like enzymes often requires sequencing, which is rather expensive and time-consuming. A specific PCR (i.e., OXA181PCR) for the detection of blaOXA-181 was validated using a global collection (n = 315) of bacteria with well-characterized carbapenemases and showed 100% sensitivity and specificity (95% confidence interval [CI], 94.1 to 100 and 98.6 to 100, respectively) for detecting bacteria with OXA-181. The OXA181PCR subsequently gave positive results on 58/160 (36%) Enterobacterales with OXA-48-like carbapenemases from the 2015 INFORM surveillance program. The blaOXA-181-positive Enterobacterales were present in 9 countries spanning 5 continents, illustrating the global distribution of OXA-181. This methodology can easily be incorporated into molecular surveillance programs to provide accurate information about the prevalence of OXA-181. A loop-mediated isothermal amplification (LAMP)-OXA48 assay overall performed well for detecting OXA-48-like enzymes but showed poor specificity due to false-positive results with non-OXA carbapenemases.


Assuntos
Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , beta-Lactamases/genética , Técnicas Bacteriológicas/economia , Análise Custo-Benefício , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/microbiologia , Reações Falso-Positivas , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/economia , Sensibilidade e Especificidade , Temperatura
6.
BMC Infect Dis ; 19(1): 565, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253101

RESUMO

BACKGROUND: To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-ß-lactamases (NDMs) was developed. METHODS: The immunochromatographic assay for New Delhi metallo-ß-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing. RESULTS: Of the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, - 4, - 5 and - 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs. CONCLUSIONS: The immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.


Assuntos
Bactérias Gram-Negativas/isolamento & purificação , Imunoensaio/métodos , beta-Lactamases/imunologia , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/isolamento & purificação , Animais , Antibacterianos/farmacologia , Anticorpos Monoclonais/imunologia , Farmacorresistência Bacteriana , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Humanos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Mianmar , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Ratos , beta-Lactamases/genética , beta-Lactamases/metabolismo
8.
Am J Trop Med Hyg ; 100(6): 1355-1362, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31017082

RESUMO

The diffusion of extended-spectrum beta-lactamase (E-ESBL)-producing Enterobacteriaceae is a major concern worldwide, especially in low-income countries, where they may lead to therapeutic failures. In hospitals, where colonization is the highest, E-ESBL transmission is poorly understood, limiting the possibility of establishing effective control measures. We assessed E-ESBL-acquisition routes in a neonatalogy ward in Madagascar. Individuals from a neonatology ward were longitudinally followed-up (August 2014-March 2015). Newborns' family members' and health-care workers (HCWs) were stool-sampled and tested for E-ESBL colonization weekly. Several hypothetical acquisition routes of newborns-e.g. direct contact with family members and HCWs and indirect contact with other newborns through environmental contamination, colonization pressure, or transient hand carriage-were examined and compared using mathematical modeling and Bayesian inference. In our results, high E-ESBL acquisition rates were found, reaching > 70% for newborns, > 55% for family members, and > 75% for HCWs. Modeling analyses indicated transmission sources for newborn colonization to be species dependent. Health-care workers' route were selected for Klebsiella pneumoniae and Escherichia coli, with respective estimated transmission strengths of 0.05 (0.008; 0.14) and 0.008 (0.001; 0.021) ind-1 day-1. Indirect transmissions associated with ward prevalence, e.g. through hand carriage or environment, were selected for Enterobacter cloacae, E. coli, and K. pneumoniae (range 0.27-0.41 ind-1 day-1). Importantly, family members were not identified as transmission source. To conclude, E-ESBL acquisition sources are strongly species dependent. Escherichia coli and E. cloacae involve more indirect contamination, whereas K. pneumoniae also spreads through contact with colonized HCWs. These findings should help improve control measures to reduce in-hospital transmission.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/transmissão , Enterobacteriaceae/efeitos dos fármacos , beta-Lactamases/metabolismo , Portador Sadio , Enterobacteriaceae/enzimologia , Infecções por Enterobacteriaceae/epidemiologia , Pessoal de Saúde , Humanos , Recém-Nascido , Madagáscar/epidemiologia , Modelos Biológicos , Método de Monte Carlo , Berçários Hospitalares , Pais , beta-Lactamases/genética
9.
Euro Surveill ; 24(9)2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30862330

RESUMO

A survey on the epidemiological situation, surveillance and containment activities for carbapenemase-producing Enterobacteriaceae (CPE) was conducted in European countries in 2018. All 37 participating countries reported CPE cases. Since 2015, the epidemiological stage of CPE expansion has increased in 11 countries. Reference laboratory capability, dedicated surveillance and a specific national containment plan are in existence in 33, 27 and 14 countries, respectively. Enhanced control efforts are needed for CPE containment in Europe.


Assuntos
Proteínas de Bactérias/metabolismo , Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Enterobacteriaceae/epidemiologia , Enterobacteriaceae/enzimologia , Controle de Infecções , beta-Lactamases/metabolismo , Notificação de Doenças , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Europa (Continente)/epidemiologia , Inquéritos Epidemiológicos , Humanos , Testes de Sensibilidade Microbiana , Vigilância da População , Prevalência , Reação em Cadeia da Polimerase em Tempo Real
10.
Clin Microbiol Infect ; 25(10): 1286.e9-1286.e15, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30898725

RESUMO

OBJECTIVES: The aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories. METHODS: The immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric ß-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2). RESULTS: The overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6-89.2%)/100% (CI 91.8-100%) for RESIST-4, 88.2% (CI 82.1-92.4%)/100% (CI 91.8-100%) for CARBA-5, 88.2% (CI 82.1-92.4%)/100% (CI 91.8-100%) for Xpert Carba-R, 73.7% (CI 66.2-80.0%)/100% (CI 93.4-99.0%) for ß-CARBA, and 97.4% (CI 87.9-99.6%)/97.7% (CI 87.9-99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for ß-CARBA (76.6% (CI 68.4-83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3-98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed. CONCLUSIONS: Except for ß-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.


Assuntos
Algoritmos , Proteínas de Bactérias/análise , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimologia , beta-Lactamases/análise , Técnicas Bacteriológicas/economia , Custos e Análise de Custo , Humanos , Sensibilidade e Especificidade
11.
Jpn J Infect Dis ; 72(2): 124-126, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30381688

RESUMO

We hypothesized that quick Sequential Organ Failure Assessment (qSOFA) would be associated with 30-day mortality in bacteremia caused by extended-spectrum ß-lactamase (ESBL)-producing bacteria and might be a selection criterion for the use of carbapenem as initial empirical therapy. A multicenter retrospective study was conducted in six hospitals. All patients who had bacteremia due to ESBL-producing bacteria were included in the study. Multivariable logistic regression analysis was performed to analyze 30-day mortality as the main outcome. A total of 203 adult patients were identified with bacteremia caused by ESBL-producing Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis. In multivariate logistic regression analysis, bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis (odds ratio [OR] 5.07, 95% confidence interval [CI] 1.64-15.56), underlying liver disease (OR 3.38, 95% CI 1.09-10.00), and underlying solid cancer (OR 3.45, 95% CI 1.27-9.69) were associated with 30-day mortality. In a subgroup analysis, empirical non-carbapenem therapy was associated with 30-day mortality in bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis. Our results suggest that the qSOFA score is not a selection criterion for the use of carbapenem in initial empirical therapy.


Assuntos
Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/patologia , Carbapenêmicos/uso terapêutico , Enterobacteriaceae/enzimologia , Escores de Disfunção Orgânica , beta-Lactamases/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/microbiologia , Bacteriemia/mortalidade , Estudos de Casos e Controles , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/mortalidade , Infecções por Enterobacteriaceae/patologia , Feminino , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Análise de Sobrevida , Resultado do Tratamento , Adulto Jovem
12.
J Hosp Infect ; 102(1): 31-36, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30557588

RESUMO

BACKGROUND: The spread of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) in healthcare environments has become a major public health threat in recent years. AIM: To assess how healthcare workers (HCWs) manage excreta and the possible association with the incidence of ESBL-PE. METHODS: Eight hundred HCWs and 74 nurse-supervisors were questioned through two self-report questionnaires in order to assess their knowledge and practices, and to determine the equipment utilized for excreta management in 74 healthcare departments. Performance on equipment utilized, knowledge and practices were scored as good (score of 1), intermediate (score of 2) or poor (score of 3) on the basis of pre-established thresholds. Linear regression was performed to evaluate the association between HCWs' knowledge/practices and the incidence of ESBL-PE. FINDINGS: Six hundred and eighty-eight HCWs (86%) and all nurse-supervisors participated in the survey. The proportions of respondents scoring 1, 2 and 3 were: 14.8%, 71.6% and 17.6% for equipment; 30.1%, 40.6 % and 29.3% for knowledge; and 2.0%, 71.9% and 26.1% for practices, respectively. The single regression mathematic model highlighted that poor practices (score of 3) among HCWs was significantly associated with increased incidence of ESBL-PE (P = 0.002). CONCLUSIONS: A positive correlation was found between HCWs' practices for managing excreta and the incidence of ESBL-PE, especially in surgical units. There is an urgent need for development of public health efforts to enhance knowledge and practices of HCWs to better control the spread of multi-drug-resistant bacteria, and these should be integrated within infection control programmes.


Assuntos
Transmissão de Doença Infecciosa/prevenção & controle , Infecções por Enterobacteriaceae/prevenção & controle , Enterobacteriaceae/enzimologia , Conhecimentos, Atitudes e Prática em Saúde , Pessoal de Saúde/psicologia , Controle de Infecções/métodos , beta-Lactamases/metabolismo , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Pesquisa sobre Serviços de Saúde , Humanos , Modelos Estatísticos , Inquéritos e Questionários
13.
J Microbiol Methods ; 147: 26-35, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29486226

RESUMO

A new phenotypic method for detecting carbapenemases has been adapted (assembling of two MAST® kits, including one that contains faropenem to which a temocillin disk has been added) then assessed using 101 bacterial strains (Enterobacteriaceae with assays on Pseudomonas aeruginosa and Acinetobacter baumannii) including 62 which produce genetically identified carbapenemases. Concerning Carbapenemase-Producing Enterobacteriaceae (CPE), there is 100% sensitivity for Klebsiella pneumoniae carbapenemase (KPC, Ambler class A) and OXA-48 (Ambler class D), and 91% for metallo-beta-lactamase (MBL, Ambler class B), with a 97% sensitivity for all carbapenemases, with a specificity of 100%. The test is also efficient for detecting Pseudomonas aeruginosa carbapenemases (sensitivity between 82 and 100% and 100% specificity). The major innovation is the combined use of faropenem and temocillin for reliable detection (excellent performance with 100% sensitivity and specificity) of OXA-48. This study has led to the development of a new algorithm to detect the different classes of carbapenemases, for first-line diagnosis, by combining this modified MAST® test with immunochromatographic methods and molecular biology techniques.


Assuntos
Acinetobacter baumannii/enzimologia , Algoritmos , Proteínas de Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/enzimologia , Kit de Reagentes para Diagnóstico , beta-Lactamases/isolamento & purificação , Proteínas de Bactérias/classificação , Biologia Molecular/métodos , Penicilinas/farmacologia , Fenótipo , Sensibilidade e Especificidade , beta-Lactamases/classificação , beta-Lactamas/farmacologia
15.
Ann Clin Microbiol Antimicrob ; 16(1): 48, 2017 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-28693493

RESUMO

BACKGROUND: Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). METHODS: 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. RESULTS: Among the carbapenem-resistant isolates the genes bla KPC, bla VIM, bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC, bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. CONCLUSION: The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.


Assuntos
Proteínas de Bactérias/genética , Enterobacteriaceae/enzimologia , Bactérias Gram-Negativas/enzimologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real/economia , beta-Lactamases/metabolismo
16.
Rev Soc Bras Med Trop ; 50(2): 173-178, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28562752

RESUMO

INTRODUCTION:: In this study, we used phenotypic methods to screen carbapenem-resistant Enterobacteriaceae (CREs) and evaluated their antimicrobial sensitivity profile. METHODS:: One hundred and seventy-eight CREs were isolated at a university hospital in south Brazil in a one-year period. Samples were assessed using disk diffusion tests with inhibitors of ß-lactamases such as phenylboronic acid (AFB), cloxacillin (CLOXA), and ethylenediaminetetraacetic acid (EDTA). Strains with differences in zone diameters ≥ 5mm for disks supplemented or not were considered producers of carbapenemases. RESULTS:: Klebsiella pneumoniae was the most prevalent CRE, which appeared in 80.3% cases (n = 143). Among clinical materials, the rectal swab was responsible for 43.4% of the isolations (n = 62), followed by urine (18.9%; n = 27). Among the CREs identified in this study, the growth of 56.7% (n = 101) isolates, which were putative producers of Klebsiella pneumoniae carbapenemase (KPC), were inhibited by AFB, whereas 7.3% (n = 13) isolates were inhibited by both AFB and CLOXA and were considered as putative producers of plasmid-mediated AmpC; approximately 3.4% (n = 6) were inhibited by EDTA, which possibly produced metallo-ß-lactamase. Lastly, 32.6% (n = 58) cases showed negative results for AFB, CLOXA, and EDTA sensitivity, and represented another class of ß-lactamases and/or mechanism of resistance. CONCLUSIONS:: Phenotypic screening of CREs is important for clinical laboratories that monitor outbreaks of resistant microbes. Phenotypic tests that use carbapenemase inhibitors and enhancers such as AFB, CLOXA, and EDTA are necessary since they are good screening methods for the detection of carbapenemases.


Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Resistência beta-Lactâmica/genética , Enterobacteriaceae/classificação , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Hospitais Universitários , Humanos , Testes de Sensibilidade Microbiana , Fenótipo
17.
J Med Microbiol ; 66(7): 884-887, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28639543

RESUMO

PURPOSE: Rapid and inexpensive tests for detecting extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae are needed, particularly in low-resource countries where infections with these bacteria constitute a major public health issue. The recently described ESBL NDP test performed well in developed countries. This study was designed to assess performance, cost and feasibility of this test in positive blood cultures, in Cotonou, Benin (West Africa). METHODOLOGY: The test was performed on 175 positive Bactec broth blood cultures containing Enterobacteriaceae, and blindly compared with the double-disc synergy test (DDST) for the phenotypic detection of ESBL producers. RESULTS: There was a complete agreement between the ESBL NDP test and the DDST. On average, the time to give results was 37 min for a sample and the cost was US$ 7.3. CONCLUSION: The ESBL NDP test is rapid, relatively affordable and performed well in our setting.


Assuntos
Bacteriemia/diagnóstico , Hemocultura , Testes Diagnósticos de Rotina/métodos , Infecções por Enterobacteriaceae/diagnóstico , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , beta-Lactamases/análise , Adolescente , Bacteriemia/microbiologia , Benin , Criança , Pré-Escolar , Custos e Análise de Custo , Testes Diagnósticos de Rotina/economia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Fatores de Tempo
18.
J Antimicrob Chemother ; 72(9): 2519-2527, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28535303

RESUMO

Objectives: To develop a simple gold nanoparticle (AuNP)-based colorimetric test, GoldNano Carb (GoldC), for detecting carbapenemase production in Gram-negative bacteria, compared with updated Carba NP (CNP) and CarbAcineto NP (CAcNP) tests by using PCR methods as gold standard. Methods: Ninety-nine carbapenemase-producing Enterobacteriaceae (CPE), Pseudomonas spp. and Acinetobacter spp. isolates and 89 non-CPE isolates were tested by the GoldC and CNP. Additionally, the CAcNP was performed in the Acinetobacter spp. isolates. The final imipenem (imipenem/cilastatin form) concentration was 5 mg/mL for all three tests. For the GoldC, the imipenem powder was added directly to bacterial cell suspension in distilled water prior to detection of acid product by the citrate-capped AuNP solution. An AuNP change from red to purple, blue or green indicates carbapenemase activity. Results: The GoldC detected all carbapenemase producers except one OXA-23-like producer (99.0% sensitivity), whereas 11 carbapenemase producers (10 Acinetobacter and 1 P. aeruginosa) were CNP negative (88.9% sensitivity). However, the GoldC and CNP provided 100% and 98.6% sensitivity, respectively, for the CPE and Pseudomonas spp. Both tests gave one false positive from CTX-M-1-like-producing Enterobacter spp. (98.9% specificity). The GoldC and CAcNP detected 96.7% and 93.3% of the Acinetobacter spp. isolates, respectively. Interestingly, times to positivity by the GoldC were markedly shorter than those by the CNP (76.8% versus 36.2% positive at 5 min) and CAcNP (43.3% at 5 min versus 20% within 30 min). Conclusions: The GoldC is fast, easy, highly sensitive and inexpensive (∼$0.25 per test), suggesting that it may be suitable for routine carbapenemase detection in low-resource settings for infection control or epidemiological purposes.


Assuntos
Acinetobacter/enzimologia , Proteínas de Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimologia , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/isolamento & purificação , Acinetobacter/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Técnicas Bacteriológicas/economia , Colorimetria/métodos , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Ouro , Humanos , Imipenem/farmacologia , Nanopartículas Metálicas , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Sensibilidade e Especificidade , beta-Lactamases/biossíntese
19.
J Clin Microbiol ; 55(6): 1954-1960, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28404676

RESUMO

Eighty Gram-negative bacilli (54 Enterobacteriaceae and 26 nonfermenting Gram-negative bacilli) obtained from multiple institutions in the United States were distributed in a blinded manner to seven testing laboratories to compare their performance of a test for detection of carbapenemase production, the Carba NP test. The Carba NP test was performed by all laboratories, following the Clinical and Laboratory Standards Institute (CLSI) procedure. Site-versus-site comparisons demonstrated a high level of consistency for the Carba NP assay, with just 3/21 site comparisons yielding a difference in sensitivity (P < 0.05). Previously described limitations with blaOXA-48-like carbapenemases and blaOXA carbapenemases associated with Acinetobacter baumannii were noted. Based on these data, we demonstrate that the Carba NP test, when implemented with the standardized CLSI methodology, provides reproducible results across multiple sites for detection of carbapenemases.


Assuntos
Proteínas de Bactérias/análise , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimologia , beta-Lactamases/análise , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados Unidos
20.
BMC Microbiol ; 17(1): 54, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28274205

RESUMO

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE), particularly carbapenemase-producing Klebsiella pneumoniae isolates, are important causative agents of nosocomial infections associated with significant mortality rates mostly in critical wards. The rapid detection and typing of these strains is critical either for surveillance purposes and to prevent outbreaks and optimize antibiotic therapy. In this study, the MALDI-TOF MS method was used to detect rapidly these isolates from blood cultures (BCs) and to obtain proteomic profiles enable to discriminate between carbapenemase-producing and non-carbapenemase-producing strains. RESULTS: Fifty-five K. pneumoniae strains were tested. Identification and carbapenemase-production detection assay using Ertapenem were performed both from bacterial pellets extracted directly from BCs flasks and from subcultures of these strains. For all isolates, a complete antimicrobial susceptibility testing and a genotypic characterization were performed. We found 100% agreement between the carbapenemase-producing profile generated by MALDI TOF MS and that obtained using conventional methods. The assay detected and discriminated different carbapenemase-producing K. pneumoniae isolates within 30 min to 3 h after incubation with Ertapenem. CONCLUSIONS: MALDI-TOF MS is a promising, rapid and economical method for the detection of carbapenemase-producing K. pneumoniae strains that could be successfully introduced into the routine diagnostic workflow of clinical microbiology laboratories.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Hemocultura/métodos , Klebsiella pneumoniae/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , beta-Lactamases/isolamento & purificação , Proteínas de Bactérias/análise , Técnicas de Cultura de Células , Infecção Hospitalar , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Ertapenem , Humanos , Itália , Infecções por Klebsiella/diagnóstico , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Laboratórios , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Fatores de Tempo , beta-Lactamases/análise , beta-Lactamas/farmacologia
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