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1.
Ann Agric Environ Med ; 26(3): 405-408, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559794

RESUMO

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae have spread rapidly through the countries and continents to become a global concern. One of the main reservoirs of NDM-1 positive strains from the Enterobacteriaceae family is the Indian subcontinent (Bangladesh, Pakistan, India). MATERIAL AND METHODS: During June 2017 - June 2018, rectal swab samples were collected routinely in all patients returning to Poland from South and South-East Asia. During molecular examinations gene blaNDM-1 encoding NDM-1 carbapenemase was detected. RESULTS: 31 patients were examined after returning to Poland from a trip to South and South-East Asia. The presence of New Delhi Metallo-ß-lactamase-1 producing Escherichia coli and Klebsiella pneumoniae was confirmed in three patients (9.7%) returning to Poland from travels to India. All the positive patients were hospitalized during the trip in a New Delhi hospital. CONCLUSIONS: Digestive tract carriage of NDM in a group of Polish travelers is a significant health and epidemiological problem. The study confirms the necessity for screening for carbapenemase-producing Enterobacteriaceae (CPE), particularly among travellers. Rectal swabs should be collected in every case of patients returning from international trips, and the possibility of environment-associated infections should be emphasized.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/enzimologia , Klebsiella pneumoniae/enzimologia , Viagem , beta-Lactamases/metabolismo , Adulto , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Humanos , Índia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Polônia , beta-Lactamases/genética
2.
Klin Lab Diagn ; 64(8): 497-502, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31479607

RESUMO

The growing prevalence of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa in nosocomial pathogen populations has been attributed to their clonal spread, and/or horizontal transfer of MBL determinants in mobile genetic elements, including integrons. To characterize the genetic background of the beta-lactamase VIM-2 encoding gene in the population of carbapenemresistant (Carba-R) P. aeruginosa clinical isolates.The detection of class 1 integrons was performed by PCR. Typing of the class 1 integrons containing the blaVIM gene cassette was performed by the PCR-restriction fragment length polymorphism (RFLP) approach followed by sequencing of variable regions of class 1 integrons. Five types of the blaVIM-2-carrying integrons were identified: ST654-isolates accounting for more than 50% of the Carba-R population harbored In56; ST235-isolates contained In559 (26% Carba-R isolates); ST111-isolates (19% Carba-R isolates) were characterized by carrying In59-like integron; two ST235-isolates harbored In59 and In249 each. Except In56, carrying the only blaVIM-2-gene cassette, all other identified integron types harbored the genes of resistance to trimethoprim and/or aminoglycosides. No new types of integrons were identified in the P. aeruginosa clinical isolates. The observed correlation of the integron type with specific STs indicates a clonal dissemination of significant resistance determinant producers - ST111, ST654 and ST235 epidemic lines. The features of the integron variable regions can be used for the epidemiological characterization of clinical P. aeruginosa isolates.


Assuntos
Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana , Integrons , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos
3.
Phys Chem Chem Phys ; 21(32): 17821-17835, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31373340

RESUMO

The rise of New Delhi metallo-beta-lactamase-1 (NDM-1) producers is a major public health concern due to carbapenem resistance. Infections caused by carbapenem-resistant enterobacteria (CRE) are classified as a serious problem. To understand the structure and function of NDM-1, an amino acid replacement approach is considered as one of the methods to get structural insight. Therefore, we have generated novel mutations (N193A, S217A, G219A and T262A) near active sites and an omega-like loop to study the role of conserved residues of NDM-1. The minimum inhibitory concentrations (MICs) of ampicillin, imipenem, meropenem, cefotaxime, cefoxitin and ceftazidime for all mutants were found to be reduced 2 to 6 fold, compared to a wild type NDM-1 producing strain. The Km values increased while Kcat and Kcat/Km values were decreased compared to wild type. The affinity as well as the catalysis properties of these mutants were reduced considerably for imipenem, meropenem, cefotaxime, cefoxitin, and ceftazidimem compared to wild type, hence the catalytic efficiencies (Kcat/Km) of all mutant enzymes were reduced owing to the poor affinity of the enzyme. The IC50 values of these mutants with respect to each drug were reduced compared to wild type NDM-1. MD simulations and docking results from the mutant protein models, along with the wild type example, showed stable and consistent RMSD, RMSF and Rg behavior. The α-helix content values of all mutant proteins were reduced by 13%, 6%, 14% and 9% compared to NDM-1. Hence, this study revealed the impact role of active sites near residues on the enzyme catalytic activity of NDM-1.


Assuntos
Antibacterianos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , beta-Lactamases/química , Antibacterianos/farmacologia , Biocatálise , Domínio Catalítico , Farmacorresistência Bacteriana , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica , beta-Lactamases/genética , beta-Lactamases/metabolismo
4.
Epidemiol Mikrobiol Imunol ; 68(2): 99-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398983

RESUMO

The increasing incidence of multiresistant bacterial strains is currently a serious health concern. These pathogens are often the cause of nosocomial infections with limited treatment options and high fatality rates. A case report is presented of an uncommon detection of four different species (Citrobacter freundii, Klebsiella pneumoniae, Escherichia coli, and Morganella morganii) producing the same type of carbapenemase, KPC-2, in a female patient during her complicated long-term hospital stay. Resistance was probably spread to other species by horizontal transmission of plasmids carrying the blaKPC-2 genes. The implementation of strict anti-epidemic measures prevented further spread of these carbapenem-resistant bacteria.


Assuntos
Antibacterianos , Bactérias , Infecções Bacterianas , Infecção Hospitalar , beta-Lactamases , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/genética , Coinfecção/microbiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana/genética , Feminino , Transferência Genética Horizontal , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética
5.
BMC Infect Dis ; 19(1): 678, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370804

RESUMO

BACKGROUND: Fecal colonization with carbapenem-resistant Enterobacteriaceae (CRE) is a risk factor for bacterial translocation resulting in subsequent endogenous infections. The purpose of this study is to investigate the prevalence of CRE strains colonization in stool samples of outpatient in a tertiary pediatric hospital of Shanghai, China. METHODS: In a retrospective study, fecal samples were consecutively obtained from patients in 2016 and screening test for CRE was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the broth microdilution method and ß-lactamases were characterized by polymerase chain reaction (PCR) assays and DNA sequencing. Multilocus sequence typing (MLST) was performed for the genetic relationships of the isolates. RESULTS: A total of 880 fecal samples were included for this screening test and 32 CRE strains were identified in 32 non-duplicate fecal samples from 32 children (1.3 ± 1.5 years), with a carriage rate of 3.6%. These strains mainly distributed in Klebsiella pnuemoniae (37.5%) and Escherichia coli (37.5%). All CRE strains showed high resistance to most of the routinely used antibiotics (> 90%) except for polymyxin B and tigecycline. The blaNDM gene was the major carbapenemase gene harbored by gastrointestinal CRE strains, followed by blaKPC-2, blaIMP-26, and blaIMP-4. Other ß-Lactamase genes including blaCTX-M, blaSHV, blaTEM-1, and blaDHA-1 were also detected. MLST analysis revealed that various sequence types (STs) were detected in these strains, with ST11 and ST37 being more prevalent in K.pneumoniae and ST101 in E.coli. CONCLUSIONS: This study revealed the prevalence of CRE fecal carriage in children from outpatient and urgent implementation of infection control measure should be conducted to limit the spread of CRE strains.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/patogenicidade , Infecções por Enterobacteriaceae/epidemiologia , Fezes/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Pré-Escolar , China/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/genética , Feminino , Humanos , Lactente , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Pacientes Ambulatoriais/estatística & dados numéricos , Prevalência , Estudos Retrospectivos , beta-Lactamases/genética
6.
BMC Vet Res ; 15(1): 268, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31357996

RESUMO

BACKGROUND: This study investigated changes over time in the epidemiology of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish blaCTX-M-1 and blaCTX-M-15 within CTX-M-1 group. RESULTS: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). In 2017, 314 faecal samples were collected from 74 horses with ESBL-producing E. coli identified in 157 samples (50.0, 95% CI 44.5-55.5). There were 135 and 187 non-duplicate ESBL-producing isolates from 2008 and 2017, respectively. In 2008, 12.6% of isolates belonged to CTX-M-1 group, all carrying blaCTX-M-1, whilst in 2017, 94.1% of isolates were CTX-M-1 group positive and of these 39.2 and 60.8% of isolates carried blaCTX-M-1 and blaCTX-M-15, respectively. In addition, the prevalence of doxycycline, gentamicin and 3rd generation cephalosporin resistance increased significantly from 2008 to 2017 while a decreased prevalence of phenotypic resistance to potentiated sulphonamides was observed. CONCLUSIONS: The real-time PCR proved a reliable and high throughput method to distinguish between blaCTX-M-1 and blaCTX-M-15. Furthermore, its use in this study demonstrated the emergence of faecal carriage of CTX-M-15 in hospitalised horses, with an increase in prevalence of ESBL-producing E. coli as well as increased antimicrobial resistance to frequently used antimicrobials.


Assuntos
Infecções por Escherichia coli/veterinária , Fezes/microbiologia , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/microbiologia , beta-Lactamases/metabolismo , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Cavalos , Hospitais Veterinários/estatística & dados numéricos , Prevalência , Reino Unido/epidemiologia , beta-Lactamases/genética
7.
Rev Soc Bras Med Trop ; 52: e20180348, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31271614

RESUMO

We report the occurrence in Brazil of the bla NDM-1 gene in Acinetobacter pittii, prior to the previously described first reports regarding the species Providencia rettgeri and Enterobacter hormaechei. Clinical isolates were investigated by polymerase chain reaction followed by bidirectional sequencing, and species was confirmed by 16S rDNA sequencing and matrix-assisted laser desorption-ionization time-of-flight spectrometry. A. pittii carrying bla NDM-1 was confirmed in a patient with no national or international travel history, or transfer from another hospital. The findings warn of the possibility of silent spread of bla NDM-1 to the community.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter/isolamento & purificação , Antibacterianos/uso terapêutico , beta-Lactamases/isolamento & purificação , Infecções por Acinetobacter/tratamento farmacológico , Idoso de 80 Anos ou mais , Brasil , Feminino , Humanos , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
8.
BMC Infect Dis ; 19(1): 620, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299909

RESUMO

BACKGROUND: Escherichia coli lineage ST131 predominates across various spectra of extra-intestinal infections, including urinary tract infection (UTI). The distinctive resistance profile, diverse armamentarium of virulence factors and rapid global dissemination of ST131 E. coli makes it an intriguing pathogen. However, not much is known about the prevalence and genetic attributes of ST131 lineage in Pakistan. METHODS: We estimated prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed for phylogenetic grouping, O-typing and fumC/fimH typing. Isolates were further tested for the ESBL and virulence factors using PCR. RESULTS: Overall, 59% of the UPEC isolates belonged to the phylogenetic group B2, followed by D = 28%, B1 = 8% and A = 5%. Among 18 different Sequence-types, ST131 was the dominant lineage (n = 71; 46%) out of which 72% of the isolates were assigned to the phylogenetic group B2, while 61% adhered to the serogroup O25b. FumC/fimH typing confirmed 49% of the ST131 as H30 sub-types. In this study, significant numbers of the identified ST131 isolates were MDR and 42% showed ESBL phenotypes, out of which 37% carried bla-CTX-M-15. Moreover, different virulence factors were detected in following percentages: fimH,155(100%), iutA 86 (55%), feoB 76 (49%), papC 75 (48%), papGII 70 (45%), kpsMTII 40 (26%), papEF 37 (24%), fyuA 37 (24%), usp 22 (14%), papA 20 (13%), sfa/foc20 (13%), hlyA 18 (12%), afa 15 (10%), cdtB 11 (7%), papGI 6 (4%), papGIII 6 (4%), kpsMTIII 4 (3%) and bmaE2 (1%). CONCLUSION: Conclusively, this study provides important insight into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights higher prevalence of ST131-O25b-H30 UPEC isolates in patients, which was previously unreported from this part of globe.


Assuntos
Infecções por Escherichia coli/microbiologia , Infecções Urinárias/diagnóstico , Escherichia coli Uropatogênica/classificação , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Masculino , Paquistão/epidemiologia , Filogenia , Prevalência , Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/isolamento & purificação , Escherichia coli Uropatogênica/patogenicidade , Virulência , Fatores de Virulência/classificação , Fatores de Virulência/genética , beta-Lactamases/classificação , beta-Lactamases/genética
9.
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
10.
BMC Infect Dis ; 19(1): 575, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31269912

RESUMO

BACKGROUND: Hospital environment in patient care has been linked on healthcare-associated infections (HAI). No touch disinfection technologies that utilize pulsed xenon ultraviolet light has been recognized to prevent infection in contaminated environments. The purpose of this study was: 1) to evaluate the effectiveness of pulsed-xenon ultraviolet light (PX-UV) disinfection for the reduction of bacteria on environmental surfaces of Hospital General Enrique Garcés, and 2) to evaluate the in-vitro efficacy against multi-drug resistance microorganisms. METHODS: This was a quality-improvement study looking at cleaning and disinfection of patient areas. During the study, a total of 146 surfaces from 17 rooms were sampled in a secondary 329-bed public medical center. Microbiological samples of high-touch surfaces were taken after terminal manual cleaning and after pulsed xenon ultraviolet disinfection. Cleaning staff were blinded to the study purpose and told clean following their usual protocols. For positive cultures PCR identification for carbapenemase-resistance genes (blaKPC, blaIMP, blaVIM, and blaNDM) were analyzed and confirmed by sequencing. The total number of colony forming units (CFU) were obtained and statistical analyses were conducted using Wilcoxon Rank Sum tests to evaluate the difference in CFU between terminal manual cleaning and after pulsed xenon ultraviolet disinfection. RESULTS: After manual disinfection of 124 surfaces showed a total of 3569 CFU which dropped to 889 CFU in 80 surfaces after pulsed xenon disinfection (p < 0.001). Overall, the surface and environmental contamination was reduced by 75% after PX-UV compared to manual cleaning and disinfection. There were statistically significant decreases in CFU counts of high touch surfaces in OR 87% (p < 0.001) and patient rooms 76% (p < 0.001). Four rooms presented serine carbapenemases blaKPC, and metallo beta-lactamases blaNDM, blaVIM, blaIMP. confirmed by PCR and sequencing. The in-vitro testing with endemic strains found that after five minutes of pulsed xenon ultraviolet exposure an 8-log reduction was achieved in all cases. CONCLUSION: This study is one of the first of its kind in an Ecuador Hospital. We found that pulsed-xenon ultraviolet disinfection technology is an efficacious complement to the established manual cleaning protocols and guidelines in the significant reduction of MDRO.


Assuntos
Desinfecção/métodos , Hospitais , Bactérias/genética , Proteínas de Bactérias/genética , Contagem de Colônia Microbiana , Infecção Hospitalar/prevenção & controle , Desinfecção/instrumentação , Farmacorresistência Bacteriana/genética , Equador , Humanos , Quartos de Pacientes , Raios Ultravioleta , Xenônio , beta-Lactamases/genética
11.
J Med Microbiol ; 68(7): 1021-1032, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188094

RESUMO

INTRODUCTION: The spread of carbapenem-resistant Acinetobacter baumannii has led to a worldwide healthcare problem. Carbapenem resistance in A. baumannii is mainly mediated by the acquisition of the carbapenem-hydrolyzing oxacillinase OXA-23. The phenotypic detection of carbapenem-producing A. baumannii is challenging and time-consuming. Hence, there is an unmet medical need for reliable and rapid diagnostic tools to detect OXA-23-producing Acinetobacter isolates to enable successful patient management. AIM: Development of an immunochromatographic lateral flow test (ICT) for the rapid and reliable detection of OXA-23-producing carbapenem-resistant Acinetobacter isolates. METHODOLOGY: For the development of an antibody-based ICT, we generated anti-OXA-23 monoclonal antibodies (MoAbs) and screened them sequentially for their ability to bind native OXA-23. Selected OXA-23-specific MoAbs were tested in different combinations for their capacity to capture and detect OXA-23His6 by sandwich enzyme-linked immunosorbent assay (ELISA) and ICT. A well-characterized collection of carbapenem-resistant Acinetobacter isolates with defined carbapenem resistance mechanisms were used to evaluate the specificity of the final OXA-23 ICT prototype. RESULTS: The antibody pairs best suited for the sandwich ELISA format did not match the best pairs in the ICT format selected during the development process of the final prototype OXA-23 ICT. This prototype was able to differentiate between OXA-23 subfamily-mediated carbapenem resistance and carbapenem-resistant Acinetobacter isolates overexpressing other OXAs with 100  % specificity and a turnaround time of 20 min from culture plate to result. CONCLUSION: With this rapid detection assay one can save 12-48 h of diagnostic time, which could help avoid inappropriate use of carbapenems and enable earlier intervention to control the transmission of OXA-23-producing carbapenem-resistant Acinetobacter isolates to other patients and healthcare workers.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Imunoensaio/métodos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/imunologia , Animais , Antibacterianos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias , Carbapenêmicos/metabolismo , Clonagem Molecular , Feminino , Regulação Bacteriana da Expressão Gênica , Camundongos , Testes de Sensibilidade Microbiana , beta-Lactamases/genética , beta-Lactamases/imunologia , beta-Lactamases/metabolismo
12.
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
13.
J Med Microbiol ; 68(8): 1219-1226, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31237534

RESUMO

PURPOSE: The new third-generation sequencing platform MinION is an attractive maintenance-free and disposable portable tool that can perform long-read and real-time sequencing. In this study, we validated this technology for the identification of pathogens from positive blood culture (BC) bottles. METHODOLOGY: A total of 38 positive BC bottles were collected from patients with bloodstream infections, and 18 isolates of Gram-negative (GN) bacteria and 20 isolates of Gram-positive (GP) bacteria were identified from these using 16S rRNA sequencing and then used in this study. DNA was extracted from each aliquot using an extraction protocol that combined glass bead beating and chemical lysis. Up to 200 ng of each purified DNA sample was processed for library preparation and whole-genome sequencing was performed on up to 12 samples through a single MinION flow cell. RESULTS: All GN bacteria identifications made by MinION sequencing for 30 min using the What's In My Pot? (WIMP) workflow via EPI2ME on the basis of the most frequent classified reads were consistent with those made by 16S rRNA sequencing. On the other hand, for GP bacteria specimens, the identification results for 16S rRNA sequencing and MinION were only in agreement in 12 out of 20 (60.0 %) cases. ARMA analysis was able to detect extended-spectrum ß-lactamase (ESBL)-associated genes among various antimicrobial resistance-related genes. CONCLUSION: We demonstrated the potential of the MinION sequencer for the identification of GN bacteria from positive BC bottles and the confirmation of an ESBL phenotype. This innovative sequence technology and its application could lead to a breakthrough in the diagnosis of infectious diseases.


Assuntos
Bacteriemia/microbiologia , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , beta-Lactamases/genética , Antibacterianos/farmacologia , DNA Bacteriano/genética , Genoma Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Nanoporos , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/normas , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas , Fatores de Tempo
14.
Future Microbiol ; 14: 691-704, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31148474

RESUMO

Aim: To determine the prevalence of New Delhi metallo-ß-lactamase (NDM)-producing Gram-negative pathogens isolated from children's samples. Materials & methods: Carbapenem-resistant clinical isolates (n = 117) were confirmed by VITEK® 2 compact system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing. MIC (µg/ml) of various antibiotics was determined by VITEK 2 compact system. Molecular characterization of the isolates was performed by PCR, DNA sequencing, PFGE and DNA hybridization. Results: Out of 117 carbapenemase producers, 37 (31.6%) and 29 (24.7%) were Klebsiella pneumoniae and Acinetobacter baumannii, respectively. 72 (61.5%) isolates were NDM positive and among these 60, 9 and 3 were NDM-1, -5 and -7, respectively. Majority of the NDM-producing K. pneumoniae belonged to ST11 and ST273 while most of the Escherichia coli belonged to ST405 and ST101. blaNDM were mainly located on 150kb plasmids. MIC displayed high resistance against ß-lactams drugs including carbapenems, and the most sensitive drugs were tigecycline and colistin. Conclusion: Dissemination of blaNDM-producing pathogens, particularly in children clinical settings, is a matter of great public health concern.


Assuntos
Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , beta-Lactamases/biossíntese , beta-Lactamases/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Criança , DNA Bacteriano/análise , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Paquistão/epidemiologia , Plasmídeos , Análise de Sequência de DNA
15.
Vet Microbiol ; 233: 52-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176413

RESUMO

The spread of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli is a major public health issue and ESBL-producing bacteria are frequently reported in livestock. For the assessment of the role of the foodborne transmission pathway in Germany, detailed data on the prevalence and characteristics of isolates of food origin are necessary. The objective of this study was to describe the prevalence of cefotaxime resistant E. coli as well as ESBL/pAmpC-producing E. coli and their characteristics in foods in Germany. Out of 2256 food samples, the highest prevalence of cefotaxime resistant E. coli was observed in chicken meat (74.9%), followed by turkey meat (40.1%). Prevalence in beef, pork and minced meat was considerably lower (4.2-15.3%). Whereas 18.0% of the raw milk samples, collected at farm level were positive, this was true only for few cheese samples (1.3%). In one out of 399 vegetable samples a cefotaxime-resistant E. coli was isolated. ESBL resistance genes of the CTX-M-group (10.1% of all samples) were most frequently detected, followed by genes of the pAmpC (2.6%), SHV (2.0%) and TEM (0.8%) families. Distribution of ESBL/AmpC-encoding E. coli resistance genes and E. coli phylogroups was significantly different between the chicken related food samples and all other food items. Our study results reflect that consumers might get exposed to ESBL/pAmpC-producing E. coli through several food chains. These results together with those collected at primary production and in the human population in other studies will allow more detailed analysis of the foodborne pathways, considering transmission from livestock populations to food at retail and to consumers in Germany.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Microbiologia de Alimentos , Carne/microbiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cefotaxima/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/enzimologia , Infecções por Escherichia coli/transmissão , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Alemanha , Gado/microbiologia , Aves Domésticas/microbiologia , Prevalência , Carne Vermelha/microbiologia , Verduras/microbiologia , beta-Lactamases/biossíntese
16.
Vet Microbiol ; 233: 61-67, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176414

RESUMO

The incidence of infections with extended spectrum ß-lactamase producing Escherichia coli (ESBL-E) is increasing both in humans and animals. There is a paucity of data about the rate of faecal carriage of ESBL-E in pets. In this study, faecal swabs collected from 586 pets (225 cats; 361 dogs) in Auckland, New Zealand, were analysed for the presence of ESBL-E by culture, and a questionnaire was delivered to the owners. The ESBL-E were characterised and data elicited by the questionnaires were used for a multivariable analysis, to investigate the factors associated with faecal ESBL-E carriage. The prevalence of ESBL-E in faecal swabs was 6.4%. The ß-lactamase genes detected in the ESBL-E were the blaCTX-M-14 (n = 2) and blaCMY-2 (n = 34). Several isolates displayed multilocus sequence types (ST) associated with human and animal infections. Multiple isolates sharing the same ST displayed different antibiograms and ß-lactamase genes, reflecting horizontal gene transfer between and within ST. Variables independently associated with increased odds of ESBL-E carriage were: animal received systemic antimicrobial treatment in the six months before the sampling; presence of household members working in veterinary clinics; presence of household members travelling overseas in the six months before the sampling. We conclude that pets are colonised by ESBL-E which are genotypically similar to the bacteria found to infect humans and animals. The statistical analysis suggested a number of eco-epidemiological factors associated with ESBL-E carriage. In particular, they suggest veterinary clinics may represent hot-spots of antimicrobial resistance.


Assuntos
Portador Sadio/veterinária , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/veterinária , Escherichia coli/genética , Animais de Estimação/microbiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Portador Sadio/epidemiologia , Gatos/microbiologia , Cães/microbiologia , Escherichia coli/enzimologia , Infecções por Escherichia coli/epidemiologia , Fezes/microbiologia , Feminino , Transferência Genética Horizontal , Genética Populacional , Genótipo , Hospitais Veterinários , Humanos , Masculino , Tipagem de Sequências Multilocus , Nova Zelândia/epidemiologia , Prevalência , beta-Lactamases/biossíntese
17.
Mem Inst Oswaldo Cruz ; 114: e190020, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31166421

RESUMO

BACKGROUND: The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens. OBJECTIVES: The present study aimed to verify the occurrence of metallo-ß-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region. METHODS: Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing. FINDINGS: A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86. MAIN CONCLUSIONS: To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.


Assuntos
Acinetobacter/química , Acinetobacter/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/isolamento & purificação , Acinetobacter/efeitos dos fármacos , Antibacterianos/farmacologia , Brasil , Carbapenêmicos/farmacologia , DNA Bacteriano , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Unidades de Terapia Intensiva , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
19.
Environ Sci Pollut Res Int ; 26(22): 22305-22311, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31154643

RESUMO

The dissemination of extended spectrum beta-lactamases (ESBL) genes through gene transfer attracts wide attention. Bentonite is widely used as a feed additive in an animal-breeding environment. In order to obtain a better understanding of the effect of bentonite on Escherichia coli carrying ESBL gene, proteomic analysis was carried out to screen the key proteins. The results showed that a total of 31 proteins were differentially expressed, including 21 up-regulated proteins and 10 down-regulated proteins. These proteins were involved in biosynthetic process, metabolic process, stress response, transport, anaerobic respiration, proteolysis, hydrolase, protein folding, transcription, salvage, and other. The transcriptional level of four genes (mipA, gntY, tldD, and arcA) was in consensus with proteomic results. This study revealed the differentially expressed proteins involved when E. coli was incubated under bentonite and PBS condition, which implied the possibility that bentonite may promote the transfer of ESBL gene between E. coli.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , beta-Lactamases/genética , Animais , Bentonita/química , Bentonita/farmacologia , Escherichia coli/química , Infecções por Escherichia coli , Proteínas de Escherichia coli/química , Humanos , Proteômica , beta-Lactamases/análise
20.
Rev Soc Bras Med Trop ; 52: e20180352, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31141048

RESUMO

INTRODUCTION: The emergence of New Delhi metallo-ß-lactamase (NDM) is concernig because it reduces the antibiotic therapy options for bacterial infections. METHODS: Resistant and virulent genes from an isolate of Klebsiella pneumoniae derived from a patient with sepsis in a hospital in Recife-PE, Brazil, were investigated using PCR and DNA sequencing. RESULTS: bla NDM-1, aac(6')-Ib-cr and acrB resistance genes, and cps and mrkD virulence genes were detected. CONCLUSIONS: To our knowledge, this is the first report on bla NDM-1 in Recife-PE. This detection alerts researchers to the need to control the spread of bla NDM-1 resistance gene by this bacterium in Brazil.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Klebsiella pneumoniae/genética , Virulência/genética , beta-Lactamases/genética , Feminino , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Sepse/microbiologia , Análise de Sequência de DNA
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