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Braz. j. microbiol ; 49(4): 914-918, Oct.-Dec. 2018. tab
Article in English | LILACS | ID: biblio-974286


ABSTRACT The global emergence of carbapenemases led to the need of developing new methods for their rapid detection. The aim of this study was to evaluate the performance of the rapid tests for carbapenemase-producing and non-producing Enterobacteriaceae. Carbapenem non-susceptible Enterobacteriaceae from a surveillance study submitted to a multiplex real time PCR for carbapenemase detection were included in this study. The isolates were subjected to the rapid phenotypic tests Carba NP, Blue-Carba and Carbapenem Inactivation Method (CIM). A total of 83 carbapenemase-producing (43) and non-producing (40) isolates were included in the study. The sensitivity/specificity were 62.7%/97.5%, 95.3%/100%, and 74.4%/97.5% for Carba NP, Blue-Carba and CIM, respectively. Both Carba NP and Blue-Carba presented their final results after 75 min of incubation; the final results for CIM were obtained only after 8 h. Failure to detect OXA-370 carbapenemase was the main problem for Carba NP and CIM assays. As the Blue-Carba presented the highest sensitivity, it can be considered the best screening test. Conversely, CIM might be the easiest to perform, as it does not require special reagents. The early detection of carbapenemases aids to establish infection control measures and prevent carbapenemases to spread reducing the risk of healthcare associated infections and therapeutic failure.

Humans , Bacterial Proteins/analysis , beta-Lactamases/analysis , Enterobacteriaceae/enzymology , Enterobacteriaceae Infections/microbiology , Enzyme Assays/methods , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolism , Brazil , Carbapenems/pharmacology , Polymerase Chain Reaction , Sensitivity and Specificity , Enterobacteriaceae/isolation & purification , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae Infections/diagnosis , Anti-Bacterial Agents/pharmacology
Braz. j. microbiol ; 49(4): 885-890, Oct.-Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-974312


ABSTRACT In this study, the performance of the "RESIST-3 O.K.N. K-SeT" (Coris BioConcept, Gembloux, Belgium) immunochromatographic assay was evaluated in 132 Klebsiella pneumoniae comprising 102 carbapenem resistant and 30 carbapenem susceptible isolates. Genotypically known isolates of Gram negative bacteria (n = 22) including various species were also tested by the assay as controls. The isolates tested by the immunochromatographic assay and also were run PCR for bla KPC, bla IMP, bla VIM, bla NDM, and bla OXA-48. The rates of bla NDM, bla OXA-48, and bla KPC in carbapenem resistant isolates were found at 52.9%, 39.2%, and 2.0%, respectively. Both bla NDM and bla OXA-48 were found in six (5.9%) isolates. The results of the assay showed 100% concordance with those obtained by PCR in 132 K. pneumoniae. The agreement between the two methods was found to be identical at the isolate level. The assay also correctly detected all genotypically known isolates of Escherichia coli, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, K. pneumoniae carrying bla KPC, bla NDM, and/or bla OXA-48. On the other hand, the assay did not exhibit any cross-reaction in control isolates harboring bla IMP and bla VIM. We conclude that the RESIST-3 O.K.N. K-SeT is a reliable, rapid, and user friendly test and we recommend it for routine diagnostic laboratories.

Humans , Bacterial Proteins/analysis , beta-Lactamases/analysis , Klebsiella Infections/microbiology , Immunoassay/methods , Klebsiella pneumoniae/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Turkey , beta-Lactamases/metabolism , Carbapenems/pharmacology , Polymerase Chain Reaction , Klebsiella pneumoniae/isolation & purification , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/chemistry , Anti-Bacterial Agents/pharmacology
Braz. j. microbiol ; 48(2): 242-245, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839378


Abstract The modified Carba NP test presented here may be a valuable tool for laboratories interested in investigating a large number of carbapenemase-producing bacteria in a less-costly way. The test was evaluated against 48 carbapenemase-producing and carbapenemase-non-producing gram-negative bacteria. No false–positive results were obtained, but false-negative results were observed with OXA-23- and GES-carbapenemase-producing isolates. Aeromonas sp. are not testable by Modified Carba NP.

Bacterial Proteins/analysis , beta-Lactamases/analysis , Bacteriological Techniques/methods , Gram-Negative Bacteria/enzymology , False Negative Reactions
Braz. j. microbiol ; 48(2): 211-217, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839365


Abstract Pseudomonas aeruginosa is an opportunistic pathogen that causes frequently nosocomial infections, currently becoming more difficult to treat due to the various resistance mechanisms and different virulence factors. The purpose of this study was to determine the risk factors independently associated with the development of bacteremia by carbapenem-resistant P. aeruginosa, the frequency of virulence genes in metallo-β-lactamases producers and to evaluate their ability to produce biofilm. We conducted a case–control study in the Uberlândia Federal University – Hospital Clinic, Brazil. Polymerase Chain Reaction was performed for metallo-β-lactamases and virulence genes. Adhesion and biofilm assays were done by quantitative tests. Among the 157 strains analyzed, 73.9% were multidrug-resistant, 43.9% were resistant to carbapenems, 16.1% were phenotypically positive for metallo-β-lactamases, and of these, 10.7% were positive for blaSPM gene and 5.3% positive for blaVIM. The multivariable analysis showed that mechanical ventilation, enteral/nasogastric tubes, primary bacteremia with unknown focus, and inappropriate therapy were independent risk factors associated with bacteremia. All tested strains were characterized as strongly biofilm producers. A higher mortality was found among patients with bacteremia by carbapenem-resistant P. aeruginosa strains, associated independently with extrinsic risk factors, however it was not evident the association with the presence of virulence and metallo-β-lactamases genes.

Humans , Male , Female , Adult , Middle Aged , Aged , Pseudomonas aeruginosa/genetics , Pseudomonas Infections/epidemiology , Bacterial Proteins/genetics , beta-Lactamases/genetics , Bacteremia/epidemiology , Biofilms/growth & development , beta-Lactam Resistance , Virulence Factors/genetics , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Pseudomonas Infections/microbiology , Bacterial Proteins/analysis , beta-Lactamases/analysis , Brazil/epidemiology , Case-Control Studies , Survival Analysis , Polymerase Chain Reaction , Risk Factors , Bacteremia/microbiology
Braz. J. Pharm. Sci. (Online) ; 53(2): e16111, 2017. tab
Article in English | LILACS | ID: biblio-839487


ABSTRACT The present study evaluated the antimicrobial susceptibility profile, ß-lactamase production, and genetic diversity of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. using phenotypic identification, antimicrobial susceptibility testing, and ß-lactamase phenotypic detection. Isolates were obtained from patients in an intensive care unit in a hospital in southern Brazil. Bacterial genomic DNA was extracted, followed by the genotypic detection of carbapenemases and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). Fifty-six isolates (26 Klebsiella pneumoniae, five Escherichia coli, three Enterobacter aerogenes, nine P. aeruginosa, and 13 Acinetobacter spp.) were evaluated. The phenotypic extended spectrum ß-lactamase (ESBL) test was positive in 53.8% of the K. pneumoniae isolates, 100.0% of the E. coli isolates, and 100.0% of the E. aerogenes isolates. Phenotypic and genotypic testing of K. pneumoniae carbapenemase (KPC) was positive in 50.0% of the K. pneumoniae isolates. Phenotypic and genotypic testing showed that none of the P. aeruginosa or Acinetobacter spp. isolates were positive for metallo- ß-lactamase (MBL). The bla OXA gene was detected only in Acinetobacter spp. The lowest genetic diversity, determined by ERIC-PCR, was observed among the KPC-producing K. pneumoniae isolates and OXA-producing Acinetobacter spp. isolates, indicating the inadequate dissemination control of multidrug-resistant bacteria in this hospital environment.

Humans , Male , Female , beta-Lactamases/analysis , Gram-Negative Bacteria/classification , Intensive Care Units/statistics & numerical data , Pseudomonas aeruginosa/metabolism , Acinetobacter/metabolism , Microbiology , Bacterial Typing Techniques/instrumentation , Enterobacteriaceae/metabolism
Rev. argent. microbiol ; 48(4): 320-324, dic. 2016. ilus, tab
Article in Spanish | LILACS | ID: biblio-1041768


En este trabajo se evalúa una prueba rápida in house para la detección de enterobacterias sensibles a cefotaxima, basada en el cambio de pH del rojo fenol debido a la hidrólisis de este antibiótico. Las cepas de enterobacterias procedentes de 1.947 urocultivos se evaluaron mediante los paneles MicroScan y esta prueba in house. Mediante los paneles de MicroScan se estudiaron 499 aislados de enterobacterias, entre los cuales había 27 aislados de Escherichia coli productora de β-lactamasa de espectro extendido (BLEE), 16 de Klebsiella pneumoniae BLEE y una de Klebsiella oxytoca BLEE. La prueba in house mostró una sensibilidad del 98% y una especificidad del 97%, con un valor predictivo negativo del 100% y un valor predictivo positivo del 78%. La prueba in house basada en el cambio de pH es útil en nuestro medio para detectar presuntivamente de forma rápida cepas de enterobacterias con cierta resistencia a cefotaxima.

In this work an "in house" rapid test based on the change in pH that is due to hydrolysis for detecting Enterobacteriaceae susceptible to cefotaxime is evaluated. The strains of Enterobacteriaceae from 1947 urine cultures were assessed using MicroScan panels and the "in house" test. This rapid test includes red phenol solution and cefotaxime. Using MicroScan panels, 499 Enterobacteriaceae isolates were evaluated, which included 27 isolates of Escherichia coli producing extended-spectrum beta-lactamases (ESBL), 16 isolates of Klebsiella pneumoniae ESBL and 1 isolate of Klebsiella oxytoca ESBL. The "in house" test offers the following values: sensitivity 98% and specificity 97%, with negative predictive value 100% and positive predictive value 78%. The "in house" test based on the change of pH is useful in our area for detecting presumptively cefotaxime-resistant Enterobacteriaceae strains.

Humans , Male , Female , Microbial Sensitivity Tests/methods , Cefotaxime/therapeutic use , Enterobacteriaceae/drug effects , Phenolsulfonphthalein/analysis , beta-Lactamases/analysis , Cefotaxime/pharmacology , Enterobacteriaceae/isolation & purification
Braz. j. microbiol ; 47(1): 191-195, Jan.-Mar. 2016. graf
Article in English | LILACS | ID: lil-775110


Abstract The aim of this study was to investigate the occurrence of multidrug-resistant, extended spectrum beta-lactamase (ESBL) producing Salmonella spp. isolated from conveyor belts of broiler cutting rooms in Brazilian broiler processing plants. Ninety-eight strains of Salmonella spp. were analyzed. Multidrug resistance was determined by the disk diffusion test and the susceptibility of the isolated bacteria was evaluated against 18 antimicrobials from seven different classes. The double disk diffusion test was used to evaluate ESBL production. Of the 98 strains tested, 84 were multidrug resistant. The highest rates of resistance were against nalidixic acid (95%), tetracycline (91%), and the beta-lactams: ampicillin and cefachlor (45%), followed by streptomycin and gentamicin with 19% and 15% of strain resistance, respectively. By contrast, 97% of the strains were sensitive to chloramphenicol. 45% of the strains were positive for the presence of ESBL activity. In this study, high rates of multidrug resistance and ESBL production were observed in Salmonella spp.

Animals , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial , Environmental Microbiology , Food-Processing Industry , Salmonella/drug effects , Salmonella/enzymology , beta-Lactamases/analysis , Brazil , Chickens , Disk Diffusion Antimicrobial Tests , Salmonella/isolation & purification
Braz. oral res. (Online) ; 29(1): 1-6, 2015. tab
Article in English | LILACS | ID: lil-777189


This study aimed to determine the presence of Prevotella strains and genes associated with resistance to lactamics in different oral niches from patients with/without primary endodontic infections. Saliva (S) and supragingival biofilm (SB) were collected from three patient groups: Group I – no endodontic infection (n = 15); Group II – acute endodontic infection (n = 12); and Group III – chronic endodontic infection (n = 15). Root canal (RC) samples were collected from Groups II and III. The presence of P. intermedia, P nigrescens, P. tannerae and cfxA/cfxA2 gene was assessed by PCR. The cfxA/cfxA2 gene was not detected in all environments within the same patient. The cfxA/cfxA2 gene was present in 23.81% of S samples, 28.57% of SB samples, and 7.41% of RC samples. Prevotella species were detected in 53.97%, 47.62% and 34.56% of the S, SB, and RC samples, respectively. P. intermedia had a high frequency in saliva samples from Group 3. Saliva samples from Group 1 had higher detection rates of P. nigrescens than did Groups 2 and 3. Patients without endodontic disease had high frequencies of P. nigrescens in the SB samples. The presence or absence of spontaneous symptoms was not related to the detection rates for resistance genes in the RC samples. Saliva, supragingival biofilm and root canals can harbor resistant bacteria. The presence of symptomatology did not increase the presence of the cfxA/cfxA2 gene in the supragingival biofilm and inside root canals.

Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Biofilms , Dental Pulp Cavity/microbiology , Gingiva/microbiology , Prevotella/isolation & purification , Saliva/microbiology , beta-Lactam Resistance/genetics , beta-Lactamases/genetics , Chi-Square Distribution , DNA, Bacterial/analysis , Polymerase Chain Reaction , Prevotella/genetics , Sequence Analysis, DNA , beta-Lactamases/analysis
Rev. argent. microbiol ; 46(3): 175-181, oct. 2014. tab
Article in Spanish | LILACS | ID: lil-734579


En este estudio se determinó el perfil de distribución de grupos filogéneticos y la detección genética de factores de virulencia en cepas de Escherichia coli uropatógena (ECUP) productoras de ß-lactamasa CTX-M-15. Veintiocho cepas fueron aisladas de pacientes con infección del tracto urinario (ITU) que asistieron al Laboratorio de Salud Pública del estado Mérida, Venezuela, durante el lapso comprendido entre enero 2009 y julio 2011. La determinación de los grupos filogenéticos y la detección de seis genes de virulencia, fimH, fyuA, kpsMTII, usp, PAI y papAH, se realizó mediante amplificación por PCR. Quince cepas de 28 se ubicaron principalmente en el filogrupo A, seguidos por el B2 (12/28) y D (1/28). No se observó una relación directa entre la recurrencia o gravedad de la ITU y la distribución de los filogrupos. Todos los factores de virulencia estudiados se encontraron con la frecuencia más alta en el grupo B2. El perfil de virulencia prevalente estuvo conformado por la asociación de tres genes principales: fimH, fyuA y kpsMTII y en menor frecuencia, por la presencia de otros determinantes como usp, PAI y/o papAH. Estos resultados indican que la mayoría de ECUP estuvieron dotadas de tres propiedades virulentas importantes: adhesión, captación de hierro y evasión de la fagocitosis, las cuales favorecieron la producción de ITU recurrentes. Este es el primer trabajo que describe la asociación de grupos filogenéticos con el potencial de virulencia de cepas de ECUP productoras de ß-lactamasa CTX-M-15 en Venezuela.

In this study, the distribution of phylogenetic groups and the genetic detection of virulence factors in CTX-M-15 ß-lactamase-producing uropathogenic Escherichia coli (UPEC) strains were analyzed. Twenty eight strains were isolated between January 2009 and July 2011 from patients with urinary tract infection (UTI) who attended the Public Health Laboratory at Mérida, Venezuela. Determination of phylogenetic groups and detection of six virulence genes, fimH, fyuA, kpsMTII, usp, PAI and papAH, were performed by PCR amplification. Fifteen of the 28 isolates were mainly located in the phylogenetic group A, followed by B2 (12/28) and D (1/28). No direct relationship between the severity or recurrence of UTI and the distribution of phylogroups was observed. All studied virulence factors were found in group B2 strains with the highest frequency. The prevalent virulence profile included the combination of three main genes: fimH, kpsMTII and fyuA and, to a lesser extent, the presence of other determinants such as usp, PAI and/or papAH. These results indicate that virulent UPEC incorporated three important properties: adhesion, iron uptake and evasion of phagocytosis, which favored the production of recurrent UTI. This is the first report describing the association of phylogenetic groups with the potential virulence of CTX-M-15 ß-lactamase producing UPEC strains in Venezuela.

Adolescent , Adult , Aged , Aged, 80 and over , Child , Female , Humans , Male , Middle Aged , Pregnancy , Young Adult , Community-Acquired Infections/microbiology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/analysis , Escherichia coli/classification , Urinary Tract Infections/microbiology , beta-Lactamases/analysis , Bacterial Adhesion/genetics , Comorbidity , Community-Acquired Infections/epidemiology , Disease Susceptibility , DNA, Bacterial/genetics , Escherichia coli Infections/epidemiology , Escherichia coli Proteins/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/isolation & purification , Genes, Bacterial , Iron/metabolism , Phagocytosis , Phylogeny , Pregnancy Complications, Infectious/microbiology , Recurrence , Urinary Tract Infections/epidemiology , Venezuela/epidemiology , Virulence/genetics , beta-Lactamases/genetics
Invest. clín ; 55(1): 32-43, mar. 2014. ilus, tab
Article in Spanish | LILACS | ID: lil-746283


En este estudio se determinó la prevalencia de b-lactamasas de espectro extenso (BLEEs) en grupos filogenéticos de E. coli uropatógena (ECUP) aislados en pacientes de la comunidad. Durante Enero 2009 a Julio 2010, se coleccionaron 21 cepas de ECUP, con susceptibilidad disminuida a las cefalosporinas de amplio espectro, provenientes de pacientes que asistieron al Laboratorio de Salud Pública del estado Mérida, Venezuela con diagnóstico de infección del tracto urinario (ITU). La caracterización genotípica determinó que todas las cepas ECUP albergaban genes blaBLEEs. En el 76,2% de las cepas se observó la presencia de un único gen productor de BLEE, representado por blaCTX-M-15, mientras que el 23,8% estuvo conformado por ECUP con diversas combinaciones de genes bla (blaCTX-M-15 + blaTEM-1, blaCTX-M-15 + blaSHV y blaSHV + blaTEM-1). El 61,9% de los aislados se ubicó en el filogrupo A y el resto de las cepas en el grupo B2 (38,1%). No se evidenció la diseminación de una clona de ECUP particular, solo 7 cepas demostraron pertenecer a un grupo clonal con un índice de similitud de más de 85%. De acuerdo a nuestro conocimiento, esta es la primera descripción de blaCTX-M-15 en ECUP causantes de ITU en pacientes de la comunidad, lo que evidencia que Venezuela también forma parte de la llamada pandemia CTX-M-15. Los hallazgos obtenidos en este estudio y las implicaciones clínicas y epidemiológicas que de ello derivan, conllevan a la necesidad de controlar y vigilar la diseminación de ECUP productora de CTX-M-15 no sólo en el ámbito regional sino también nacional.

In this study we determined the prevalence of extended-spectrum b-lactamases (ESBLs) in phylogenetic groups of uropathogenic E. coli (UPEC) isolated from patients in the community. Twenty one UPEC strains with reduced susceptibility to broad-spectrum cephalosporins were collected between January 2009 and July 2010, from patients with urinary tract infection who attended the Public Health Laboratory in Mérida, Venezuela. Genotypic characterization determined that all UPEC strains harbored blaBLEEs genes: 76.2% of the strains showed the presence of a single ESBL-producer gene, represented by blaCTX-M-15, whereas 23.8% of UPEC showed various combinations of bla genes (blaCTX-M-15 + blaTEM-1, blaCTX-M-15 + blaSHV and blaSHV + blaTEM-1). In this study, 61.9% of the isolates were placed in phylogroup A and the remaining strains were assigned to group B2 (38.1%). There was no evidence of spread of a particular UPEC clone; only seven strains belonged to a clonal group with an index of similarity greater than 85%. To our knowledge, this is the first description of blaCTX-M-15 in UPEC from patients with community-acquired urinary tract infections, which shows that Venezuela is also part of the so-called CTX-M-15 pandemic. The findings in this study, as well as its clinical and epidemiological implications, lead to the need for monitoring and controlling the spread of CTX-M-15 producing UPECs, not only regionally, but also nationwide.

Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Community-Acquired Infections/microbiology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Genes, Bacterial , Urinary Tract Infections/microbiology , beta-Lactam Resistance/genetics , beta-Lactamases/genetics , Community-Acquired Infections/epidemiology , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli Infections/epidemiology , Escherichia coli Proteins/analysis , Escherichia coli/classification , Escherichia coli/enzymology , Escherichia coli/isolation & purification , Gene Frequency , Phylogeny , Recurrence , Urinary Tract Infections/epidemiology , Venezuela/epidemiology , beta-Lactamases/analysis
Rev. argent. microbiol ; 46(1): 30-33, mar. 2014.
Article in English | LILACS | ID: biblio-1009598


Salmonellaenterica serovar Heidelberg es uno de los principales agentes causantes de salmonelosis en humanos en Estados Unidos y Canadá, sin embargo, resulta infrecuente en los países de Sudamérica y Europa. En este trabajo se caracterizó un aislamiento de S. Heidelberg resistente a oximino-cefalosporinas recuperado de un paciente internaen un hospital de la Ciudad de Buenos Aires. Se evidenció la presencia de un plásmido de 97 kbperteneciente al grupo de incompatibilidad IncN, portador del gen blaCMY-2. ISEcp1 fue localizado corriente arriba de blaCMY-2, promoviendo su expresión y movilización.El aislamiento de S. Heidelberg correspondió al secuenciotipo 15 y en la virotipifi cación se detectó el gen sopE. En este trabajo describimos por primera vez la producción de CMY-2 en una cepa de S. Heidelberg en nuestro país y América Latina

Salmonellaenterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries.Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the ß-lactam resistance profi le. S. Heidelberg contained a 97 kb plasmid belonging to the Inc N groupharboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization.The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identifi ed the fi rst CMY-2 producing isolate of S. Heidelberg in Argentina and even in South Americ

Humans , Male , South America/epidemiology , beta-Lactamases/analysis , Salmonella enterica/isolation & purification , Plasmids/analysis , Salmonella enterica/pathogenicity
Article in English | IMSEAR | ID: sea-157562


Bacteria of genus acinetobacter, invariably susceptible to many antibiotics earlier have emerged as a multidrug resistant opportunistic pathogen in recent years. Unwarranted and unrestricted usage of an antibiotic is associated with drug resistance in thereat in infections by acinetobacter species. Rapid detection of Metallo – Betalactamase starins can be done to prevent their dissemtination. The present study is undertaken to know antibiotic sensitivity pattern of acinetobacter species and screen the imipenem resistant starins for Metallo Beta Lactamase (MBL) production by various phenotypic methods.

Acinetobacter/drug therapy , Acinetobacter/genetics , Acinetobacter/isolation & purification , Carbapenems/pharmacology , Drug Resistance, Multiple, Bacterial , Microbial Sensitivity Tests , beta-Lactamases/analysis
Article in English | IMSEAR | ID: sea-162936


Aims: Beta-lactamase production and subsequent resistance to β-lactam drugs has been a global concern in the treatment of Gram negative anaerobes. The aim of this study was to identify F. nucleatum strains producing Class D β-lactamase through the detection of FUS-1 (OXA-85) resistance gene. Place and Duration of Study: Department of Preventive Dentistry, Lagos University Teaching Hospital, Idi-Araba, between February 2010 and November 2010. Methodology: Twenty two oral clinical samples were obtained from patients with chronic periodontitis who admitted to previous use of amoxicillin. Antibacterial susceptibility of the bacterial isolates was determined by E-test on Brucella Blood agar. Amplification of the bacterial DNA was carried out by PCR using F. nucleatum species-specific primer, FUS-1 specific for blaFUS-1 and strain-specific primers for subspecies nucleatum,, fusiforme, polymorphum and vincentii. Results: From the 19 samples collected, F. nucleatum was isolated, and the identity of the isolates was confirmed by PCR. Four of the isolates produced similar bands with the control strain, 3 (15.7%) strains were able to produce amplication with FUS-1 primer specific for blaFUS-1 gene found in β-lactamase producing F. nucleatum subsp. polymorphum. Conclusion: This study shows the presence of class D β-lactamase producing F. nucleatum species in Nigeria.

Adult , Bacteria/genetics , Child , Female , Fusobacterium nucleatum/classification , Fusobacterium nucleatum/genetics , Genes, Bacterial , Humans , Male , Middle Aged , Nigeria , beta-Lactamases/analysis , beta-Lactamases/drug effects , beta-Lactamases/isolation & purification
Braz. j. infect. dis ; 16(6): 521-526, Nov.-Dec. 2012. ilus, tab
Article in English | LILACS | ID: lil-658921


INTRODUCTION: Acinetobacter spp. have emerged as notorious pathogens involved in healthcareassociated infections. Carbapenems are important antimicrobial agents for treating infections due to multidrug resistant Acinetobacter spp. Different mechanisms may confer resistance to these drugs in the genus, particularly production of class D carbapenemases. OXA-23-like family has been pointed out as one of the predominant carbapenamases among Acinetobacter. The present work aimed to investigate the occurrence of OXA-23-like carbapenemases among Acinetobacter isolates recovered from patients of a university hospital in Niterói, RJ, Brazil. METHODS: Antimicrobial susceptibility profiles were determined by disk-diffusion. Imipenem resistant isolates were submitted to Modified Hodge Test in order to screen for carbapenemase production, and later to polymerase chain reaction (PCR) to investigate the presence of blaOXA-23. RESULTS: Imipenem and meropenem resistance rates were 71.4% and 69.7%, respectively. The Modified Hodge Test revealed carbapenemase production among 76 (89.4%) of the 85 imipenem resistant isolates analyzed; according to PCR results, 81 isolates (95.4%) carried the blaOXA-23 gene. CONCLUSIONS: OXA-23-like enzymes may be an important mechanism of carbapenem resistance among isolates present in the hospital studied.

Humans , Acinetobacter/enzymology , beta-Lactamases/analysis , Acinetobacter/drug effects , Acinetobacter/isolation & purification , Anti-Bacterial Agents/pharmacology , Brazil , Disk Diffusion Antimicrobial Tests , Drug Resistance, Bacterial , Hospitals, University , Microbial Sensitivity Tests , Polymerase Chain Reaction , beta-Lactamases/genetics
Einstein (Säo Paulo) ; 10(4): 439-441, Oct.-Dec. 2012.
Article in English | LILACS | ID: lil-662468


OBJECTIVE: To evaluate ertapenem disk performance to predict Klebsiella pneumonie carbapenemase production by Gram-negative bacilli. METHODS: All Gram-negative bacilli isolated between January 2010 and June 2011 were tested by disk diffusion (OxoidTM) for sensitivity to ertapenem, meropenem and imipenem. Resistant or intermediate sensitivity strains (diameter <22 mm for ertapenem) were also tested for the blaKPC gene by polymerase chain reaction. Disk predictive positive value for Klebsiella pneumoniae carbapenemase and specificity were calculated. RESULTS: Out of the 21839 cultures performed, 3010 (13.78%) were positive, and Gram-negative bacilli were isolated in 708 (23.52%) of them. Zone of inhibition diameter for ertapenem disk was <22 mm for 111 isolates, representing 15.7% of all Gram-negative isolates. The PCR assay for blaKPC detected 40 Klebsiella pneumoniae carbapenemase-producing strains. No strains intermediate or resistant to meropenem and imipenem were sensitive to ertapenem. The ertapenem disk presented a positive predictive value of 36% to predict blaKPC and 89% specificity. CONCLUSION: The resistance of Gram-negative bacilli detected by disk diffusion against ertapenem does not predict Klebsiella pneumoniae carbapenemase production. Other mechanisms, such as production of other betalactamases and porin loss, may be implicated. The need to confirm the presence of the blaKPC is suggested. Therefore, ertapenem was a weak predictor for discriminating strains that produce Klebsiella pneumoniae carbapenemase.

OBJETIVO: Avaliar o desempenho do disco de ertapenem para predizer micro-organismos produtores de Klebsiella pneumoniae carbapenemase. MÉTODOS: Bacilos Gram-negativos isolados em cultura entre janeiro de 2010 e junho de 2011 foram testados por disco-difusão (OxoidTM) para ertapenem, meropenem e imipenem. As cepas consideradas intermediárias ou resistentes (halo<22mm) para ertapenem foram encaminhadas para a pesquisa do blaKPC por reação em cadeia da polimerase. Calcularam-se o valor preditivo positivo e a especificidade do disco. RESULTADOS: Foram realizadas 21.839 culturas nesse período, sendo 3.010 (13,78%) positivas. Bacilos Gram-negativos foram isolados em 708 (23,52%) destas. A zona de inibição do disco de ertapenem foi <22mm para 111 (15,67%) dos isolados. A pesquisa do blaKPC caracterizou 40 cepas produtoras de Klebsiella peneumoniae carbapenemase. Não houve nenhum caso de disco intermediário ou resistente para meropenem ou imipenem com ertapenem sensível. O valor preditivo positivo foi de 36% e a especificidade calculada do disco de ertapenem para produção de Klebsiella pneumoniae carbapenemase foi de 89% em nosso serviço. CONCLUSÃO: A resistência ao disco de ertapenem não define bacilo produtor de Klebsiella pneumoniae carbapenemase. Mecanismos, como produção de outras betalactamases e perda de porinas, podem estar implicados. Sugerese a necessidade da confirmação da presença do gene blaKPC. O ertapenem, portanto, mostrou-se fraco preditor para discriminar cepas produtoras de Klebsiella pneumoniae carbapenemase.

Humans , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/biosynthesis , Disk Diffusion Antimicrobial Tests/methods , Klebsiella pneumoniae/enzymology , beta-Lactamases/biosynthesis , beta-Lactams/pharmacology , beta-Lactam Resistance , Brazil , Bacterial Proteins/analysis , Gram-Negative Bacteria/isolation & purification , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Polymerase Chain Reaction , Predictive Value of Tests , beta-Lactamases/analysis
Rev. argent. microbiol ; 44(4): 290-302, dic. 2012. ilus
Article in Spanish | LILACS | ID: lil-663679


Se investigó el desempeño de diversos métodos fenotípicos para la detección de carbapenemasas KPC en 44 aislamientos de Klebsiella pneumoniae con sensibilidad disminuida a los carbapenems, 30 productores y 14 no productores de KPC. Se determinó la sensibilidad a imipenem, meropenem y ertapenem por dilución en agar y difusión por discos. Se evaluaron los siguientes métodos fenotípicos: sinergia entre discos de carbapenems y discos con los inhibidores amoxicilina/ácido clavulánico (AMC ) y ácido aminofenilborónico (APB); inhibición por "discos combinados" (según una técnica de predifusión diseñada a tal fin en nuestro laboratorio), comparando el efecto de los carbapenems solos o asociados con los inhibidores mencionados; y el test de Hodge modificado, para el cual se propone una lectura cuantificada. El método de Hodge detectó todos los aislamientos KPC positivos con los tres carbapenems evaluados, mientras que fue negativo para todos los aislamientos KPC negativos con imipenem y meropenem, y produjo dos resultados falsos positivos con ertapenem. Cuantificando la lectura de este método se pudieron discriminar objetivamente los resultados verdaderos positivos (≥ 8 mm) de los falsos positivos (< 5 mm). Se observó sinergia entre carbapenems y APB con todos los aislamientos KPC positivos, aunque esto requirió ajustar la distancia entre discos. En los aislamientos KPC negativos no se observó sinergia entre carbapenems y APB. Empleando el método con discos combinados con imipenem o meropenem más APB se detectaron la mayoría de los aislamientos KPC positivos y no se observaron falsos positivos. Por el contrario, las combinaciones carbapenem más AMC no fueron sensibles ni específicas.

We evaluated phenotypic methods for the detection of KPC carbapenemases in 44 clinical isolates of K. pneumoniae having reduced susceptibility to carbapenems, 30 of which were KPC-positive and 14 KPC-negative. Both the agar dilution and disk diffusion methods were performed for imipenem, meropenem and ertapenem. The following phenotypic methods were assayed: the double disk synergy test, using boronic acid or clavulanic acid as inhibitors, "combined" disks of carbapenem plus inhibitor (boronic acid, clavulanic acid and both boronic plus clavulanic acid), by using a pre-diffusion technique and the modified Hodge test. The double disk diffusion test using boronic acid could detect all KPC-positive isolates, but adjustment of disk distance was necessary for achieving such performance. The simulation of combined disks by our pre-diffusion technique detected all KPCpositive strains for all 3 carbapenems when using boronic acid as inhibitor, clavulanic acid was less susceptible and specific as compared with boronic acid. The modified Hodge test using any carbapenem was clearly positive for all KPC-producing isolates. This test was negative for all KPC-negative strains when imipenem or meropenem were used, but 2/14 isolates yielded a weak positive result when using ertapenem. By measuring the enhanced growth of E. coli ATCC 25922 observed in this test, we could objectively discriminate true-positive (≥ 8 mm) from false-positive results (< 5 mm). Our results show that the use of phenotypic methods is effective for the rapid detection of KPC producers in K. pneumoniae isolates with reduced susceptibility to carbapenems.

Bacterial Proteins/analysis , Bacterial Proteins/genetics , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , beta-Lactamases/analysis , beta-Lactamases/genetics , Phenotype
Rev. Soc. Bras. Med. Trop ; 45(6): 707-712, Nov.-Dec. 2012. ilus, graf, tab
Article in English | LILACS | ID: lil-661071


INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options. METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL) genes, and clonal relationship among isolates. RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive) and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates) were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29) were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13) were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles. CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

INTRODUÇÃO: A emergência de mecanismos de resistência aos carbapenêmicos em Pseudomonas aeruginosa tem se destacado devido ao amplo espectro de degradação de antimicrobianos, reduzindo as opções terapêuticas. MÉTODOS: Sessenta e um isolados de P. aeruginosa procedentes de cinco hospitais públicos de Recife, Pernambuco, Brasil, entre 2006 e 2010, foram analisadas, com o objetivo de avaliar o perfil de virulência, resistência aos antimicrobianos, a presença de genes metalo-β-lactamase (MBL) e a relação clonal entre os isolados. RESULTADOS: Foi observada uma elevada produção de fatores de virulência na amostra (34,4% colônias mucoides; 70,5% piocianina; 93,4% gelatinase e 72,1% hemolisina), bem como um elevado percentual de resistência (4,9% isolados panresistentes e 54,1% multirresistentes). Dentre os 29 isolados resistentes ao imipenem e/ou ceftazidima, 44,8% (13/29) apresentaram MBL por meio da pesquisa fenotípica, e destes, 46,2% (6/13) foram positivos para o gene blaSPM-1, não havendo detecção dos genes blaIMP e blaVIM. A tipagem molecular revelou 21 perfis genéticos dos quais sete foram detectados em hospitais e períodos distintos, e dos isolados blaSPM-1 positivos, três apresentaram o mesmo perfil clonal e foram procedentes do mesmo hospital, enquanto que os outros três isolados blaSPM-1 positivos apresentaram perfis clonais distintos. CONCLUSÕES: Estes resultados revelam que a P. aeruginosa é capaz de acumular diferentes fatores de virulência e resistência, dificultando o tratamento das infecções. A identificação de genes blaSPM-1 e disseminação de clones sugere a necessidade de aplicação mais rigorosa de medidas de controle de infecção nos hospitais de Recife, visando reduzir custos e danos provocados por este tipo de infecção.

Humans , Drug Resistance, Multiple, Bacterial , Pseudomonas aeruginosa/drug effects , Virulence Factors/analysis , beta-Lactamases/biosynthesis , Anti-Bacterial Agents/pharmacology , Brazil , Electrophoresis, Gel, Pulsed-Field , Microbial Sensitivity Tests , Molecular Typing , Phenotype , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/isolation & purification , beta-Lactamases/analysis
Indian J Med Microbiol ; 2012 Oct-Dec; 30(4): 456-461
Article in English | IMSEAR | ID: sea-144010


Background: One leading factor responsible for resistance in Acinetobacter baumannii, an important opportunist in health care institutions globally, is the production of carbapenamases like metallo-β-lactamases (MBLs), which hydrolyze a variety of β-lactams including penicillin, cephalosporins and carbapenems. However, neither any standard guidelines are available nor any method has been found to be perfect for their detection. Various methods have shown discordant results, depending upon the employed methodology, β-lactamase substrate and MBL inhibitor used. This study aims to evaluate two phenotypic methods against PCR as gold standard among carbapenem resistant A. baumannii for identifying MBL producers. Materials and Methods: A total of 130 A. baumannii were screened for imipenem and meropenem resistance by Kirby-Bauer disc diffusion method. Phenotypic expression of MBL was detected by EDTA-imipenem-microbiological (EIM) assay and extended EDTA disc synergy (eEDS) test and presence of bla-IMP and bla-VIM was detected by PCR in all the carbapenem resistant isolates. Results: Of the 43 imipenem and/or meropenem resistant A. baumannii isolates, 4 (9.3%) were found to be MBL producers by EIM and 3 (6.97%) by eEDS. Only bla-VIM gene was detected in 7 (16.28%) by PCR. In addition EIM detected 14 (32.56%) carbapenem resistant non-metallo enzyme producers. Conclusion: Of the two MBL genes targeted, bla-VIM was only detected and that too in isolates resistant to both imipenem and meropenem. Further, EIM was useful in differentiating MBL from non-metalloenzymes producers.

Acinetobacter baumannii/metabolism , Acinetobacter baumannii/physiology , Biological Assay/methods , Microbial Sensitivity Tests/methods , Polymerase Chain Reaction/methods , beta-Lactamases/analysis , beta-Lactamases/classification , beta-Lactamases/genetics , beta-Lactams/physiology
Braz. j. microbiol ; 43(2): 766-769, Apr.-June 2012. tab
Article in English | LILACS | ID: lil-644494


Emerging antimicrobial resistance rates and Extended-spectrum beta-lactamase producing Escherichia coli recovered from urinary tract infections (UTI) is an increasing problem in specific regions, limiting therapeutic options. One hundred E. coli isolates causing UTI in patients with age from 2 months to 12 years admitted at CMC in the period of April 2009 to March 2010 were tested for antibiotic susceptibility using the disk diffusion method. Surprisingly high resistance rates were recorded for E. coli against TMP/SMX (84%), cefalotin (66%), cefuroxime (50%), cefixime (50%) and ceftriaxone (45%). Antimicrobial susceptibility of E. coli isolates was followed by meropenem (98%), amikacin (95%), nitrofurantoin (91%) and gentamicin (68%). Extended spectrum beta-lactamase production, was observed in 32% of community and 42% of nosocomial isolates. The results of this study and numerous observations regarding the increasing resistance to these antibiotics, in several countries, emphasize the need for local population-specific surveillance for guiding empirical therapy for UTI in children.

Humans , Infant, Newborn , Adolescent , Diagnostic Techniques and Procedures , Disease Susceptibility , Drug Resistance, Microbial , Escherichia coli Infections , Escherichia coli/isolation & purification , beta-Lactamases/analysis , beta-Lactamases/isolation & purification , Enzyme Activation , Methods , Patients , Surveillance , Urine