Resistencia de cepas de Staphylococcus aureus aislados en ambientes nosocomiales / Resistance of Staphylococcus aureus strains isolated in nosocomial environments / Resistência de cepas de Staphylococcus aureus isoladas em ambientes nosocomiais

Staphylococcus aureus es un microorganismo que posee características particulares de virulencia y resistencia a los antibióticos. Las infecciones que produce, ocurren mayormente en personas inmunodeprimidas que podrían presentar severas consecuencias, a pesar de la terapia antimicrobiana. Objetivo. Determinar la resistencia de Staphylococcus aureus aislados en ambientes nosocomiales mediante métodos convencionales y moleculares. Materiales y métodos. el estudio presenta un enfoque cuantitativo, investigación de campo, observacional de corte transversal. Se analizó 200 muestras de aislados de ambientes nosocomiales mediante métodos fenotípicos (antibiograma por la técnica de Kirby Bauer) y genotípicos (genes de resistencia blaZ, mecA, y vanA por PCR punto final). Resultados. Los resultados de las pruebas de susceptibilidad antibiótica mostraron que el 100% de las cepas de S. aureus aisladas, fueron resistentes a oxacilina y penicilina g; y sensibles a vancomicina. Conclusiones. los resultados obtenidos en este estudio evidenciaron la presencia de SARM en las diferentes áreas hospitalarias, constituyéndose en un factor de riesgo de transmisión horizontal entre el personal sanitario y los pacientes, razón por la cual es de gran importancia evaluar su prevalencia y establecer medidas rigurosas de prevención y control de su diseminación, para disminuir el riesgo de nuevas infecciones.

Staphylococcus aureus is a microorganism with particular characteristics of virulence and resistance to antibiotics. The infections it produces occur mostly in immunocompromised individuals who may present severe consequences, despite antimicrobial therapy. Objective. To determine the resistance of Staphylococcus aureus isolated in nosocomial environments by conventional and molecular methods. Materials and methods. The study presents a quantitative, field research, observational, cross-sectional approach. Two hundred samples of isolates from nosocomial environments were analyzed by phenotypic (antibiogram by Kirby Bauer technique) and genotypic (resistance genes blaZ, mecA, and vanA by endpoint PCR) methods. Results. The results of the antibiotic susceptibility tests showed that 100% of the S. aureus strains isolated were resistant to oxacillin and penicillin g; and sensitive to vancomycin. Conclusions. the results obtained in this study showed the presence of MRSA in the different hospital areas, constituting a risk factor for horizontal transmission between health personnel and patients, which is why it is of great importance to evaluate its prevalence and establish rigorous measures for the prevention and control of its dissemination, in order to reduce the risk of new infections.

O Staphylococcus aureus é um microorganismo com características particulares de virulência e resistência a antibióticos. As infecções ocorrem principalmente em indivíduos imunocomprometidos que podem ter conseqüências graves, apesar da terapia antimicrobiana. Objetivo. Para determinar a resistência de Staphylococcus aureus isolado em ambientes nosocomiais usando métodos convencionais e moleculares. Materiais e métodos. O estudo apresenta uma abordagem quantitativa, pesquisa de campo, observacional, transversal. Duzentas amostras de isolados de ambientes nosocomiais foram analisadas pelos métodos fenotípico (antibiograma pela técnica de Kirby Bauer) e genotípico (genes de resistência blaZ, mecA, e vanA pelo método de PCR de ponto final). Resultados. Os resultados dos testes de suscetibilidade aos antibióticos mostraram que 100% das cepas de S. aureus isoladas eram resistentes à oxacilina e penicilina g; e sensíveis à vancomicina. Conclusões. Os resultados obtidos neste estudo mostraram a presença de MRSA nas diferentes áreas hospitalares, constituindo um fator de risco para a transmissão horizontal entre o pessoal de saúde e os pacientes, razão pela qual é de grande importância avaliar sua prevalência e estabelecer medidas rigorosas para a prevenção e controle de sua disseminação, a fim de reduzir o risco de novas infecções.

Vigilancia epidemiológica de Staphylococcus aureus y resistencia antibiótica en ambientes nosocomiales / Epidemiological surveillance of Staphylococcus aureus and the antibiotic resistence in nosocomial environments / Vigilância epidemiológica de Staphylococcus aureus e resistência a antibióticos em ambientes nosocomiais

Uno de los microrganismos más importantes en Infecciones Asociadas a la Atención en Salud (IAAS) es Staphylococcus aureus, una bacteria aerobia Gram positiva, resistente a diferentes condiciones ambientales. Objetivo. Identificar Staphylococcus aureus y su resistencia a los principales antibióticos Betalactámicos, aislada en áreas inertes. Materiales y métodos. Se realizó análisis fenotípico, antibiograma y métodos moleculares como: Extracción de ADN mediante Lisis Alcalina, identificación molecular para la amplificación de los genes tanto de identificación de la bacteria (nucA y femB), como de resistencia de antibióticos (blaZ, mecA y vanA) mediante PCR punto final, la separación de los amplicones se realizó mediante electroforesis en gel de Agarosa, los productos de la PCR se revelaron mediante la utilización de transiluminador UV. Resultados. De 200 muestras tomadas se obtuvo dos muestras positivas (1%) para Staphylococcus aureus, con el 100% de resistencia a penicilina y sensible a todos los demás antibióticos testeados. Conclusiones. La identificación de la bacteria y su resistencia hoy en día se realiza mayormente mediante métodos moleculares, lo cual no descarta la identificación fenotípica que, en este caso, determinó resultados importantes como lo es la prueba D-test positivo. La resistencia a fármacos betalactámicos se considera como un serio problema de salud, por lo tanto, se requiere de una vigilancia epidemiológica constante.

Staphylococcus aureus is one of the most important microorganisms related to Health Care Associated Infections (HCAI), it is a Gram-positive aerobic bacterium, highly resistant to the outer environment. Objective. Identify Staphylococcus aureus and its resistance to the most common beta-lactam antibiotics in isolated, inert areas. Materials and methods. Phenotypic analysis, antibiogram and molecular testing such as: DNA extraction by Alkaline Lysis, molecular identification for the amplification of genes both for identification of Staphylococcus aureus (nucA and femB), and for antibiotic resistance (blah, mega and vanA) by PCR, the disassociation of the amplicons was preforming by Agarose gel electrophoresis and results were shown in a UV translluminator. Results. From the 200 samples taken, two showed positive (1%) for Staphylococcus aureus, with 100% penicillin-resistant and sensitive to all other antibiotics tested. Conclusions. Nowadays identification of the bacteria and its resistance is carried out mostly through molecular testing, ruling out phenotypic identification, which in this case it determined important results such as the positive D-test. Resistance to beta-lactam drugs is considered a serious health issue, therefore it requires a safer epidemiological vigilance, an increase in sensitivity testing and the accuracy of results. It is recommended to carry out the D-test in laboratories to identify resistance mechanisms and to guide health personnel to select the best option regarding specific and effective treatment for patients affected with MRSA.

Um dos microrganismos mais importantes em Infecções Associadas à Saúde (HAIs) é o Staphylococcus aureus, uma bactéria aeróbica gram-positiva resistente a diferentes condições ambientais. Objetivo. Identificar Staphylococcus aureus e sua resistência aos principais antibióticos beta-lactam, isolados em áreas inertes. Materiais e métodos. Análise fenotípica, antibiograma e métodos moleculares foram realizados tais como: extração de DNA por Alkaline Lysis, identificação molecular para a amplificação dos genes tanto da identificação da bactéria (nucA e femB), e resistência a antibióticos (blaZ, mecA e vanA) pelo ponto final da PCR, a separação dos amplicons foi realizada por eletrofose em gel de Agarose, os produtos PCR foram revelados usando transiluminador UV. Resultados. De 200 amostras colhidas, duas amostras positivas (1%) foram obtidas para o Staphylococcus aureus, com 100% de resistência à penicilina e sensível a todos os outros antibióticos testados. Conclusões. A identificação da bactéria e sua resistência hoje é realizada principalmente por métodos moleculares, o que não exclui a identificação fenotípica que, neste caso, determinou resultados importantes como o teste D positivo. A resistência aos medicamentos beta-lactam é considerada um grave problema de saúde, portanto, é necessária uma vigilância epidemiológica constante.

Prevalence of blaZ Gene and Performance of Phenotypic Tests to Detect Penicillinase in Staphylococcus aureus Isolates from Japan

Guidelines recommend that clinical laboratories perform phenotypic tests (nitrocefin-based test and penicillin 10-U [P10] or 1-U [P1] zone edge tests) to detect penicillinase in Staphylococcus aureus isolates. This study aimed to assess the prevalence of blaZ encoding penicillinase and perform various phenotypic tests in S. aureus isolates from Japan. We prospectively collected 200 methicillin-susceptible S. aureus isolates from June 2015 to January 2016 and performed six phenotypic tests (nitrocefin-based test, P10 zone edge test/P10 diffusion test, penicillin 2-U [P2] zone edge test/P2 diffusion test, and cloverleaf test) on each sample. We confirmed the presence of blaZ (two blaZ-positive isolates) using PCR. Using blaZ PCR as a standard, we observed a low sensitivity (50%) and positive predictive value (PPV, 50%) of the nitrocefin-based test, low PPV (18.2%) of the P10 zone edge test, low sensitivity (50%) of the P10 diffusion test, low PPV (50% and 22.2%) of the P2 zone edge test and P2 diffusion test, respectively, and low sensitivity (50%) of the cloverleaf test. These data suggest a low performance (sensitivity and PPV) of these six phenotypic tests because of the low prevalence (1%) of blaZ in S. aureus isolates from Japan.

Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase

Abstract Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28 mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29 mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

Antibiotic resistance profiles of Staphylococcus pseudintermedius isolated from dogs and cats

Aims: Antibiotic resistance in Staphylococcus pseudintermedius is increasing gradually towards those antibiotics that are frequently used leading to limited therapeutic options due to multidrug resistance. The objectives of the study were to investigate the antibiotic resistance profiles of S. pseudintermedius isolates from pet and stray dogs and cats in Selangor, Malaysia and to detect the resistance genes (mecA and BlaZ) within the isolates. Methodology and results: A total of 200 stray and pet dogs and cats were sampled. The samples were cultured onto Mannitol Salt agar and all the presumptive colonies were subcultured, then identified using biochemical tests and confirmed by PCR assay targeting the nuc gene. The isolates were subjected to antibiotic susceptibility test against 12 antibiotics. Twenty three isolates (11.5%) were positive to S. pseudintermedius (stray cats, 11/50; stray dogs, 9/50; pet dogs, 3/50 and pet cats, 0/50). One hundred percent (100%) of the S. pseudintermedius isolates were found to be resistant to penicillin, erythromycin and tetracycline while they showed 100% susceptible to oxacillin, amoxicillinclavulanic acid, gentamicin, chloramphenicol, vancomycin, ciprofloxacin, enrofloxacin, cephalexin and rifampicin. The blaZ gene which codes for β-lactamases production was found in all of the isolates that were resistant to penicillin but not to methicillin. Conclusion, significance and impact of study: A high number of S. pseudintermedius from dogs and cats developed antibiotic resistance which is a public health concern.

The inhibition effect of Chlorpromazine against the β-lactam resistance of MRSA

OBJECTIVE@#To investigate the gene related to β-lactam resistance and to confirm the mechanism about a synergy effect between CPZ and β-lactam antibiotics.@*METHODS@#To measure antibacterial activity, we performed a minimum inhibitory concentration (MIC) and synergy test. Transmission electron microscopy (TEM) was used in morphological analysis. To analyze gene expression, we conducted reverse transcriptase polymerase chain reaction (PCR).@*RESULTS@#We confirmed a synergy effect between CPZ and β-lactam antibiotics. Furthermore, we observed that CPZ affect the cell envelope of MRSA by using TEM. At the gene level, CPZ reduced the expression of resistance genes.@*CONCLUSIONS@#Through this result, we hypothesize that a decrease of resistance factor expressions was caused by CPZ because it disrupts the activity of a sensor protein located in the cell membrane.

The inhibition effect of Chlorpromazine against the β-lactam resistance of MRSA

Objective To investigate the gene related to β-lactam resistance and to confirm the mechanism about a synergy effect between CPZ and β-lactam antibiotics. Methods To measure antibacterial activity, we performed a minimum inhibitory concentration (MIC) and synergy test. Transmission electron microscopy (TEM) was used in morphological analysis. To analyze gene expression, we conducted reverse transcriptase polymerase chain reaction (PCR). Results We confirmed a synergy effect between CPZ and β-lactam antibiotics. Furthermore, we observed that CPZ affect the cell envelope of MRSA by using TEM. At the gene level, CPZ reduced the expression of resistance genes. Conclusions Through this result, we hypothesize that a decrease of resistance factor expressions was caused by CPZ because it disrupts the activity of a sensor protein located in the cell membrane.

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis / Detecção de beta-lactamase em Staphylococcus aureus e Staphylococcus coagulase negativa isolados de mastite bovina

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Os objetivos do presente estudo foram avaliar a presença/produção de beta-lactamases por ambos os métodos fenotípicos e genotípicos, verificar se os resultados são dependentes do tipo de bactéria (Staphylococcus aureus contra Staphylococcus coagulase negativa - CNS) e verificar a concordância entre os testes. Um total de 200 amostras bactérianas oriundas de 21 rebanhos distintos foram incluídos, sendo 100 CNS e 100 S. aureus. A presença/detecção de beta-lactamase foi realizada por diferentes testes (PCR, teste trevo (clover leaf test) - CLT, disco Nitrocefin e resistência in vitro à penicilina). Os resultados de todos os testes não foram dependentes do tipo de bactérias (CNS ou S. aureus). Vários isolados de S. aureus produtores de beta-lactamase eram de um mesmo rebanho. Testes fenotípicos excluindo resistência in vitro à penicilina mostraram uma forte associação medida pelo coeficiente kappa para ambas as espécies de bactérias. Nitrocefina e CLT são testes mais confiáveis para detectar a produção de beta-lactamase em estafilococos.

Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis / Resistência antimicrobiana e detecção dos genes mec e blaZ em Staphylococcus coagulase-negativos isolados de mastite bovina

The present study evaluated the pheno- and genotypical antimicrobial resistance profile of coagulase-negative Staphylococcus (CNS) species isolated from dairy cows milk, specially concerning to oxacillin. Of 100 CNS isolates, the S. xylosus was the prevalent species, followed by S. cohnii, S. hominis, S. capitis and S. haemolyticus. Only 6% were phenotypically susceptible to the antimicrobial agents tested in disk diffusion assay. Penicillin and ampicillin resistance rates were significantly higher than others antimicrobials. Four isolates were positive to mecA gene (4%), all represented by the S. xylosus species. The blaZ gene was detected in 16% of the isolates (16/100). It was noticed that all mecA + were also positive to this gene and the presence of both genes was correlated to phenotypic beta-lactamic resistance. We conclude that CNS species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic and genotypic tests, which has implications for treatment and management decisions.

O presente estudo avaliou o perfil fenogenotípico de resistência aos antimicrobianos em espécies de Staphylococcus coagulase-negativo (ECN) isoladas do leite de vacas com mastite, em especial considerando a oxacilina. Dos 100 isolados de ECN, S.xylosus foi a espécie predominante, seguida por S. cohnii, S. hominis, S. capitis and S. haemolyticus Apenas 6% dos isolados foram fenotipicamente suscetíveis aos agentes antimicrobianos testados no ensaio de difusão em disco. O percentual de resistência à penicilina e ampicilina foi significativamente maior que aos outros antimicrobianos. Quatro isolados foram positivos para o gene mecA (4%), sendo todos representados pela espécie S.xylosus. O gene blaZ foi detectado em 16% dos isolados (16/100), sendo todos mecA positivos e a presença de ambos os genes foi correlacionada com a resistência fenotípica aos beta-lactâmicos. Foi possível concluir que as espécies de ECN provenientes de leite bovino apresentaram distintos perfis de resistência antimicrobiana, avaliados por testes fenotípicos e genotípicos, podendo dificultar a adoção de medidas de tratamento e manejo dos animais.

Detection of mecA, blaZ and aac(6')-aph(2") gene of staphylococci by PCR / 대한임상병리학회지

BACKGROUND: Staphylococci are major nosocomial pathogens and reveal an increase in resistant strains such as methicillin-resistant Staphylococcus aureus. For treatment of infection and prevention of dissemination, rapid and reliable identification methods are required but the conventional bacterial identification and susceptibility tests require at least 24 hours. In this study, we evaluated the polymerase chain reaction (PCR) of the antibiotic resistant genes by comparing with the disk diffusion susceptibility test for the detection of resistance to penicillin, oxacillin and gentamicin. METHODS: A hundred-thirty-five staphylococci including 95 S. aureus and 40 S. epidermidis were from clinical specimens from June to December 2000. Antimicrobial susceptibility tests were done using the NCCLS disk diffusion method. PCRs were performed with primer sets specific for mecA, blaZ and aac(6')-aph(2"). The species-specific PCR was also used to identify S. aureus and S. epidermidis. RESULTS: All four penicillin-susceptible staphylococci were negative for blaZ and 108 of 131 penicillin resistant-staphylococci were positive for blaZ. The concordance rate for PCR of the blaZ gene and penicillin disk diffusion test was 83.0%. 110 of 115 oxacillin-resistant staphylococci were positive for mecA and all five mecA negative oxacillin-resistant strains were positive for blaZ and have the phenotype beta-Lactamase hyperproducer. One of the oxacillin-susceptible S. aureus was positive for mecA. The concordance rate of PCR for the mecA gene and oxacillin disk diffusion test and those of the aac(6')-aph(2") gene and gentamicin disk diffusion test was 95.6% and 97.8%, respectively. CONCLUSTIONS: The disk diffusion tests misdiagnosed 25% of the mecA negative staphylococci as methicillin-resistant staphylococci (MRS) and lost one of the mecA positive strain. We considered that the detection of the mecA and blaZ gene using the PCR was more useful than the disk diffusion test for detection of methicillin-resistant staphylococci.