Detection of coagulase gene in Staphylococcus aureus from several dairy farms in East Java, Indonesia, by polymerase chain reaction.

AIM: This study was conducted to study the coagulase (coa) gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle from three different regions in East Java Province, Indonesia. MATERIALS AND METHODS: A total of 160 raw milk samples collected in East Java Province, Indonesia, were screened for the presence of S. aureus. The presumptive isolates were confirmed by coa test. The confirmed S. aureus isolates were subjected to coa gene polymerase chain reaction. RESULTS: Of 160 different samples, 20 (12.5%) isolates of S. aureus were confirmed by positive coa test. Of 20 S. aureus isolates, 19 (95%) isolates carried coa gene. Six different genotypes of coa gene, i.e., 440 bp, 510 bp, 547 bp, 680 bp, 740 bp, and 820 bp were obtained. One coa genotypes, 510 bp (10 isolates) were observed in polymorphism to be more prevalent than the others, and the genotype was present in at least one isolates from every region. CONCLUSION: It can be concluded that coa gene is easily epidemiological tool for detection of variation strain from S. aureus.

Characterization of Staphylococcus spp. strains in milk from buffaloes with mastitis in Brazil: the need to identify to species level to avoid misidentification / Caracterização de cepas de Staphylococcus spp. isoladas de leite de búfalas com mastites em Brasil: a necessidade de se identificar até nível de espécie para evitar erros de identificação

Mastitis is an inflammation of the mammary gland that affects dairy cattle worldwide causing economic losses. Coagulase-negative staphylococci (CNS) are the predominant cause of this type of infection. We have recently showed that coagulase-positive staphylococci could be misidentified. So, the aim of this study was to characterize the Staphylococcus spp. strains initially classified as coagulase-negative Staphylococci, isolated from buffalo with subclinical mastitis. Milk of buffaloes with mastitis in herds was collected and 9 strains were identified as CNS by phenotypic tests. Molecular methodologies latter identified the strains as coagulase-negative Staphylococcus chromogenes (5), coagulase-positive Staphylococcus hyicus (2) and coagulase-positive Staphylococcus aureus (2). Our results strongly support the need to identify the isolates to a species level in order to avoid misidentification and to be aware of the classification using the coagulase test alone.(AU)

A mastite é uma inflamação da glândula mamária que afeta o gado leiteiro em todo o mundo, causando perdas econômicas. Staphylococcus coagulase-negativa (SCN) são a causa predominante desse tipo de infecção. Mostrou-se recentemente que Staphylococcus coagulase-positiva podem ser identificados erroneamente. Assim, o objetivo deste estudo foi caracterizar cepas de Staphylococcus spp. inicialmente classificados como Staphylococcus coagulase-negativa, isolados de búfalas com mastite subclínica. O leite de búfalas com mastite foi coletado, e nove cepas foram identificadas como SCN por testes fenotípicos. Metodologias moleculares identificaram as cepas como Staphylococcus chromogenes coagulase-negativa (5) Staphylococcus hyicus coagulase-positiva (2) e Staphylococcus aureus coagulase-positiva (2). Os resultados reforçam a necessidade de identificar as cepas em termos de espécie, a fim de se evitarem erros de identificação e estar atento à classificação utilizando o teste de coagulase sozinho.(AU)

First reported case of Staphylococcus condimenti infection associated with catheter-related bacteraemia.

We report a case of a patient who experienced a catheter-related bloodstream infection caused by Staphylococcus condimenti, which was first isolated from soy sauce mash. This is the first reported case of human infection. Although blood culture isolates and the catheter tip tube did not reveal coagulase or clumping factor, false-positive results were obtained from latex agglutination tests for clumping factor and protein A due to self-agglutination. Care is needed when performing only latex agglutination test without a coagulase test. Further studies are needed to determine the pathogenic potential of S. condimenti based on appropriate identification.

Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

Evaluation of direct tube coagulase test in diagnosing staphylococcal bacteremia.

BACKGROUND: Blood Stream Infections (BSIs) are one of the most common nosocomial infections encountered in a hospital. It occurs 2 to 7 times more often in ICU patients than in ward patients and is associated with high rates of mortality and morbidity. Staphylococci is the most commonly encountered blood culture isolates for which, rapid and reliable methods for detection are warranted. AIM: To study the efficiency of direct tube coagulase test (DTC) in comparison with slide/tube coagulase test for early detection of Staphylococcal bacteraemia. MATERIALS AND METHODS: This was a prospective study conducted in a tertiary care hospital, between January 2009 to December 2009. Two sets of blood cultures were obtained from each patient with a suspicion of Staphylococcal bacteremia. Both, direct tube coagulase test and conventional biochemical tests were done on the samples, to identify the species. STATISTICAL ANALYSIS USED: Cohen's kappa coefficient of agreement was used to detect the differences between the two modes of detection. RESULTS: Ninety four samples (out of 460) yielded the growth of Gram positive cocci in clusters. The commonest isolates were Staphylococcus aureus (42.5%), Staphylococcus epidermidis (36.2%), Staphylococcus haemolyticus (8.6%), Staphylococcus cohnii (4.3%). Cohen's kappa coefficient which implies the extent of agreement of the results between the Direct Tube Coagulase test and conventional methods, was 95.6% along with sensitivity of 95.5% and specificity of 100%. CONCLUSION: Direct Tube Coagulase test (DTC) can be used for early detection of Staphylococcal bacteremia. It shows an almost perfect agreement with conventional coagulase test.

Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis / Avaliação de uma chave de identificação simplificada para Staphylococcus coagulase-positivos isolados de mastite bovina

Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulase-positive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypic tests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and three microorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis.

Visando testar a eficiência de uma chave simplificada baseada em testes fenotípicos para a discriminação de Staphylocococcus coagulase positivos (SCP) isolados de infecções intramamárias de bovinos, 344 amostras destes microrganismos foram submetidas a testes fenotípicos e genotípicos. Estes consistiram na amplificação do gene femA, na observação de hemólise em ágar sangue, produção de acetoína e fermentação de maltose, manitol e trealose. Amostras que apresentaram resultado negativo na amplificação do gene femA ou que foram identificadas com não Staphylococcus aureus (S. aureus) por meio dos testes fenotípicos foram submetidas ao kit APISTAPH (Biomériux-França) para identificação mais precisa. Os testes fenotípicos utilizados na chave simplificada permitiram identificar 338 amostras (98,25%) como S. aureus, três amostras (0,86%) como Staphylococcus hyicus e três (0,86%) como Staphylococcus intermedius. Por meio da reação em cadeia da polimerase (PCR) 338 (98,25%) amostras foram identificadas como S. aureus, ratificando os resultados para 336 das 338 amostras identificadas por meio da chave fenotípica simplificada. Observou-se elevada concordância (98,83%) entre os resultados dos testes genotípicos e fenotípicos para a identificação de S. aureus, demonstrando a aplicabilidade da chave de identificação proposta para a discriminação deste microrganismo entre SCP isolados de casos de mastite bovina.

Identificación rápida de Staphylococcus aureus en hemocultivos por medio de la prueba directa de la coagulasa / Rapid identification of Staphylococcus aureus in blood cultures by means of the direct tube coagulase test

La diferenciación rápida del tipo de estafilococo hallado en los hemocultivos permite establecer tempranamente un tratamiento apropiado y evita el inicio de antibióticos de tipo glicopéptido. Objetivo: determinar el rendimiento de la prueba de la coagulasa, hecha directamente con el contenido de los viales de hemocultivos, en los cuales se detectaba crecimiento de cocos grampositivos compatibles con estafilococos. Métodos: en caso de observar en el vial de hemocultivo solo cocos grampositivos compatibles con estafilococos, se procedía a hacer la prueba directa de la coagulasa, interpretándola cuatro horas después. Se hacían subcultivo e identificación de la bacteria y se comparaba el resultado con el de la prueba directa. Resultados: se hicieron 1.518 pruebas de coagulasa directa de las que 411 (27,1%) fueron positivas y 1.107 (72,9%), negativas. Se cultivaron 446 cepas de S. aureus de las que 410 tuvieron la prueba de coagulasa directa positiva, para una sensibilidad del 91,9%. Solo en un vial de los 411 con la prueba positiva se aisló un Staphylococcus spp., coagulasa negativa, lo cual determinó una especificidad del 99,8%. El valor predictivo positivo fue 99,7% y el negativo, 96,7%. Conclusiones: los resultados confirman la utilidad de la prueba de la coagulasa directa en los viales de hemocultivos para diferenciar oportunamente el tipo de estafilococo cultivado. Se puede emplear como una prueba presuntiva para decidir el inicio o no del tratamiento empírico del paciente infectado por cocos grampositivos, al obtener el reporte preliminar de los hemocultivos.

Rapid differentiation of Staphylococcus spp. strains grown in bood cultures is the basis for early and appropriate treatment of S. aureus infections. Objective: To evaluate the usefulness of the direct tube coagulase test done from blood culture vials with presumptive growth of Staphylococci. Methods: Direct tube coagulase test was carried out if only clusters of gram-positive cocci were observed in the blood culture vials; interpretation was done four hours later and results were compared with those obtained with colonies from subcultures. Results: 1.518 direct tube coagulase tests were carried out; of them, 411 (27.1%) were positive and 1.107 (72.9%), negative. Out of the 446 strains of S. aureus that were isolated, 410 had positive direct tube coagulase test (sensitivity 91.9%). An additional strain of S. aureus was positive in the direct test but negative in the subculture (specificity 99.9%). Predictive values were 99.7% (positive) and 96.7% (negative). Conclusions: Our results confirm the usefulness of the direct tube coagulase test done from blood cultures to opportunely differentiate staphylococci grown in blood cultures. Information so obtained may be used to decide the beginning of empirical treatment.

Identification and Differentiation of Coagulase-Negative Staphylococcus aureus by Polymerase Chain Reaction.

Staphylococcus aureus is an etiological agent of a wide variety of human and animal infections. The majority of S. aureus are coagulase-positive; however, some may be atypical in that they do not produce coagulase. Incorrect identification of an isolate can impact implementation of effective treatment and/or control measures. In this study, polymerase chain reaction-based DNA fingerprinting was used to differentiate coagulase-positive Staphylococcus aureus (CPSA) from coagulase-negative Staphylococcus aureus (CNSA). A total of 29 CNSA and 50 CPSA were evaluated. PCR-based DNA fingerprinting differentiated CNSA from CPSA on the basis of visible observation and densitometric evaluation. The method is rapid and accurate, eliminating variability associated with conventional techniques.

Modification of the Conventional Procedure for the Test of Staphylococcal Coagulase.

The coagulase test is routinely used for the confirmation of suspect Staphylococcus aureus on Baird-Parker agar (BPA). Overnight (18 to 24 h) preincubation of suspect colonies in brain heart infusion (BHI) broth is generally practiced prior to the coagulase test. In order to shorten the test protocol, different preincubation times of S. aureus in BHI were evaluated. Stock cultures (257 strains) of S. aureus were subcultured on BPA, and single colonies of each strain on BPA were analyzed for coagulase activities by the conventional procedure except that the preincubation times in BHI were varied (0, 4, and 24 h). The formation of a clot was examined at 2-h intervals over a 6-h period and at 24 h, and any degree of clot formation (1+ to 4+) was considered a positive reaction. Low sensitivities were found for tests without preincubation in BHI prior to the enzyme test. However, for 4- and 24-h preincubation, there was no difference in the degree of clot formation and in the sensitivities of the coagulase tests with incubation periods from 6 (98.1%) to 24 h (99.6%). Compared with the conventional procedures which may need two days, the modified protocol (4-h preincubation in BHI) can identify a large majority (97.7-98.1 %) of suspect S. aureus on BPA within one working day.