Development of a PCR Test for the Identification of Staphylococcus intermedius Based on the 16S rDNA Sequence.

Wakita, Yoshihisa; Kawano, Junichi; Shimizu, Akira; Hájek, Václav; Tomisaka, Emi; Yasuda, Ryo; Matsuo, Eisuke

doi:10.1292/jvms.64.603

Cited by 9 publications

(7 citation statements)

References 18 publications

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“…schleiferi, as reported by Frank et al [5], it is easy to differentiate these two subspecies on the basis of their tube coagulase test [8] and urease production [21]. Previously, we [22] designed a primer set for PCR specific for 16S rDNA in order to identify the strains of canine pathogenic S. intermedius. These two primer sets for PCR Fig.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Staphylococci from External Auditory Meatus of Dogs with or without Otitis Externa with Special Reference to Staphylococcus schleiferi subsp. coagulans Isolates

et al. 2005

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ABSTRACT. Staphylococci were isolated from the external auditory meatus in 14 (48.3%) of 29 dogs affected with otitis externa (OE dogs) and 28 (68.3%) of 41 dogs without OE (non-OE dogs). Twenty-two OE isolates were identified as belonging to 12 species, and 42 non-OE isolates were identified as belonging to 13 species. The predominant species found in both OE and non-OE isolates were S. intermedius, and S. epidermidis. Thirty-eight (59.4%) of 64 isolates were resistant to one or more of the 17 antimicrobial agents tested. Resistance to PCG and ABPC was most frequent. S. schleiferi subsp. coagulans, a recent etiologic agent of canine OE, was isolated from OE and non-OE dogs. All of the 5 S. schleiferi subsp. coagulans isolates showed typical characteristics. No clear difference in the extracellular enzyme or toxin profiles, nor in the PFGE patterns, was demonstrated between the OE and non-OE isolates of S. schleiferi subsp. coagulans. A new PCR primer set specific for 16S rDNA was designed to identify strains of S. schleiferi subsp. coagulans. The amplified fragment was detected in all of the 5 isolates as well as the type strain GA 211 (=JCM 7470) and a reference strain GA 11, but was not detected in any strains of the related species, S. aureus, S. intermedius and S. hyicus. The PCR may allow a simple, rapid and precise identification of S. schleiferi subsp. coagulans, in addition to the standard tube test for free coagulase. Otitis externa (OE) is common in dogs and is a disease of multifarious etiology. The yeast and bacterium most frequently isolated from OE dogs are Malassezia pachydermatis and Staphylococcus intermedius [2,11,20,23], respectively. Staphylococci including S. intermedius are present in the nasal vestibulum, external auditory meatus, intestinal and urinogenital tracts, anal mucosa, and on the skin surface of dogs. Knowledge of the composition of the normal staphylococcal flora in the external auditory meatus is an important factor in determining the etiology and epidemiology of staphylococcal OE in dogs. However, the ecology of the staphylococci inhabiting the external auditory meatus of healthy dogs has not yet been studied sufficiently.Determination of the susceptibility of Staphylococcus species to antimicrobial agents is of great importance in the selective use of chemotherapeutics, the evaluation of new antimicrobial agents, and in the development of drug resistance through continuous use of antimicrobial agents against field isolates. However, there is little information concerning the antimicrobial susceptibility patterns of Staphylococcus species isolated from OE and non-OE dogs in Japan [18].Recently, coagulase-positive S. schleiferi subsp. coagulans has also been isolated from the external auditory meatus of OE dogs [8]. Subsequently, the organism was cultured from dogs with pyoderma [1,5,7] and from a finger-wound infection in a human patient [21]. Recognition of the clinical importance of S. schleiferi subsp. coagulans in veterinary and human medicine is growing. To our knowledge...

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Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Staphylococci from External Auditory Meatus of Dogs with or without Otitis Externa with Special Reference to Staphylococcus schleiferi subsp. coagulans Isolates

et al. 2005

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“…c Descrito por Forsman et al (1997). d Descrito por Wakita et al (2002 Com a identificação da espécie S. chromogenes pelo sequenciamento do rDNA 16S, a prova da coagulase em tubo foi refeita para estes isolados, confirmando a positividade em alguns deles. Nestes últimos isolados deverá ser pesquisada a atividade de coagulação, que pode também ser causada por interferência de proteases, conforme estudos realizados por Wegrzynowicz et al (1979), Chomarat & Flandrois (1984) e Vandenesch et al (1994.…”

Section: Resultsunclassified

“…Entretanto, falhou em identificar outros dois isolados identificados como S. hyicus pelo sequenciamento. Portanto neste estudo os oligonucleotídeos iniciadores descritos por Wakita et al (2002) para S. intermedius não foram específicos, pois amplificaram também S. chomogenes; e os empregados para S. hyicus (Forsman et al 1997) não foram sensíveis, pois falharam na identificação de dois isolados de S. hyicus.…”

Section: Resultsunclassified

“…Para identificação de S. aureus foram usados oligonucleotídeos descritos por Mehrotra et al (2000), que amplificam um fragmento do gene femA, de 132 pares de bases (pb). Para a identificação de S. intermedius foram usados oligonucleotídeos descritos por Wakita et al (2002), que amplificam um fragmento do rDNA 16S, de 901 pb. E para a identificação de S. hyicus foram usados oligonucleotídeos descritos por Forsman et al (1997), que amplificam um fragmento do rDNA 16S-23S, de 250 pb.…”

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Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero Staphylococcus isoladas de mastite bovina

Lange¹,

Brito²,

Brito

et al. 2011

Pesq. Vet. Bras.

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O objetivo deste trabalho foi identificar espécies de Staphylococcus (n=100) isoladas de mastite em rebanhos bovinos do Estado de Minas Gerais. Para esta finalidade foram utilizadas reações de PCR empregando oligonucleotídeos iniciadores descritos anteriormente para amplificar genes específicos de S. aureus (femA), S. intermedius (rDNA 16S) e S. hyicus (rDNA 16S-23S) e o sequenciamento do rDNA 16S. De acordo com as reações de PCR, 83 isolados foram identificados como S. aureus, 13 isolados como S. intermedius, dois como S. hyicus e dois isolados não foram identificados. Foram submetidos ao sequenciamento do rDNA 16S seis isolados identificados como S. aureus e os 17 restantes. Os seis isolados identificados como S. aureus confirmaram essa identificação. Dos outros 17 isolados, 13 foram identificados como S. chromogenes e quatro como S. hyicus, com similaridade igual ou superior a 99%. Baseando-se nos resultados da reação de PCR do gene femA e do sequenciamento do rDNA 16S, foram identificados 83 S. aureus, 13 S. chromogenes e quatro S. hyicus. Neste estudo os oligonucleotídeos iniciadores empregados na reação de PCR para S. intermedius não foram específicos, pois amplificaram também S. chromogenes; e os empregados na reação de PCR para S. hyicus não foram sensíveis, pois falharam na identificação de dois isolados de S. hyicus. A identificação definitiva das duas últimas espécies somente foi possível pelo sequenciamento do rDNA 16S.

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“…Biochemical activities of the isolates were examined using API Staph test (bioMerieux). Additionally, the PCR assay based on the amplification of 16S rRNA gene of S. intermedius was used for confirmation of identification of SIG strains (Wakita et al 2002). Staphylococcus intermedius ATCC 29663 and methicillin-resistant Staphylococcus aureus MIK-ROBANK 14.002 were used as the control form PCR and determination of MICs.…”

Section: Isolation and Identification Of Sig Strainsmentioning

confidence: 99%

Antibiotic resistance of canine Staphylococcus intermedius group (SIG) - practical implications

Chrobak¹,

Kizerwetter-Świda²,

Rzewuska³

et al. 2011

Polish Journal of Veterinary Sciences

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A total of 221 SIG strains were isolated from clinical samples of canine origin submitted to the Diagnostic Laboratory of the Division of Bacteriology and Molecular Biology at the Warsaw University of Life Sciences in Warsaw during the period 2006-2010. The aim of the study was to investigate the frequency of prevalence of methicillin-resistant SIG strains and to determine the MIC values of cephalotin, amoxicillin/clavulanic acid, ciprofloxacin, clindamycin, gentamicin, chloramphenicol, mupirocin for a collection of randomly selected 79 strains belonging to Staphylococcus intermedius group (SIG), including 23 mecA-positive and 56 mecA-negative strains. All isolates were identified as belonging to SIG based on their phenotypic properties and PCR amplification of S. intermedius-specific fragment of the 16S rRNA gene. The mecA gene was detected in 26 (12%) of 221 SIG strains. All tested mecA-negative SIG strains were susceptible to amoxicillin/clavulanic acid and cephalotin. One of the 56 mecA-negative SIG strains was resistant to ciprofloxacin, six (11%) to gentamicin. It was found that sixteen (29%) of 56 mecA-negative SIG strains were resistant to clindamycin. Most of the mecA-positive SIG strains were resistant to ciprofloxacin (96%), clindamycin (96%), and gentamicin (96%). Only one MRSIG strain was resistant to chloramphenicol. All examined mecA-positive SIG strains were found to be susceptible to mupirocin. Our results imply that staphylococcal multidrug resistance has become more prevalent, which could lead to difficulties in effective treatment. With some resistant strains the only therapeutic possibility are antimicrobial agents important in human medicine. New regulations for veterinary medicine concerning appropriate therapy of infections caused by multidrug-resistat staphylococci are needed.

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Development of a PCR Test for the Identification of Staphylococcus intermedius Based on the 16S rDNA Sequence.

Cited by 9 publications

References 18 publications

Isolation and Characterization of Staphylococci from External Auditory Meatus of Dogs with or without Otitis Externa with Special Reference to Staphylococcus schleiferi subsp. coagulans Isolates

Isolation and Characterization of Staphylococci from External Auditory Meatus of Dogs with or without Otitis Externa with Special Reference to Staphylococcus schleiferi subsp. coagulans Isolates

Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero Staphylococcus isoladas de mastite bovina

Antibiotic resistance of canine Staphylococcus intermedius group (SIG) - practical implications

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