Genetic Classification and Distinguishing of Staphylococcus Species Based on Different Partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf Gene Sequences

Otto-von-Guericke University, Clinical Microbiology, Leipziger Str. 44, Magdeburg, Germany.
Journal of clinical microbiology (Impact Factor: 3.99). 04/2008; 46(3):1019-25. DOI: 10.1128/JCM.02058-07
Source: PubMed


The analysis of 16S rRNA gene sequences has been the technique generally used to study the evolution and taxonomy of staphylococci.
However, the results of this method do not correspond to the results of polyphasic taxonomy, and the related species cannot
always be distinguished from each other. Thus, new phylogenetic markers for Staphylococcus spp. are needed. We partially sequenced the gap gene (∼931 bp), which encodes the glyceraldehyde-3-phosphate dehydrogenase, for 27 Staphylococcus species. The partial sequences had 24.3 to 96% interspecies homology and were useful in the identification of staphylococcal
species (F. Layer, B. Ghebremedhin, W. König, and B. König, J. Microbiol. Methods 70:542-549, 2007). The DNA sequence similarities
of the partial staphylococcal gap sequences were found to be lower than those of 16S rRNA (∼97%), rpoB (∼86%), hsp60 (∼82%), and sodA (∼78%). Phylogenetically derived trees revealed four statistically supported groups: S. hyicus/S. intermedius, S. sciuri, S. haemolyticus/S. simulans, and S. aureus/epidermidis. The branching of S. auricularis, S. cohnii subsp. cohnii, and the heterogeneous S. saprophyticus group, comprising S. saprophyticus subsp. saprophyticus and S. equorum subsp. equorum, was not reliable. Thus, the phylogenetic analysis based on the gap gene sequences revealed similarities between the dendrograms based on other gene sequences (e.g., the S. hyicus/S. intermedius and S. sciuri groups) as well as differences, e.g., the grouping of S. arlettae and S. kloosii in the gap-based tree. From our results, we propose the partial sequencing of the gap gene as an alternative molecular tool for the taxonomical analysis of Staphylococcus species and for decreasing the possibility of misidentification.

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    • "ntify coagulase-positive and coagulase-negative Staphylococcus spp. isolates of bovine mastitis at the species level. All type and reference strains were correctly identified. However, in three cases, S. capitis 35661, S. warneri 49454 and S. xylosus 29971 T , the fragment sequenced showed 99% or 100% similarity with the sequences of other species.Ghebremedhin et al. (2008)reported that some Staphylococcus taxa have the same 16S rRNA gene sequences in variable regions, including S. capitis subsp. urealyticus and S. caprae. According to the CLSI (2008), 16S rRNA provides a poor separation between S. capitis, S. caprae and S. epidermidis, as S. warneri shares approximately 98.7% identity with S. pasteuri and"
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    ABSTRACT: Bacteria of the genus Staphylococcus are one of the major pathogens causing bovine mastitis. In recent decades, resistance of this genus to oxacillin (methicillin) has been a matter of concern due to the possibility of reducing the effectiveness of mastitis treatments and the transfer of resistance determinants to other bacteria. Oxacillin resistance was studied in 170 staphylococci from bovine milk samples, including 79 Staphylococcus aureus and 91 coagulase-negative staphylococci (CNS). The susceptibility profile of 10 antimicrobial agents used in veterinary practice was determined by the Etest method. In addition to the Etest, the phenotypic characterization of oxacillin resistance was tested using the cefoxitin disk diffusion test. All isolates were screened by PCR to detect the mecA gene in 2 different regions of the gene. The isolates with an oxacillin minimum inhibitory concentration ≥0.5 µg/mL or resistant to cefoxitin were identified by sequencing a 536-bp fragment of the 16S rRNA gene. This group of isolates was also evaluated for the presence of blaZ and mecC genes. Molecular analysis of the mecA gene was carried out by typing of the staphylococcal cassette chromosome mec (SCCmec). The relatedness of the mecA-positive isolates was evaluated by macrorestriction of chromosomal DNA followed by pulsed-field gel electrophoresis. With the exception of penicillin and oxacillin, 86% of the isolates showed susceptibility to cephalothin, gentamicin, erythromycin, sulfonamide, trimethoprim-sulfamethoxazole, and tetracycline. All S. aureus isolates were susceptible to oxacillin, whereas 47% (n = 43) of the CNS isolates were resistant. The CNS isolates showed a higher resistance to cephalothin, erythromycin, tetracycline, and gentamicin in comparison with S. aureus. The mecA gene was only detected in 10 CNS isolates, identified as Staphylococcus epidermidis, and classified into 3 pulsotypes (A, B, and C) and 4 subtypes (A1, B1, B2, and B3). Among the isolates with an oxacillin resistance phenotype, 12 were positive for the blaZ gene, and 9 of them were mecA-positive. Two of the oxacillin-resistant isolates amplified the mecA homolog gene of Staphylococcus sciuri and none amplified mecC. Three SCCmec types, I, IV, and V, were found. Our results suggest that Staphylococcus epidermidis can be a reservoir for mecA for other Staphylococcus species. Studies investigating the molecular and phenotypic profile of antimicrobial resistance in staphylococcal species should be performed for controlling the spread of resistance and the selection of appropriate therapeutic measures.
    Full-text · Article · Dec 2015 · Journal of Dairy Science
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    • "Staphylococci species are not usually identified at the species level by routine laboratory testing and commercial kits, since phenotypic discrimination cannot reliably identify these species due to the variable expression of some phenotypic traits [13]. For this purpose, molecular techniques, including nucleotide sequencing within the 16S rDNA, hsp60, tuf, sodA, and rpoB genes, have been successfully used to identify Staphylococcus species [14]. Depending on the conditions, some species of coagulasenegative staphylococci can present health risks, since they have shown resistance to several antibiotics of therapeutic importance, such as í µí»½-lactams [15]. "

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    • "The study of genes other than 16S rRNA, has been considered for species discrimination. Thus, the 23S rRNA, the DNA gyrase b subunit (gyrB) (Chen and Tsen, 2002; La Duc et al., 2004), the RNA polymerase b subunit (rpoB) (Drancourt and Raoult, 2002; Qi et al., 2001) and the TU elongation factor (tuf) (Ghebremedhin et al., 2008) genes have been considered for the identification of certain bacterial taxons. Such genes may provide well conserved regions with potential usefulness for the design of primers and molecular probes for identification purposes in Bacillus spp. "
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    ABSTRACT: Bacillus genus includes foodborne pathogenic and spoilage-associated species, such as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus. Bacillus is also a heterogeneous genus that includes closely related species that are difficult to discriminate among, especially when well-conserved genes such as 16S rRNA and 23S rRNA are considered. The main goal of the present work was to study the usefulness of three housekeeping genes, the TU elongation factor (tuf), the DNA gyrase β subunit (gyrB) and the RNA polymerase β subunit (rpoB) genes, for use in differentiating among the most important foodborne Bacillus spp. sequences from 20 foodborne isolated Bacillus strains, and sequences belonging to different Bacillus spp. retrieved from the GenBank were analysed. In general terms, gyrB, rpoB and tuf gene regions for the strains considered in this study exhibited interspecific similarities of 57.8%, 67.23% and 77.66% respectively. Novel tufGPF and tufGPR universal primers targeted to the tuf gene were designed and proved to be useful for the amplification of all Bacillus spp considered. In conclusion, the tuf gene can be considered to be a good target for the differential characterisation of foodborne Bacillus species, especially for differentiating B. subtilis and B. cereus from other closely related species.
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