Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food

AIMS: To develop a multiplex PCR that allows the identification of bacteria belonging to the Staphylococcus genus and in particular to the species Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus isolated from food manufacturing plants. METHODS AND RESULTS: Five primer pairs were used in the multiplex PCR, one specific to the Staphylococcus genus and four specific to S. xylosus, S. saprophyticus, S. epidermidis and S. aureus species. All the 31 Staphylococcus reference strains yielded a specific PCR product with the genus-specific primers. Staphylococcus xylosus, S. saprophyticus, S. epidermidis and S. aureus gave a specific PCR fragment with the corresponding species-specific primers. No amplification with the Kocuria, Macrococcus and Micrococcus strains was observed in our conditions. This multiplex PCR was performed on 30 strains of Gram-positive cocci isolated from different workshops and fermented sausages. Among them, 28 belonged to the Staphylococcus genus and 14 were identified to S. saprophyticus, four to S. xylosus, two to S. aureus and one to S. epidermidis. CONCLUSIONS: This multiplex PCR provided reliable and repeatable PCR results. It allowed the identification of a major part of the isolates, highlighting the predominance of the S. saprophyticus species in the workshops studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This tool is a useful way to screen the strains isolated from foodstuff and food environment and to monitor these species during the food processing.     

Monitoring of staphylococcal starters in two French processing plants manufacturing dry fermented sausages

AIMS: The growth and survival of Staphylococcus xylosus and Staphylococcus carnosus were monitored during sausage manufacture in two processing plants. METHODS AND RESULTS: The gram-positive, catalase-positive cocci isolated from the processing plants F10 and F11 were identified by Staphylococcus-specific PCR and species-specific oligonucleotide array. In the inoculated products with starter cultures, 90% of staphylococcal strains isolated in F10 were identified as S. xylosus and 10% as S. carnosus. In F11, 77% were identified as S. xylosus and 20% as S. carnosus. Staphylococcus xylosus dominated the staphylococcal microbiota while S. carnosus survived during the process. The pulse-field gel electrophoresis analysis revealed that all S. xylosus and S. carnosus strains isolated corresponded to the starter strains inoculated. The two starter strains of S. xylosus co-dominated in the isolates from sausages of F11, whereas the strain with pattern A1 was dominant in the isolates from sausages of F10. In the environments, no S. carnosus and S. xylosus were found, whereas Staphylococcus equorum and Staphylococcus saprophyticus were the main species isolated. CONCLUSIONS: This work highlighted the domination of S. xylosus starter strains, which showed a strong capacity to grow during sausage process, while S. carnosus survived during the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Successful implantation of starter cultures is obviously a prerequisite for their contribution to sensorial qualities. Thus, the monitoring of the growth and the survival of S. xylosus and S. carnosus are required to guarantee a well-adapted starter culture. This study revealed that the two Staphylococcus species are suitable for manufacturing sausages in processing plants with very different capacities of production.     

The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of d-serine

Human urine contains a relatively high concentration of d -serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d -serine deaminase gene ( dsd A) of UPEC. We found the gene in several clinical isolates of S. saprophyticus ; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii , phylogenetically close relatives of S. saprophyticus , and could also not be detected in isolates of Staphylococcus aureus , Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d -serine, whereas S. aureus , S. epidermidis and other staphylococcal species could not. The association of the dsd A gene with growth in media including d -serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d -serine utilization and detoxification may be a general property of uropathogenic bacteria.     

Pulsed-field gel electrophoresis of the genomic restriction fragments of coagulase-negative staphylococci

Abstract The genomes of 47 coagulase-negative staphylococcal strains assigned to different species were analysed by pulsed-field electrophoresis. The strains were clustered on the basis of their similarity in the Sma I restriction patterns into various groups, each group consisting of the type strain and the strains whose Sma I restriction patterns were similar to that of the type strain. The Sma I restriction groups seem to correspond to the following species: Staphylococcus warneri, S. hominis, S. xylosus, S. lugdunensi, S. kloosii, S. haemolyticus, S. lentus, S. cohnii, S. equorum, S. chromogenes, S. saprophyticus, S. simulans, S. carnosus, S. capitis and S. auricularis . The species S. sciuri, S. caseolyticus, S. gallinarum, S. epidermidis and S. schleiferi were represented only by their type strains and showed no similarity in their Sma I restriction patterns neither to each other nor to all the species investigated here. Thus, the classification of coagulase-negative staphylococcal strains into the above species seems to be confirmed also by genome restriction analysis carried out by pulsed-field gel electrophoresis.     

Ribotyping and rapid identification of Staphylococcus xylosus by 16-23S spacer amplification

Ninety-five strains of Staphylococcus xylosus isolated from goat milk, French sausage or mice were analyzed together with 35 Staphylococcus type strains by 16-23S spacer amplification and ribotyping. The results obtained by PCR amplification of the 16-23S spacer region permitted the distinction of each type strain and additionally generated a DNA banding pattern characteristic for 93 of the 95 Staphylococcus xylosus strains. Ribotyping proved to be an efficient epidemiological tool for Staphylococcus xylosus species as it clustered the 95 strains into 23 distinct types.     

Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.     

代表性差异分析比较两株来自不同地区的猪源Lactobacillus菌株

[目的]对分离自猪肠道的乳酸杆菌S1菌株进行鉴定,并比较该菌株与同种的001T菌株的基因差异.[方法]对S1菌株进行16S rRNA基因序列分析和种特异PCR检测,并且对S1菌株和Lactobacillus sobrius 001T进行代表性差异分析(Representational difference analysis,RDA).[结果]16S rRNA基因序列分析表明,与S1菌株最相似的已知菌为L.sobrius.变性梯度凝胶电泳分析显示,仔猪空、回肠细菌图谱中有一与S1菌株有相同迁移位置的优势条带,克隆、测序鉴定表明,与该条带相匹配的16S rRNA基因克隆(Clone S)的最相似已知菌也为L.sobrius.16S rRNA基因系统进化分析表明,S1菌株与Clone S和L.sobrius 16S rRNA基因序列同源性分别为99.8%和99.6%.L.sobrius特异性引物也可以扩增S1株菌的16S rRNA基因的特定片段.因此S1菌株可被确定为Lsobrius.RDA对菌株S1和同种的猪源L.sobrius 001T菌株的基因差异进行分析,未发现这两株菌的基因组差异.[结论]猪肠道乳杆酸菌S1菌株属于L.sobrius,其与猪源L sobrius 001T菌株为相似菌株.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

Identification of Staphylococcus from French dry sausage

Fifty-one strains of staphylococci isolated from French dry sausages were mainly identified with Staphylococcus carnosus, S. xylosus, S. warneri and S. saprophyticus. The API Staphylococcus identification system proved to be reliable for S. xylosus and S. carnosus. The identification of S. warneri and S. saprophyticus was performed by DNA-DNA hybridization. These species are better identified by taking into account not only the API Staphylococcus system but also the following characters: novobiocin and lysozyme susceptibilities, production of D-lactate. hydrolysis of tri-olein.     

Characterization of Staphylococcus species by ribosomal RNA gene restriction patterns

The rRNA gene restriction pattern sof 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct HindIII patterns and 44 distinct EcoRI patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed with some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcus xylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g.S.aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.     

Ecology and dynamics of coagulase-negative cocci isolated from naturally fermented Italian sausages

Coagulase-negative cocci (CNC) ecology in naturally fermented sausages from Friuli Venezia Giulia region, in the North East of Italy, was investigated. A total of 617 CNC strains, isolated from three different plants during the fermentation process, were identified by traditional methods (biochemical tests) and molecular methods based on species specific PCR, PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of the V3 region of the 16S rRNA gene. The identification, by using biochemical tests, was not successful for 130 strains. Moreover, incongruent results were observed comparing the traditional with the molecular identifications. The same species of CNC were found in all three processing plants, but their contribution to the fermentations was different. In two plants Staphylococcus xylosus was the main species involved in fermentation process, while in the third the maturation was carried out equally by three species: S. xylosus, Staphylococcus warneri and Staphylococcus pasteuri.     

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.     

Development of specific PCR primers for a rapid and accurate identification of Staphylococcus xylosus, a species used in food fermentation

Twenty-seven Staphylococcus strains isolated from food and food environments were assigned to Staphylococcus xylosus by API-Staph system. But only seven isolates had similar patterns to this species when compared to the pulse-field gel electrophoresis patterns of 12 S. xylosus strains. To perform a rapid identification of the S. xylosus species, a random amplified polymorphic DNA product of 539-bp shared by all of the S. xylosus strains was used to design a pair of primers. These primers were species-specific for S. xylosus when tested by PCR on 21 staphylococci species. This specific PCR assay confirms the identification of the seven isolates identified by PFGE to S. xylosus. In conclusion, we developed specific PCR primers for a rapid and accurate identification of the S. xylosus species.     

Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: a contribution to the study of these free-living nitrogen-fixing bacteria

A 16S rRNA gene-based fingerprinting method was developed for the identification of Azotobacteraceae and tested onto 48 soil isolates and 28 reference strains belonging to the free-living nitrogen-fixing bacterial group and to the most common species found in soil samples. According to this method, the 16S rRNA gene was amplified using universal primers for Eubacteria and PCR products were subsequently digested with RsaI, HhaI, HpaII, FnuDII, and AluI. The analysis of the restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the eight Azotobacteraceae species tested. Cluster analysis was successfully employed for the identification of members of the family Azotobacteraceae, being assignation into species of the isolates confirmed by means of partial 16S rRNA gene sequencing.     

Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.     

Design and evaluation of specific PCR primers for rapid and reliable identification of Staphylococcus xylosus strains isolated from dry fermented sausages

Rapid and reliable identification of Staphylococcus xylosus was achieved by species-specific PCR assays. Two sets of primers, targeting on xylulokinase (xylB) and 60 kDa heat-shock protein (hsp60) genes of S. xylosus, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 27 reference strains of the DSM collection, representing 23 different species of the Staphylococcus genus and 3 species of the Kocuria genus. Moreover, 90 wild strains isolated from different fermented dry sausages were included in the analysis. By using primers xylB-F and xylB-R the expected PCR fragment was obtained only when DNA from S. xylosus was used. By contrast, amplification performed by using primers xylHs-F and xylHs-R produced a single PCR fragment, of the expected length, when DNA from S. xylosus, S. haemolyticus, S. intermedius and S. kloosii were used as template. Nevertheless, AluI digestion of the xylHs-F/xylHs-R PCR fragment allowed a clear differentiation of these 4 species. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. xylosus strains.     

Multiplex PCR assay to identify methicillin-resistant Staphylococcus haemolyticus

Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.     

Identification by 16S-23S rDNA intergenic region amplification, genotypic and phenotypic clustering of Staphylococcus xylosus strains from dry sausages

AIMS: To ascertain the identification and typing of the Gram-positive, coagulase-negative cocci present in 'Salsiccia Sotto Sugna', an Italian artisanal sausage. METHODS AND RESULTS: Fifty-one strains were isolated and genotypically identified by amplification of the 16S-23S rDNA intergenic region with universal primers. Most isolates were identified as Staphylococcus xylosus and one strain as Staph. condimenti. Isolates were clustered by numerical analysis of both RAPD (Random Amplified Polymorphic DNA) PCR profiles and physiological characters. Genotypic clustering allowed the separation of strains showing nitrate reduction and amino acid decarboxylase activities. Phenotypic clustering distinguished strains isolated at diverse ripening stages. CONCLUSION: The predominance of Staph. xylosus in Italian dry sausages was confirmed. Genotypic similarities related to the possession of single phenotypic traits. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a rapid method of Staphylococcus and Kocuria species distinction was proposed. The suitability of RAPD PCR to discriminate strains of Staph. xylosus with technologically relevant activities was reported.     

Cross protection between a strain of Staphylococcus epidermidis and eight other species of coagulase-negative staphylococci

Passive protective activity of rabbit antiserum prepared by a representative capsular type II strain of Staphylococcus epidermidis in mice was absorbed out with homologous capsular type strains of S. simulans, S. cohnii, S. xylosus, S. hominis, S. capitis, S. hyicus, S. haemolyticus, and S. saprophyticus in addition to the homologous strain. The minimum amount of the strains required for absorption differed greatly, depending upon the strain. No absorption of the activity was shown with a strain of capsular type I and III of S. epidermidis, S. simulans, and S. cohnii, and a strain of capsular type III of S. hominis. These results suggest a possible capsular type specificity in the cross protection between strains of S. epidermidis and other species of coagulase-negative staphylococci.     

Surface microflora of four smear-ripened cheeses

The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.