Multiplex PCR and RPLA Identification of Staphylococcus aureus enterotoxigenic strains from bulk tank milk

Enterotoxigenic Staphylococcus aureus in raw milk poses a potential health hazard to consumers, and the identification of such strains should be used as part of a risk analysis of milk and milk products. The primary purpose of this study was to investigate the occurrence of enterotoxigenic S. aureus strains in raw milk supplied for dairy processing in the Czech Republic. A further aim was to compare the production of staphylococcal enterotoxins (SEs) with the presence of the corresponding genes. This was undertaken using multiplex polymerase chain reaction (PCR) and reversed passive latex agglutination (RPLA). Out of 440 bulk tank milk samples from 298 dairy herds, 70 proved positive for S. aureus (15.9%). Staphylococcal enterotoxin genes (ses) were detected in 39 (55.7%) isolates. The genes most commonly detected were sei (38.6%), seg (31.4%) and sea (27.1%). Genes seb, seh, sed, sej and sec were observed in 10%, 4.3%, 2.9%, 2.9% and 1.4% of strains respectively. Genes see and sel did not occur. The most frequently detected genotypes were seg, sei at 11.4%; sea at 10.0%; and sea, seg, sei at 8.6%. Toxin production was observed in nine (12.9%) S. aureus isolates. Seven strains were detected as SEB- (10%) and two as SED- (2.9%) producing. A relatively high number (32%) of discrepancies between the results with multiplex PCR and RPLA assays was obtained, particularly on account of SEA. Nineteen strains were sea positive by PCR but SEA negative by RPLA, and one strain was sec positive and SEC negative. The results of both methods were identical concerning SEB and SED. It was concluded that detection of ses by PCR was a useful additional tool to support identification of enterotoxigenic strains.     

Pattern of enterotoxin genes seg, seh, sei and sej positive Staphylococcus aureus isolated from bovine mastitis

PCR detection of the genes encoding the newly described staphylococcal enterotoxins (SE) SEG, SEH, SEI and SEJ was carried out for 104 randomly selected Staphylococcus aureus field strains isolated from cases of bovine mastitis. Sixty-one (58.7%) isolates were positive for one or more of these novel enterotoxin genes. Thirty-six field strains were classified as carrier of seg, 22 of sei gene and 23 were positive for sej gene. None of the 104 investigated ruminant S. aureus strains carried the seh gene. Thirty-seven of these S. aureus strains showed a combination of genes encoding enterotoxin types SEA to SEE or toxic shock syndrome toxin 1 (TSST 1). Thirteen cultures harboured only one, 28 two, 12 three and 8 four enterotoxin genes. Among the 61 S. aureus field strains 14 (23.0%) were positive for the genes encoding SEJ and SED and 10 (16.4%) isolates for those encoding SEG and SEI. Isolates harbouring the sed/sej genes were further characterized by macrorestriction analysis and pulsed-field-gelelectrophoresis (Pfge). Macrorestriction analysis revealed six patterns. Nine of these14 S. aureus isolates (64.3%) exhibited two patterns with a high degree of relationship (>80%).     

Molecular typing of Staphylococcus aureus isolated from cows, goats and sheep with intramammary infections on the basis of gene polymorphisms and toxins genes

We investigated 116 Staphylococcus aureus isolates from cows, goats and sheep with intramammary infections (IMI) in Italy to provide information about the spread of enterotoxigenic strains and to compare strains isolated from different ruminant species. The isolates were typed by restriction fragment length polymorphism (RFLP) analysis of the coagulase (coa) gene, by analysis of polymorphisms of the X region of protein A (spa) gene and by detection of genes encoding enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej and sel). Seven different coa types and 12 different spa types were distinguished. On the basis of polymerase chain reaction-RFLP, 29 different coa subtypes were identified. Two different coa subtypes accounted for 49% and 67% of bovine and ovine isolates respectively. Only seven coa subtypes were observed in isolates from more than one host species and no coa subtype was present in isolates from all three ruminant species. Furthermore, 85 of the isolates (73%) harboured at least one enterotoxin gene (se) with a predominance of sea, sed and sej among isolates from bovine IMI, and sec and sel among isolates from caprine and ovine IMI. Comparing the S. aureus isolates on the basis of gene polymorphisms and presence of se genes, significant differences were found in distributions of genotypes among isolates from cows, goats and sheep.     

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk

Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety.     

Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds

To determine the distribution of genes that encode enterotoxins A, B and C, 36 strains of Staphylococcus aureus isolated from goat mastitis and 64 isolated from bovine mastitis were analyzed by Multiplex PCR. Of the total strains studied, 37 (37%) were detected to have some of the SEs genes. From the bovine mastitis strains, 4 (6.3%) co-amplified the sea and seb genes and 2 (3.1%) were positive for the sec gene. From the goat mastitis strains, 31 (86%) tested positive to the Multiplex, and the sec gene was detected in all of them. The production of SE was detected in all strains harboring the corresponding gene. The results demonstrated that S. aureus isolated from goat mastitis had a higher enterotoxigenic potential than those isolated from bovine mastitis. Additionally, the presence of the sec gene in the majority of goat mastitis strains suggests a possible involvement of SEC in goat mastitis pathogenesis.     

Production of staphylococcal enterotoxins and TSST-1 by coagulase negative staphylococci isolated from ruminant mastitis.

The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S. xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.     

Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep

Two hundred and ninety-three isolates of Staphylococcus aureus obtained from 127 bulk-tank milk samples of goats and sheep from Switzerland were characterised by pheno- and genotypic traits. Of the 293 S. aureus isolates, 193 (65.9%) were egg yolk-negative and 15 (5.1%) were negative for clumping factor and/or protein A determined by a latex agglutinating test system. For 285 isolates, PCR amplification of the 3' end of the coagulase gene showed a single amplicon. Five differently sized PCR products of 500, 580, 660, 740 and 820 bp were distinguished. In 191 isolates (n = 293) staphylococcal enterotoxin (SE) genes were detected: 123 isolates tested positive for SEC gene, 31 for SEG gene, 28 for SEA gene, 26 for SEJ gene, 24 for SEI gene, 4 for SEB gene and 4 for SED gene. Furthermore, 126 isolates were positive for the gene encoding the toxic shock syndrome toxin 1. Coagulase gene restriction profile analysis of the 145 isolates harbouring SEA or SEC genes revealed six different patterns using AluI and five different patterns using HaeIII. In summary, within these two groups, high genotypic uniformity within the different sized coagulase gene amplicons was demonstrated. This is the first study providing comprehensive characterisation data of S. aureus strains originating from bulk-tank milk samples of goats and sheep. Remarkable differences in phenotypic traits between S. aureus originating from goats and sheep and bovine milk were found. Moreover, the high prevalence of toxin-producing S. aureus may be important as it is relevant to food hygiene.     

spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.     

Distribution of staphylococcal enterotoxin genes among Staphylococcus aureus isolates from poultry and humans with invasive staphylococcal disease

Food poisoning by Staphylococcus aureus affects hundreds of thousands of people each year. Staphylococcus aureus also causes invasive diseases such as arthritis (in poultry) and septicemia (in poultry and humans). Foodborne disease is caused by the ingestion of a staphylococcal enterotoxin (SE). Enterotoxin has also been associated with other S. aureus illnesses in humans and domestic animals. In this study, polymerase chain reaction was used to detect the staphylococcal enterotoxin genes, SEA, SEB, SEC, SED, and SEE, in S. aureus isolates associated with invasive disease in poultry and humans. In the 34 poultry isolates, only one isolate was found to contain a SE gene, sec. In the 41 human isolates, over 51% tested positive for an SE gene with 12.2% positive for the gene for SEA, 2.4% for SEB, 22% for SEC, 24.4% for SED, and 0 for SEE. The disparity between the rates for SE gene(s) in poultry and human isolates suggests a lesser role for the enterotoxins in invasive poultry disease than in human disease.     

Lack of Staphylococcal Enterotoxin Production among Strains of Staphylococcus aureus from Bovine Mastitis in Denmark

Staphylococcal enterotoxins (SE’s) are a group of small exoproteins produced by some strains of Staphylococcus aureus. The SE’s, designated A to E according to their antigenic specificities, are important causes of food poisoning worldwide. Milk and dairy products are frequently associated with S. aureus enter-otoxin food poisoning, and it is supposed that infected milk from mastitic animals constitute the main source of enterotoxigenic S. aureus of animal origin (Bryon 1983, Gilmour & Harvey 1990, Bergdoll 1989). Indeed, S. aureus is the most common cause of bovine mastitis worldwide, and if mastitis strains produce SE this makes up an enormous reservoir of potential enterotoxin producers. The production of SE by S. aureus isolated from bovine mastitis have been investigated in several countries (Matsunaga et al. 1993, Kenny et al. 1993, Olson et al 1970, Orden et al. 1992, Olsvik et al. 1981, Adekeye 1980, Garcia et al. 1980, Abbar 1986, Harvey & Gilmour 1985). Since no studies have been performed on the prevalence of enterotoxigenic strains of S. aureus isolated from bovine mastitis in Denmark, a well characterized collection of S. aureus (Aarestrup et al. 1995) was investigated with respect to this property.     

原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析

对临床乳房炎（57株）和原料乳（44株）金黄色葡萄球菌菌株,用PCR方法检测mecA基因、PVL基因、ETs基因、SEs基因和TSST-1基因;采用CLSI指导说明执行琼脂稀释法药敏性试验。结果显示原料乳菌株中,84.09%携带有毒素基因,其中PVL的检出率为84.09%,肠毒素的检出率为52.27%,主要流行的肠毒素基因为sea（56.82%）,均未检测到携带mecA、ETs、TSST-1、sei和sej基因的菌株;同时得到10种毒素基因型,其主要流行的毒素基因型为PVL＋sea（29.55%）和PVL（27.27%）。临床菌株中,78.95%携带有毒素基因,其中PVL的检出率为28.07%,肠毒素的检出率为77.19%,主要流行的肠毒素基因为sea（47.37%）,没有检测到携带ETs、TSST-1和seh基因菌株;同时得到25种毒素基因型,其主要流行的毒素基因型为sea（19.30%）,其次是seb（7.02%）,sea＋sed＋sej（3.51%）和PVL＋sea＋seb＋sec＋seg＋sei（3.51%）。6株（10.53%）携带有mecA基因菌株均含有较多毒素基因。原料乳分离株对甲氧苄啶和头孢西丁的耐药率较高,分别为100%和86.36%,其次对氯霉素、红霉素、苯唑西林、头孢哌酮和庆大霉素的耐药率分别为11.36%、4.55%、2.27%、2.27%和6.82%,所有原料乳菌株均对环丙沙星敏感,同时得到8种耐药谱,多重耐药率达22%;临床乳房炎菌株对红霉素和甲氧苄啶的耐药率较高,分别为100%和71.93%,其次对氯霉素、庆大霉素、环丙沙星、头孢西丁和苯唑西林的耐药率分别为28.07%、26.07%、24.56%、19.30%和7.02%,临床乳房炎菌株对头孢哌酮和四环素的敏感率为100%,同时得到13种耐药谱,多重耐药率达77.19%。所有原料乳和临床乳房炎菌株均对万古霉素和阿米卡星敏感。临床乳房炎菌株携带的毒素基因和多重耐药率比原料奶菌株高,同时在临床乳房炎乳中检测到MRSA菌株,提示我们应加强乳及其乳制品的管理,并对奶牛乳房炎加以重视。     

Resistance situation and enterotoxin production capacity of Staphylococcus aureus strains from bovine mastitis milk samples]

In total, 63 S. aureus strains from mastitis milk samples of different animals in 57 farms were isolated. In 14 (22%) of the S. aureus strains resistancies against one or several of the examined antibiotics could be observed whereby six resistance patterns were found. 14.3% of the strains were penicillin resistant. 34 (54%) of the 63 S. aureus produced enterotoxins (SE). Three strains formed SEA, 21 SEC, three SED and seven strains 2 SE, SEAC, SEAD or SEBD.     

Detection of staphylococcal enterotoxins in milk and milk products

For food evaluation the determination of the number of Staphylococcus aureus (hereinafter S. aureus) colonies is insufficient in view of present scientific knowledge. The results, advantages and shortcomings of diagnostic methods are demonstrated on an example of three methods of detection of staphylococcal enterotoxins in milk and milk products. 133 strains were investigated by the method of biotyping of S. aureus strains. Four strains of S. aureus were included in biotype A, seven xin-producing strains were isolated seventeen times by detection of 96 S. aureus strains were not included in any biotype, the other strains belonged to biotypes C and E. This method can be used as an auxiliary method of evaluation of foods containing S. aureus bacteria. The agar-gel precipitation method of enterotoxin detection in isolated strains of S. aureus has just restricted validity. The enteroto-strains. The main shortcoming of this method is a fact that the result concerning the isolated strains need not be identical with the result of enterotoxin detection in food. Direct assays of staphylococcal enterotoxins in milk and milk products using an enzymoimmunological method seem to be the most promising, mainly due to their high sensitivity (0.0001-0.001 micrograms.ml1-) and other advantages. Positive and negative results are presented on an example of two model trials with winter sheep milk cheese.     

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from birds

The present study was designed to comparatively investigate 19 Staphylococcus aureus strains isolated from specimens of 19 different birds during routine microbiological diagnostics. The S. aureus strains were characterized genotypically by polymerase chain reaction (PCR) amplification using 62 different oligonucleotide primers amplifying genes encoding staphylococcal cell surface proteins, exoproteins and two classes of the accessory gene regulator agr. All 19 investigated S. aureus were positive for the gene segment encoding a S. aureus-specific part of the 23S rRNA, the genes encoding thermostable nuclease (nuc), clumping factor (clfA) and coagulase (coa) and the gene segments encoding the Xr-repetitive region and the immunoglobulin G (IgG)-binding region of protein A (spa). In addition, all tested strains were positive for the genes hla and fnbA and negative for the genes seb, sec, sed, see, sej, tst, eta and etb. The remaining genes, including sbi, hlb, fnbB, ebpS, cna (domains A and B), cap5, cap8, set1, agr class I, agr class II, sea, seg, seh and sei were detected in a variable number of isolates. The presented data give an overview on the distribution of virulence determinants of S. aureus strains isolated from birds. This might be useful to understand the role of these virulence determinants in bird infections.     

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.     

Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis

In the present study, 188 coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for antimicrobial substance production. Twelve CNS strains (6.4%) exhibited antagonistic activity against a Corynebacterium fimi indicator strain. Most antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). Amongst the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. The Bac(+) strains presented either no or few antibiotic resistance phenotypes. Three strains were shown to produce a bacteriocin either identical or similar to aureocin A70, a bacteriocin previously isolated from an S. aureus strain isolated from food. The remaining Bac(+) strains produce antimicrobial peptides that seem to be distinct from the best characterised staphylococcal bacteriocins described so far. Some of them were able to inhibit Listeria monocytogenes, an important food-borne pathogen, and several strains of Streptococcus agalactiae associated with bovine mastitis, suggesting a potential use of these bacteriocins either in the prevention or in the treatment of streptococcal mastitis.     

Genetic differences among Staphylococcus aureus isolates from dairy ruminant species: a single-dye DNA microarray approach

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating sheep, goats and cows. The present study was carried out to compare 65 S. aureus isolates mainly obtained from nasal carriage and subclinical mastitis in dairy sheep and 43 isolates obtained from subclinical mastitis from 22 goats and 21 cows. A DNA microarray, containing probes against 190 true or putative virulence factors, was used to detect the presence of the virulence genes. Their presence/absence was independently assessed by PCR for the genes of interest. Sheep isolates obtained from the nostrils or the udders did not show any significant tissue specific virulence factor. The dominant pulse-field electrophoresis profile (OV/OV'), associated with spa clonal complex spa-CC 1773, matched mainly with the agr group III and was only found in ovine and caprine isolates. This clone was more specifically characterized by the prevalence of the following virulence genes: lpl4, ssl6, bsaA1, bsaB, bsaP, SAV0812. Moreover, seven virulence-associated genes (lpl1, sel, sec, tst, lukF-PV-like component, lukM, SAV0876) were associated with isolates from small ruminants, while the egc cluster, fhuD1, abiF and SAV2496 with bovine isolates. This genomic study suggests the existence of lineage- and host-specific genes leading to the development of host-specific pathogenic traits of S. aureus isolates.     

Identification of Staphylococcus aureus strains negative for enterotoxins A, B and C isolated from bovine mastitis in México

A total of 41 isolates of Staphylococcus aureus obtained from bovine mastitis in 7 different states in Mexico were analyzed by the polymerase chain reaction (PCR) to determine the presence of encoding genes for enterotoxins A, B and C. The oligonucleotides were designed by specific regions of the sea, seb, sec genes. Surprisingly, none of the isolates presented the prospective amplification bands when they were run on agarose gels. On the contrary, reference strains CECT 976 SEA; CECT 5191 SEB; and CECT 4465 SEC showed the prospective amplification products. In order to confirm these results, enterotoxin production A, B, C, D, and E was determined by enzyme linked fluorescent assay (ELFA) using a MiniVIDAS system, on 15 Staphylococcus aureus selected at random from among the 41 isolates. None of the analyzed strains was positive to the test, whereas reference strains enterotoxins producing: CECT 976 SEA; CECT 5191 SEB; CECT 4465 SEC, CECT 4466 SED; CECT 5192 SEE produced concentrations of the toxins detected for this technique. The role of enterotoxins in the pathogenicity of S. aureus in bovine mastitis in Mexico is discussed.     

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals and prevalence of lukM-lukF-PV genes by bacteriophages in bovine isolates

Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.     

Molecular typing of Staphylococcus aureus isolated from bovine mastitic milk on the basis of toxin genes and coagulase gene polymorphisms

A total of 270 strains of Staphylococcus aureus, isolated from mastitic milk, were investigated by the polymerase chain reaction for the presence of genes encoding enterotoxins (sea to sej) and a toxic shock syndrome toxin (tst). One hundred eighty three (67.8%) bovine isolates possessed either one or more toxin genes and the most common pattern that coexisted in S. aureus was tst, sec, seg, and sei. Coagulase genotyping revealed 15 patterns, and 161 of the 270 isolates (59.6%) belonged to the coagulase genotype B1. Further, these 161 isolates possessed at least two enterotoxin genes. However, the role of these toxins in udder pathogenicity remains unclear. Moreover, the predominant isolate possessed the enterotoxin genes supporting the theory that superantigenic toxins are important for the udder pathogenesis.