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.     

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。     

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.     

Antimicrobial drug susceptibility of Staphylococcus aureus strains isolated from bovine milk

The minimum inhibitory concentration (MIC) of ten antimicrobial drugs for 287 S. aureus strains recently isolated from bovine mastitic milk in different herds all over Sweden was determined. The minimum bactericidal concentration (MBC) of benzylpenicillin for 20 strains was determined. Thirty strains (10%) produced beta-lactamase. All strains were susceptible to oxacillin and neomycin, and more than 90% to streptomycin, trimethoprim-sulphamethoxazole chloramphenicol, erythromycin and tetracycline, whereas all were resistant to sulphamethoxazole. None of 20 strains investigated was tolerant to benzylpenicillin. However, S. aureus strains, isolated from bovine milk, should be tested for beta-lactamase production prior to treating mastitic cases with beta-lactam drugs.     

Phenotypic and genotypic characterization of Staphylococcus aureus isolated from raw camel milk samples

In the present study 320 milk samples collected from 160 apparently healthy camels of three different locations in Sudan were investigated for the presence of Staphylococcus aureus resulting in the isolation of this bacterial pathogen from 28 milk samples from 24 camels. Twenty-five S. aureus were identified phenotypically and by PCR mediated amplification of species-specific genes or gene segments. Investigation of the S. aureus for toxinogenic potential revealed that three S. aureus strains were positive for the enterotoxin encoding gene sec and the genes seg, sei, sem, sen and seo, representing the egc gene cluster. In addition all 25 S. aureus were positive for the superantigen-like encoding gene ssl7 (set1). Partial sequencing of gene sec of the three S. aureus strains yielded an almost complete sequence identity to the sequence of the sec variant sec2. However, all three sec2 genes of the present study showed a deletion of one base causing a frame shift and a corresponding earlier stop codon.According to the present results, the raw camel milk collected from three locations in Sudan seems to be, at least at this stage, of minor importance as vector causing staphylococcal food poisoning.     

荧光定量PCR检测牛乳中金黄色葡萄球菌肠毒素A基因方法的建立

为建立检测牛乳中金黄色葡萄球菌(S.aureus)肠毒素A基因(SEA)定性定量的检测方法,本研究针对S.aureus SEA基因片段设计1对引物,将构建的重组质粒作为阳性对照,建立了S.aureus SEA DNA的SYBR Green I real-time PCR检测方法.结果显示,特异性产物Tm值为78.2℃～78.5℃,最低可检测到49.5 fg/μL(16.5拷贝)的阳性质粒.标准曲线的相关系数为0.99.与其他常见的产SEB的S.aureus、产SEC的S.aureus、无乳链球菌、大肠杆菌、嗜热链球菌、伤寒沙门氏菌、大肠杆菌DH5 α及JM109均无交叉反应.该检测方法具有较好的特异性和敏感性,为牛乳中S.aureus的快速检测提供了新的技术手段.     

Phage typing of Staphylococcus aureus strains isolated in Sweden from bovine milk.

Both the human and the bovine international sets of phages were used for typing of 372 bovine Staphylococcus aureus (Sa) strains, whereas the bovine set alone was used for typing of a further 1183 strains. In addition, 338 of the strains were tested for antibiotic sensitivity. Out of 372 Sa strains 85.5% could be typed with the human and 89.8% with the bovine phage set. Of all the 1555 Sa strains used 92.4% were lysed by the bovine phage set. Several phage types can be present in one and the same herd and some of them can predominate. Resistance to most of the tested antibiotics was very low. The incidence of resistance to penicillin and ampicillin was 10.0% and 4.4% respectively.     

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.     

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.     

Antibiogram of staphylococcal strains isolated from milk and milk-products

A study of the resistance patterns of 248 staphylococcal isolates from milk and milk products to eight antimicrobial agents using the disc method showed that 80 (32.3%) were resistant to sulphafurazole, 75 (30.2%) to penicillin G, 63 (25.4%) to ampicillin, 23 (9.3%) to cloxacillin, 19 (7.7%) to tetracycline, 17 (6.9%) to streptomycin, 14 (5.6%) to erythromycin and 12 (4.8%) to chloramphenicol. One hundred and sixteen (46.8%) of the 248 staphylococcal isolates were sensitive to all the agents tested. A significant percentage (P less than 0.05) of the isolates from raw milk were resistant to erythromycin, sulphafurazole, cloxacillin, penicillin G and streptomycin compared to isolates from fermented milk. The minimum inhibitory concentrations (MIC) as determined by the tube method for isolates resistant by the disc method, were greater than or equal to 16 micrograms for ampicillin in 5 (11.9%) isolates; greater than or equal to 40 micrograms for cloxacillin, 9 (36.0%) and greater than or equal to 12 international units for penicillin G amongst 12 (22.2%) isolates. MIC values of greater than or equal to 40 micrograms were recorded for 9 (90.0%), 9 (69.2%), 8 (73.7%) and 7 (70.0%) isolates to chloramphenicol, streptomycin, erythromycin and tetracycline respectively. The relatively high level of resistance to antimicrobial agents is a reflection of misuse or abuse of these agents in the environment.     

Detection of classical enterotoxins and identification of enterotoxin genes in Staphylococcus aureus from milk and dairy products

Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.     

兔源金黄色葡萄球菌的耐药性及耐药基因分析

为了解四川地区规模化兔场金黄色葡萄球菌(S.aureus)的耐药情况,本研究从四川地区部分规模化兔场共分离鉴定出41株S.aureus,采用纸片扩散法对分离株进行了耐药表型鉴定,同时采用PCR方法对相关耐药基因进行了检测。药敏试验结果显示:分离株对β-内酰胺类、氨基糖苷类、四环素类和大环内脂类药物的耐药率分别为87.80%、97.56%、78.05%和56.10%,对多肽类、喹诺酮类及磺胺类耐药率较低。耐药基因检测结果显示:所有分离株均检测到耐药基因,并且29株分离株具有多重耐药性,其中耐药基因mec A、tet、erm、aac(6')/aph(2')、ant(4',4')及aph(3')-III的检出率分别为75.61%、70.73%、46.34%、92.68%、80.49%及53.66%。结合耐药表型与耐药基因结果分析显示兔源S.aureus的耐药表型与耐药基因检出率基本呈正相关。本研究通过检测41株兔源S.aureus对临床常用抗生素的耐药性及相关耐药基因,初步掌握了四川地区规模化兔场S.aureus的耐药情况,为该地区规模化兔场预防及治疗葡萄球菌病临床合理用药提供了依据。     

Determination of beta-lactamase production in strains of Staphylococcus aureus isolated from the milk of cows

Determination of sensitivity to penicillin G by a standard disk assay diffusion method was compared with a iodometric method of test papers to determine beta-lactamase production after Jorgensen in Staphylococcus aureus strains isolated from cow's milk from different farms. Out of 179 test strains, 32 strains 17.8%) were found to be well sensitive in the diffusion test; eight of these strains (25.0%) were demonstrated to produce beta-lactamase. 54 strains (30.2%) were sensitive. 27 strains (50%) of this group produced beta-lactamase. 93 strains (51.9%) were resistant to penicillin G in the diffusion test. 86 strains (92.5%) were found to produce beta-lactamase and seven strains were negative in this test. Using the diffusion test of sensitivity in these cultures, 86 strains were sensitive (48.1%) and 93 strains were resistant (51.9%). Beta-lactamase was produced by 121 strains (67.6%) and no beta-lactamase production was recorded in 58 strains (32.4%). Differences in the results of both tests were manifest mainly in the set of strains qualified as sensitive (inhibition zone diameter 24 to 16 mm) and well sensitive (inhibition zone diameter larger than 25 mm). The results indicate that the currently performed diffusion test of sensitivity to penicillin G should be accompanied by an assay of beta-lactamase production. The iodometric method of test papers is simple, rapid and cheap and can be made in any bacteriological laboratory. The high resistance of Staphylococcus aureus strains to penicillin G documents that this antibiotic is little efficient in the treatment of mastitis of this etiology in the given region.     

Detection of virulence-associated genes in Staphylococcus aureus isolated from bovine clinical mastitis milk samples in Guangxi

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals and is one of the most common etiological agents of clinical and subclinical bovine mastitis. The purpose of this study was to determine the presence of genes encoding clfA, fnbA, fnbB, cap5, cap8, hla, hlb, nuc, sea, and tst of S. aureus strains (n?=?39) isolated from bovine clinical mastitis in Guangxi by polymerase chain reaction amplification. The results of the present study indicated that all isolates were found to contain one or more virulence-associated genes. The most frequently encountered genes were fnbA (97?%) and nuc (90?%), followed by hla (85?%) and hlb (82?%), respectively. None of the investigated S. aureus strains harbored fnbB and sea genes. The data in the present study showed a relatively wide distribution of the genes fnbA and nuc among the investigated isolates, indicating that they play an important role on bovine mastitis pathogenesis. The study provides a valuable insight into the virulence-associated genes of this important pathogen.     

Pseudomonas aeruginosa in raw and pasteurized milk

Eighteen strains of Pseudomonas aeruginosa, i.e. 8.87%, were isolated during a year from 203 samples of raw milk. Two Pseudomonas aeruginosa strains, i.e. 4%, were isolated from 50 samples of pasteurized milk. The strains were isolated using propagation techniques in meat-peptone broth with malachite green and on selective media--on centrimide agar (CEM) and on Pseudomonas F agar. All the isolated strains produced protease, whereas lipase was produced by only five strains. The strains were devitalized when exposed to pasteurization temperatures (72 degrees C) for 20 seconds. At cold store temperatures (4 degrees C), Pseudomonas aeruginosa strain cells propagated on average by two orders, inhibitory effects of low temperatures were recorded only with one strain. Inhibitory effects of milk cultures (cream, yogurt) on Pseudomonas aeruginosa were observed; their effects were more clear-cut at the temperature of 4 degrees C. The strains were markedly susceptible to gentamycin.     

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%).     

Antimicrobial resistance and resistance genes in Staphylococcus aureus strains from rabbits

Fifty-six Staphylococcus aureus isolates recovered between 1998 and 2003 from 31 rabbit farms with and without problems of chronic staphylococcosis, were screened for resistance to enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, penicillin and tetracyclines using the agar dilution test. For penicillin, a disk diffusion test was also performed. The detection of tetP(B), tet(K), tet(L), tet(M), tet(O), tet(T), tet(W), erm(A), erm(B), erm(C) and mec(A) genes was done via a PCR assay. Four isolates showed resistance to erythromycin and lincomycin. These isolates were positive for the erm(C) gene in the PCR. Eleven strains were resistant to tetracyclines and all harboured the tet(K) gene. In the agar dilution test, five isolates showed resistance to penicillin, whereas in the disk diffusion test 12 isolates showed resistance. None of these 12 resistant isolates carried the mec(A) gene. Only one strain showed resistance to gentamicin, and all strains were susceptible to enrofloxacin and neomycin. This study demonstrates that resistance to antimicrobial agents in S. aureus isolates originating from rabbits is relatively rare compared to resistance in S. aureus isolates originating from other animals and humans.