Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.     

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.     

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.     

Short communication: High frequency of β-lactam-resistant Staphylococcus aureus in artisanal coalho cheese made from goat milk produced in northeastern Brazil

Reports of β-lactam-resistant Staphylococcus aureus in artisanal goat cheese are increasing, and this phenomenon is relevant to public health. The objective of the present study was to determine the prevalence of S. aureus strains carrying the blaZ and mecA resistance genes, as well as the genes encoding the staphylococcal enterotoxins SEA, SEB, SEC, SED, SEE, and TSST-1 in artisanal coalho cheese made from goat milk produced in northeastern Brazil. We used biochemical and molecular tests to characterize 54 S. aureus isolates found in artisanal coalho cheese collected from commercial establishments producing animal products in 11 municipalities of Pernambuco State, Brazil. A PCR analysis revealed that 42.6% (23/54) of the isolates were positive for the blaZ gene, and 7.4% (4/54) were resistant to methicillin by phenotypic testing. We did not detect mecA or any genes encoding enterotoxins. The presence of S. aureus carriers of the blaZ gene and the identification of methicillin-resistant S. aureus strains are of concern for the health of consumers of this type of cheese.     

Antibiogram and coagulase diversity in staphylococcal enterotoxin-producing Staphylococcus aureus from bovine mastitis

We investigated antibiogram and coagulase gene diversity in staphylococcal enterotoxin (StE)-producing Staphylococcus aureus isolated from raw milk samples of cows infected with mastitis from 140 dairy farms in Korea between 1997 and 2004. Of the 696 Staph. aureus isolates collected in this study, 164 isolates (23.6%) produced one or more staphylococcal enterotoxins (A to D), and 19 isolates (2.7%) were methicillin-resistant. The percentage of StE-producing Staph. aureus (SES) isolates resistant to methicillin, kanamycin, neomycin, amikacin, and tetracycline was greater than that of non-SES. Ten coagulase genotype patterns were observed, including 4 main types comprising I (25.4%), II (13.9%), VII (13.2%), and VIII (17.8%). More than 4 Staph. aureus types were isolated from each of 82 dairy farms in different geographic locations, and only 1 coagulase genotype pattern was observed in 39 of the herds (47.6%). There was no significant correlation between coagulase genotypes harbored by Staph. aureus and their specific StE type. The percentage of isolates producing major StE types (A, B, AC, and ABCD) and being resistant to cephalothin and methicillin was greater among the Staph. aureus isolates with the 4 predominant coagulase genotypes (I, II, VII, and VIII) than among the isolates harboring the 6 rare coagulase types (III, IV, V, VI, IX, and X). Based on coagulase gene polymorphisms, our data indicate that a broad distribution of identical or closely related enterotoxin-producing Staph. aureus strains seem to contribute to bovine mastitis in the Republic of Korea.     

Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples

Mastitis is a prevalent disease in dairy cattle, and staphylococci are among the most common causative pathogens. Staphylococci can express resistance to a range of antimicrobials, of which methicillin resistance is of particular public health concern. Additionally, Staphylococcus aureus carries a variety of virulence factors, although less is understood about the virulence of non-aureus staphylococci (NAS). The aim of our study was to identify and characterize 3 collections of staphylococcal isolates from bovine milk samples regarding antimicrobial resistance, with emphasis on methicillin resistance, and their carriage of virulence genes typically displayed by Staph. aureus. A total of 272 staphylococcal isolates collected in Norway and Belgium in 2016 were included, distributed as follows: group 1, Norway, 100 isolates; group 2, Flanders, Belgium, 64 isolates; group 3, Wallonia, Belgium, 108 isolates. Species identification was performed by use of MALDI-TOF mass spectrometry. Phenotypic resistance was determined via disk diffusion, and PCR was used for detection of methicillin resistance genes, mecA and mecC, and virulence genes. Antimicrobial resistance was common in Staphylococcus epidermidis and Staphylococcus haemolyticus from all different groups, with resistance to trimethoprim-sulfonamide frequently occurring in Staph. epidermidis and Staph. haemolyticus as well as in Staph. aureus. Resistance to penicillin was most frequently observed in group 1. Ten Belgian isolates (1 from group 2, 9 from group 3) carried the methicillin resistance determinant mecA: 5 Staph. aureus from 2 different farms and 5 NAS from 3 different farms. Almost all Staph. aureus isolates were positive for at least 3 of the screened virulence genes, whereas, in total, only 8 NAS isolates harbored any of the same genes. Our study contributes to the continuous need for knowledge regarding staphylococci from food-producing animals as a basis for better understanding of occurrence of resistance and virulence traits in these bacteria.     

Short communication: Detection of antibiotic resistance,mecA, and virulence genes in coagulase-negative Staphylococcus spp. from buffalo milk and the milking environment

The aim of this study was to determinate whether coagulase-negative staphylococci (CNS) from buffalo milk or the milking environment possess virulence factors that are associated with intramammary infections or antimicrobial resistance. Milk samples (n = 320) from 80 lactating buffalo were evaluated for clinical and subclinical mastitis by physical examination, the strip cup test, California Mastitis Test (CMT), and somatic cell count (SCC) over a 4-mo period. In addition, swabs were obtained from the hands of consenting milkers (16), liners (64), and from the mouths (15) and nostrils (15) of buffalo calves. No clinical cases of mastitis were observed; however, CMT together with SCC results indicated that 8 animals had subclinical mastitis. Eighty-four CNS isolates were identified by MALDI-TOF MS and cydB real-time PCR (qPCR) and then evaluated by qPCR for presence of the eta, etb, sea, sec, cna, seb, sei, seq, sem, seg, see, and tst toxin genes, adhesion- and biofilm-associated genes (eno, ebps, fib, fnbA, coa), and the methicillin resistance gene (mecA). Resistance to antibiotics commonly used for mastitis treatment in Brazil was determined using the Kirby-Bauer test. Two strains were positive for the see and eta toxin genes; and mecA (1), eno (27), ebps (10), fnbA (10), and coa (5) genes were also detected. A notable number of isolates were resistant to erythromycin (30), penicillin (26), and cotrimoxazole (18); importantly, 10 vancomycin-resistant isolates were also detected. A smaller number of isolates were resistant to rifampicin (8), oxacillin (7), clindamycin (5), cefepime (4), tetracycline (3), ciprofloxacin (2), and chloramphenicol (1), and none were resistant to gentamicin or ciprofloxacin. Isolates with resistance to 2 (13 isolates), 3 (3), 4 (3), 5 (1), and 6 (1) antibiotics were detected. Taken together, our findings suggest that CNS isolates may not be a significant cause of clinical or even subclinical mastitis in buffaloes, but they may be a reservoir of virulence and antibiotic resistance genes.     

Detection of genes for enterotoxins (ent) and toxic shock syndrome toxin-1 (tst) in mammary isolates of Staphylococcus aureus by polymerase-chain-reaction

During a two-year-period (1997–1998) 94 field strains of Staphylococcus aureus isolated from cases of bovine mastitis were investigated for the presence of genes for staphylococcal enterotoxin (ent) and toxic shock syndrome toxin-1 (TSST-1; tst). Polymerase-chain reaction (PCR) was performed using six pairs of relevant oligonucleotide primers. Thirty-four isolates were positive for one (18 strains) or two (16 strains) toxin genes. Three field strains were positive for enterotoxin A gene (sea), two for enterotoxin B gene (seb), 22 for enterotoxin C gene (sec), four for enterotoxin D gene (sed) and 19 for TSST1 gene (tst). The enterotoxin E gene (see) sequence was not found. The combination of sec and tst showed the highest incidence. A high correlation between PCR results and toxin protein detection by a commercial enzyme linked immunosorbent assay (ELISA) system and a reversed passive latex-agglutination (RPLA) test was observed. In these immunoassays, only one strain of S. aureus produced equivocal results for production of enterotoxin A, giving an overall concordance between genotypic and phenotypic identification of 98.9%.     

Enterotoxin genes,enterotoxin production,and methicillin resistance in Staphylococcus aureus isolated from milk and dairy products in Central Italy

A total of 227 Staphylococcus aureus colonies, isolated from 54 samples of raw milk and dairy products of bovine, ovine, caprine and bubaline origin were tested for the presence of genes coding for staphylococcal enterotoxins (SEs/SEls) and for methicillin resistance. Ninety-three colonies, from 31 of the 54 samples (57.4%) and from 18 (69.2%) of the 26 farms of origin tested positive for SEs/SEls genes. Most isolates harboured more than one toxin gene and 15 different toxinotypes were recorded. The most frequent were “sec” gene alone (28.6%), “sea, sed, ser, selj” (20%), “seg, sei” and “seh” (8.6%). The 77 colonies harbouring “classical enterotoxins” genes (sea-sed) were further tested for enterotoxin production, which was assessed for 59.2% of the colonies. Three methicillin-resistant S. aureus (MRSA) isolates were detected in three different ovine milk/dairy product samples (1.3%). All isolates belonged to spa type t127, sequence type 1, clonal complex 1, SCCmec type IVa.     

Characterization of coagulase-positive staphylococci isolated from tank and silo ewe milk

The prevalence of coagulase-positive staphylococci (CPS) was studied among 390 samples of ewe milk. Fifty-seven (14.85%) samples of tank milk and all samples (6) of silo milk gave a positive result on Baird-Parker agar base supplemented with rabbit plasma fibrinogen, whereas amplification of the coagulase (coa) gene was successful in 6 (1.56%) samples of tank milk and in all silo samples. Phenotypic and genetic analysis of 153 isolates identified 151 (98.69%) as Staphylococcus aureus. Amplification of the coa gene was positive for 149 isolates (97.39%). The staphylococcal enterotoxin (SE) C gene was detected in 116 strains (75.82%), whereas more than one SE gene was carried in 5 strains (3.26%). None of the isolates harbored the genes for SEE or for methicillin resistance. A high prevalence of CPS carrying enterotoxin genes should be of concern because ewe milk is mainly processed into raw milk cheeses. The detection of the coa gene from milk samples could help to assess the microbiological safety of raw milk intended for direct use in the dairy industry.     

Short communication: Antimicrobial resistance and virulence characterization of methicillin-resistant staphylococci isolates from bovine mastitis cases in Portugal

Methicillin-resistant staphylococci (MRS) have already been reported as mastitis agents. Such bacterial species are a public health concern, and the characterization of their antimicrobial resistance and virulence profile is important to better control their dissemination. The present work evaluated the distribution of methicillin-resistance among 204 staphylococci from clinical (n = 50) and subclinical (n = 154) bovine mastitis. The presence ofthe mecA gene was determined by PCR. Phenotypic expression of coagulase, DNase, lipase, gelatinase, hemolytic enzymes, and biofilm production was evaluated. The presence of biofilm-related genes, icaA, icaD, and bap, was also determined. Antimicrobial resistance patterns for aminoglycosides, lincosamides, macrolides, fluoroquinolones, sulphonamides, tetracyclines, and fusidic acid were determined. Nineteen (9.3%) isolates were identified as MRS, and the presence of mecA in these isolates was confirmed by PCR. Virulence factors evaluation revealed that gelatinase was the most frequently detected (94.7%), followed by hemolysins (73.7%) and lipase (68.4%); 84.2% of the MRS isolates produced biofilm and icaA and icaD were detected in almost half of the MRS isolates (52.6%), but all were bap-negative. Resistance against other antimicrobial agents ranged from 0 (fusidic acid, ciprofloxacin, norfloxacin, enrofloxacin) to 100% (nalidixic acid). Resistance to nalidixic acid and nalidixic acid-tetracycline were the most common antimicrobial resistance profiles (31.6%). This study confirms that despite the low prevalence of MRS, isolates frequently express other virulence traits, especially biofilm, that may represent a serious challenge to clinicians.     

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) ST8 in raw milk and traditional dairy products in the Tizi Ouzou area of Algeria

Staphylococcus aureus is one of the leading causes of food-borne illness worldwide. Raw milk and dairy products are often contaminated with enterotoxigenic strains of this bacterium. Some of these strains carry antimicrobial resistance, leading to a potential risk for consumers. The aim of this study was to characterize S. aureus strains circulating in raw milk and traditional dairy products for carriage of staphylococcal enterotoxin (se) genes and antimicrobial resistance. Overall, 62 out of 270 samples (23%) were contaminated with S. aureus, and 69 S. aureus strains were identified. We studied the enterotoxin genes using 2 multiplex PCR targeting 11 se genes. Seventeen (24.6%) isolates carried one or more genes encoding for staphylococcal enterotoxins. The most commonly detected se genes were seb and sep, followed by seh, sea, and see. Using the disk diffusion method, we found that resistance to penicillin G and tetracycline was the most common. Eleven isolates of methicillin-resistant S. aureus (MRSA) carried the mecA gene. All MRSA isolates belonged to the same spa type (t024) and sequence type (ST8), and carried the seb and sep enterotoxin genes. However, none of them carried the Panton Valentine leukocidin gene (lukF/S-PV). The presence of enterotoxigenic S. aureus strains, including MRSA, in raw milk and dairy products, raises a serious public health concern, because these strains may cause food poisoning outbreaks, be disseminated to the population, or both.     

Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik

Sunsik, a ready-to-eat food in Korea, is comprised of various agricultural and marine products, and has been an important concern in Bacillus cereus food poisoning. The aim of this study was to investigate the toxin profiles, genotypic and phenotypic patterns as well as antibiotic resistance of B. cereus strains isolated from Sunsik. A subtyping method known as automated repetitive sequence-based PCR system (DiversiLab™) was used to assess the intraspecific biodiversity of these isolates. Thirty-five B. cereus strains were isolated from 100 commercial Sunsik samples, all of which harbored at least 1 enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin gene among all isolates were 97%, 86%, 77%, and 100%, respectively. Most strains also produced corresponding enterotoxins such as HBL (83%) and NHE (94%). One strain (2.9%) carried the emetic toxin genes, including ces and EM1, and was positive for the HEp-2 cell emetic toxin assay. Most strains were positive for various biochemical tests such as salicin hydrolysis (86%), starch fermentation (89%), hemolysis (89%), motility test (100%) and lecithinase hydrolysis (89%). All isolates were susceptible to most antibiotics although they were highly resistant to β-lactam antibiotics. By using the automated rep-PCR system, all isolates were successfully differentiated, indicating the diversity of B. cereus strains present in Sunsik.     

Biodiversity and characterization of indigenous coagulase-negative staphylococci isolated from raw milk and cheese of North Italy

The aim of the current study was to detect coagulase-negative staphylococci (CNS) in raw milk and cheeses produced in North Italy, and to analyze isolates for their biodiversity, safety aspects and technological properties. Molecular identification methods revealed a high biodiversity among isolates and assigned them to 17 species. The most recovered species were Staphylococcus equorum (12%), Staphylococcus lentus (12%), Staphylococcus simulans (12%), Staphylococcus sciuri (10%), and Staphylococcus xylosus (9%). The presence of ten transferable antibiotic resistance (AR) genes was verified by PCR and 19% of isolates were positive, with tet(K) being the most frequent gene (10%); interestingly, no strain carried multiple AR genes. Twenty-four isolates displayed hemolytic activity; tyrosine decarboxylase gene (tdcA) was found in two isolates, while histidine decarboxylase gene (hdcA) and enterotoxin genes (se) were not detected. Isolates were further characterized for the presence of some relevant technological properties; 16% of isolates displayed proteolytic activity and 39% lipolytic activity, while no one of the isolates was found to exhibit antimicrobial activity against Staphylococcus aureus. This study provided evidence of a low occurrence of safety hazards in CNS isolated from dairy products.     

Production of antilisterial bacteriocins by staphylococci isolated from bovine milk

A collection of 111 staphylococcal isolates recovered from healthy cows in 41 dairy herds in Brazil was surveyed for the production of bacteriocins. The group included 94 coagulase-positive and 17 coagulase-negative strains of staphylococci. All cultures were grown in tryptic soy broth for 18 h at 37°C, and cell-free supernatants were tested for antimicrobial activity against several target organisms by using the agar diffusion method. Filtrates of 57 staphylococci showed strong activity against Listeria monocytogenes Scott A, and 52 isolates also inhibited the growth of Stapylococcus aureus Newbould 305, a major causative agent of bovine mastitis in the United States. The plasmid profiles of staphylococci invariably included an 8-kb plasmid. Staphylococcal isolates were tested for the production of aureocins A70 and A53, 2 bacteriocins of coagulase-positive staphylococci known to be associated with 8-kb and 10.2-kb plasmids, respectively. The presence of the A70 or A53 bacteriocin gene was checked by PCR techniques using primers based on nucleotide sequences flanking the structural gene of each bacteriocin. Agarose gel analysis of amplified PCR products of plasmid templates from all 58 isolates showed only a 525-bp fragment corresponding to the structural gene of the bacteriocin aureocin A70. The results indicated that the apparently widespread association of A70-producing staphylococci with healthy cows in Brazil may be beneficial in controlling undesirable bacteria in dairy herds.     

Molecular characterisation and typing the methicillin resistance of Staphylococcus spp. isolated from raw milk and cheeses in northwest Spain: A mini survey

During the last decade, the occurrence of methicillin-resistant staphylococci in animals and foods derived from them has been increasingly reported, but the dairy sector still requires further surveillance. For this, 150 samples of raw bovine milk, ovine and caprine chesses were analysed for identification of Staphylococcus spp. isolates based on 16S rRNA gene sequencing and additionally, typing of methicillin resistance (MR) via PCR-targeting of mec-region genes. Accordingly, 84 staphylococci isolates were identified and their 16S rRNA gene sequences were deposited in GenBank. Among these, 2 isolates of each of Staphylococcus chromogenes and Staphylococcus epidermidis were methicillin-resistant. Phenotypically, a variable multidrug-resistance was noticed for the mecA-positive isolates against eleven selected antibiotics. Results demonstrated that coagulase-negative staphylococci can be a carrier for MR, as Staphylococcus aureus does, and could exist in raw milk from lactating animals; which represents a source for MR transmission to the surrounding environment.     

Comparison of the enterotoxigenic types,toxic shock syndrome toxin I (TSST-1) strains and antibiotic susceptibilities for enterotoxigenicStaphylococcus aureusstrains isolated from food and clinical samples

The distribution of enterotoxin types and toxic shock syndrome toxin I (TSST-1) strains for 176Staphylococcus aureusstrains obtained from food samples and 62S. aureusstrains isolated from clinical samples were compared. It was found that for both the food and clinical isolates, staphylococcal enterotoxin A (SEA) strains accounted for the major part (75% and 45% of the total enterotoxigenic strains for food and clinical isolates, respectively) followed by SEB or SEAB, SEC and SED strains. For food isolates, none of theS. aureusstrains was TSST-1 strain while for clinical isolates, three strains (1 SEC, 1 SED and 1 SEAB strain) were found to be TSST-1 strains. When susceptibilities for these enterotoxigenicS. aureusstrains to antibiotics, such as penicillin, oxacillin, vancomycin, methicillin, streptomycin, tetracycline, gentamycin and kanamycin were compared, results showed that 51.6% of the food isolates were resistant to penicillin G only but sensitive to the other antibiotics tested. Also, 8 of the 64 enterotoxigenic strains isolated from food samples were all sensitive to antibiotics while none of the enterotoxigenic strains from clinical samples showed this antibiotic susceptibility pattern. No methicillin resistantS. aureus(MRSA) strains could be found among the food isolates. On the other hand, the majority (42.4%) of the enterotoxigenicS. aureusstrains from clinical samples were penicillin and/or other antibiotics resistant MRSA strains. Since MRSA strains often posed the therapeutic problem, the MRSA strains were further confirmed by PCR assay using the primers specific formecA gene. It was found that results obtained from the disc agar diffusion method and the PCR method were the same.     

Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants

Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n = 40) and from udder tissue (n = 7) and foremilk (n = 24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.     

Short communication: First detection of Panton-Valentine leukocidin–positive methicillin-resistant Staphylococcus aureus ST30 in raw milk taken from dairy cows with mastitis in South Korea

We identified 199 Staphylococcus aureus isolates from quarter milk samples of 1,289 dairy cattle between 2014 and 2018. About 66% of the isolates were resistant to at least 1 antimicrobial agent; the highest rate of resistance was to penicillin, followed by resistance to ampicillin, erythromycin, and sulfadimethoxine. We obtained 30 methicillin-resistant S. aureus (MRSA) strains from 6 farms in 3 provinces. The MRSA strains exhibited a significantly higher resistance rate to most of the tested antimicrobials than the oxacillin-susceptible strains. The MRSA strains represented 5 genotypes: ST72-t324-SCCmec IV (n = 14), ST30-t1752-SCCmec IV (n = 8), ST188-t189-SCCmec NT (n = 6), ST188-t2284-SCCmec NT (n = 1), and NT-NT-SCCmec IV (n = 1). One of the ST188 MRSA strains represented a novel staphylococcal protein A (spa) type (t2284). In addition, 7 of the 8 ST30 MRSA strains were Panton-Valentine leukocidin (PVL)–positive and carried various staphylococcal enterotoxin encoding genes. This is the first report of PVL-positive ST30 MRSA-t1752-SCCmec IV from bovine mastitis in Korea. All of ST72-t324-SCCmec IV MRSA strains carried staphylococcal enterotoxin and leukotoxin encoding genes. They were also sensitive to most of the tested non-β-lactam antimicrobials. In contrast, ST188-t189 MRSA strains were resistant to multiple antimicrobials and predominantly carried the leukotoxin encoding gene. Taken together, these findings may indicate that dairy cows could be a major source for spreading MRSA strains, and contaminated milk could be a vehicle for transmission. Suitable hygienic measures should be established in dairy farms and processing plants to limit the likelihood of introducing MRSA into the food chain.     

2009—2020年北京市朝阳区113株食源性金黄色葡萄球菌耐甲氧西林和肠毒素/类肠毒素基因携带与分子分型分析

目的 了解北京市朝阳区2009—2020年113株食源性金黄色葡萄球菌甲氧西林耐药、肠毒素/类肠毒素基因携带和分子分型情况.方法 采用纸片扩散法进行头孢西丁药物敏感性实验,结合mecA基因扩增鉴定耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)...