Analysis of antibiotic resistance patterns and detection of mecA gene in Staphylococcus aureus isolated from packaged hamburger

Presence of Staphylococcus aureus, antibiotic resistance pattern and PCR detection of mecA gene in isolated strains were investigated in total of 256 packaged hamburgers in Iran-Tehran. For this purpose we used standard disk-diffusion method and sensitive and specific PCR technique, respectively. Results showed that 25% of samples were positive for S. aureus. Resistance to meticillin, erythromycin, penicillin G, cefazolin, ciprofloxasin, vacomycin and amoxiclave was determined 89%, 20.3%, 18.7%, 15.6%, 14%, 26.6% and 12.5%, respectively. According to the obtained results from PCR analysis of methicillin-resistant S. aureus (MRSA), mecA gene was present in 100% of the resistant isolates, 0% of intermediate-resistance isolates and 25% of susceptible isolates. The results obtained from PCR detection of mecA gene showed high correlation with standard disk diffusion test.     

Short communication: Occurrence of methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci in dairy goat herds in Ohio,United States

In light of the scarcity of information about the occurrence and epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MRCNS) in small ruminants in general, and particularly dairy goats, we launched this limited-scope study. The findings reported here show the detection of MRSA and MRCNS in goat milk and teat skin samples from dairy goat herds in the state of Ohio. A total of 120 milk samples and 120 teat-swab samples were collected from 5 farms. After conventional isolation and phenotypic characterization of the staphylococci colonies, bacterial isolates were tested by PCR assay targeting the genes nuc to identify Staphylococcus aureus and mecA to detect MRSA and MRCNS. The clonal complexes of MRSA isolates was also determined by multiloccus sequence typing. Fifteen (6.2%) positive S. aureus samples were found in this study: 9 from milk and 6 from teat skin samples. Four (2%) MRSA isolates were detected and, using multiloccus sequence typing genotyping, these were designated to clonal complexes CC133 (n = 2; milk samples) and CC5 (n = 2; teat skin). Three (1.25%) coagulase-negative staphylococci isolates from the teat skin also harbored the mecA gene. Although, the MRSA isolated from milk samples is not a typical human-associated lineage, the CC5 clone isolated from teat skin is a common and widespread clonal complex associated with humans, suggesting that this extramammary niche could be a relevant reservoir of methicillin-resistant staphylococci. Furthermore, the fact that 75% of MRSA were recovered from 1 farm showing poor hygiene practices strengthens the hypothesis that good hygiene practices could be useful to prevent persistence and spread of MRSA at a farm level.     

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.     

Distribution of Methicillin-resistant Staphylococcus aureus (MRSA) in RAW meat and fish samples in Korea

Methicillin-resistant Staphylococcus aureus (MRSA) in raw meat and fish samples in Korea was investigated. A total of 209 samples was analyzed. Antimicrobial disk susceptibility testing with oxacillin (OX) was used to detect MRSA in 74 S. aureus isolates (35.4%), 7 of which showed resistance towards OX. The mecA gene was identified in the 7 isolates with OX resistance and was expressed in all 7 isolates. Pulsed-field gel electrophoresis was used to analyze genetic homologies among the 7 MRSA isolates. No genetic correlations were detected among the isolates.     

Prevalence and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from retail yak butter in Tibet,China

This study aimed to investigate the prevalence, molecular characteristics and antibiotic resistance of Staphylococcus aureus isolates from yak butter in Tibet, China. A total of 218 yak butter samples were collected from retail stores in Tibet and screened for Staph. aureus. Furthermore, the virulence genes, resistance genes, antimicrobial susceptibility, and molecular typing [pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and staphylococcal protein A (spa) typing] of Staph. aureus isolates were detected. The results showed that 12.4% of yak butter samples were contaminated with Staph. aureus, including 5 samples positive for methicillin-resistant Staph. aureus (MRSA). Among all isolates, 96.3% harbored one or more virulence genes, including classical (sea and sec), novel enterotoxin-encoding genes (seh, sek, sel, and seq), and hemolysin genes (hla and hld). All isolates were resistant to at least 2 different antibiotic classes, and the isolates were most commonly resistant to sulfonamides, β-lactams, and erythromycin. For resistance genes, blaZ (74.1%) was most frequently detected, followed by dfrG (51.9%), erm(B) (22.2%), mecA (18.5%), tet(K) (14.8%), aph(2″)-Ia, aph(3′)-III, and ant(6)-Ia (11.1% for each), and erm(C) (7.4%). We detected 8 spa types, 6 sequence types (ST), and 5 clonal complex (CC) types. In addition, 1 isolate of Staph. aureus was nontypeable. We found that CC1-ST1-t559 (55.6%) was the most predominant clone, followed by CC59-ST59-t437 (11.1%), CC5-ST5-t002 (7.4%), CC1-ST1, CC1-ST1-t114, CC1-ST573-t4938, CC1-ST573-t8915, CC30-ST30-t021, and CC25-ST25-t167 (3.7% for each). For PFGE typing, a total of 5 clusters and 15 pulsotypes were generated, and some isolates from different samples showed indistinguishable pulsotypes. Our findings suggest that yak butter produced in Tibet, China, could be contaminated by Staph. aureus strains, including MRSA strains, carrying various virulence and resistance genes, representing multiple antimicrobial resistance phenotypes. The presence of potentially virulent and antibiotic-resistant Staph. aureus strains in yak butter poses a potential threat to consumers, and appropriate measures need to be taken in the production chain to reduce the occurrence of Staph. aureus in yak butter.     

Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals’ health, because β-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.     

Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Multiple cases of methicillin-resistant CC130 Staphylococcus aureus harboring mecC in milk and swab samples from a Bavarian dairy herd

The discovery of a new mecA homolog, mecC, necessitates a modification of diagnostic procedures for the identification of methicillin-resistant Staphylococcus aureus (MRSA), as most assays used for the genotypic and phenotypic mecA detection cannot currently recognize mecC. Although the prevalence, distribution, and importance of mecC are not yet completely understood, an exchange of mecC-MRSA between humans and animals seems possible. All previously reported observations of mecC-positive strains have been sporadic. To the best of our knowledge, this is the first report about multiple cases of mecC-positive Staph. aureus in 1 dairy herd. Clonal complex 130 Staph. aureus harboring mecC were found in milk samples from 16 of 56 lactating cows kept in a herd in Bavaria, Germany. Almost all quarter milk samples positive for mecC-MRSA had the lowest possible California Mastitis Test score; composite somatic cell counts obtained from monthly milk recordings showed a mean of 51,600 cells/mL in mecC-MRSA affected cows. Additionally, mecC-positive clonal complex 130 Staph. aureus were detected in swab samples from the mammary skin and a teat lesion of 1 cow from this herd. This report suggests that mecC-carrying strains are able to spread among livestock, and that they have the ability to cause multiple cases in single herds. Therefore, future studies targeting MRSA in dairy cows need to consider mecC.     

Antimicrobial resistance of Staphylococcus spp. isolated from organic and conventional Minas Frescal cheese producers in São Paulo,Brazil

The genus Staphylococcus is recognized worldwide as a cause of bacterial infections in humans and animals. Antibiotics used in dairy cattle combined with ineffective control can increase antimicrobial resistance. The objective of this study was to characterize 95 Staphylococcus strains isolated from organic and conventional Minas Frescal cheese production regarding antibiotic resistance (phenotype and genotype), presence of sanitizer-resistant genes and biofilm-formation genes, and SCCmec typing. Most strains (25.3%) showed higher resistance to penicillin, followed by oxacillin (21.1%) and clindamycin (11.6%). Among antibiotic resistance genes, the most prevalent were blaZ (25.3%), mecA (13.7%), lsaB (6.3%), msrA (4.2%), ant4 (3.2%), and tetM (2.1%); among sanitizer-resistance genes they were qacA/B (5.3%) and qacC (6.3%); and among biofilm, bap (4.2%), icaA (29.5%), icaD (41.1%). However, there was no statistically significant difference between organic and conventional dairy products, possibly due to the lack of synthetic antibiotic use on conventional farms during the sample collection period. Methicillin-resistant Staphylococcus aureus (MRSA) had their SCCmec identified as types I and IVc, and the methicillin-resistant coagulase-negative staphylococci had nontypeable SCCmec. These results suggest that there are antibiotic-resistant strains in both organic and conventional Minas Frescal cheese production in the state of São Paulo, Brazil. This supports the idea that improved quality control is needed from the milking stage up to the final product.     

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.     

Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis

Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.     

Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa,Algeria

This study was designed to determine antimicrobial resistance phenotypes and genotypes and virulence factors in Staphylococcus aureus and coagulase-negative staphylococci (CNS) in unpasteurized milk sold in Djelfa, Algeria. Eighty-two unpasteurized cow milk samples were randomly obtained from 82 retail stores in Djelfa and tested to detect staphylococci. Species were identified by biochemical tests and MALDI-TOF. Antimicrobial resistance phenotypes and genotypes were determined by disk diffusion test, PCR, and sequencing. The Staph. aureus isolates were subjected to spa typing, multilocus sequence typing, and detection of virulence genes and the scn gene by PCR and sequencing. Forty-five (54.9%) milk samples were contaminated by staphylococci and 45 isolates were recovered: 10 Staph. aureus (12.2% of total samples) and 35 CNS (42.7%). Resistance to penicillin (blaZ), tetracycline (tetL/tetK), and erythromycin (ermB/msrA/ermC) were the most common phenotypes (genotypes). Three CNS were methicillin-resistant and all were mecA-positive. The Staph. aureus isolates were ascribed to the following lineages [spa type/sequence type/associated clonal complex (number of isolates)]: t267/ST479/CC479 (n = 6), t1510/ST5651/CC45 (n = 1), t359/ST97/CC97/ (n = 1), t346/ST15/CC15 (n = 1), and t044/ST80 (n = 1). The mecA gene was detected in the cefoxitin-susceptible t044/ST80 isolate and co-harbored the lukF/lukS-PV and scn genes. The detection of mecA-PVL-positive Staph. aureus, methicillin-resistant CNS, and multidrug-resistant staphylococcal species indicates a potentially serious health issue and reveals that unpasteurized milk sold in Djelfa city could be a potential vehicle for pathogenic and antimicrobial-resistant staphylococci.     

Short communication: Methicillin-resistant Staphylococcus aureus detection in US bulk tank milk

Staphylococcus aureus is a major cause of mastitis in dairy cattle. This study estimated the herd prevalence of methicillin-resistant Staph. aureus (MRSA) among US dairy herds by testing bulk tank milk (BTM) samples using genotypic and phenotypic methods. A nationally representative sample of 542 operations had BTM cultured for Staph. aureus, and 218 BTM samples were positive upon initial culture. After 4 wk to 4 mo of frozen storage, 87% of 218 samples (n = 190) were still culture positive for Staph. aureus on blood agar, but none were positive for MRSA on the selective indicator medium CHROMagar MRSA. A duplex PCR was used to detect the Staph. aureus-specific nuc gene and the methicillin resistance gene, mecA, in mixed staphylococcal isolates from the 190 BTM samples that were positive for Staph. aureus after storage. Seven samples tested positive for nuc and mecA, and 2 samples tested positive for mecA only. MecA-positive Staphylococcus spp., but not MRSA, were subsequently isolated from 5 samples, whereas neither mecA-positive Staphylococcus spp. nor MRSA was isolated from the remaining 4 samples. Presence of methicillin-resistant, coagulase-negative Staphylococcus spp. may complicate the detection of MRSA by means of PCR on BTM. Bulk tank milk in the United States is not a common source of MRSA.     

First report of methicillin‐resistant Staphylococcus aureus in tank cultured dusky kob (Argyrosomus japonicus), and evaluation of three phenotypic methods in the detection of MRSA

This study was conducted to ascertain the occurrence of methicillin‐resistant Staphylococcus aureus (MRSA) in marine finfish (Argyrosomus japonicus) harvested from the wild and two re‐circulatory aquaculture systems, and evaluating the reliability of three phenotypic methods in the detection of methicillin resistance. A total of 120 dusky kob fish were sampled for S. aureus detection using conventional methods and confirmed by polymerase chain reaction (PCR) targeting the nuc gene. Methicillin resistance was determined by molecular detection of the mecA gene. Using mecA as the defining standard, the specificities and sensitivities of cefoxitin disc diffusion, oxacillin screen agar, and growth of S. aureus on Brilliance MRSA II Agar were evaluated. A total of 321 presumptive S. aureus isolates were recovered by culture, out of which 202 (62.9%) were confirmed by PCR. Of these, 33 (16.3%) strains were mecA positive while 169 (83.7%) were mecA negative. The sensitivities and specificities of MRSA detection was 93.9 and 91.7%, 81.8 and 92.3%, and 87.9 and 94.1% for cefoxitin disc, oxacillin screen agar test, and Brilliance MRSA II agar, respectively. This is so far the first report of MRSA in dusky kob aquaculture in South Africa. In the absence of molecular techniques, cefoxitin disc diffusion test is recommended along with any other phenotypic method to improve MRSA detection from samples of veterinary origin.

Practical implications

Methicillin‐resistant Staphylococcus aureus currently presents one of the greatest challenges for medical research worldwide, as well as is one of the most important causes of bacteria gastroenteritis due to preformed toxins in foods. There is a dearth of knowledge on marine foods as carriers/sources of MRSA infection. There are also major discrepancies obtained with MRSA detection methods, making effective detection of this pathogen complicated. The results from this study show that healthy aquaculture fish are reservoirs of MRSA, thus it is necessary to regularly monitor marine foods. Cefoxitin disc diffusion test is recommended as the preferred method for detection of MRSA from fish/food samples where molecular methods are lacking.     

The occurrence of methicillin-resistant non-aureus staphylococci in samples from cows,young stock,and the environment on German dairy farms

This study aimed to determine the occurrence of methicillin-resistant (MR) non-aureus staphylococci (NAS) on 20 preselected German dairy farms. Farms were selected based on the detection of methicillin-resistant Staphylococcus aureus (MRSA) during previous diagnostic investigations. Bacterial culture of presumptive MR-NAS was based on a 2-step enrichment method that has been recommended for MRSA detection. Quarter milk samples (QMS), bulk tank milk, swab samples from young stock, and environmental samples were collected for bacterial culture. Methicillin-resistant NAS were detected on all study farms. The MR-NAS positive test rate was 3.3% (77/2,347) in QMS, 42.1% (8/19) in bulk tank milk, 29.1% (59/203) in nasal swabs from milk-fed calves, 18.3% (35/191) in postweaning calves, and 7.3% (14/191) in nasal swabs from prefresh heifers. In the environment, MR-NAS were detected in dust samples on 25% (5/20) of the dairy farms as well as in teat liners and suckers from automatic calf feeders. The geometric mean somatic cell count in QMS affected by MR-NAS (183,000 cells/mL) was slightly higher compared with all QMS (114,000 cells/mL). Nine MR-NAS species were identified; Staph. sciuri, Staph. lentus, Staph. fleurettii, Staph. epidermidis, and Staph. haemolyticus were the most common species. In addition, 170 NAS isolates were identified that showed reduced cefoxitin susceptibility (4 mg/L) but did not harbor the mecA or mecC genes. On some farms, similar mobile genetic elements were detected in MR-NAS and MRSA. It was suggested that resistance genes may be transferred between NAS and Staph. aureus on the respective farms.     

Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea

Staphylococcus aureus belongs to the group of major contagious mastitis pathogens, whereas the coagulase-negative staphylococci (CNS) are also capable of causing opportunistic bovine mastitis. Many of these strains are resistant to penicillin or ampicillin because of the long-term use of β-lactam antibiotics in agricultural and healthcare settings. Based on the simple and highly specific coagulase genotyping by PCR-RFLP used for discriminating among Staph. aureus strains, the relationship between phenotypic antibiogram and the polymorphism of coagulase gene was determined in this study. The staphylococci strains (835 Staph. aureus and 763 CNS) were isolated from 3,047 bovine mastitic milk samples from 153 dairy farms in 8 provinces from 1997 to 2004 in the Republic of Korea. Twenty-one (2.5%) Staph. aureus and 19 (2.4%) CNS strains were resistant to methicillin [oxacillin minimum inhibitory concentration (MIC) ≥4 μg/mL]. The mecA gene was also found in 13 methicillin-resistant Staph. aureus (MRSA) and 12 methicillin-resistant CNS (MRCNS) isolates with a significantly higher detection rate of the mecA gene in MRSA with high MIC (≥16 μg/mL) compared with those with MIC ≤ 8 μg/mL. Methicillin-resistant Staph. aureus and MRCNS were also more resistant to other antibiotics (ampicillin, cephalothin, kanamycin, and gentamicin) than methicillin-susceptible staphylococci. Among 10 different coa PCR-RFLP patterns (A to J) in 706 Staph. aureus strains, the main types were A (26.9%), B (17.0%), G (10.5%), and H (15.4%), with the frequent observation of the A and H types (6 and 10 isolates) in MRSA. This study indicates that major epidemic Staph. aureus clones may be spread between different dairy farms, and the profile of coa genotype can be applied for epidemiological investigations and control of bovine mastitis, particularly one caused by MRSA with specific prevalent coa types.     

Short communication: β-Lactam resistance and vancomycin heteroresistance in Staphylococcus spp. isolated from bovine subclinical mastitis

The use of antimicrobial agents has led to the emergence of resistant bacterial strains over a relatively short period. Furthermore, Staphylococcus spp. can produce β-lactamase, which explains the survival of these strains in a focus of infection despite the use of a β-lactam antibiotic. The aim of this study was to evaluate the resistance of Staphylococcus spp. isolated from bovine subclinical mastitis to oxacillin and vancomycin (by minimum inhibitory concentration) and to detect vancomycin heteroresistance by a screening method. We also evaluated β-lactamase production and resistance due to hyperproduction of this enzyme and investigated the mecA and mecC genes and performed staphylococcal cassette chromosome mec typing. For this purpose, 181 Staphylococcus spp. isolated from mastitis subclinical bovine were analyzed. Using the phenotypic method, 33 (18.2%) of Staphylococcus spp. were resistant to oxacillin. In contrast, all isolates were susceptible to vancomycin, and heteroresistance was detected by the screening method in 13 isolates. Production of β-lactamase was observed in 174 (96%) of the Staphylococcus spp. isolates. The mecA gene was detected in 8 isolates, all of them belonging to the species Staphylococcus epidermidis, and staphylococcal cassette chromosome mec typing revealed the presence of type I and type IV isolates.     

Characterisation of Staphylococcus aureus strains from milk and goat cheese and evaluation of their inhibition by gallic acid,nisin and velame of the Brazilian caatinga

Twenty isolates from milk and goat cheese were confirmed as Staphylococcus aureus. These isolates were characterised for phenotypic properties related to cell adhesion and for the presence of enterotoxin production, intercellular adhesion and β‐lactam resistance genes. Staphylococcus aureus L47 showed cell adhesion ability and positivity for the sec, sed, icaD, mecA and blaZ genes. Three antimicrobial compounds were tested singly or in pairs for growth control of strain L47: gallic acid (GA), nisin and essential oil (EO) of Croton heliotropiifolius (velame). At 24 h, EO and EO + nisin showed higher inhibitory activity against S. aureus L47 in goat milk.     

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.