Genetic characterization and antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Tunisia.

Staphylococcus aureus is a major agent of bovine mastitis in dairy herds, causing economic losses in dairy industry worldwide. In addition, milk and milk-products contaminated by Staph. aureus can cause harmful human diseases. The aim of this study was to characterize Staph. aureus strains isolated from dairy farms in Tunisia. Bulk tank milk (n = 32) and individual cow milk (n = 130) samples were collected during the period of 2013-2014. Forty-three Staph. aureus isolates were recovered and typed by spa typing, 16S-23S rRNA intergenic spacer (RS-PCR) and multiplex PCRs for 22 virulence genes. Antimicrobial resistance was also investigated with a disc diffusion test. A selected subsample of 22 strains was additionally genotyped by multilocus sequence typing. Seventeen spa types were recovered, and t2421 (n = 10), t521 (n = 6) and t2112 (n = 5) were the most common. Fourteen different RS-PCR genotypes grouped into 11 clusters were detected in our study, with predominance of the RVI genotype (n = 24). Eight sequence types were identified and Clonal Complex 97, corresponding to RS-PCR cluster R, was the most common (n = 10), followed by CC1 (n = 4), CC15 (n = 3) and other four accounting for one or two strains. Different combinations of virulence genes were reported, and enterotoxin genes were present in few strains (seh, n = 4; sea, n = 2; sea and seh, n = 2; sec and sel, n = 2). The majority of strains were resistant only to penicillin; only one strain was found to be multiresistant and no methicillin-resistant Staph. aureus was demonstrated. Our study reported the isolation of CC97 from bovine milk in Tunisia for the first time and confirmed the relevance of this lineage in intramammary infection in cows. This paper describes the characteristics of Staphylococcus aureus isolated from bulk tank and individual cow milk in Tunisia. All strains were genotyped by spa typing and RS-PCR, a method based on the amplification of the 16S-23S rRNA intergenic spacer region, and multiplex PCRs for 22 virulence genes. A selected subsample of strains was also genotyped by multilocus sequence typing. All strains were tested for antimicrobial resistance. Our study evidences a predominance of strains belonging to Clonal Complex 97. Methicillin-resistant strains were not detected, and overall low level of antimicrobial resistance was reported.

Ben Said M ，Abbassi MS ，Bianchini V ，Sghaier S ，Cremonesi P ，Romanò A ，Gualdi V ，Hassen A ，Luini MV ... -  《-》

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil.

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.

Silva NCC ，Guimarães FF ，Manzi MP ，Budri PE ，Gómez-Sanz E ，Benito D ，Langoni H ，Rall VLM ，Torres C ... -  《-》

Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary.

Peles F ，Wagner M ，Varga L ，Hein I ，Rieck P ，Gutser K ，Keresztúri P ，Kardos G ，Turcsányi I ，Béri B ，Szabó A ... -  《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》

Staphylococcus aureus related to bovine mastitis in Switzerland: Clonal diversity, virulence gene profiles, and antimicrobial resistance of isolates collected throughout 2017.

Staphylococcus aureus can be associated with subclinical, acute, chronic, and toxic cases of bovine intramammary infections, leading to considerable financial losses for the dairy industry in Switzerland and worldwide. In addition, milk products are one of the most common food categories implicated in staphylococcal food poisoning in humans. Detailed information on the population structure, as well as the virulence and resistance characteristics of Staph. aureus originating from bovine mastitis milk is needed to allow for source attribution and risk assessment of Staph. aureus in a food poisoning context and to improve therapeutic approaches in cattle. Our objective was to assess the population structure, phenotypic resistance patterns, and virulence and resistance gene profiles of Staph. aureus isolates from bovine mastitis milk in Switzerland. To this end, 58 Staph. aureus strains were characterized. The DNA microarray was used to test for the presence or absence of virulence and resistance genes. In addition, minimum inhibitory concentrations of various antimicrobial agents were determined by microdilution. To assess the population structure of the isolates, we determined clonal complexes (CC) using DNA microarray hybridization profiles and performed multilocus sequence typing and spa typing. The strains were assigned to 7 clonal complexes, 10 sequence types, and 11 spa types, with CC705 (43%), CC97 (33%), and CC20 (12%) representing the most common lineages and t529 (43%) and t267 (21%) representing the most common spa types. Only 1 isolate was assigned to CC8, a clonal lineage linked to high within-herd prevalence of mastitis. A total of 14% (n = 8) of strains were classified as resistant to penicillin, and 1 strain each was classified as oxacillin and pirlimycin resistant. Although no clinical breakpoints are available for the combination of kanamycin/cefalexin, growth of all strains was inhibited by the lowest combination of kanamycin/cefalexin concentrations tested (4 µg/mL of kanamycin and 0.4 µg/mL of cefalexin). One strain assigned to CC20, ST389, and t2094 exhibited resistance to penicillin, oxacillin, and pirlimycin as well as intermediate susceptibility to erythromycin and high minimum inhibitory concentration for several antimicrobial agents, for which no breakpoints were available.

Käppeli N ，Morach M ，Corti S ，Eicher C ，Stephan R ，Johler S ... -  《-》

Short communication: Characterization of Staphylococcus aureus from bulk tank milk of dairy cattle in Lombardy (northern Italy).

Staphylococcus aureus is one of the most important pathogens causing mastitis in cattle, and it is responsible for economic losses in dairy herds worldwide. The PCR amplification of the 16S-23S rRNA intergenic spacer (ribosomal spacer PCR, RS-PCR) allows a rapid classification of the strains in genotypes and genotypic clusters (CL), which are characterized by different epidemiological and clinical properties. Both RS-PCR and multi-locus sequence typing (MLST) were performed on strains isolated from bovine bulk tank milk (BTM) collected from dairy herds located in the Lombardy region (northern Italy), to outline the distribution of Staph. aureus genotypes in this geographical area. Out of 844 examined samples, 398 were positive for Staph. aureus, with a variable count (cfu/mL) Up to 8 colonies from each sample were genotyped. A total of 1,101 Staph. aureus strains were analyzed with RS-PCR, and only a selection of them (n = 86), in relation to their frequency and geographical origin, underwent MLST. This study revealed 8 major genotypic clusters (CLB, CLC, CLR, CLS, CLI, CLF, CLAO, and CLZ), of which Staph. aureus CLB (29.3%) was the most common. Samples of BTM positive for CLB had a Staph. aureus cfu/mL count significantly higher than the non-CLB positive ones. Our MLST analysis showed genotypes already known as bovine-associated in literature, such as clonal complexes CC8, CC97, and CC151. The same selection of 86 strains was also analyzed for the presence of the adlb gene, which was recently proposed as a possible marker of contagiousness. Most Staph. aureus belonging to CLB or CC8 carried the adlb gene (85%), whereas this gene was detected in only 9% of non-CLB strains (CLAA, CLBI, CLBJ, CLS). In conclusion, the present study confirms that Staph. aureus CLB, which is recognized as a contagious genotype, is a particularly relevant agent of intramammary infection in dairy cows in Lombardy, and indirectly supports the idea that adlb can be a possible marker of contagiousness of isolates.

Gazzola A ，Maisano AM ，Bianchini V ，Vezzoli F ，Romanò A ，Graber HU ，Cremonesi P ，Zanardi G ，Cappa V ，Luini M ... -  《-》