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.

Short communication: Characterization of Staphylococcus aureus isolated along the raw milk cheese production process in artisan dairies in Italy.

Staphylococcus aureus is a common cause of food-borne intoxications. Several staphylococcal food poisoning outbreaks have been linked to consumption of raw milk cheeses and artisanal cheese production. However, information on Staph. aureus isolated from artisanal raw milk cheeses and small-scale dairy production environments is very limited. Therefore, we aimed to characterize Staph. aureus isolated along the artisanal raw milk production chain by determining (1) the population structure, and (2) the presence/absence of enterotoxin genes, mecA/C, and pvl. We collected 276 samples from different production stages (raw milk, whey, curd, brine, drying worktops, and cheese) at 36 artisan dairies in Italy. A total of 102 samples from 25 dairies tested positive for Staph. aureus, with 80% positive samples among the tested artisan cheeses. All isolates were further characterized by spa typing and PCR screening for staphylococcal enterotoxin genes, the mecA/mecC genes characteristic for methicillin-resistant Staph. aureus, and the pvl gene encoding Panton-Valentine leukocidin. The 102 isolates represented 15 different spa types and were assigned to 32 different Staph. aureus strains. The spa type most frequently detected was t2953 (30%), which is associated with genotype B strains causing high within-herd levels of bovine mastitis. In addition, 3 novel spa types (t13269, t13277, and t13278) were identified. Although none of the strains harbored mecA/mecC or pvl, 55% of the isolates exhibited at least one enterotoxin gene. Many strains were present in samples from multiple dairies from different regions and years, highlighting the spread of Staph. aureus in small-scale cheese production plants. Our findings demonstrate that enterotoxigenic Staph. aureus and in particular t2953 (genotype B) isolates commonly occur in artisanal dairies and raw milk cheeses in Italy. It is particularly alarming that 80% of the artisan cheeses sampled in our study were positive for Staph. aureus. These findings stress the need for effective measures preventing staphylococcal food poisoning by limiting Staph. aureus growth and enterotoxin formation along the production chain and in the final product.

Tracing and inhibiting growth of Staphylococcus aureus in barbecue cheese production after product recall.

Staphylococcal food poisoning is one of the most prevalent causes of foodborne intoxication worldwide. It is caused by ingestion of enterotoxins formed by Staphylococcus aureus during growth in the food matrix. Following a recall of barbecue cheese due to the detection of staphylococcal enterotoxins in Switzerland in July 2015, we analyzed the production process of the respective dairy. Although most cheese-making processes involve acidification to inhibit the growth of pathogenic bacteria, barbecue cheese has to maintain a pH >6.0 to prevent undesired melting of the cheese. In addition, the dairy decided to retain the traditional manual production process of the barbecue cheese. In this study, therefore, we aimed to (1) trace Staph. aureus along the barbecue cheese production process, and (2) develop a sustainable strategy to inhibit growth of Staph. aureus and decrease the risk of staphylococcal food poisoning without changing the traditional production process. To this end, we traced Staph. aureus in a step-wise blinded process analysis on 4 different production days using spa (Staphylococcus protein A gene) typing, DNA microarray profiling, and pulsed-field gel electrophoresis analysis. We subsequently selected a new starter culture and used a model cheese production including a challenge test assay to assess its antagonistic effect on Staph. aureus growth, as well as its sensory and technological implications. We detected Staph. aureus in 30% (37/124) of the collected samples taken from the barbecue cheese production at the dairy. This included detection of Staph. aureus in the final product on all 4 production days, either after enrichment or using quantitative detection. We traced 2 enterotoxigenic Staph. aureus strains (t073/CC45 and t282/CC45) colonizing the nasal cavity and the forearms of the cheesemakers to the final product. In the challenge test assay, we were able to show that the new starter culture inhibited growth of Staph. aureus while meeting the sensory and technological requirements of barbecue cheese production.

Success of interventions in mastitis problems with Staphylococcus aureus after the introduction of an automatic milking system.

Staphylococcus aureus (S. aureus) is often the cause of mastitis problems in dairy herds and causes great economic losses. In this study, isolates from a dairy herd with a known S. aureus mastitis problem were examined by means of molecular methods (spa typing, PFGE, and DNA microarray) to investigate their epidemiological relationship and the success of intervention measures. The investigated dairy farm has a herd size of 60 cows and uses a fully automated milking system for milk production. A S. aureus strain, which contaminated the automated milking system and was subsequently spread among the herd through the latter, was suspected to be the origin of the mastitis problem within the herd. Thanks to the applied molecular methods, the common origin of the S. aureus isolates from the collected milk and swab samples could be shown. By culling chronically infected cows, optimising dry cow management and ensuring reliable intermediate cluster disinfection, the bulk milk somatic cell count improved.

Species distribution and resistance profiles of coagulase-negative staphylococci isolated from bovine mastitis in Switzerland.

Coagulase-negative staphylococci (CNS) are the predominant cause of bovine intra-mammary infections. They can lead to chronic infections and were reported to significantly increase milk somatic cell counts. The goal of our study was to determine the species distribution and antimicrobial susceptibility profiles of CNS in bovine mastitis milk samples in Switzerland. Between March 2011 and February 2012, a total of 120 CNS were isolated from mastitis milk samples from 117 different animals at 77 farms. The isolates were identified by matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS) and subsequently tested for sensitivity to various antibiotic agents by disk diffusion. Antimicrobial agents were selected mainly based on their relevance to the treatment of bovine mastitis in Switzerland. MALDI-TOF MS assigned the 120 isolates to 12 different staphylococcal species - S. chromogenes (33 %), S. xylosus (28 %), S. sciuri (13 %), S. haemolyticus (9 %), S. epidermidis (4 %), S. simulans (4 %), S. warneri (3 %), S. equorum (2 %), S. hyicus (2 %), S. cohnii (1 %), S. succinus (1 %), and S. fleuretti (1 %). Resistance rates in CNS were high, with 39% of isolates exhibiting resistance to ampicillin and penicillin, 6% of isolates being resistant to amoxicillin with clavulanic acid, ceftiofur, cephalothin, and cefoxitin, and 5 % being resistant to erythromycin. In rare cases resistance to gentamicin (2 %), kanamycin (2 %), and kanamycin-cefalexin (1 %) was detected.

Les staphylocoques coagulase négatifs (CNS) sont la cause la plus fréquente d'infections intra mammaires chez les bovins. Ils peuvent causer des infections chroniques et augmenter nettement le nombre de cellules somatiques du lait. Le but de la présente étude était de déterminer la répartition des espèces et les profils de réséstance des CNS dans des échantillons de lait mammiteux bovins. De mars 2011 à février 2012, 120 CNS ont été isolé à partir d'échantillons de lait issus de 117 vaches provenant de 77 exploitations. Les isolats ont été identifiés par la technologie MALDI-TOF MS (matrix-assisted laser desorption ionization ­ time of flight mass spectrometry) puis testés par diffusion sur gel d'agar quant à leur sensibilité vis à vis de divers antibiotiques. Les antibiotiques ont été choisis principalement par rapport à leur importance quant aux traitements des mammites bovines en Suisse. Le test MALDI-TOF MS a classifié les 120 isola en 12 espèces différentes de staphylocoaques: S. chromogenes (33 %), S. xylosus (28 %), S. sciuri (13 %), S. haemolyticus (9 %), S. epidermidis (4 %), S. simulans (4 %), S. warneri (3 %), S. equorum (2 %), S. hyicus (2 %), S. cohnii (1 %), S. succinus (1 %) et S. fleuretti (1 %). D'une façon générale, la proportion de CNS résistants était élevée, 39 % des échantillons étant résistants à l'ampicilline et à la pénicilline, 6 % à l'amoxicilline avec acide clavulanique, au ceftiofur, à la céphalothine et à la céfoxitine et 5 % à l'érythromycine. Dans de rares cas on a trouvé des resistances à la gentamicine (2 %), à la kanamycine (2 %) et à la kanamycine-céfalexine (1 %).

Virulence and antimicrobial resistance genes in human MRSA ST398 isolates in Austria.

This study determined the genetic background of virulence and resistance genes of MRSA ST398 in Austria. From 2004 up to 2008 a total of 41 human isolates of MRSA ST398 were investigated for virulence and resistance gene patterns using DNA microarray chip analysis. Highly similar virulence gene profiles were found in 29 (70·7%) of the isolates but genes encoding Panton-Valentine leukocidin, enterotoxins, or toxic shock syndrome toxin were not detected. Genes conferring resistance to tetracycline and erythromycin-lincosamide were common as all but one of the isolates exhibited tetM and/or tetK, which are involved in tetracycline resistance, and 12 (29·9%) were positive for ermC, conferring resistance to erythromycin/lincosamide. SplitsTree analysis showed that 40 isolates were closely related. Changes in virulence and resistance gene patterns were minimal over the observed time period.

Comparison of genomic and antimicrobial resistance features of latex agglutination test-positive and latex agglutination test-negative Staphylococcus aureus isolates causing bovine mastitis.

The dairy industry suffers massive economic losses due to staphylococcal mastitis in cattle. The Staphaureux latex agglutination test (Oxoid, Basel, Switzerland) was reported to lead to negative results in 54% of bovine Staphylococcus aureus strains, and latex-negative strains are thought to be less virulent than Staphaurex latex-positive strains. However, comparative information on virulence and resistance profiles of these 2 groups of Staph. aureus is scarce. Our objective was to associate the latex agglutination phenotype of Staph. aureus strains isolated from bovine mastitis milk with data on clonal complexes, virulence genes, and antibiotic resistance to (1) determine the virulence profiles of the Staphaureux test positive and Staphaurex test negative groups, and (2) provide data needed to improve treatment of bovine mastitis and to identify potential vaccine targets. Seventy-eight Staph. aureus strains isolated from 78 cows on 57 Swiss farms were characterized. Latex agglutination was tested by Staphaureux kit, and resistance profiles were generated by disk diffusion. A DNA microarray was used to assign clonal complexes (CC) and to determine virulence and resistance gene profiles. By the Staphaureux test, 49% of the isolates were latex-positive and 51% were latex-negative. All latex-negative strains were assigned to CC151, whereas latex-positive strains were assigned to various clonal complexes, including CC97 (n=16), CC8 (n=10), CC479 (n=5), CC20 (n=4), CC7 (n=1), CC9 (n=1), and CC45 (n=1). Although the latex-negative isolates were susceptible to all antimicrobial agents tested, 24% of latex-positive isolates were classified as intermediate with regard to cefalexin-kanamycin and 13% were resistant to both ampicillin and penicillin. Microarray profiles of latex-negative isolates were highly similar, but differed largely from those of latex-positive isolates. Although the latex-negative group lacked several enterotoxin genes and sak, it exhibited significantly higher prevalence rates of genes encoding enterotoxin C, toxic shock syndrome toxin, and leukocidins (lukM/lukF-P83, lukD). Our findings suggest that latex-negative isolates represent a group of closely related strains with specific resistance and virulence gene patterns.

Comparison of Staphylococcus aureus isolates associated with food intoxication with isolates from human nasal carriers and human infections.

Staphylococcus aureus represents an organism of striking versatility. While asymptomatic nasal colonization is widespread, it can also cause serious infections, toxinoses and life-threatening illnesses in humans and animals. Staphylococcal food poisoning (SFP), one of the most prevalent causes of foodborne intoxication worldwide, results from oral intake of staphylococcal enterotoxins leading to violent vomiting, diarrhea and cramps shortly upon ingestion. The aim of the present study was to compare isolates associated with SFP to isolates collected from cases of human nasal colonization and clinical infections in order to investigate the role of S. aureus colonizing and infecting humans as a possible source of SFP. Spa typing and DNA microarray profiling were used to characterize a total of 120 isolates, comprising 50 isolates collected from the anterior nares of healthy donors, 50 isolates obtained from cases of clinical infections in humans and 20 isolates related to outbreaks of staphylococcal food poisoning. Several common spa types were found among isolates of all three sources (t015, t018, t056, t084). DNA microarray results showed highly similar virulence gene profiles for isolates from all tested sources. These results suggest contamination of foodstuff with S. aureus colonizing and infecting food handlers to represent a source of SFP.