Identification of staphylococcal cassette chromosome mec in Staphylococcus aureus and non-aureus staphylococci from dairy cattle in Belgium: Comparison of multiplex PCR and whole genome sequencing.

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

Whole Genome Sequencing of Staphylococci Isolated From Bovine Milk Samples.

Staphylococci are among the commonly isolated bacteria from intramammary infections in bovines, where Staphylococcus aureus is the most studied species. This species carries a variety of virulence genes, contributing to bacterial survival and spread. Less is known about non-aureus staphylococci (NAS) and their range of virulence genes and mechanisms, but they are the most frequently isolated bacteria from bovine milk. Staphylococci can also carry a range of antimicrobial resistance genes, complicating treatment of the infections they cause. We used Illumina sequencing to whole genome sequence 93 staphylococcal isolates selected from a collection of staphylococcal isolates; 45 S. aureus isolates and 48 NAS isolates from 16 different species, determining their content of antimicrobial resistance genes and virulence genes. Antimicrobial resistance genes were frequently observed in the NAS species as a group compared to S. aureus. However, the lincosamide resistance gene lnuA and penicillin resistance gene blaZ were frequently identified in NAS, as well as a small number of S. aureus. The erm genes conferring macrolide resistance were also identified in several NAS isolates and in a small number of S. aureus isolates. In most S. aureus isolates, no antimicrobial resistance genes were detected, but in five S. aureus isolates three to six resistance genes were identified and all five of these carried the mecA gene. Virulence genes were more frequently identified in S. aureus, which contained on average five times more virulence genes compared to NAS. Among the NAS species there were also differences in content of virulence genes, such as S. chromogenes with a higher average number of virulence genes. By determining the content of a large selection of virulence genes and antimicrobial resistance genes in S. aureus and 16 different NAS species our results contribute with knowledge regarding the genetic basis for virulence and antimicrobial resistance in bovine staphylococci, especially the less studied NAS. The results can create a broader basis for further research into the virulence mechanisms of this important group of bacteria in bovine intramammary infections.

Penicillin-binding protein PBP2a provides variable levels of protection toward different ß-lactams in Staphylococcus aureus RN4220.

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to most ß-lactams due to the expression of an extra penicillin-binding protein, PBP2a, with low ß-lactam affinity. It has long been known that heterologous expression of the PBP2a-encoding mecA gene in methicillin-sensitive S. aureus (MSSA) provides protection towards ß-lactams, however, some reports suggest that the degree of protection can vary between different ß-lactams. To test this more systematically, we introduced an IPTG-inducible mecA into the MSSA laboratory strain RN4220. We confirm, by growth assays as well as single-cell microfluidics time-lapse microscopy experiments, that PBP2a expression protects against ß-lactams in S. aureus RN4220. By testing a panel of ten different ß-lactams, we conclude that there is also a great variation in the level of protection conferred by PBP2a. Expression of PBP2a resulted in an only fourfold increase in minimum inhibitory concentration (MIC) for imipenem, while a 32-fold increase in MIC was observed for cefaclor and cephalexin. Interestingly, in our experimental setup, PBP2a confers the highest protection against cefaclor and cephalexin-two ß-lactams that are known to have a high specific affinity toward the transpeptidase PBP3 of S. aureus. Notably, using a single-cell microfluidics setup we demonstrate a considerable phenotypic variation between cells upon ß-lactam exposure and show that mecA-expressing S. aureus can survive ß-lactam concentrations much higher than the minimal inhibitory concentrations. We discuss possible explanations and implications of these results including important aspects regarding treatment of infection.

Haematological and serum biochemical values in Norwegian sled dogs before and after competing in a 600 km race.

BACKGROUND: Long-distance racing is known to cause alterations in haematological and serum biochemical parameters in sled dogs. Given that finishing status reflects the physical condition in dogs completing a race, such variations will mainly be the result of physiological adaption achieved during endurance exercise. However, changes observed in withdrawn dogs may indicate pathological conditions. The aim of this study was to reveal changes in haematological and serum biochemical values in sled dogs participating in a long-distance race, with emphasis on the withdrawn dogs. Sixty-five sled dogs participated in a clinical prospective cohort study: 46 dogs competed in the 600 km race (25 finishing and 21 withdrawn dogs), and 19 dogs served as controls. Blood sampling was performed early in the training season and after the race. RESULTS: When compared to control dogs, both withdrawn and finishing dogs showed significant increases in neutrophil count, C-reactive protein, blood urea nitrogen and sodium/potassium ratio. Significant decreases were found in erythrocytes and eosinophil cell count, and in haematocrit, haemoglobin, total protein, albumin, globulin, creatinine, potassium and calcium levels. Finishing dogs presented significant increases in white blood cells, large unstained cells, monocyte count and cortisol level compared to control dogs. In contrast, withdrawn dogs had significant elevations in alanine aminotransferase and alkaline phosphatase activity, as well as parameters associated with muscle metabolism, such as aspartate aminotransferase, creatine kinase and phosphorus concentration. CONCLUSIONS: Competing sled dogs experienced minor changes in blood parameters in general, mainly revealing the same pattern among withdrawals and finishers. This might indicate that numerous changes simply reflect physiological adaption due to endurance exercise. However, the serum concentration of muscle enzymes was significantly increased only in the withdrawals, and were well above reference ranges. This reflects muscle degradation, which could be the main cause of performance failure in some of the withdrawals.

Serum concentration of gastrin, cortisol and C-reactive protein in a group of Norwegian sled dogs during training and after endurance racing: a prospective cohort study.

BACKGROUND: High incidences of gastritis and gastric ulceration are observed in sled dogs participating in endurance races. Exercise-induced increases in hormones like gastrin and cortisol have been suggested as possible contributing factors. An increase in C-reactive protein (CRP) has also been observed in canines during physical exercise. The aim of this study was to evaluate the effect of long distance racing on the serum concentration of gastrin, cortisol and CRP in a group of sled dogs, by comparing the results achieved early in the training season and after participating in a long distance race; "Femundløpet". Dogs that only trained to the race, but did not compete in the race, were used as control dogs. Sixty-five sled dogs participated in the study; 46 competing dogs (25 completing and 21 non-completing the race) and 19 non-racing dogs (control dogs). The blood samples were collected in October 2012 and February 2013. RESULTS: The post-race serum concentration of gastrin, cortisol and CRP was significantly elevated in sled dogs participating in the race (both completing and non-completing dogs) when compared to the results from training. However, no significant differences were observed between the two sampling dates in the control dogs. Post-race results for completing and non-completing dogs were also compared. This demonstrated a significant elevation in gastrin in non-completing versus completing dogs, and a significant elevation in cortisol in completing compared to non-completing dogs. CONCLUSIONS: Participation in a long distance race was associated with a significant increase in serum gastrin, cortisol and CRP in sled dogs.