Mechanisms of ß-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases.

A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for ß-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for ß-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased ß-lactam MIC had SNPs leading to pbp2x E381K and Q554E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased ß-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E381K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to ß-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis.

Papillomaviral DNA and increased p16CDKN2A protein are frequently present within feline cutaneous squamous cell carcinomas in ultraviolet-protected skin.

Squamous cell carcinomas (SCCs) are common feline skin tumours. While exposure to ultraviolet (UV) light causes some SCCs, a subset develop in UV-protected skin. In cats, papillomaviruses (PVs) cause viral plaques and Bowenoid in situ carcinomas (BISCs). As both may progress to SCC, it was hypothesized that SCCs in UV-protected skin may represent neoplastic transformation of a PV-induced lesion. To investigate this hypothesis, PCR was used to amplify PV DNA from 25 UV-protected and 45 UV-exposed SCCs. Oncogenic human PVs cause neoplasia by mechanisms that also increase p16(CDKN2A) protein (p16). As increased p16 is present in feline viral plaques and BISCs, immunohistochemistry was used to detect p16 within the SCCs. Papillomaviral DNA was amplified from 76% of UV-protected SCCs, but only 42% of UV-exposed SCCs. Increased p16 was present in 84% of UV-protected SCCs, but only 40% of UV-exposed SCCs. The more frequent detection of PV DNA and increased p16 within UV-protected SCCs supports the hypothesis that some develop from a PV-induced plaque or BISC. Felis domesticus PV-2 is thought to cause viral plaques and BISCs. This PV was detected most frequently within the UV-protected SCCs, supporting development from a PV-induced lesion. Increased p16 and PV DNA were less frequent within UV-exposed SCCs, presumably because these developed from actinic keratosis rather than a PV-induced lesion. The results support the hypothesis that some feline cutaneous SCCs are caused by PV infection and suggest that PVs may cause neoplasia by mechanisms that also increase p16.