Production of icaADBC-encoded polysaccharide intercellular adhesin, or poly-N-acetylglucosamine (PIA/ PNAG), represents an important biofilm mechanism in staphylococci. We previously described a glucoseinduced, ica-independent biofilm mechanism in four methicillin-resistant Staphylococcus aureus (MRSA) isolates. Here, biofilm regulation by NaCl and glucose was characterized in 114 MRSA and 98 methicillinsensitive S. aureus (MSSA) isolates from diagnosed device-related infections. NaCl-induced biofilm development was significantly more prevalent among MSSA than MRSA isolates, and this association was independent of the isolate's genetic background as assessed by spa sequence typing. Among MSSA isolates, PIA/PNAG production correlated with biofilm development in NaCl, whereas in MRSA isolates grown in NaCl or glucose, PIA/PNAG production was not detected even though icaADBC was transcribed and regulated. Glucose-induced biofilm in MRSA was ica independent and apparently mediated by a protein adhesin(s). Experiments performed with strains that were amenable to genetic manipulation revealed that deletion of icaADBC had no effect on biofilm in a further six MRSA isolates but abolished biofilm in four MSSA isolates. Mutation of sarA abolished biofilm in seven MRSA and eight MSSA isolates. In contrast, mutation of agr in 13 MRSA and 8 MSSA isolates substantially increased biofilm (more than twofold) in only 5 of 21 (23%) isolates and had no significant impact on biofilm in the remaining 16 isolates. We conclude that biofilm development in MRSA is ica independent and involves a protein adhesin(s) regulated by SarA and Agr, whereas SarA-regulated PIA/PNAG plays a more important role in MSSA biofilm development.

Phenotypic biotyping has traditionally been used to differentiate bacteria occupying distinct ecological niches such as host species. For example, the capacity of Staphylococcus aureus from sheep to coagulate ruminant plasma, reported over 60 years ago, led to the description of small ruminant and bovine S. aureus ecovars. The great majority of small ruminant isolates are represented by a single, widespread clonal complex (CC133) of S. aureus, but its evolutionary origin and the molecular basis for its host tropism remain unknown. Here, we provide evidence that the CC133 clone evolved as the result of a human to ruminant host jump followed by adaptive genome diversification. Comparative whole-genome sequencing revealed molecular evidence for host adaptation including gene decay and diversification of proteins involved in host–pathogen interactions. Importantly, several novel mobile genetic elements encoding virulence proteins with attenuated or enhanced activity in ruminants were widely distributed in CC133 isolates, suggesting a key role in its host-specific interactions. To investigate this further, we examined the activity of a novel staphylococcal pathogenicity island (SaPIov2) found in the great majority of CC133 isolates which encodes a variant of the chromosomally encoded von Willebrand-binding protein (vWbpSov2), previously demonstrated to have coagulase activity for human plasma. Remarkably, we discovered that SaPIov2 confers the ability to coagulate ruminant plasma suggesting an important role in ruminant disease pathogenesis and revealing the origin of a defining phenotype of the classical S. aureus biotyping scheme. Taken together, these data provide broad new insights into the origin and molecular basis of S. aureus ruminant host specificity.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

Staphylococcus aureus is an important pathogen of man, but is also able to colonize and cause disease in a wide variety of mammals and birds. An extended multilocus sequencing approach, involving multilocus sequence typing (MLST), sas typing, spa typing and agr typing, was used to examine the molecular diversity of 118 S. aureus isolates recovered from a range of host species and to compare these data with the known diversity of human-derived isolates. MLST revealed that the commonest animal-associated MLST types were ST133, ST5, ST71, ST97, ST126 and ST151. ST133 appears to be an ungulate-animal-specific genotype, as no evidence of ST133 associating with humans has yet been found in the literature. Novel and unique sas alleles were identified in the animal-associated strains that may represent animal-associated sas alleles. However, sas typing exhibited a lower typeability than MLST for the animal strains (91.3 %). Phylogenetic analyses using neighbour-joining and maximum-parsimony trees localized ruminantassociated MLST lineages to both previously identified S. aureus subspecies aureus subgroups, thus explaining the finding of all four agr types within the ruminant-associated strains. S. aureus isolates recovered from chickens and rabbits were genotypically more similar to known human genotypes than the ruminant-associated lineages.

In recent years several new staphylococcal enterotoxins (SEs) have been described, which currently have largely unknown frequencies of occurrence and roles in human or animal disease. One hundred and ninety-one Staphylococcus aureus isolates from cows (99), goats (39), sheep (23), rabbits (15), chickens (15) and a cat (1) were screened for SE genes sea-see, seg-seo and seq and for the tst gene encoding staphylococcal toxic shock syndrome toxin-1 using multiplex PCRs and individual PCRs for the seb and sek genes. One hundred and ten isolates tested positive for at least one of these 16 superantigen (SAg)-encoding genes. There were statistically significant differences in the frequencies of some of these SAg genes between isolates from different animals. No strain possessed either the sea or see gene. The sec gene was present in 51 isolates, the sed gene in eight and the seb gene in one. The seh gene was found in four strains and the sek and seq genes together in one isolate. The most common combinations of genes were the egc cluster, bearing the seg, sei, sem, sen and seo genes, in 47 isolates, the sec, sel and tst gene combination typical of the SaPIbov pathogenicity island in 44 isolates, the egc cluster lacking the seg gene in 11 isolates, the sed and sej genes in nine isolates, and the sec and tst genes without the sel gene in seven isolates. The higher frequencies of the sec and tst genes together and the lower frequencies of the egc gene cluster among the SAg gene-positive sheep or goat isolates compared to bovine isolates were statistically significant. Of 36 bovine isolates that were mitogenic for human T lymphocytes, four were negative for the 16 SAg genes tested for, while a further 14 gave borderline results in the mitogenicity assay, 12 of which were SAg gene-negative. Twenty-nine strains lacking all the SAg genes did not induce T-cell proliferation. This survey indicates that novel SE genes seg, sei, sel, sem, sen and seo along with the sec and tst genes predominate in S. aureus from animal hosts. The mitogenicity assays indicate that further uncharacterized SAgs may be present in bovine isolates. INTRODUCTIONStaphylococcus aureus is a major human pathogen that causes a wide variety of diseases ranging in severity from food poisoning (McCormick et al., 2001;Le Loir et al., 2003) and life-threatening toxic shock syndrome (Llewelyn & Cohen, 2002;Proft & Fraser, 2003) to less serious infections, e.g. boils (Stulberg et al., 2002). S. aureus can also cause a number of infections in animals, such as tick-associated pyaemia in lambs (Webster & Mitchell, 1989), staphylococcosis in rabbits (Hermans et al., 2003), oedematous and necrotic dermatitis, septicaemia, abcesses and chondronecrosis in chickens McNamee et al., 1998;Takeuchi et al., 2002) S. aureus is the most frequent cause of bovine mastitis, a disease that is of economic importance worldwide (Beck et al., 1992;Miles et al., 1992). Typically staphylococcal mastitis is chronic in nature, with subclinical mastitis being the most common form (Gruet et a...