The HIV-1 latent reservoir is largely sensitive to circulating T cells.

HIV-1-specific CD8+ T cells are an important component of HIV-1 curative strategies. Viral variants in the HIV-1 reservoir may limit the capacity of T cells to detect and clear virus-infected cells. We investigated the patterns of T cell escape variants in the replication-competent reservoir of 25 persons living with HIV-1 (PLWH) durably suppressed on antiretroviral therapy (ART). We identified all reactive T cell epitopes in the HIV-1 proteome for each participant and sequenced HIV-1 outgrowth viruses from resting CD4+ T cells. All non-synonymous mutations in reactive T cell epitopes were tested for their effect on the size of the T cell response, with a≥50% loss defined as an escape mutation. The majority (68%) of T cell epitopes harbored no detectable escape mutations. These findings suggest that circulating T cells in PLWH on ART could contribute to control of rebound and could be targeted for boosting in curative strategies.

The RM test for determining methicillin-resistant Staphylococcus aureus lineages.

Staphylococcus aureus lineages evolve independently and differ in hundreds of genes. Identification of lineages can be useful for epidemiological typing and infection control at the local or global level, and can also be useful when investigating differences in pathogenesis between strains. MLST (multilocus sequence typing) and spa typing (polymorphisms in the protein A gene) are useful methods for identifying lineages but can be time-consuming and expensive. Here, we describe a method for identifying lineages using PCR, which is very easy to perform and can generate results within hours. It can also be adapted to commercial or real-time platforms. The RM (restriction modification) test is based on unique sequences found in each lineage that determine the specificity of an RM system, which detects and digests foreign DNA, thereby controlling the independent evolution of the lineages; thus, it is the ideal single gene to target for a rapid lineage test.

Staphylococcus aureus host specificity: comparative genomics of human versus animal isolates by multi-strain microarray.

Staphylococcus aureus is a commensal and pathogen of several mammalian species, particularly humans and cattle. We aimed to (i) identify S. aureus genes associated with host specificity, (ii) determine the relatedness of human and animal isolates, and (iii) identify whether human and animal isolates typically exchanged mobile genetic elements encoding virulence and resistance genes. Using a well-validated seven-strain S. aureus microarray, we compared 56 UK S. aureus isolates that caused infection in cows, horses, goats, sheep and a camel with 161 human S. aureus isolates from healthy carriers and community acquired infections in the UK. We had previously shown that human isolates are clustered into ten dominant and a few minor lineages, each with unique combinations of surface proteins predicted to bind to human proteins. We found that the animal-associated S. aureus clustered into ten lineages, with 61 % assigned to four lineages, ST151, ST771, ST130 and ST873, that were unique to animals. The majority of bovine mastitis was caused by isolates of lineage ST151, ST771 and ST97, but a few human lineages also caused mastitis. S. aureus isolated from horses were more likely to cluster into human-associated lineages, with 54 % of horse-associated S. aureus assigned to the human clusters CC1, CC8 and CC22; along with the presence of some multi-drug resistant strains, this suggests a human origin. This is the most comprehensive genetic comparison of human versus animal S. aureus isolates conducted, and because we used a whole-genome approach we could estimate the key genes with the greatest variability that are associated with host specificity. Several genes conserved in all human isolates were variable or missing in one or more animal lineages, including the well-characterized lineage specific genes fnbA, fnbB and coa. Interestingly, genes carried on mobile genetic elements (MGEs) such as chp, scn and sak were less common in animal S. aureus isolates, and bap was not found. There was a lot of MGE variation within lineages, and some evidence that exchange of MGEs such as bacteriophage and pathogenicity islands between animal and human lineages is feasible, but there was less evidence of antibiotic resistance gene transfer on the staphylococcal cassette chromosomes (SCC) or plasmids. Surprisingly, animal lineages are closely related to human lineages and only a handful of genes or gene combinations may be responsible for host specificity.

Staphylococcus aureus strains that are hypersusceptible to resistance gene transfer from enterococci.

We identified naturally occurring Staphylococcus aureus mutants of the restriction modification pathway SauI, including bovine lineage ST151. In a model of vancomycin resistance transfer from Enterococcus faecalis, ST151 isolates are 500 times more susceptible than human S. aureus isolates. The eradication of "hyperrecipient" strains may reduce the evolution of vancomycin-resistant S. aureus.

First report of multiresistant, mecA-positive Staphylococcus intermedius in Europe: 12 cases from a veterinary dermatology referral clinic in Germany.

Resistance to cephalosporins and/or fluoroquinolones by Staphylococcus intermedius has remained low in Europe, with effective drugs generally available for systemic therapy in pets. However, multiresistant, mecA-positive S. intermedius isolated from dogs and cats is now emerging in Europe. Twelve S. intermedius isolates, highly resistant to at least five antimicrobial classes, were isolated from skin and ear infections in 11 dogs and a cat. The 12 isolates represented 23% of all S. intermedius submissions from one veterinary dermatology referral clinic in northern Germany to veterinary diagnostic laboratories during an 18-month period and resistance included cefalexin, methicillin and enrofloxacin. The animals had been referred to the clinic with recurrent superficial pyoderma, deep pyoderma, pododermatitis or chronic otitis, all unresponsive to systemic beta-lactam-antibiotics or fluoroquinolones. Infection resolved in 10 dogs and the cat on a combination of antimicrobial treatment and correction of underlying causes. Four dogs and a cat required systemic and topical therapy; in six dogs topical antimicrobial therapy alone was successful. Phenotypic and genotypic characteristics of the S. intermedius isolates were determined; species identification was confirmed by polymerase chain detection of thermonuclease genes (nuc) and the presence and expression of the gene conferring resistance to all beta-lactam antibiotics (mecA) were demonstrated in all; based on pulsed-field gel electrophoresis, six were indistinguishable, the others closely or possibly related. The emergence of multiresistant, mecA-positive S. intermedius in Europe is alarming. Zoonotic implications, awareness among veterinary laboratories and strategies for the use of antimicrobials in small animal practice need to be considered.

Accessory gene regulator locus of Staphylococcus intermedius.

The accessory gene regulator (agr) locus, a candidate system for the regulation of the production of virulence factors in Staphylococcus intermedius, has been characterized. Using PCR-based genome walking, we have obtained the first complete sequence (3,436 bp) of the accessory gene regulator (agr) gene in this organism. Sequence analysis of the agr gene has identified five open reading frames (ORFs), agrB, agrD, agrC, agrA, and hld. The translated ORF contained amino acid motifs characteristic of the response regulator and histidine protein kinase signal transducer of the classic two-component regulatory system. Sequencing of the agrD PCR products amplified from DNA from 20 different isolates has facilitated detection of genetic variation in the putative autoinducing peptide (AIP) within the agr gene of S. intermedius, revealing the presence of at least three agr specificity groups within this species. Classification of the agr gene from S. intermedius was supported by phylogenetic analysis. Real-time PCR also revealed that the effector molecule of the agr system, RNAIII, was regulated in an autocrine manner in S. intermedius and demonstrated positive correlation with the temporal gene expression patterns of luk and entC. Transcription of RNAIII was also dependent on self secreted cues. Cyclic self and nonself peptides were synthesized on the basis of the novel AIPs produced by S. intermedius, which lack the cysteine necessary to form the thiolactone ring in analogous peptides from Staphylococcus aureus and Staphylococcus epidermidis. Experiments with these synthetic cyclic peptides indicated that self peptides led to up-regulation of RNAIII--findings in support of the assumption that activation of the agr gene is initiated by growth- and species-specific factors generated during bacterial growth.