Infectious titres of human papillomaviruses (HPVs) in patient lesions, methodological considerations in evaluating HPV infectivity and implications for the efficacy of high-level disinfectants.

BACKGROUND: Recent publications from a single research group have suggested that aldehyde-based high-level disinfectants (HLDs), such as ortho-phthalaldehyde (OPA), are not effective at inactivating HPVs and that therefore, patients may be at risk of HPV infection from medical devices. These results could have significant public health consequences and therefore necessitated evaluation of their reproducibility and clinical relevance. METHODS: We developed methods and used standardised controls to: (1) quantify the infectious levels of clinically-sourced HPVs from patient lesions and compare them to laboratory-derived HPVs, (2) evaluate experimental factors that should be controlled to ensure consistent and reproducible infectivity measurements of different HPV genotypes, and (3) determine the efficacy of select HLDs. FINDINGS: A novel focus forming unit (FFU) infectivity assay demonstrated that exfoliates from patient anogenital lesions and respiratory papillomas yielded infectious HPV burdens up to 2.7 × 103 FFU; therefore, using 2.2 × 102 to 1.0 × 104 FFU of laboratory-derived HPVs in disinfection assays provides a relevant range for clinical exposures. RNase and neutralising antibody sensitivities were used to ensure valid infectivity measures of tissue-derived and recombinant HPV preparations. HPV infectivity was demonstrated over a dynamic range of 4-5 log10; and disinfection with OPA and hypochlorite was achieved over 3 to >4 log10 with multiple genotypes of tissue-derived and recombinant HPV isolates. INTERPRETATION: This work, along with a companion publication from an independent lab in this issue, address a major public health question by showing that HPVs are susceptible to HLDs. FUNDING: Advanced Sterilization Products; US NIH (R01CA207368, U19AI084081, P30CA118100).

Dynamics of papillomavirus in vivo disease formation & susceptibility to high-level disinfection-Implications for transmission in clinical settings.

BACKGROUND: High-level disinfection protects tens-of-millions of patients from the transmission of viruses on reusable medical devices. The efficacy of high-level disinfectants for preventing human papillomavirus (HPV) transmission has been called into question by recent publications, which if true, would have significant public health implications. METHODS: Evaluation of the clinical relevance of these published findings required the development of novel methods to quantify and compare: (i) Infectious titres of lab-produced, clinically-sourced, and animal-derived papillomaviruses, (ii) The papillomavirus dose responses in the newly developed in vitro and in vivo models, and the kinetics of in vivo disease formation, and (iii) The efficacy of high-level disinfectants in inactivating papillomaviruses in these systems. FINDINGS: Clinical virus titres obtained from cervical lesions were comparable to those obtained from tissue (raft-culture) and in vivo models. A mouse tail infection model showed a clear dose-response for disease formation, that papillomaviruses remain stable and infective on fomite surfaces for at least 8 weeks without squames and up to a year with squames, and that there is a 10-fold drop in virus titre with transfer from a fomite surface to a new infection site. Disinfectants such as ortho-phthalaldehyde and hydrogen peroxide, but not ethanol, were highly effective at inactivating multiple HPV types in vitro and in vivo. INTERPRETATION: Together with comparable results presented in a companion manuscript from an independent laboratory, this work demonstrates that high-level disinfectants inactivate HPV and highlights the need for standardized and well-controlled methods to assess HPV transmission and disinfection. FUNDING: Advanced Sterilization Products, UK-MRC (MR/S024409/1 and MC-PC-13050) and Addenbrookes Charitable Trust.

Comparison of Staphylococcus aureus strains for ability to cause infective endocarditis and lethal sepsis in rabbits.

Staphylococcus aureus is a major cause of infective endocarditis (IE) and sepsis. Both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains cause these illnesses. Common S. aureus strains include pulsed-field gel electrophoresis (PFGE) types USA200, 300, and 400 types where we hypothesize that secreted virulence factors contribute to both IE and sepsis. Rabbit cardiac physiology is considered similar to humans, and rabbits exhibit susceptibility to S. aureus superantigens (SAgs) and cytolysins. As such, rabbits are an excellent model for studying IE and sepsis, which over the course of four days develop IE vegetations and/or fatal septicemia. We examined the ability of MRSA and MSSA strains (4 USA200, 2 USA300, 2 USA400, and three additional common strains, FRI1169, Newman, and COL) to cause vegetations and lethal sepsis in rabbits. USA200, TSST-1(+) strains that produce only low amounts of α-toxin, exhibited modest LD(50) in sepsis (1 × 10(8) - 5 × 10(8)) colony-forming units (CFUs), and 3/4 caused significant IE. USA200 strain MNPE, which produces high-levels of α-toxin, was both highly lethal (LD(50) 5 × 10(6) CFUs) and effective in causing IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD(50)s 1 × 10(6) and 5 × 10(7) CFUs) but were minimally capable of causing IE. Strain Newman, which is phylogenetically related to USA300 strains, was not highly lethal (LD(50) of 2 × 10(9) CFUs) and was effective in causing IE. USA400 strains were both highly lethal (LD(50)s of 1 × 10(7) and 5 × 10(7) CFUs) and highly effective causes of IE. The menstrual TSS isolate FRI1169, that is TSST-1(+), produces high-levels of α-toxin, but is not USA200, was both highly lethal and effective in causing IE. Additional studies showed that phenol soluble modulins (PSMs) produced by FRI1169 were important for sepsis but did not contribute to IE. Our studies show that these clonal groups of S. aureus differ in abilities to cause IE and lethal sepsis and suggest that secreted virulence factors, including SAgs and cytolysins, account for some of these differences.

Biofilm growth alters regulation of conjugation by a bacterial pheromone.

Conjugation is an important mode of horizontal gene transfer in bacteria, enhancing the spread of antibiotic resistance. In clinical settings, biofilms are likely locations for antibiotic resistance transfer events involving nosocomial pathogens such as Enterococcus faecalis. Here we demonstrate that growth in biofilms alters the induction of conjugation by a sex pheromone in E. faecalis. Mathematical modelling suggested that a higher plasmid copy number in biofilm cells would enhance a switch-like behaviour in the pheromone response of donor cells with a delayed, but increased response to the mating signal. Alterations in plasmid copy number, and a bimodal response to induction of conjugation in populations of plasmid-containing donor cells were both observed in biofilms, consistent with the predictions of the model. The pheromone system may have evolved such that donor cells in biofilms are only induced to transfer when they are in extremely close proximity to potential recipients in the biofilm community. These results may have important implications for development of chemotherapeutic agents to block resistance transfer and treat biofilm-related clinical infections.

Generation of virulence factor variants in Staphylococcus aureus biofilms.

Several serious diseases are caused by biofilm-associated Staphylococcus aureus. Colonial variants occur in biofilms of other bacterial species, and S. aureus variants are frequently isolated from biofilm-associated infections. Thus, we studied the generation of variants with altered expression of virulence factors in S. aureus biofilms. We observed that the number of variants found in biofilms, as measured by hemolytic activity, varied for different strains. Further study of hemolytic activity and signaling by the accessory gene regulator (Agr) quorum-sensing system in one S. aureus strain revealed three primary biofilm subpopulations: nonhemolytic (Agr deficient), hemolytic (Agr positive), and hyperhemolytic (also Agr positive). The nonhemolytic variant became the numerically dominant subpopulation in the biofilm. The nonhemolytic variant phenotype was stable and heritable, indicating a genetic perturbation, whereas the hyperhemolytic phenotype was unstable, suggesting a phase variation. Transcription profiling revealed that expression of the agr locus and many extracellular virulence factors was repressed in the nonhemolytic variant. Expression of the agr-activating gene, sarU, was also repressed in the nonhemolytic variant, suggesting one potential regulatory pathway responsible for the Agr-deficient phenotype. We suggest that the development of these variants in biofilms may have important clinical implications.

Delays in Pseudomonas aeruginosa quorum-controlled gene expression are conditional.

Acyl-homoserine lactone (acyl-HSL) signaling is thought to mediate quorum sensing in many species of Proteobacteria. The opportunistic human pathogen Pseudomonas aeruginosa uses acyl-HSLs to regulate hundreds of genes, including many that code for extracellular virulence factors. The idea that the P. aeruginosa acyl-HSLs serve as quorum-sensing signals has been questioned recently because microarray experiments show that the addition of signals to cultures of P. aeruginosa does not advance the onset of transcription for most acyl-HSL-dependent genes. We show that, under specific conditions, the expression of many acyl-HSL-dependent genes can be triggered at low culture density by signal addition. If complex medium is conditioned by growth of a non-acyl-HSL-producing P. aeruginosa, signals can eliminate the delay in expression of a battery of acyl-HSL-dependent genes. Furthermore, for one representative gene, lasB, there is no delay when signals are added to P. aeruginosa growing in conditioned complex medium or in minimal medium. We conclude that complex medium contains an inhibitor or inhibitors that can prevent induction of many, but not all, acyl-HSL-regulated genes and that the inhibitor is consumed by P. aeruginosa. Our results show that acyl-HSL signals can trigger expression of a large number of acyl-HSL-dependent genes regardless of growth phase. In this way, signaling in P. aeruginosa appears similar to quorum signaling in other Proteobacteria.

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

Quorum sensing in Staphylococcus aureus biofilms.

Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.

Quorum sensing in Staphylococcus infections.

Quorum sensing via the accessory gene regulator (agr) system has been assigned a central role in the pathogenesis of staphylococci, particularly Staphylococcus aureus. While the control of virulence gene expression in vitro by agr has been relatively straightforward to describe, regulation of both the quorum response itself and virulence genes in vivo is considerably more complex. The quorum response is highly dependent upon the environment in which the organism is grown and is strongly influenced by additional regulators that respond to signals other than cell density. There is increasing evidence that the agr phenotype may influence the behavior and pathogenesis of biofilm-associated S. aureus and S. epidermidis and may contribute to the chronic nature of some biofilm-associated infections.

Characterization and expression analysis of Staphylococcus aureus pathogenicity island 3. Implications for the evolution of staphylococcal pathogenicity islands.

We describe the complete sequence of the 15.9-kb staphylococcal pathogenicity island 3 encoding staphylococcal enterotoxin serotypes B, K, and Q. The island, which meets the generally accepted definition of pathogenicity islands, contains 24 open reading frames potentially encoding proteins of more than 50 amino acids, including an apparently functional integrase. The element is bordered by two 17-bp direct repeats identical to those found flanking staphylococcal pathogenicity island 1. The island has extensive regions of homology to previously described pathogenicity islands, particularly staphylococcal pathogenicity islands 1 and bov. The expression of 22 of the 24 open reading frames contained on staphylococcal pathogenicity island 3 was detected either in vitro during growth in a laboratory medium or serum or in vivo in a rabbit model of toxic shock syndrome using DNA microarrays. The effect of oxygen tension on staphylococcal pathogenicity island 3 gene expression was also examined. By comparison with the known staphylococcal pathogenicity islands in the context of gene expression described here, we propose a model of pathogenicity island origin and evolution involving specialized transduction events and addition, deletion, or recombination of pathogenicity island "modules."

Repression of the Staphylococcus aureus accessory gene regulator in serum and in vivo.

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (agr locus) as well as multiple virulence-associated genes in Staphylococcus aureus. Gene expression was examined during growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcutaneous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxin B. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and in vivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome (TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profiles in the various experimental conditions demonstrated no positive correlation between the expression of agr and any staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on the expression in vivo of the virulence factors examined. An effect of immunization on the expression of agr and virulence factors was also observed. These results suggest that agr activation is not necessary for development of staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity.