Different bacterial gene expression patterns and attenuated host immune responses are associated with the evolution of low-level vancomycin resistance during persistent methicillin-resistant Staphylococcus aureus bacteraemia

BACKGROUND: Low-level vancomycin resistance in Staphylococcus aureus (vancomycin-intermediate S. aureus (VISA) and hetero-VISA [hVISA]) emerges during persistent infection and failed vancomycin therapy. Up-regulation of genes associated with the "cell wall stimulon" and mutations in the vraSR operon have both been implicated in the development of resistance, however the molecular mechanisms of resistance are not completely understood. To further elucidate the mechanisms leading to resistance transcriptome comparisons were performed using multiple clinical pairs of vancomycin-susceptible S. aureus (VSSA) and hVISA/VISA (n = 5), and three VSSA control pairs from hospitalized patients with persistent bacteraemia that did not develop hVISA/VISA. Based on the transcriptome results multiple genes were sequenced and innate immune system stimulation was assessed in the VSSA and hVISA/VISA pairs. RESULTS: Here we show that up-regulation of vraS and the "cell wall stimulon" is not essential for acquisition of low-level vancomycin resistance and that different transcriptional responses occur, even between closely related hVISA/VISA strains. DNA sequencing of vraSR, saeSR, mgrA, rot, and merR regulatory genes and upstream regions did not reveal any differences between VSSA and hVISA/VISA despite transcriptional changes suggesting mutations in these loci may be linked to resistance in these strains. Enhanced capsule production and reduced protein A expression in hVISA/VISA were confirmed by independent bioassays and fully supported the transcriptome data. None of these changes were observed in the three control pairs that remained vancomycin-susceptible during persistent bacteremia. In a macrophage model of infection the changes in cell surface structures in hVISA/VISA strains were associated with significantly reduced NF-kappaB activation resulting in reduced TNF-alpha and IL-1beta expression. CONCLUSION: We conclude that there are multiple pathways to low-level vancomycin resistance in S. aureus, even among closely related clinical strains, and these can result in an attenuated host immune response. The persistent infections associated with hVISA/VISA strains may be a consequence of changes in host pathogen interactions in addition to the reduced antibiotic susceptibility

Mycobacterium ulcerans in Mosquitoes Captured during Outbreak of Buruli Ulcer, Southeastern Australia

Mosquitoes positive for M. ulcerans were linked to outbreaks of Buruli ulcer in humans

The Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93-IV [2B] Is Highly Virulent and Genetically Distinct

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source

In-vitro Activity of Avermectins against Mycobacterium ulcerans

Mycobacterium ulcerans causes Buruli ulcer (BU), a debilitating infection of subcutaneous tissue. There is a WHO-recommended antibiotic treatment requiring an 8-week course of streptomycin and rifampicin. This regime has revolutionized the treatment of BU but there are problems that include reliance on daily streptomycin injections and side effects such as ototoxicity. Trials of all-oral treatments for BU show promise but additional drug combinations that make BU treatment safer and shorter would be welcome. Following on from reports that avermectins have activity against Mycobacterium tuberculosis, we tested the in-vitro efficacy of ivermectin and moxidectin on M. ulcerans. We observed minimum inhibitory concentrations of 4-8 mu g/ml and time-kill assays using wild type and bioluminescent M. ulcerans showed a significant dose-dependent reduction in M. ulcerans viability over 8-weeks. A synergistic killing-effect with rifampicin was also observed. Avermectins are well tolerated, widely available and inexpensive. Based on our in vitro findings we suggest that avermectins should be further evaluated for the treatment of BU

Buruli Ulcer (M. ulcerans Infection): New Insights, New Hope for Disease Control

Buruli ulcer is a disease of skin and soft tissue caused by Mycobacterium ulcerans. It can leave affected people scarred and disabled. What are the prospects for disease control

Mycobacterium ulcerans DNA Not Detected in Faecal Samples from Buruli Ulcer Patients: Results of a Pilot Study

It has recently been shown that in a Buruli ulcer (BU) endemic region of southeastern Australia, significant numbers of possums (native tree-dwelling marsupials) have clinical BU disease. Furthermore, based on quantitative PCR (qPCR) analysis, animals with BU lesions (and some without) shed M. ulcerans DNA in their faeces, indicative of bacterial loads of up to 108 organisms/gram. These findings led us to propose that humans might also harbour M. ulcerans in their gastrointestinal tract and shed the bacterium in their faeces. We conducted a pilot study and collected faecal swabs from 26 patients with confirmed BU and 31 healthy household controls. Faecal samples were also collected from 10 healthy controls from non-endemic regions in Ghana. All 67 specimens were negative when tested by IS2404 PCR. The detection sensitivity of this method was ≥104 bacteria per gram (wet-weight) of human faecal material. We conclude that the human gastrointestinal tract is unlikely to be a significant reservoir of M. ulcerans

Genomic insights to control the emergence of vancomycin-resistant enterococci.

UNLABELLED: Nosocomial outbreaks of vancomycin-resistant Enterococcus faecium (VREfm) are thought to occur by transmission of VREfm between patients, predicting that infection control interventions will limit cross-transmission. Despite implementation of such strategies, the incidence of VREfm infections continues to rise. We aimed to use genomics to better understand the epidemiology of E. faecium within a large hospital and investigate the reasons for failure of infection control strategies. Whole-genome sequencing was performed on 61 E. faecium (36 VREfm) isolates, predominately from blood cultures collected at a single hospital between 1998 and 2009, and on five vanB-positive anaerobic commensal bacteria isolated from human feces. Phylogenomic analysis and precise mapping of the vanB gene, which contains the Tn1549 transposon, showed that at least 18 of the 36 VREfm isolates had acquired the transposon via independent insertion events, indicating de novo generation of VREfm rather than cross-transmission. Furthermore, Tn1549 sequences found in 15 of the 36 VREfm isolates were the same as the Tn1549 sequence from one of the gut anaerobes. National and international comparator E. faecium isolates were phylogenetically interspersed with isolates from our hospital, suggesting that our findings might be globally representative. These data demonstrate that VREfm generation within a patient is common, presumably occurring in the human bowel during antibiotic therapy, and help explain our inability to reduce VREfm infections. A recommendation from our findings is that infection control practices should include screening patients for specific hospital clones of vancomycin-susceptible E. faecium rather than just VREfm. IMPORTANCE: Enterococcus faecium is an increasingly important human pathogen causing predominantly antibiotic-resistant infections in hospitalized patients. Large amounts of health care funding are spent trying to control antibiotic-resistant bacteria in hospitals globally, yet in many institutions around the world, vancomycin-resistant E. faecium (VREfm) infections continue to rise. The new findings from this study help explain the failures of our current approaches to controlling vanB VREfm in health care institutions. Given the importance of this bacterium as a cause of hospital-acquired infections and the difficulties faced by infection control units in trying to prevent colonization in their institutions, the novel findings from this study provide evidence that a new approach to controlling VREfm in hospitals is required. In particular, more attention should be given to understanding the epidemiology of hospital-adapted vancomycin-susceptible E. faecium, and patients at higher risk for de novo generation of VREfm need to be identified and optimally managed

Serological Evaluation of Mycobacterium ulcerans Antigens Identified by Comparative Genomics

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans

Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans

Adaptive Change Inferred from Genomic Population Analysis of the ST93 Epidemic Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem around the world. In Australia, ST93-IV[2B] is the dominant CA-MRSA clone and displays significantly greater virulence than other S. aureus. Here, we have examined the evolution of ST93 via genomic analysis of 12 MSSA and 44 MRSA ST93 isolates, collected from around Australia over a 17-year period. Comparative analysis revealed a core genome of 2.6 Mb, sharing greater than 99.7% nucleotide identity. The accessory genome was 0.45 Mb and comprised additional mobile DNA elements, harboring resistance to erythromycin, trimethoprim, and tetracycline. Phylogenetic inference revealed a molecular clock and suggested that a single clone of methicillin susceptible, Panton-Valentine leukocidin (PVL) positive, ST93 S. aureus likely spread from North Western Australia in the early 1970s, acquiring methicillin resistance at least twice in the mid 1990s. We also explored associations between genotype and important MRSA phenotypes including oxacillin MIC and production of exotoxins (α-hemolysin [Hla], δ-hemolysin [Hld], PSMα3, and PVL). High-level expression of Hla is a signature feature of ST93 and reduced expression in eight isolates was readily explained by mutations in the agr locus. However, subtle but significant decreases in Hld were also noted over time that coincided with decreasing oxacillin resistance and were independent of agr mutations. The evolution of ST93 S. aureus is thus associated with a reduction in both exotoxin expression and oxacillin MIC, suggesting MRSA ST93 isolates are under pressure for adaptive chang