Rachael A. Lilliebridge

Community-Associated Strains of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible S. aureus in Indigenous Northern Australia: Epidemiology and Outcomes

BACKGROUND Some strains of non-multidrug-resistant, methicillin-resistant Staphylococcus aureus (nmMRSA) in Australia are likely to have emerged from strains of methicillin-susceptible S. aureus (MSSA) in remote Aboriginal communities. OBJECTIVE To describe the clinical epidemiology of infection due to community-associated MRSA strains in an Australian tropical hospital setting with a significant Aboriginal population and to compare infections caused by community-associated strains of MRSA, health-care-associated strains of MRSA, and MSSA strains with respect to demographic risk factors and clinical outcomes. Methods. We queried the microbiology database for the Top End of the Northern Territory, Australia, to determine population incidences for S. aureus infection and conducted a prospective matched case-control study to compare infection due to nmMRSA, MSSA, or multidrug-resistant MRSA at the Royal Darwin Hospital. RESULTS The annual incidence of S. aureus bacteremia was 65 cases per 100,000 population, but in the Aboriginal population the incidence was 172 cases per 100,000 population (odds ratio [OR] compared with non-Aboriginal population, 5.8 [95% confidence interval {CI}, 3.8-8.9). Female sex (adjusted OR [aOR], 1.5 [95% CI, 1.1-2.0) and remote residence (aOR, 1.8 [95% CI, 1.2-2.5]) were associated with the isolation of nmMRSA rather than MSSA, but disease spectrum and outcomes were similar. Among those from whom nmMRSA was isolated, Aboriginal patients were younger (aOR for each additional year, 0.94 [95% CI, 0.92-0.96]), more likely to be female (aOR, 3.8 [95% CI, 1.7-8.5]), and more likely to reside in a remote community (aOR, 29 [95% CI, 8.9-94]) than non-Aboriginal patients. The presence of Panton-Valentine leukocidin in nmMRSA was associated with double the odds of sepsis (aOR, 2.2 [95% CI, 1.1-4.6]). CONCLUSIONS The association of nmMRSA infection with female sex and remote residence supports the hypothesis that nmMRSA arose from MSSA strains in remote Aboriginal communities where staphylococcal disease is highly prevalent. The similar clinical spectrum and outcomes for nmMRSA infection and MSSA infection suggest that virulence is not correlated with resistance phenotype.

Clinical Correlates of Panton-Valentine Leukocidin (PVL), PVL Isoforms, and Clonal Complex in the Staphylococcus aureus Population of Northern Australia

BACKGROUND Regional differences in the prevalence of Panton-Valentine leukocidin (PVL) and PVL isoform-harboring strains as well as in the local population structure of Staphylococcus aureus may influence the clinical spectrum of S. aureus infections. METHODS Using a prospective collection of S. aureus isolates from northern Australia, we determined differences between infections caused by (1) PVL(+) and PVL(-) isolates, (2) PVL histidine (H) isoform- and PVL arginine (R) isoform-harboring isolates, and (3) different lineages, including the genetically divergent clonal complex (CC) 75 and the PVL(+) CC93. RESULTS PVL(+) isolates comprised 54% (128/239) of community-associated methicillin-resistant isolates and 40% (95/239) of methicillin-susceptible S. aureus (MSSA) isolates. There were 113 H isoform- and 110 R isoform-harboring isolates. PVL was associated with truly community-acquired disease, younger age, and presentation with sepsis. We found no differences in infections due to H isoform-harboring isolates, compared with R isoform-harboring isolates. CC93 was the most prevalent lineage. The genetically divergent CC75 caused clinical disease similar to that of other S. aureus clones. CONCLUSIONS PVL(+) and PVL(-) infections are clearly distinct. MSSA contributes a large but underrecognized burden of PVL(+) disease. Compared with elsewhere in the world, there is a relative abundance of the clade that contains CC93 and CC121 in both northern Australia and Asia.

Phylogenetically Distinct Staphylococcus aureus Lineage Prevalent among Indigenous Communities in Northern Australia

ABSTRACT The aim was to determine the evolutionary position of the Staphylococcus aureus clonal complex 75 (CC75) that is prevalent in tropical northern Australia. Sequencing of gap, rpoB, sodA, tuf, and hsp60 and the multilocus sequence typing loci revealed a clear separation between conventional S. aureus and CC75 and significant diversity within CC75.

The Utility of High-Resolution Melting Analysis of SNP Nucleated PCR Amplicons—An MLST Based Staphylococcus aureus Typing Scheme

High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpsons Index of Diversity. Only G↔A, G↔T, C↔A, C↔T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 “melt types” (MelTs), and provides a Simpsons Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs.

Sequences of multiple bacterial genomes and a Chlamydia trachomatis genotype from direct sequencing of DNA derived from a vaginal swab diagnostic specimen

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen.

High-resolution melting analysis of the spa locus reveals significant diversity within sequence type 93 methicillin-resistant Staphylococcus aureus from northern Australia

High-resolution melting analysis is an inherently robust, easy and inexpensive approach to the examination of genomic regions containing single-nucleotide polymorphisms and hypervariable loci. Staphylococcus aureus sequence type (ST) 93 is a singleton, Panton-Valentine leukocidin-positive clone unique to Australia. A high-resolution melting-based method for the identification of ST93 was developed, and a similar approach was used to reveal diversity within the spa locus of this lineage. Statistical and graphical methods that account for instrumental and operator-dependent variation in high-resolution melting curves were developed, to allow greater confidence and reproducibility in deciding whether another curve is truly different from the baseline curve of an amplicon with known sequence. The data support a very early acquisition, or multiple independent acquisitions, of SCCmec by ST93 methicillin-susceptible S. aureus (MSSA), and the coexistence of MSSA and methicillin-resistant S. aureus versions of the same lineage within northern Australia.

Rapid detection of H and R Panton–Valentine leukocidin isoforms in Staphylococcus aureus by high-resolution melting analysis

We designed a single-step, closed-tube, real-time polymerase chain reaction and high-resolution melting assay to simultaneously detect the presence of the Panton-Valentine leukocidin gene and discriminate histidine and arginine isoforms. Of 223 Staphylococcus aureus isolates from northern Australia, isoforms clustered by clonal complex (CC). All CC93 isolates harbored the arginine isoform.

Chlamydia trachomatis genotypes in a cross-sectional study of urogenital samples from remote Northern and Central Australia

Objectives The objective was to determine the frequency of trachoma genotypes of Chlamydia trachomatis-positive urogenital tract (UGT) specimens from remote areas of the Australian Northern Territory (NT). Setting The setting was analysis of remnants of C. trachomatis positive primarily UGT specimens obtained in the course of clinical practice. The specimens were obtained from two pathology service providers. Participants From 3356 C. trachomatis specimens collected during May 2012–April 2013, 439 were selected for genotyping, with a focus on specimens from postpubescent patients, in remote Aboriginal communities where ocular trachoma is potentially present. Primary and secondary outcome measures The primary outcome measure was the proportion of successfully genotyped UGT specimens that were trachoma genotypes. The secondary outcome measures were the distribution of genotypes, and the frequencies of different classes of specimens able to be genotyped. Results Zero of 217 successfully genotyped UGT specimens yielded trachoma genotypes (95% CI for frequency=0–0.017). For UGT specimens, the genotypes were E (41%), F (22%), D (21%) and K (7%), with J, H and G and mixed genotypes each at 1–4%. Four of the five genotyped eye swabs yielded trachoma genotype Ba, and the other genotype J. Two hundred twenty-two specimens (50.6%) were successfully genotyped. Urine specimens were less likely to be typable than vaginal swabs (p<0.0001). Conclusions Unlike in some other studies, in the remote NT, trachoma genotypes of C. trachomatis were not found circulating in UGT specimens from 2012 to 2013. Therefore, C. trachomatis genotypes in UGT specimens from young children can be informative as to whether the organism has been acquired through sexual contact. We suggest inclusion of C. trachomatis genotyping in guidelines examining the source of sexually transmitted infections in young children in areas where trachoma genotypes may continue to circulate, and continued surveillance of UGT C. trachomatis genotypes.

Multisite Direct Determination of the Potential for Environmental Contamination of Urine Samples Used for Diagnosis of Sexually Transmitted Infections.

Background The detection of a sexually transmitted infection (STI) agent in a urine specimen from a young child is regarded as an indicator of sexual contact. False positives may conceivably arise from the transfer of environmental contaminants in clinic toilet or bathroom facilities into urine specimens. Methods The potential for contamination of urine specimens with environmental STI nucleic acid was tested empirically in the male and female toilets or bathrooms at 10 Northern Territory (Australia) clinics, on 7 separate occasions at each. At each of the 140 experiments, environmental contamination with Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis nucleic acid contamination was determined by swabbing 10 locations, and urine collection was simulated 5 times, using a (1) synthetic urine surrogate and (2) a standardized finger contamination procedure. Results The most contaminated toilets and bathrooms were in remote Indigenous communities. No contamination was found in the Northern Territory Government Sexual Assault Referral Centre clinics, and intermediate levels of contamination were found in sexual health clinics and in clinics in regional urban centres. The frequency of surrogate urine sample contamination was low but non-zero. For example, 4 of 558 of the urine surrogate specimens from remote clinics were STI positive. Conclusions This is by far the largest study addressing the potential environmental contamination of urine samples with STI agents. Positive STI tests arising from environmental contamination of urine specimens cannot be ruled out. The results emphasize that urine specimens from young children taken for STI testing should be obtained by trained staff in clean environments, and duplicate specimens should be obtained if possible.

Whole genome sequencing to investigate a putative outbreak of the virulent community-associated methicillin-resistant Staphylococcus aureus ST93 clone in a remote Indigenous community

We report two cases of severe pneumonia due to clone ST93 methicillin-resistant Staphylococcus aureus (MRSA) presenting from a remote Australian Indigenous community within a 2-week period, and the utilization of whole genome sequences to determine whether these were part of an outbreak. S. aureus was isolated from 12 of 92 nasal swabs collected from 25 community households (including the two index households); one isolate was ST93. Three of five skin lesion S. aureus isolates obtained at the community were ST93. Whole genome sequencing of the ST93 isolates from this study and a further 20 ST93 isolates from the same region suggested that recent transmission and progression to disease had not taken place. The proximity in time and space of the two severe pneumonia cases is probably a reflection of the high burden of disease due to ST93 MRSA in this population where skin infections and household crowding are common.