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

Phenotypic and genomic comparisons of community-associated Staphylococcus aureus Clone ST93

© 2015 Justin StepnellStaphylococcus aureus is an opportunistic bacterial pathogen that primarily colonises the anterior nares of 30-50% of individuals at any one time, without causing disease. S. aureus does however cause a wide range of diseases including skin and soft tissue infections, pneumonia, osteoarticular infections and bacteraemia or septicaemia, frequently resulting in hospitalisation and a high mortality rate for invasive disease. S. aureus strains have acquired resistance to many classes of antibiotics, most importantly resistance to beta-lactam antibiotics (methicillin-resistant S. aureus, MRSA). While traditionally a health care associated issue, community-acquired MRSA (CA-MRSA) clones are increasing worldwide resulting in severe, antibiotic resistant infections occurring in patients without healthcare contact. The ST93 clone of CA-MRSA is essentially unique to Australia, and has been associated with severe, invasive S. aureus infections in otherwise healthy individuals. ST93 CA-MRSA is also the most common CA-MRSA clone in Australia. The overall aims of this study were to determine the virulence characteristics of a collection of ST93 S. aureus isolates from around Australia, and to uncover the molecular determinants of virulence in ST93 S. aureus. Fifty-eight ST93 isolates were assessed for virulence using the Galleria mellonella invertebrate virulence model and by measuring expression of key virulence factors. Whole genome sequencing and genomic analysis of all isolates was used to uncover genetic differences that might account for differences in virulence characteristics. In this study ~50% of isolates (n=28) were avirulent when compared to a virulent reference ST93 isolate JKD6159 using the G. mellonella model. This study also revealed that the G. mellonella model does not respond to exotoxin expression of isolates with no significant differences in G. mellonella mortality between culture supernatant of virulent and avirulent isolates used in the G. mellonella model. The expression levels of PSMα3 and δ-toxin varied significantly amongst the isolate collection, and the concentration of δ-toxin was found not to correlate with that of PSMα3, indicating that systems other than the quorum-sensing agr system must be controlling the expression of PSMα3 in ST93 S. aureus

Novel <i>Wolbachia</i>-transinfected <i>Aedes aegypti</i> mosquitoes possess diverse fitness and vector competence phenotypes

<div><p><i>Wolbachia pipientis</i> from <i>Drosophila melanogaster</i> (<i>w</i>Mel) is an endosymbiotic bacterium that restricts transmission of human pathogenic flaviviruses and alphaviruses, including dengue, Zika, and chikungunya viruses, when introduced into the mosquito vector <i>Aedes aegypti</i>. To date, <i>w</i>Mel-infected <i>Ae</i>. <i>aegypti</i> have been released in field trials in 5 countries to evaluate the effectiveness of this strategy for disease control. Despite the success in establishing <i>w</i>Mel-infected mosquitoes in wild populations, and the well-characterized antiviral capabilities of <i>w</i>Mel, transinfecting different or additional <i>Wolbachia</i> strains into <i>Ae</i>. <i>aegypti</i> may improve disease impact, and perhaps more importantly, could provide a strategy to account for the possible evolution of resistant arboviruses. Here, we report the successful transinfection of <i>Ae</i>. <i>aegypti</i> with the <i>Wolbachia</i> strains <i>w</i>MelCS (<i>D</i>. <i>melanogaster</i>), <i>w</i>Ri (<i>D</i>. <i>simulans</i>) and <i>w</i>Pip (<i>Culex quinquefasciatus)</i> and assess the effects on <i>Ae</i>. <i>aegypti</i> fitness, cytoplasmic incompatibility, tissue tropism and pathogen blocking in a laboratory setting. The results demonstrate that <i>w</i>MelCS provides a similar degree of protection against dengue virus as <i>w</i>Mel following an infectious blood meal, and significantly reduces viral RNA levels beyond that of <i>w</i>Mel following a direct challenge with infectious virus in mosquitoes, with no additional fitness cost to the host. The protection provided by <i>w</i>Ri is markedly weaker than that of <i>w</i>MelCS, consistent with previous characterisations of these lines in <i>Drosophila</i>, while <i>w</i>Pip was found to substantially reduce the fitness of <i>Ae</i>. <i>aegypti</i>. Thus, we determine <i>w</i>MelCS as a key candidate for further testing in field-relevant fitness tests and viremic blood feeding challenges in a clinical setting to determine if it may represent an alternative <i>Wolbachia</i> strain with more desirable attributes than <i>w</i>Mel for future field testing.</p></div

Maternal transmission of <i>Wolbachia</i> assessed from progeny of crosses between infected females and uninfected males.

<p>Maternal transmission of <i>Wolbachia</i> assessed from progeny of crosses between infected females and uninfected males.</p

<i>w</i>MelCS provides superior blocking of DENV-3 genome replication in a mosquito injection challenge model.

<p>(A) DENV-3 was injected into the thorax of 6 or 7-day old female mosquitoes at 2.5 x10⁶ TCID₅₀/ml (undiluted) or 10, 100 or 1000-fold dilutions thereof. RNA was extracted from whole mosquito bodies 7-days post infection and virus replication was quantified by qRT-PCR. Data are the mean number of genome copies per mosquito ± SEM. Number of DENV-3 positive mosquitoes/total <i>n</i> are indicated above each bar. ** p < 0.01, ****p<0.0001, Mann-Whitney test. (B) The mean DENV genome copies and SEM from (A) are replotted as a function of virus concentration injected. Significant differences in the mean RNA copies of <i>w</i>Mel and <i>w</i>MelCS lines are indicated by ** (p<0.01). Significant differences in the mean RNA copies of <i>w</i>Mel.Tet and <i>w</i>MelCS.Tet lines are indicated by <b>‡</b> (p<0.05), Mann-Whitney test. Injection dilutions were performed as independent experiments, with the data combined to produce the final data series.</p

CI, fecundity and egg diapause viability in <i>w</i>MelCS, <i>w</i>Ri, and <i>w</i>Pip transinfected <i>Ae</i>. <i>aegypti</i>.

<p>(A) Fecundity was determined as a measure of eggs laid per female. Bars are the mean number of eggs laid ± SEM from >40 females (individual data points are superimposed). Symbols for tetracycline-treated mosquitoes (uninfected) are in black, <i>Wolbachia</i>-infected are in red. Asterisks indicate significance compared to Tet x Tet controls (Kruskal-Wallis, Dunn's test, * for <0.05, ** for <0.01, *** for <0.001, **** for <0.0001). (B) Hatch rates for crosses between infected and uninfected mosquitoes show CI and successful hatching. Symbol codes and statistics are as per (A). Data are the mean ± SEM from >40 females (individual data points are superimposed). (C) Eggs from gravid females were collected over 72 h, from 3-days post blood meal. Eggs were dried slowly over 3–5 days then stored in a humid, airtight container. Batches of 100–500 eggs were hatched after 1, 2, 3, 4, 6, 8, 10 and 12 weeks. Hatched larvae were counted at 2nd instar stage until no hatch was observed for a week then the percent hatch calculated. Statistical analysis was performed using 2way ANOVA Sidak's test (n = 4 at each time point). <i>w</i>Pip hatch rate was significantly reduced at all weeks compared to <i>w</i>Pip.Tet (p<0.0001). <i>w</i>Mel hatch rate was significantly reduced at weeks 4, 8, 10, (p<0.05) and 12 (p<0.0001) compared to <i>w</i>Mel.Tet. <i>w</i>MelCS had a significantly reduced hatch rate at weeks 10 and 12 compared to <i>w</i>MelCS.Tet (p<0.01 and p<0.0001, respectively). <i>w</i>Ri had a significantly reduced hatch rate at week 12 only, compared to <i>w</i>Ri.Tet (p<0.0001).</p

<i>Wolbachia</i> density and distribution in transinfected <i>Ae</i>. <i>aegypti</i> lines.

<p>(A) Density of <i>Wolbachia</i> within 5-, 10- and 15-day old whole female mosquitoes was determined by qPCR using primers directed to the conserved <i>16S</i> rRNA gene. Density is expressed as the mean ratio between <i>16S</i> and the <i>Ae</i>. <i>aegypti</i> host <i>rps17 gene</i>. Data are the mean and SEM of 24 mosquitoes. Asterisks indicate significance compared to <i>w</i>Mel at each time point (Kruskal-Wallis, Dunn's test with multiple test corrections; *p<0.05, **p<0.01, ***p<0.001, n.s. not significant). (B) The distribution of <i>w</i>MelCS, <i>w</i>Ri and <i>w</i>Pip <i>Wolbachia</i> strains in mosquitoes was determined in sections of paraffin-embedded female mosquitoes (5 to 7-day old) using fluorescence <i>in situ</i> hybridisation (FISH). The fluorescently labelled 16S probe detects the <i>16S</i> rRNA gene from all four <i>Wolbachia</i> strains. Total DNA was stained in blue using DAPI and a green filter was included to increase contrast with surrounding tissues. <i>Sg</i> indicates salivary gland tissue, <i>m</i> indicates muscle, and <i>c</i> indicates cardia. White arrows identify select regions of <i>Wolbachia</i> staining.</p

Longevity in transinfected <i>Ae</i>. <i>aegypti</i> lines.

<p>Triplicate cages of age-controlled (emergence within 24 h) adults (~150 males and ~150 females/cage) were maintained at 26°C, 65% relative humidity and a 12:12 h light:dark cycle in a climate controlled room. The number of dead males and/or females was recorded and carcasses removed daily until all mosquitoes in the cages were dead. Data are the total % survival from the three cages/line. Significant differences were observed for <i>w</i>Ri females (p<0.0001), <i>w</i>Mel (p<0.05) and <i>w</i>MelCS (p<0.05) males, relative to their respective Tet control line. <i>w</i>Pip had a significantly shorter lifespan for both males and females compared its matched Tet-control (p<0.0001). Statistical analysis was performed using a Log-rank (Mantel-Cox) test.</p