The Molecular Epidemiology of the Highly Virulent ST93 Australian Community Staphylococcus aureus Strain

In Australia the PVL - positive ST93-IV [2B], colloquially known as ‘‘Queensland CA-MRSA’’ has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage WSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable.The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVLpositive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known

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

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

Evolution of Multidrug Resistance during Staphylococcus aureus Infection Involves Mutation of the Essential Two Component Regulator WalKR

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen

Two Novel Point Mutations in Clinical Staphylococcus aureus Reduce Linezolid Susceptibility and Switch on the Stringent Response to Promote Persistent Infection

Staphylococcus aureus frequently invades the human bloodstream, leading to life threatening bacteremia and often secondary foci of infection. Failure of antibiotic therapy to eradicate infection is frequently described; in some cases associated with altered S. aureus antimicrobial resistance or the small colony variant (SCV) phenotype. Newer antimicrobials, such as linezolid, remain the last available therapy for some patients with multi-resistant S. aureus infections. Using comparative and functional genomics we investigated the molecular determinants of resistance and SCV formation in sequential S. aureus isolates from a patient who had a persistent and recurrent S. aureus infection, after failed therapy with multiple antimicrobials, including linezolid. Two point mutations in key staphylococcal genes dramatically affected clinical behaviour of the bacterium, altering virulence and antimicrobial resistance. Most strikingly, a single nucleotide substitution in relA (SACOL1689) reduced RelA hydrolase activity and caused accumulation of the intracellular signalling molecule guanosine 3′, 5′-bis(diphosphate) (ppGpp) and permanent activation of the stringent response, which has not previously been reported in S. aureus. Using the clinical isolate and a defined mutant with an identical relA mutation, we demonstrate for the first time the impact of an active stringent response in S. aureus, which was associated with reduced growth, and attenuated virulence in the Galleria mellonella model. In addition, a mutation in rlmN (SACOL1230), encoding a ribosomal methyltransferase that methylates 23S rRNA at position A2503, caused a reduction in linezolid susceptibility. These results reinforce the exquisite adaptability of S. aureus and show how subtle molecular changes cause major alterations in bacterial behaviour, as well as highlighting potential weaknesses of current antibiotic treatment regimens

The effects of sport organization’s volunteer management : exploring the Mehrabian-Russell model

With the complexity of staging a sport event, large numbers of sport volunteers are needed to deliver services and help meet the organization’s objectives. Understanding the motivation and satisfaction of volunteers allow organizations to build an effective volunteer management structure which will influence the behavioural intentions of sport volunteers. This paper explores the Mehrabian-Russell Model by applying it to the sport event context. Using the online questionnaire platform, data were collected from 307 sport volunteers (Male - 51.9%, Female - 48.1%) across several sport events. The study identifies the volunteer management factors that impact motivation and satisfaction and followed by testing the relationship between satisfaction and behavioural intentions. Each construct has several questions which were measured with 5-point Likert Scales anchored by “strongly agree” to “strongly disagree”. SPSS version 24.0 was used for data screening and AMOS 25.0 was used to perform Structural Equation Modeling to measure the relationship among constructs. Findings include that there is a positive relationship between volunteer management and motivation (t=3.376, p <.001), but volunteer management has no direct impact on satisfaction (t=1.135, p =.256). Satisfaction is indirectly influenced by volunteer management through motivation (t=2.464, p=.014). Also, the study shows that volunteers who are satisfied are most probably going to remain in the same organization, donate financially to the organization, recommend the volunteer experience to their family and friends, and participate in sports for fun and to keep healthy (t=5.483, p <.001). Outcomes of the study help ensure the sustainability of the sport organization.Bachelor of Science (Sport Science and Management

Virulence in Australian community-associated methicillin-resistant staphylococcus aureus

The global phenomenon of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections is a key public health issue. These CA-MRSA strains can cause severe, fulminant sepsis in previously healthy individuals. Unlike other regions, in Australia, the dominant CA-MRSA strain is the Queensland ST93-IV [2B] clone, a unique strain that is genetically distinct from other S. aureus. This project aimed to characterise a representative isolate from this clone (JKD6159) in comparison with other Australian strains and also the USA300 epidemic CAMRSA strain. Initially, functional virulence analysis using an invertebrate Galleria killing assay and a mouse skin infection assay demonstrated that JKD6159 was the most virulent strain tested. Whole genome sequencing was then used to uncover molecular mechanisms for this increased virulence. The complete genome of JKD6159 was determined, and annotated. Comparative genomics demonstrated that although there were unique regions of difference in the JKD6159 genome compared to other S. aureus genome sequences, there were no novel virulence factors to explain the heightened virulence. The JKD6159 genome did contain lukSF-PV, the genes encoding for Panton- Valentine leukocidin and like other S. aureus genomes, also contained hla (encoding α-hemolysin) and psmα genes (encoding the α-type phenol soluble modulins) in the core staphylococcal genome. These exotoxins have been demonstrated to be important virulence factors in the pathogenesis of USA300. In order to better understand virulence of ST93 MRSA, JKD6159 was compared with another three ST93 MRSA strains with varying virulence phenotypes, using both comparative and functional genomics approaches. This demonstrated that ST93 virulence is primarily dependent on high-level expression of α-hemolysin and an intact agr system. However, other regulators, including a previously uncharacterised AraC/XylS family regulator (AryK) that acts as a positive regulator of exotoxin expression, also influence virulence of this clone. This project has characterised the virulence phenotype of ST93 CA-MRSA and used comparative and functional genomics to determine the key molecular determinants of virulence in this clone

Individual and community predictors of urinary ceftriaxone-resistant Escherichia coli isolates, Victoria, Australia

Abstract Background Ceftriaxone-resistant Enterobacteriaceae are priority pathogens of critical importance. Escherichia coli is the most commonly isolated Enterobacteriaceae. There are few data regarding non-invasive ceftriaxone-resistant E. coli (CR-EC) isolates in the Australian community. We aimed to describe the prevalence, phenotype, geographic variation, and sociodemographic predictors of ceftriaxone-resistance among E. coli isolates recovered from urine specimens. Methods In August 2017, we prospectively analysed E. coli isolates recovered from urine specimens submitted to Dorevitch Pathology (Victoria, Australia), a laboratory that services patients in the community and hospitals. In addition to patient-level predictors of ceftriaxone resistance, we mapped patient postcodes to community-level indicators including Index of Relative Socioeconomic Deprivation, remoteness, and proportion of residents born overseas. We used Poisson regression with log link and robust standard errors to quantify the association between ceftriaxone resistance and patient- and community-level factors. Results We included 6732 non-duplicate E. coli isolates. Most (89.2%, 6008/6732) were obtained from female patients. Median age was 56 years (IQR, 32–74). Most patients (90.5%, 5789/6732) were neither referred from a hospital nor residing in a residential aged care facility (RACF). Among the 6732 isolates, 5.7% (382) were CR-EC, ranging from 3.5% (44/1268) in inner regional areas to 6.3% (330/5267) in major cities. Extended spectrum ß–lactamase (ESBL) -production was the most common mechanism for ceftriaxone resistance (89%, 341/382). Nitrofurantoin was the most active oral agent against CR-EC. Eight CR-EC isolates (2.4%) were susceptible only to amikacin, meropenem and nitrofurantoin. None were resistant to meropenem. On multivariable analysis, ceftriaxone resistance was associated with age, residence in a RACF (adjusted relative risk [aRR] 2.94, 95% confidence interval [CI] 2.10–4.13), specimen referral from hospital (aRR 2.05, 95% CI 1.45–2.9), and the proportion of residents born in North Africa and the Middle East (aRR 1.30 for each 5% absolute increase, 95% CI 1.09–1.54), South-East Asia (aRR 1.14, 95% CI 1.02–1.27), and Southern and Central Asia (aRR 1.16, 95% CI 1.04–1.28). Conclusions These results provide insights into sociodemographic variation in CR-EC in the community. A better understanding of this variation may inform empiric treatment guidelines and strategies to reduce community dissemination of CR-EC

Assessing the effect of electronic word of mouth and firm strategy on business performance as perceived by selected online sellers

The main focus of this study, which was conducted by the researchers, was to identify the effects of electronic word of mouth and firm strategy on the firm performance of selected online businesses in the Philippines. The research approach is quantitative, descriptive, correlational, and causal-explanatory because the purpose of this study is to characterize and portray the participants\u27 views and opinions. Mean response scores and standard deviations were used to determine the extent of electronic word of mouth, business performance, and firm strategy of online businesses in Metro Manila. The researchers also analyzed Pearson\u27s correlation coefficient to see if there was a link between electronic word of mouth and firm strategy and online business performance in Metro Manila. Finally, the effect of electronic word of mouth and firm strategy on the firm performance of online businesses in Metro Manila was investigated using the p-value and significance f methods. A survey of 100 online sellers in Metro Manila was undertaken. For their businesses, these respondents use digital markets. The study\u27s findings reveal the survey\u27s and comparable research’s similarities and differences. That said, electronic word of mouth and firm strategy may not have as big of an impact on the firm performance of online businesses which is in contrast with the findings of previous research on similar studies. This is for a variety of reasons, and the researchers offered suggestions on how aspiring entrepreneurs, online businesses, other businesses, academe, and future researchers can continue to use electronic word of mouth and firm strategy in their businesses, maximize the knowledge gained from this study, and, most importantly, take this study forward