A novel molecular assay conducted on the BD Max system to facilitate reflex testing for vanA and vanB in clinical isolates of enterococci.

Infections caused by vancomycin-resistant enterococci (VRE) are common. Real-time PCR assays targeting vanA and vanB facilitate screening of patients in healthcare settings to limit the risk of dissemination, especially amongst those at high-risk of infection or with limited treatment options. Such assays are commonly performed as reflex testing procedures where they augment phenotypic techniques and shorten turnaround time to benefit timely clinical management. 'Random access' and 'sample-to-result' real-time PCR platforms are suited for this application as they are of low complexity and less technically demanding. Modelled on these attributes, we configured a real-time PCR assay (VRE BD) for detection of vanA/B in clinical isolates of enterococci, adapted for the BD Max System (Becton Dickinson). We applied an unconventional approach by testing suspensions of microorganisms in water to circumvent the traditional pre-analytical genomic extraction process. Our objective of this study was to assess the performance of this assay for detection of VRE in cultures by validating against a traditional real-time PCR assay based on the LightCycler 2.0 platform (Roche, VRE RO). A high level of analytical sensitivity and specificity (≥99.0%) for both genes was obtained when testing suspensions derived from blood agar. Results for suspensions obtained from chromID VRE (Edwards Group) showed a similar level of performance for vanA detection (100%), but not for the vanB target (≥90.9%) where a lesser number of isolates were available for testing. However, our results for VRE detection in isolates from these media were repeatable and reproducible, and equated to positive and negative predictive values of ≥95.2% and ≥97.8%, respectively. Furthermore, the VRE BD assay was also able to accurately detect VRE in clinical and spiked BacT/ALERT (bioMérieux) blood cultures. Thus, the technical simplicity, short turnaround time and robustness of this high performing assay for VRE is suitable for reflex testing. In addition, the format developed for the BD Max platform has potential application for reflex testing other molecular targets of clinical importance.

A vanA vancomycin-resistant Enterococcus faecium ST80 outbreak resulting from a single importation event.

BACKGROUND: A marked genotype shift among vancomycin-resistant Enterococcus faecium (VREfm) from vanB to vanA in Australia between 2011 and 2015 is a well-known phenomenon. It is hypothesized that this was caused by multiple independent clones emerging simultaneously in different settings and/or regions. OBJECTIVES: To gain insights into the circumstances surrounding the shift from vanB to vanA VREfm in one Australian hospital. METHODS: The genomes of 69 vanA VREfm isolates from St George Hospital collected between 2009 and 2018 were studied. An expansion of ST80 vanA VREfm was noted following a single introduction. ST80 isolates were thus further characterized using hybrid sequencing and contextualized through comparisons with other published Australian ST80 isolates. Phylogenies were constructed with plasmid sequences compared with the index isolate. RESULTS: The 2011 expansion of ST80 vanA VREfm isolates in our institution originated from the 2009 index isolate, from a patient transferred from overseas. Phylogenetic analysis with other Australian ST80 vanA VREfm isolates showed that the 2011 expansion event was unique, with limited spread to adjacent local health districts. Plasmid analysis showed multiple variants, which can also be traced back to the 2009 isolate, consistent with ongoing plasmid adaptation over time. CONCLUSIONS: These findings confirm an expansion event following a VREfm introduction event leading to a sustained clonal and plasmid outbreak over several years. Moreover, it demonstrates the complexity of countrywide replacement events. This study also highlights the use of hybrid sequencing in establishing an epidemiological relationship to the index isolate that was initially inapparent.

A simple and expedient PCR format for rapid molecular screening of methicillin-resistant Staphylococcus aureus in Amies gel swabs.

Screening patients for carriage of methicillin-resistant Staphylococcus aureus (MRSA) is commonly undertaken in hospital laboratories using phenotypic methods. This work is labour-intensive, costly and may take several days to complete. We report on the validation of a novel rapid screening approach for direct testing of Amies gel swabs for MRSA. The method is based on two quantitative real time-PCR (qRT-PCR) assays for the detection of the nuc and mecA genes of MRSA. Based on SYBR Green technology, the assays use significantly less reagents than conventional qRT-PCR methods and are applied to testing templates derived directly from aqueous suspensions of swabs. Notwithstanding the occurrence of false-positives due to non-specific fluorescence generated by the SYBR Green dye, the novel assays showed a high negative predictive value enabling earlier reporting of negative findings and selection of swabs for confirmatory phenotypic testing for MRSA. In a blinded trial of 461 swabs, of which 34 (7.4%) were previously shown to be culture-positive for MRSA, the novel assays selected 121 (26.2%) swabs (inclusive of the known MRSA-positive swabs) for phenotypic testing. This enabled early reporting of negative findings for 340 (73.8%) of the 461 swabs tested. Application of this method has implications for screening strategies for large laboratories whilst achieving cost benefits.

Keratitis antimicrobial resistance surveillance program, Sydney, Australia: 2016 Annual Report.

IMPORTANCE: Antimicrobial resistance data from bacterial keratitis in Australia are lacking. BACKGROUND: Antimicrobial resistance is a global health threat. Bacterial keratitis is an ophthalmic emergency requiring immediate and effective treatment. DESIGN: Retrospective cohort study of bacterial isolates and antibiotic susceptibility profiles at a quaternary hospital in Sydney, Australia. PARTICIPANTS: Two hundred and twenty-four corneal scrapes from patients from January 1 to December 31, 2016. METHODS: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identified bacteria. The Calibrated Dichotomous Sensitivity (CDS) method determined antibiotic susceptibilities. MAIN OUTCOME MEASURES: Isolated organisms and antibiotic susceptibilities. RESULTS: One hundred and sixty-eight scrapes of 224 (75%) were culture positive. One hundred and thirty-one patients had a single organism isolated and 21 had mixed bacterial growth. Of the 157 organisms isolated, 131 (83%) were Gram-positive and 27 (17%) Gram-negative. Of the Gram-positive organisms, 75 (57%) were coagulase-negative Staphylococci (CoNS), 15 (11%) Staphylococcus aureus (including one methicillin-resistant Staphylococcus aureus [MRSA]) and 8 (6%) Corynebacterium spp. Of the Gram-negative organisms, 15 (58%) were Pseudomonas aeruginosa. With methicillin-sensitive Staphylococcus aureus (MSSA) resistance to chloramphenicol was 21%, ciprofloxacin 7% and gentamicin 7%. With CoNS resistance to cefalotin was 9%, gentamicin 9% and ciprofloxacin 9%. With Corynebacterium spp. resistance was 40% to cefalotin, chloramphenicol 25% and ciprofloxacin 14%. CONCLUSIONS AND RELEVANCE: Staphyloccocus spp. and Pseudomonas spp. were the most common microorganisms isolated. There was low resistance to cefalotin and ciprofloxacin for these isolates. More than 90% of these would be covered by current therapeutic recommendations for empiric therapy in Australia.

Comparative genomics of Cylindrospermopsis raciborskii strains with differential toxicities.

BACKGROUND: Cylindrospermopsis raciborskii is an invasive filamentous freshwater cyanobacterium, some strains of which produce toxins. Sporadic toxicity may be the result of gene deletion events, the horizontal transfer of toxin biosynthesis gene clusters, or other genomic variables, yet the evolutionary drivers for cyanotoxin production remain a mystery. Through examining the genomes of toxic and non-toxic strains of C. raciborskii, we hoped to gain a better understanding of the degree of similarity between these strains of common geographical origin, and what the primary differences between these strains might be. Additionally, we hoped to ascertain why some cyanobacteria possess the cylindrospermopsin biosynthesis (cyr) gene cluster and produce toxin, while others do not. It has been hypothesised that toxicity or lack thereof might confer a selective advantage to cyanobacteria under certain environmental conditions. RESULTS: In order to examine the fundamental differences between toxic and non-toxic C. raciborskii strains, we sequenced the genomes of two closely related isolates, CS-506 (CYN+) and CS-509 (CYN-) sourced from different lakes in tropical Queensland, Australia. These genomes were then compared to a third (reference) genome from C. raciborskii CS-505 (CYN+). Genome sizes were similar across all three strains and their G + C contents were almost identical. At least 2,767 genes were shared among all three strains, including the taxonomically important rpoc1, ssuRNA, lsuRNA, cpcA, cpcB, nifB and nifH, which exhibited 99.8-100% nucleotide identity. Strains CS-506 and CS-509 contained at least 176 and 101 strain-specific (or non-homologous) genes, respectively, most of which were associated with DNA repair and modification, nutrient uptake and transport, or adaptive measures such as osmoregulation. However, the only significant genetic difference observed between the two strains was the presence or absence of the cylindrospermopsin biosynthesis gene cluster. Interestingly, we also identified a cryptic secondary metabolite gene cluster in strain CS-509 (CYN-) and a second cryptic cluster common to CS-509 and the reference strain, CS-505 (CYN+). CONCLUSIONS: Our results confirm that the most important factor contributing to toxicity in C. raciborskii is the presence or absence of the cyr gene cluster. We did not identify any other distally encoded genes or gene clusters that correlate with CYN production. The fact that the additional genomic differences between toxic and non-toxic strains were primarily associated with stress and adaptation genes suggests that CYN production may be linked to these physiological processes.