Epidemiology: Open Access
Open Access

Methicillin; leukocidin; phylogenetic; prognosis

The impact of S. aureus toxins on the severity, management, and prognosis of SAB illness is still under investigation and needs more research. The most extensively researched S. aureus toxin, Panton- Valentine leukocidin (PVL), is controversial since it typically occurs at higher levels in non-invasive than invasive S. aureus isolates and does not seem to have an impact on outcomes for all clinical characteristics. Further paediatric-specific S. aureus molecular analyses are required to better our understanding of complicated host-pathogen dynamics, as there is few research exploring additional virulence determinants for paediatric SAB [1]. We performed a prospective genomic investigation of paediatric SAB in light of the illness burden, shifting patterns of antibiotic resistance, and ambiguities regarding the incidence and consequences of particular virulence factors. In order to understand the molecular epidemiology of SAB in children, we looked at how clinical phenotypes, severity, and outcomes were related to antibiotic resistance genes and virulence factors.

Isolate Collection

This prospective, multi-site, cross-sectional research of paediatric SAB in Australia and New Zealand included ten newborn intensive care units (NICUs), seven tertiary and secondary paediatric hospitals, and seven tertiary and level paediatric hospitals. The clinical outcomes that were looked at included 90-day all-cause mortality or a composite outcome that was predetermined to be 90-day all-cause mortality, 90- day relapse, ICU admission, or duration of stay. The procedures for the ISAIAH dataset are further discussed below and further information is provided [2]. Children under the age of 18 who presented to the study hospitals with a positive blood culture for S. aureus were prospectively enrolled. Local infectious disease or microbiology services detected episodes throughout a 24-month period (2017–2018). Also included were children with SAB who had been moved from outlying hospitals to the research locations. Blood cultures with several microbes were left out of the study [3].

Laboratories used semi-automated susceptibility platforms, typical commercial blood culture methods, and bacterial identification. Each laboratory reported its own antimicrobial susceptibility data while using the CLSI M100 minimum inhibitory concentration (MIC) breakpoints. Isolates with resistant or intermediate susceptibility were labelled as non-susceptible. Isolates of multi-resistant MRSA (mrMRSA) were identified as those that were resistant to three or more non-beta-lactam antibiotic classes. For whole genome sequencing, all isolates were sent to the national AGAR S. aureus reference laboratory (WGS) [4]. On sequencing was done using 150-bp paired-end chemistry. With the use of a QubitTM 3 Fluorometer and the MagMAXTM Express-96 deep well magnetic particle processor, genomic DNA was recovered. As advised by the manufacturer, DNA libraries were created and normalised using the Nextera XT DNA Library Preparation Kit. With an estimated coverage of >60, each genome was sequenced.

Using the SPAdes version, raw sequence reads were reconstructed from scratch. Contigs with less than 200 bp were discarded. The Supplementary Appendix S1 contains statistics on the quality of genome assembly. The assembled contigs for each isolate are accessible via a special identifier for each isolate and were uploaded to the Bacterial Isolate Genome Sequence Database (BIGSdb) on the aureus website. Genomes were annotated using the default Prokka parameters. Using Roary version, the core genes from this set were concatenated and aligned. On MEGA version X, phylogenetic trees were created using the neighbour-joining algorithm, and iTOL version 6.3 was used to visualise them [Figure 1].

Figure 1: Molecular Analysis Pediatric Staphylococcus Aureus Bacteremia.

85 percent of SAB events were caused by MSSA as the pathogen. One isolate, which the MALDI-TOF initially classified as MSSA, was later classified by the WGS as Staphylococcus argenteus. Every MRSA isolate included mecA. Only one isolate (0.2 percent) that was ST22- MRSA-IV, which was connected to the healthcare industry, was recognised as a multi-resistant MRSA, making up the majority of MRSA bacteraemia cases. Thirteen percent (38/301) of MSSA isolates were penicillin-susceptible S. aureus (PSSA); nevertheless, four of these PSSA isolates (11%) encoded a penicillinase. Between phenotypic and genotypic penicillin susceptibility testing, there was an overall 86 concordance (Supplementary Appendix S5). Cotri- moxazole resistance was found in 5% (18/353) of isolates overall, primarily in MSSA (78%) and MRSA bacteremia (22%) compared to each other.

The percentages of isolates with clindamycin inducible and constitutive resistance were 9 percent and 0.6 percent (2/353), respectively. As contrast to MRSA (10 percent, 3/31), MSSA bacteremia had a higher percentage of inducible clindamycin resistance (90 percent, 28/31); this corresponds to 84 percent concordance with genotypic testing. Results of additional antimicrobial susceptibility tests are displayed in Supplementary Appendix S5. It was shown that MSSA isolates had more STs and CCs than MRSA isolates, indicating greater variety. MRSA and MRSA isolates were genetically related, and no clusters peculiar to Australia or New Zealand were found. CCs 45, 15 and 121 were the only CCs connected to MSSA bacteraemia [7-10]. CC15 and CC8 were two popular MSSA clones related with healthcare. Eight MSSA STs were present but could not be categorised as a CC. 92 percent (24/26) of CC93 were MRSA, even though there were no CCs that contained just MRSA.

When compared using the 90-day all-cause mortality endpoint, no changes in the frequency of any CC, virulence, or antibiotic resistance genes were found using univariate or multivariable analysis. On univariate analysis, factors for community-onset SAB included CC93 and methicillin resistance that were predictive of the composite result. However, LOS >30 days had a significant impact on this composite outcome's lone microbiological variable, PVL positive, which was the only microbiological variable included in multivariable analysis (Supplementary Appendix S6). Multifocal disease, endovascular infection, multiorgan dysfunction, surgical source control, number of surgeries, peak C-reactive protein (CRP), days with SAB and fever,LOS, and all clinical indicators of S. aureus disease severity examined on univariate analysis were all significantly associated with PVL+ SAB compared to PVL SAB. Antibacterial treatment. These findings held true for pulmonary, osteoarticular, and SSTI foci, among other SAB clinical phenotypes. S. argenteus, a coagulase-positive species in the S. aureus complex that was previously believed to be a divergent S. aureus clonal lineage, was identified as one MSSA isolate using WGS.

The eight-year-old from regional Australia with a communityonset single focus osteoarticular infection and bacteraemia lasting longer than the median of 1 day for the entire cohort was the source of the S. argenteus isolate found in this investigation. No relapse or death was noted after the patient had antibiotic medication for a total of five weeks. S. argenteus has been found in clinical infections in humans, non-human primates, and African bats. There is a dearth of information on the epidemiology, clinical importance, and risk of transmission of S. argenteus. New research indicates that S. argenteus's pathogenicity is comparable to that of A. aureus only one other instance of paediatric S. argenteus has been documented, and that was a Japanese child who had lymphadenitis. Our case adds significantly to the body of knowledge about the pathogenicity of S. argenteus bacteremia in paediatric settings. We further draw attention to the fact that only a small percentage of S. argenteus is found in Australian and New Zealand children who have SAB.

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