Shakeel Mowlaboccus

Daptomycin is a cyclic lipopeptide antimicrobial used against gram-positive pathogens, including Staphylococcus aureus. Daptomycin relies on physiological calcium to anchor onto bacterial membranes and sequester lipid II, overproducing reactive oxygen species resulting in cell death. Although daptomycin is thought to be effective against approximately 80% of S. aureus, treatment failure can arise from resistant or tolerant cells. Daptomycin-resistant S. aureus typically acquires mutations in the multiple peptide resistance factor (MprF) to overproduce cationic lysyl-phosphatidylglycerol located on the outer membrane. The increase in positive cell surface charge inhibits daptomycin binding via electrostatic repulsion. Upon exposure to higher daptomycin concentrations, S. aureus may undergo further peptidoglycan modifications to attenuate daptomycin activity. These alterations can also be driven by the crosstalk in the signal transduction systems. Furthermore, the pathways in daptomycin resistance appear to overlap with tolerance which is understudied in S. aureus. In this review, we explore the current understanding of the complex interplay of molecular mechanisms involved in daptomycin resistance and tolerance in S. aureus.

From 1 January to 31 December 2024, fifty-five institutions across Australia participated in the Australian Enterococcal Surveillance Outcome Program (AESOP). The aim of AESOP 2024 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to determine the molecular epidemiology of the reported Enterococcus faecium isolates. Of the 1,461 unique episodes of enterococcal bacteraemia investigated, 92.5% were caused by either E. faecalis (51.5%) or E. faecium (41.0%). Ampicillin and vancomycin resistance were not detected in E. faecalis but were detected in 96.8% and 44.5% of E. faecium respectively. Five linezolid-resistant E. faecalis isolates were identified, for which the linezolid minimum inhibitory concentrations (MICs) ranged from 6.0 mg/L to 8.0 mg/L. All five isolates harboured the linezolid resistance optrA gene and were vancomycin susceptible. One linezolid-resistant E. faecium was confirmed with an MIC of 6.0 mg/L. The isolate was vancomycin and teicoplanin resistant and harboured vanA and optrA genes.

Overall, 49.8% of E. faecium isolates harboured the vanA and/or the vanB gene: within these isolates, 40.2% harboured vanA, 58.8% harboured vanB, and 1.0% harboured vanA and vanB. The percentage of vancomycin-resistant E. faecium bacteraemia isolates in Australia remains substantially higher than that recorded in most European countries. The E. faecium isolates consisted of 56 multi-locus sequence types (STs); 85.7% of isolates were classified into eight STs, each containing ten or more isolates. The eight STs (ST17, ST78, ST80, ST117, ST555, ST796, ST1421 and ST1424) belonged to clonal complex (CC) 17, a global hospital-adapted polyclonal E. faecium CC, and were found in most Australian jurisdictions. Overall, 54.6% of E. faecium isolates belonging to the eight predominant STs harboured the vanA or vanB gene. AESOP 2024 has shown that enterococcal bacteraemia episodes in Australia continue to be frequently caused by polyclonal ampicillin-resistant high-level gentamicin-resistant vanA- or vanB-positive E. faecium which have limited treatment options.

From 1 January to 31 December 2024, fifty-five institutions across Australia participated in the Australian Staphylococcus aureus Surveillance Outcome Program (ASSOP). The aim of ASSOP 2024 was to determine the proportion of Staphylococcus aureus bacteraemia (SAB) isolates in Australia that were antimicrobial resistant, with particular emphasis on methicillin resistance, and to characterise the molecular epidemiology of methicillin-resistant S. aureus (MRSA). A total of 3,358 SAB episodes were reported, of which 78.5% were community-onset. Overall, 14.9% of S. aureus were methicillin resistant. The 30-day all-cause mortality associated with methicillin-resistant SAB was 13.7%, which was not significantly different to the 14.1% 30-day all-cause mortality associated with methicillin-susceptible SAB (p = 0.9). With the exception of the β-lactams and erythromycin, antimicrobial resistance in methicillin-susceptible S. aureus (MSSA) was infrequent. However, in addition to the β-lactams, 34.8% of MRSA were resistant to erythromycin; 28.9% to ciprofloxacin; 13.1% to gentamicin; 11.0% to tetracycline; and 2.7% to cotrimoxazole. A daptomycin-resistant MRSA from New South Wales was identified. The isolate had a daptomycin minimum inhibitory concentration (MIC) of 6.0 mg/L, and was identified as ST5-V, with a S337L MprF mutation. When applying the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, teicoplanin resistance was detected in three MSSA isolates. Linezolid or vancomycin resistance was not detected. Resistance to the non-β-lactam antimicrobials was largely attributable to the predominant healthcare-associated MRSA (HA-MRSA) clone ST22-IV [2B] (EMRSA-15), and to the community-associated MRSA (CA-MRSA) clone ST45-V [5C2&5], which has acquired resistance to multiple antimicrobials including ciprofloxacin, clindamycin, erythromycin, gentamicin, and tetracycline. Overall, 89.6% of methicillin-resistant SAB episodes were caused by CA-MRSA clones. Although polyclonal, approximately 72% of CA-MRSA clones were characterised as ST93-IV [2B] (Queensland clone), ST5-IV [2B], ST45-V [5C2&5], ST8-IV [2B], ST30-IV [2B], ST1-IV [2B], ST6-IV [2B], ST97-IV [2B] and Panton-Valentine leucocidin positive ST22-IV [2B]. As CA-MRSA is well established in the Australian community, it is important to monitor antimicrobial resistance patterns in community- and healthcare-associated SAB, as this information will guide therapeutic practices in treating S. aureus bacteraemia.

Between January 2022 and December 2023, there were 1,827 bloodstream infection (BSI) isolates in 1,745 children and adolescents reported to the Australian Group on Antimicrobial Resistance (AGAR) surveillance outcome programs, with 40% of episodes in children aged < 12 months. Two-thirds of BSIs were community-onset.

Of 1,034 gram-negative isolates, 932 (90%) were Enterobacterales. Gram-negative BSI episodes were more commonly community-onset and in children < 12 months of age. Of Enterobacterales isolates, 17.9% were ciprofloxacin resistant; 14.0% were ceftriaxone and/or ceftazidime resistant; 9.5% were gentamicin and/or tobramycin resistant; and 8.9% were piperacillin-tazobactam resistant. Increasing ciprofloxacin resistance was noted, primarily due to the increase in Salmonella Typhi BSI. Overall, 13% of Enterobacterales were extended spectrum β-lactamase producers, and 18.5% were multi-drug resistant (MDR).

Of 601 Staphylococcus aureus isolates, 13.6% were methicillin-resistant (MRSA), and 5.5% were MDR. Overall, 14.4% of S. aureus isolates were erythromycin resistant; 10.3% were clindamycin resistant; and 5.0% were ciprofloxacin resistant. Erythromycin, clindamycin, and ciprofloxacin resistance in MRSA were significantly higher than in methicillin-sensitive isolates. No co-trimoxazole resistant S. aureus was isolated.

There were 192 enterococcal isolates reported; 70.8% were E. faecalis and 17.2% were E. faecium. All ampicillin-resistant, vancomycin-resistant, and MDR enterococci were E. faecium.

The 2022–2023 AGAR Kids Biennial Report shows relative stability in the antimicrobial resistance landscape within the Australian paediatric population, with few significant differences detected when compared to the 2020–2021 report. Small increases in the proportion of resistant Enterobacterales and Enterococcus spp. isolates highlight the importance of ongoing surveillance to inform stewardship and infection prevention interventions.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in Western Australia (WA) in the 1990s. Although ST8-IVa [2B] (WA-5) was the first identified CA-MRSA in WA, ST1-IVa [2B] (WA-1) soon emerged as the dominant clone. To investigate the genomic epidemiology of clonal complex (CC) CC1 S. aureus in WA Aboriginal communities from 1995 to 2003 and assess the acquisition and diversity of the SCCmec element, whole genome sequencing was performed. Three sequence types (STs) were identified: ST1 (81.4%), ST761 (0.9%) and ST762 (17.8%). MRSA constituted 78% (n = 92) of the collection and all harboured SCCmec Type IVa [2B]. Panton–Valentine leukocidin (PVL)–encoding genes were identified in seven closely related isolates. The phylogenetic tree topology suggests the acquisition of the same SCCmec IV into the CC1 lineage occurred on two occasions. Bayesian coalescence analysis predicts the CC1 S. aureus lineage originated in WA more than 150 years ago. Dissemination of the CC1 S. aureus lineage, as well as the horizontal acquisition of SCCmec IV, may have been aided by the concurrent movement of Aboriginal inhabitants across different remote communities.

Objectives
Benzylpenicillin with ceftriaxone is used for outpatient antimicrobial therapy of Enterococcus faecalis infective endocarditis (EFIE) due to poor stability of aminopenicillins. The aim was to correlate the impact of benzylpenicillin-ceftriaxone synergy on EFIE treated with benzylpenicillin-ceftriaxone, and investigate whether simpler phenotypic methods can predict synergy.

Methods
Clinical outcomes of a retrospective cohort of EFIE patients were correlated with treatment and synergy. Isolates were assessed for synergy using the checkerboard method, compared with double disc diffusion (DDD) and layered and crossed gradient diffusion strip (GDS) tests, with sensitivity and specificity of these methods calculated.

Results
Thirty-eight episodes of EFIE in 34 patients were included; the majority received benzylpenicillin-ceftriaxone alone (n = 16) or in sequence (n = 12; any benzylpenicillin-ceftriaxone n = 28), and 10 received other regimens. There was no statistical difference between any benzylpenicillin-ceftriaxone versus other therapies on outcomes, nor between benzylpenicillin-ceftriaxone synergy and outcome. GDS was an unreliable predictor of checkerboard synergy; DDD was reasonable. Five isolates lacked benzylpenicillin-ceftriaxone synergy (one also lacking ampicillin-ceftriaxone synergy); these all had high-level ceftriaxone resistance [zone diameter 6 mm, or broth microdilution (BMD) MIC >512 mg/L]. Most isolates from relapse cases developed reduced zone diameters to ceftriaxone. Two isolates lacking synergy had the same mutation near the ceftriaxone response regulator (CroR) binding site in the pbp4 promoter region.

Conclusions
Ceftriaxone susceptibility, either MIC <512 mg/L by BMD or disc zone diameter >6 mm, is the best predictor of ampicillin-ceftriaxone and particularly benzylpenicillin-ceftriaxone synergy in E. faecalis. There was no clear relationship between the absence of benzylpenicillin-ceftriaxone synergy and outcome in this highly comorbid cohort.

Neisseria meningitidis is a colonizer of the human nasopharynx which occasionally causes invasive meningococcal disease. Despite numerous reports of penicillin-resistant isolates, antimicrobial-resistant meningococcal clones have historically not persisted over long time periods or become globally distributed, presumably due to the imposed fitness cost associated with antimicrobial resistance. One exception is a penicillin-resistant clade of serogroup W clonal complex 11 (MenW:cc11) isolates identified in Western Australia in 2013, which has since caused disease globally. Here, we investigated the genomic changes associated with penicillin resistance in MenW:cc11 isolated during the 2013–2020 Western Australian meningococcal outbreak. Seventy-six MenW:cc11 disease-causing isolates underwent short-read whole genome sequencing. Reference genomes were generated for three isolates. In accordance with previous analysis, two phylogenetically distinct clusters were identified: cluster A (12 penicillin-susceptible isolates) and cluster B (63 penicillin-resistant isolates). Genomic comparison of the cluster A and cluster B isolates revealed 128 allelic differences present at the branching point between the two lineages. The differences included polymorphisms in genes associated with cell wall regulation, pilus biogenesis and the MtrR transcriptional regulator. A further 60 allelic changes were identified in the Western Australian isolates which were not identified in globally distributed cluster B isolates. In a search of the PubMLST Neisseria database, all allelic variants associated with the emergence of cluster B were found exclusively in other hypervirulent lineages. Taken together, the data suggest the global success of the penicillin-resistant N. meningitidis is due to compensatory mutations acquired through horizontal exchange from other hypervirulent lineages.

Introduction
Enterococcus faecium clade B has recently been re-classified as Enterococcus lactis. Although E. lactis was previously associated with food products and probiotics, the recent re-classification has prompted the need for the accurate identification of this species and re-interpretation of its disease-causing ability. Since the re-classified E. lactis can currently only be identified by molecular techniques such as whole-genome sequencing, we constructed a MALDI Biotyper® custom database to rapidly identify and differentiate E. lactis causing bacteraemia from E. faecium.

Hypothesis/Gap statement
The re-classification of E. faecium clade B as E. lactis warrants the development of rapid and accurate identification methods to distinguish these species, particularly in clinical settings where E. lactis may be misidentified as E. faecium.

Aim
The aim of this study was to construct a MALDI Biotyper® custom database to rapidly identify and differentiate E. lactis causing bacteraemia from E. faecium.

Methodology
A total of 97 enterococcal isolates, including 38 E. lactis, 51 E. faecium and 8 non-E. faecium non-E. lactis enterococci (E. avium, E. casseliflavus, E. cecorum, E. durans, E. faecalis, E. faecium, E. gallinarum, E. lactis, E. mundtii and E. raffinosus) were investigated. Whole-genome sequence analysis was used to confirm the species of each isolate. A MALDI Biotyper® in-house database was constructed using 29 E. lactis isolates and the ethanol/formic acid/acetonitrile preparation protocol. The in-house database was validated using the 97 enterococcal isolates and the extended direct transfer preparation protocol.

Results
Our in-house database correctly identified all isolates at the species level, including the E. lactis isolates, all of which were misidentified as E. faecium by the BioTyper® MBT Compass reference library (2022). Of the 38 E. lactis isolates, 84.2% (n=32) were identified at the high probable species level (score ≥2.300), while the remaining 15.8% (n=6) were identified at the probable species level (score 2.000–2.299). Similarly, all E. faecium isolates (n=51) were accurately identified, including 84.3% (n=43/51) identified at the high probable species level and 15.7% (n=8/51) identified at the probable species level.

Conclusion
Our study provides a ready-to-use custom MALDI spectral database that can be implemented in clinical diagnostic and research laboratories to accurately identify E. lactis, which is currently misidentified as E. faecium by the standard spectrum database available on commercial platforms.

Background
The incidence, risks and organisms causing bloodstream infections (BSI) differ between children and adults due to distinct comorbidities, procedures and antibiotic exposures. Age-specific data are required to inform targeted interventions, empiric treatment and guideline development. We aimed to compare the incidence, risk factors and resistance patterns of bacteria causing BSI in children and adults (2020-21).

Methods
The Australian Group on Antimicrobial Resistance (AGAR) is a national hospital-based BSI surveillance program reporting on Staphylococcus aureus, Enterococcus spp. and key gram-negative pathogens.

Results
Data from 25,958 isolates were assessed (children: 1,679; adults: 24,279). The most common organisms in children and adults were Escherichia coli (20.9 vs 39.1%) and S. aureus (36.2 vs 20.9%). E. faecalis and non-typhoidal Salmonella spp. were more frequent in children (7.3% and 4.4% of surveyed organisms in children vs 5.1% and 0.5% in adults). BSI were more often community onset (69.0% children; 76.4% adults). 30-day mortality was significantly lower in children (3.3% vs 9.8%).

Enterobacterales resistance was more common in children: e.g. to gentamicin/tobramycin (11.6% of child isolates; 8.5%, adult isolates; rate ratio [RR]: 1.4 [95%CI: 1.1-1.7]) and piperacillin-tazobactam resistance (11.2%; 8.6%; RR: 1.3 [1.0-1.6]). However, there was no difference observed in Enterobacterales resistance to cephalosporins, ciprofloxacin, meropenem or multi-drug resistance status. AMR prevalence in Pseudomonas and Acinetobacter spp isolates were also similar between children and adults.

Methicillin-resistant S. aureus isolates were less common in children (13.2 vs 17.7%; RR: 0.7 [0.6-0.9]). Rates of clindamycin and cotrimoxazole resistance in S. aureus were similar to adults.

Children had a lower proportion of E. faecium infections (24.1 vs 39.8%) and the rate of vancomycin-resistant E. faecium in children was half that in adults (19.5 vs 37.2%; RR; 0.5, [0.2-1.0]).

Conclusion
Analysis of national AMR data identifies unique trends in children. Ongoing clinical surveillance, targeted prevention, antimicrobial stewardship strategies and research to evaluate AMR drivers in children are required.

Disclosures
All Authors: No reported disclosures

Background
Gram-negative bloodstream infections are associated with significant morbidity and mortality in children. Increasing antimicrobial resistance (AMR) is reported globally, yet efforts to track pediatric AMR at a national level over time are lacking.

Methods
The Australian Group on Antimicrobial Resistance (AGAR) surveillance program captures clinical and microbiological data of isolates detected in blood cultures across Australia. EUCAST 2022 was used for MIC interpretation and the AMR package in R for data analysis.

Results
Over a nine-year period, there were 3,145 bloodstream infections with 3,266 gram-negative isolates reported in hospitalized children aged <18 years; 21.0% were from neonates. The median length of stay was 9 days, and 30-day all-cause mortality was 5.2%. A greater odds of death was observed in those with a multi-drug resistant organism (aOR: 2.1, 95%CI: 1.3, 3.3, p: 0.001).

Escherichia coli (44.5%) and Klebsiella pneumoniae complex (12.6%) were the two most frequently reported organisms. Overall resistance in Enterobacterales to gentamicin/tobramycin was 11.6%, to ceftazidime/ceftriaxone was 12.9%, and 13.2% to ciprofloxacin. Resistance increased over time.

Of 201 Pseudomonas aeruginosa isolates reported, 19.7% were resistant to piperacillin-tazobactam, 13.1% resistant to cefepime/ceftazidime and 9.8% to ciprofloxacin. Of 108 Acinetobacter spp. isolates, one was resistant to meropenem, and two were resistant to ciprofloxacin. Resistance did not increase over time.

Conclusion
AMR in gram-negative organisms causing bloodstream infections in Australian children is increasing which should be considered when updating guidelines and empiric treatment regimens. Ongoing pediatric-specific national surveillance with pediatric reporting must remain a priority to strengthen antimicrobial stewardship and infection control programs.