Communicable Diseases Intelligence

From 1 January to 31 December 2022, fifty-five institutions across Australia participated in the Australian Enterococcal Surveillance Outcome Program (AESOP). The aim of AESOP 2022 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the Enterococcus faecium isolates. Of the 1,535 unique episodes of enterococcal bacteraemia investigated, 92.8% were caused by either E. faecalis (52.9%) or E. faecium (39.9%). Ampicillin and vancomycin resistance were not detected in E. faecalis but were detected in 95.4% and 46.9% of E. faecium respectively. One E. faecalis isolate, with a daptomycin minimum inhibitory concentration (MIC) of 8.0 mg/L, harboured the F478L GdpD mutation. One E. faecium with a daptomycin MIC of 24.0 mg/L harboured the A20D Cls mutation; both mutations are known to be associated with daptomycin resistance. Two E. faecium isolates , one with a linezolid MIC ≥ 256 mg/L and the other with a linezolid MIC of 16 mg/L, harboured the 23S rRNA G2576T mutation, a mutation associated with linezolid resistance in enterococci. Overall, 48.8% of E. faecium harboured either the vanA or the vanB gene, of which 28.0% harboured vanA and 72.0% harboured 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 62 multi-locus sequence types (STs); 85.5% of isolates were classified into eight major STs each containing ten or more isolates. All major STs belonged to clonal complex (CC) 17, a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST78, ST80, ST117, ST555, ST796, ST1421, and ST1424) were each found across most regions of Australia. The predominant ST was ST17, which was identified in all regions. Overall, 53.7% of isolates belonging to the eight major STs harboured the vanA or vanB gene. AESOP 2022 has shown that enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin resistant vanA - or vanB -positive E. faecium which have limited treatment options.


Background
Globally, Staphylococcus aureus is one of the most frequent causes of hospital-acquired and community-acquired bloodstream infections. 1 Although there are a wide variety of manifestations of serious invasive infection caused by S. aureus, in the majority of cases the organism can be detected in blood cultures.Therefore, S. aureus bacteraemia (SAB) is considered a very useful marker for serious invasive infection. 2In 2009, the Infectious Diseases Society of America highlighted S. aureus as one of the key problem bacteria or ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) requiring new therapies. 36][7][8] Mortality rates, however, are known to vary significantly with patient age, clinical manifestation, comorbidities and methicillin resistance.A prospective study of SAB conducted in 27 laboratories in Australia and New Zealand found a 30-day all-cause mortality of 20.6%. 9On univariate analysis, increased mortality was significantly associated with: older age; European ethnicity; methicillin resistance; infections not originating from a medical device; sepsis syndrome; pneumonia/ empyema; and treatment with a glycopeptide or other non-β-lactam antibiotic.
The Australian Group on Antimicrobial Resistance (AGAR), a network of laboratories located across Australia, commenced surveillance of antimicrobial resistance in S. aureus in 1986. 10In 2013, AGAR commenced the Australian Staphylococcus aureus Sepsis Outcome Program, now known as the Australian Staphylococcus aureus Surveillance Outcome Program (ASSOP). 11The primary objective of ASSOP 2022 was to determine the proportion of SAB isolates displaying antimicrobial resistance, with particular emphasis on: 1. susceptibility to methicillin; and 2. molecular epidemiology of methicillin-resistant S. aureus (MRSA).

Methodology Participants
Thirty-three laboratories servicing 55 institutions from all Australian states and mainland territories.

Collection period
From 1 January to 31 December 2022, the 33 laboratories collected all S. aureus isolated from blood cultures.When isolated from a patient's blood culture within 14 days of the first positive culture, S. aureus isolates with the same antimicrobial susceptibility profiles were excluded.
A new SAB episode in the same patient was recorded if it was identified by a culture of blood collected more than 14 days after the last positive culture.Data were collected on age, sex, dates of admission and discharge (if admitted), and mortality at 30 days from date of first positive blood culture.To avoid interpretive bias, no attempt was made to assign attributable mortality.Each SAB episode was designated healthcare onset if the first positive blood culture(s) in the episode were collected > 48 hours after admission.

Laboratory testing
Participating laboratories performed antimicrobial susceptibility testing using the Vitek2  12 and European Committee on Antimicrobial Susceptibility Testing (EUCAST) 13 MIC breakpoints were utilised for interpretation.Linezolid and daptomycin non-susceptible isolates were retested by Etest ® (bioMérieux) using the Mueller-Hinton agar recommended by the manufacturer.The control strain used was S. aureus ATCC ® 29213.High-level mupirocin resistance was determined by the BD Phoenix™ or by using a mupirocin 200 μg disk according to CLSI guidelines on all isolates with a mupirocin MIC > 8 mg/L by Vitek2 ® .Multi-resistance was defined as resistance to three or more of the following non-β-lactam antimicrobials: ciprofloxacin, co-trimoxazole, erythromycin/clindamycin, fusidic acid, gentamicin, linezolid, high-level mupirocin, rifampicin, tetracycline, teicoplanin, and vancomycin.
Molecular testing was performed by whole genome sequencing (WGS) using the NextSeq 500 platform (Illumina, San Diego, USA).Sequence reads were analysed using the Nullarbor pipeline. 14[17][18] Confidence intervals for proportions, Fisher's exact test for categorical variables, and chisquare test for trend were calculated, if appropriate, using MedCalc for Windows, version 12.7 (MedCalc Software, Belgium).
Approval to conduct the prospective data collection was given by the research ethics committee associated with each participating laboratory.

Methicillin-susceptible Staphylococcus aureus (MSSA) antimicrobial susceptibility
Overall, 2,733 of the 3,214 isolates (85.0%) were methicillin susceptible.Where results were available, 1,962/2,721 MSSA isolates (72.1%) were penicillin resistant (MIC > 0.12 mg/L).All penicillin-susceptible isolates (MIC ≤ 0.12 mg/L) were tested either by blaZ PCR or by penicillin disc diffusion (zone-edge test).On testing, a further 74 phenotypically penicillinsusceptible isolates were considered penicillin resistant.Twelve penicillin-susceptible isolates were not available for confirmation.Apart from erythromycin resistance (13.2% and 13.9% using CLSI and EUCAST breakpoints respectively), resistance to the non-β-lactam antimicrobials amongst MSSA was rare (Table 1).There were nine isolates reported by Vitek2 ® as non-susceptible to daptomycin (MIC > 1.0 mg/L).By Etest ® , eight of these nine isolates were considered daptomycin susceptible (MICs 0.25 -1.0 mg/L), whilst the remaining one isolate was unavailable for confirmation.By Vitek2 ® , three isolates were reported as linezolid resistant (MIC > 4 mg/L).By Etest ® , the three isolates had a linezolid MIC of 0.75 mg/L and were therefore considered linezolid susceptible.Using EUCAST interpretive criteria, 21 isolates were reported by Vitek2 ® or BD Phoenix™ as teicoplanin resistant (MIC > 2.0 mg/L).By Etest ® , all isolates had a teicoplanin MIC ≤ 2.0 mg/L and were therefore considered teicoplanin susceptible.All MSSA were vancomycin susceptible.Overall, 2,098 of the 2,733 MSSA (76.7%) had mupirocin susceptibility testing performed, of which 25 (1.2%) were high-level mupirocin resistant.Eleven of the 25 high-level mupirocin-resistant MSSA isolates were referred from Queensland.The remainder of the isolates were from New South Wales (n = 8), Victoria (n = 3), and South Australia (n = 3).Fourteen of the 25 mupirocin resistant MSSA were also resistant to fusidic acid.Of the 2,722 MSSA isolates tested, 45 (1.7%) were constitutively resistant to clindamycin; however, 312 (11.5%) were classified as having both constitutive and inducible clindamycin resistance.Only 3.9% of MSSA were multi-resistant.By Vitek2 ® or BD Phoenix TM , fifty-five isolates were reported as non-susceptible to cotrimoxazole.5).Typically PVL positive, 88.5% of ST93-IV [2B] were community-onset.ST5-IV [2B] accounted for 12.4% of CA-MRSA and was isolated in all regions of Australia except the Australian Capital Territory (Table 5).All ST5-IV [2B] were PVL negative and 81.3% of ST5-IV [2B] were community-onset.ST45-V [5C2&5] accounted for 9.8% of CA-MRSA and was isolated primarily in New South Wales and Victoria (Table 5).Of the ST45-V [5C2&5] isolates, 47.4% were PVL positive and 71.1% were community-onset.ST1-IV [2B] accounted for 6.4% of CA-MRSA and was isolated in all regions of Australia except Victoria, the Northern Territory and the Australian Capital Territory (Table 5).Of the ST1-IV [2B] isolates, 8.0% were PVL positive and 68.0% were community-onset.ST30-IV [2B] accounted for 5.4% of CA-MRSA and was isolated in all regions of Australia except Tasmania and the Northern Territory (Table 5).Of the ST30-IV [2B] isolates, 76.2% were PVL positive and 85.7% were community-onset.ST97-IV [2B] accounted for 5.4% of CA-MRSA and was isolated in all regions except South Australia and the Northern Territory (Table 5).All ST97-IV [2B] isolates were PVL negative and 61.9% of ST97-IV [2B] were community-onset.ST953-IV [2B] accounted for 2.8% of CA-MRSA and was only isolated in Western Australia (Table 5).Overall 81.8% of ST953-IV [2B] were PVL positive and 90.9% were community-onset.ST8-IV [2B] accounted for 2.8% of CA-MRSA and was only isolated in New South Wales, Victoria and Queensland.All ST8-IV [2B] were PVL negative and 81.8% were community-onset.

Discussion
The AGAR surveillance programs collect data on antimicrobial resistance, focussing on bloodstream infections caused by S. aureus, Enterococcus and gram-negative bacilli including the Enterobacterales, Pseudomonas aeruginosa and Acinetobacter species.All data collected in the AGAR programs are generated as part of routine patient care in Australia, with most data available through laboratory and hospital bed management information systems.Isolates are referred to a central laboratory where strain and antimicrobial resistance determinant characterisation are performed.As the programs are similar to those conducted in Europe, comparison of Australian antimicrobial resistance data with other countries is possible. 19,20 ASSOP 2022, methicillin resistance was found in 15.0% (95% CI: 13.8-26.3) of the 3,214 SAB episodes.In the 2021 European Centre for Disease Prevention and Control (ECDC) SAB surveillance program, methicillin resistance ranged from 0.9% (95% CI: 0.5-1.5) in Norway to 42.9% (95% CI: 35.5-50.5) in Cyprus. 21decrease in methicillin-resistant SAB has been reported in several parts of the world, 22,23 and is believed to be due to the implementation of antimicrobial stewardship and a package of improved infection control procedures including hand hygiene; MRSA screening and decolonisation; patient isolation; and infection prevention care bundles.[24][25][26][27] The percentage of methicillin-resistant SAB in Australia has decreased significantly over the ten years of ASSOP, ranging from 18.5% in 2013 to 15.1% in 2022 (Χ 2 for linear trend = 12.993; p = 0.0003).i There have also been significant decreases in HA-MRSA from 41.0% to 13.6% (p < 0.0001) and i Rates include only those laboratories that participated in all years 2013-2022.
in hospital-onset MRSA from 38.0% to 24.5% (p < 0.0001) over the ten ASSOP surveys.Approximately 21.6% of SAB caused by CA-MRSA was hospital-onset.As transmission of CA-MRSA in Australian hospitals is thought to be rare, 36,37 it is likely that many of the hospital-onset CA-MRSA SAB infections reported in ASSOP 2022 were caused by the patient's own colonising strains acquired prior to admission.In Australia, CA-MRSA clones such as PVLpositive ST93-IV [2B] (Queensland clone) are well established in the community and therefore it is important to monitor antimicrobial resistance patterns in both community-and healthcare-associated SAB, as this information will guide therapeutic practices in treating S. aureus sepsis.
In conclusion, ASSOP 2022 has demonstrated antimicrobial resistance in SAB in Australia continues to be a significant problem and is associated with a high mortality.This may be due, in part, to the high prevalence of community-associated methicillin-resistant SAB in Australia, which is higher than in most EU/EEA countries.Consequently, MRSA must remain a public health priority; continuous surveillance of SAB and its outcomes, and the implementation of comprehensive MRSA management strategies targeting hospitals and long-term care facilities are essential.

Table 1 :
The number and proportion of methicillin-susceptible Staphylococcus aureus (MSSA) isolates non-susceptible to penicillin and the non-β-lactam antimicrobials, AGAR, 2022 a No category defined.bBeta-lactamaseadjusted.cNon-susceptible;resistance not defined (DAP).dNoguidelinesforindicated species (FUSc).eMupirocinhigh-levelresistancescreen.fTherifampicinconcentration range on cards restricts category interpretation to non-resistant or resistant.gDoxycycline concentration range (Phoenix panel) restricts ability to accurately identify intermediate and resistant category.

Table 2 :
The number and proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates non-susceptible to penicillin and the non-β-lactam antimicrobials, AGAR, 2022

Table 4 :
The number and proportion of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) clones, AGAR, 2022, by state and territory.