Resistome Analysis of Klebsiella pneumoniae Complex from Residential Aged Care Facilities Demonstrates Intra-facility Clonal Spread of Multidrug-Resistant Isolates.

Antimicrobial-resistant Klebsiella pneumoniae is one of the predominant pathogens in healthcare settings. However, the prevalence and resistome of this organism within residential aged care facilities (RACFs), which are potential hotspots for antimicrobial resistance, remain unexplored. Here, we provide a phenotypic and molecular characterization of antimicrobial-resistant K. pneumoniae isolated from RACFs. K. pneumoniae was isolated from urine, faecal and wastewater samples and facility swabs. The antimicrobial susceptibility profiles of all the isolates were determined and the genomic basis for resistance was explored with whole-genome sequencing on a subset of isolates. A total of 147 K. pneumoniae were isolated, displaying resistance against multiple antimicrobials. Genotypic analysis revealed the presence of beta-lactamases and the ciprofloxacin-resistance determinant QnrB4 but failed to confirm the basis for the observed cephalosporin resistance. Clonal spread of the multidrug-resistant, widely disseminated sequence types 323 and 661 was observed. This study was the first to examine the resistome of K. pneumoniae isolates from RACFs and demonstrated a complexity between genotypic and phenotypic antimicrobial resistance. The intra-facility dissemination and persistence of multidrug-resistant clones is concerning, given that residents are particularly vulnerable to antimicrobial resistant infections, and it highlights the need for continued surveillance and interventions to reduce the risk of outbreaks.

Lytic activity of phages against bacterial pathogens infecting diabetic foot ulcers.

Complications of diabetes, such as diabetic foot ulcers (DFUs), are common, multifactorial in origin, and costly to treat. DFUs are the cause of nearly 90% of limb amputations among persons with diabetes. In most chronic infections such as DFU, biofilms are involved. Bacteria in biofilms are 100-1000 times more resistant to antibiotics than their planktonic counterparts. Multidrug-resistant (MDR) Staphylococcus aureus and Pseudomonas aeruginosa infections in DFUs may require alternative therapeutic agents such as bacteriophages ("phages"). This study describes the lytic activity of phage cocktails AB-SA01 (3-phage cocktail) and AB-PA01 (4-phage cocktail), which target S. aureus and P. aeruginosa, respectively. The host range and lytic effect of AB-SA01 and AB-PA01 on a planktonic culture, single-species biofilm, and mixed-species biofilm were evaluated. In vitro testing showed that 88.7% of S. aureus and 92.7% of P. aeruginosa isolates were susceptible to AB-SA01 and AB-PA01, respectively, in the planktonic state. The component phages of AB-SA01 and AB-PA01 infected 66% to 94.3% of the bacterial isolates tested. Furthermore, AB-SA01 and AB-PA01 treatment significantly (p < 0.05) reduced the biofilm biomass of their hosts, regardless of the antibiotic-resistant characteristics of the isolates and the presence of a non-susceptible host. In conclusion, the strong lytic activity, broad host range, and significant biofilm biomass reduction of AB-SA01 and AB-PA01 suggest the considerable potential of phages in treating antibiotic-resistant S. aureus and P. aeruginosa infections alone or as coinfections in DFUs.

Population pharmacokinetics of posaconazole in allogeneic haematopoietic stem cell transplant patients.

BACKGROUND: Invasive fungal disease (IFD) in the early post-allogeneic HSCT (alloHCT) period is associated with increased likelihood of catastrophic outcomes. The utility of oral modified release (MR) posaconazole tablets is limited by reduced drug absorption from gastrointestinal toxicity induced by cytotoxic chemotherapy, necessitating a switch to the IV posaconazole formulation. OBJECTIVES: To describe the population pharmacokinetics of posaconazole for oral MR and IV formulations in alloHCT patients and determine dosing regimens likely to achieve therapeutic exposures. METHODS: We performed a prospective observational pharmacokinetic study in adult patients in the early post-alloHCT period requiring a change in posaconazole formulation (oral to IV). Samples were analysed using a validated LC-MS/MS method. Population pharmacokinetic analysis and Monte Carlo simulations (n = 1000) were performed using Pmetrics for R. RESULTS: Twenty patients aged between 21 and 70 years were included in the study. A two-compartment model, incorporating mucositis/diarrhoea to modify the bioavailability for oral administration best described the data. To achieve ≥90% PTA, simulations showed that higher than currently recommended doses of oral MR posaconazole were required for prophylaxis Cmin targets (≥0.5 and ≥0.7 mg/L), while increased doses of both formulations were required for IFD treatment PK/PD targets, with patients experiencing oral mucositis/diarrhoea unlikely to achieve these. CONCLUSIONS: Increased doses of posaconazole should be considered for both prophylaxis and treatment of IFD to increase the proportion of alloHCT patients achieving therapeutic exposures, particularly the oral formulation in patients with mucositis and/or diarrhoea. Posaconazole therapeutic drug monitoring should be considered for all formulations in this setting.

Nocardia species distribution and antimicrobial susceptibility within Australia.

BACKGROUND: Nocardia is a ubiquitous saprophyte capable of causing human disease. Disease is primarily respiratory or cutaneous, usually acquired via inhalation or inoculation. Under the influence of environmental and host factors, Nocardia incidence and species distribution demonstrate geographical variation. AIMS: To examine for differences in Nocardia incidence within Western Australia (WA) and analyse species distribution in the context of prior published studies. To analyse antibiogram data from a nationwide passive antimicrobial resistance surveillance program. METHODS: Retrospective extraction of laboratory data for Western Australian Nocardia isolates over a 21-year period. Analysis of Nocardia antimicrobial susceptibility testing data submitted to the Australian Passive Antimicrobial Resistance Surveillance (APAS) program between 2005 and 2022. RESULTS: Nine hundred sixty WA isolates were identified, giving an annual incidence of 3.03 per 100 000 population with apparent latitudinal variation. The four most common species identified within WA and amongst APAS isolates were N. nova, N. cyriacigeorgica, N. brasiliensis and N. farcinica. APAS data demonstrated that all species exhibited high rates of susceptibility to linezolid (100%) and trimethoprim-sulfamethoxazole (98%). Amikacin (>90% susceptibility for all species except N. transvalensis) was the next most active parenteral agent, superior to both carbapenems and third-generation cephalosporins. Susceptibility to oral antimicrobials (other than linezolid) demonstrated significant interspecies variation. CONCLUSIONS: We demonstrate geographical variation in the distribution of Nocardia incidence. Four species predominate in the Australian setting, and nationwide data confirm a high in vitro susceptibility to trimethoprim-sulphamethoxazole and linezolid, justifying their ongoing role as part of first-line empiric therapy.

Refractory Helicobacter pylori infection in Australia: updated multicentre antimicrobial resistance.

BACKGROUND AND AIM: Helicobacter pylori infection is responsible for considerable morbidity and mortality worldwide and eradication rates are falling globally because of increasing antimicrobial resistance. However, there is a paucity of local data to guide the choice of eradication therapy in Australia. This study aimed to evaluate current Australian rates of H. pylori antibiotic resistance in patients who had failed prior eradication therapy. METHODS: A retrospective analysis of routine culture and antibiotic susceptibility data from two pathology laboratories servicing multiple tertiary referral hospitals in Western Australia (WA) and South Australia (SA), between 2018 and 2022, was performed. Rates of antimicrobial resistance and prevalence of multiresistant isolates in both SA and WA were calculated and comparison of temporal trends and differences between the two states was conducted. RESULTS: A total of 796 H. pylori isolates revealed a clarithromycin resistance rate of 82%, metronidazole 68%, amoxicillin 4.4% and tetracycline 0.5%. Resistance to levofloxacin was observed in 22% and rifampicin 14%. Rates of resistance to clarithromycin were lower in SA compared with WA (incidence rate ratio [IRR]: 0.69, P = 0.0001). Multiresistant isolates were discovered in 63% of patients, with lower rates in SA compared with WA (IRR: 0.74, P = 0.002). CONCLUSION: This first multicentre, multistate study of H. pylori resistance in Australian patients exposed to prior therapy demonstrated high rates of antimicrobial resistance, including levofloxacin (>20%). This raises concern about recommending levofloxacin in empirical second-line therapies. Increased monitoring and awareness of current H. pylori resistance rates in Australia are needed to guide local eradication practices.

Longer-term Mortality and Kidney Outcomes of Participants in the Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus (CAMERA2) Trial: A Post Hoc Analysis.

Background: The Combination Antibiotic Therapy for Methicillin-Resistant Staphylococcus aureus (CAMERA2) trial ceased recruitment in July 2018, noting that a higher proportion of patients in the intervention arm (combination therapy) developed acute kidney injury (AKI) compared to the standard therapy (monotherapy) arm. We analyzed the long-term outcomes of participants in CAMERA2 to understand the impact of combination antibiotic therapy and AKI. Methods: Trial sites obtained additional follow-up data. The primary outcome was all-cause mortality, censored at death or the date of last known follow-up. Secondary outcomes included kidney failure or a reduction in kidney function (a 40% reduction in estimated glomerular filtration rate to <60 mL/minute/1.73 m2). To determine independent predictors of mortality in this cohort, adjusted hazard ratios were calculated using a Cox proportional hazards regression model. Results: This post hoc analysis included extended follow-up data for 260 patients. Overall, 123 of 260 (47%) of participants died, with a median population survival estimate of 3.4 years (235 deaths per 1000 person-years). Fifty-five patients died within 90 days after CAMERA2 trial randomization; another 68 deaths occurred after day 90. Using univariable Cox proportional hazards regression, mortality was not associated with either the assigned treatment arm in CAMERA2 (hazard ratio [HR], 0.84 [95% confidence interval [CI], .59-1.19]; P = .33) or experiencing an AKI (HR at 1 year, 1.04 [95% CI, .64-1.68]; P = .88). Conclusions: In this cohort of patients hospitalized with methicillin-resistant S aureus bacteremia, we found no association between either treatment arm of the CAMERA2 trial or AKI (using CAMERA2 trial definition) and longer-term mortality.

Population Pharmacokinetics of Ganciclovir in Allogeneic Hematopoietic Stem Cell Transplant Patients.

Treatment of cytomegalovirus (CMV) infection in allogeneic hematopoietic stem cell transplantation (alloHCT) patients with ganciclovir is complicated by toxicity and resistance. This study aimed to develop an intravenous ganciclovir population pharmacokinetic model for post-alloHCT patients and to determine dosing regimens likely to achieve suggested therapeutic exposure targets. We performed a prospective observational single-center pharmacokinetic study in adult alloHCT patients requiring treatment with intravenous ganciclovir for CMV viremia or disease. Samples were analyzed using a validated ultraperformance liquid chromatography method. Population pharmacokinetic analysis and Monte Carlo simulations (n = 1000) were performed using Pmetrics for R. Twenty patients aged 18 to 69 years were included in the study. A 2-compartment model with linear elimination from the central compartment and between occasion variability best described the data. Incorporating creatinine clearance (CLCR) estimated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation and presence of continuous renal replacement therapy as covariates for ganciclovir clearance improved the model. Compared to current dosing recommendations, simulations demonstrated loading doses were required to achieve a target AUC24 of 80 to 120 mg.h/L on day 1 of induction therapy. Increased individualization of post-loading induction and maintenance doses based on CLCR is required to achieve the suggested exposures for efficacy (AUC24 >80/>40 mg.h/L for induction/maintenance) while remaining below the exposure thresholds for toxicity (AUC24 <120/<60 mg.h/L for induction/maintenance). Intravenous ganciclovir dosing in alloHCT patients can be guided by CLCR estimated by CKD-EPI. Incorporation of loading doses into induction dosing regimens should be considered for timely achievement of currently suggested exposures.

Optimizing antifungal prophylaxis in allogeneic stem cell transplantation: A cohort study of two different approaches.

BACKGROUND: Limited consensus exists on the optimal use of antifungal agents to prevent invasive fungal infection in the early post allogeneic hematopoietic stem cell transplant (alloHCT) period, particularly when patients cannot tolerate oral medication administration. METHODS: We undertook a retrospective observational cohort study to assess the tolerability, efficacy, and cost of a new antifungal prophylaxis pathway at a major tertiary alloHCT centre. Patients aged ≥16 years who underwent alloHCT between February 2018 and October 2019 (cohort 1) or between April 2020 and November 2021 (cohort 2) were included. In both cohorts, first line prophylactic therapy was oral posaconazole. The second line drugs where oral therapy was unable to be administered were intravenous voriconazole (cohort 1) versus intravenous posaconazole (cohort 2). RESULTS: There were 142 patients enrolled in the study, 71 in each cohort. The proportion of patients remaining on first-line prophylaxis or progressing to second-, third-, and fourth-line options was 22.5%, 39.4%, 29.6%, and 8.5% in cohort 1 and 39.4%, 59.2%, 1.4%, and 0% in cohort 2, respectively. The frequency of neuropsychiatric adverse events was significantly higher in cohort 1 compared to cohort 2 (49.3% vs. 19.8%, p = .0004). Occurrence of proven and probable fungal infections was not significantly different between cohorts. Antifungal drug expenditure was $359 935 (AUD) more in cohort 1 ($830 486 AUD) compared to cohort 2 ($477 149 AUD). CONCLUSION: The antifungal prophylaxis pathway used in cohort 2 resulted in reduced antifungal-associated adverse effects, less patients requiring progression to 3rd and 4th line prophylaxis and reduced antifungal drug costs.

Genomic characterisation of CC398 MRSA causing severe disease in Australia.

Clonal complex 398 (CC398) livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been reported worldwide in a variety of food-animal species. Although CC398 is synonymous with LA-MRSA, community-associated MRSA (CA-MRSA) variants have emerged, including the Panton-Valentine leukocidin (PVL)-positive ST398-V and ST398 single-locus variant ST1232-V, and the PVL-negative ST398-V clones. Using comparative genomic analysis, we determined whether ten CC398 MRSA bacteraemia episodes recently identified in Australia were due to LA-MRSA or CA-MRSA CC398. Isolates were sourced from the Australian Group on Antimicrobial Resistance S. aureus surveillance programme and episodes occurred across Australia. Whole-genome sequencing (WGS) and phylogenetic comparison of the ten CC398 bacteraemia isolates with previously published CC398 MRSA whole-genome sequences identified that the Australian CC398 isolates were closely related to the human-associated II-GOI clade and the livestock-associated IIa clade. The identified CC398 MRSA clones were: PVL-positive ST1232-V (5C2&5), PVL-negative community-associated ST398-V (5C2&5) and livestock-associated ST398-V (5C2&5). Our findings demonstrate the importance of using WGS and comparing the sequences with international sequences to distinguish between CC398 CA-MRSA and LA-MRSA and to determine the isolates' origin. Furthermore, our findings suggest that CC398 CA-MRSA has become established in the Australian community and that ST398-V (5C2&5) LA-MRSA is now widespread in Australian piggeries. Our study emphasises the need for national One Health antimicrobial resistance surveillance programmes to assist in monitoring the ongoing epidemiology of MRSA and other clinically significant antimicrobial-resistant organisms.

Geospatial analysis of Helicobacter pylori infection in South Australia: Should location influence eradication therapy?

BACKGROUND AND AIM: Rates of antimicrobial-resistant Helicobacter pylori infection are rising globally; however, geospatial location and its interaction with risk factors for infection have not been closely examined. METHODS: Gastric biopsy specimens were collected to detect H. pylori infection at multiple centers in Adelaide, South Australia, between 1998 and 2017. The geospatial distribution of antibiotic-resistant H. pylori in the Greater Adelaide region was plotted using choropleth maps. Moran's I was used to assess geospatial correlation, and multivariate linear regression (MLR) was used to examine associations between migration status, socioeconomic status, age, gender, and rates of H. pylori positivity and antibiotic resistance. Geographically weighted regression (GWR) was used to determine the extent to which the associations varied according to geospatial location. RESULTS: Of 20 108 biopsies across 136 postcodes within the Greater Adelaide region, 1901 (9.45%) were H. pylori positive. Of these, 797 (41.9%) displayed clarithromycin, tetracycline, metronidazole, or amoxicillin resistance. In MLR, migration status was associated with the rate of H. pylori positivity (ß = 3.85% per 10% increase in a postcode's migrant population; P < 0.001). H. pylori positivity and resistance to any antibiotic were geospatially clustered (Moran's I = 0.571 and 0.280, respectively; P < 0.001 for both). In GWR, there was significant geospatial variation in the strength of the migrant association for both H. pylori positivity and antibiotic resistance. CONCLUSION: Our study demonstrates the heterogeneous geospatial distribution of H. pylori positivity and antibiotic resistance, as well as its interaction with migrant status. Geographic location and migrant status are important factors to consider for H. pylori eradication therapy.

Standardised treatment and monitoring protocol to assess safety and tolerability of bacteriophage therapy for adult and paediatric patients (STAMP study): protocol for an open-label, single-arm trial.

INTRODUCTION: There has been renewed interest in the therapeutic use of bacteriophages (phages); however, standardised therapeutic protocols are lacking, and there is a paucity of rigorous clinical trial data assessing efficacy. METHODS AND ANALYSIS: We propose an open-label, single-arm trial investigating a standardised treatment and monitoring protocol for phage therapy. Patients included will have exhausted other therapeutic options for control of their infection and phage therapy will be administered under Australia's Therapeutic Goods Administration Special Access Scheme. A phage product with high in vitro activity against the targeted pathogen(s) must be available in line with relevant regulatory requirements. We aim to recruit 50-100 patients over 5 years, from any public or private hospitals in Australia. The standardised protocol will specify clinical assessments and biological sampling at scheduled time points. The primary outcome is safety at day 29, assessed by the frequency of adverse events, and overseen by an independent Data Safety Monitoring Board. Secondary outcomes include long-term safety (frequency of adverse events until at least 6 months following phage therapy), and feasibility, measured as the proportion of participants with>80% of minimum data available for analysis. Additional endpoints assessed include clinical response, patient/guardian reported quality of life measures, phage pharmacokinetics, human host immune responses and microbiome analysis. All trial outcomes will be summarised and presented using standard descriptive statistics. ETHICS AND DISSEMINATION: Participant inclusion will be dependent on obtaining written informed consent from the patient or guardian. The trial protocol was approved by the Sydney Children's Hospitals Network Human Research Ethics Committee in December 2021 (Reference 2021/ETH11861). In addition to publication in a peer-reviewed scientific journal, a lay summary of study outcomes will be made available for participants and the public on the Phage Australia website (https://www.phageaustralia.org/). TRIAL REGISTRATION NUMBER: Registered on ANZCTR, 10 November 2021 (ACTRN12621001526864; WHO Universal Trial Number: U1111-1269-6000).

Increasing Helicobacter pylori clarithromycin resistance in Australia over 20 years.

BACKGROUND: Helicobacter pylori infection is responsible for considerable morbidity and mortality worldwide, and eradication rates are falling in many countries, primarily due to clarithromycin and metronidazole resistance. AIMS: There is a paucity of contemporary Australian data, which we sought to address by evaluating local rates of resistance of H. pylori to amoxicillin, clarithromycin, metronidazole and tetracycline over the past 20 years. METHODS: All gastric biopsy specimens collected at endoscopy to detect H. pylori infection at a single centre underwent routine culture and antibiotic susceptibility testing between 1998 and 2017. Specimens from 12 842 patients were cultured for H. pylori, of which 1473 positive cultures were tested for antibiotic susceptibility. RESULTS: Antibiotic resistance to clarithromycin increased by 3.7% per year (incidence rate ratio [IRR] 1.037; P = 0.014) over 20 years, with a corresponding 5.0% annual increase in minimum inhibitory concentration (MIC) (odds ratio 1.050; P < 0.001). Since 2010, average clarithromycin resistance has exceeded 20%, with >25% of isolates resistant in the past 2 years of data capture. In contrast, rates of resistance to metronidazole (35.3%), amoxicillin (0.14%) and tetracycline (0.34%) and their MIC have remained stable. Review of a representative sample (n = 120; 8%) of these patients revealed that only 5% had documented prior H. pylori eradication therapy. CONCLUSIONS: Over the past 20 years there has been a substantial rise in clarithromycin resistance, with stable metronidazole resistance and low rates of resistance to amoxicillin and tetracycline. Current first-line H. pylori eradication therapy may fail to achieve adequate eradication rates, and optimal first-line therapy in Australia should be revisited.

Environmental dynamics of hospital microbiome upon transfer from a major hospital to a new facility.

BACKGROUND: Infection control is critical to safe hospital care. However, how bacteria within nosocomial environments relate to space utilisation and occupancy remains poorly understood. Our aim was to characterise the hospital microbiome in the context of the closure of a tertiary hospital and the opening of a new facility. METHODS: Environmental swabs were collected from common and inpatient areas in the old and new hospitals during a 12-month transition period. Microbiota characteristics were determined by 16S rRNA gene sequencing and quantitative (q)PCR. Targeted assays were used to detect Methicillin-resistant Staphylococcus aureus (MRSA) and vanB-positive Vancomycin-Resistant Enterococci (VRE). RESULTS: The transition to full occupancy in the new facility was associated with an increase in bacterial load (inpatient areas, 3 months p = 0.001; common areas, 6 months p = 0.039) and a change in microbiota composition (baseline-12 months, PERMANOVA p = 0.002). These changes were characterised by an increase in human microbiota-associated taxa, including Acinetobacter and Veillonella. Closure of the existing facility was associated with a decrease in bacterial load (p = 0.040). Detection of MRSA did not differ significantly between sites. CONCLUSIONS: Occupancy is a major determinant of bacterial dispersion within hospital environments. Steady-state bacterial levels and microbiota composition provide a basis for assessment of infection control measures.

Investigating potential transmission of antimicrobial resistance in an open-plan hospital ward: a cross-sectional metagenomic study of resistome dispersion in a lower middle-income setting.

BACKGROUND: Antimicrobial resistance (AMR) represents a profound global health threat. Reducing AMR spread requires the identification of transmission pathways. The extent to which hospital wards represent a venue for substantial AMR transmission in low- and middle-income countries settings is poorly understood. METHODS: Rectal swabs were obtained from adult male inpatients in a "Nightingale" model general medicine ward in Yangon, Myanmar. Resistome characteristics were characterised by metagenomic sequencing. AMR gene carriage was related to inter-patient distance (representing inter-patient interaction) using distance-based linear models. Clinical predictors of AMR patterns were identified through univariate and multivariate regression. RESULTS: Resistome similarity showed a weak but significant positive correlation with inter-patient distance (r = 0.12, p = 0.04). Nineteen AMR determinants contributed significantly to this relationship, including those encoding ß-lactamase activity (OXA-1, NDM-7; adjusted p < 0.003), trimethoprim resistance (dfrA14, adjusted p = 0.0495), and chloramphenicol resistance (catB3, adjusted p = 0.002). Clinical traits of co-located patients carrying specific AMR genes were not random. Specifically, AMR genes that contributed to distance-resistome relationships (OXA-1, catB3, dfrA14) mapped to tuberculosis patients, who were placed together according to ward policy. In contrast, patients with sepsis were not placed together, and carried AMR genes that were not spatially significant or consistent with shared antibiotic exposure. CONCLUSIONS: AMR dispersion patterns primarily reflect the placement of particular patients by their condition, rather than AMR transmission. The proportion of AMR determinants that varied with inter-patient distance was limited, suggesting that nosocomial transmission is a relatively minor contributor to population-level carriage.