Phage Therapy in Korea: A Prescribers' Survey of Attitudes Amongst Korean Infectious Diseases Specialists Towards Phage Therapy.

BACKGROUND: Concerns about the rise in antimicrobial resistance have led to renewed interest in phage therapy worldwide, but perceptions among relevant medical professionals in Korea remain largely unknown. MATERIALS AND METHODS: We conducted a semi-quantitative online survey to evaluate the Korean infectious disease specialists' perception of phage therapy. RESULTS: We sent out the link to the questionnaire to 380 subjects and received 91 replies, with 90/91 respondents identifying as Korean infectious diseases specialists or trainees. Ten out of 91 (11.0%) respondents scored themselves as well-informed about phage therapy. The majority (93.4%) of respondents would consider using phage therapy if the safety of the phage formulation is guaranteed, and 80% of respondents would consider participating in clinical trials with phage therapy given adequate support. The biggest concern was uncertainty about safety (73.6%) and efficacy (65.9%). Acinetobacter baumannii was ranked as a high priority for phage therapy research, as were bone and joint infections. CONCLUSION: Korean infectious diseases specialists are receptive to phage therapy, but a better understanding of safety, efficacy and clinical trials are warranted to progress phage therapy within the Korean healthcare system.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Surveillance Outcome Program (GnSOP) Bloodstream Infection Annual Report 2022.

The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2022 survey was the tenth year to focus on blood stream infections caused by Enterobacterales, and the eighth year where Pseudomonas aeruginosa and Acinetobacter species were included. Fifty-five hospitals Australia-wide participated in 2022. The 2022 survey tested 9,739 isolates, comprising Enterobacterales (8,773; 90.1%), P. aeruginosa (840; 8.6%) and Acinetobacter species (126; 1.3%), using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2023). Key resistances included resistance to the third-generation cephalosporin ceftriaxone in 12.7%/12.7% (CLSI/EUCAST criteria) of Escherichia coli and in 6.6%/6.6% of Klebsiella pneumoniae complex. Resistance rates to ciprofloxacin were 13.7%/13.7% for E. coli; 7.8%/7.8% for K. pneumoniae complex; 5.3%/5.3% for Enterobacter cloacae complex; and 4.3%/10.0% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.8%/5.9%; 2.9%/8.7%; 18.3%/27.2%; and 6.1%/14.7% for the same four species, respectively. Twenty-nine Enterobacterales isolates from 28 patients were shown to harbour a carbapenemase gene: 18 blaIMP-4; four blaNDM-5; three blaNDM-1; one blaOXA-181; one blaOXA-244; one blaNDM-1 + blaOXA-181; and one blaNDM-5 + blaOXA-181. Transmissible carbapenemase genes were also detected among two Acinetobacter baumannii complex isolates (blaOXA-23) and one P. aeruginosa (blaNDM-1) in the 2022 survey.

Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series.

BACKGROUND: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. METHODS: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. FINDINGS: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once- to twice-daily regimen. CONCLUSIONS: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials. FUNDING: The study was funded in part by The Israeli Science Foundation IPMP (ISF_1349/20), Rosetrees Trust (A2232), United States-Israel Binational Science Foundation (2017123), and the Milgrom Family Support Program.

Quantifying the Economic and Clinical Value of Reducing Antimicrobial Resistance in Gram-negative Pathogens Causing Hospital-Acquired Infections in Australia.

INTRODUCTION: Antimicrobial resistance (AMR) is a global public health challenge requiring a global response to which Australia has issued a National Antimicrobial Resistance Strategy. The necessity for continued-development of new effective antimicrobials is required to tackle this immediate health threat is clear, but current market conditions may undervalue antimicrobials. We aimed to estimate the health-economic benefits of reducing AMR levels for drug-resistant gram-negative pathogens in Australia, to inform health policy decision-making. METHODS: A published and validated-dynamic health economic model was adapted to the Australian setting. Over a 10-year time horizon, the model estimates the clinical and economic outcomes associated with reducing current AMR levels, by up to 95%, of three gram-negative pathogens in three hospital-acquired infections, from the perspective of healthcare payers. A willingness-to-pay threshold of AUD$15,000-$45,000 per quality-adjusted life-year (QALY) gained and a 5% discount rate (for costs and benefits) were applied. RESULTS: Over ten years, reducing AMR for gram-negative pathogens in Australia is associated with up to 10,251 life-years and 8924 QALYs gained, 9041 bed-days saved and 6644 defined-daily doses of antibiotics avoided. The resulting savings are estimated to be $10.5 million in hospitalisation costs, and the monetary benefit at up to $412.1 million. DISCUSSION: Our results demonstrate the clinical and economic value of reducing AMR impact in Australia. Of note, since our analysis only considered a limited number of pathogens in the hospital setting only and for a limited number of infection types, the benefits of counteracting AMR are likely to extend well beyond the ones demonstrated here. CONCLUSION: These estimates demonstrate the consequences of failure to combat AMR in the Australian context. The benefits in mortality and health system costs justify consideration of innovative reimbursement schemes to encourage the development and commercialisation of new effective antimicrobials.

The evolution and international spread of extensively drug resistant Shigella sonnei.

Shigella sonnei causes shigellosis, a severe gastrointestinal illness that is sexually transmissible among men who have sex with men (MSM). Multidrug resistance in S. sonnei is common including against World Health Organisation recommended treatment options, azithromycin, and ciprofloxacin. Recently, an MSM-associated outbreak of extended-spectrum ß-lactamase producing, extensively drug resistant S. sonnei was reported in the United Kingdom. Here, we aimed to identify the genetic basis, evolutionary history, and international dissemination of the outbreak strain. Our genomic epidemiological analyses of 3,304 isolates from the United Kingdom, Australia, Belgium, France, and the United States of America revealed an internationally connected outbreak with a most recent common ancestor in 2018 carrying a low-fitness cost resistance plasmid, previously observed in travel associated sublineages of S. flexneri. Our results highlight the persistent threat of horizontally transmitted antimicrobial resistance and the value of continuing to work towards early and open international sharing of genomic surveillance data.

A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry.

Bacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding the transfer dynamics and epidemiology of conjugative plasmids. In this study, we present a streamlined experimental approach for fluorescence labelling of low-copy-number conjugative plasmids that allows plasmid transfer frequency during filter mating to be measured by flow cytometry. A blue fluorescent protein gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescent protein gene with a toxin-antitoxin system that functions as a plasmid stability module, is used to label the recipient bacterial strain. This offers the dual advantage of circumventing chromosomal modifications of recipient strains and ensuring that the red fluorescent protein gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescent protein genes to be strongly and constitutively expressed from the plasmids, thus allowing flow cytometers to clearly distinguish between donor, recipient, and transconjugant populations in a conjugation mix for monitoring conjugation frequencies more precisely over time.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Surveillance Outcome Program (GnSOP).

Abstract: The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2021 survey was the ninth year to focus on bloodstream infections caused by Enterobacterales, and the seventh year where Pseudomonas aeruginosa and Acinetobacter species were included. The 2021 survey tested 8,947 isolates, comprising Enterobacterales (8,104; 90.6%), P. aeruginosa (745; 8.3%) and Acinetobacter species (98; 1.1%), using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2022). Of the key resistances, resistance to the third-generation cephalosporin ceftriaxone was found in 12.5%/12.5% (CLSI/EUCAST criteria) of Escherichia coli and in 6.1%/6.1% of Klebsiella pneumoniae. Resistance rates to ciprofloxacin were 12.3%/12.3% for E. coli; 7.2%/7.2% for K. pneumoniae; 5.4%/5.4% for Enterobacter cloacae complex; and 3.7%/8.0% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.8%/6.5%; 2.9%/9.9%; 18.4%/28.1%; and 6.9%/12.8% for the same four species, respectively. Seventeen Enterobacterales isolates from 17 patients were shown to harbour a carbapenemase gene: 12 blaIMP-4; two blaNDM-7; one blaNDM-1; one blaOXA-181; and one blaKPC-2. No transmissible carbapenemase genes were detected among P. aeruginosa or Acinetobacter isolates in the 2021 survey.

Seeking patterns of antibiotic resistance in ATLAS, an open, raw MIC database with patient metadata.

Antibiotic resistance represents a growing medical concern where raw, clinical datasets are under-exploited as a means to track the scale of the problem. We therefore sought patterns of antibiotic resistance in the Antimicrobial Testing Leadership and Surveillance (ATLAS) database. ATLAS holds 6.5M minimal inhibitory concentrations (MICs) for 3,919 pathogen-antibiotic pairs isolated from 633k patients in 70 countries between 2004 and 2017. We show most pairs form coherent, although not stationary, timeseries whose frequencies of resistance are higher than other databases, although we identified no systematic bias towards including more resistant strains in ATLAS. We sought data anomalies whereby MICs could shift for methodological and not clinical or microbiological reasons and found artefacts in over 100 pathogen-antibiotic pairs. Using an information-optimal clustering methodology to classify pathogens into low and high antibiotic susceptibilities, we used ATLAS to predict changes in resistance. Dynamics of the latter exhibit complex patterns with MIC increases, and some decreases, whereby subpopulations' MICs can diverge. We also identify pathogens at risk of developing clinical resistance in the near future.

A Novel Plasmid Entry Exclusion System in pKPC_UVA01, a Promiscuous Conjugative Plasmid Carrying the blaKPC Carbapenemase Gene.

Conjugative plasmids are the principal mediator in the emergence and spread of antibiotic resistance genes in Enterobacterales. Plasmid entry exclusion (EEX) systems can restrict their transfer into the recipient bacteria carrying closely related plasmids. In this study, we identified and characterized a novel plasmid entry exclusion system in a carbapenem resistance plasmid pKPC_UVA01, which is responsible for widespread dissemination of the blaKPC carbapenemase gene among Enterobacterales in the United States. The identified eex gene in the recipient strain of different Enterobacterales species inhibited the conjugation transfer of pKPC_UVA01 plasmids at a range of 200- to 400-fold, and this inhibition was found to be a dose-dependent function of the EEX protein in recipient cells. The C terminus truncated version of eex or eex with an early termination codon at the C terminus region alleviated the inhibition of conjugative transfer. Unlike the strict specificity of plasmid exclusion by the known EEX protein, the newly identified EEX in the recipient strain could inhibit the transfer of IncP and IncN plasmids. The eex gene from the plasmid pKPC_UVA01 was not required for conjugative transfer but was essential in the donor bacteria for entry exclusion of this plasmid. This was a novel function of a single protein that is essential in both donor and recipient bacteria for the entry exclusion of a plasmid. This eex gene is found to be distributed in multidrug resistance plasmids similar to pKPC_UVA01 in different Enterobacterales species and may contribute to the stability of this plasmid type by controlling its transfer.

Bacterial lysis, autophagy and innate immune responses during adjunctive phage therapy in a child.

Adjunctive phage therapy was used in an attempt to avoid catastrophic outcomes from extensive chronic Pseudomonas aeruginosa osteoarticular infection in a 7-year-old child. Monitoring of phage and bacterial kinetics allowed real-time phage dose adjustment, and along with markers of the human host response, indicated a significant therapeutic effect within two weeks of starting adjunctive phage therapy. These findings strongly suggested the release of bacterial cells or cell fragments into the bloodstream from deep bony infection sites early in treatment. This was associated with transient fever and local pain and with evidence of marked upregulation of innate immunity genes in the host transcriptome. Adaptive immune responses appeared to develop after a week of therapy and some immunomodulatory elements were also observed to be upregulated.

Biological Functions of Type II Toxin-Antitoxin Systems in Bacteria.

After the first discovery in the 1980s in F-plasmids as a plasmid maintenance system, a myriad of toxin-antitoxin (TA) systems has been identified in bacterial chromosomes and mobile genetic elements (MGEs), including plasmids and bacteriophages. TA systems are small genetic modules that encode a toxin and its antidote and can be divided into seven types based on the nature of the antitoxin molecules and their mechanism of action to neutralise toxins. Among them, type II TA systems are widely distributed in chromosomes and plasmids and the best studied so far. Maintaining genetic material may be the major function of type II TA systems associated with MGEs, but the chromosomal TA systems contribute largely to functions associated with bacterial physiology, including the management of different stresses, virulence and pathogenesis. Due to growing interest in TA research, extensive work has been conducted in recent decades to better understand the physiological roles of these chromosomally encoded modules. However, there are still controversies about some of the functions associated with different TA systems. This review will discuss the most current findings and the bona fide functions of bacterial type II TA systems.

The higBA-Type Toxin-Antitoxin System in IncC Plasmids Is a Mobilizable Ciprofloxacin-Inducible System.

A putative type II toxin-antitoxin (TA) module almost exclusively associated with conjugative IncC plasmids is homologous to the higBA family of TA systems found in chromosomes and plasmids of several species of bacteria. Despite the clinical significance and strong association with high-profile antimicrobial resistance (AMR) genes, the TA system of IncC plasmids remains largely uncharacterized. In this study, we present evidence that IncC plasmids encode a bona fide HigB-like toxin that strongly inhibits bacterial growth and results in cell elongation in Escherichia coli. IncC HigB toxin acts as a ribosome-dependent endoribonuclease that significantly reduces the transcript abundance of a subset of adenine-rich mRNA transcripts. A glycine residue at amino acid position 64 is highly conserved in HigB toxins from different bacterial species, and its replacement with valine (G64V) abolishes the toxicity and the mRNA cleavage activity of the IncC HigB toxin. The IncC plasmid higBA TA system functions as an effective addiction module that maintains plasmid stability in an antibiotic-free environment. This higBA addiction module is the only TA system that we identified in the IncC backbone and appears essential for the stable maintenance of IncC plasmids. We also observed that exposure to subinhibitory concentrations of ciprofloxacin, a DNA-damaging fluoroquinolone antibiotic, results in elevated higBA expression, which raises interesting questions about its regulatory mechanisms. A better understanding of this higBA-type TA module potentially allows for its subversion as part of an AMR eradication strategy. IMPORTANCE Toxin-antitoxin (TA) systems play vital roles in maintaining plasmids in bacteria. Plasmids with incompatibility group C are large plasmids that disseminate via conjugation and carry high-profile antibiotic resistance genes. We present experimental evidence that IncC plasmids carry a TA system that functions as an effective addiction module and maintains plasmid stability in an antibiotic-free environment. The toxin of IncC plasmids acts as an endoribonuclease that targets a subset of mRNA transcripts. Overexpressing the IncC toxin gene strongly inhibits bacterial growth and results in cell elongation in Escherichia coli hosts. We also identify a conserved amino acid residue in the toxin protein that is essential for its toxicity and show that the expression of this TA system is activated by a DNA-damaging antibiotic, ciprofloxacin. This mobile TA system may contribute to managing bacterial stress associated with DNA-damaging antibiotics.

Third generation cephalosporins and piperacillin/tazobactam have distinct impacts on the microbiota of critically ill patients.

Effective implementation of antibiotic stewardship, especially in critical care, is limited by a lack of direct comparative investigations on how different antibiotics impact the microbiota and antibiotic resistance rates. We investigated the impact of two commonly used antibiotics, third-generation cephalosporins (3GC) and piperacillin/tazobactam (TZP) on the endotracheal, perineal and faecal microbiota of intensive care patients in Australia. Patients exposed to either 3GC, TZP, or no ß-lactams (control group) were sampled over time and 16S rRNA amplicon sequencing was performed to examine microbiota diversity and composition. While neither treatment significantly affected diversity, numerous changes to microbiota composition were associated with each treatment. The shifts in microbiota composition associated with 3GC exposure differed from those observed with TZP, consistent with previous reports in animal models. This included a significant increase in Enterobacteriaceae and Enterococcaceae abundance in endotracheal and perineal microbiota for those administered 3GC compared to the control group. Culture-based analyses did not identify any significant changes in the prevalence of specific pathogenic or antibiotic-resistant bacteria. Exposure to clinical antibiotics has previously been linked to reduced microbiota diversity and increased antimicrobial resistance, but our results indicate that these effects may not be immediately apparent after short-term real-world exposures.

Phage Biobank: Present Challenges and Future Perspectives.

After a century of use in human infection, the preparation and administration of therapeutic bacteriophages (phages) still relies on ad hoc partnerships of researchers, biotech companies, clinicians and regulators. There is a clear need to improve the reproducibility, safety and speed of the provision of suitable phages. Here we discuss the specific characteristics and challenges of a sustainable phage biobank and, as we build a national consortium aimed at delivering phage therapeutics, suggest a roadmap toward national biobanking and phage therapy initiatives using the Australian context as a model.

COVID-19 in Australia: our national response to the first cases of SARS-CoV-2 infection during the early biocontainment phase.

BACKGROUND: On 31 December 2019, the World Health Organization recognised clusters of pneumonia-like cases due to a novel coronavirus disease (COVID-19). COVID-19 became a pandemic 71 days later. AIM: To report the clinical and epidemiological features, laboratory data and outcomes of the first group of 11 returned travellers with COVID-19 in Australia. METHODS: This is a retrospective, multi-centre case series. All patients with confirmed COVID-19 infection were admitted to tertiary referral hospitals in New South Wales, Queensland, Victoria and South Australia. RESULTS: The median age of the patient cohort was 42 years (interquartile range (IQR), 24-53 years) with six men and five women. Eight (72.7%) patients had returned from Wuhan, one from Shenzhen, one from Japan and one from Europe. Possible human-to-human transmission from close family contacts in gatherings overseas occurred in two cases. Symptoms on admission were fever, cough and sore throat (n = 9, 81.8%). Co-morbidities included hypertension (n = 3, 27.3%) and hypercholesterolaemia (n = 2, 18.2%). No patients developed severe acute respiratory distress nor required intensive care unit admission or mechanical ventilation. After a median hospital stay of 14.5 days (IQR, 6.75-21), all patients were discharged. CONCLUSIONS: This is a historical record of the first COVID-19 cases in Australia during the early biocontainment phase of the national response. These findings were invaluable for establishing early inpatient and outpatient COVID-19 models of care and informing the management of COVID-19 over time as the outbreak evolved. Future research should extend this Australian case series to examine global epidemiological variation of this novel infection.

Specialised functions of two common plasmid mediated toxin-antitoxin systems, ccdAB and pemIK, in Enterobacteriaceae.

Toxin-antitoxin systems (TAS) are commonly found on bacterial plasmids and are generally involved in plasmid maintenance. In addition to plasmid maintenance, several plasmid-mediated TAS are also involved in bacterial stress response and virulence. Even though the same TAS are present in a variety of plasmid types and bacterial species, differences in their sequences, expression and functions are not well defined. Here, we aimed to identify commonly occurring plasmid TAS in Escherichia coli and Klebsiella pneumoniae and compare the sequence, expression and plasmid stability function of their variants. 27 putative type II TAS were identified from 1063 plasmids of Klebsiella pneumoniae in GenBank. Among these, ccdAB and pemIK were found to be most common, also occurring in plasmids of E. coli. Comparisons of ccdAB variants, taken from E. coli and K. pneumoniae, revealed sequence differences, while pemIK variants from IncF and IncL/M plasmids were almost identical. Similarly, the expression and plasmid stability functions of ccdAB variants varied according to the host strain and species, whereas the expression and functions of pemIK variants were consistent among host strains. The specialised functions of some TAS may determine the host specificity and epidemiology of major antibiotic resistance plasmids.

Fine capsule variation affects bacteriophage susceptibility in Klebsiella pneumoniae ST258.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18 Kumoniae target strains and tested them against novel lytic bacteriophages (n = 65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

CCMetagen: comprehensive and accurate identification of eukaryotes and prokaryotes in metagenomic data.

There is an increasing demand for accurate and fast metagenome classifiers that can not only identify bacteria, but all members of a microbial community. We used a recently developed concept in read mapping to develop a highly accurate metagenomic classification pipeline named CCMetagen. The pipeline substantially outperforms other commonly used software in identifying bacteria and fungi and can efficiently use the entire NCBI nucleotide collection as a reference to detect species with incomplete genome data from all biological kingdoms. CCMetagen is user-friendly, and the results can be easily integrated into microbial community analysis software for streamlined and automated microbiome studies.