Using a human colonoid-derived monolayer to study bacteriophage translocation.

Bacteriophages (phages) are estimated to be the most abundant microorganisms on Earth. Their presence in human blood suggests that they can translocate from non-sterile sites such as the gastrointestinal tract where they are concentrated. To examine phage translocation ex vivo, we adapted a primary colonoid monolayer model possessing cell diversity and architecture, and a thick layer of mucus akin to the colonic environment in vivo. We show that the colonoid monolayer is superior to the Caco-2 cell-line model, possessing intact and organized tight junctions and generating a physiologically relevant mucus layer. We showed, using two different phages, that translocation across the colonoid monolayer was largely absent in differentiated monolayers that express mucus, unlike Caco-2 cultures that expressed little to no mucus. By stimulating mucus production or removing mucus, we further demonstrated the importance of colonic mucus in preventing phage translocation. Finally, we used etiological drivers of gut permeability (alcohol, fat, and inflammatory cytokines) to measure their effects on phage translocation, demonstrating that all three stimuli have the capacity to amplify phage translocation. These findings suggest that phage translocation does occur in vivo but may be largely dependent on colonic mucus, an important insight to consider in future phage applications.

Host transcriptomics and machine learning for secondary bacterial infections in patients with COVID-19: a prospective, observational cohort study.

BACKGROUND: Viral respiratory tract infections are frequently complicated by secondary bacterial infections. This study aimed to use machine learning to predict the risk of bacterial superinfection in SARS-CoV-2-positive individuals. METHODS: In this prospective, multicentre, observational cohort study done in nine centres in six countries (Australia, Indonesia, Singapore, Italy, Czechia, and France) blood samples and RNA sequencing were used to develop a robust model of predicting secondary bacterial infections in the respiratory tract of patients with COVID-19. Eligible participants were older than 18 years, had known or suspected COVID-19, and symptoms of a recent respiratory infection. A control cohort of participants without COVID-19 who were older than 18 years and with no infection symptoms was also recruited from one Australian centre. In the pre-analysis phase, data were filtered to include only individuals with complete blood transcriptomics and patient data (ie, age, sex, location, and WHO severity score at the time of sample collection). The dataset was then divided randomly (4:1) into a training set (80%) and a test set (20%). Gene expression data in the training set and control cohort were used for differential expression analysis. Differentially expressed genes, along with WHO severity score, location, age, and sex, were used for feature selection with least absolute shrinkage and selection operator (LASSO) in the training set. For LASSO analysis, samples were excluded if gene expression data were not obtained at study admission, no longitudinal clinical information was available, a bacterial infection at the time of study admission was present, or a fungal infection in the absence of a bacterial infection was detected. LASSO regression was performed using three subsets of predictor variables: patient data alone, gene expression data alone, or a combination of patient data and gene expression data. The accuracy of the resultant models was tested on data from the test set. FINDINGS: Between March, 2020, and October, 2021, we recruited 536 SARS-CoV-2-positive individuals and between June, 2013, and January, 2020, we recruited 74 participants into the control cohort. After prefiltering analysis and other exclusions, samples from 158 individuals were analysed in the training set and 47 in the test set. The expression of seven host genes (DAPP1, CST3, FGL2, GCH1, CIITA, UPP1, and RN7SL1) in the blood at the time of study admission was identified by LASSO as predictive of the risk of developing a secondary bacterial infection of the respiratory tract more than 24 h after study admission. Specifically, the expression of these genes in combination with a patient's WHO severity score at the time of study enrolment resulted in an area under the curve of 0·98 (95% CI 0·89-1·00), a true positive rate (sensitivity) of 1·00 (95% CI 1·00-1·00), and a true negative rate (specificity) of 0·94 (95% CI 0·89-1·00) in the test cohort. The combination of patient data and host transcriptomics at hospital admission identified all seven individuals in the training and test sets who developed a bacterial infection of the respiratory tract 5-9 days after hospital admission. INTERPRETATION: These data raise the possibility that host transcriptomics at the time of clinical presentation, together with machine learning, can forward predict the risk of secondary bacterial infections and allow for the more targeted use of antibiotics in viral infection. FUNDING: Snow Medical Research Foundation, the National Health and Medical Research Council, the Jack Ma Foundation, the Helmholtz-Association, the A2 Milk Company, National Institute of Allergy and Infectious Disease, and the Fondazione AIRC Associazione Italiana per la Ricerca contro il Cancro.

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.

Single-arm, open-labelled, safety and tolerability of intrabronchial and nebulised bacteriophage treatment in children with cystic fibrosis and Pseudomonas aeruginosa.

INTRODUCTION: Cystic fibrosis (CF) is a multisystem condition that is complicated by recurrent pulmonary infections requiring aggressive antibiotic treatment. This predisposes the patient to complications such as sensorineural hearing loss, renal impairment, hypersensitivity and the development of antibiotic resistance. Pseudomonas aeruginosa is one of the more common organisms which cause recurrent infections and result in greater morbidity and mortality in people living with CF. Bacteriophages have been identified as a potential alternative or adjunct to antibiotics. We hypothesise that bacteriophage therapy is a safe and well-tolerated treatment in children with CF infected with P. aeruginosa infection in their airways. METHODS: This single-arm, open-labelled, non-randomised trial will run for a maximum period of 36 months with up to 10 participants. Adolescents (≥12 years and <18 years of age) who continue to shed P.aeruginosa (within 3 months of enrolment) despite undergoing eradication therapy previously, will be considered for this trial. Non-genetically modified bacteriophages that have demonstrated obligate lytic activity against each of the study participants' P. aeruginosa strains will be selected and prepared according to a combination of established protocols (isolation, purification, sterility testing and packaging) to achieve close to good manufacturing practice recommendations. The selected bacteriophage will be administered endo-bronchially first under direct vision, followed by two times a day nebulisation for 7 days in addition to standard CF treatment (intravenous antibiotics, physiotherapy to be completed as inpatient for 10-14 days). Safety and tolerability will be defined as the absence of (1) fever above 38.5°C occurring within 1 hour of the administration of the nebulised bacteriophage, (2) a 10% decline in spirometry (forced expiratory volume in 1 s %) measured preadministration and postadministration of the first dose of nebulised bacteriophage. Clinical reviews including repeat sputum cultures and spirometry will be performed at 3, 6, 9 and 12 months following bacteriophage treatment. ETHICS AND DISSEMINATION: Our clinical trial is conducted in accordance with (1) good clinical practice, (2) Australian legislation, (3) National Health and Medical Research Council guidelines for the ethical conduct of research. TRIAL REGISTRATION NUMBER: Australia and New Zealand Clinical Trial Registry (ACTRN12622000767707).

Translating phage therapy into the clinic: Recent accomplishments but continuing challenges.

Phage therapy is a medical form of biological control of bacterial infections, one that uses naturally occurring viruses, called bacteriophages or phages, as antibacterial agents. Pioneered over 100 years ago, phage therapy nonetheless is currently experiencing a resurgence in interest, with growing numbers of clinical case studies being published. This renewed enthusiasm is due in large part to phage therapy holding promise for providing safe and effective cures for bacterial infections that traditional antibiotics acting alone have been unable to clear. This Essay introduces basic phage biology, provides an outline of the long history of phage therapy, highlights some advantages of using phages as antibacterial agents, and provides an overview of recent phage therapy clinical successes. Although phage therapy has clear clinical potential, it faces biological, regulatory, and economic challenges to its further implementation and more mainstream acceptance.

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.

Pathway and Network Analyses Identify Growth Factor Signaling and MMP9 as Potential Mediators of Mitochondrial Dysfunction in Severe COVID-19.

Patients with preexisting metabolic disorders such as diabetes are at a higher risk of developing severe coronavirus disease 2019 (COVID-19). Mitochondrion, the very organelle that controls cellular metabolism, holds the key to understanding disease progression at the cellular level. Our current study aimed to understand how cellular metabolism contributes to COVID-19 outcomes. Metacore pathway enrichment analyses on differentially expressed genes (encoded by both mitochondrial and nuclear deoxyribonucleic acid (DNA)) involved in cellular metabolism, regulation of mitochondrial respiration and organization, and apoptosis, was performed on RNA sequencing (RNASeq) data from blood samples collected from healthy controls and patients with mild/moderate or severe COVID-19. Genes from the enriched pathways were analyzed by network analysis to uncover interactions among them and up- or downstream genes within each pathway. Compared to the mild/moderate COVID-19, the upregulation of a myriad of growth factor and cell cycle signaling pathways, with concomitant downregulation of interferon signaling pathways, were observed in the severe group. Matrix metallopeptidase 9 (MMP9) was found in five of the top 10 upregulated pathways, indicating its potential as therapeutic target against COVID-19. In summary, our data demonstrates aberrant activation of endocrine signaling in severe COVID-19, and its implication in immune and metabolic dysfunction.

Pharmacokinetics/pharmacodynamics of phage therapy: a major hurdle to clinical translation.

BACKGROUND: The increasing emergence of antimicrobial resistance worldwide has led to renewed interest in phage therapy. Unlike antibiotics, the lack of pharmacokinetics/pharmacodynamics (PK/PD) information represents a major challenge for phage therapy. As therapeutic phages are biological entities with the ability to self-replicate in the presence of susceptible bacteria, their PK/PD is far more complicated than that of antibiotics. OBJECTIVES: This narrative review examines the current literature on phage pharmacology and highlights major pharmacological challenges for phage therapy. SOURCES: Included articles were identified by searching PubMed and Google Scholar till June 2022. The search terms were 'bacteriophage', 'antimicrobial', 'pharmacokinetics' and 'pharmacodynamics'. Additional relevant references were obtained from articles retrieved from the primary search. CONTENT: In this review, phage PK is first discussed, focusing on absorption, distribution, metabolism, and elimination. Key factors affecting phage antimicrobial activities are reviewed, including multiplicity of infection, passive and active phage therapy, and the involvement of the human immune system. Importantly, we emphasize the impact of phage self-replication on the PK/PD and the fundamental phage characteristics that are required for PK/PD modelling and clinical translation. IMPLICATIONS: Recent progress in phage pharmacology has shown that we are in a far better position now to treat infections with phage therapy than a century ago. However, phage therapy is still in its infancy when compared to antibiotics due to the scarce pharmacological knowledge (e.g. PK/PD). Optimization of phage PK/PD is key for translation of phage therapy in patients.

Chest CT in COVID-19 patients: A clinical need.

INTRODUCTION: The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has resulted in a global healthcare crisis. The provision of computed tomography (CT) imaging services by radiology departments for COVID-19 patients poses multiple challenges. Consequently, it is important to explore the clinical need and indications for thoracic CT and whether they subsequently alter patient management. METHODS: We report our experience in this single-centre retrospective cohort study of all confirmed COVID-19 cases admitted during the peak of the 'Delta' variant wave in Australia, and who underwent a chest CT. Clinical indication and patient management plan pre- and post-CT were ascertained. RESULTS: A total of 92 out of 1403 patients who were admitted with COVID-19 underwent a thoracic CT (73 CT pulmonary angiogram (CTPA), 14 CT Chest and five high-resolution CT (HRCT) studies). 72.8% of studies were to evaluate for pulmonary emboli, 16.2% for assessment of COVID-19 pneumonia complications, 5.4% for tuberculosis and 6.5% for other indications. 21 (23%) of these studies resulted in a change in management with two patients having a major change in management (thrombolysis, CT-guided aspiration). Management was altered due to diagnosis of pulmonary embolism (PE), pneumonia, cryptogenic organising pneumonia and other reasons. Of 73 CTPA studies, 11 (15%) patients had evidence of PE. CONCLUSION: In our centre, thoracic CT in COVID-19 patients were predominantly for the evaluation of PE with other indications being for COVID-19 complications and other cardiopulmonary pathologies. 23% of studies subsequently altered patient management, suggesting there is good clinical need for CT chests for these indications.

Association between timing and adequacy of antibiotics and adverse outcomes in patients with sepsis and septic shock: A multicentre retrospective cohort study.

OBJECTIVES: To investigate the association between the timing and adequacy of antibiotics administered to patients presenting with culture-positive sepsis and septic shock to the ED and in-hospital mortality and/or intensive care unit (ICU) admission. METHODS: Multicentre retrospective cohort study of ED presentations at four metropolitan hospitals in Sydney, Australia between January 2017 and November 2019. Encounters for patients aged ≥16 years meeting specified criteria for sepsis or septic shock with antibiotic administration within the first 6 h of presentation were included. RESULTS: Of 7611 encounters included in the study, 2328 (31%) were culture positive, and 2228 (29%) met the criteria for septic shock. In culture-positive sepsis encounters, partial or inadequate antibiotic coverage was associated with higher risk of death or ICU admission (adjusted odds ratio [AOR] 1.50, 95% confidence interval [CI] 1.04-2.06 and 1.95, 95% CI 1.28-2.99, respectively). This effect was not significant in septic shock encounters (AOR 1.10, 95% CI 0.64-1.88) with partial coverage and (AOR 1.63, 95% CI 0.81-3.3) inadequate coverage. Time to antibiotics was not significantly associated with the risk of mortality/ICU admission. This inference remained the same when analysis was restricted to cases with adequate antibiotic coverage. CONCLUSIONS: In a large multicentre sample of patients with culture-positive sepsis, inadequacy of antibiotics was associated with higher risk of in-hospital mortality or ICU admission.

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

BACKGROUND: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. METHODS: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. RESULTS: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. CONCLUSIONS: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

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.

L-Form Switching in Escherichia coli as a Common ß-Lactam Resistance Mechanism.

Cell wall deficient bacterial L-forms are induced by exposure to cell wall-targeting antibiotics and immune effectors such as lysozyme. L-forms of different bacteria (including Escherichia coli) have been reported in human infections, but whether this is a normal adaptive strategy or simply an artifact of antibiotic treatment in certain bacterial species remains unclear. Here we show that members of a representative, diverse set of pathogenic E. coli readily proliferate as L-forms in supratherapeutic concentrations of the broad-spectrum antibiotic meropenem. We report that they are completely resistant to antibiotics targeting any penicillin-binding proteins in this state, including PBP1A/1B, PBP2, PBP3, PBP4, and PBP5/6. Importantly, we observed that reversion to the cell-walled state occurs efficiently, less than 20 h after antibiotic cessation, with few or no changes in DNA sequence. We defined for the first time a logarithmic L-form growth phase with a doubling time of 80 to 190 min, followed by a stationary phase in late cultures. We further demonstrated that L-forms are metabolically active and remain normally susceptible to antibiotics that affect DNA torsion and ribosomal function. Our findings provide insights into the biology of L-forms and help us understand the risk of ß-lactam failure in persistent infections in which L-forms may be common. IMPORTANCE Bacterial L-forms require specialized culture techniques and are neither widely reported nor well understood in human infections. To date, most of the studies have been conducted on Gram-positive and stable L-form bacteria, which usually require mutagenesis or long-term passages for their generation. Here, using an adapted osmoprotective growth media, we provide evidence that pathogenic E. coli can efficiently switch to L-forms and back to a cell-walled state, proliferating aerobically in supratherapeutic concentrations of antibiotics targeting cell walls with few or no changes in their DNA sequences. Our work demonstrates that L-form switching is an effective adaptive strategy in stressful environments and can be expected to limit the efficacy of ß-lactam for many important infections.

Biological foundations of successful bacteriophage therapy.

Bacteriophages (phages) are selective viral predators of bacteria. Abundant and ubiquitous in nature, phages can be used to treat bacterial infections (phage therapy), including refractory infections and those resistant to antibiotics. However, despite an abundance of anecdotal evidence of efficacy, significant hurdles remain before routine implementation of phage therapy into medical practice, including a dearth of robust clinical trial data. Phage-bacterium interactions are complex and diverse, characterized by co-evolution trajectories that are significantly influenced by the environments in which they occur (mammalian body sites, water, soil, etc.). An understanding of the molecular mechanisms underpinning these dynamics is essential for successful clinical translation. This review aims to cover key aspects of bacterium-phage interactions that affect bacterial killing by describing the most relevant published literature and detailing the current knowledge gaps most likely to influence therapeutic success.

Co-Occurrence of Multidrug Resistant Klebsiella pneumoniae Pathogenic Clones of Human Relevance in an Equine Pneumonia Case.

The global epidemiology of multidrug resistant Klebsiella pneumoniae, a serious threat to both animal and human health, is dominated by the spread of pathogenic clones, each separately evolving via acquisition of transferable antibiotic resistance or niche-specific virulence determinants. In horses, K. pneumoniae infection can lead to severe respiratory illness. Here, we characterized multiple isolates recovered from bronchial aspirates of a mare with pneumonia refractory to antibiotics. First, we used a combination of standard microbiology, bacteriophage cross-susceptibility and antibiotic resistance testing to profile the infecting K. pneumoniae population. The genomes of isolates with distinct fingerprints (pulsed-field gel electrophoresis) and unique combined bacteriophage/antibiotic profiles were then further analyzed using whole-genome sequencing. Adhesion to human epithelial cells and biofilm production were also measured as virulence indicators. Although it is commonly expected for one clone to dominate an infection episode, we identified five coexisting multidrug resistant K. pneumoniae sharing the same niche. One was a novel sequence type (ST4656), while the other four were all members of emerging human pathogenic clonal groups (ST307, ST628, ST893 and ST392). These isolates did not display significant differences from one another in terms of virulence or resistance and differed only in plasmid content from isolates implicated in severe human infections, with equal potential to prolong duration and severity of infection when sharing the same niche. This study highlights the importance of more precise surveillance and detection measures to uncover bacterial heterogeneity, reminding us that the "single clone" concept is not an absolute in invasive bacterial infections. IMPORTANCE Multidrug resistant Klebsiella pneumoniae are agents of life-threatening infections in animals and humans, with several multidrug resistant clones causing outbreaks of disease worldwide. It is generally accepted that only one clone will be dominant in an infection episode. In this study, we investigated K. pneumoniae isolates from a horse with severe pneumonia and demonstrated co-occurrence of multiple sequence types previously identified as emerging human pathogens. The equine isolates are not significantly different from one another in terms of virulence or resistance, with equal potential to prolong duration and severity of infection, and are indistinguishable from isolates recovered from humans, except for plasmid content. Our study highlights how the "one dominant clone" concept is not an absolute in severe infection, illustrating the need for improved diagnostics to track heterogeneity of infection, and reinforces the importance of cross-monitoring of environmental and human reservoirs of multidrug resistant pathogens.