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Heteroresistance is a transient resistance phenotype characterized by the presence of small subpopulations of bacterial cells with elevated antibiotic resistance within a susceptible main population. In Gram-negative pathogens, heteroresistance is frequently caused by tandem amplification of genes encoding resistance proteins with low activity towards the antibiotic, a process commonly mediated by homologous recombination between flanking repeated sequences. However, the specific roles of individual recombination proteins in this mechanism remain largely undefined. In this study, we systematically evaluated the contribution of 19 recombination-associated genes to tandem amplification-driven heteroresistance in Escherichia coli. A clinical plasmid causing tobramycin heteroresistance by tandem amplification of aac(3)-IId gene was conjugated into recombination gene deficient mutants and the wild-type parental strain. While heteroresistance was observed with all mutants, the frequency of resistant subpopulations was decreased in recA and recB mutants, and a shift in resistance mechanism towards increased plasmid copy number and resistance mutations was observed. Partially reduced frequencies of tandem amplifications and a shift towards other heteroresistance mechanisms were also observed with recC, recJ, ruvA, and ruvC mutants, whereas other deletions of recombination genes had no or little impact on tandem amplifications. These findings identify RecABC as a key pathway in heteroresistance via tandem amplification, but even when these genes are deleted resistant subpopulations can still be generated by other mechanisms. 

Antimicrobial resistance (AMR) has become a global public health concern, particularly in developing countries where antibiotics are often overused and misused. In Bolivia, the indiscriminate use of antibiotics, including (fluoro-)quinolones, has led to the proliferation of multidrug-resistant (MDR) Campylobacter spp., increasing the risk of resistance gene dissemination to other bacteria, and further deepening the AMR problem. To help mitigate the proliferation of MDR bacteria, bacteriophages can be a valuable complementary treatment to antibiotics. In the present study, we isolated and characterized three novel lytic bacteriophages with activity against (fluoro-)quinolone-resistant Campylobacter isolates and C. jejuni strains. The isolated bacteriophages, BMBo_CjP_006, BMBo_CjP_007, and BMBo_CjP_009, belong to the class Caudoviricetes and possess a linear double-stranded DNA genome. Their genome size ranges from 59 to 77 kb, with a GC-content between 42 to 46%. The 90, 144, and 146 predicted coding sequences (CDSs) of the different bacteriophages did not encode any antibiotic resistance, virulence, or lysogenic-associated genes, confirming their genetic safety and lytic nature. The isolated bacteriophages showed a narrow host range and lytic activity against nine (fluoro-)quinolone-resistant Campylobacter spp., including C. jejuni, with lytic activity varying at MOIs from 0.1 to 100, dependent on bacteriophage and host isolate. In addition, the bacteriophages were stable across a pH range of 4 to 10 and a temperature range of -20 degrees C to 70 degrees C. These characteristics make them promising for biotechnological applications due to their lytic activity, lack of resistance and virulence genes, and potential utility for product preservation.

Inherently antibacterial materials could be an effective method to reduce the spread and impact of bacterial infections when incorporated into healthcare settings. The aim of this study was to examine whether additively manufactured PVDF-graphene nanoplatelet composites could confer antibacterial effects. The composites and reference filaments were produced with thermal compounding extrusion, which is a scalable method commonly used in industry, and were successfully printed using fused filament fabrication. The composites reduced bacterial attachment by 21 % and 81 % within the first hour of exposure for Escherichia coli and Staphylococcus aureus respectively, when graphene flakes were exposed on the surface of the samples. E. coli strains were also examined for biofilm formation on the developed materials, but no additional antibacterial effect was seen, most likely because of the limited exposure of the graphene nanoplatelets on the surface of the samples. It was found that the surface topology resulting from different printing configurations, as well as the exposure time to bacteria had a significant influence on the biological response to the samples.

Acrylic bone cement is widely used in vertebroplasty to treat osteoporosis-induced vertebral compression fractures. However, infection after vertebroplasty is problematic and previous work has suggested loading the bone cement with an antibiotic for prophylaxis. Linoleic acid (LA) has been investigated as a promising additive to improve the mechanical properties of bone cement for vertebroplasty, but LA could potentially also have an antibacterial effect. In this study, we evaluated the antibacterial properties of LA-loaded bone cement by comparing its antibiofilm properties with that of original bone cement through quantification of bacterial growth using viable cell count and scanning electron microscopy. The released monomer (MMA) concentration and the monomer minimum inhibitory concentration were determined to clarify the monomer's potential role in inhibiting bacterial growth. The LA release profile was measured, and a checkerboard assay was done to determine any synergistic effects of LA and the commonly used antibiotic gentamicin. Results show that LA-loaded bone cement could significantly inhibit Staphylococcus aureus biofilm formation, including gentamicin-resistant strains, but with limited effect on Escherichia coli. Furthermore, the released MMA did not have a significant influence on bacterial growth. The checkerboard assay results show that the LA and gentamicin combination could broaden the antibacterial spectrum and increase gentamicin efficacy. In conclusion, LA merits further investigation as an antibacterial agent in bone cement, alone or in combination with antibiotics.

Background

Heteroresistance (HR) is a significant type of antibiotic resistance observed for several bacterial species and antibiotic classes where a susceptible main population contains small subpopulations of resistant cells. Mathematical models, animal experiments and clinical studies associate HR with treatment failure. Currently used susceptibility tests do not detect heteroresistance reliably, which can result in misclassification of heteroresistant isolates as susceptible which might lead to treatment failure. Here we examined if whole genome sequence (WGS) data and machine learning (ML) can be used to detect bacterial HR.

Methods

We classified 467 Escherichia coli clinical isolates as HR or non-HR to the often used β-lactam/inhibitor combination piperacillin-tazobactam using pre-screening and Population Analysis Profiling tests. We sequenced the isolates, assembled the whole genomes and created a set of predictors based on current knowledge of HR mechanisms. Then we trained several machine learning models on 80% of this data set aiming to detect HR isolates. We compared performance of the best ML models on the remaining 20% of the data set with a baseline model based solely on the presence of β-lactamase genes. Furthermore, we sequenced the resistant sub-populations in order to analyse the genetic mechanisms underlying HR.

Findings

The best ML model achieved 100% sensitivity and 84.6% specificity, outperforming the baseline model. The strongest predictors of HR were the total number of β-lactamase genes, β-lactamase gene variants and presence of IS elements flanking them. Genetic analysis of HR strains confirmed that HR is caused by an increased copy number of resistance genes via gene amplification or plasmid copy number increase. This aligns with the ML model's findings, reinforcing the hypothesis that this mechanism underlies HR in Gram-negative bacteria.

Interpretation

We demonstrate that a combination of WGS and ML can identify HR in bacteria with perfect sensitivity and high specificity. This improved detection would allow for better-informed treatment decisions and potentially reduce the occurrence of treatment failures associated with HR.

Bacterial pathogens adapt to host niches because of selective within-host pressures, an evolutionary process that offers invaluable insights into host-pathogen interactions. Here, we retrospectively track the evolution of a single multiresistant Klebsiella pneumoniae clone in 110 patients over a 5-year nosocomial outbreak. We combine comparative genomics with phenotypic characterization of mucoviscosity, serum survival, iron utilization, biofilm formation, and infection potential in Galleria mellonella for all isolates. Strong positive selection within patients targeted key virulence factors. Notably, convergent evolutionary trajectories were dominated by reduced acute virulence and recurrent changes in iron uptake regulation, capsule and lipopolysaccharide, and enhanced biofilm formation. These phenotypic changes likely represent clinical niche adaptations, with some resulting in trade-offs during gastrointestinal colonization. This study underscores the dynamic nature of within-host evolution and its role in shaping virulence in opportunistic pathogens, even on short time scales.

Background

Antibiotic heteroresistance is a common bacterial phenotype characterised by the presence of small resistant subpopulations within a susceptible population. During antibiotic exposure, these resistant subpopulations can be enriched and potentially lead to treatment failure. In this study, we examined the prevalence, misclassification, and clinical effect of heteroresistance in Escherichia coli bloodstream infections for the clinically important antibiotics cefotaxime, gentamicin, and piperacillin–tazobactam.

Methods

We conducted a retrospective cohort analysis of patients (n=255) admitted to in-patient care and treated for E coli bloodstream infections within the Uppsala region in Sweden between Jan 1, 2014, and Dec 31, 2015. Patient inclusion criteria were admission to hospital on suspicion of infection, starting systemic antibiotics at the time of admission, positive blood cultures for the growth of E coli upon admission, and residency in the Uppsala health-care region at the time of admission. Exclusion criteria were growth of an additional pathogen than E coli in blood cultures taken at admission or previous inclusion of the patients in the study for another bloodstream infection. Antibiotic susceptibility of preserved blood culture isolates of E coli was assessed for cefotaxime, gentamicin, and piperacillin–tazobactam by disk diffusion and breakpoint crossing heteroresistance (BCHR) was identified using population analysis profiling. The clinical outcome parameters were obtained from patient records. The primary outcome variable was length of hospital stay due to the E coli bloodstream infection, defined as the time between admission and discharge from inpatient care as noted on the physician’s notes. Secondary outcomes were time to fever resolution, admission to intermediary care unit or intensive care unit during time in hospital, switching or adding another intravenous antibiotic treatment, re-admission to hospital within 30 days of original admission, recurrent E coli infection within 30 days of admission to hospital, and all-cause mortality within 90 days of admission.

Findings

A total of 255 participants with a corresponding E coli isolate (out of 500 screened for eligibility) met the inclusion criteria, with 135 female patients and 120 male patients. One (<1%) of 255 strains was BCHR for cefotaxime, 109 (43%) of 255 strains were BCHR for gentamicin, and 22 (9%) of 255 strains were BCHR for piperacillin–tazobactam. Clinical susceptibility testing misclassified 120 (96%) of 125 heteroresistant bacterial strains as susceptible. The BCHR phenotypes had no correlation to length of hospital stay due to the E coli bloodstream infection. However, patients with piperacillin–tazobactam BCHR strains who received piperacillin–tazobactam had 3·1 times higher odds for admittance to the intermediate care unit (95% CI 1·1–9·6, p=0·041) than the remainder of the cohort, excluding those treated with gentamicin. Similarly, those infected with gentamicin BCHR who received gentamicin showed higher odds for admittance to the intensive care unit (5·6 [1·1–42·0, p=0·043]) and mortality (7·1 [1·2–49·2, p=0·030]) than patients treated with gentamicin who were infected with non-gentamicin BCHR E coli.

Interpretation

In a cohort of patients with E coli bloodstream infections, heteroresistance is common and frequently misidentified in routine clinical testing. Several negative effects on patient outcomes are associated with heteroresistant strains.

Background

Piperacillin/tazobactam is a β-lactam/β-lactamase inhibitor combination with a broad spectrum of activity that is often used as empirical and/or targeted therapy among hospitalized patients. Heteroresistance (HR) is a form of antibiotic resistance in which a minority population of resistant cells coexists with a majority susceptible population that has been found to be a cause of antibiotic treatment failure in murine models.

Objectives

To determine the prevalence of HR and mechanisms of HR to piperacillin/tazobactam among Klebsiella pneumoniae bloodstream infection (BSI) isolates.

Materials

From July 2018 to June 2021, K. pneumoniae piperacillin/tazobactam-susceptible BSI isolates were collected from two tertiary hospitals in Atlanta, GA, USA. Only first isolates from each patient per calendar year were included. Population analysis profiling (PAP) and WGS were performed to identify HR and its mechanisms.

Results

Among 423 K. pneumoniae BSI isolates collected during the study period, 6% (25/423) were found to be HR with a subpopulation surviving above the breakpoint. WGS of HR isolates grown in the presence of piperacillin/tazobactam at concentrations 8-fold that of the MIC revealed copy number changes of plasmid-located β-lactamase genes bla_CTX-M-15, bla_SHV33, bla_OXA-1 and bla_TEM-1 by tandem gene amplification or plasmid copy number increase.

Conclusions

Prevalence of HR to piperacillin/tazobactam among bloodstream isolates was substantial. The HR phenotype appears to be caused by tandem amplification of β-lactamase genes found on plasmids or plasmid copy number increase. This raises the possibility of dissemination of HR through horizontal gene transfer and requires further study.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.