Journal of Applied Microbiology

BackgroundResistance to colistin, a last-reserve antibiotic used for treating drug-resistant infections, is increasing globally. This study evaluated six diagnostic tests designed to detect colistin-resistant pathogens. MethodsPCR and broth microdilution assays (BMD) were used to respectively characterize the molecular mechanisms and phenotypic colistin resistance of 142 Gram-negative bacterial isolates and controls. The sensitivity, specificity, positive- and negative-predictive values, major (ME) and very major errors (VME), categorical and essential agreements (EA) of ComASP Colistin, CHROMagar COL-APSE, Rapid NP Test, Sensititre, MicroScan, and Vitek 2 were determined with these isolates; the BMD was used as gold standard. ResultsThe Vitek 2, Sensititre, and ComASP tests were more efficient, albeit with concerning ME and VMEs and low EAs. Sensititre was 100% specific with 0% ME and 3.61% VME; Vitek 2 had the least VME (1.25% and 0%) and a low EA (57.50%). ComASP had an EA of 75.35%. MicroScan was highly sensitive (96.55%) but less specific (87.50%), with very below-accetable EAs (48.11%). The CHROMAgar COL-APSE efficiently identified the species with their unique colours but was the least specific (67.80%), with the highest ME (32.20%) and high VME (7.23%). The Rapid NP test had the highest VME (7.84%), producing results within 4 hours with 92.16% sensitivity and 96.08% specificity. ConclusionVitek 2, MicroScan, ComASP colistin, and Sensititre are good for determining colistin resistance; the latter two tests are recommendable for low-resourced laboratories. The in-house Rapid NP test has short turnaround time with high efficiency for initial resistance screening.

Dairy calves, particularly pre-weaned calves have been identified as a common source of multidrug (MDR) resistant E. coli. However, the strains and whether their resistance genes are plasmid or chromosomally located have not been well characterised. Our study examined the phenotype and genotype of antimicrobial resistant E. coli isolated from young calves ([≤] 14 days old). Recto-anal swab enrichments from 40 dairy calves located on four dairy farms were examined for tetracycline, streptomycin, ciprofloxacin, and third-generation cephalosporin resistant E. coli. Fifty-eight percent (23/40) of calves harboured antimicrobial resistant E. coli: 18/40 (45%) harboured tetracycline resistant and 25% (10/40) harboured chromosomal mediated AmpC producing E. coli. Whole genome sequencing of 27 isolates revealed five sequence types, with ST88 being the dominant ST (17/27, 63% of the sequenced isolates) followed by ST1308 (3/27, 11%), along with the extraintestinal pathogenic E. coli lineages ST69 (3/27), ST10 (2/27, 7%), and ST58 (1/27, 4%). Additionally, 16 isolates were MDR, harbouring additional resistance genes that were not tested phenotypically. Oxford Nanopore long-read sequencing technologies enabled the location of multiple resistant gene cassettes in IncF plasmids to be determined. A phylogenetic comparison of the ST10 and ST69 isolates demonstrated that the calf derived isolates were distinct from other New Zealand animal, human, and environmental isolates. and highlights the importance of understanding the sources of antimicrobial resistance.

Foodborne outbreaks caused by fecal contamination of fresh produce represent a serious concern to public health and the economy. As the consumption of fresh produce increases, public health officials and organizations have pushed for improvements in food safety procedures and environmental assessments to reduce the risk of contamination. Visual inspections and the establishment of "buffer zones" between animal feeding operations and producing fields are the current best practices for environmental assessments. However, a generalized distance guideline and visual inspections may not be enough to account for all environmental risk variables. Here, we report a baseline measurement surveying the background Bacteroidales concentration, as a quantitative fecal contamination indicator, in Californias Salinas Valley. We collected a total of 1632 samples from two romaine lettuce commercial fields at the time of harvesting through two seasons in a year. The Bacteroidales concentration was very low (0 - 2.00 copies/cm2). Furthermore, we established a practical methodology for evaluating the risk of fecal contamination in a real-world setting, complementing the current environmental assessment practices. This method can identify site-specific risks and offer fresh produce stakeholders a more comprehensive understanding of their fields. We anticipate this work can encourage the use of Bacteroidales in the fresh produce industry to monitor fecal contamination and prevent future foodborne outbreaks.

Enterococci are opportunistic, potentially life-threatening pathogens of humans that are difficult to manage due to antimicrobial resistance. Historically, enterococci entering the food-chain through livestock have been viewed as a likely source of antimicrobial resistance in humans. Australian human-derived clinical enterococci have a propensity to be resistant to multiple classes of antimicrobials including vancomycin. Recent Australian studies involving pigs and chicken have virtually excluded these species as reservoirs of infection for resistant enterococci in humans. However, the Australian bovine population has not been similarly assessed. This study investigates the antimicrobial resistance profiles of enterococci from Australian cattle and the phylogenetic relationship between E. faecium isolated from cattle and human sepsis cases. Minimum inhibitory concentration assays were performed for E. faecium (n=343), E. faecalis (n=92), and E. hirae (n=284) against a panel of 15 antimicrobials. The majority of isolates were sensitive to all tested antimicrobials. Erythromycin resistance was most prevalent for E. faecium isolates (18.7%), daptomycin for E. faecalis (12.1%) and tetracycline for E. hirae (13.3%). Phenotypically, 1 E. faecalis was resistant to vancomycin and 9 were resistant to linezolid (E. faecium n=4, E. faecalis n=2, E. hirae n=3) but this was not confirmed with any know genotype. A subset of 67 E. faecium isolates selected for comparative phylogenetic analysis revealed that bovine isolates clustered with other livestock-derived and van-negative human isolates. In conclusion, there is a low prevalence of antimicrobial resistance amongst enterococci from Australian cattle which are unlikely to be precursor strains to vancomycin-resistant strains currently circulating in Australian hospitals. ImportanceEnterococci resistant to critically important antimicrobials such as vancomycin and linezolid are difficult to manage in health care settings. Historically, there has been the belief that livestock can act as a reservoir of resistance for human infections. Previous studies in poultry and pork have demonstrated that isolates derived from these livestock are unlikely to be precursor strains for currently circulating vancomycin resistant-Enterococci causing infection in humans. To date, there has been no study looking at the genetic similarity of bovine derived Enterococci and the relationship to human pathogenic strains. In this study we performed phenotypic and genotypic characterization of bovine derived Enterococci along with comparative phylogenetic analysis with other livestock derived and human sepsis derived E. faecium isolates. We found that while non-vancomycin resistant strain sharing occurs between hosts, cattle are unlikely to be precursor strains for vancomycin resistant human E. faecium infections.

Biofilms pose a significant challenge in medical settings, leading to persistent infections. Phage therapy has shown promise in biofilm eradication, but its effectiveness under dynamic flow conditions remains unclear. Here we use two novel phages isolated on Klebsiella, Llofrudd and Samara, and characterized their genomes, host range and virulence. In this study, we built a simple catheterised bladder model with flow to investigate the impact of phage treatment on biofilm viability in a flow-based catheter model. Our analyses demonstrate that phages Llofrudd and Samara are the same species and infect a limited number of strains (3/222), but across three species: Klebsiella aerogenes, Klebsiella pneumoniae and E. coli. Phage treatment significantly reduced E. coli biofilm viability in catheters both in static conditions and under flow, highlighting the potential of phage therapy as an intervention strategy for catheter associated urinary tract infections (CAUTI).

BackgroundAntimicrobial resistance in Neisseria gonorrhoeae is an urgent public health threat. Resistance-guided therapy can assure appropriate treatment and reintroduce alternative therapeutic options by identifying genetic predictors of resistance. Mosaicism at codons 375-377 of the penA gene are associated with cefixime resistance. Rapid, field-deployable assays for predicting cefixime susceptibility are lacking. MethodsWe used a machine-learning algorithm to develop a CRISPR Cas13a-based assay to detect the absence of mosaicism at codons 375-377 of the penA gene combined with isothermal amplification in a single reaction. We integrated the assay onto a portable fluorescence-based platform. We evaluated performance using cultured isolates and compared results with PCR genotyping and phenotypic antimicrobial susceptibility testing. We also assessed feasibility of reagent lyophilization for cold-chain-independent deployment. ResultsAmong 40 N. gonorrhoeae isolates, the Cas13a penA assay demonstrated 100% concordance with PCR genotyping and 92{middle dot}5% (95% CI 79.6-98.4%) concordance with phenotypic cefixime susceptibility. Median time to detection was 12 minutes (IQR 5 minutes). The lyophilized detection system detected all 12 isolates with a median time to detection of 45{middle dot}0 minutes (IQR 40-45) compared to 45{middle dot}0 minutes (IQR 35-50) for the positive aqueous control, although peak fluorescence was higher for the aqueous control (p<0.01). ConclusionThe Cas13a assay was rapid and demonstrated strong correlation with genotypic and phenotypic cefixime susceptibility in N. gonorrhoeae, while a lyophilized assay retained functionality.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n=2), healthcare facility wastewater (n=2), wastewater treatment plant influent (n=1) and integrated constructed wetland influent (piggery farm waste) (n=1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 {+/-} 4.1 (10-61), including the carbapenemases, blaOXA-48 (n=2) and blaNDM-1 (n=1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

AimsBacteriophage (phage) propagation has traditionally relied on bacterial culture media containing animal-derived ingredients; however, safety concerns with animal-derived materials for production of phages for therapeutic use limit their acceptability. We compared animal-free and traditional media formulations, and evaluated their effects on phage yield, bactericidal activity, and genomic characteristics, hypothesizing no significant differences would be observed. Methods and ResultsPhages targeting Pseudomonas aeruginosa (n=8) and Staphylococcus aureus (n=1) were propagated in solid and liquid media containing animal-free (AF) or animal-derived (LB) peptones. Kinetic assays were used to assess phage suppression of host bacterial growth. In a mock therapeutic phage screen, spot tests, Efficiency of Plating (EOP) and kinetic assays were performed against novel bacterial targets. Whole genome sequencing of phages and their bacterial hosts propagated in AF or LB broth was used to observe genomic differences between formulations. Animal-free peptone did not impact phage yield, with both AF and LB phage stocks growing to high titers ([&ge;]108 PFU/mL). Kinetic assay results showed similar suppression indices for AF and LB-grown phages. Likewise, phage screen spot test, EOP, and kinetic assay results were similar between AF and LB phages. Comparisons of phage and bacterial genome annotations showed no major differences arising from media formulation. ConclusionsFindings suggest animal-free peptones do not significantly alter phage yield, bactericidal activity, or genomic characteristics, supporting use of animal-free medium for medicinal phage manufacture. This is one of the first studies to systematically combine phenotypic and genomic assessment of phages and hosts across animal-free and traditional media. Impact StatementPhage therapy is increasingly used to treat antimicrobial resistance infections. Emerging guidelines and regulations for the manufacture of phage therapeutics will impact laboratory processes and materials used for phage production. Here, we explored the use of an animal-free medium for medicinal phage propagation, providing data on phage yield and metrics of phage activity.

Antimicrobial resistance (AMR) has increased at an alarming pace in the recent years. Molecular-based methods such as whole genome sequencing (WGS) offer a potential alternative to the conventional labour-intensive methods traditionally used to characterise AMR phenotypes. The aim of this study was to investigate whether WGS could be used as a predictor of AMR in Escherichia coli isolates of bovine origin. Genomes of 143 E. coli cultured from cattle presenting with diarrhoea or mastitis were sequenced on an Illumina MiSeq platform. AMR genes were identified using the ResFinder and AMRFinder databases. Antimicrobial susceptibility testing by disk diffusion was performed on a panel of 10 antibiotics, covering 7 antimicrobial classes. Minimum inhibitory concentration (MIC) measurements were made using the Sensititre plate with 6 antibiotics, covering 5 antimicrobial classes. Correlation between genotype and phenotype was assessed statistically by means of a two-by-two table analysis and Cohens kappa ({kappa}) test. The overall {kappa} correlation between WGS and disk diffusion was 0.81, indicating a near perfect agreement, and the average positive predicted value was 77.4 %. Correlation for individual antimicrobial compounds varied, with five yielding near perfect agreement ({kappa} = 0.81-1.00; amoxicillin, florfenicol, gentamicin, tetracycline and trimethoprim-sulfamethoxazole), one showing substantial agreement ({kappa} = 0.65; nalidixic acid), and four showing moderate agreement ({kappa} = 0.41- 0.60). The overall {kappa} correlation between WGS and MIC was 0.55 indicating moderate agreement, and the average positive predicted value was 68.6 %. Three antibiotics yielded near perfect agreement (gentamicin, tetracycline and trimethoprim-sulfamethoxazole) and a further three showed fair agreement ({kappa} = 0.21-0.40). WGS is a useful tool that can be used for the prediction of AMR phenotypes, and correlates well with disk diffusion results. MIC measurements may be necessary for antimicrobial compounds with a high proportion of intermediately resistant isolates recorded, such as cephalothin. HighlightsO_LICulture based antimicrobial susceptibility testing is used to identify therapeutics in the treatment of clinical veterinary isolates C_LIO_LIWhole genome sequencing is increasingly adopted for surveillance, epidemiological traceback investigations, and detection of antimicrobial resistance genes C_LIO_LILittle is known in correlations between antimicrobial resistance genotypes and disk diffusion antimicrobial susceptibility testing C_LIO_LIThis study finds that whole genome sequencing is a useful predictor for antimicrobial susceptibility however, minimum inhibitory concentration measurements may still be needed for intermediately resistant isolates C_LI

The pre-emergence herbicide trifluralin is widely used in the minimum-tillage cropping systems of Australia, with the result that resistance to trifluralin is increasing in the major weed of the region, annual ryegrass (Lolium rigidum). Repeated exposure to low herbicide rates is also known to result in the rapid evolution of resistance in weed populations. As trifluralin is highly volatile, readily photo-decomposed, metabolised by soil microbes and to bind strongly to soil organic matter, there are many factors that could result in weed populations receiving reduced (even sub-lethal) rates of the herbicide. To investigate whether trifluralin dissipation could play a role in the increasing levels of trifluralin resistance in annual ryegrass, resistance levels of populations from 18 Western Australian farms were compared with the dissipation rate of trifluralin applied to soil collected from these farms. Although there was no direct correlation between resistance level and trifluralin half-life, there were links between resistance and soil properties which suggest that higher rates of trifluralin dissipation could make a minor contribution to the development of resistance.

The excessive and often unregulated use of antibiotics in livestock production and human health has led to the dissemination of antibiotic residues and antibiotic resistance genes (ARGs) posing a serious threat to the environment and public health. This study investigates the co-occurrence and spatial distribution of antibiotic residues and ARGs in livestock manure and agricultural soils from Machakos, Kiambu, and Kajiado counties in Kenya, regions characterized by intensive livestock farming. A total of 180 samples from 30 farms across the three counties were collected and pooled into 18 samples. Antibiotic residues were extracted and quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Nine ARGs (aadA, ermB, sul1, tetQ, tetW, dfrA1, blaMOX, blaOXA and qnrB were quantified via absolute and relative qPCR, normalized to 16s rRNA gene copy numbers. Multivariate analyses, including PCoA and Spearman Correlation, were conducted to explore ARG structure and co-occurrence. Tetracycline, particularly oxytetracycline, was the most abundant antibiotic (up to 1150 ng/mL), especially in pig manure from Kiambu. Sulfadimethoxin was undetectable in nearly all samples except in soil mixed with pig manure from Kajiado county, which showed a concentration above 70 ng/mL, the pig manure from the same county had levels below 2 ng/mL. Sulfadimidin, sulfamethoxanol and erythromycin were not detected in all samples. A three-way ANOVA showed that animal source and sample type were generally not significant factors in antibiotic concentration, but oxytetracycline (p= 0.0421, 0.0901) and sulfadimethoxin (P =0.0904, 0.044, 0.054) showed marginal significance by sample type (P=0.0785). Antibiotic resistance genes were widely distributed, with aadA, ermB and sul1 being the most prevalent, especially in soils from Kiambu and Machakos. TetQ showed extremely high relative abundance, indicating intense tetracycline selection pressure. Strong positive correlations were observed between co-occurring ARGs, including tetQ and tetW and erm and sul1. The concurrent detection of persistent antibiotic residues and high ARG loads in both manure and soils underscores the urgent need for improved antibiotic stewardship, sustainable manure management, and environmental monitoring in Kenyan agroecosystems.

Faster, and simpler methods that determine the susceptibility of bacterial pathogens to antibiotics are needed to enable more effective treatment of bacterial infections and reduce the overuse of antibiotics. Here, we demonstrate a simple method for rapidly detecting bacteria and simultaneously determining their antibiotic susceptibility status. We demonstrate the method with E. coli, a high-impact pathogen, and meropenem, a member of the carbapenem class of antibiotics which is usually reserved for multi-drug resistant infections. Inclusion of a fluorogenic oligonucleotide substrate for endonuclease I, a well-conserved nuclease of the Enterobacteriaceae family, in a tryptic soy agar-based media enabled fluorescence-based detection of E. coli growth within 9 hours. Plating established carbapenem-resistant and -susceptible E. coli strains on this media with and without inclusion of meropenem, followed by fluorescent imaging yielded a clear phenotypic measure of the antibiotic susceptibility status of the strains in 9 hours. In addition to its simplicity and fast turnaround time, advantages of this approach include its ability to provide a measure of the bacterial load of the tested samples, and its easy integration into current microbiology laboratory workflows.

Application of a beneficial microbial cocktail of Bacillus spp. and Pediococcus spp. was evaluated first for adherence to polyethylene calf hutch material, and second, to determine if application in situ to individual calf hutches post-cleaning influenced surface recolonization by enteric pathogens. Three treatments were utilized: 1) no application (NC), 2) chlorine-free, distilled water (DW), or 3) an application of a microbial inoculant containing Bacillus spp. and Pediococcus spp. at a concentration of 0.4 g/m2 of hutch space (LF). Thirty-six 15 x 15 cm pieces of naive, sterile polyethylene calf hutch material received either NC or LF, were incubated at 28{degrees}C, and bacterial growth was evaluated by total aerobic plate counts at 24, 48, and 72 h post-application. Thirty polyethylene calf hutches (n = 10/treatment) were randomized to either NC, DW, or LF 24 h after cleaning. Calves were placed in the hutches 24 h after treatment application and monitored daily for 28 d. In situ surface samples were randomized by time from five unique locations within the calf hutch interior: 24 h post-cleaning, then 24 h, 7 d, 14 d, and 21 d post-application. Total aerobic plate counts and culture-independent approaches RT-qPCR and 16S amplicon sequencing were used to detect and identify the composition of the bacterial community in situ. The bacteria in the inoculant were able to successfully colonize on polyethylene, and application to individual polyethylene calf housing in situ influenced microbial diversity and reduced the presence of some undesirable bacteria on high-contact interior surfaces. IMPORTANCEDue to its multifactorial nature, neonatal calf diarrhea can be difficult to manage on farms. Clean housing environments are a critical disease control point, especially for calves less than one month of age. Application of a beneficial biofilm-forming bacterial product after cleaning of neonatal calf housing may influence the microbial communities present on the surface, particularly those that may present disease risk to calves in early life.

Antimicrobial-resistant (AMR) bacteria and genes are continually exchanged among humans, animals, and environmental reservoirs. Disparate and siloed surveillance methods present a major challenge for tracing and disrupting AMR transmission, with clinical monitoring focusing on detecting specific pathogens or specific genes of interest, and environmental surveys often relying on inferences drawn from indicator organisms. Here, we demonstrate that, following sample-specific pre-processing, common surveillance approaches can be applied consistently to profile AMR abundance, distribution, and phenotypes across diverse reservoirs, including soil, sediment, water, wastewater, and faecal samples from both urban and agricultural settings. Across all sample types, three core methods provided complementary insights: (i) quantitative PCR (qPCR) arrays to measure multiple AMR genes, (ii) gene- and genome-centric metagenomics for comprehensive resistome profiling, and (iii) culture-based genomics with susceptibility testing to link genotypes to phenotypes. We applied this approach to profile 1,032 metagenome-assembled genomes, 66 bacterial isolate genomes, and 78 and 6,442 AMR genes/reference sequences via qPCR and metagenomics, respectively. This integrated framework revealed a moderate prevalence but high diversity of resistance mechanisms in both pathogens and non-pathogenic bacteria with potentially transmissible genes, with wastewater especially enriched in AMR genes. We detected mismatches between genotype and phenotype predictions and a prevalence of intermediate resistance phenotypes, highlighting how many mechanisms of environmental resistance remain poorly understood. Overall, this study demonstrates that unified field-leading surveillance methods can be extended beyond clinical contexts into diverse environmental and animal samples, while highlighting that multiple methods are needed to capture the diverse AMR genotypes and phenotypes in these settings to enable comprehensive monitoring and adaptive solutions to restrict transmission.

Antibiotic resistant bacteria present an emerging challenge to human health as the pressure instituted on the microbial world through the liberal use of antibiotics has resulted in their emergence across the globe. Those bacteria that acquire mobile genetic elements such as plasmids are especially concerning because those plasmids may be shared readily with other microbes that then can also become antibiotic resistant. Serious infections have recently been related to contamination of preservative-free eyedrops with extensively drug resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for lysis of these XDR isolates, and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the 3 separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

The Genomic Enumeration of Antibiotic Resistance in Space (GEARS) flight project seeks to characterize the frequency and genomic identity of antibiotic-resistant organisms on the ISS and expand in-space sequencing-based diagnostic capabilities. This project selects for antibiotic-resistant microbes on space station surfaces using agar plates containing antibiotics, which can require storage of these plates for several months. However, little published data is available on the longevity of antibiotics in agar plates. Here, we describe the process of antibiotic selection and validation of antibiotic stability for GEARS. A literature review was conducted on several antibiotics for their intrinsic and acquired resistance in Enterococcus and Staphylococcus and stability in solution and in agar. Gentamicin and streptomycin were selected for a long-term study tracking changes in bactericidal ability of the antibiotics in agar plates. After 6.5 months of storage at 4{degrees}C, we found that both antibiotic types remained stable throughout the test period, although they both exhibited evidence of slight degradation. These findings indicated that either antibiotic would be sufficient for the GEARS study based on stability under cold stowage conditions. Furthermore, this study generated information on long-term stability of antibiotics in agar, which can be useful for microbiology applications beyond spaceflight studies.

In Aotearoa New Zealand, urinary tract infections in humans are commonly caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. This group of antimicrobial resistant bacteria are often multidrug resistant. However, there is limited information on ESBL-producing E. coli found in the environment and their link with human clinical isolates. In this study, we examined the genetic relationship of environmental and human clinical ESBL-producing E. coli and isolates collected in parallel within the same area over 14 months. Environmental samples were collected from treated effluent, stormwater and multiple locations along an Aotearoa New Zealand river. Treated effluent, stormwater and river water sourced downstream of the treated outflow point were the main sources of ESBL-producing E. coli (7/14 samples, 50.0%; 3/6 samples, 50%; and 15/28 samples, 54% respectively). Whole genome sequence comparison was carried out on 307 human clinical and 45 environmental ESBL-producing E. coli isolates. Sequence type 131 was dominant for both clinical (147/307, 47.9%) and environmental isolates (11/45, 24.4%). The most prevalent ESBL genes were both blaCTX-M-27 and blaCTX-M-15 for the clinical isolates (134/307, 43.6%) and blaCTX-M-15 for the environmental isolates (28/45, 62.2%). A core single nucleotide polymorphism analysis of these isolates suggested that some strains were shared between humans and the local river. These results highlight the importance of understanding different transmission pathways for the spread of ESBL-producing E. coli. 2. Impact statementExtended spectrum beta lactamase (ESBL)-producing E. coli frequently cause urinary tract infections that exhibit multidrug resistance. Surveillance studies have identified the predominant strains and resistance genes associated with urinary tract infections. However, there is limited information on the extent of spread beyond the patient. We describe the genetic relatedness of ESBL-producing environmental and clinical E. coli isolated during the same temporal-spatial period in Aotearoa New Zealand. Comparative genomic analyses of these bacteria provide evidence of clonal spread between humans and the environment, highlighting the need to integrate environmental surveillance into antimicrobial resistance monitoring. 3. Data summaryAll Illumina sequence reads for this study have been deposited in GenBank under BioProject PRJNA1032159, except for strain SB0283h1, whose data can be found under BioProject PRJNA715472. The sequence read accessions for each genome are provided in the supplementary material. The code used for the genomic and statistical analyses is available from the GitHub repository https://github.com/sburgess1/Manawat-_ESBL. The authors confirm all supporting data and protocols have been provided within the article or through supplementary data files.

SynopsisO_ST_ABSBackgroundC_ST_ABSGlobally, there is a large gap in our understanding of the prevalence, ecology and transmission dynamics of antibiotic resistance (ABR) in extensively reared ruminants, despite these animals contributing to the food chain and frequently sharing land with humans. MethodsFive hundred and seventy one visits to 33 Welsh beef cattle and/or sheep farms resulted in 1874 samples being collected at faecally contaminated sites from April 2022 to March 2023 (ADGC1) and September 2023 to December 2024 (ADGC2). Samples were tested for resistant Escherichia coli using amoxicillin, streptomycin, spectinomycin, cefotaxime and ciprofloxacin. WGS used Illumina technology. Clonal relationships were determined following core-genome alignment. ResultsA significant reduction in positivity for spectinomycin-resistant E. coli in sheep samples from ADGC1 to ADGC2 was observed, coincident with market withdrawal of a spectinomycin-containing preparation widely used in sheep. Reductions were seen in 19/22 sheep flocks with nine seeing a >50% reduction. Resistance to other tested antibiotics was unchanged. Phenotypic analysis and WGS for 713 E. coli showed that resistance to antibacterials important for human medicine was rare and genetically diverse. We identified 77 E. coli clones (<100 SNP cutoff) circulating among study farms with mixed farms contributing most; clones were also shared between animal species on mixed farms. ConclusionsFor extensively reared ruminants, ABR-reducing efforts can have significant impacts on antibiotic resistance on farms. Focusing these efforts onto farms contributing to the most animal movement and mixing events may generate the greatest reductions in overall on-farm ABR prevalence at regional and national levels.

The widespread of antibiotic resistance (ABR) among bacteria has become a global health concern for humans, animals and the environment. In this respect, beta-lactams and colistin are of particular interest due to the emergence of multidrug-resistant gram-negative bacteria. Households provide a habitat for bacteria originating from humans, animals, foods, contaminated clothes or other sources in which detergents and biocides are frequently used. Thus, bacteria carrying antibiotic resistance genes (ARGs) might be introduced into private households and may consequently be also released from households to the environment via domestic wastewater. Since data on ABR in the domestic environment is limited, this study aimed to determine the abundance and correlation of beta-lactamase, mobile colistin resistance and class 1 integron genes and to characterize phenotypic resistant strains in private households in Germany. Additionally, the persistence of ABR bacteria to laundering and automated dishwashing was assessed. Shower drains, washing machines and dishwashers were sampled and analyzed using quantitative real-time PCR. Resistant strains were isolated, followed by identification and antibiotic susceptibility testing using VITEK 2. The results show a significantly higher occurrence of ARGs in shower drains compared to washing machines and dishwashers. Several beta-lactamase genes co-occurred and resistance of bacterial isolates correlated positively with genotypic resistance. Laundering and automated dishwashing reduced ABR bacteria significantly and the efficacy increased with increasing duration and temperature. Overall, the domestic environment seems to represent a potential reservoir of beta-lactamase genes and beta-lactam resistant bacteria with shower drains as the dominant source of ABR. ImportanceThe abundance of ABR bacteria and ARGs is steadily increasing and has been comprehensively analyzed in natural environments, animals, foods or wastewater treatment plants. Despite of their connection to these environments, private households seem to be neglected. Therefore, the present study investigated shower drains, washing machines and dishwashers as possible sites of ARGs and ABR bacteria. The analysis of the domestic environment as a potential reservoir of resistant bacteria is crucial to determine whether households contribute to the spread of ABR or are a habitat where resistant bacteria from the environment, humans, food or water accumulate.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) causes severe and potentially life-threatening infections in hospitalized patients with mortality rates of more than 40%. To detect CRPA carriage in humans for surveillance purposes or to prevent spread and outbreaks in hospitals, a highly-sensitive culture method for CRPA carriage in humans is needed. We aimed to develop such a highly-sensitive method, that would be feasible in laboratories with limited resources. In this study, seven well-defined CRPA strains belonging to high-risk clones were used, including one CRPA without a carbapenemase gene and six carbapenem-resistant isolates with carbapenemase genes. We applied a stepwise approach wherein we included four enrichment broths and eight Pseudomonas aeruginosa-selective culture media. Spiking experiments were performed to further evaluate the combination of the most sensitive enrichment broths and selective agar plates in human samples. The two most sensitive enrichments broths were TSB-vancomycin and TSB-vancomycin with 2 mg/L imipenem and the most sensitive selective agar plates were Pseudomonas isolation agar Becton Dickinson, Pseudomonas isolation agar Sigma-Aldrich, and M-PA-C (Becton Dickinson). After the spiking experiment, the best method for detecting CRPA based on the sensitivity and the selectivity was the combination of TSB-vancomycin with 2 mg/L imipenem as an enrichment broth for overnight incubation, followed by subculturing the broth on M-PA-C agar plate. We have thus developed a highly-sensitive selective method to detect CRPA carriage in humans, which can also be applied in limited-resource laboratories. This may contribute to an overall effort to control CRPA.