Journal of Applied Microbiology

Antimicrobial resistance (AMR) is a multifactorial issue involving an intertwining relationship between animals, humans and the environment. The environment can harbour bacteria that are pathogenic to human health, including Escherichia coli, an indicator of environmental faecal contamination. Through culture dependent approaches this study identified 46 E. coli isolates in porcine and bovine manure, non-manured and manured soil, and the phyllosphere of manured grass. The grass isolation highlights grass as an environmental reservoir for E. coli. Whole genome sequencing identified 11 different multi-locus sequence types. We also identified a diverse plasmidome with 23 different plasmid replicon types. The E. coli isolates were phenotypically antibiotic resistance, predominantly multidrug resistant. Additionally, whole genome sequencing identified 31 antibiotic resistance genes, and mutations in the gyrA, parC, and parE genes, conferring fluoroquinolone resistance. The main virulence genes were associated actin mediated locomotion (icsP/sopA), siderophore production and alginate production (algA), which suggest adaptation to survive in the non-gut environment or the UV environment of grass surfaces. These results suggest that E. coli in soils and grasses may adapt to their new environments evolving novel strategies. This study demonstrates grass as an understudied environmental niche of AMR E. coli, which directly links the environment to the grass grazing animal and vice-versa via the circular economy of manure application. Impact statementEscherichia coli is capable of surviving across biomes within One Health. This study sheds light on the genomic elements present in AMR E. coli in the understudied niche of agricultural grassland. Data summaryThe genome sequences have been deposited in Genbank. Bioproject number PRJNA1080214 and SRP491607 in the sequence read archive https://www.ncbi.nlm.nih.gov/sra/?term=SRP491607.

IntroductionAntimicrobial resistance has existed in the environment long before its rapid emergence and detection in clinically relevant pathogens. Studying the resistance of environmental bacterial strains may allow novel resistance mechanisms to be identified before they appear in pathogenic strains. Gap StatementSearching for antimicrobial resistance genes in environmental bacteria represents an understudied research area compared to resistance within clinically relevant pathogens. AimTo evaluate resistance genes present within environmental non-aeruginosa Pseudomonas spp. isolates. MethodologyWe screened a set of bacterial isolates from untreated wastewater from Liverpool, UK, for the presence of extended spectrum {beta}-lactamases and carbapenemases. A sub-set of three resistant Pseudomonas spp. isolates were selected for whole-genome sequencing. We performed minimum inhibitory concentration assays against several {beta}-lactams, and ectopic expression of four novel resistance genes within Escherichia coli. ResultsHere, we report the discovery of novel class C {beta}-lactamase genes blaPFL7, blaPFL8 and blaPFL9, as well as a novel subclass B2 metallo-{beta}-lactamase blaPFM5 present within these strains. The class C genes encoded proteins with between 61-71% amino acid identity to the closest known match, blaPFL-1. These novel {beta}-lactamases degraded the cephalosporin nitrocefin and confer piperacillin and ceftazidime resistance to susceptible Escherichia coli when ectopically expressed. The {beta}-lactamase inhibitor tazobactam was effective at inhibiting these enzymes. The sub-class B2 metallo-{beta}-lactamase had 88% amino acid identity to its closet match blaPFM-1 and conferred carbapenem resistance to susceptible E. coli. The {beta}-lactamase inhibitors relebactam, vaborbactam, xeruborbactam and captopril had no impact on the carbapenem resistance phenotype. Analogues of all these novel genes (>95% nucleotide sequence identity) were identified within publicly available whole-genome sequencing data, suggesting they are found sporadically. ConclusionOur analysis adds to the growing number of {beta}-lactamase genes found from environmental Pseudomonas spp. and suggests that continued surveillance of this environmental reservoir for novel, clinically relevant, {beta}-lactamase genes is warranted.

In low- and middle-income countries, living in homes with soil floors and animal cohabitation may expose children to fecal organisms, increasing risk of enteric and antimicrobial-resistant infections. Our objective was to understand whether cow cohabitation in homes with soil floors in rural Bangladesh contributed to the presence and diversity of potential pathogens and antimicrobial resistance genes (ARGs) in the home. In 10 randomly selected households in rural Sirajganj District, we sampled floor soil and cow dung, which is commonly used as sealant in soil floors. We extracted DNA and performed shotgun metagenomic sequencing to explore potential pathogens and ARGs in each sample type. We detected 6 potential pathogens in soil only, 49 pathogens in cow dung only, and 167 pathogens in both soil and cow dung. Pathogen species with relative abundances >5% in both soil floors and cow dung from the same households included E. coli (N=8 households), Salmonella enterica (N=6), Klebsiella pneumoniae (N=2), and Pseudomonas aeruginosa (N=1). Cow dung exhibited modestly higher pathogen genus richness compared to soil floors (Wilcoxon signed-rank test p=0.002). Using Bray-Curtis dissimilarity, pathogen species community composition differed between floors and cow dung (PERMANOVA p<0.001). All soil floors and cow dung samples contained ARGs against antibiotic classes including sulfonamides, rifamycin, aminoglycosides, lincosamides, and tetracycline. Paired floor and cow dung samples shared ARGs against rifamycin. Our findings support the development of interventions to reduce soil and animal feces exposure in rural, low-income settings. ImportanceIn low-income countries, inadequate housing materials and animal cohabitation can lead to fecal contamination of rural homes. Contaminated soil floors are difficult to clean and may harbor organisms causing illness and antibiotic resistance, especially in young children, who frequently ingest soil. We sequenced soil floor and cow dung samples from households in Sirajganj district, Bangladesh and identified pathogens and antibiotic resistance genes. We detected 167 pathogens in both soil and cow dung; pathogens present in both sample types at the highest relative abundances were E. coli, Salmonella enterica, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Antibiotic resistance genes were found in all samples. In cow dung, the most common genes conferred resistance to the antibiotics lincosamide, rifamycin, cephamycin, and tetracycline. In soil floors, the most common genes conferred resistance to rifamycin, sulfonamides, and aminoglycosides. Household soil and cow dung may be important reservoirs of pathogens and antimicrobial resistance in low-income countries.

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.

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.

Furfural residues s influence on maize rhizosphere microbiota subjected to long-term monocropping is poorly understood. In this study, high-throughput sequencing was employed to investigate the rhizosphere microbiota composition and its variation under long-term field monocropping for maize seed production. The results showed that unplanted, chemical fertilizer (only) treated soil, and furfural residue treated soil as well as seasonal soil batches recruited distinct rhizosphere microbiota. Microbial community diversity increased, and many operational taxonomic units (OTUs) were induced in the rhizosphere soil. Maize plants grown under field conditions were preferentially colonized by Ascomycota and Zygomycota in the unplanted soil, Ascomycota and Mortierellomycota in chemical fertilizer-treated soil, and Ascomycota and Basidiomycota in frufural residue-treated soil. Some potential pathogens, such as Alternaria, Trichocladium, Bipolaris, Solicoccozyma and Cladosporium were not detected, while beneficial microbes, such as Penicillium, Schizothecium and Rhizophlyctis were present. Acidobacteria and Bacteroidetes increased, Actinobacteria and Firmicutes decreased in furfural residue treated soil. The core bacteria detected after long-term cropping were MND1, RB41, UTCFX1, Nitrospira, Cellvibrio, Adhaeribacter etc. The relative abundances of Clostridium, Pseudarthrobacter and Roseiflexus decreased; Haliangium, Nitrospira and MND1 increased; Pirellula, Ellin6067 and Luteimonas reduced in different seasonal soil batches. Amendment with furfural residue promoted the development of beneficial microbes and decreased the abundance of pathogens after different continuous cropping years. The amendment increased cellulose-degrading and complex carbon-decomposing microbes, decreased the number of reductive substance-decomposing microbes, which led to microbial community structure shift over time. Amendment with furfural residue improved the rhizosphere environment, which will in turn improve plant growth. Furfural residue can be used as a soil amendment to control soil-borne diseases and to establish beneficial soil microbes. ImportanceContinuous monoculture of maize seed production led to reduction in plant nutrient absorption, destruction of soil structure, high incidence of soil-borne plant diseases, and decrease in crop yield. Traditional organic fertilizers are either unavailable or unbalanced for intensive cultivation. Furfural residues are acidic and carbon-rich making it a promising organic alternative to chemical fertilizers to improve seed corn production. Amendment with furfural residue may promote the microbiota in rhizosphere soil. In addition, the amendment may increase cellulose-degrading and complex carbon-decomposing microbes, which led to a shift in microbial community structure over time. Amendment with furfural residue may improve the rhizosphere environment, which will in turn improve plant growth, control soil-borne diseases and establish beneficial soil microbes.

Nucleic acid extraction is the first step in molecular biology studies of soil bacterial communities. The most common used soil DNA extraction method is the direct, hard extraction Mobio method, which uses bead beating to lyse bacteria. In this study we compared the Mobio method with a soft, enzymatic lysis extraction method. Next generation sequencing (Illumina and Pyrosequencing) of amplicons generated from four 16S primer pairs and DNA from 12 soils and 3 composts was used to compare the two extraction methods. Four bacterial orders, the delta proteobacterial Desulfuromonadales and gamma proteobacterial Pseudomonadales, Enterobacteriales, and Alteromonadales were more common in amplicons from soft extracted DNA, sometimes by two orders of magnitude. These groups can be a significant fraction of the bacterial population. For example the Pseudomonadales made up to 16 % and Enterobacteriales 10% of amplicons from Soft extracted DNA. The JG30-KF-CM45 order was under extracted by the enzymatic lysis extraction method. Results differed more by primer choice than extraction method and the phylogenetic resolution of differences between extraction methods changed with primer choice. Given how often Mobio extraction is used, these proteobacterial orders are probably under-represented in the studies of soil bacteria that use nucleic acid methods. Further improvements in soil DNA extraction are needed. Amplicons sequencing studies should use a range of different primers to confirm the phylogenetic resolution of their results. ImportanceSeveral large scale studies of soil bacteria that compare thousands of soil samples across continents have used the Mobio method for DNA extraction. Large scale studies like these are increasing with the recent establishment of the Global Soil Biodiversity Observation Network (Soil BON), which also uses the Mobio method. The results of this work will be used to make policy decisions about how to manage the soil and may be a guide for bioprospectors. As the Mobio method is so widely used, it is important to know its limitations. Studies that use the Mobio method underestimate the fraction of several proteobacterial groups. Most notably the Enterobacteria and Pseudomonas can be under extracted by 10-100 fold. The degree of under extraction varies with different soils.

BackgroundIn this study, we aimed to identify bacteria able to grow in the presence of several antibiotics including the ultra-broad-spectrum antibiotic meropenem in a British agricultural soil, by combining DNA stable isotope probing (SIP) with high throughput sequencing. Soil was incubated with cefotaxime, meropenem, ciprofloxacin and trimethoprim in 18O-water. Metagenomes and the V4 region of the 16S rRNA gene from the labelled "heavy" and the unlabelled "light" SIP fractions were sequenced. ResultsAfter incubations, an increase of the 16S rRNA copy numbers in the "heavy" fractions of the treatments with 18O-water compared with their controls was detected. The treatments resulted in differences in the community composition of bacteria. Members of the phyla Acidobacteriota (formally Acidobacteria) are highly abundant after two days of incubation with antibiotics. Several Pseudomonadota (formally Proteobacteria) including Stenotrophomonas were prominent after four days of incubation. Furthermore, a metagenome-assembled genome (MAG-1) from the genus Stenotrophomonas (90.7% complete) was retrieved from the heavy fraction. Finally, 11 antimicrobial resistance genes (ARGs) were identified in the unbinned-assembled heavy fractions, and 10 ARGs were identified in MAG-1. On the other hand, only two ARGs from the unbinned-assembled light fractions were identified. ConclusionsThe results indicate that both non-pathogenic soil-dwelling bacteria as well as potential clinical pathogens are present in this agricultural soil, and several ARGs were identified from the labelled communities, but it is still unclear if horizontal gene transfer between these groups can occur.

The goal of this study was to determine minimum selection concentrations of various antibiotics using four manure-originated multi-drug resistant plasmids in a surrogate Escherichia coli host. These plasmids carried genes conferring resistance phenotypes to several antibiotic classes including {beta}-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined: 14.1-28.2 mg/L for penicillin G, 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, 2 mg/L for lincomycin, 1 mg/L for florfenicol, 1.3-4 mg/L for azithromycin, 0.13-0.25 mg/L for tetracycline, 0.004-0.01 mg/L for cefotaxime. Penicillin G, oxytetracycline, chlortetracycline, lincomycin and florfenicol had minimum selection concentrations in nutrient-defined medium slightly changed within 3.5-fold range compared to those in nutrient-rich medium. The minimum selection concentrations of antibiotics interfering folic acid synthesis in bacteria were also determined: 63 mg/L for sulfamethoxazole, 11.2 mg/L for sulfisoxazole and 0.06 mg/L for trimethoprim. Mixing two antibiotics changed minimum selection concentrations within 3.7-fold range compared to those in single antibiotic tests. Relatively high plasmid loss rates (> 90%) were observed when culturing plasmid-bearing strains in antibiotic-free nutrient-rich and nutrient-defined media. Overall results suggested that these plasmids can be maintained at concentrations environmentally relevant in waste water treatment plants, sewage, manure and manured soil although they are not stable in antibiotic-free environments. IMPORTANCEAntibiotic resistance crisis is a grave concern in healthcare systems around the world. To combat this crisis, we sought to find out how likely manure-originated multi-drug resistant plasmids are to be selected and maintained in different environment matrices. Our study showed that these plasmids conferring resistance to {beta}-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines can be selected at minimum selection concentrations which are lower than minimum inhibition concentrations of the E. coli host strain. Lincomycin, oxytetracycline, chlortetracycline, tetracycline, chloramphenicol, trimethoprim had minimum selection concentrations lower than the antibiotic concentrations in several environment matrices reported previously. Our findings suggest that despite the burden and the high rate of plasmid loss, these plasmids can still be selected, maintained and circulated well in some polluted environments.

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.

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.

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 ([&le;] 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.

This study was to assess the gene diversity and characterize a large set of plasmids harboring extended {beta}-lactamase (ESBL) genes from raw and digested dairy manure. A total of eighty-four plasmids that were captured in this E. coli recipient were sequenced using Illumina MiSeq sequencing technology. Twenty-four plasmids of interest were subsequently sequenced using MinION technology in order that a hybrid assembly could be performed on short-and long-read sequences to circularize and complete these plasmids. The size of sequenced plasmids ranged between 40 and 260 kb with various incompatibility groups: IncC, IncI1, IncN, IncY, IncB/O/K/Z, IncX1, IncHI2, IncHI2A, IncFIB(K), IncFII. A variety of extended {beta}-lactamase genes were identified: blaCTXM -1, blaCTXM -14, blaCTXM -15, blaCTXM-27, blaCTXM-55, blaCTXM-61, blaPER-1, blaIMP-27. Interestingly, the blaIMP-27 gene, a novel metallo-{beta}-lactamase discovered in the last decade, was found located on an integrated region in the host chromosome. And one plasmid carrying the blaCMY-2 gene, an AmpC gene, also expressed ESBL phenotype. Four virulence factors, including cia, cib, traT and terC, were detected on some of these plasmids. In addition, six type-2 toxin-antitoxin systems were detected: MazF/E, PemK/I, HipA/B, YdcE/D, RelB/E and HigB/A. Twenty-two out of twenty-four complete plasmids carried putative prophage regions; and most of prophage hits were marked as incomplete, except that the largest plasmid pT525A and the IncY plasmid pT415A had prophage hits with higher scores. IMPORTANCEThe widespread of antibiotic resistant bacteria is largely due to the exchange of mobile genetic elements such as plasmids. Plasmids harboring extended {beta}-lactamase (ESBL) genes originated from dairy manure potentially become entrained in manured soil, which subsequently enter the human food chain. Currently there is a lack of detailed information on these plasmids in the environment, specifically in dairy manure. This study unveils the abundance and diversity of ESBL-carrying plasmids from both raw and digested manures which were captured in gfp-labelled E. coli CV601. In addition, the study provides insightful information of plasmid characteristics including incompatibility groups, ESBL genes combined with other resistance genes, mobile genetic elements (transposons, insertion sequence), toxin-antitoxin systems, virulence factors and prophage sequences.

BackgroundSoil naturally harbours antibiotic resistant bacteria and is considered to be a reservoir for antibiotic resistance. The overuse of antibiotics across human, animal, and environmental sectors has intensified this issue leading to an increased acquisition of antibiotic resistant genes by bacteria in soil. Various biogeographical factors, such as soil pH, temperature, and pollutants, play a role in the spread and emergence of antibiotic resistance in soil. In this study, we utilised publicly available metagenomic datasets from four different soil types (rhizosphere, urban, natural, and rural areas) sampled from nine distinct geographic locations to explore the patterns of antibiotic resistance in soils from different regions. ResultsBradyrhizobium was predominant in vegetation soil types regardless of soil pH and temperature. ESKAPE pathogen Pseudomonas aeruginosa was prevalent in rural soil samples. Antibiotic resistance gene families such as 16s rRNA with mutations conferring resistance to aminoglycoside antibiotics, OXA {beta}-lactamase, ANT(3), and the RND and MFS efflux pump gene were identified in all soil types, with their abundances influenced by anthropogenic activities, vegetation, and climate in different geographical locations. Plasmids were more abundant in rural soils and were linked to aminoglycoside resistance. Integrons and integrative elements identified were associated with commonly used and naturally occurring antibiotics, showing similar abundances across different soil types and geographical locations. ConclusionAntimicrobial resistance in soil may be driven by anthropogenic activities and biogeographical factors, increasing the risk of bacteria developing resistance and leading to higher morbidity and mortality rates in humans and animals.

1.BackgroundEnterococcus species are Gram-positive, facultative anaerobic bacteria commonly found in soil, water, and the gastrointestinal tracts of humans and animals. Their ability to acquire antibiotic resistance has made them significant opportunistic pathogens in both clinical and environmental settings. The presence of antibiotic-resistant Enterococcus strains in aquatic environments reflects contamination from human activities and poses a potential risk of horizontal gene transfer to other bacterial species. AimsThis study aims to assess the antibiotic and heavy metal resistance patterns of Enterococcus strains isolated from water bodies in Vladivostok and evaluate their potential environmental risks. Study designExperimental study. MethodsWater samples were collected from the Vtoraya Rechka River and Zolotoy Rog Bay in Vladivostok. 30 enterococcus strains were identified by polymerase chain reaction (PCR) technique. The presence of antibiotic and heavy metal resistance genes was determined using specific primers. ResultsGenetic analysis of the isolated Enterococcus strains revealed that 23% of strains carried the tetL gene, 33% harbored the ermB gene, and the heavy metal resistance genes tcrB and cadA were detected in 40% and 27% of strains, respectively. Notably, the ermB and tcrB genes co-occurred in 13.3% of strains, while none of the tetL-positive strains exhibited simultaneous presence of ermB, tcrB, and cadA. ConclusionEnterococcus strains isolated from Vladivostoks water bodies exhibit resistance to multiple antibiotics and heavy metals, with a notable co-occurrence of copper and antibiotic resistance genes. These findings contribute to understanding microbial contamination in aquatic environments and its public health implications, providing valuable insights for future research in clinical and environmental microbiology. 2. IMPORTANCEThe dissemination of antibiotic and heavy metal resistance genes in environmental reservoirs poses a serious public health threat, as these genes may be transferred to clinically relevant pathogens. Enterococcus species, widely present in aquatic ecosystems, serve as both reservoirs and indicators of such resistance. This study identifies patterns of co-occurrence among resistance genes and highlights a potential genetic linkage between macrolide (ermB) and copper (tcrB) resistance in environmental Enterococcus strains. Notably, none of the tetL-positive strains carried all three of ermB, tcrB, and cadA, suggesting a mutually exclusive distribution pattern and a potentially distinct mechanism of acquisition for tetL. These findings reveal novel gene distribution dynamics in environmental Enterococcus populations and underscore the importance of integrated surveillance of antimicrobial and metal resistance in human-impacted ecosystems.

ObjectivesIncreasing resistance to antimicrobials used for the treatment of Clostridioides difficile infections necessitates reproducible antimicrobial susceptibility testing. Current guidelines take a one-size-fits-all approach and/or offer limited guidance. We investigated how the choice of medium affects measured MIC values across two sites. MethodsWe determined MIC values for the antimicrobials fidaxomicin, metronidazole and vancomycin for a representative collection of European C. difficile strains (n=235) using agar dilution on three different media: Brucella Blood Agar (BBA), Fastidious Anaerobe Agar supplemented with horseblood (FAA-HB) and Wilkins-Chalgren (WC) agar. The study was conducted at two sites to compare reproducibility. Useability (ease of preparation of the media as well as read-out of the assay) was assessed through a survey. ResultsWe found that all media result in highly consistent aggregated MIC data for all antibiotics, with MIC50 and MIC90 within 2-fold of each other across sites. For fidaxomin, MIC values on WC were lower than on the other media. Metronidazole showed the lowest MIC on BBA, and the highest on WC. For vancomycin, there was little difference between media. Though absolute values for individual isolates differed between sites, identified resistant isolates were similar. Results obtained on FAA-HB were most consistent between sites and results obtained on WC showed the most divergence. FAA-HB was positively evaluated in the usability survey. ConclusionsThis study shows medium-dependent differences in C. difficile MICs for at least two antimicrobials across two sites. We suggest the use of FAA-HB to align with general EUCAST recommendations for susceptibility testing of anaerobic bacteria and deposited reference strains for standard susceptibility testing of C. difficile to increase interlaboratory reproducibility.

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.

Reducing the environmental impact of Canadian field crop agriculture, including the reliance on conventional synthesised fertilisers, are key societal targets for establishing long-term sustainable practices. Municipal biosolids (MSB) are an abundant, residual organic material, rich in phosphate, nitrogen and other oligo-nutrients, that could be used in conjunction with conventional fertilisers to decrease their use. Though MBS have previously been shown to be an effective fertiliser substitute for different crops, including corn and soybean, there remain key knowledge gaps concerning the impact of MBS on the resident soil bacterial communities in agro-ecosystems. We hypothesised that the MBS fertiliser amendment would not significantly impact the structure or function of the soil bacterial communities, nor contribute to the spread of human pathogenic bacteria, in corn or soybean agricultural systems. In field experiments, fertiliser regimes for both crops were amended with MBS, and compared to corn and soybean plots with standard fertiliser treatments. We repeated this across four different agricultural sites in Quebec, over 2021 and 2022. We sampled MBS-treated, and untreated soils, and identified the composition of the soil bacterial communities via 16S rRNA metabarcoding. We found no indication that the MBS fertiliser amendment altered the structure of the soil bacterial communities, but rather that the soil type and crop identities were the most significant factors in structuring the bacterial communities. Moreover, there was no evidence that the MBS-treated soils experienced a shift in functions, nor contributed potential human bacterial pathogens over the two years of our study. Our analysis indicates that not only can MBS function as substitutes for conventional, synthesised fertilisers, but that they also do not disrupt the structure, or function, of the resident soil bacterial communities in the short term. Finally, we suggest that the use of MBS in agro-ecosystems poses no greater concern to the public than existing soil bacterial communities. HighlightsO_LIMunicipal biosolids may represent a sustainable fertiliser substitute C_LIO_LIBut, the impact of biosolids on soil bacteria in agricultural fields is unknown C_LIO_LIUsing 16S rRNA metabarcoding we analysed community structure and functions C_LIO_LIWe found no disruption of soil bacterial communities fertilised with biosolids C_LIO_LIBiosolids are safe, sustainable fertilisers with little impact on soil bacteria C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/571735v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b9ca2corg.highwire.dtl.DTLVardef@8818d2org.highwire.dtl.DTLVardef@1158864org.highwire.dtl.DTLVardef@ad952f_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundWe showed that exposure of an AcrB D408A mutant to efflux inhibitors applied evolutionary pressure to select bacteria with the wild type acrB sequence. This suggested that reversion to wild type can differentiate between efflux inhibitors. Thus, we hypothesized that this experiment could identify inhibitors of the primary RND pump, AcrB or its homologues in other species. ObjectivesTo construct three mutants, Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A and expose the mutants to substrates and non-substrates of AcrB and/or MexB and determine the rate of reversion to wildtype acrB/mexB sequence. MethodsMutant Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A were constructed with site-directed mutagenesis of the relevant nucleotide in the acrB/mexB gene. Mutants were exposed on agar to substrates and the mutation frequency and mutation rate determined. The MIC of antibiotics and the presence/absence of the D408A substitution was determined for mutants. ResultsExposure to the AcrB substrates chlorpromazine and minocycline reverted the D408A genotypes to wild type in a species-dependent manner. Exposure to a non-AcrB substrate, spectinomycin, did not select wild type acrB. Chlorpromazine selected for wild type acrB K. pneumoniae as it had for S. Typhimurium, whereas minocycline selected for wild type E. coli acrB. None of the antibiotics selected wild type mexB, including the tested MexB substrates. ConclusionsEvolutionary paths depend upon the genetic background of the species and availability of alternative routes/genetic pathways that can confer resistance/decreased susceptibility to antibiotics.