Plasmid Replicon Research Articles

The dynamics of blaTEM resistance genes in Salmonella Typhi

Salmonella Typhi (S. Typhi) is an important pathogen causing typhoid fever worldwide. The emergence of antibiotic resistance, including that of blaTEM genes encoding to TEM \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\:\\beta\\:$$\\end{document}-lactamases has been observed. This study aimed to investigate the dynamics of blaTEM genes in S. Typhi by analyzing the phylogeny and flanking region patterns and phylogenetic associating them with metadata (year, country) and genomic data (genotypes, antibiotic resistance genes (ARGs), plasmids). Genomic sequences of publicly available S. Typhi harboring blaTEM (n = 6079), spanning from 1983 to 2023, were downloaded and analyzed using CSIPhylogeny for phylogeny, Flankophile for identifying genetic contexts around blaTEM genes and GenoTyphi for determining genotypes, ARGs and plasmid replicons. We found that blaTEM-positive isolates occurred most commonly in specific location, especially in Asia and Africa and clustered among a limited number of genotypes. Flankophile identified 740 isolates (12.2%) with distinct flanking region patterns, which were categorized into 13 patterns. Notably, 7 patterns showed a predominantly phylogenetic association with genotypes. Additionally, these 7 patterns exhibited relation to the country, ARGs and plasmid replicons. Further examination of the flanking region patterns provided association with mobile genetic elements (MGEs). Taken together, this study suggests that blaTEM has been acquired by S. Typhi isolates a limited number of times and subsequently spread clonally with specific genotypes.

Tn4661-mediated transfer of bla CTX-M-15 from Klebsiella michiganensis to an outbreak clone of Pseudomonas aeruginosa.

Carriage of CTX-M-type extended-spectrum β-lactamase (ESBL) is rare in Pseudomonas aeruginosa. During routine surveillance of an endemic ST-621 P. aeruginosa at a large hospital, isolate MRSN 100690 carrying bla CTX-M-15 was cultured from a patient (P2). This was the first detection of this ESBL in the endemic ST-621 lineage. All 1 488 bacterial isolates collected from the same facility in the 12 months prior to the incidence of 100 690 were screened for the presence of bla CTX-M-15. A set of 183 isolates was identified, in which corresponding patient metadata was evaluated for spatiotemporal overlaps with P2. The resulting three isolates, along with 100 690, were long-read sequenced using the Oxford Nanopore MinION platform to determine a potential donor of bla CTX-M-15. The screen revealed a single Klebsiella michiganensis isolate, MRSN 895358, which carried an IncA/C2 plasmid harbouring bla CTX-M-15. Notably, the patient harbouring 895358, P1, occupied the same hospital room as P2 9 months prior. Genomic alignment revealed that both isolates shared an identical 80.8 kb region containing the IncA/C2 plasmid replicon and bla CTX-M-15. This region was plasmid bound in 895 358, but chromosomally bound in 100 690 due to Tn4661-mediated transposition. ESBL bla CTX-M-15 was acquired and subsequently integrated into the chromosome of a ST-621 P. aeruginosa, likely initiated by plasmid transfer from a K. michiganensis strain.

Whole genome analysis of Pantoea species identified from sepsis patients in selected Ethiopian referral hospitals: emerging pathogens

BackgroundThe burden of sepsis worsens due to the continuation of emerging pathogens such as multidrug-resistant Pantoea species.MethodsA multicenter study was conducted between October 2019 and September 2020 at four hospitals located in central, southern, and northern parts of Ethiopia. A total of 1416 sepsis patients were recruited and blood cultures were performed. At each study site, positive cultures were characterized by their colony characteristics, gram stain, and conventional biochemical tests. All Pantoea species were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF) and subjected to whole genome sequencing (WGS) using Illumina HiSeq 2500. The phylogeny structure of Pantoea isolates was calculated using IQ-TREE v1.6.12 from single-nucleotide polymorphisms detected by Snippy v.4.6.0 and filtered by Gubbins v.2.3.4. Average nucleotide identity was estimated by using OrthoANI v.0.93.1 on Shovill v.1.1.0 assemblies. Antimicrobial resistance genes and plasmid replicons were detected using ARIBA v.2.14.6. Phylogenetic trees were visualized using iTOLv.6.5.2.ResultsMultiple Pantoea species include: P. dispersa (n = 19), P. septica (n = 1), and a novel Pantoea spp. (n = 1), were identified among sepsis patients. All P. dispersa isolates and the novel Pantoea species were isolated at Dessie Referral Hospital and displayed phylogenetic clonality, including the ubiquity of an IncM1 plasmid and identical antimicrobial resistance (AMR) gene profiles, encoding blaCTX−M−15, blaTEM−1D, blaSCO−1, and aac(3)-lla. The novel Pantoea spp. isolate harboured blaCTX−M−9 and blaTEM−1D and carried an IncN3 plasmid replicon. The P. septica was isolated at Tikur Anbessa Specialized Hospital in Addis Ababa and carried no detectable acquired AMR genes.ConclusionThe emerging Pantoea spp. carrying multiple AMR genes were identified from sepsis patients. Implementation of strong infection prevention strategies and building surveillance capacity with advanced bacteriology laboratories capable of identifying multidrug-resistant emerging pathogens is strongly recommended.

Serotype Distribution and Antimicrobial Resistance of Salmonella Isolates from Poultry Sources in China.

Salmonella is an important zoonotic pathogen, of which poultry products are important reservoirs. This study analyzed the prevalence, antimicrobial resistance, and characterization of Salmonella from broiler and laying hen sources in China. A total of 138 (12.27%) strains of Salmonella were isolated from 1125 samples from broiler slaughterhouses (20.66%, 44/213), broiler farms (18.21%, 55/302), and laying hen farms (6.39%, 39/610). Multiplex PCR was used to identify the serotypes. Antibiotic susceptibility testing to a set of 21 antibiotics was performed and all strains were screened by PCR for 24 selected antimicrobial resistance genes (ARGs). In addition, 24 strains of Salmonella were screened out by whole-genome sequencing together with 65 released Salmonella genomes to evaluate phylogenetic characteristics, multilocus sequence typing (MLST), and plasmid carriage percentages. A total of 11 different serotypes were identified, with the dominance of S. Enteritidis (43/138, 31.16%), S. Newport (30/138, 21.74%), and S. Indiana (19/138, 13.77%). The results showed that S. Enteritidis (34.34%, 34/99) and S. Newport (51.28%, 20/39) were the dominant serotypes of isolates from broilers and laying hens, respectively. The 138 isolates showed the highest resistance to sulfisoxazole (SXZ, 100%), nalidixic acid (NAL, 54.35%), tetracycline (TET, 47.83%), streptomycin (STR, 39.86%), ampicillin (AMP, 39.13%), and chloramphenicol (CHL, 30.43%), while all the strains were sensitive to both tigacycline (TIG) and colistin (COL). A total of 45.65% (63/138) of the isolates were multidrug-resistant (MDR) strains, and most of them (61/63, 96.83%) were from broiler sources. The results of PCR assays revealed that 63.77% of the isolates were carrying the quinolone resistance gene qnrD, followed by gyrB (58.70%) and the trimethoprim resistance gene dfrA12 (52.17%). Moreover, a total of thirty-four ARGs, eighty-nine virulence genes, and eight plasmid replicons were detected in the twenty-four screened Salmonella strains, among which S. Indiana was detected to carry the most ARGs and the fewest plasmid replicons and virulence genes compared to the other serotypes. This study revealed a high percentage of multidrug-resistant Salmonella from poultry sources, stressing the importance of continuous monitoring of Salmonella serotypes and antimicrobial resistance in the poultry chain, and emergency strategies should be implemented to address this problem.

Analyzing Antibiotic Resistance in Bacteria from Wastewater in Pakistan Using Whole-Genome Sequencing.

Background: Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. Objectives: This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. Methods: We analyzed 32 bacterial isolates, including 18 Escherichia coli, 4 Klebsiella pneumoniae, and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Results: Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All E. coli isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The E. coli isolates had 11 different STs, with ST410 predominating (28%). Notably, the E. coli phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of dfrA12-aadA2-qacEΔ1 on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in K. pneumoniae 22EB1 and Acinetobacter baumannii 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. Conclusions: These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates.

Mobile genetic elements contributing to horizontal gene transfer of blaNDM among Escherichia coli in the community setting

ObjectiveTo investigate the distribution of carbapenem-resistant Enterobacterales (CRE) in the community and to describe the genomic characteristics. MethodsCRE screened from fecal samples in healthy people at the health examination center of a tertiary hospital in China underwent Whole genome sequencing (WGS) to analyze genotypic characteristics of CRE. The flanking DNA sequence of blaNDM-5 and mcr1.1 genes were analyzed by Gcluster software. ResultsA total of 7187 fecal samples were screened, and CRE carriage was detected in 0.4 % of the sampled population. In total, 30 Escherichia coli, one Citrobacter freundii and one Klebsiella aerogene were screened. The 30 carbapenem-resistant Escherichia coli (CREC) isolates displayed slight resistance to amikacin (13.3 %) and aztreonam (20.0 %). All the CRE isolates contained blaNDM, and blaNDM-5 (84.4 %) was the most common one. B1 (n = 11) and A (n = 7) were predominant phylogroups. Furthermore, 34 distinct plasmid replicons, 67 different VFs, 22 distinct STs, 17 different FimH types, 26 O:H serotypes as well as 74 MGEs including 61 insertion sequences and 13 transposons were identified. The flanking DNA sequence analysis of blaNDM-5 and mcr1.1 genes indicates the key role of horizontal transfer of blaNDM-5 in the CRE development evidenced by diverse STs and phylogenetic tree. ConclusionE. coli was the most predominant CRE isolates in community setting, and blaNDM (blaNDM-5) was the main CHβL encoding genes. The high prevalence of ARGs was associated with high resistance to commonly used antimicrobials. Besides, the genetic diversity of these isolates suggested the key role of blaNDM horizontal transfer in the CRE development. Thus, active screening of blaNDM in communities is particularly important for the prevention and control of CRE.

Genome characterization of Shewanella algae in Hainan Province, China.

Shewanella algae is an emerging marine zoonotic pathogen. In this study, we first reported the Shewanella algae infections in patients and animals in Hainan Province, China. Currently, there is still relatively little known about the whole-genome characteristics of Shewanella algae in most tropical regions, including in southern China. Here, we sequenced the 62 Shewanella algae strains isolated from Hainan Province and combined with the whole genomes sequences of 144 Shewanella algae genomes from public databases to analyze genomic features. Phylogenetic analysis revealed that Shewanella algae is widely distributed in the marine environments of both temperate and tropical countries, exhibiting close phylogenetic relationships with genomes isolated from patients, animals, and plants. Thereby confirming that exposure to marine environments is a risk factor for Shewanella algae infections. Average nucleotide identity analysis indicated that the clonally identical genomes could be isolated from patients with different sample types at different times. Pan-genome analysis identified a total of 21,909 genes, including 1,563 core genes, 8,292 strain-specific genes, and 12,054 accessory genes. Multiple putative virulence-associated genes were identified, encompassing 14 categories and 16 subcategories, with 171 distinct virulence factors. Three different plasmid replicon types were detected in 33 genomes. Eleven classes of antibiotic resistance genes and 352 integrons were identified. Antimicrobial susceptibility testing revealed a high resistance rate to imipenem and colistin among the strains studied, with 5 strains exhibiting multidrug resistance. However, they were all sensitive to amikacin, minocycline, and tigecycline. Our findings clarify the genomic characteristics and population structure of Shewanella algae in Hainan Province. The results offer insights into the genetic basis of pathogenicity in Shewanella algae and enhance our understanding of its global phylogeography.

Characterization of colistin-resistant carbapenemase producing Klebsiella pneumoniae in a river receiving wastewater treatment plant effluent.

Genes conferring antibiotic resistance phenotype, particularly to last resort antibiotics, pose a significant concern globally. Wastewater treatment plant (WWTP) effluent substantially contributes to antibiotic resistance in receiving rivers, threatening human health. Globally, colistin- and carbapenem-resistant Klebsiella pneumoniae infections cause high morbidity and mortality. We investigated colistin-resistant carbapenemase-producing K. pneumoniae (Co-CRKP) isolates in Kathajodi river receiving WWTP effluent, their resistance genes, and pathogenic potential. Four isolates (Co-CRKP-7, Co-CRKP-8, Co-CRKP-10, and Co-CRKP-15) exhibited extensively drug-resistant (XDR) phenotype, harbouring blaTEM-1, blaCTX-M-15, blaNDM-5, and blaOXA-48 genes. Colistin resistance was attributed to mutations in the pmrA and pmrB genes. Virulence genes (fimH, mrkD, entB, iucA, iutA, and irp1), capsular serotypes (K1, K2) and biofilm formation in the isolates explicated their pathogenicity. Furthermore, Inc plasmid replicons (Y, FrepB, P, K/B, L/M, N, FIA, A/C, and FIB) indicated the dissemination potential of the resistance genes in Co-CRKP isolates. The multi-locus sequence typing showed that Co-CRKP-7 and Co-CRKP-8 belonged to ST42, while Co-CRKP-10 and Co-CRKP-15 were ST16 and ST231, respectively. These high-risk clones carrying multidrug resistance and virulence genes, implicated in numerous outbreaks, have spread worldwide. Our findings emphasize the necessity for effective treatment of hospital wastes to restrict the spread of clinical isolates into aquatic environments.

Molecular epidemiology of extended-spectrum beta-lactamase-producing-Klebsiella species in East Tennessee dairy cattle farms.

The rising prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Klebsiella species (spp.) poses a significant threat to human and animal health and environmental safety. To address this pressing issue, a comprehensive study was undertaken to elucidate the burden and dissemination mechanisms of ESBL-Klebsiella spp. in dairy cattle farms. Fifty-seven Klebsiella species were isolated on CHROMagar™ ESBL plates and confirmed with MADLI-TOF MS and whole genome sequenced from 14 dairy farms. Six families of beta-lactamase (bla) (bla CTX-M, bla SHV, bla TEM, bla OXY, bla OXA, and bla SED) were detected in ESBL-Klebsiella spp. genomes. Most (73%) of isolates had the first three types of beta-lactamase genes, with bla SHV being the most frequent, followed by bla CTX-M. Most (93%) isolates harbored two or more bla genes. The isolates were genotypically MDR, with 26 distinct types of antibiotic resistance genes (ARGs) and point mutations in gyrA, gyrB, and parC genes. The genomes also harbored 22 different plasmid replicon types, including three novel IncFII. The IncFII and Col440I plasmids were the most frequent and were associated with bla CTXM-27 and qnrB19 genes, respectively. Eighteen distinct sequence types (STs), including eight isolates with novel STs of K. pneumoniae, were detected. The most frequently occurring STs were ST353 (n = 8), ST469 (n = 6), and the novel ST7501 (n = 6). Clusters of ESBL-Klebsiella strains with identical STs, plasmids, and ARGs were detected in multiple farms, suggesting possible clonal expansion. The same ESBL variant was linked to identical plasmids in different Klebsiella STs in some farms, suggesting horizontal spread of the resistance gene. The high burden and dual spread mechanism of ESBL genes in Klebsiella species, combined with the emergence of novel sequence types, could swiftly increase the prevalence of ESBL-Klebsiella spp., posing significant risks to human, animal, and environmental health. Immediate action is needed to implement rigorous surveillance and control measures to mitigate this risk.

Genomic insights into prevalence of virulence and multi drug resistance genes in milk borne Klebsiella pnuemoniae: Face of emerging resistance to last resort antibiotics

Spread of hypervirulent and multi-drug resistant Klebsiella pneumoniae in raw milk is public health concern due to its potential impact on food safety and public health. Therefore, this study investigated antibiotic susceptibility test (AST), antibiotic resistance genes (ARGs), mutations conferring ARGs, virulence factor and plasmid replicons to check prevalence of fosfomycin resistant MDR K. pneumoniae isolated from raw milk samples collected from Saurashtra region of Gujarat, India. K. pneumoniae isolated from raw milk and subjected to disk diffusion assay. From that, MDR along with fosfomycin resistant isolates were analysed for multi locus sequence typing, presence of ARGs, mutations conferring resistance, virulence factors and plasmid replicon types by using its whole genome sequence. Results shows that, among 32 K. pneumoniae, 8 were phenotypically resistant to fosfomycin. As per WGS analysis, 8 MDR isolates were assigned into different sequence types such as ST3321, ST37, ST2715, ST1087, ST3157, ST299 and ST29. Among that, ST37 is well recognized MDR high risk clone reported worldwide and first time reported from raw milk of Saurashtra region of Gujarat, India. ARGs responsible for resistance to fosfomycin (fosA) were found in all 8 isolates. Other ARGs such as blaSHV, kdeA, OqxA, OqxB, dfrA1, sul1, qnrB4, aadA2 and ere(A) were also detected. High diversity of virulence factors was also identified by detection of genes encoding virulence factors related to iron uptake such as entE, fepD, entA, entB, Irp2, fepG, ybtU, ybtP, fepC, ybtA, ybtE, fepB, ybtS, fyuA, ybtQ, ybtT, ybtX, Irp1, adherence such as yagZ/ecpA, yagV/ecpE, yagX/ecpC, yagV/ecpE, ykgK/ecpR and invasion such as fimA, pla, fimC, fimH, fimB, fimE were detected in eight genomes. Mutations in murA, uhpT and glpT conferring a fosfomycin resistance were also present in genomes of 8 K. pneumoniae. IncF was the most common plasmid replicon type detected in all 8 genomes. The study reports high diversity of virulent and multidrug resistant K. pneumoniae in raw milk. Hence, genomic surveillance plans are urgently required for food borne pathogens.

Whole genome analysis of Salmonella Gallinarum strains isolated from a fowl typhoid outbreak in southern Brazil

ABSTRACT 1. Salmonella Gallinarum strains isolated from a southern Brazil fowl typhoid outbreak were subjected to phenotypic and genotypic analyses to identify genetic elements that could improve prevention and control strategies. 2. Whole-genome sequencing revealed the presence of the aac(6’)-Iaa gene, conferring aminoglycoside resistance, along with novel chromosomal point mutations, including the first detection of parE p.S451F in Salmonella Gallinarum. 3. Additionally, IncFII(S) plasmid replicons, Salmonella pathogenicity islands and 105 virulence genes associated with cell adhesion, invasion and antimicrobial peptide resistance were identified. 4. These findings shed light on the molecular mechanisms of fowl typhoid and provide crucial insights into emerging antimicrobial resistance and virulence factors.

Emergence of multidrug-resistant Providencia rettgeri clone in food-producing animals: A public health threat

The occurrence of carbapenemases encoding genes in Providencia rettgeri is a critical public health concern since this species has intrinsic resistance to several antimicrobials, including polymyxins. The identification of this multidrug-resistant (MDR) pathogen outside the hospital setting has become increasingly frequent, and raises an alert for the global health agencies, as they indicate a possible spread of such pathogens. Herein, we described three MDR P. rettgeri isolates carrying a diversity of antimicrobial resistance genes (ARGs) isolated from stool samples of swine and bovine in Brazil. Molecular analysis revealed that all isolates belonged to the same clone. The whole genome sequencing (WGS) of a representative isolate (PVR-188) was performed by MiSeq Illumina® platform, while the assembling and annotation was achieved using SPAdes and Prooka, respectively. The WGS analyses indicated the presence of ARGs that confer resistance to β-lactams (blaNDM-1, blaCTX-M-2), quinolones (qnrD1), aminoglycosides (aadA2, aadA1, aph(3′)-Via), phenicol (catB2), sulfonamides (sul1, sul2), and trimethoprim (dfrA12, dfrA1). The presence of three plasmid replicons (Col3M, IncQ1, and IncT) was detected, but no phage sequences were found. The phylogenetic analyses confirmed the genomic relationship of the PVR-188 with P. rettgeri isolates recovered from animals and humans in the USA and Malaysia. In conclusion, we report the occurrence of MDR P. rettgeri clone colonizing the gut microbiota of food-producing animals in Brazil, revealing the spread of this pathogen beyond hospital boundaries.

Molecular epidemiological investigation of Salmonella isolated from the environment, animals, foods and patients in China

Salmonella as a foodborne pathogen, has a very important impact on human health. This study characterized the serotype, plasmid replicon, MLST typing, resistance phenotype and resistance gene of 282 strains of Salmonella isolated from the environment, animals, foods, and patients in China. These strains are classified into four sources: poultry farms (39/282), animals (30/282), foods (164/282), and hospitals (49/282) their resistance to ciprofloxacin, levofloxacin and ceftazidime is gradually increasing. Among them, ST11, ST19 and ST314 are dominant ST types; Salmonella enteritidis, Salmonella typhimurium and Kentucky Salmonella enteritidis serotypes are the dominant serotypes. This study conducted a comprehensive molecular epidemiological investigation of Salmonella from the unique perspective of Onehealth. It was revealed that the mcr-1 carried by Salmonella mainly relies on the horizontal transfer of Inc I2 and Inc Q plasmids. And a novel integron variable region cassette arrangement pattern aadA2-aadA23-catB3-tet(B)-dfrA12-eptB has emerged in Salmonella strains. More noteworthy is the outbreak of blaTEM-148 subtype in Salmonella from food sources, in which the Inc HI plasmid played an important role.

Genomic characterization of third-generation cephalosporin-resistant Escherichia coli strains isolated from diseased dogs and cats: Report from Japanese Veterinary Antimicrobial Resistance Monitoring

This study investigates the genomic characteristics of canine and feline cefotaxime (CTX, a third-generation cephalosporin)-resistant Escherichia coli using the JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring System, a nationwide monitoring.In this study, whole-genome sequencing (WGS) was performed on 51 canine and 45 feline CTX-resistant E. coli isolates, with certain isolates subjected to pulsed-field gel electrophoresis with S1 nuclease for plasmid–chromosome separation.The most common blaCTX-M genes were blaCTX-M-27 (dogs: 11/51 [21.6 %]; cat: 10/45 [22.2 %]), followed by blaCTX-M-14 (dogs: 10/51 [19.6 %]; cats: 10/45 [22.2 %]), and blaCTX-M-15 (dogs: 9/51 [17.6 %]; cats: 5/45 [11.1 %]). Besides β-lactamase genes, all isolates harbored mdf(A), a multidrug efflux pump, with resistance genes for aminoglycosides, sulfonamides, trimethoprims, macrolides and tetracyclines. None of the isolates had carbapenemase genes, such as blaOXA-48, blaNDM, and blaIMP, whereas most of the isolates showed double mutations in gyrA and parC, which affected quinolone resistance. For the isolates separately analyzed for plasmid and chromosomal DNA via WGS, the majority of CTX-M genes were present on the plasmids. Some plasmids also harbored the same combination of resistance genes and plasmid replicon type, although they differed from isolates derived from different areas of Japan. The predominant plasmids were blaCTX-M-27,aadA5, aph(6)-Id, aph(3”)-Ib, sul1, sul2, tet(A), dfrA17, and mph(A) on IncF. The predominant combination of ST131, O25:H4, and B2 isolates comprised the largest cluster in the minimum spanning tree and the ST131 E. coli harboring blaCTX-M-27 from human in Japan was closely related to these isolates.The results indicated that CTX-resistant canine and feline E. coli harbored multiple plasmids carrying the same combination of resistance genes and emphasizes the need to prevent the spread. Data AvailabilityAll raw short-read sequence data have been deposited in the DNA Data Bank of Japan. (DRR Run No, DRR335726–335821).

Characterization of methicillin resistant Staphylococcus Aureus in municipal wastewater in Finland

Wastewater-based surveillance (WBS) of multidrug-resistant bacteria could complement clinical data, serving as a population-level early warning tool. This study evaluated WBS as a pandemic preparedness tool, by selectively isolating and culturing methicillin-resistant Staphylococcus aureus (MRSA) with CHROMagar MRSA. Some 24-h composite wastewater samples (n = 80) were collected from ten treatment plants across Finland between February 2021 and January 2022. MRSA prevalence in wastewater samples was 27.5% (n = 22/80), showing seasonal and temporal variations. Phenotypic antimicrobial susceptibility testing (AST) with microdilution showed that over 80% of isolates were drug-resistant to clindamycin, sulfamethoxazole/trimethoprim, tetracycline, fusidic acid, and erythromycin. Four isolates (18.2%) were vancomycin-resistant. WGS revealed that 31.8% (n = 7) of the isolates belonged to the ST8-t008 and ST6-t304 spa types, respectively. In addition, two spa types (t011 and t034) belong to the CC398 complex. The mecA gene was found in all isolates (n = 22) and three tetracycline resistance determinants (tet38, tetK, and tetM) were detected with tet38 being the most abundant (81.8%, n = 18/22). Three isolates harboured the plasmid-mediated sat4 gene that confers resistance to Streptothricin. In addition, resistance determinants to macrolide antibiotics (mph (C)/msr (A) and fosfomycin (fosB) were detected in the seven isolates that belonged to spa type t008. All isolates except one harboured the SCCmec_type_IVa(2B). Six ST8 isolates harboured the LukS/F-PV genes encoding the Panton–Valentine leukocidin (PVL) and were also positive for the Arginine Catabolic Mobile Element (ACME), suggesting they belong to the USA300 clone. The Inc18 plasmid was the most abundant as it was detected in 72.7% (n = 16/22) of the isolates. Other plasmid replicons detected were the rep_trans and repA_N which were detected in 45.4% (n = 10/22) and 40.9% (n = 9/22) of the isolates respectively. Ten isolates harboured at least three plasmid replicons and no plasmid replicons were detected in four isolates (ST6/t304). The cgMLST revealed that some isolates aggregated into two genomically indistinguishable clusters: ST6/t304 belonging to cluster type CT12405 (≤20 allelic differences) and ST8/t008 belonging to cluster type CT1925 (<8 allelic differences). Overall, we found a high genotypic concordance with the national clinical bacterial resistance data. Our study demonstrates the sensitivity of culture-based wastewater surveillance for MRSA using clinical media following pre-enrichment, reliably predicting pathogen occurrence at the population level.

Klebsiella pneumoniae species complex: From wastewater to the environment

Klebsiella pneumoniae plays a significant role in nosocomial infections and spreading antibiotic resistance, and therefore forms a major threat to public health. In this study, we investigated the role of the wastewater pathway in the spread of pathogenic bacteria and more specifically, in the spread of antibiotic resistant Klebsiella pneumoniae subspecies. Whole-genome sequencing was performed of 185 K. pneumoniae isolates collected from hospital, nursing home, and community wastewater, the receiving wastewater treatment plant (WWTP), and clinical isolates from the investigated hospital. K. pneumoniae isolates from different sources were not genetically related, except for WWTP influent (46.5%) and effluent (62.5%), revealing survival of bacteria from wastewater treatment. The content of antibiotic resistance (ARGs), virulence, and plasmid replicon genes differed between K. pneumoniae subspecies and their origin. While chromosomal bla genes were specific for each K. pneumoniae subspecies, bla genes predicted in plasmid contigs were found in several K. pneumoniae subspecies, implying possible gene transfer between subspecies. Transferable ARGs were most abundant in patients and hospital isolates (70%), but the average number of plasmid replicon genes per isolate was similar across all sources, showing plasmid content being more relevant than plasmid quantity. Most patient (90%) and hospital wastewater (34%) isolates were K. pneumoniae subsp. pneumoniae, and the yersiniabactin cluster genes ybt, fyuA, and irp12 were only found in this subspecies, as were the IncFII(pECLA), IncHI2A, and IncHI2 plasmid replicon genes, suggesting the clinical origin of these type of plasmids.

Genetic Characteristics of Multidrug-Resistant Salmonella Isolated from Poultry Meat in South Korea.

Given the lack of genetic characterization data for multidrug-resistant (MDR) Salmonella in South Korean poultry, we analyzed 53 MDR Salmonella strains from 1232 poultry meat samples (723 chicken, 509 duck) using whole-genome sequencing. Five serotypes were identified: S. Infantis (30/53, 56.6%), S. Enteritidis (11/53, 20.8%), S. Virchow (9/53, 17.0%), S. Agona (2/53, 3.8%), and S. Indiana (1/53, 1.9%). Sequence types (STs) included ST32, ST11, ST16, ST13, and ST17, with three major clusters, each having two subclusters. Eight core genome sequence types (cgSTs) were identified: 225993, 2268, 58360, 150996, 232041, 96964, 117577, and 267045. Salmonella Infantis and S. Enteritidis had two (117577, 267045) and three (225993, 2268, 58360) cgSTs, respectively, whereas S. Virchow showed allelic differences in identical cgSTs. The S. Enteritidis subcluster was classified as chicken or duck. Twenty-eight antimicrobial resistance genes (ARGs), 10 plasmid replicons, 11 Salmonella pathogenicity islands (SPIs), and 230 virulence genes were identified, showing distinct profiles by cluster and subcluster. Salmonella Infantis, the primary MDR Salmonella, carried the IncFIB (pN55391) plasmid, 10-11 ARGs, nine SPIs, and approximately 163 virulence genes. Three major MDR Salmonella serotypes (S. Infantis, S. Enteritidis, and S. Virchow) had specific genetic profiles that can inform epidemiological surveillance.

Investigating the virulence-associated genes and antimicrobial resistance of Escherichia fergusonii Isolated from diseased ostrich chicks

This study investigates the presence of virulence-associated genes and antimicrobial resistance (AMR) in Escherichia fergusonii isolates obtained from ostrich chicks. A total of 287 isolates were recovered from 106 fecal samples from ostrich chicks suffering from diarrhea and subjected to molecular identification and biochemical characterization. E. fergusonii was detected in 10 samples (9.4 %) using two PCR-detection protocols. Notably, the isolates lacked various virulence genes commonly associated with pathogenic E. coli including elt, est, stx, eae, ehly, cdt, iss, iutA, iroN, hlyA, ompT, except for one isolate harboring the astA gene. Antimicrobial susceptibility testing revealed that all isolates were susceptible to ciprofloxacin, while high resistance was observed against amoxicillin clavulanate (AMC), trimethoprim-sulfamethoxazole (SXT), and doxycycline (D). Moreover, eight isolates displayed multidrug resistance (MDR) and four exhibited resistance to 9–11 antimicrobials. The most frequent resistance gene was sul2, which was present in all isolates; the other resistance genes detected consisted of int1 (4/10), int2 (3/10), blaCMY (2/10), and qnrS, blaTEM, blaCMY, blaCTX-M, and flo each were detected only in one E. fergusonii Isolate. Plasmid replicon typing identified the presence of I1 (7/10), N (5/10), and Y (1/10). This study provides valuable insights into the virulence and antimicrobial resistance of E. fergusonii isolates from ostrich chicks, highlighting the complexity of antimicrobial resistance mechanisms exhibited by these bacteria. Further research is essential to understand the transmission dynamics and clinical implications of these findings in veterinary and public health settings.

A genome-based investigation of the Priestia species isolated from anthrax endemic regions in Kruger National Park

Priestia is a genus that was renamed from the genus Bacillus based on the conserved signature indels (CSIs) in protein sequences that separate Priestia species from Bacillus, with the latter only including species closely related to B. subtilis and B. cereus. Diagnosis of anthrax, a zoonotic disease, is implicated by tripartite anthrax virulence genes (lef, pagA, and cya) and poly-γ-D-glutamic acid capsular genes cap-ABCDE of Bacillus anthracis. Due to the amplification of anthrax virulence genes in Priestia isolates, the search for homologous anthrax virulence genes within the Priestia genomes (n = 9) isolated from animal blood smears was embarked upon through whole genome sequencing. In silico taxonomic identification of the isolates was conducted using genome taxonomy database (GTDB), average nucleotide identity (ANI), and multi-locus sequence typing (MLST), which identified the genomes as P. aryabhattai (n = 5), P. endophytica (n = 2) and P. megaterium (n = 2). A pan-genome analysis was further conducted on the Priestia genomes, including the screening of virulence, antibiotic resistance genes and mobile genetic elements on the sequenced genomes. The oligoribonuclease NrnB protein sequences showed that Priestia spp. possess a unique CSI that is absent in other Bacillus species. Furthermore, the CSI in P. endophytica is unique from other Priestia spp. Pan-genomic analysis indicates that P. endophytica clusters separately from P. aryabhattai and P. megaterium. In silico BLASTn genome analysis using the SYBR primers, Taqman probes and primers that target the chromosomal marker (Ba-1), protective antigen (pagA), and lethal factor (lef) on B. anthracis, showed partial binding to Priestia regions encoding for hypothetical proteins, pyridoxine biosynthesis, hydrolase, and inhibitory proteins. The antibiotic resistance genes (ARG) profile of Priestia spp. showed that the genomes contained no more than two ARGs. This included genes conferring resistance to rifamycin and fosfomycin on P. endophytica, as well as clindamycin on P. aryabhattai and P. megaterium. Priestia genomes lacked B. anthracis plasmids and consisted of plasmid replicon types with unknown functions. Furthermore, the amplification of Priestia strains may result in false positives when qPCR is used to detect the virulence genes of B. anthracis in soil, blood smears, and/or environmental samples.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015–2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6′)-Ib-cr, aph(3′)-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.