Multilocus Sequence Typing Research Articles

Multi-locus sequence typing of Candida tropicalis among Candiduria shows an outbreak in azole-susceptible isolates and clonal cluster enriched in azole-resistant isolates.

The increasing detection rate of C.tropicalis and its azole resistance have made clinical treatment difficult. The presence of candiduria seems to correlate with invasive candida infection, especially for patients admitted to ICUs. However, the prevalence and antifungal resistance of C.tropicalis isolates in urine samples has not been well studied. To retrospectively investigate the clinical features, antifungal resistance, and genetic relatedness of C.tropicalis isolates from urine samples. A total of 107 clinical C.tropicalis isolates were retrospectively studied, including phenotypes of isolates and characteristics of patients. The genetic profiles of 107 isolates were genotyped using multi-locus sequence typing (MLST). Phylogenetic analysis was inferred using unweighted pair group method with arithmetic averages. MLST clonal clusters (CCs) were analysed by goeBURST. Of the 107 isolates, 27.1% were resistant to fluconazole, and there was a notable increasing trend of fluconazole resistance from 16.1% in 2019 to 40.0% in 2021. Forty-seven diploid sequence types (DSTs) were assigned to ten major CCs. CC1 was the predominant fluconazole-susceptible group; 24 isolates from CC1 belonged to DST333, an outbreak clone in NICU ward. The azole-resistant CC4 contained 19 isolates, accounting for 65.5% of the azole-resistant isolates in this study. CC4 belongs to a prevalent FNS CC1 globally, of which the putative founder genotype was DST225. This study revealed an outbreak of azole-susceptible C.tropicalis isolates in urine specimens and a high azole resistance rate of C. tropicalis in candiduria, and the MLST type showed clonal aggregation in azole-resistant isolates from urine samples.

Non-carbapenem-producing carbapenem-resistant Pseudomonas aeruginosa in children: Risk factors, molecular epidemiology, and resistance mechanism.

The investigation into risk factors, molecular epidemiology, and resistance mechanisms of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in pediatric populations in China is currently inadequate. To assess epidemiology, molecular characteristics, and resistance mechanisms, virulence-associated genes were analyzed, alongside multi locus sequence typing (MLST), PCR, and qRT-PCR. Multivariate analysis identified prolonged hospitalization (OR: 1.026; 95 % CI: 1.004-1.049; P = 0.023) and increased exposure to enzyme inhibitor complex preparations (OR: 3.165; 95 % confidence interval [CI]: 1.113-8.999; P = 0.031) as independent risk factors for CRPA healthcare-associated infections (HAIs). Mortality rates were significantly higher in the HAI group compared to the non-HAI group (19.1 % vs. 6.0 %, P = 0.021). Analysis of virulence-associated gene combinations revealed 10 and 15 distinct profiles among HAI and non-HAI isolates, respectively, characterized by exoS-/exoU+ or exoS+ /exoU- genotypes, with no isolates exhibiting both exoS+ and exoU+ genotypes concurrently. Infections predominantly correlated with CC244, with a significantly greater occurrence in the HAI group (72.1 % vs. 46.3 %, P = 0.002). Antimicrobial susceptibility testing identified that both CC244 + and HAI isolates demonstrated elevated resistance across all tested antibiotics. Furthermore, low oprD expression was observed in 77.9 % of HAI isolates and 67.2 % of non-HAI isolates, while increased ampC production and mexB gene overexpression were infrequently detected (all P > 0.05). Prolonged hospital stays and an increased exposure to enzyme-inhibitor complex therapies were identified as independent risk factors for CRPA HAIs. CRPA demonstrated considerable genetic diversity, with STs predominantly represented by CC244, and virulence-associated genes have spread. The primary mechanism driving carbapenem resistance involved the downregulation of outer membrane porin protein oprD, accompanied by oprD mutation inactivation.

Whole genome sequencing characterization and comparative genome analysis of Acinetobacter baumannii JJAB01: A comprehensive insights on antimicrobial resistance and virulence genotype.

The emergence of antibiotic resistance has significantly elevated the threat posed by Acinetobacter baumannii as an opportunistic pathogen. A.baumannii, a notorious bacterium, poses a serious threat to health care, leading to severe nosocomial infections, particularly in immunocompromised individuals. Whole-Genome Sequencing studies are efficient in providing accurate genetic information, aiding in detecting outbreaks, surveillance of resistance, and controlling infection transmission. In this study, we investigated the whole genome of a clinical isolate A. baumannii JJAB01 which sourced from a urine sample of an Intensive Care Unit (ICU) patient. This strain showed resistance to 24 available antibiotics, signifying Extremely Drug Resistant (XDR) and high potential for pathogenicity. Whole Genome Sequencing was performed using Illumina, and the raw reads were evaluated using the FastQC tool. Genome assembly and annotation were performed with Unicycler and the RAST server. The JJAB01 genome is 4.07Mb with a GC content of 38.9%. A total of 51 and 31 virulence factors and antimicrobial-resistant (AMR) genes were predicted using the VFDB and CARD databases. Comparative genome studies were carried out on virulence factors, resistance genes, prophages, and Multi-Locus Sequence Typing (MLST) across twelve closely related A. baumannii genomes, including JJAB01, X4-584, X4-705, 2023CK-00423, 2023CK-00890, 2023CK-00127, 2022CK-00066, B20AB01, B20AB10, F20AB03, G20AB08, and X4-65. These computational investigations in this study emphasis the multidimensional nature of the ICU strain JJAB01 and its genetic similarity to other strains, thereby enhancing our understanding of drug resistance and the pathogenicity associated with A. baumannii infections.

Evaluation of nationwide analysis surveillance for methicillin-resistant Staphylococcus aureus within Genomic Medicine Sweden.

Background. National epidemiological investigations of microbial infections greatly benefit from the increased information gained by whole-genome sequencing (WGS) in combination with standardized approaches for data sharing and analysis.Aim. To evaluate the quality and accuracy of WGS data generated by different laboratories but analysed by joint pipelines to reach a national surveillance approach.Methods. A national methicillin-resistant Staphylococcus aureus (MRSA) collection of 20 strains was distributed to nine participating laboratories that performed in-house procedures for WGS. Raw data were shared and analysed by three pipelines: 1928 Diagnostics, JASEN (GMS pipeline) and CLC-Genomics Workbench. The outcomes were compared according to quality, correct strain identification and genetic distances.Results. One isolate contained intraspecies contamination and was excluded from further analysis. The mean sequencing depth varied between sites and technologies. However, all analysis methods identified 12 strains that belonged to one of five outbreak clusters. The cut-off definition was set to <10 allele differences for core genome multilocus sequence typing (cgMLST) and <20 genetic differences for SNP analysis in a pairwise comparison.Conclusions. MRSA isolates, which are whole genome sequenced by different laboratories and analysed using the same bioinformatic pipelines, yielded comparable results for outbreak clustering for both cgMLST and SNP, using the 1928 analysis pipeline. In this study, JASEN was best suited to analyse Illumina data and CLC to analyse within respective technology. In the future, real-time sharing of data and harmonized analysis within the Genomic Medicine Sweden consortium will further facilitate investigations of outbreaks and transmission routes.

Molecular Characterization of Colistin- and Carbapenem-Resistant Klebsiella pneumoniae: mgrB Mutations and Clonal Diversity in Pediatric Intensive Care Isolates.

Colistin- and carbapenem-resistant Klebsiella pneumoniae (ColR CrKp) cause important health problems in pediatric intensive care units (PICUs) due to its ability to harbor multiple resistance genes and spread of high-risk clones. In this study, molecular epidemiological characteristics, transferable resistance genes, and mgrB alterations of ColR CrKp isolated from PICU were investigated. Isolates were identified by MALDI-TOF MS, and antimicrobial susceptibility tests were performed using disk diffusion method, gradient strip test, and broth microdilution method. Extended spectrum beta-lactamase, AmpC beta-lactamase, carbapenemase, 16S rRNA methyltransferase, plasmid-mediated quinolone resistance, and mcr-1 to -5 genes were investigated by polymerase chain reaction. Sanger sequencing was performed to obtain blaOXA-48-like and mgrB sequences. Pulsed-field gel electrophoresis and multilocus sequence typing were used to determine the clonal spread of the isolates. Ten ColR CrKp harboring blaOXA-48 (70%), blaOXA-232 (20%), blaCTX-M (90%), armA (20%), qnrB (20%), and qnrS (50%) were identified. No mcr genes were found, whereas mgrB mutations through modifications (A7T, C88T, and A121G) and insertion of an IS-1-like insertion sequence were determined. Isolates belonged to ST 14, ST 37, ST 101, ST 147, ST 661, ST 985, and ST 2096. It is crucial to determine the antimicrobial resistance properties and the clonal spread of the isolates to guide the treatment decisions, implement effective infection control measures, and develop novel antimicrobial strategies.

Whole-genome evaluation and prophages characterization associated with genome of carbapenem-resistant Acinetobacter baumannii UOL-KIMZ-24-2.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is an emerging threat to healthcare settings in many countries, principally in South Asia. The current study was aimed to identify, evaluate whole-genome and characterize the prophages in genome of CRAB strain, recovered from patients of Lahore General Hospital, Lahore. More than 200 samples were collected and identified by morphological and biochemical tests. These strains were also subjected to a comprehensive antimicrobial susceptibility evaluation using Kirby-Bauer method and further confirmed as CRAB strains by exploring blaOXA-51. In addition, the whole-genome evaluation of a Acinetobacter baumannii UOL-KIMZ-24-2 was carried out using various Bioinformatics tools. A total of 150 strains of A. baumannii were recovered and identified in the current study. Among them, 49% strains were found resistant to carbapenem. The blaOXA-51 was found prevalent in the genome of A. baumannii recovered from medical ICU (38%). In addition, the UOL-KIMZ-24-2 genome analysis based on multilocus sequence typing (MLST) highlighted that UOL-KIMZ-24-2 belonged to ST2 (Pasteur scheme) sequence type. A total of 29 antimicrobial resistance (AMR) genes were present, importantly, blaOXA-66, blaOXA-23 and blaOXA-25. The mobile genetic elements (MGEs) were identified as transposases and belonged to four classes e.g. IS15d1, ISAba24, ISEc29, and ISEc35. A total of 14 virulence factors encoded by 58 different genes were detected in UOL-KIMZ-24-2. In addition, the phage sequences were identified in genome of UOL-KIMZ-24-2, divided into 3 regions. In conclusion, UOL-KIMZ-24-2 contained a mixture of AMR genes, MGEs. prophages sequences and virulence genes.

Evolution of ceftazidime/avibactam resistance and plasmid dynamics in OXA-48-producing Klebsiella spp. during long-term patient colonization.

To prospectively monitor the evolution of the resistome of OXA-48-producing Klebsiella species in a patient with long-term colonization, with a particular focus into the plasmid dynamics and the evolution of ceftazidime/avibactam resistance. All OXA-48-producing Klebsiella spp. isolates from a single patient admitted to a hospital during seven months were prospectively collected. MICs were determined through reference broth microdilution. Multilocus sequence types, SNPs analysis, resistance mechanisms, genetic context of β-lactamases and plasmid dynamics were determined by WGS and bioinformatic analysis. The impact of β-lactamase variant obtained after ceftazidime/avibactam exposure was determined via cloning experiments. Four isolates, two before (one OXA-48-producing K. pneumoniae and one CTX-M-15-like-producing K. pneumoniae) and two after treatment with ceftazidime/avibactam (one OXA-48- and CTX-M-15-like-producing K. pneumoniae and one OXA-48- and CTX-M-15-like-producing K. aerogenes) were collected. The plasmid dynamics analysis demonstrated that the IncL and IncFIIK plasmids, in which blaOXA-48 and blaCTX-M-15-like genes were located, respectively, exhibited a high degree of conservation indicating a potential for both intra- and interspecies transmission. The K. pneumoniae isolate obtained after treatment, which differed from the previous isolate by just six SNPs, exhibited resistance to ceftazidime/avibactam through P167S substitution in CTX-M-15, which is now designated CTX-M-273. Cloning experiments demonstrated enhanced resistance to ceftazidime/avibactam. The transfer of plasmid-borne β-lactamase resistance genes between intra- and interspecies bacterial populations enables the rapid diversification of the bacterial genome. The emergence of ceftazidime/avibactam resistance through the modification of CTX-M-enzymes represents a mechanism by which OXA-48-producing Enterobacterales may evolve toward ceftazidime/avibactam resistance in vivo.

Molecular Characterization of Whole Genome Sequencing of Salmonella spp. in Shapingba District, Chongqing, China, 2016-2023.

In recent years, Salmonella infection is a major global public health concern, particularly in food safety. This study analyzed the genomes of 102 Salmonella strains isolated between 2016 and 2023 from food, foodborne disease patients, and food poisoning incidents, focusing on their molecular characteristics, antibiotic resistance genes (ARGs), and virulence genes. S. enterica serovar Enteritidis (37.3%) and S. enterica serovar Typhimurium (21.6%, including its monophasic variant 1,4,[5],12:i:-) were the main strains among 22 serotypes. Multilocus sequence typing revealed 23 sequence types (STs), with ST11, ST19, and ST34 as the most prevalent. All strains carried at least 24 ARGs. Detection rates for aac(6')-Iy, blaTEM-1, and sul2 ranged from 44.1% to 63.7%, mainly in S. Enteritidis and S. Typhimurium. Rates for qnrS1, sul1, and aadA were 12.8% to 16.7%, while mcr-1 appeared in one ST34 S. Typhimurium strain. All strains contained at least 98 virulence genes. The genes pefABCD, mig-5, and spvBCD were in 48.0% of strains, while rck was found in 36.3%, mainly linked to S. Enteritidis and S. Typhimurium. The tssM gene was found in 37.3% of the strains, exclusively in S. Enteritidis. Core genome single nucleotide polymorphisms (cgSNPs) analysis grouped the strains into nine clusters, with 75.5% belonging to three major groups. Food poisoning event 1 was correlated with cluster 3, while events 2 and 3 were linked to cluster 1. Across events, SNP differences among strains were ≤6. Strains with SNP differences ≤10 were also found in other clusters. This method is promising for tracking sporadic cases and identifying potential foodborne safety incidents. Salmonella strains in the region exhibit substantial genetic diversity, demonstrating the efficacy of cgSNPs analysis for source tracking. Ongoing surveillance is essential given the prevalence of ARGs and virulence genes. This study provides a data foundation for local Salmonella epidemiology.

Genomic detection of Panton-Valentine Leucocidins encoding genes, virulence factors and distribution of antiseptic resistance determinants among Methicillin-resistant S. aureus isolates from patients attending regional referral hospitals in Tanzania

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a formidable public scourge causing worldwide mild to severe life-threatening infections. The ability of this strain to swiftly spread, evolve, and acquire resistance genes and virulence factors such as pvl genes has further rendered this strain difficult to treat. Of concern, is a recently recognized ability to resist antiseptic/disinfectant agents used as an essential part of treatment and infection control practices. This study aimed at detecting the presence of pvl genes and determining the distribution of antiseptic resistance genes in Methicillin-resistant Staphylococcus aureus isolates through whole genome sequencing technology.Materials and methodsA descriptive cross-sectional study was conducted across six regional referral hospitals-Dodoma, Songea, Kitete-Kigoma, Morogoro, and Tabora on the mainland, and Mnazi Mmoja from Zanzibar islands counterparts using the archived isolates of Staphylococcus aureus bacteria. The isolates were collected from Inpatients and Outpatients who attended these hospitals from January 2020 to Dec 2021. Bacterial analysis was carried out using classical microbiological techniques and whole genome sequencing (WGS) using the Illumina Nextseq 550 sequencer platform. Several bioinformatic tools were used, KmerFinder 3.2 was used for species identification, MLST 2.0 tool was used for Multilocus Sequence Typing and SCCmecFinder 1.2 was used for SCCmec typing. Virulence genes were detected using virulenceFinder 2.0, while resistance genes were detected by ResFinder 4.1, and phylogenetic relatedness was determined by CSI Phylogeny 1.4 tools.ResultsOut of the 80 MRSA isolates analyzed, 11 (14%) were found to harbor LukS-PV and LukF-PV, pvl-encoding genes in their genome; therefore pvl-positive MRSA. The majority (82%) of the MRSA isolates bearing pvl genes were also found to exhibit the antiseptic/disinfectant genes in their genome. Moreover, all (80) sequenced MRSA isolates were found to harbor SCCmec type IV subtype 2B&5. The isolates exhibited 4 different sequence types, ST8, ST88, ST789 and ST121. Notably, the predominant sequence type among the isolates was ST8 72 (90%).ConclusionThe notably high rate of antiseptic resistance particularly in the Methicillin-resistant S. aureus strains poses a significant challenge to infection control measures. The fact that some of these virulent strains harbor the LukS-PV and LukF-PV, the pvl encoding genes, highlight the importance of developing effective interventions to combat the spreading of these pathogenic bacterial strains. Certainly, strengthening antimicrobial resistance surveillance and stewardship will ultimately reduce the selection pressure, improve the patient’s treatment outcome and public health in Tanzania.

Molecular Epidemiology Revealed Distinct Patterns Among Multidrug Resistant Clinical Acinetobacter baumannii Strains in Different Periods in the Main Hospital in Molise Region, Central Italy

Background: Acinetobacter baumannii is a major cause of nosocomial infections in critically ill patients, and strains are frequently multidrug resistant (MDR). This study aimed to characterize 45 clinical A. baumannii isolates collected in different periods in the main hospital in the Molise Region, central Italy. Methods: Antimicrobial susceptibility was evaluated using an automated system, and PCRs were performed to detect resistance-associated genes. Pulsed-field gel electrophoresis (PFGE) was carried out with AscI and ApaI, and Multi-locus sequence typing (MLST) was performed according to the Oxford scheme. Results: All isolates exhibited MDR profiles, showing total susceptibility towards colistin. All strains harbored the blaOXA-23, blaOXA-51, and blaAmpC genes, as well as adeB, adeJ, adeG, abeS, and soxR. Dendrogram with AscI and ApaI revealed eleven and three clusters, respectively, and twenty-three and eighteen pulsotypes (Simpson’s index 0.96 and 0.93), and isolates from different periods were clearly distinguished. MLST revealed five sequence types, which varied depending on the isolation period, and ST1720 and ST369 were prevalent, followed by ST281, ST218, and ST513. Conclusions: Molecular characterization enables the identification of distinct patterns of MDR A. baumannii over time, underscoring its usefulness for improving epidemiological surveillance and combating antimicrobial resistance. This study provides previously unavailable information regarding A. baumannii circulating in the examined setting.

Antimicrobial Susceptibility and Genomic Characterization of Vibrio cholerae Non-O1/non-O139 Isolated from Clinical and Environmental Samples in Jiaxing City, China.

Non-O1/non-O139 (NOVC) strains inhabit aquatic environments and sporadically induce human illnesses. This study involved the virulence and antimicrobial genetic characterization of 176 NOVC strains, comprising 25 from clinical samples and 151 from environmental sources, collected between 2021 and 2023. The antimicrobial susceptibility of the examined NOVC population was predominantly high, exhibiting only poor susceptibility to colistin, with 89.2% resistance. The examination of virulence genes revealed that the majority of strains were positive for glucose metabolism (als gene) (169/176, 96.0%). Through multilocus sequence typing, the 176 NOVC strains were categorised into 121 sequence types, 79 of which were novel. NOVC strains demonstrate significant genetic variability and frequently engage in recombination. This work offers genetic characterization of the pathogenicity and antimicrobial resistance of a NOVC community. Our findings offer insights that may aid in the development of preventative and treatment methods for this pathogen.

First Detection and Genomic Characterization of Linezolid-Resistant Enterococcus faecalis Clinical Isolates in Bulgaria.

Linezolid is an oxazolidinone antibiotic and is considered a last-resort treatment option for serious infections caused by problematic Gram-positive pathogens, including vancomycin-resistant enterococci. The present study aimed to explore the linezolid resistance mechanisms and genomic characteristics of two vancomycin-susceptible Enterococcus faecalis isolates from Bulgaria. The strains designated Efs2503-bg (inpatient from Pleven) and Efs966-bg (outpatient from Varna) were recovered from wounds in 2018 and 2023, respectively. Antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, and phylogenomic analysis based on 332 linezolid-resistant E. faecalis genomes were performed. Efs2503-bg was high-level resistant to linezolid (MIC > 256 mg/L) and displayed the G2576T mutation affecting three of the four 23S rDNA loci. Efs966-bg (MIC = 8 mg/L) carried a plasmid-located optrA determinant surrounded by fexA and ermA. No mutations in the genes encoding for ribosomal proteins L3, L4, and L22 were detected. The isolates belonged to the sequence types ST6 (Efs2503-bg) and ST1102 (Efs966-bg). Phylogenomic analysis revealed that Efs2503-bg and Efs966-bg are genetically distinct, with a difference of 12,051 single-nucleotide polymorphisms. To our knowledge, this is the first report of linezolid-resistant enterococci in Bulgaria. Although the global incidence of linezolid-resistant enterococci is still low, their emergence is alarming and poses a growing clinical threat to public health.

Antimicrobial resistance profiles and molecular epidemiology of Klebsiella pneumoniae isolates from Scottish bovine mastitis cases.

Klebsiella pneumoniae are opportunistic pathogens which can cause mastitis in dairy cattle. K. pneumoniae mastitis often has a poor cure rate and can lead to the development of chronic infection, which has an impact on both health and production. However, there are few studies which aim to fully characterize K. pneumoniae by whole-genome sequencing from bovine mastitis cases. Here, K. pneumoniae isolates associated with mastitis in dairy cattle were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing. Furthermore, whole-genome sequence data were used for phylogenetic analyses and both virulence and antimicrobial resistance (AMR) prediction, in parallel with phenotypic AMR testing. Forty-two isolates identified as K. pneumoniae were subject to whole-genome sequencing, with 31 multi-locus sequence types being observed, suggesting the source of these isolates was likely environmental. Isolates were examined for key virulence determinants encoding acquired siderophores, colibactin, and hypermucoidy. The majority of these were absent, except for ybST (encoding yersiniabactin) which was present in six isolates. Across the dataset, there were notable levels of phenotypic AMR against streptomycin (26.2%) and tetracycline (19%), and intermediate susceptibility to cephalexin (26.2%) and neomycin (21.4%). Of importance was the detection of two ESBL-producing isolates, which demonstrated multi-drug resistance to amoxicillin-clavulanic acid, streptomycin, tetracycline, cefotaxime, cephalexin, and cefquinome.

Characterization of Streptococcus pyogenes Strains from Tonsillopharyngitis and Scarlet Fever Resurgence, 2023-FIRST Detection of M1UK in Bulgaria.

Recently a resurgence of Streptococcus pyogenes infections has arisen, with concerns around the highly virulent M1UK lineage. Our aim was to characterize S. pyogenes, the immune responses it causes, and to determine the presence of the M1UK lineage in Sofia, Bulgaria. In our study, the infections were confirmed by culture testing or rapid antigen test. Identification was performed by MALDI-TOF and was followed up by antibiotic susceptibility testing (EUCAST). Virulence factors were identified using multiplex PCR and whole genome sequencing (WGS). Immune responses were measured through detection of serum complement levels, lymphocyte subsets, and cytokine profiling. Out of 82 children, 38 had scarlet fever and the rest had streptococcal pharyngitis. Strains were susceptible to penicillin (β-lactams), macrolides, clindamycin, tetracyclines, co-trimoxazole, fluoroquinolones, and linezolid. Superantigen profiles were identified: SpeA + SpeJ (45%), SpeC, and SpeI + SpeH (27.5% each). A novel Multilocus sequence typing (MLST) haplotype in the mutS gene (d90b) was found in four strains. The M1UK lineage was detected for the first time in Bulgaria. We observed an increase in complement fractions C3 and C4 and a decrease in T lymphocytes. A significant increase in the levels of IFN-γ, IL-6, and IL-10 with corresponding reduction in IL-17A were revealed. In conclusion, the studied S. pyogenes strains were characterized by their susceptibility to antibiotics and the predominance of SpeA superantigen; for the first time in Bulgaria the presence of M1UK and a novel SNP variation in the mutS gene (d90b) were found. A mixed pattern of pro- and anti-inflammatory immune responses in patients was observed.

Whole-genome sequencing-based characterization of Salmonella enterica Serovar Enteritidis and Kentucky isolated from laying hens in northwest of Iran, 2022–2023

BackgroundThe transmission of Salmonella spp. to human through the consumption of contaminated food products of animal origin, mainly poultry is a significant global public health concern. The emerging multidrug resistant (MDR) clones of non-typhoidal Salmonella (NTS) serovars, have spread rapidly worldwide both in humans and in the food chain. In this study NTS strains were isolated from diseased laying hens in Iran and were further studied by whole-genome sequencing (WGS) to investigate the prevalent serovars, multilocus sequence types, antimicrobial resistance and virulence genes.ResultsOut of eight isolated Salmonella spp. six were identified as S. Enteritidis serovar ST11 (n = 5) or ST5824 (n = 1), and two isolates were recognized as S. Kentucky serotype ST198 lineages. The aminoglycoside resistance gene aac(6′)-Iaa was the most frequently detected gene being present in all serovars, but it did not confer phenotypic resistance to corresponding agents (tobramycin and amikacin). All S. Enteritidis isolates carried a single GyrA D87N/Y substitution. Other identified antimicrobial resistance genes (ARGs) including tetA, floR, sul1, dfrA1, aph(3′)-Ia and double gyrA and parC mutations conferring high-level ciprofloxacin resistance (CIPR) (MIC ≥ 16mg/L) were only found in S. Kentucky isolates. The comparison of phenotypic and genotypic antimicrobial resistance (AMR) profiles revealed inconsistent results for some antibiotics. A total of 11 different Salmonella Pathogenicity Islands (SPIs) including SPIs-1, to 5, 9, 10, 13, 14, C63PI, CS54 and several virulence genes related to type III secretion system, adhesins, iron and magnesium uptake, serum and antimicrobial peptide resistance were detected among the isolates.ConclusionsOur study reports emergence of a highly MDR- CIPRS. Kentucky ST198 clone form poultry associated sources in Iran. The presence of numerous virulence determinants, SPIs and ARGs in the examined NTS isolates poses a significant risk for food safety. The inconsistencies between the genotypic and phenotypic AMR profiles indicate that WGS data alone may not be always sufficient for guiding therapeutic strategies.

Global insights into the genome dynamics of Clostridioides difficile associated with antimicrobial resistance, virulence, and genomic adaptations among clonal lineages

BackgroundClostridioides difficile is a significant cause of healthcare-associated infections, with rising antimicrobial resistance complicating treatment. This study offers a genomic analysis of C. difficile, focusing on sequence types (STs), global distribution, antibiotic resistance genes, and virulence factors in its chromosomal and plasmid DNA.MethodsA total of 19,711 C. difficile genomes were retrieved from GenBank. Prokka was used for genome annotation, and multi-locus sequence typing (MLST) identified STs. Pan-genome analysis with Roary identified core and accessory genes. Antibiotic resistance genes, virulence factors, and toxins were detected using the CARD and VFDB databases, and the ABRicate software. Statistical analyses and visualizations were performed in R.ResultsAmong 366 identified STs, ST1 (1,326 isolates), ST2 (1,141), ST11 (893), and ST42 (763) were predominant. Trends of genome streamlining included reductions in chromosomal length, gene count, protein-coding genes, and pseudogenes. Common antibiotic resistance genes—cdeA (99.46%), cplR (99.63%), and nimB (99.67%)—were nearly ubiquitous. Rare resistance genes like blaCTX-M-2, cfxA3, and blaZ appeared in only 0.005% of genomes. Vancomycin susceptibility-reducing vanG cluster genes were detected at low frequencies. Virulence factors showed variability, with highly prevalent genes such as zmp1 (99.62%), groEL (99.60%), and rpoB/rpoB2 (99.60%). Moderately distributed genes included cwp66 (54.61%) and slpA (79.02%). Toxin genes tcdE (91.26%), tcdC (89.67%), and tcdB (89.06%) were widespread, while binary toxin genes cdtA (26.19%) and cdtB (26.26%) were less common. Toxin gene prevalence, particularly tcdA and tcdB, showed a gradual decline over time, with sharper reductions for cdtA and cdtB. Gene presence patterns (GPP-1) for resistance, virulence, and toxin genes were primarily linked to ST2, ST42, and ST8.ConclusionThis study highlights C. difficile’s adaptability and genetic diversity. The decline in toxin genes reflects fewer toxigenic isolates, but the bacterium’s increasing preserved resistance factors and virulence genes enable its rapid evolution. ST2, ST42, and ST8 dominate globally, emphasizing the need for ongoing monitoring.

High Antimicrobial Susceptibility of Cloacal Enterococci and Escherichia coli from Free-Living Dalmatian and Great White Pelicans with Detection of Cefotaximase CTX-M-15 Producing Escherichia coli ST69.

Background/Objectives: In 2022, an outbreak of H5N1 highly pathogenic avian influenza (HPAI) killed 60% of the largest breeding colony of Dalmatian pelicans (DPs) in the world at Mikri Prespa Lake (Greece), prompting a multidisciplinary study on HPAI and other pathogens. This study determines the antimicrobial resistance rates of cloacal enterococci and Escherichia coli in DPs. Methods: Fifty-two blood and cloacal swab samples were collected from 31 nestlings (20 DP/11 great white pelicans) hatched after the H5N1 outbreak at the Prespa colony and 21 subadult/adult DPs captured at a spring migration stopover. The swabs were inoculated in non-selective and chromogenic-selective media. Identification was performed using MALDI-TOF, and antimicrobial susceptibility was tested. The genetic content was characterized using PCR and sequencing, and the clonality of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates was characterized using Multilocus Sequence Typing. Results: Twenty-eight non-repetitive E. coli and 45 enterococci isolates were recovered in non-selective media; most of them were susceptible to all antibiotics tested (85.7% E. coli/91.1% enterococci). Three of the fifty-two samples (6%, all adults) contained ESBL-E. coli isolates (detected in chromogenic ESBL plates), all carrying the blaCTX-M-15 gene and belonging to the lineage ST69. Conclusions: Despite the susceptibility of most fecal E. coli and enterococci isolates to all antibiotics tested, the finding that E. coli of lineage ST69 carry blaCTX-M-15 is of concern. This high-risk clone needs further investigation to elucidate its primary sources and address the growing threat of antimicrobial resistance from an integrated "One Health" perspective. Furthermore, it is imperative to study the potential impacts of ESBL-E. coli on the endangered DP further.

Genetic diversity atlas of Brucella melitensis strains from Sichuan Province, China

Human brucellosis is a re-emerging disease in Sichuan Province, China. In this study, bacteriology, conventional bio-typing, multi-locus sequence typing (MLST), and multiple locus variable-number tandem repeat analysis (MLVA) were applied to preliminarily characterize the strains in terms of genetic diversity and epidemiological links. A total of 101 Brucella strains were isolated from 16 cities (autonomous prefectures) from 2014 to 2021, and all of the strains were identified as Brucella melitensis bv. 3, suggesting that surveillance should focus on ruminants. MLST analysis identified four STs, namely, ST8 (n = 93), ST39 (n = 6), ST101 (n = 1), and ST118 (n = 1). The latter were new STs, indicating that strains displayed high population diversity. Six MLVA-8, namely, 42, 43, 45, 63, 83, and 114, and eight MLVA-11, namely, 111, 115, 116, 125, 180, 291, 298, and 342, genotypes were identified, demonstrating that all of the strains were from the Eastern Mediterranean lineage, and these strains exhibited a high genotype diversity. MLVA-16 analysis revealed that there was a co-existing transmission pattern, where sporadic cases and multiple outbreak events had a common origin. The dominant STs and MLVA genotypes of strains were epidemic in Northern, China, and 36 MLVA-16 genotypes were shared among strains (n = 51, 50.4%, 51/101) from Sichuan and strains from 22 other provinces. The findings imply that infected animals were introduced from outside the province. The surveillance and control of the disease have become public health challenges. Animal quarantines should be strengthened to prevent the spread of B. melitensis species among adjacent regions.

Pathogenic potential of ornithogenic &lt;i&gt;Escherichia coli&lt;/i&gt; strains detected in the Earth's polar regions

Introduction. Pathogenic strains of Escherichia coli are an important object of surveillance within the One Health concept in the wild, agriculture and human society. Migratory bird colonies and high latitude avian colonies may be points of active intraspecies and interspecies contact between different animal species, accompanied by the spread of pathogens. At the same time, the phylogeography of E. coli in relation to the presence of natural foci of colibacillosis in polar regions remains virtually unstudied. The aim of this study was to assess the pathogenic potential of E. coli strains from the polar regions of the Earth, based on the analysis of the genomes of these bacteria from typical ornithogenic ecosystems of the Arctic and Antarctic. Materials and methods. The study used collections of E. coli isolated from ornithogenic biological material during expeditions to high latitude areas of the Arctic (archipelagos of Novaya Zemlya, Franz Josef Land, Svalbard) and Antarctic (Haswell Archipelago). 16 cultures associated with avian E. coli (12 polar and 4 temperate strains) were selected for genome-wide sequencing using BGI technology. The annotation of the genomes focused on the identification of genes for pathogenicity factors and antimicrobial resistance, as well as the identification of strains belonging to individual genetic lineages using the cgMLST method. Results. The annotation of the genomes allowed their assignment to different sequence types in the multilocus sequencing typing and genome-wide sequencing typing schemes. The analysis of the geographical distribution of the sequence types of polar E. coli strains determined by the cgMLST method showed their global representation in geographically distant regions of the planet. For example, cgST 133718 was observed in Antarctica (strain 17_1myr) and in the UK, and sequence 11903, to which strain 32-1 from the northernmost point of Novaya Zemlya belonged, was previously identified in the USA. All strains studied were characterized by the presence of extensive virulence. Among the pathogenicity factors identified were haemolysins A, E, F, siderophores, including the yersiniabactin gene cluster, a number of adhesion, colonization and invasion factors, as well as the thermostable enterotoxin EAST-1 and genes that characterize enteroaggregative strains of E. coli (the virulence regulator gene eilA and enteroaggregative protein (air)). One of the Arctic strains (33-1) had determinants of antibiotic resistance, in particular the extended-spectrum beta-lactamase gene TEM-1b and the Tn1721 transposon, including tetracycline resistance genes (tetA-TetR), were detected in its genome. Conclusion. The results of the study indicate the circulation of E. coli strains with strong pathogenic potential in high-latitude Arctic and Antarctic ornithogenic ecosystems. The analysis of genomic data indicates the presence of geographically widespread genetic lineages in these regions, which justifies the importance of monitoring epidemic clones of E. coli, along with monitoring for other pathogens, in bird colonies in high-latitude areas.

Understanding the clinical and molecular epidemiological characteristics of carbapenem-resistant Acinetobacter baumannii infections within intensive care units of three teaching hospitals

BackgroundCarbapenem-resistant Acinetobacter baumannii (CRAB) is recognized as a common clinical conditional pathogen with blaOXA−23 gene-mediated multidrug-resistance that is a significant threat to public health safety. Timely and effective infection control measures are needed to prevent their spread.MethodsWe conducted a retrospective study of CRAB patients at three teaching hospitals from 2019 to 2022. We identified bacterial isolates, collected clinical data, and performed antimicrobial susceptibility testing. Genome characteristics of isolates were investigated by whole genome sequencing. Multilocus sequence typing and phylogenetic trees were used to assess the genetic similarity of isolates. Acquired antimicrobial resistance genes and virulence factors carried in the isolated group genome were analyzed by ResFinder, PubMLST and VFDB. Sequence alignment was used to analyze genetic environment around blaOXA−23. Phylogenetic tree was constructed to analyze the genetic relationship of isolates.ResultsA total of 92 non-repetitive CRAB isolates were collected, with sputum samples accounting for the majority (94.57%, n = 87) of samples. These were distributed into ST2, with ST2 identified to have the highest prevalence of infection, accounting for 99.99% (n = 91) of all isolates. The major resistance genes identified were blaOXA−23, blaOXA−66, blaOXA−51, and blaADC. Also, 92 CRAB strains showed high levels of resistance to common clinical antibiotics, but not minocycline. Meanwhile, most of the isolates carried virulence genes such as various ompA, csuA, csuB, csuC, csuD, abaI, abaR, lpxC, lpxA, and bmfRS. Single nucleotide polymorphism (SNP) analyses further indicated that the bacterial genome was progressively polymorphic with time. We analyzed the environment of the blaOXA−23 gene and found that CRAB accumulated in the context of prominent environmental antibiotic exposure and had longer survival times in the antibiotic environment, resulting in the tendency of bacteria to develop greater antibiotic resistance.ConclusionsWe find that CRAB is prevalent within the ICU and is progressively resistant to antibiotics over time. Enhanced clinical understanding and timely management of CRAB infections will be crucial to minimize or even eliminate the spread of CRAB within the ICU setting.