MLST Typing Research Articles

Easy analysis of bacterial whole-genome sequencing data for clinical microbiologists using open-source Galaxy platform: Characterization of ESBL-producing Enterobacterales from bloodstream infections

ObjectivesClinical microbiologists require easy-to-use open access tools with graphical interfaces to perform bacterial whole-genome sequencing (WGS) in routine practice. This study aimed to build a bioinformatics pipeline on the open-source Galaxy platform, facilitating comprehensive and reproducible analysis of bacterial WGS data in a few steps. We then used it to characterize our local epidemiology of ESBL-producing Enterobacterales isolated from patients with bacteremia. MethodsWe built a bioinformatics pipeline consisting of the following sequential tools: Fastp (input data trimming); FastQC (read quality control); SPAdes (genome assembly); Quast (quality control of genome assembly); Prokka (gene annotation); Staramr (ResFinder database) and ABRicate (CARD database) for antimicrobial resistance (AMR) gene screening and molecular strain typing. Paired-end short read WGS data from all ESBL-producing Enterobacterales strains isolated from patients with bacteremia over one year were analysed. ResultsThe Galaxy platform does not require command line tools. The bioinformatics pipeline was constructed within one hour. It only required uploading fastq files and facilitated systematization of de novo assembly of genomes, MLST typing, and AMR gene screening in one step. Among the 66 ESBL-producing strains analysed, the two most frequent ESBL genes were blaCTX−M-15 (62.1%) and blaCTX−M-27 (13.6%). ConclusionsThe open-access Galaxy platform provides a graphical interface and easy-to-use tools suitable for routine use in clinical microbiology laboratories without bioinformatics specialists. We believe that this platform will facilitate fast and low-cost analysis of bacterial WGS data, especially in resource-limited settings.

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.

Relationship between virulence and carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection: identification of a carbapenem-resistant and hypervirulent strain.

To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection, and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HVKP)strains. A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019, of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae (CRKP) and 96 were carbapenem-sensitive Klebsiella pneumoniae (CSKP). The drug susceptibility was detected by VITEK-2 automatic microbial analyzer; carbapenemase genes, virulence genes and capsule typing were detected by polymerase chain reaction; the high viscosity phenotype of strains was detected by string test, and the genome characteristics of CR-HVKP were detected by whole genome sequencing. Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP. There were significant differences in drug resistance to common antibiotics, except for minocycline between CSKP and CRKP isolates (all P<0.05). 92 out of 96 CRKP isolates carried carbapenemase genes, mainly blaKPC-2. The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP (P<0.05). The detection rates of virulence genes Kfu, aerobictin, iutA, ybtS, rmpA, magA, allS, and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group (all P<0.05). One HVKP strain was detected in the CRKP group (CR-HVKP) and 36 HVKP was detected in the CSKP group (P<0.05). The CR-HVKP strain belonged to the MLST412, serotype K57, expressed iutA, entB, mrkD, fimH, and rmpA virulence genes, and showed strong biofilm formation and significantly increased serum resistance. Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145, blaTEM-1, blaCTX-M-3, fosA6, oqxA5, oqxB26, and aac(3)-IId resistance genes, accompanied by abnormalities in outer membrane protein K (OmpK) 35 and OmpK36. The drug resistance of CRKP is significantly higher than that of CSKP, while CRKP carrying fewer virulence genes in both number and types compared to CSKP. A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected, which requires clinical awareness and epidemiological monitoring.

The genetic diversity and evolution analysis of the Hainan melioidosis outbreak strains

Melioidosis is a zoonotic disease, with its outbreaks being rare and indicative of an unusual concurrence of extreme climate and natural environmental factors. An outbreak of melioidosis cases emerged in Hainan following Typhoon “Dianmu” from October to December 2021, presenting an opportunity to identify the environmental sources of infection for these cases due to its nature as a well-defined point-source cluster. To investigate the relationship between the occurrence of these melioidosis cases and the environment, we extracted the entire genome of 25 clinical strains and conducted MLST typing, followed by whole genome sequencing and analysis of molecular genetic information for four ST46 genotypes from these strains. Phylogenetic and evolutionary relationships between Hainan sequence types (STs) and those found in other endemic regions were analyzed using IslandPath-DIMO, PHASTER, e-BURST, PHYLOViZ, and the maximum likelihood method. Notably, a total of 25 clinical strains were identified, encompassing 12 STs (ST46, ST1105, ST1991, ST30, ST1992, ST50, ST164, ST55, ST70, ST1993, ST1545, and ST58), with ST1991, ST1992, and ST1993 being newly discovered subtypes. PHYLOViZ clustering analysis divided the strains into two groups (A and B), both closely related to the Asian region. Phylogenetic tree analysis further revealed that most of the strains in this study were closely related to those found in Australia and Thailand. Analysis of patient information and visits to their residences suggested that contaminated water sources might be the primary source of infection during this outbreak.Our findings underscore that extreme weather events, such as typhoons, significantly increase the infection rate of B. pseudomallei, along with its genetic diversity, necessitating additional prevention strategies to control these B. pseudomallei infections.

Emergence of blaNDM-5 and blaOXA-232 Positive Colistin- and Carbapenem-Resistant Klebsiella pneumoniae in a Bulgarian Hospital.

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. blaNDM-5 was the only NDM variant, and it was always combined with blaOXA-232. The coexistence of blaOXA-232 and blaNDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All blaNDM-5- and blaOXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed blaOXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for blaNDM-5- and blaOXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for blaOXA-232- or blaKPC-2-positive isolates. All blaOXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation.

Campylobacteriosis: genotypic characteristics of the pathogen and immunological status of patients

Introduction. Campylobacteriosis is among the leading causes of acute gastrointestinal infections. The severity of campylobacteriosis and the development of long-term complications may be influenced by the genotype of the pathogen, whose biological properties can affect immune response parameters. The aim of the study was to identify common genotypes of epidemic clones of Campylobacter pathogens and to investigate characteristics of the immune response and severity of the disease. Materials and methods. The study included 203 patients aged from 1 month to 17 years with campylobacteriosis who underwent treatment at the clinic of the Federal State Budgetary Institution "DNKCIB FMBA" in 2019–2021. The diagnosis was confirmed using polymerase chain reaction method. Patient samples were also analyzed using culture-based methods. Total DNA was extracted using the QIAamp DNA Mini Kit. Genetic determinants encoding virulence factors and MLST typing were performed using the ResFinder program. The immune status of patients was assessed on days 1 and 7 of the illness. Immunological investigation included measurement of serum immunoglobulin concentrations (IgA, IgM, IgG), C-reactive protein, and cytokines (IL-1β, IL-1, IL-2, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-10, TNF-α, and IFN-γ). Results. When analyzing the frequency of detection of Campylobacter sequence types in children with clinical intestinal infections, it was found that the profile of isolated isolates is most similar to those from countries of North America (USA and Canada), Northern Europe (Great Britain, Holland ) and Scandinavia (Denmark, Sweden, Finland). Identification of a pathogen with the flgE+, cdtA+, cdtC+ genotype was accompanied by a statistically significant increase in the level of IL-8 and a decrease in the content of IgA in the peripheral blood serum, which reflected the low efficiency of the immune response during infection with Campylobacter and predetermined the severe course of the infectious process during the disease.

Temporal evolution of bacterial species and their antimicrobial resistance characteristics in wound infections of war-related injuries in Ukraine from 2014 to 2023

This study continues surveillance of antimicrobial resistance associated with combat injuries in Ukraine. To compare species composition, antibiotic resistance profiles, and emergence of new resistance genes between 2014-2020 and 2022-2023. This was a retrospective multi-centre microbiological survey in Ukrainian hospitals. Antibiotic susceptibility, whole-genome sequencing and multi-locus sequence typing were conducted on 154 organisms obtained from 125 casualties between 2022 and 2023. The data revealed a predominance of Gram-negative bacteria, particularly Acinetobacter baumannii (35.7%), Pseudomonas aeruginosa (14.9%) and Klebsiella pneumoniae (20.7%). High levels of carbapenem resistance were observed among A.baumannii {meropenem 72.2% [39/54, 95% confidence interval (CI) 58.4-83.5]; imipenem 66.7% (36/54, 95% CI 52.5-78.9)}, K.pneumoniae [meropenem 90.6% (29/32, 95% CI 75.0-98.0); imipenem 81.2% (26/32, 95% CI 63.6-92.8)] and P.aeruginosa [meropenem 47.8% (11/23, 95% CI 26.8-69.4); imipenem 60.8% (14/23, 95% CI 38.5-80.3)] strains. A.baumannii sequence type (ST)-78 and ST-400 were prevalent from 2014 to 2020, while five strains of ST-1077 were newly identified in 2022-2023. P. aeruginosa strains showed diversity across 16 STs, with ST-773 increasing in frequency and new STs emerging, but lacking carbapenemase genes. K.pneumoniae exhibited increased genetic diversity over time, with three STs from 2014 to 2020 and six new STs, including blaNDM-1, blaOXA-48 and blaKPC2 carriers, in 2022-2023. The prevalence of multi-drug-resistant isolates with STs associated with a high risk of global dissemination is increasing.

Molecular epidemiology of Brucella abortus isolated from the environment in Ningxia Hui autonomous region, China

Brucellosis is among the key zoonotic infectious diseases in China, and The Ningxia Hui Autonomous Region represents a major endemic area, and it is one of the main causes of poverty in the region due to illness. In Ningxia, there is substantial research on Brucella melitensis, studies on the molecular epidemiology of Brucella abortus are notably scarce. Consequently, this study aims to undertake pathogenic isolation and molecular epidemiological research on Brucella abortus isolated from the environment in Ningxia, providing insights and evidence to advance the prevention and control measures for brucellosis in the region. Building on traditional pathogenic detection methods, this research employs whole-genome sequencing(WGS) techniques and bioinformatics software to conduct a phylogenetic comparison of Ningxia strains and strains of Brucella abortus from various geographical origins. The results indicate that four Brucella abortus strains are classified as biovar 3 and MLST type ST2. It is shown that the local strains were closer phylogenetic relationships with strains from Asian and European countries. The presence of Brucella abortus in certain environmental sectors of Ningxia indicates a risk of transmission from the environment to animals and subsequently to humans. In conclusion, the Brucella abortus exists in some farming environments in Ningxia, and exists for a long time. Therefore, it is necessary to strengthen the monitoring of the disinfection effect of the farming environment to provide a basis for the forward movement of the gate of brucellosis prevention and control.

Genomic and Phenotypic Analysis of bla KPC-2 Associated Carbapenem Resistance in Klebsiella aerogenes: Insights into Clonal Spread and Resistance Mechanisms Across Hospital Departments in Beijing.

This study conducted an phenotypic and whole-genome sequencing analysis with Klebsiella aerogenes to elucidate its clinical epidemiological characteristics, antimicrobial resistance (AMR) phenotype, biofilm formation ability and hemolytic activity testing, AMR genes and phylogenetic relationships, so as to provide a further understanding of the intra-hospital strain transmission. Samples were collected from a hospital in Beijing between 2020 and 2022. All strains underwent bacterial identification, antimicrobial susceptibility testing (AST) using the VITEK-2 compact system. Biofilm formation ability and hemolytic activity were tested. Second-generation sequencing was applied to all strains, with those carrying the bla KPC gene were selected for third-generation sequencing. Whole-genome analysis identified resistance genes, plasmid types, MLST typing, and phylogenetic relationships. Plasmids were assembled to detect plasmid structures and AMR gene location. Among the 42 K. aerogenes isolates, 21 were carbapenem-resistant K. aerogenes (CRKA). All strains exhibited strong biofilm formation and no hemolytic activity. Most were sourced from sputum (83.3%). CRKA demonstrated extensive resistance to antibiotics, particularly β-lactamase inhibitors and Cefotetan. This resistance pattern was closely associated with the presence of an IncFII(pHN7A8) plasmid, which carried multiple resistance genes, including bla KPC-2, bla CTX-M-65, bla TEM-1, rmtB and a large number of mobile elements. The majority of CRKA strains clustered within the same branch of the phylogenetic tree, exhibiting minimal single nucleotide polymorphism (0-13 SNPs) differences, and they shared the same sequence type (ST292), resistance genes, and plasmids, originating from different departments, suggesting clonal transmission among the hospital. Our research reveals that the clonal transmission of CRKA occurs across various departments within the hospital. The widespread resistance observed in CRKA, attributed to the presence of bla KPC and ESBLs genes, underscores the need for heightened vigilance to prevent the further dissemination of CRKA within the hospital and, potentially, throughout the wider community.

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China

BackgroundRecently, linezolid-resistant staphylococci have become an emerging problem worldwide. Understanding the mechanisms of resistance, molecular epidemiology and transmission of linezolid-resistant CoNS in hospitals is very important.MethodsThe antimicrobial susceptibilities of all isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were determined using whole-genome sequencing and PCR.ResultsAll the strains were resistant to oxacillin and carried the mecA gene; 13 patients (36.1%) had prior linezolid exposure. Most S. epidermidis and S. hominis isolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated most linezolid-resistant CoNS strains were genetically related. In this study, we revealed that distinct CoNS strains have different mechanisms of linezolid resistance. Among ST22-type S. epidermidis, acquisition of the T2504A and C2534T mutations in the V domain of the 23 S rRNA gene, as well as mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S), were linked to the development of linezolid resistance. In S. cohnii isolates, cfr, S158Y and D159Y mutations in the ribosomal protein L3 were detected. Additionally, emergence of the G2576T mutation and the cfr gene were major causes of linezolid resistance in S. hominis isolates. The cfr gene, G2576T and C2104T mutations, M156T change in L3 protein, and I188S change in L4 protein were found in S. capitis isolates.ConclusionThe emergence of linezolid-resistant CoNS in the environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms.

Prevalence and characterization of community-associated Staphylococcus aureus isolates from human mastitis in Beijing, China

ObjectivesStaphylococcus aureus (S. aureus) spreads worldwide and occurrence of mastitis caused by it holds significant implications for public health. We aim to reveal the molecular typing, antibiotic resistance and virulence gene profile of S. aureus causing mastitis through investigation. MethodsA total of 200 isolates of S. aureus were collected from outpatients infected with mastitis in a hospital in Beijing from 2020.7 to 2021.7. The molecular characteristics were analyzed by MLST and spa typing, virulence genes were screened by PCR, antibiotic susceptible test was performed by VITEK® 2 Compact system and phylogenetic analysis was performed by MEGA11 and iTOL. ResultsNineteen sequence types (STs) belonging to 9 clone complexes (CCs) were identified. ST22 was the most dominant clone (77.0%, 154/200). MRSA accounted for 19.0% (38/200) and 89.5% (34/38) of MRSA isolates belonged to CC22 and CC59. The isolates had relatively low levels of antibiotic resistance, with the exception of β-lactams and macrolides with resistance rates above 50.0%. The carrying rate of pvl in the ST22-MRSA strains were 84.2% and the detection rates of seb and pvl in the MRSA isolates were significantly higher than those in the MSSA isolates, while the hlg, fnbA and sdrD showed opposite results. Whole genome sequenced specimens of MRSA strains X4 and B5 show the same evolutionary origin as ST22 EMRSA-15 (HE681097), which is popular in Europe. ConclusionsThe method based on molecular epidemiology is an important tool for tracking the spread of S. aureus infections. We need to be alert to the major MRSA clones CC22 and CC59 in the region and be vigilant to the possible pandemic and spread of ST22 EMRSA-15.

Molecular and epidemiological characterization of Staphylococcus aureus causing bovine mastitis in China

BackgroundStaphylococcus aureus is one of the most prevalent pathogens in bovine mastitis, which leads to substantial financial losses for the dairy industry. ResultsIn this study, S. aureus (n = 72) was isolated from 18 dairy farms in 15 provinces across China in 2021. The identification of these isolates at the species level was achieved by employing 16S rRNA sequencing. An isothermal amplification method for auxiliary detection of S. aureus was established, which can be employed not only for laboratory detection but also for point-of-care testing (POCT). Molecular characteristics of S. aureus mastitis in Chinese dairy cows were determined through MLST and spa typing. Finally, methicillin-resistant Staphylococcus aureus (MRSA) and MRSA resistance genes were detected using MIC and PCR amplification techniques. 72 isolates were identified as 12 sequence types (STs) and 7 clonal complexes (CC). ST1/CC1 was the dominant prevalent accounting for 33.3 % of the total, and exhibiting a wide distribution range. In terms of spa types, t114 was the dominant type, accounting for 31.9 % of the total, followed by t529 as the second major type. Four S. aureus strains were classified as MRSA according to their levels of oxacillin resistance (MIC ≥4 μg/mL). Among these four MRSA strains, one of them was found to be mecA positive. However, the presence of drug-resistance genes mecA and mecC was not detected in the remaining three MRSA strains, indicating the possible existence of new resistance genes. ConclusionsOur study investigated the prevalence of S. aureus mastitis in dairy cows in China, while also examined the molecular characteristics and MRSA strains. This information will help with the clinical monitoring, prevention, and control of S. aureus mastitis in dairy cattle.

Antibiotic resistance, biofilm formation, and molecular epidemiology of Staphylococcus aureus in a tertiary hospital in Xiangyang, China.

Staphylococcus aureus is a common clinical pathogen that causes various human infections. The aim of this study was to investigate the antibiotic susceptibility pattern, molecular epidemiological characteristics, and biofilm formation ability of S. aureus isolates from clinical specimens in Xiangyang and to analyze the correlation among them. A total of 111 non-duplicate S. aureus isolates were collected from the Affiliated Hospital of Hubei University of Arts and Science. All isolates were tested for antibacterial susceptibility. Methicillin-resistant S. aureus (MRSA) was identified by the mecA gene PCR amplification. All isolates were analyzed to determine their biofilm-forming ability using the microplate method. The biofilm-related gene was determined using PCR. SCCmec, MLST, and spa types of MRSA strains were performed to ascertain the molecular characteristics. Among the 111 S. aureus isolates, 45 (40.5%) and 66 (59.5%) were MRSA and MSSA, respectively. The resistance of MRSA strains to the tested antibiotics was significantly stronger than that of MSSA strains. All isolates were able to produce biofilm with levels ranging from strong (28.9%, 18.2%), moderate (62.2%, 62.1%), to weak (8.9%, 19.7%). Strong biofilm formation was observed in MRSA strains than in MSSA strains, based on percentages. There were dynamic changes in molecular epidemic characteristics of MRSA isolates in Xiangyang. SCCmecIVa-ST22-t309, SCCmecIVa-ST59-t437, and SCCmecIVa-ST5-t2460 were currently the main epidemic clones in this region. SCCmecIVa-ST5-t2460 and SCCmecIVa/III-ST22-t309 have stronger antibiotic resistance than SCCmecIVa-ST59-t437 strains, with resistance to 6 ~ 8 detected non-β-lactam antibiotics. The molecular epidemic and resistance attributes of S. aureus should be timely monitored, and effective measures should be adopted to control the clinical infection and spread of the bacteria.

Genome stability of &lt;i&gt;Bacillus velezensis&lt;/i&gt; after two-year exposure in open space

Spore-forming bacteria have a unique resistance to negative environmental conditions, including aggressive space factors, and are an excellent model for studying adaptation mechanisms and survival strategies at the molecular level. The study analyzed the genome of Bacillus velezensis, which remained viable after a two-year exposure in outer space on the outer surface of the ISS as part of the Test space experiment. A comparative analysis of the draft genomes of the exhibit strain and the ground control did not reveal significant changes, the average nucleotide identity was 99.98%, which indicates the ability of microorganisms to maintain genome stability in space conditions, due to both increased stress resistance of bacterial spores and efficient operation of the system. repair of accumulated changes. The study of single nucleotide polymorphism in the genome of Bacillus velezensis revealed nine point substitutions, three of which are in intergenic regions, 6 in protein-coding genes, three of them are missense mutations, two nucleotide deletions leading to a shift in the reading frame, and one synonymous substitution. The profiles of the housekeeping genes were determined during MLST typing, and it was found that the allelic profiles obtained for B. velezensis T15.2 and 924 strains do not correspond to any of the previously described sequence types. The presented results indicate the ability of Bacillus velezensis bacteria to maintain the viability of spores and the integrity of the genome for a long time under extreme conditions of outer space, which is important for the problem of planetary protection, as well as the potential possibility of implementing biotechnological processes based on Bacillus velezensis during space exploration.

Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

The emergence of multi-drug resistant (MDR) pathogens linked to healthcare-associated infections (HCAIs) is an increasing concern in modern veterinary practice. Thus, rapid bacterial typing for real-time tracking of MDR hospital dissemination is still much needed to inform best infection control practices in a clinically relevant timeframe. To this end, the IR Biotyper using Fourier-Transform InfraRed (FTIR) spectroscopy has the potential to provide fast cluster analysis of potentially related organisms with substantial cost and turnaround time benefits. A collection of MDR bacterial isolates (n = 199, comprising 92 Klebsiella pneumoniae and 107 Pseudomonas aeruginosa) obtained from companion animal (i.e., dogs, cats and horses) clinical investigations, faecal and environmental screening from four veterinary facilities between 2012 and 2019 was analysed retrospectively by FTIR spectroscopy. Its performance was compared against MLST extracted from whole genomes of a subset of clustering isolates (proportionally to cluster size) for investigation of potential nosocomial transmission between patients and the surrounding hospital environments. Concordance between the FTIR and MLST types was overall high for K. pneumoniae (Adjusted Rand Index [ARI] of 0.958) and poor for P. aeruginosa (ARI of 0.313). FTIR K. pneumoniae clusters (n = 7) accurately segregated into their respective veterinary facility with evidence of intra-hospital spread of K. pneumoniae between patients and environmental surfaces. Notably, K. pneumoniae ST147 intensely circulated at one Small Animal Hospital ICU. Conversely, Pseudomonas aeruginosa FTIR clusters (n = 18) commonly contained isolates of diversified hospital source and heterogeneous genetic background (as also genetically related isolates spread across different clusters); nonetheless, dissemination of some clones, such as P. aeruginosa ST2644 in the equine hospital, was apparent. Importantly, FTIR clustering of clinical, colonisation and/or environmental isolates sharing genomically similar backgrounds was seen for both MDR organisms, highlighting likely cross-contamination events that led to clonal dissemination within settings. FTIR spectroscopy has high discriminatory power for hospital epidemiological surveillance of veterinary K. pneumoniae and could provide sufficient information to support early detection of clonal dissemination, facilitating implementation of appropriate infection control measures. Further work and careful optimisation need to be carried out to improve its performance for typing of P. aeruginosa veterinary isolates.

Longitudinal study of meningococcal carriage in adolescents and young adults in South Australia 2017-2020

This analysis investigated longitudinal changes in meningococcal carriage in adolescents in South Australia over 4 years. Data from the "B Part of It" study, which included a state-wide cluster randomized controlled trial in secondary-school students (n=34,489 in 2017 and 2018) and serial cross-sectional studies in school leavers aged 17-25 years (n=4028 in 2019-2020). Individuals had oropharyngeal swabs collected annually. This study included two unique cohorts: (1) individuals enrolled in 2019, with three consecutive annual swabs taken in 2017, 2018 and 2019; and (2) individuals enrolled in 2020, with swabs taken in 2017, 2018, and 2020. Disease-associated N. meningitidis genogroups were identified using PCR and whole genome sequencing. Univariate analysis identified risk factors for recurrent carriage (≥2). Among school leavers, 50 (1.7%, total n=2980) had carriage detected at successive visits. In participants with meningococcal carriage at successive visits, 38/50 (76.0%) had the same genogroup detected by porA PCR. Of those, 19 had the same MLST type and demonstrated minimal variation, indicating they most likely had sustained carriage of the same isolate (range 226 to 490 days, mean duration 352 [SD 51] days). In the 2019 school leaver cohort, 6.7% acquired carriage in their first year out of school compared to 3.3% in their final school year. Compared to single carriage detection, recurrent carriage was potentially more likely in older adolescents (16 compared to ≤15 years; OR=1.97 (95%CI 1.0, 3.86); p=0.048). Whilst carriage is typically transient, some adolescents/young adults may have persistent carriage and are likely to be an important group in the transmission of meningococci.

In Vitro Antimicrobial Activity of Five Newly Approved Antibiotics against Carbapenemase-Producing Enterobacteria-A Pilot Study in Bulgaria.

To solve the problem with pan-drug resistant and extensively drug-resistant Gram-negative microbes, newly approved drugs such as ceftazidime/avibactam, cefiderocol, plazomicin, meropenem/vaborbactam, and eravacycline have been introduced in practice. The aim of the present study was to collect carbapenemase-producing clinical Enterobacterales isolates, to characterize their carbapenemase genes and clonal relatedness, and to detect their susceptibility to commonly used antimicrobials and the above-mentioned newly approved antibiotics. Sixty-four carbapenemase producers were collected in a period of one year from four Bulgarian hospitals, mainly including Klebsiella pneumoniae (89% of the isolates) and also single Proteus mirabilis, Providencia stuartii and Citrobacter freundii isolates. The main genotype was blaNDM-1 (in 61%), followed by blaKPC-2 (23%), blaVIM-1 (7.8%) and blaOXA-48 (7.8%). Many isolates showed the presence of ESBL (blaCTX-M-15/-3 in 76.6%) and AmpC (blaCMY-4 in 37.5% or blaCMY-99 in 7.8% of isolates). The most common MLST type was K. pneumoniae ST11 (57.8%), followed by ST340 (12.5%), ST258 (6.3%) and ST101 (6.3%). The isolates were highly resistant to standard-group antibiotics, except they were susceptible to tigecycline (83.1%), colistin (79.7%), fosfomycin (32.8%), and aminoglycosides (20.3-35.9%). Among the newly approved compounds, plazomicin (90.6%) and eravacycline (76.3%) showed the best activity. Susceptibility to ceftazidime/avibactam and meropenem/vaborbactam was 34.4% and 27.6%, respectively. For cefiderocol, a large discrepancy was observed between the percentages of susceptible isolates according to EUCAST susceptibility breakpoints (37.5%) and those of CLSI (71.8%), detected by the disk diffusion method. This study is the first report to show patterns of susceptibility to five newly approved antibiotics among molecularly characterized isolates in Bulgaria. The data may contribute to both the improvement of treatment of individual patients and the choice of infection control strategy and antibiotic policy.

Genomic diversity of Campylobacter jejuni and Campylobacter coli isolated from the Ethiopian dairy supply chain.

Campylobacteriosis outbreaks have previously been linked to dairy foods. While the genetic diversity of Campylobacter is well understood in high-income countries, it is largely unknown in low-income countries, such as Ethiopia. This study therefore aimed to conduct the first genomic characterization of Campylobacter isolates from the Ethiopian dairy supply chain to aid in future epidemiological studies. Fourteen C. jejuni and four C. coli isolates were whole genome sequenced using an Illumina platform. Sequences were analyzed using the bioinformatics tools in the GalaxyTrakr platform to identify MLST types, and single nucleotide polymorphisms, and infer phylogenetic relationships among the studied isolates. Assembled genomes were further screened to detect antimicrobial resistance and virulence gene sequences. Among 14 C. jejuni, ST 2084 and ST 51, which belong to the clonal complexes ST-353 and ST-443, respectively, were identified. Among the 4 sequenced C. coli isolates, two isolates belonged to ST 1628 and two to ST 830 from the clonal complex ST-828. The isolates of C. jejuni ST 2084 and ST 51 carried β-lactam resistance gene blaOXA-605, a fluoroquinolone resistance-associated mutation T86I in the gryA gene, and a macrolide resistance-associated mutation A103V in 50S L22. Only ST 2084 isolates carried the tetracycline resistance gene tetO. Conversely, all four C. coli ST 830 and ST 1628 isolates carried tetO, but only ST 1628 isolates also carried blaOXA-605. Lastly, C. jejuni ST 2084 isolates carried a total of 89 virulence genes, and ST 51 isolates carried up to 88 virulence genes. Among C. coli, ST 830 isolates carried 71 genes involved in virulence, whereas two ST 1628 isolates carried up to 82 genes involved in virulence. Isolates from all identified STs have previously been isolated from human clinical cases, demonstrating a potential food safety concern. This finding warrants further monitoring of Campylobacter in dairy foods in Ethiopia to better understand and manage the risks associated with Campylobacter contamination and transmission.

Impact and Diversity of ESBL-Producing Klebsiella pneumoniae Recovered from Raw Chicken Meat Samples in Türkiye.

The interrelationship between human, animal and environmental sectors leads to the spread of antibiotic resistance due to selective pressures, evolutionary traits and genomic evolution. In particular, the frequent use of antibiotics in livestock inevitably influences the emergence of specific resistance determinants in human strains, associated with reduced treatment options in clinical therapy. In this study, ESBL-producing Klebsiella pneumoniae strains isolated from chicken meat samples were evaluated for public health implications in Türkiye. Whole-genome sequencing was used for genetic dissection and phylogenetic comparison of their genomes. The isolates were assigned to four MLST types (ST147, ST37, ST2747 and ST219); two of them were found to represent the ST147 clone associated with severe human infections worldwide. In addition to cephalosporins, high resistance levels to quinolones/fluoroquinolones were identified phenotypically, caused by acquired resistance genes and chromosomal point variations. One isolate was also found to carry the qacE∆1 efflux transporter gene, which confers tolerance to quaternary ammonium compounds. The detection of virulence genes (i.e., that coding for enterobactin) associated with the pathogenicity of K. pneumoniae suggests a public health impact. Thus, comprehensive information on the occurrence and impact of K. pneumoniae from livestock is needed to derive appropriate management strategies for consumer protection. In this study, it was shown that poultry meat serves as a reservoir of clinically emerging multidrug-resistant high-risk clones.