Pulsed-field Gel Electrophoresis Research Articles

AdeABC, AdeFGH, and AdeIJK efflux pumps as key factors in tigecycline resistance of Acinetobacter baumannii: a study from Western Balkan hospitals.

The present study investigated the role of resistance-nodulation-cell division (RND) efflux pumps in tigecycline resistance of Acinetobacter baumannii clinical isolates recovered from three Western Balkan countries (Serbia, Bosnia and Herzegovina and Montenegro). A total of 37 A. baumannii isolates recovered from seven tertiary care hospitals in 2016 and 2022 were tested against tigecycline using broth microdilution method. Then, efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used to determine the involvement of efflux pumps in tigecycline resistance. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multiplex PCR-based determination of clonal lineage. Regulators of efflux pumps were analyzed for amino acid substitutions, while reverse transcription-quantitative PCR (RT-qPCR) enabled quantification of RND efflux pumps expression. All tested isolates were interpreted as resistant to tigecycline and showed reduced tigecycline minimum inhibitory concentration (MIC) values in the presence of CCCP. PFGE analysis showed significant diversity among isolates grouped in cluster I including IC2 (n = 32) and IC3 (n = 1) isolates, while cluster II was comprised of four IC1 isolates. The most prevalent substitutions in AdeR were V120I and A136V and in AdeS G186V and N268H (n = 33). The Q262R substitution was detected in AdeL proteins of IC1 isolates, whereas no alterations were observed within AdeN. The expression of the adeB, adeG, and adeJ genes in selected isolates was upregulated in five (1.16- to 3-fold), sixteen (1.35- to 2.82-fold), and twelve isolates (1.62- to 4-fold) compared to ATCC19606, respectively. This study revealed that overexpression of RND efflux pumps underlies tigecycline resistance in A. baumannii clinical isolates from the Western Balkans.

Genomic characterization of a blaKPC-2–producing IncM2 plasmid harboring transposon ΔTn6296 in Klebsiella michiganensis

Klebsiella michiganensis is an emerging hospital-acquired bacterial pathogen, particularly strains harboring plasmid-mediated carbapenemase genes. Here, we recovered and characterized a multidrug-resistant strain, blaKPC-2–producing Klebsiella michiganensis LS81, which was isolated from the abdominal drainage fluid of a clinical patient in China, and further characterized the co-harboring plasmid. K. michiganensis LS81 tested positive for the blaKPC-2 genes by PCR sequencing, with blaKPC-2 located on a plasmid as confirmed by S1 nuclease pulsed-field gel electrophoresis combined with Southern blotting. In the transconjugants, the blaKPC-2 genes were successfully transferred to the recipient strain E. coli EC600. Whole-genome sequencing and bioinformatics analysis confirmed that this strain belongs to sequence type 196 (ST196), with a complete genome comprising a 5,926,662bp circular chromosome and an 81,451bp IncM2 plasmid encoding blaKPC-2 (designated pLS81-KPC). The IncM2 plasmid carried multiple β-lactamase genes such as blaTEM-1B, blaCTX-M-3, and blaKPC-2 inserted in truncated Tn6296 with the distinctive core structure ISKpn27–blaKPC-2–ISKpn6. A comparison with 46 K. michiganensis genomes available in the NCBI database revealed that the closest phylogenetic relative of K. michiganensis LS81 is a clinical isolate from a wound swab in the United Kingdom. Ultimately, the pan-genomic analysis unveiled a substantial accessory genome within the strain, alongside significant genomic plasticity within the K. michiganensis species, emphasizing the necessity for continuous surveillance of this pathogen in clinical environments.

Etiological characteristics and whole genome sequence analysis of Clostridium perfringens causing a food poisoning outbreak

This study investigated the etiological characteristics and whole genome sequencing of clostridium perfringens (CP) from a food poisoning outbreak. Multiplex real-time PCR was employed to screen pathogens in collected samples. Based on the preliminary screening results, the isolation, cultivation, and identification of suspected pathogenic CP bacteria were performed. The nucleic acid tests were conducted for CP-related virulence genes on CP isolates, anal swab specimens and food samples. The molecular typing of CP isolates was analyzed by using pulsed-field gel electrophoresis (PFGE). A phylogenetic tree was established to analyze nucleotide and amino acid homology after sequencing the PLC gene. The whole genome sequencing and gene annotation on the representative strain QD2022FB4-CP4 were performed to explore its drug resistance, toxin genes and biological characteristics. The analysis revealed that the food poisoning was triggered by F-type Clostridium perfringens, which infected the consumers by contaminating'roujiamo' sandwiches. The whole genome sequencing of the strain QD2022FB4-CP found that it had active metabolic processes, multiple virulence genes and multidrug resistance characteristics.

First documentation of a clinical multidrug-resistant Enterobacter chuandaensis ST2493 isolate co-harboring blaNDM-1 and two blaKPC-2 bearing plasmids.

The increasing prevalence of carbapenem-resistant Enterobacter cloacae complex (CREC) poses great challenges to infection treatment in the clinical setting. In this study, we reported the emergence of carbapenemase in a rare species, Enterobacter chuandaensis, belonging to the Enterobacter cloacae complex (ECC). We elucidated the genetic characteristics of carbapenem-resistant isolate FAHZZU5885, co-harboring blaNDM-1 and blaKPC-2. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and average nucleotide identity (ANI) analysis were used to identify E. chuandaensis. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were used to clarify the number and size of the plasmids in FAHZZU5885. Antimicrobial phenotypes were identified by antimicrobial susceptibility testing (AST), and the characteristics of the strain were examined with whole-genome sequencing (WGS). The conjugation experiment and stability assay were conducted to verify the transferability and stability of the plasmid carrying carbapenemase-encoding genes. E. chuandaensis FAHZZU5885 was isolated from a perianal swab of a patient admitted to the ICU. This strain simultaneously carried blaNDM-1 and two blaKPC-2 genes. FAHZZU5885 was resistant to most of the tested antibiotics except for amikacin, tigecycline, and colistin. Two blaKPC-2 were located separately on two different plasmids, the ~ 120kb IncFIA-IncFII plasmid and the ~ 80kb IncR plasmid. Both plasmids shared the conserved sequence klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. The blaNDM-1-bearing plasmid had the potential to transfer and can remain stable after successive passages. In addition, the blaNDM-1 was carried on a ~ 80kb IncFII plasmid with the conserved sequence ISAba125-blaNDM-1-ble-trpF-dsbD-cutA-groS-groL. In summary, this study marks the first report of the multidrug-resistant E. chuandaensis strain FAHZZU5885 harboring two blaKPC-2-bearing plasmids, indicating the potential for the further dissemination of carbapenemase-encoding genes in novel species. The findings contribute to enhancing our understanding of CREC strains, emphasizing the need for continued and comprehensive surveillance of this species.

Molecular Characteristics Demonstrate the Occurrence of Phylogenetic Similar Isolates of <i>Vibrio alginolyticus</i> and <i>Vibrio parahaemolyticus</i> in Aquatic Environments

Vibrio alginolyticus is an opportunistic fish pathogen with a potential to cause septicemia and often associated with other Vibrio infections, particularly Vibrio parahaemolyticus, and can pose a significant threat to aquatic health. This study investigated the biochemical and phylogenetic characteristics of V. alginolyticus isolated from various aquaculture farms, focusing on its prevalence, hemolysin genes, genetic relatedness, and antibiotic susceptibility. Out of 92 brackish water aquaculture farm samples, 16 isolates were biochemically confirmed as V. alginolyticus, with 12 subsequently confirmed by polymerase chain reaction targeting collagenase gene. Molecular analysis of the thermolabile hemolysin gene (tlh) via specific PCR amplified a 450 bp fragment in 8 isolates, confirming the presence of the tlh gene. Pulsed Field Gel Electrophoresis (PFGE) typing differentiated tlh-positive and tlh-negative V. alginolyticus isolates with 92% genetic similarity. The isolates exhibited proteolytic, lipolytic, and lecithinase activities. Notably, the isolates showed intermediate resistance to most of the tested antibiotics, indicating exposure to antimicrobial agents. This study provides evidence of the presence of similar phylogenetical isolates of V. alginolyticus and V. parahaemolyticus in aquatic environments. Furthermore, this study emphasizes the importance of expanding surveillance programs by incorporating strain-specific characterization to better understand and control vibriosis in aquaculture.

Molecular surveillance of A. baumannii in intensive care units: exploration of transmission chains

Abstract Background Multidrug-resistant Acinetobacter baumannii (MDR-Ab) is one of the main causes of healthcare associated infections (HAIs). During the SARS-CoV-2 pandemic there was an increase in MDR-Ab infections, especially in intensive care units (ICUs). This study aimed to assess the potential spread or emergence of specific clusters of MDR-Ab across four different ICUs at the Umberto I teaching hospital of Rome. Methods From January 2020 to January 2022 microbiological surveillance was conducted in four ICUs: two dedicated to COVID-19 patients (ICU-1C, ICU-2C) and two to non-COVID-19 patients (ICU-1R, ICU-2R). The genetic relatedness between A. baumannii isolates was assessed using pulsed-field gel electrophoresis (PFGE). Illumina whole genome sequencing was conducted on 26 representative isolates. Results In total, 178 A. baumannii isolates were obtained from 129 COVID-19 patients and 49 non-COVID-19 patients. The isolates were classified into 17 PFGE pulsotypes, being two major (A, B) and five intermediate (C, D, E, H, Q). Clone A was present in all ICUs, while Clone B was present only in ICU-1C and ICU-2R. Overall, 117 isolates belonged to clone A and exhibited a MDR phenotype; all of them were placed within the international clonal lineage II. All isolates showed carbapenems resistance primarily attributed to the presence of the blaOXA-23 gene, while aminoglycosides resistance observed in almost all isolates was attributed to the presence of the armA gene. Small outbreaks involving intermediate pulsotypes were detected between ICU-1R and ICU-2R, ICU-1C and ICU-2R, ICU-1C and ICU-2C, ICU-1C and ICU-1R. Conclusions The observed outbreaks could be attributed to a decline in attention to normal care practices for the prevention of HAIs during the COVID-19 pandemic, favouring the spread of MDR-Ab. Therefore, it is recommended the strengthen of control measures and the implementation of long-term strategies targeting MDR microorganisms in the ICUs. Key messages • Acinetobacter baumannii was frequently found infecting patients across four different ICUs at the Umberto I teaching hospital of Rome. • Acinetobacter baumanni outbreaks and multidrug resistance were observed. Therefore, the implementation of long-term strategies targeting MDR microorganisms in the ICUs is highly recommended.

Clinical epidemiology and impact of Haemophilus influenzae airway infections in adults with cystic fibrosis

BackgroundHaemophilus influenzae is prevalent within the airways of persons with cystic fibrosis (pwCF). H. influenzae is often associated with pulmonary exacerbations (PEx) in pediatric cohorts, but in adults, studies have yielded conflicting reports around the impact(s) on clinical outcomes such as lung function decline. Accordingly, we sought to discern the prevalence, natural history, and clinical impact of H. influenzae in adult pwCF.MethodsThis single-centre retrospective cohort study reviewed all adult pwCF with H. influenzae sputum cultures between 2002 and 2016. From this cohort, persistently infected subjects (defined as: ≥2 samples with the same pulsotype and > 50% sputum culture-positive for H. influenzae in each year) were matched (1:2) to controls without H. influenzae. Demographic and clinical status (baseline health or during periods of PEx) were obtained at each visit that H. influenzae was cultured. Yearly biobank isolates were typed using pulsed-field gel electrophoresis (PFGE) to assess relatedness.ResultsOver the study period, 30% (n = 70/240) of pwCF were culture positive for H. influenzae, of which 38 (54%) were culture-positive on multiple occasions and 12 (17%) had persistent infection. One hundred and thirty-seven isolates underwent PFGE, with 94 unique pulsotypes identified. Two (1.5%) were serotype f with the rest non-typeable (98.5%). H. influenzae isolation was associated with an increased risk of PEx (RR = 1.61 [1.14–2.27], p = 0.006), however, this association was lost when we excluded those who irregularly produced sputum (i.e. only during a PEx). Annual lung function decline did not differ across cohorts.ConclusionsIsolation of H. influenzae was common amongst adult pwCF but often transient. H. influenzae infection was not associated with acute PEx or chronic lung function decline.

Ten Years Later: Still Unchanged Susceptibility to Antibiotics of Listeria monocytogenes from Poultry Processing Environments.

Listeria monocytogenes is still recognized as being commonly susceptible to antibiotics; however, there have been reports of reduced susceptibility in recent years. The significance of this resistance is not clear, in part due to the disparity in the antimicrobial susceptibility testing methods used. EUCAST (European Committee on Antimicrobial Susceptibility Testing) has recently proposed a standardized method for antibiotic susceptibility testing of L. monocytogenes. The aim of this work was to evaluate the susceptibility to 11 antibiotics in clinical use of 50 pulsed-field gel electrophoresis types of L. monocytogenes representing 347 isolates from a poultry industry setting using the EUCAST method and to compare the results with those obtained 10 years before. All poultry strains were sensitive to all the antibiotics tested but one strain was resistant to benzylpenicillin according to the EUCAST criteria. The current findings supported the previous study and confirmed that in certain food-associated L. monocytogenes populations, antibiotic sensitivity has remained stable.

Molecular characterization of the tet (M)-carrying transposon Tn7124 and plasmids in Escherichia coli isolates recovered from swine.

Here, we report the genetic features and evolutionary mechanisms of two tet (M)-bearing plasmids (pTA2 and pTA7) recovered from swine Escherichia coli isolates. The genetic profiles of pTA2 and pTA7 and corresponding transconjugants were accessed by S1 nuclease pulsed-field gel electrophoresis and Southern hybridization, followed by whole genome sequencing and bioinformatics analysis. The biological influences of pTA2 and pTA7 were determined by stability and direct competition assays. Both pTA7 and pTA2 had the IncR backbone sequences but differed in the multidrug resistance region (MDR). The MDR of pTA2 consisted of sul3, tet (M), qnrS1, bleO, oqxAB, floR, aadA1, cmlA1, aadA2, and tet (A)-tetR (A) in addition to 22 insertion sequences. Notably, pTA2 carried the novel complex Tn7124 (IS26-ctp-lp-tet (M)-hp-IS406tnp-IntI4-IS26) harboring tet (M). The fragment carrying tet (M) (IS26-ctp-lp-tet (M)-IS406 tnp-ctp-aadA1-cmlA1-aadA2-dfrA12-IntI1), named Tn6942-like, and the two resistance modules ISVsa3-VirD2-floR-lysR and tet (A)-tetR (A) were located in the MDR of pTA7. Both pTA2 and pTA7 were highly stable in E. coli DH5α cells with no fitness cost to the host or disadvantage in growth competition. These results indicate that transposons carrying tet (M) continuously integrate via mediation with an insertion sequence, which accelerates the transmission of tet (M) in E. coli isolates through integration of other drug-resistant genes, thereby posing a potential serious threat to the efficacy of clinical treatment.

Disruption of mgrB gene by ISkpn14 sourced from a blaKPC−2 carrying plasmid mediating polymyxin resistance against carbapenem-resistant Klebsiella pneumoniae during treatment: study on the underlying mechanisms

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) infections poses global challenges, with limited options available for targeted therapy. Polymyxin was been regarded as one of the most important last-resort antimicrobial agents. Many factors could accelerate the resistance evolution of polymyxin. Insertion sequence (IS) inserted into mgrB is the main polymyxin resistance mechanism in K. pneumoniae. In this study, two CRKPs (KP31157 and KP31311) were isolated from the urine of a patient, shifting from susceptible to resistant as the mgrB inserted by ISkpn14. We intended to explore the origin of the IS and underlying mechanisms resulting in polymyxin resistance.MethodsThe within-host evolution relationship and molecular features of both CRKPs were determined by pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). pKP31311_KPC-2 plasmid genome structures contained in the above two CRKPs were aligned with the homologic plasmids, retrieved from the NCBI genome database via comparative genomic analysis. The plasmids encoding ISkpn14 elements flanked by direct repeat (DR) or not were analyzed. The mRNA expression, plasmid curing and in vitro antibiotics inducing experiment were employed to understand the potential mechanism of polymyxin resistance.ResultsBoth strains, sharing homology, exhibited polymyxin resistance due to the insertion of ISkpn14 into the mgrB gene, influenced by minocycline exposure. Minocycline and tigecycline could accelerate polymyxin resistance (P < 0.05), validated by an in vitro induction experiment. The ISkpn14 without DR flanked expressed about 4 times higher than that with DR. The frequency of the mgrB insertion induced by polymyxin was significantly reduced (0 strain detected) after the blaKPC−2-carrying plasmid was eliminated.ConclusionsThis study provides direct experimental evidence that the ISkpn14 element causing mgrB inactivation and polymyxin resistance in K. pneumoniae originates from blaKPC−2-carrying plasmids. Minocycline exposure will accelerate the evolution of polymyxin resistance. Understanding the dynamics of IS transposition and its association with antibiotic exposure is crucial for developing effective strategies to reduce the emergence of polymyxin resistance in CRKP.

Molecular Patterns and Antimicrobial Resistance Characterization of Salmonella enterica Non-Typhoidal from Human, Food, and Environment Samples Isolated in Luanda, Angola

The aim of this study was to characterize the antimicrobial resistance phenotype and genotype of non-typhoidal Salmonella spp. isolated in Luanda, Angola. Between 2013 and 2015, human clinical samples, food, and environmental samples (n = 290) were collected at different regions of Luanda city and screened for the presence of Salmonella spp. Bacterial isolates were preliminarily identified using the API 20E Kit, and their identification was confirmed using PCR and serotyping. All Salmonella spp. isolates were tested by minimum inhibitory concentration against 19 antimicrobials. The isolates were also screened using PCR for the presence of resistance genes (blaOXA-1, blaSHV, blaTEM, sul1, sul2, sul3, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6′)-Ib, dfrIa [targeting dfrA1, dfrA5, dfrA15, dfrA15b, dfrA16, dfrA16b] and dfrA12, cmlA, and floR) and typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Salmonella enterica non-typhoidal was detected in 21.3% of the clinical samples (n = 32/150), 11.1% of the food samples (n = 10/90), and 26% of the environmental samples (n = 13/50). Serotyping revealed that the monophasic variant of Salmonella Typhimurium (Salmonella enterica serovar 4,[5],12:i:-) was detected in 38.1% of the samples. Moreover, serovar Salmonella Enteritidis was the second most frequent. Only 7.3% of the isolates were resistant to at least one antimicrobial. Furthermore, isolates from different origins (clinical, environmental, and food) were associated with the same lineages, Salmonella Enteritidis ST11 and S. enterica ser. Typhimurium ST313. The detection of S. enterica serovar 4,[5],12:i:- in different settings reinforces the need for a One Health approach to control this zoonosis in Angola.

Cryopreservation of bovine sperm causes single-strand DNA breaks that are localized in the toroidal regions of chromatin

BackgroundSperm cryopreservation is widely used in the cattle industry, as it allows for disassociating the localization of sires and the collection of semen from the timing of artificial insemination. While freeze-thawing is known to impair sperm DNA integrity, whether the damage induced consists of single- (SSB) or double-strand breaks (DSB) has not been determined. In addition, no previous study has addressed if DNA breaks preferentially reside in specific genome regions such as those forming the toroid linker regions, or are rather spread throughout the regions linked to protamines. The main aim of the present work, therefore, was to elucidate the type and localization of the DNA damage generated by cryopreservation and to evaluate its impact on artificial insemination outcomes in cattle.ResultsThe incidence of SSB and DSB was evaluated in 12 ejaculates before and after cryopreservation with the Comet assay, and the localization of the DNA breaks was assessed using pulsed-field gel electrophoresis (PFGE). Before cryopreservation, the incidence of SSB was 10.99% ± 4.62% and involved 20.56% ± 3.04% of sperm cells, whereas these figures significantly (P < 0.0001) increased up to 34.11% ± 3.48% and 53.36% ± 11.00% in frozen-thawed sperm. In contrast, no significant differences in the incidence of DSB were observed (P > 0.990) before and after cryopreservation (before: incidence of 13.91% ± 1.75% of sperm DNA affecting 56.04% ± 12.49% of sperm cells; after: incidence of 13.55% ± 1.55% of sperm DNA involving 53.36% ± 11.00% of sperm cells). Moreover, PFGE revealed that the percentage of sperm DNA fragments whose length was shorter than a toroid (< 31.5 kb) was greater (P < 0.0001) after (27.00% ± 4.26%) than before freeze-thawing (15.57% ± 4.53%). These differences indicated that the DNA breaks induced by cryopreservation affect the regions condensed in protamines, which are structured in toroids. On the other hand, in vivo fertility rates were associated to the incidence of SSB and DSB in frozen-thawed sperm (P = 0.032 and P = 0.005), but not with the size of the DNA fragments resulting from these breaks (P > 0.05).ConclusionCryopreservation of bovine sperm generates single-strand DNA breaks, which are mainly located in protamine-condensed toroidal regions. The incidence of DNA breaks in cryopreserved sperm has an impact on cattle fertility, regardless of the size of generated fragments.

Discovery of clinical isolation of drug-resistant Klebsiella pneumoniae with overexpression of OqxB efflux pump as the decisive drug resistance factor.

Background emergence of multidrug-resistant (MDR) bacterial strains is a public health concern that threatens global and regional security. Efflux pump-overexpressing MDR strains from clinical isolates are the best subjects for studying the mechanisms of MDR caused by bacterial efflux pumps. A Klebsiella pneumoniae strain overexpressing the OqxB-only efflux pump was screened from a clinical strain library to explore reverse OqxB-mediated bacterial resistance strategies. We identified non-repetitive clinical isolated K. pneumoniae strains using a matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry clinical TOF-II (Clin-TOF-II) and susceptibility test screening against levofloxacin and ciprofloxacin. And the polymorphism analysis was conducted using pulsed-field gel electrophoresis. Efflux pump function of resistant strains is obtained by combined drug sensitivity test of phenylalanine-arginine beta-naphthylamide (PaβN, an efflux pump inhibitor) and detection with ethidium bromide as an indicator. The quantitative reverse transcription PCR was performed to assess whether the oqxB gene was overexpressed in K. pneumoniae isolates. Additional analyses assessed whether the oqxB gene was overexpressed in K. pneumoniae isolates and gene knockout and complementation strains were constructed. The binding mode of PaβN with OqxB was determined using molecular docking modeling. Among the clinical quinolone-resistant K. pneumoniae strains, one mediates resistance almost exclusively through the overexpression of the resistance-nodulation-division efflux pump, OqxB. Crystal structure of OqxB has been reported recently by N. Bharatham, P. Bhowmik, M. Aoki, U. Okada et al. (Nat Commun 12:5400, 2021, https://doi.org/10.1038/s41467-021-25679-0). The discovery of this strain will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and builds on the foundation for addressing the threat posed by quinolone resistance.IMPORTANCEThe emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of K. pneumoniae infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the K. pneumoniae strain GN 172867 exhibited a higher oqxB gene expression compared to the reference strain ATCC 43816. Deletion of oqxB led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with oqxB rescued antibiotic resistance in the oqxB mutant strain. We demonstrated that the overexpression of the OqxB efflux pump plays an important role in quinolone resistance. The discovery of strain GN 172867 will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and promotes further study of antimicrobial resistance.

Genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China

ObjectivesIn this study, we report the complete genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China. The aim was to investigate its molecular characteristics and resistant mechanism. MethodsA colistin-resistant hvKP was isolated from an inpatient in China. The whole genome was sequenced on Illumina NovaSeq 6000 and long-read ONT platforms. de novo assembly was conducted using SPAdes and Unicycler. S1 nuclease pulsed-field gel electrophoresis, Southern-blot, and antimicrobial susceptibility testing were performed. Sequence type, antimicrobial resistance and virulence-related genes were predicted from the sequence. The circular maps of multiple plasmids comparisons were drawn by the BLAST Ring Image. ResultsThe complete genome sequence of K. pneumoniae ACESH00926 consists of one chromosome and two plasmids. ACESH00926 belongs to K2 ST65 according to the MLST scheme. ACESH00926 showed high resistance to colistin (MIC > 8 µg/mL). Several ARGs were identified, including mobile colistin-resistant gene mcr-8 which was located in an IncFIl(K)/IncFIA(HI1) type plasmid. The bigger plasmid was a pK244-like virulence plasmid. It carrying a series of virulence genes, such as the regulator of the mucoid phenotype (rmpA and rmpA2), salmochelin siderophore biosynthesis (iroB), ABC transporter (iroC), ferric aerobactin receptor (iutA), aerobactin siderophore biosynthesis protein (iucC), and aerobactin synthetase (iucA) encoded genes. And another plasmid carrying mcr-8 with a conserved genetic context (dgkA-sasA-copR-mcr-8-ccdA-ccdB-xerD-repE-parM-umuC-lexA-klcA). ConclusionsIt is necessary to emphasize the necessity of monitoring a combination of colistin-resistant and hypervirulent Klebsiella pneumoniae strains in the future.

Phenotypic and Genetic Characteristics of Carbapenemase-Producing Enterobacterales Isolates at Okayama University Hospital.

Spread of carbapenemase-producing Enterobacterales (CPE) is an ongoing public health issue worldwide, including in Japan. In this study, we investigated the phenotypic and genetic characteristics of CPE isolates at Okayama University Hospital over the 5 years (2013-2018) prior to the outbreak of the 2019 coronavirus pandemic. Of 24 carbapenem-resistant Enterobacterales isolated during the study period, we identified 8 CPE isolates harboring blaIMP-1 (5 isolates) and blaIMP-6 genes (3 isolates). Bacterial species and carbapenem susceptibility patterns exhibited diversity. Minimum inhibitory concentrations (MICs) of meropenem were generally higher than those of imipenem and biapenem. Results of pulsed-field gel electrophoresis demonstrated that neither clonal nor plasmid-mediated outbreaks of blaIMP-harboring CPE isolates have developed at our hospital. One Klebsiella oxytoca isolate showed a high MIC (128 μg/mL) of meropenem, which could be explained by the high plasmid copy number. Subsequent analysis of this isolate may elucidate the intricacies of carbapenem resistance profiles among CPE isolates. Collectively, our findings underscore the necessity for ongoing genetic surveillance of CPE, complemented by tailored approaches for infection prevention and control.

Investigation of the outbreak of Clostridium perfringens using single nucleotide polymorphism analysis for genotyping in Toyama, Japan, 2023.

Clostridium perfringens, which produces C. perfringens enterotoxin (CPE), is a major causative agent of food poisoning owing to its gastrointestinal symptoms. Genotyping is important for identifying the etiological agent in outbreaks of C. perfringens. We attempted to genotype strains isolated from an outbreak of food poisoning in Toyama in 2023 using single nucleotide polymorphism (SNP) analysis. The strains of C. perfringens were isolated from a piece of curry food consumed by all patients and from the feces of the patients and employees. The cpe gene was detected in isolates from patients and curry food. The cpe-negative isolates were found in patients who consumed curry foods and in employees. The results of the SNP analysis suggest that the patient- and curry-derived isolates were likely from the same source but were unlikely to be related to the employee-derived isolates. The results of the SNP and pulsed-field gel electrophoresis (PFGE) analyses were consistent, indicating that the patient- and curry-derived isolates came from the same source. SNP analysis, a whole-genome-based genotyping method, is a promising alternative to the traditional PFGE method. Further studies are needed to accumulate more experience with genotyping using SNP analysis for the epidemiological investigation of outbreaks of C. perfringens.

Isolation of Virulent Leptospira Serogroup Australis Field Strains from Symptomatic Dogs for Canine Leptospiral Vaccine Development.

Leptospirosis is a widespread zoonosis caused by spirochaetes belonging to the pathogenic species of Leptospira, which are classified into more than 25 serogroups and 250 serovars. Vaccination can prevent the disease in dogs but offers incomplete efficacy because of a lack of cross-protection between serogroups. The aim of this study was to validate a robust recruitment and sampling process, with the objectives of isolating and typing circulating Leptospira pathogenic strains and then selecting those of proven virulence and pathogenicity for vaccine development. Blood and urine samples from dogs with clinical syndromes compatible with acute leptospirosis were sterilely collected and transported to a reference laboratory for a micro-agglutination test (MAT), PCR, and bacterial isolation. Isolated strains underwent molecular typing using RNA16S, variable-number tandem repeat (VNTR), and pulsed-field gel electrophoresis (PFGE). Subtyping was performed using core genome multilocus sequence typing (CgMLST). Among 64 included dogs, 41 had MAT and/or PCR results compatible with Leptospira infection, and 14 Leptospira strains were isolated. Based on molecular typing, 11 isolates were classified as L. interrogans serogroup Australis, serovar Bratislava, and 3 as serogroup Icterohaemorrhagiae, serovar Icterohaemorrhagiae. CgMLST subtyping revealed a diversity of clonal groups (CGs) distributed in several regional clusters. Besides validating a robust recruitment and sampling process, this study outlines the value of combining PCR and serological testing when suspecting leptospirosis and the usefulness of implementing molecular typing methods to identify circulating field strains. It also confirms the epidemiological importance of the Australis serogroup and allows for the collection of different highly pathogenic strains for vaccine development.

Antibiotic-resistant Escherichia coli from treated municipal wastewaters and Black-headed Gull nestlings on the recipient river

Wastewaters belong among the most important sources of environmental pollution, including antibiotic-resistant bacteria. The aim of the study was to evaluate treated wastewaters as a possible transmission pathway for bacterial colonisation of gulls occupying the receiving river. A collection of antibiotic-resistant Escherichia coli originating both from treated municipal wastewaters discharged to the river Svratka (Czech Republic) and nestlings of Black-headed Gull (Chroicocephalus ridibundus) living 35 km downstream of the outlet was obtained using selective cultivation. Isolates were further characterised by various phenotyping and genotyping methods.From a total of 670 E. coli isolates (450 from effluents, 220 from gulls), 86 isolates (41 from effluents, 45 from gulls) showed identical antibiotic resistance phenotype and genotype and were further analysed for clonal relatedness using pulsed-field gel electrophoresis (PFGE). Despite the overall high diversity of the isolates, 21 isolates from both sources showed similar PFGE profiles. Isolates belonging to epidemiologically important sequence types (ST131, 15 isolates; ST23, three isolates) were subjected to whole-genome sequencing. Subsequent phylogenetic analysis did not reveal any close clonal relationship between the isolates from the effluents and gulls' nestlings with the closest strains showing 90 SNPs difference.Although our study did not provide direct evidence of transmission of antibiotic-resistant E. coli to wild gulls via treated wastewaters, we observed gull chicks as carriers of diverse multi-resistant E. coli, including high-risk clones, posing risk of further bacterial contamination of the surrounding environment.

Prevalence and Characteristics of Plasmid-Mediated Fosfomycin Resistance Gene fosA3 among Salmonella Enteritidis Isolates from Retail Chickens and Children with Gastroenteritis in China.

A total of 265 Salmonella Enteritidis isolates collected from retail markets and children's hospitals in Shanghai were used to investigate the prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes. Nine of the isolates-7 from the 146 (4.79%) retail chicken-related samples and 2 from the 119 (1.68%) samples from clinical children-were fosfomycin-resistant (FosR). The fosA3 gene was detected in all of the nine FosR isolates, which were located on Inc F-type (8/9, 88.9%) and unknown-type (1/9, 11.1%) transferable plasmids. In total, five plasmid types, namely Inc HI2 (1/9, 11.1%), Inc I1 (3/9, 33.3%), Inc X (8/9, 88.9%), Inc FIIs (9/9, 100%), and Inc FIB (9/9, 100%), were detected in these FosR isolates, which possessed five S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) profiles. The extended-spectrum β-lactamase determinant blaCTX-M-14 subtype was identified in one FosRS. Enteritidis isolate, which was located in a transferable unknown-type plasmid co-carrying fosA3 and tetR genes. Sequence homology analysis showed that this plasmid possessed high sequence similarity to previously reported blaCTX-M-14- and fosA3-positive plasmids from E. coli strains, implying that plasmids carrying the fosA3 gene might be disseminated among Enterobacterales. These findings highlight further challenges in the prevention and treatment of Enterobacteriaceae infections caused by plasmids containing fosA3.

A 5-year study of bloodstream infections caused by carbapenemase-producing Gram-negative bacilli in southern Spain

Introduction. The aim of this study was to evaluate the microbiological epidemiology of carbapenemase-producing Gram-negative bacilli (CPGNB) isolated from blood during a 5-year period. Methods. A total of 80 isolates from 78 patients were finally included; fifty-five (70.5%) were men and the mean age was 60 years. Detection of carbapenemase production was performed by immunocromatography (IC) and polymerase chain reaction (PCR). Genotyping was carried-out by pulsedfield gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), and characterization of carbapenemase-producing isolates was performed by whole genome sequencing (WGS). Results. The main microorganisms isolated were K. pneumoniae (29.4%), E. cloacae (28.2%), A. baumannii (17.9%) and P. aeruginosa (15.3%). Overall, the most common carbapenemase in Enterobacterales was OXA-48 group (57.7%). The most common carbapenemase in non-fermenting bacilli was OXA-23 (60.8%). The most common ST in K. pneumoniae producing OXA-48 types was ST45 and in E. cloacae ST114, while in E. cloacae producing VIM types was ST78. In OXA-23 types, the most common clone in A. baumannii was ST2, whereas in P. aeruginosa producing IMP types was ST253. Conclusions. There was an increase in cases recorded in the years of highest incidence and severity of the SARS-CoV-2 pandemic, with a significant number of cases in patients admitted to the ICU. All bacteremias caused by A. baumannii were caused by the same clone, and 12 of the 14 cases caused by A. baumannii were part of outbreaks in the ICU.