Distinct Sequence Types Research Articles

A novel shiga toxigenic E. coli sequence type harbouring multidrug resistance isolated from raw milk

Abstract The objective was to examine the prevalence of extended-spectrum β-lactam (ESBL) resistance among Shiga toxigenic Escherichia coli (STEC) isolated from raw milk. The findings from disc diffusion analysis and polymerase chain reaction revealed a high occurrence of ESBL resistance, specifically to penicillins, cephalosporins, and carbapenems, in isolated STEC strains. Additionally, a distinct sequence type of STEC was also identified in milk through comparative analysis.

Epidemiological and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae from pediatric patients in Henan, China

PurposeCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an emerging global threat, whereas its epidemiological characteristics in children are rarely reported. This study aims to analyze clinical and epidemiological characteristics of CRKP from children in Henan, China.MethodsCRKP strains were isolated from pediatric patients, and the antimicrobial susceptibility of CRKP was determined using broth microdilution methods. The epidemiological characteristics of CRKP, including specimen sources, clinical data, carbapenemase types, virulence factors, MLST and PBRT typing were analyzed.ResultsIn total, 108 CRKP isolates were isolated from specimens including sputum, blood and urine, mainly from preterm pediatric department and internal medical intensive care unit (ICU). Newborns and staying in the ICU were risk factors for crude mortality. 107 isolates exhibited a multi-drug resistant (MDR) phenotype, and one isolate was extensively drug-resistant (XDR). Bacterial susceptibility to colistin, tigecycline and trimethoprim/sulfamethoxazole was 98.10%, 78.50% and 91.43%, respectively. Carbapenemase blaKPC (86.11%) was predominant, followed by blaNDM (5.56%) and blaIMP (2.78%). Two strains co-harbored blaKPC-blaNDM, one had blaKPC-blaIMP, whereas three isolates did not carry any of the analyzed carbapenemase genes. All strains possessed fimH, and 98% of the isolates possessed mrkD. Hypervirulent factors rmpA2 and iucA showed high positive rates (71.30% and 49.07%), with 48.15% of strains containing both genes. MLST analysis identified nine distinct sequence types (STs), with ST11 (82.41%) being the most common, followed by ST2154 (4.63%) and ST307 (3.70%). PBRT analysis revealed IncFII (85.19%) as the most prevalent plasmid.ConclusionIn summary, this study reported the epidemiological features of CRKP in pediatric patients in Henan, China, highlighting the high prevalence of multi-drug-resistant and hypervirulent strains, and underscoring the significance of continuous surveillance.

Occurrence and diversity of Campylobacter species in diarrheic children and their exposure environments in Ethiopia

Campylobacter is a major zoonotic foodborne pathogen that poses a significant public health threat, particularly among children and immunocompromised individuals. However, data on the occurrence and sources of Campylobacter infection remain scarce in Ethiopia. This study assessed the occurrence, diversity, and relationships between Campylobacter from diarrheic children and potential exposure sources using whole-genome sequencing. Through case-based tracing, animal, food, and environmental samples were collected from Harar town and Kersa district between November 2021 and January 2023. Campylobacter was identified using selective media, and DNA was extracted and sequenced with the Illumina NextSeq 550 instrument. Sequence reads were analysed using bioinformatics tools. The overall Campylobacter prevalence in the exposure sources was 5.5%, with 6.0% in urban and 5.0% in rural settings. Campylobacter detection was 1.8 times more likely in household samples (8.7%; OR = 1.8; 95% CI: 0.7–4.5) than in samples from marketplaces. The occurrence of Campylobacter in food was 4.2%, with no significant differences across the meat, milk, and other food categories. The likelihood of Campylobacter contamination in the environment was 5.8 times higher in the presence of poultry (17.7%; OR = 5.8; CI: 1.1–30.6) compared to shoats. Sequence analysis identified a low Campylobacter spp. diversity comprising only C. jejuni and C. coli, which were characterized by 8 distinct sequence types (STs). Phylogenetically, the majority of the sequenced case isolates were clustered with isolates from either caretakers, environmental exposures, or both. In conclusion, Campylobacter was detected in various exposure sources of diarrheic children, and its occurrence did not differ significantly between Kersa and Harar or among food items. The majority of isolates shared MLST profiles and clustered together, demonstrating the involvement of multiple vectors in the transmission of the pathogen. Genome-based integrated studies supported by an attribution model are recommended to determine the relative contribution of each source.

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

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

The emergence of transposon-driven multidrug resistance in invasive nontypeable Haemophilus influenzae over the last decade

Nontypeable Haemophilus influenzae (NTHi), once considered a harmless commensal, has emerged as a significant concern due to the increased prevalence of multidrug-resistant (MDR) strains and their association with invasive infections. This study aimed to explore the epidemiology and molecular resistance mechanisms of 51 NTHi isolates collected from patients with invasive infections in northern Taiwan between 2011 and 2020. This investigation revealed substantial genetic diversity, encompassing 29 distinct sequence types and 18 clonal complexes. Notably, 68.6% of the isolates exhibited ampicillin resistance, with 28 categorised as MDR and four isolates were even resistant to up to six antibiotic classes. Among the MDR isolates, 18 pulsotypes were identified, indicating diverse genetic lineages. Elucidation of their resistance mechanisms revealed 18 β-lactamase-producing amoxicillin–clavulanate-resistant (BLPACR) isolates, 12 β-lactamase-producing ampicillin-resistant (BLPAR) isolates, and 5 β-lactamase-nonproducing ampicillin-resistant (BLNAR) isolates. PBP3 analysis revealed 22 unique substitutions in BLPACR and BLNAR, potentially contributing to cephem resistance. Notably, novel transposons, Tn7736-Tn7739, which contain critical resistance genes, were discovered. Three strains harboured Tn7739, containing seven resistance genes [aph(3′)-Ia, blaTEM-1, catA, sul2, strA, strB, and tet(B)], while four other strains carried Tn7736, Tn7737, and Tn7738, each containing three resistance genes [blaTEM-1, catA, and tet(B)]. The emergence of these novel transposons underscores the alarming threat posed by highly resistant NTHi strains. Our findings indicated that robust surveillance and comprehensive genomic studies are needed to address this growing public health challenge.

Nationwide surveillance and characterization of the third-generation cephalosporin-resistant Salmonella enterica serovar infantis isolated from chickens in South Korea between 2010 and 2022

The occurrence of extended-spectrum β-lactamase (ESBL)/AmpC β-lactamase-producing Salmonella conferring resistance to third-generation cephalosporin has emerged as a global public health concern. In this study, we aimed to investigate the prevalence and molecular characterization of third-generation cephalosporin-resistant Salmonella enterica serovar Infantis. In total, 409 S. Infatis isolates were collected from the feces and carcasses of healthy and diseased food animals, including chickens (n = 348), pigs (n = 48), cattle (n = 8), and ducks (n = 5) between 2010 and 2022 nationwide in South Korea. Among them, 61.9 % (253/409) of S. Infantis strains displayed resistance to ceftiofur, with the most resistant isolates obtained from chickens (98.4 %, 249/253). Moreover, S. Infantis isolates showed high resistance (47.7–67.2 %) to streptomycin, ampicillin, nalidixic acid, sulfisoxazole, chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole. Additionally, the multidrug resistance (MDR) was significantly greater in the ceftiofur-resistant isolates compared to the ceftiofur-susceptible isolates (p < 0.05). All the ceftiofur-resistant S. Infantis strains produced CTX-M/CMY-2 β-lactamase enzymes, with blaCTX-M-65 comprising the most (98.4 %, 249/253), followed by blaCTX-M-15 (1.2 %, 3/253), and blaCMY-2 (0.4 %, 1/253). The ceftiofur-resistant S. Infantis belonged to 37 different pulsotypes, with X1A1 (26.1 %, 66/253), X1A2 (20.9 %, 53/253), and X5A3 (9.1 %) being the most prevalent, representing a total of 56.1 % (142/253). Furthermore, the S. Infantis sequence type (ST)32 was the most common, accounting for 91.9 % (34/37) of the three distinct STs (ST32, ST16, and ST11) detected across farms located in various provinces nationwide. Most of the blaCMX-M-65 genes (77.5 %, 193/249), all of the blaCTX-M-15 genes (100 %, 3/3), and the blaCMY-2 gene (100 %, 1/1) were transferred to the recipient E. coli RG488 by conjugation. In addition, the majority of the transconjugants (98.9 %, 191/193) containing blaCTX-M-65 genes belong to the IncFIB replicon type, playing an important role in the quick and widespread dissemination of S. Infantis. Thus, ceftiofur-resistant S. Infantis carrying the β-lactamase genes in chickens has the potential to be transmitted to humans.

Multi-locus sequence typing and genetic diversity of antibiotic-resistant genes and virulence-associated genes in Burkholderia pseudomallei: Insights from whole genome sequencing of animal and environmental isolates in Thailand

Burkholderia pseudomallei is a Gram-negative bacillus and the etiological agent of melioidosis in humans and animals. The disease is highly endemic in northern Australia and Southeast Asia. Comprehensive genomic data are essential for understanding the bacteria's dissemination and genetic relationships among strains from different geographical regions. In this study, we conducted antimicrobial susceptibility testing and whole-genome sequencing of 54 B. pseudomallei isolates obtained from environmental and animal sources in southern Thailand between 2011 and 2018. Their genomics were determined of antibiotic-resistant genes (ARGs), virulence-associated genes, mobile genetic elements (MGEs), sequence types (STs), and single nucleotide polymorphisms (SNPs) to evaluate their epidemiological relatedness. Remarkably, all 54 isolates displayed sensitivity to antimicrobial agents typically used for melioidosis treatment. We identified nine distinct sequence types: ST392, ST51, ST409, ST508, ST376, ST1721, ST389, ST395, and ST289. Oxacillinase genes and the resistance nodulation family of efflux pumps (RND) were identified as contributors to antimicrobial resistance. Phylogenetic analysis demonstrated close genetic relations with other strains isolated from Southeast Asia. Furthermore, 172 virulence-associated genes were identified among the isolates, suggesting variations in clinical presentations. These findings underscore the importance of ongoing molecular genetic surveillance of B. pseudomallei for effective healthcare management and reducing melioidosis mortality.

The Genomic Epidemiology of Clinical Burkholderia pseudomallei Isolates in North Queensland, Australia.

Background:Burkholderia pseudomallei, the causative agent of melioidosis, is highly genetically recombinant, resulting in significant genomic diversity. Multiple virulence factors have been associated with specific disease presentations. To date, there are limited data relating to genomic diversity and virulence factors associated with melioidosis cases in North Queensland, Australia. Aim: To describe the genetic diversity of B. pseudomallei and identify virulence factors associated with clinical risk factors and patient outcomes. Methods: Whole genome sequencing of clinical isolates was performed and analysed with clinical data obtained from a retrospective melioidosis cohort study. Results: Fifty-nine distinct sequence types (STs) were identified from the 128 clinical isolates. Six STs comprised 64/128 (50%) isolates. Novel STs accounted for 38/59 (64%) STs, with ST TSV-13 as the most prevalent (n = 7), and were less likely to possess an LPS A genotype or YLF gene cluster (p < 0.001). These isolates were most likely to be found outside the inner city (aOR: 4.0, 95% CI: 1.7-9.0, p = 0.001). ST TSV-13 was associated with increased mortality (aOR: 6.1, 95% CI: 1.2-30.9, p = 0.03). Patients with a history of alcohol excess were less likely to be infected by fhaB3 (aOR 0.2, 95% CI: 0.1-0.7, p = 0.01) or YLF (aOR: 0.4, 95% CI: 0.2-0.9, p = 0.04) positive isolates. Conclusions: There are a significant number of novel sequence types in Townsville, Australia. An emerging novel ST appears to have an association with geographic location and mortality. Ongoing investigation is required to further understand the impact of this ST on the Townsville region.

New Insights into Molecular Characterization, Antimicrobial Resistance and Virulence Factors of Methicillin-Sensitive Coagulase-Positive Staphylococcus spp. from Dogs with Pyoderma and Otitis Externa

The first Tunisian national molecular survey of coagulase-positive staphylococci (CoPS) isolated from dogs with pyoderma and otitis externa was conducted to evaluate the prevalence of CoPS and identify its phenotypic and genotypic diversities. A total of 99 out of the 195 samples collected from 39 sick dogs were identified across multiple sites as methicillin-susceptible CoPS belonging to the species S. pseudintermedius (64.4%), S. aureus (20.2%), S. coagulans (10.1%), and S. hyicus (5%). Fifteen sampled dogs carried more than one Staphylococcus species. Their antibiotic resistance and virulence factors were determined using conventional and molecular methods. Of the S. pseudintermedius isolates found, 17.4% were multidrug-resistant, whereas high rates of virulence genes were observed among the S. aureus isolates. On polystyrene surfaces, 75% of S. aureus isolates were biofilm producers, of which 15% were classified as strong producers. The capsular polysaccharide cap8 genotype was predominant among them. A MultiLocus Sequence Typing (MLST) analysis clustered the S.aureus isolates into five distinct sequence types (STs), with four assigned for the first time. Our findings highlight the spread of CoPS among diseased dogs and, especially, the emergence of S. hyicus, S. coagulans, multidrug-resistant S. pseudintermedius and S. aureus isolates with high genetic variability. The precise characterization of these strains, as well as their continuous monitoring, is necessary for the implementation of preventive strategies given the significant public health risk.

Prevalence, antibiotic susceptibility, and genomic analysis of Vibrio alginolyticus isolated from seafood and freshwater products in China.

This study characterized Vibrio alginolyticus isolated from seafood and freshwater products in China (2020). In total, 122 (95.31%) V. alginolyticus isolates were resistant to at least 1 antibiotic category, and 2 (1.56%) isolates were resistant to at least 3 antibiotic categories and belong to multi-drug resistance (MDR) isolates. A high prevalence rate was observed to be blaCARB (98.04%) encoding beta-lactam resistance, followed by tet (97.06%) encoding tetracycline resistance and fos (4.90%) encoding resistance to fosfomycin. Among the 57 V. alginolyticus isolates, the commonest virulence genes were type III secretion system translocated gene vopD, vopB, and vcrH (54.4%, 31/57), type III secretion system regulated gene tyeA (54.39%), followed by vscI and vscF (50.88%) encoded type III secretion system inner rod protein and needle protein, respectively. Multilocus sequence typing (MLST) showed considerable genetic diversity, with 34 distinct sequence types (STs) identified among 55 isolates. ST421 (n = 5), ST166 (n = 4), ST523 (n = 3), ST516 (n = 3), and ST507 (n = 3) were dominant STs among 55 V. alginolyticus isolates. These findings highlight the widespread occurrence of V. alginolyticus in both freshwater and seafood products, underscoring the critical need for vigilant monitoring of these bacteria. Such measures are essential for ensuring effective food safety management and safeguarding public health.

Resistance to aztreonam-avibactam among clinical isolates of Escherichia coli is primarily mediated by altered penicillin-binding protein 3 and impermeability

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25–4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Rapid Dissemination of blaNDM-5 Gene among Carbapenem-Resistant Escherichia coli Isolates in a Yellow-Feather Broiler Farm via Multiple Plasmid Replicon.

Although carbapenems have not been approved for animal use, carbapenem-resistant Escherichia coli (CREC) strains are increasingly being detected in food-producing animals, posing a significant public health risk. However, the epidemiological characteristics of CREC isolates in yellow-feather broiler farms remain unclear. We comprehensively investigated the genetic features of carbapenem-resistance genes among E. coli isolates recovered from a yellow-feather broiler farm in Guangdong province, China. Among the 172 isolates, 88 (51.2%) were recovered from chicken feces (88.5%, 54/61), the farm environment (51.1%, 24/47), and specimens of dead chickens (15.6%, 41/64). All CREC isolates were positive for the blaNDM-5 gene and negative for other carbapenem-resistance genes. Among 40 randomly selected isolates subjected to whole-genome sequencing, 10 belonged to distinct sequence types (STs), with ST167 (n = 12) being the most prevalent across different sources, suggesting that the dissemination of blaNDM-5 was mainly due to horizontal and clonal transmission. Plasmid analysis indicated that IncX3, IncHI2, and IncR-X1-X3 hybrid plasmids were responsible for the rapid transmission of the blaNDM-5 gene, and the genetic surrounding of blaNDM-5 contained a common mobile element of the genetic fragment designated "IS5-△ISAba125-blaNDM-5-bleMBL-trpF-dsbC". These findings demonstrate a critical role of multiple plasmid replicons in the dissemination of blaNDM-5 and carbapenem resistance.

Global Emergence and Genomic Epidemiology of blaNDM-Carrying Klebsiella variicola.

Klebsiella variicola has emerged as a human pathogen in the past decade. Here, we present findings related to a K. variicola strain carrying the blaNDM-1 gene, which was isolated from a urinary tract infection in China. Global transmission dynamics and genomic epidemiology of blaNDM-carrying K. variicola were further investigated. The complete genome sequence of the strain was determined using the Illumina NovaSeq 6000 and Nanopore MinION sequencer. Genomic features and resistance mechanisms were analyzed through diverse bioinformatics approaches. Additionally, genome sequences of K. variicola strains carrying blaNDM were retrieved from the NCBI database, and a comprehensive analysis of the global dissemination trends of these strains was conducted. K. variicola strain 353 demonstrated resistance to multiple antimicrobials, including carbapenems. Within its genome, we identified fourteen antimicrobial resistance genes associated with β-lactam, aminoglycoside, fosfomycin, quinolone, trimethoprim, rifamycin, and sulfonamide resistance. The carbapenem-resistant gene blaNDM-1 was located on an IncU-type plasmid spanning 294,608 bp and flanked by ISCR1 and IS26. Downstream of blaNDM-1, we identified an Intl1 element housing numerous antibiotic resistance genes. A comprehensive search of the NCBI database revealed 72 K. variicola strains carrying blaNDM from twelve different countries, predominantly from clinical sources, with the highest prevalence observed in the USA and China. A total of 28 distinct sequence types (STs) were identified, with ST115 being the most prevalent, followed by ST60. In summary, this study presents the genomic characterization of a K. variicola strain carrying blaNDM-1 on an IncU-type plasmid. The research highlights the global dissemination of blaNDM-carrying K. variicola, observed in both healthcare settings and natural environments. Our data have revealed a diverse array of antimicrobial resistance determinants in K. variicola, providing valuable insights that could aid in the development of strategies for the prevention, diagnosis, and treatment of K. variicola infections.

Sequence-subtype association of multi-drug-resistant diarrheagenic Escherichia coli

Background and Objectives: Multi-drug-resistant pathogens pose a significant threat as they can rapidly spread, leading to severe healthcare-associated invasive infections. In developing countries, diarrheagenic Escherichia coli (DEC) is a major bacterial pathogen responsible for causing diarrhea. However, the outbreak of resistant strains has made the treatment of DEC infections much more challenging. This study aimed to investigate the relationship between antibiotic resistance genes and other virulence categories in E. coli strains that cause diarrhea, particularly DEC. Materials and Methods: The phylogenetic grouping was defined using PCR and multi-locus sequence type (MLST) meth- ods. Results: Among the isolates analyzed, 14 were identified as resistant and were classified into eight distinct sequence types: ST3, ST53, ST77, ST483, ST512, ST636, ST833, and ST774, indicating genetic diversity among the resistant strains. Certain sequence types, notably ST512 and ST636, were found to be associated with multiple antibiotic resistance in DEC. Regard- ing antibiotic susceptibility, strains showed the highest resistance to amoxicillin, suggesting that this antibiotic may not be effective in treating DEC infections. On the other hand, the isolates demonstrated susceptibility to amikacin and chloram- phenicol, implying that these antibiotics could be more suitable treatment options for DEC infections. Conclusion: The findings underscore the importance of promptly identifying antibiotic resistance patterns and their correla- tion with specific pathogenic virulence categories, as this knowledge can aid in selecting the most appropriate antibiotics for treating DEC infections. Considering the antibiotic resistance profiles and associated resistance genes is crucial in managing and containing diarrheal outbreaks and in selecting effective antibiotic therapies for DEC infections.

Genomic characteristics of clinical non-toxigenic Vibrio cholerae isolates in Switzerland: a cross-sectional study.

Although non-toxigenicVibrio choleraelack thectxABgenes encoding cholera toxin, they can cause diarrhoeal disease and outbreaks in humans. In Switzerland,V. choleraeis a notifiable pathogen and all clinical isolates are analysed at the National Reference Laboratory for Enteropathogenic Bacteria and Listeria. Up to 20 infections are reported annually. In this study, we investigated the population structure and genetic characteristics of non-toxigenicV. choleraeisolates collected over five years. V. choleraeisolates were serotyped and non-toxigenic isolates identified using actxA-specific PCR. Following Illumina whole-genome sequencing, genome assemblies were screened for virulence and antibiotic resistance genes. Phylogenetic analyses were performed in the context of 965 publicly availableV. choleraegenomes. Out of 33V. choleraeinfections reported between January 2017 and January 2022 in Switzerland, 31 were caused byctxA-negative isolates. These non-toxigenic isolates originated from gastrointestinal (n = 29) or extraintestinal (n = 2) sites. They were phylogenetically diverse and belonged to 29 distinct sequence types. Two isolates were allocated to the lineage L3b, actxAB-negative buttcpA-positive clade previously associated with regional outbreaks. The remaining 29 isolates were placed in lineage L4, which is associated with environmental strains. Genes or mutations associated with reduced susceptibility to the first-line antibiotics fluoroquinolones and tetracyclines were identified in 11 and 3 isolates, respectively. One isolate was predicted to be multidrug resistant. V. choleraeinfections in Switzerland are rare and predominantly caused by lowly virulentctxAB-negative andtcpA-negative strains. AsV. choleraeis not endemic in Switzerland, cases are assumed to be acquired predominantly during travel. This assumption was supported by the phylogenetic diversity of the analysed isolates.

Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant Enterobacter cloacae complex from ICU patients in Kolkata, India, during 2017-2022: emergence of phylogenetically heterogeneous Enterobacter hormaechei subsp. xiangfangensis.

Blood-borne infections caused by the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (n = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified Enterobacter hormaechei subsp. xiangfangensis (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum β-lactamase genes found were blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of ampC, overexpression of acrAB, and loss of ompF. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed Enterobacter hormaechei subsp. xiangfangensis as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.

Emergence of KPC-2 and NDM-5-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae with high-risk sequence types ST11 and ST15.

The emergence of Klebsiella pneumoniae carbapenemase-2 (KPC-2) and New Delhi metallo-β-lactamase (NDM)-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae (KPC-2-NDM-hv-CRKP) poses a certain threat to public health. Currently, only a few sporadic reports of such double-positive hv-CRKPs were available. In this study, we isolated two KPC-2-NDM-5-hv-CRKPs from elderly patients with serious underlying diseases and poor prognoses. We found both FK3122 and FK3127 were typical multidrug-resistant (MDR) isolates, exhibiting high-level resistance to both carbapenems and novel β-lactamase inhibitors ceftazidime/avibactam. Notably, FK3122 is even resistant to cefiderocol due to multiple blaNDM-5 elements. Besides the MDR phenotype, A549 human lung epithelial cells and Galleria mellonella infection model all indicated that FK3122 and FK3127 were highly pathogenic. According to the whole-genome sequencing analysis, we observed over 10 resistant elements, and the uncommon co-existence of blaKPC-2, blaNDM-5, and virulence plasmids in both two isolates. Both virulence plasmids identified in FK3122 and FK3127 shared a high identity with classical virulence plasmid pK2044, harboring specific hypervirulent factors: rmpA and iuc operon. We also found that the resistance and virulence plasmids in FK3127 could not only be transferred to Escherichia coli EC600 independently but also together as a co-transfer, which was additionally confirmed by the S1-pulsed-field gel electrophoresis plasmid profile. Moreover, polymorphic mobile genetic elements were found surrounding resistance genes, which may stimulate the mobilization of resistance genes and result in the duplication of these elements. Considering the combination of high pathogenicity, limited therapy options, and easy transmission of KPC-2-NDM-5-hv-CRKP, our study emphasizes the need for underscores the imperative for ongoing surveillance of these pathogens.IMPORTANCEHypervirulent Klebsiella pneumoniae drug resistance has increased gradually with the emergence of carbapenem-resistant hypervirulent K. pneumoniae (hv-CRKP). However, little information is available on the virulence characteristics of the New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase-2 (KPC-2) co-producing K. pneumoniae strains. In this study, we obtained two KPC-2-NDM-hv-CRKPs from elderly patients, each with distinct capsule types and sequence types: ST11-KL64 and ST15-KL24; these ST-type lineages are recognized as classical multidrug-resistant (MDR) K. pneumoniae. We found these KPC-2-NDM-hv-CRKPs were not only typical MDR isolates, including resistance to ceftazidime/avibactam and cefiderocol, but also displayed exceptionally high levels of pathogenicity. In addition, these high-risk factors can also be transferred to other isolates. Consequently, our study underscores the need for ongoing surveillance of these isolates due to their heightened pathogenicity, limited therapeutic options, and potential for easy transmission.

Sequence Stratigraphic Analysis of the Volcanic Rock–Evaporite–Clastic Rock Symbiotic Zone in the Fourth Member of the Shahejie Formation in the South of Laizhou Bay

Laizhou Bay Sag is a typical continental rift lake basin, which is affected by strike–slip activity, salt rock activity, volcanic activity, and paleoclimate change in the Tanlu fault zone, where three major lithologies—volcanic rock, clastic rock, and evaporite—mainly develop. In order to determine the sequence correspondence between the different lithologies in the study area based on drilling, logging data, and seismic data, it is determined that the fourth member of the Shahejie Formation in the southern Laizhou Bay mainly develops three types of sequences: clastic rock type sequences, salt lake type sequences, and volcanic rock/clastic rock type sequences. Two second-order sequence boundaries (SBs3L, SBs4L) and one third-order sequence boundary (SBs4U) were identified, and the principle of three divisions of system tracts (LST, TST, HST) and sedimentary processes in different types of sequences were determined by analyzing the stacking patterns of different sequences. Compared with the previous research results, this study provides a more detailed analysis of the sequence boundary definition of different blocks and different lithologic strata, and it improves the previous general sequence stratigraphic division method. Based on the study of distinct types of sequences, a unified stratigraphic framework is established in the whole zone, and the stratum thickness distribution of the fourth member of Shahejie Formation is smaller in the southwest and larger in the northeast. The study area is generally filled with pyroclastic rocks–evaporite–clastic rocks, changing from fan delta–saline lake filling to braided river delta–freshwater lake–fan delta filling from bottom to top. A sequence stratigraphic model for the comparison of steep slope–gentle slope differences is established to provide guidance for future oil and gas field exploration and development.

Infection with Carbapenem-resistant Hypervirulent Klebsiella Pneumoniae: clinical, virulence and molecular epidemiological characteristics

BackgroundCarbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is gradually becoming the dominant nosocomial pathogens in the healthcare setting.MethodsA retrospective study was conducted on patients with CR-KP from July 2021 to May 2022 in a teaching hospital. We identified bacterial isolates, collected the clinical data, and performed antimicrobial susceptibility testing, hypermucoviscosity string test, antimicrobial and virulence-associated genotype, as well as multi-locus sequence typing. CR-hvKP was defined as the presence of some combination of rmpA and/or rmpA2 with iucA, iroB, or peg-344. SPSS was used for data analysis. Univariate logistic regression analyses were used for risk factor and all statistically significant variables were included in the multivariate model. Statistical significance was taken to be P < 0.05.ResultsA total of 69 non-duplicated CR-KP isolates were collected, 27 of which were CR-hvKP. Out of the 69 CR-KP strains under investigation, they were distributed across 14 distinct sequence types (STs), wherein ST11 exhibited the highest prevalence, constituting 65.2% (45/69) of the overall isolates. The principal carbapenemase genes identified encompassed blakpc−2, blaNDM−1, and blaOXA−48, with blakpc−2 prevailing as the predominant type, accounting for 73.9% (51/69). A total of 69 CR-KP strains showed high resistance to common clinical antibiotics, with the exception of ceftazidime/avibactam. The ST11 (P = 0.040), ST65 (P = 0.030) and blakpc−2 ST11 clones (P = 0.010) were found to be highly related to hvKp. Regarding the host, tracheal intubation (P = 0.008), intracranial infection (P = 0.020) and neutrophil count (P = 0.049) were significantly higher in the patients with CR-hvKP. Multivariate analysis showed tracheal intubation to be an independent risk factor for CR-hvKP infection (P = 0.030, OR = 4.131). According to the clinical data we collected, tracheal intubation was performed mainly in the elderly with severe underlying diseases, which implied that CR-hvKP has become prevalent among elderly patients with comorbidities.ConclusionsThe prevalence of CR-hvKP may be higher than expected in the healthcare setting. CR-hvKP is gradually becoming the dominant nosocomial pathogen, and its prevalence and treatment will be a major challenge. It is essential to enhance clinical awareness and management of CR-hvKP infection.

Resistome, mobilome, virulome analysis and phylogenomics of Enterococcus faecalis isolated from raw muscle foods of beef origin in Gauteng, South Africa

Enterococcus faecalis is a ubiquitous bacterium found in various environments, including processed beef meat, and is known for its importance in both food safety and public health. This pivotal significance stems not solely from its virulence but also from its adeptness in eliciting multidrug-resistant infections in humans. The aim of this study was to investigate the population structure, resistome, mobilome, and virulome of E. faecalis obtained from processed beef meat sources in South Africa. A total of eight genomes sequenced in this study were examined, alongside 78 publicly available, high-quality genomes of E. faecalis, with a comprehensive analysis conducted to identify antimicrobial resistance (AMR) determinants, virulence factors, and mobile genetic elements (MGE). Six distinct sequence types (STs) (ST79, ST860, ST40, ST238, ST21, and ST700) and 41 core virulence factors were found across all the genomes. The virulence factors included genes encoding adherence (ace, asa1, Ef0485, ebpA, ebpB, ebpC, srtC); exoenzyme (Ef3023, Ef0818, gelE, sprE); immunomodulation (cpsA, cpsB, cpsC, cpsD, cpsE, cpsF, cpsG, cpsH, cpsI, cpsK), and biofilm formation (bopD, fsrA, fsrB, fsrC). In addition, AMR genes were identified across all genomes, which include aminoglycoside resistance (ant(6)-Ia), trimethoprim resistance (dfrA), drug and biocide resistance (efrA and efrB), multidrug efflux pump (emeA), clindamycin quinupristin-dalfopristin, dalfopristin resistance (lsaA), and tetracycline resistance (tetM). The genomes of E. faecalis sequenced here contained a variety of MGEs, including Insertion Sequences (ISs), transposons, prophages, and plasmids, which may have facilitated genetic exchange within and between these species. The results highlight that beef meat products act as a reservoir for virulent E. faecalis strains possessing antibiotic-resistance traits. This study provides insight into the genomic characteristics, antimicrobial resistance genes, virulence factors, and genetic mobile elements associated with eight E. faecalis isolates from processed beef meat in the Gauteng province of South Africa.