Multilocus Sequence Typing Research Articles

Genetic diversity, biofilm formation, and Vancomycin resistance of clinical Clostridium innocuum isolates

BackgroundClostridium innocuum, previously considered a commensal microbe, is a spore-forming anaerobic bacterium. C. innocuum displays inherent resistance to vancomycin and is associated with extra-intestinal infections, antibiotic-associated diarrhea, and inflammatory bowel disease. This study seeks to establish a multilocus sequence typing (MLST) scheme to explore the correlation between C. innocuum genotyping and its potential pathogenic phenotypes.MethodsFifty-two C. innocuum isolates from Linkou Chang Gung Memorial Hospital (CGMH) in Taiwan and 60 sequence-available C. innocuum isolates from the National Center for Biotechnolgy Information Genome Database were included. The concentrated sequence of housekeeping genes in C. innocuum was determined by amplicon sequencing and used for MLST and phylogenetic analyses. The biofilm production activity of the C. innocuum isolates was determined by crystal violet staining.ResultsOf the 112 C. innocuum isolates, 58 sequence types were identified. Maximum likelihood estimation categorized 52 CGMH isolates into two phylogenetic clades. These isolates were found to be biofilm producers, with isolates in clade I exhibiting significantly higher biofilm production than isolates in clade II. The sub-inhibitory concentration of vancomycin seemed to minimally influence biofilm production by C. innocuum isolates. Nevertheless, C. innocuum embedded in the biofilm structure demonstrated resistance to vancomycin treatments at a concentration greater than 256 µg/mL.ConclusionsThis study suggests that a specific genetic clade of C. innocuum produces a substantial amount of biofilm. Furthermore, this phenotype assists C. innocuum in resisting high concentrations of vancomycin, which may potentially play undefined roles in C. innocuum pathogenesis.

Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms

BackgroundThe salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187).ResultsThe Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint.ConclusionThe prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Draft genomes of multidrug-resistant Campylobacter jejuni and Campylobacter coli strains from Brazil representing novel sequence types.

Whole-genome sequencing identified three previously unidentified multilocus sequence types of Campylobacter jejuni (ST-12332) and Campylobacter coli (ST-12333 and ST-12663), harboring resistance genes for multiple antimicrobial classes. The sources of isolation highlight the circulation of resistance strains within animals and humans, emphasizing the need for preventive measures.

Case Report: Soft tissue infection with Burkholderia thailandensis capsular variant: case report from the Lao PDR

Background Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandednisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.

Co-Occurrence of Two Plasmids Encoding Transferable blaNDM-1 and tet(Y) Genes in Carbapenem-Resistant Acinetobacter bereziniae.

Acinetobacter bereziniae has emerged as a significant human pathogen, acquiring multiple antibiotic resistance genes, including carbapenemases. This study focuses on characterizing the plasmids harboring the blaNDM-1 and tet(Y) genes in two carbapenem-resistant A. bereziniae isolates (UCO-553 and UCO-554) obtained in Chile during the COVID-19 pandemic. Methods: Antibiotic susceptibility testing was conducted on UCO-553 and UCO-554. Both isolates underwent whole-genome sequencing to ascertain their sequence type (ST), core genome multilocus sequence-typing (cgMLST) profile, antibiotic resistance genes, plasmids, and mobile genetic elements. Conjugation experiments were performed for both isolates. Results: Both isolates exhibited broad resistance, including resistance to carbapenems, third-generation cephalosporins, fluoroquinolones, tetracycline, cotrimoxazole, and aminoglycosides. Both isolates belong to sequence type STPAS1761, with a difference of 17 out of 2984 alleles. Each isolate carried a 47,274 bp plasmid with blaNDM-1 and aph(3')-VI genes and two highly similar plasmids: a 35,184 bp plasmid with tet(Y), sul2, aph(6)-Id, and aph(3″)-Ib genes, and a 6078 bp plasmid containing the ant(2″)-Ia gene. Quinolone-resistance mutations were identified in the gyrA and parC genes of both isolates. Importantly, blaNDM-1 was located within a Tn125 transposon, and tet(Y) was embedded in a Tn5393 transposon. Conjugation experiments successfully transferred blaNDM-1 and tet(Y) into the A. baumannii ATCC 19606 strain, indicating the potential for horizontal gene transfer. Conclusions: This study highlights the critical role of plasmids in disseminating resistance genes in A. bereziniae and underscores the need for the continued genomic surveillance of this emerging pathogen. The findings emphasize the importance of monitoring A. bereziniae for its potential to cause difficult-to-treat infections and its capacity to spread resistance determinants against clinically significant antibiotics.

Phylogenetic Analysis Based on Whole Genome Sequences, Antibiotic Resistance, and Virulence of Salmonella enterica Clinical Isolates from South Korea.

Salmonella is a major cause of foodborne disease and frequently causes human salmonellosis in South Korea. In this study, we investigated the genome diversity, antimicrobial resistance, and virulence of clinical isolates of Salmonella enterica from South Korea. We collected 42 S. enterica subsp. enterica isolates from two hospitals in South Korea. Whole genome sequences were determined. Serovars and sequence types (STs) based on multilocus sequence typing (MLST) were identified from whole genome sequences. Phylogenetic trees based on whole genome sequences and a minimum spanning tree based on MLST were constructed. Human serum resistance assays and gentamicin protection assays were performed to assess in vitro virulence. Nineteen serovars were identified among 42 clinical isolates, including nine Salmonella Typhi isolates. There were inconsistencies between serogroups and phylogenetic clusters in the phylogenetic tree and minimum spanning tree, but high clonality of S. Typhi was observed. Salmonella Typhi isolates were divided into two clusters, corresponding to ST1 and ST2. Isolates of serovars Typhimurium and I4,[5],12:i:- clustered into a group, and a hybrid isolate between the two serovars was identified. Four ciprofloxacin-resistant isolates were identified among nine S. Typhi isolates, and all isolates of S. Enteritidis and S. Panama were resistant to colistin. The gentamicin protection assay revealed that serogroup D1 was significantly less virulent than the other serogroups. Our study suggests high diversity of S. enterica clinical isolates from South Korea and non-monophyly of serogroups. In addition, subgroups of S. Typhi isolates and a hybrid isolate between serovars Typhimurium and I4,[5],12:i:- were identified.

Molecular epidemiology of Streptococcus pneumoniae isolates causing invasive and noninvasive infection in Ethiopia

Streptococcus pneumoniae, a medically important opportunistic bacterial pathogen of the upper respiratory tract, is a major public health concern, causing a wide range of pneumococcal illnesses, both invasive and noninvasive. It is associated with significant global morbidity and mortality, including pneumonia, meningitis, sepsis, and acute otitis media. The major purpose of this study was to determine the molecular epidemiology of Streptococcus pneumoniae strains that cause invasive and noninvasive infections in Ethiopia. A prospective study was undertaken in two regional hospitals between January 2018 and December 2019. Whole-genome sequencing was used to analyze all isolates. Serotypes and multilocus sequence types (MLST) were derived from genomic data. The E-test was used for antimicrobial susceptibility testing. Patient samples obtained 54 Streptococcus pneumoniae isolates, 33 from invasive and 21 from noninvasive specimens. Our findings identified 32 serotypes expressed by 25 Global Pneumococcal Sequence Clusters (GPSCs) and 42 sequence types (STs), including 21 new STs. The most common sequence types among the invasive isolates were ST3500, ST5368, ST11162, ST15425, ST15555, ST15559, and ST15561 (2/33, 6% each). These sequence types were linked to serotypes 8, 7 C, 15B/C, 16 F, 10 A, 15B, and 6 A, respectively. Among the noninvasive isolates, only ST15432, associated with serotype 23 A, had numerous isolates (4/21, 19%). Serotype 14 was revealed as the most resistant strain to penicillin G, whereas isolates from serotypes 3, 8, 7 C, and 10 A were resistant to erythromycin. Notably, all serotype 6 A isolates were resistant to both erythromycin and penicillin G. Our findings revealed an abnormally significant number of novel STs, as well as extremely diversified serotypes and sequence types, implying that Ethiopia may serve as a breeding ground for novel STs. Recombination can produce novel STs that cause capsular switching. This has the potential to influence how immunization campaigns affect the burden of invasive pneumococcal illness. The findings highlight the importance of continuous genetic surveillance of the pneumococcal population as a vital step toward enhancing future vaccine design.

Molecular Characterization of Listeria monocytogenes in the Food Chain of the Republic of Kosovo from 2016 to 2022.

The present study describes the genetic characterization of L. monocytogenes strains found in the Republic of Kosovo's food chain. From 2016 to 2022, 995 samples were collected. Overall, 648 samples were from ready-to-eat (RTE) food products, 281 from food products consumed cooked (FPCC), 60 from raw materials, and 6 from environmental samples. Overall, 11.76% (117 out of 995) of the samples were contaminated by L. monocytogenes, comprising 6.33% (41 out of 648) from RTE products, 14.95% (42 out of 281) from FPCC, 55.00% (33 out of 60) from raw materials, and 16.66% (1 out of 6) from environmental samples. All isolates were subjected to molecular serotyping and clonal complex (CC) identification by using real-time PCR, as well as multilocus sequence typing. All isolates were grouped into four molecular serotypes, IIa (34.19%), IIb (3.48%), IIc (32.48%), and IVb (29.91%), as well as Lineage I (33.33%) and Lineage II (66.66%). In total, 14 CCs were identified from 41 RTE isolates; however, CC29 (7), CC2 (6), and CC6 (6) were the most dominant. By contrast, CC9 was by far the most represented CC in both FPCC (21) and RM (14). Moreover, 30 isolates expressed CC1, CC2, CC4, or CC6, which are particularly associated with severe human infections.

Prevalence of Wolbachia infection in field natural population of the apricot seed wasp Eurytoma samsonowi (Hymenoptera: Eurytomidae).

Obligate endosymbiont bacteria associated with insects are naturally providing their hosts with essential nutrients such as vitamins and amino acids and biological services including protection from pathogens. In this study, we aimed to investigate the presence of Wolbachia infection among males and females of the parasitic apricot seed wasp (ASW) Eurytoma samsonowi Vassiliev (Vassiliev Petrograd 11: 1-15, 1915) (Hymenoptera: Eurytomidae), a very harmful pest of apricot (Prunus armeniaca), in the oasis of Gafsa, Southern-West of Tunisia. The detection of Wolbachia infection was assessed based on the amplification of the Wolbachia surface protein (wsp) gene and a multilocus sequence typing (MLST) as a universal genotyping tool for Wolbachia involving the analyses of genes gatB, coxA, hcpA, fbpA, and ftsz. Confirming the screening results, Wolbachia was detected in the natural apricot wasp for the first time, with a significant difference between males (5%) and females (59%) based on wsp gene. All Wolbachia strains identified in E. samsonowi were clustered among supergroups B of Wolbachia.

Multilocus sequence typing and phenotypic properties of Streptococcus mutans from Thai children with different caries statuses

BackgroundStreptococcus mutans is studied for its acidogenic and aciduric characteristics, notably its biofilm formation in the presence of sucrose, toward its role in the caries process. Variations in both genotype and phenotype have been reported among clinical isolates of S. mutans. This study aimed to examine genotypic and phenotypic characteristics of S. mutans obtained from Thai children with varying caries statuses.MethodsWe determined the presence of S. mutans and caries status in 395 children aged 3–4 years. From 325 children carrying S. mutans, we selected 90 with different caries statuses—caries-free (CF; n = 30), low severity of caries (LC; n = 30), or high severity of caries (HC; n = 30). Three isolates of S. mutans were taken from each child, thus, a total of 270 isolates were obtained. Multilocus sequence typing (MLST) was used to genotype the isolates and assess their clonal relationships. The properties, including biofilm formation, collagen binding, and acid production and tolerance were also evaluated.ResultsChildren with carious lesions showed a higher detection rate and number of S. mutans in saliva than those without caries. S. mutans from individuals with HC status showed the lowest biofilm formation ability, while this group had the highest detection rate of collagen-binding isolates. There was no difference in acid production or tolerance by caries status. Genotyping by MLST did not reveal any clone of S. mutans specific to CF status. This result remained even when we included MLST data from the open-access PubMLST database. MLST did identify clones containing only strains from caries-affected hosts, but tests of their phenotypic properties did not reveal any differences between S. mutans from these clones and clones that were from both caries-free and caries-affected children.ConclusionsThe clonal relationships of S. mutans indicated by MLST were not associated with the status of dental caries in the host.

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

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

Genotypes and Phylogenetic Analysis of Helicobacter pylori Clinical Bacterial Isolates

Helicobacter pylori is a human pathogen bacterium associated with gastritis, peptic ulcer, and gastric cancer. It can be identified through the 16S rRNA gene and characterized through cagA and vacA virulence genes. Clinical cultures of H. pylori were isolated and identified from human stomach biopsies. The isolates were characterized according to their colonial and microscopic morphology, and molecular genotyping was conducted to determine the bacterial virulence. A phylogenetic analysis of the 16S rRNA gene sequencing was performed. In addition, multilocus sequence typing analysis was performed to determine the phylogeographic nature of the isolated strains. Three bacterial isolates were selected from 22 gastric biopsies, identified as H. pylori through colonial morphology, Gram staining, urease, catalase, and oxidase tests and identification of the ureC gene through end-point PCR. Amplification of 16S rRNA, urea, and tonB genes was performed, as well. Differences between the cagA and vacA genotypes were determined among the isolates. The phylogenetic analysis confirmed the identity of the three isolates as the specie Helicobacter pylori. Different genotypes were obtained for each H. pylori strain, and all the clinical isolates showed the vacA s2/m2 genotype, indicating an absence of the VacA cytotoxin. Only HCGDL-MR01 is a cagA gene carrier with a greater risk to develop a serious disease, such as stomach cancer and peptic ulcer. The multilocus sequence typing placed all the strains within the hpEurope population structure.

Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Genomic approach to determine sources of neonatal Staphylococcus aureus infection from carriage in the Gambia

Staphylococcus aureus is a major cause of neonatal infections in various anatomical sites, resulting in high morbidity and mortality in The Gambia. These clinical infections are often preceded by nasal carriage of S. aureus, a known risk factor. To determine whether potential sources of newborn S. aureus infections were from carriage, and to characterize S. aureus present in different anatomical sites (blood, ear, eye, umbilical cord, skin, pus, oropharynx, breast milk and vagina), we performed whole-genome sequencing of 172 isolates from clinical sites as well as from healthy and unhealthy carriage. A random selection of mothers (n = 90) and newborns (n = 42) participating in a clinical trial and testing positive for S. aureus were considered for this study. Sequence data were analyzed to determine S. aureus multilocus sequence types and selected antimicrobial and virulence gene profiles. Our findings revealed that in The Gambia, ST15 is the dominant sequence type associated with both carriage and clinical infection. In addition, S. aureus isolates causing clinical infection among neonates were genetically similar to those colonizing their oropharynx, and the different anatomical sites were not found to be uniquely colonized by S. aureus of a single genomic profile. Furthermore, while S. aureus associated with clinical infection had similar antimicrobial resistance gene profiles to carriage isolates, only hemolysin and adhesive factor virulence genes were significantly higher among clinical isolates. In conclusion, this study confirmed S. aureus oropharyngeal colonization among neonates as a potential source of clinical infection in The Gambia. Hence, interventions aiming to reduce neonatal clinical infections in The Gambia should consider decreasing oropharyngeal S. aureus carriage.Trial registration The trial was registered at ClinicalTrials.gov NCT03199547.

Penicillin susceptibility among Staphylococcus aureus skin and soft tissue infections at a children's hospital.

Shortly after its introduction into clinical practice, Staphylococcus aureus isolates gained resistance to penicillin via the acquisition of β-lactamases. A number of centers have recently described an increase in the proportion of invasive methicillin-susceptible S. aureus (MSSA), which are also susceptible to penicillin (PSSA). Little data are available regarding the prevalence or impact of PSSA in skin and soft tissue infections (SSTI). Community-acquired MSSA SSTI isolates were obtained through a surveillance study at Texas Children's Hospital from January 2017 to December 2021. A total of 200 random isolates underwent PCR for blaZ β-lactamase; blaZ-negative isolates then underwent penicillin susceptibility testing using macrobroth dilution. Isolates which were blaZ negative and had a penicillin MIC ≤0.125 µg/mL were regarded as PSSA with the remainder regarded as penicillin-resistant MSSA (PR-MSSA). All PSSA underwent multilocus sequence typing. Medical records were reviewed. The median age of subjects was 4.2 years (IQR: 1.6-10.5). PSSA accounted for 9% of isolates during the study period. PSSA and PR-MSSA cases were similar with respect to age, demographics, and rates of prior antibiotic exposure. PSSA isolates less often had vancomycin MIC ≥1.5 µg/mL. Furthermore, 39% of PSSA were variants of sequence type 1. In multivariable analyses, penicillin susceptibility was independently associated with both hospital admission and surgical intervention. PSSA account for a small but significant proportion of MSSA SSTI in children. Clinically distinguishing patients with PSSA and PR-MSSA SSTI is challenging. However, PSSA SSTI were independently associated with higher rates of hospital admission as well as the need for surgical intervention suggesting a significant clinical impact.IMPORTANCEThe vast majority of Staphylococcus aureus in the US are penicillin resistant with most clinical labs no longer reporting penicillin susceptibility for this organism. A number of centers, however, have reported increasing penicillin susceptibility among invasive S. aureus infections. Skin and soft tissue infections (SSTI) are far more common than invasive infections, yet the frequency and impact of penicillin-susceptible S. aureus (PSSA) in this population are uncertain. Through active surveillance at a children's hospital, we found that 9% of methicillin-susceptible S. aureus SSTI isolates were PSSA. PSSA were independently associated with hospital admission for the management of SSTI as well as the need for debridement in the operating room. Given that most SSTI are managed in the outpatient setting, these findings suggest a clinical impact of this phenotype and the need for a reassessment of the value in susceptibility testing and potentially even treatment with penicillin.

Genomic characterization and cluster analysis of Carbapenem-resistant Klebsiella pneumoniae

To investigate the genomic features and perform cluster analysis of Carbapenem-resistant Klebsiella pneumoniae (CRKP) to provide an experimental basis for guiding the prevention and treatment of CRKP infections.A retrospective case-cohort study was conducted on 19 non-redundant CRKP strains isolated from the Tenth Affiliated Hospital of Southern Medical University between January and June 2023. Whole genome sequencing (WGS) and multilocus sequence typing (MLST) were performed to compare genomic features and analyze the resistance genes and homology of the strains.The results showed that the 19 CRKP strains were isolated from 8 different clinical departments, mainly from respiratory specimens. The whole genome sequencing revealed that the genomic lengths of CRKP ranged from 4.90 to 5.85 Mbp, with contigs N50 values>20 kb for each genome. The median overall GC content was 57.0% (50.4%-57.1%). Comparative genomic analysis identified three regions with high genomic variability. WGS detected 32 resistance genes across 11 categories. All 19 strains carried carbapenem resistance genes (blaKPC-2 and blaOXA-48), blaTEM-1B extended-spectrum β-lactamase resistance genes, qnrS1 quinolone resistance gene, and fosA fosfomycin resistance gene, with each strain carrying only one carbapenemase gene. The detection rate of blaKPC-2 was 94.7% (18/19). MLST identified three sequence types: ST11, ST437 and ST147, with ST11 being predominant (89.5%, 17/19). Clustering analysis based on acquired resistance genes revealed three clonal transmission patterns among strains 72 and 90, and strains 88, 84, 66 and 79.In conclusion, CRKP strains carry multiple resistance genes, and clustering analysis indicating that nosocomial clonal transmission is closely related to acquired resistance genes. The ST11-blaKPC-2 type strain is the predominant clone. Strengthened surveillance and effective control strategies are necessary to reduce nosocomial transmission of CRKP.

Genomics of Staphylococcus aureus and Enterococcus faecalis isolated from the ocular surface of dry eye disease sufferers

Ocular surface inflammatory disorders, such as dry eye, are becoming increasingly prevalent. Developing new treatment strategies targeting harmful bacteria could provide significant therapeutic benefits. The purpose of this study was to characterize the common ocular pathogen Staphylococcus aureus and the rarer endophthalmitis-associated species Enterococcus faecalis isolated from the ocular surface of dry eye disease patients in Norway. Together the 7 isolates (5 S. aureus and 2 E. faecalis) comprise the complete set of members of each species isolated in our previous study of the ocular microbiome of 61 dry eye sufferers. We aimed to investigate the pathogenic potential of these isolates in relation to ocular surface health. To this end, we used whole genome sequencing, multiplex PCR directed at virulence genes and antibiotic susceptibility tests encompassing clinically relevant agents. The E. faecalis isolates showed resistance to only gentamicin. S. aureus isolates displayed susceptibility to most of the tested antibiotics, except for two isolates which showed resistance to trimethoprim/sulfamethoxazole and three isolates which were resistant to ampicillin. Susceptibilities included sensitivity to several first-line antibiotics for treatment of ocular infections by these species. Thus, treatment options would be available if required. However, spontaneous resistance development to gentamicin and rifampicin occurred in some S. aureus which could be a cause for concern. Whole genome sequencing of the isolates showed genome sizes ranging from 2.74 to 2.83 Mbp for S. aureus and 2.86 Mbp for E. faecalis, which is typical for these species. Multilocus sequence typing and phylogenetic comparisons with previously published genomes, did not suggest the presence of eye-specific clusters for either species. Genomic analysis indicated a high probability of pathogenicity among all isolates included in the study. Resistome analysis revealed the presence of the beta-lactamase blaZ gene in all S. aureus isolates and the dfrG gene in two of them; while E. faecalis isolates carried the lsa(A) gene which confers intrinsic resistance to lincosamides and streptogramin A in this species. Screening for virulence factors revealed the presence of various pathogenicity associated genes in both S. aureus and E. faecalis isolates. These included genes coding for toxin production and factors associated with evading the host immune system. Some of the identified genes (tst, hylA &hylB) are suggested to be linked to the pathophysiology of dry eye disease. Lastly, the presence of specific S. aureus virulence genes was confirmed through multiplex PCR analysis.

Molecular epidemiology of brucellosis in Asia: insights from genotyping analyses.

Brucellosis infects humans and animals worldwide but is particularly prevalent in Asia. In many Asian countries, molecular diagnostic tools for accurate molecular diagnostics and molecular epidemiology are lacking. Nonetheless, some countries have conducted in-depth molecular epidemiological studies. The objective of this study was to reveal the genetic relationships, geographic origins, and distributions of Brucella strains across Asia for two primary species, B. abortus and B. melitensis. For this, we systematically searched genotyping data from published studies on the molecular epidemiology of Brucella species for both humans and livestock in Asia. We used data from multilocus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing analysis of Brucella strains. We also analyzed the MLVA genotypes of 129 B. abortus isolates and 242 B. melitensis isolates with known origins in Asia from an online MLVA database using MLVA-11 data in minimum spanning trees and MLVA-16 data in neighbor-joining trees. We found that the B. melitensis East Mediterranean lineage is predominant across the continent, with only a small number of samples from the Africa and Americas lineages, and none from the West Mediterranean lineage. The "abortus C" genotype was the most common group of B. abortus in Asia, with limited genetic variation for this species. Several studies also reported that Near Eastern countries frequently encounter human brucellosis cases of B. abortus from genotypes 42 and 43. Our study highlights the inconsistent collection of genetic data for Brucella species across Asia and a need for more extensive sampling in most countries. Finally, a consistent nomenclature is necessary to define various groupings of strains within a lineage (i.e., clade) so uniform terminology should denote particular genetic groups that are understood by all researchers.

Virulence factors, antibiotic susceptibility and sequence type distribution of hospital-associated Clostridioides difficile isolates in Israel, 2020–2022

Biofilm formation and toxin production are some of the virulence factors of Clostridioides difficile (C. difficile), which causes hospital-acquired C. difficile infection (HA-CDI). This work investigated the prevalence and distribution of different strains recovered from HA-CDI patients hospitalized in 4 medical centres across Israel, and characterized strains' virulence factors and antibiotic susceptibility. One-hundred and eighty-eight faecal samples were collected. C. difficile 's toxins were detected by the CerTest Clostridium difficile GDH + Toxin A + B combo card test kit. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Multi-locus sequence typing (MLST) was performed to classify strains. Biofilm production was assessed by crystal violet. Antibiotic susceptibility was determined using Etest. Fidaxomicin susceptibility was tested via agar dilution. Sequence type (ST) 42 was the most (13.8%) common strain. All strains harboured the 2 toxins genes; 6.9% had the binary toxin. Most isolates were susceptible to metronidazole (98.9%) and vancomycin (99.5%). Eleven (5.85%) isolates were fidaxomicin-resistant. Biofilm production capacity was associated with ST (p < 0.001). In conclusion, a broad variety of C. difficile strains circulate in Israel's medical centres. Further studies are needed to explore the differences and their contribution to HA-CDI epidemiology.

Multilocus sequence typing database for Streptococcus agalactiae contains a spurious allele of the transketolase gene.

The tkt (transketolase) gene is one of the seven gene fragments used in the multilocus sequence typing (MLST) system for Streptococcus agalactiae. We discovered that the tkt_134 allele is derived from a homologous gene (which we designate tktX) that is not present in all S. agalactiae; all known strains that contain a match to the tkt_134 allele also contain a gene sequence that is much closer in sequence identity to the other non-tkt_134 alleles (i.e., the canonical tkt gene) in the database. Based on these data, the tkt_134 allele has been removed from the MLST database as of September 2021, and all sequence types containing tkt_134 have also been removed.IMPORTANCEMultilocus sequence typing (MLST) databases are a common good and remain important for research, medical, and epidemiological purposes. This remains true even in the context of widespread whole-genome sequencing. We discovered a contaminating allele of the tkt gene in the S. agalactiae MLST database that led to unstable, ambiguous, or erroneous MLST assignment. The allele has since been removed from the public database based on the results presented in this manuscript.