Showing papers on "Typing published in 2021"

Evaluation of the IR Biotyper for Klebsiella pneumoniae typing and its potentials in hospital hygiene management

Abstract: Klebsiella pneumoniae has emerged as one of the most important pathogens that frequently encounter in community-acquired or hospital-acquired infections. Timely epidemiological surveillance could greatly facilitate infection control of K. pneumoniae and many deadly pathogens alike. In this study, we evaluated the performance of the IR Biotyper, a Fourier transform infrared (FTIR) spectroscopy system for K. pneumoniae isolates typing through (i) optimizing the culture scheme and defining the cutoff value (COV) range and (ii) comparing with commonly used typing tools such as multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). We found that a non-selective and non-chromogenic medium with 24 ± 2 h incubation gives the best discriminatory power for the IR Biotyper (IRBT). COV evaluation indicated that the IRBT is a robust typing method with good reproducibility. Besides, we observed that the modified H2 O-EtOH suspensions preparation method could enhance the quality of the spectrum, especially for those hypermucoviscous strains. For the method comparison study, our data demonstrated that FTIR spectroscopy could accurately cluster K. pneumoniae strains. The typing results of the IRBT were almost entirely in concordance with those from PFGE and WGS. Together with the advantages such as low costs and short turnaround time (less than 3h), the IRBT is a promising tool for strain typing that could make real-time outbreak investigation a reality.

Multi Locus Sequence Typing and spa Typing of Staphylococcus Aureus Isolated from the Milk of Cows with Subclinical Mastitis in Croatia.

Abstract: Background: The bacterial species S. aureus is the most common causative agent of mastitis in cows in most countries with a dairy industry. The prevalence of infection caused by S. aureus ranges from 2% to more than 50%, and it causes 10–12% of all cases of clinical mastitis. Aim: The objective was to analyze 237 strains of S. aureus isolated from the milk of cows with subclinical mastitis regarding the spa, mecA, mecC and pvl genes and to perform spa and multi-locus sequence typing (MLST). Methods: Sequencing amplified gene sequences was conducted at Macrogen Europe. Ridom StaphType and BioNumerics software was used to analyze obtained sequences of spa and seven housekeeping genes. Results: The spa fragment was present in 204 (86.1%) of strains, while mecA and mecC gene were detected in 10 strains, and the pvl gene was not detected. Spa typing successfully analyzed 153 tested isolates (64.3%), confirming 53 spa types, four of which were new types. The most frequent spa type was t2678 (14%). MLST typed 198 (83.5%) tested strains and defined 32 different allele profiles, of which three were new. The most frequent allele profile was ST133 (20.7%). Six groups (G) and 15 singletons were defined. Conclusion: Taking the number of confirmed spa types and sequence types (STs) into account, it can be concluded that the strains of S. aureus isolated from the milk of cows with subclinical mastitis form a heterogenous group. To check the possible zoonotic potential of isolates it would be necessary to test the persons and other livestock on the farms.

Hidden Diversity within Common Protozoan Parasites as Revealed by a Novel Genomotyping Scheme.

Abstract: Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is the causative agent of giardiasis, one of the most common diarrheal infections in humans. Evolutionary relationships among G. duodenalis genotypes (or subtypes) of assemblage B, one of two genetic assemblages causing the majority of human infections, remain unclear due to poor phylogenetic resolution of current typing methods. In this study, we devised a methodology to identify new markers for a streamlined multilocus sequence typing (MLST) scheme based on comparisons of all core genes against the phylogeny of whole-genome sequences (WGS). Our analysis identified three markers with resolution comparable to that of WGS data. Using newly designed PCR primers for our novel MLST loci, we typed an additional 68 strains of assemblage B. Analyses of these strains and previously determined genome sequences showed that genomes of this assemblage can be assigned to 16 clonal complexes, each with unique gene content that is apparently tuned to differential virulence and ecology. Obtaining new genomes of Giardia spp. and other eukaryotic microbial pathogens remains challenging due to difficulties in culturing the parasites in the laboratory. Hence, the methods described here are expected to be widely applicable to other pathogens of interest and advance our understanding of their ecology and evolution. IMPORTANCEGiardia duodenalis assemblage B is a major waterborne pathogen and the most commonly identified genotype causing human giardiasis worldwide. The lack of morphological characters for classification requires the use of molecular techniques for strain differentiation; however, the absence of scalable and affordable next-generation sequencing (NGS)-based typing methods has prevented meaningful advancements in high-resolution molecular typing for further understanding of the evolution and epidemiology of assemblage B. Prior studies have reported high sequence diversity but low phylogenetic resolution at standard loci in assemblage B, highlighting the necessity of identifying new markers for accurate and robust molecular typing. Data from comparative analyses of available genomes in this study identified three loci that together form a novel high-resolution typing scheme with high concordance to whole-genome-based phylogenomics and which should aid in future public health endeavors related to this parasite. In addition, data from newly characterized strains suggest evidence of biogeographic and ecologic endemism.

Establishment and Evaluation of a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Pseudomonas aeruginosa.

Abstract: The environmental bacterium Pseudomonas aeruginosa, particularly multidrug-resistant clones, is often associated with nosocomial infections and outbreaks. Today, core genome multilocus sequence typing (cgMLST) is frequently applied to delineate sporadic cases from nosocomial transmissions. However, until recently, no cgMLST scheme for a standardized typing of P. aeruginosa was available. To establish a novel cgMLST scheme for P. aeruginosa, we initially determined the breadth of the P. aeruginosa population based on MLST data with a Bayesian approach (BAPS). Using genomic data of representative isolates for the whole population and all 12 serogroups, we extracted target genes and further refined them using a random data set of 1,000 P. aeruginosa genomes. Subsequently, we investigated reproducibility and discriminatory ability with repeatedly sequenced isolates and isolates from well-defined outbreak scenarios, respectively, and compared clustering applying two recently published cgMLST schemes. BAPS generated seven P. aeruginosa groups. To cover these and all serogroups, 15 reference strains were used to determine genes common in all strains. After refinement with the data set of 1,000 genomes, the cgMLST scheme consisted of 3,867 target genes, which are representative of the P. aeruginosa population and highly reproducible using biological replicates. We finally evaluated the scheme by reanalyzing two published outbreaks where the authors used single-nucleotide polymorphism (SNP) typing. In both cases, cgMLST was concordant with the previous SNP results and the results of the two other cgMLST schemes. In conclusion, the highly reproducible novel P. aeruginosa cgMLST scheme facilitates outbreak investigations due to the publicly available cgMLST nomenclature.

Serotyping, MLST, and Core Genome MLST Analysis of Salmonella enterica From Different Sources in China During 2004-2019.

Abstract: Salmonella enterica (S. enterica) is an important foodborne pathogen, causing food poisoning and human infection, and critically threatening food safety and public health. Salmonella typing is essential for bacterial identification, tracing, epidemiological investigation, and monitoring. Serotyping and multilocus sequence typing (MLST) analysis are standard bacterial typing methods despite the low resolution. Core genome MLST (cgMLST) is a high-resolution molecular typing method based on whole genomic sequencing for accurate bacterial tracing. We investigated 250 S. enterica isolates from poultry, livestock, food, and human sources in nine provinces of China from 2004 to 2019 using serotyping, MLST, and cgMLST analysis. All S. enterica isolates were divided into 36 serovars using slide agglutination. The major serovars in order were Enteritidis (31 isolates), Typhimurium (29 isolates), Mbandaka (23 isolates), and Indiana (22 isolates). All strains were assigned into 43 sequence types (STs) by MLST. Among them, ST11 (31 isolates) was the primary ST. Besides this, a novel ST, ST8016, was identified, and it was different from ST40 by position 317 C → T in dnaN. Furthermore, these 250 isolates were grouped into 185 cgMLST sequence types (cgSTs) by cgMLST. The major cgST was cgST235530 (11 isolates), and only three cgSTs contained isolates from human and other sources, indicating a possibility of cross-species infection. Phylogenetic analysis indicated that most of the same serovar strains were putatively homologous except Saintpaul and Derby due to their multilineage characteristics. In addition, serovar I 4,[5],12:i:- and Typhimurium isolates have similar genomic relatedness on the phylogenetic tree. In conclusion, we sorted out the phenotyping and genotyping diversity of S. enterica isolates in China during 2004-2019 and clarified the temporal and spatial distribution characteristics of Salmonella from different hosts in China in the recent 16 years. These results greatly supplement Salmonella strain resources, genetic information, and traceability typing data; facilitate the typing, traceability, identification, and genetic evolution analysis of Salmonella; and therefore, improve the level of analysis, monitoring, and controlling of foodborne microorganisms in China.

Molecular Markers Reveal Epidemiological Patterns and Evolutionary Histories of the Human Pathogenic Cryptococcus.

Abstract: The human pathogenic Cryptococcus species are the main agents of fungal meningitis in humans and the causes of other diseases collectively called cryptococcosis. There are at least eight evolutionary divergent lineages among these agents, with different lineages showing different geographic and/or ecological distributions. In this review, we describe the main strain typing methods that have been used to analyze the human pathogenic Cryptococcus and discuss how molecular markers derived from the various strain typing methods have impacted our understanding of not only cryptococcal epidemiology but also its evolutionary histories. These methods include serotyping, multilocus enzyme electrophoresis, electrophoretic karyotyping, random amplified polymorphic DNA, restriction fragment length polymorphism, PCR-fingerprinting, amplified fragment length polymorphism, multilocus microsatellite typing, single locus and multilocus sequence typing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and whole genome sequencing. The major findings and the advantages and disadvantages of each method are discussed. Together, while controversies remain, these strain typing methods have helped reveal (i) the broad phylogenetic pattern among these agents, (ii) the centers of origins for several lineages and their dispersal patterns, (iii) the distributions of genetic variation among geographic regions and ecological niches, (iv) recent hybridization among several lineages, and (v) specific mutations during infections within individual patients. However, significant challenges remain. Multilocus sequence typing and whole genome sequencing are emerging as the gold standards for continued strain typing and epidemiological investigations of cryptococcosis.

Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates.

Abstract: Background Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains. Methodology/principal findings Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction. Conclusions/significance Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.

The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates.

Abstract: Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

A whole genome-based gene-by-gene typing system for standardised high resolution strain typing of Bacillus anthracis

Abstract: Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.

Investigations of sequencing data and sample type on HLA class Ia typing with different computational tools.

Abstract: Human leukocyte antigen (HLA) can encode the human major histocompatibility complex (MHC) proteins and play a key role in adaptive and innate immunity. Emerging clinical evidences suggest that the presentation of tumor neoantigens and neoantigen-specific T cell response associated with MHC class I molecules are of key importance to activate the adaptive immune systemin cancer immunotherapy. Therefore, accurate HLA typing is very essential for the clinical application of immunotherapy. In this study, we conducted performance evaluations of 4 widely used HLA typing tools (OptiType, Phlat, Polysolver and seq2hla) for predicting HLA class Ia genes from WES and RNA-seq data of 28 cancer patients. HLA genotyping data using PCR-SBT method was firstly obtained as the golden standard and was subsequently compared with HLA typing data by using NGS techniques. For both WES data and RNA-seq data, OptiType showed the highest accuracy for HLA-Ia typing than the other 3 programs at 2-digit and 4-digit resolution. Additionally, HLA typing accuracy from WES data was higher than from RNA-seq data (99.11% for WES data versus 96.42% for RNA-seq data). The accuracy of HLA-Ia typing by OptiType can reach 100% with the average depth of HLA gene regions >20x. Besides, the accuracy of 2-digit and 4-digit HLA-Ia typing based on control samples was higher than tumor tissues. In conclusion, OptiType by using WES data from control samples with the high average depth (>20x) of HLA gene regions can present a probably superior performance for HLA-Ia typing, enabling its application in cancer immunotherapy.

Molecular Characterization Based on MLST and ECDC Typing Schemes and Antibiotic Resistance Analyses of Treponema pallidum subsp. pallidum in Xiamen, China.

Abstract: In total, 49 clinical samples were analyzed using two typing schemes, Enhanced Centers for Disease Control and Prevention (ECDC) and multilocus sequence typing (MLST), to describe the molecular characteristics of circulating Treponema pallidum isolates in Xiamen between 2016 and 2017. In addition, genetic mutations potentially related to antibiotic resistance of T. pallidum were also analyzed. Forty five samples were fully typed by ECDC, and 14 different subtypes were detected. The most common subtype was 16d/f (24.4%), followed by 14d/f (20.0%). All forty nine samples were successfully typed by MLST, while only four allelic profiles were identified, including three SS14-like profiles and one Nichols-like profile. Among them, the major allelic profile was 1.1.8 (85.7%). Interestingly, the allelic profile 1.3.1 widespread in Europe and North America was not detected in this region. Additionally, A2058G mutation in 23S rRNA was found in all detectable samples (38/38), and no mutation in 16S rRNA was observed (36/36). Four non-synonymous single-nucleotide polymorphisms in penicillin-binding protein genes were found in the 35 samples eligible for Sanger sequencing. Among them, the variant in tp0500 (P564I) can only be found in the SS14-like isolates. Homoplastic changes in tp0760 (I415F/I415M) and tp0705 (A506V/A506T) were found. Moreover, the variant tp0705 A506V and the variant tp0705 A506T separately appeared in the SS14-like isolates and Nichols-like isolates, respectively. This study showed that the genotypes of T. pallidum isolates in Xiamen between 2016 and 2017 were different from those in other geographic areas. The resistance-related variants of T. pallidum isolates identified in this study could provide awareness for clinicians in the treatment of syphilis.

Variation in Accessory Genes Within the Klebsiella oxytoca Species Complex Delineates Monophyletic Members and Simplifies Coherent Genotyping

Abstract: Members of the Klebsiella oxytoca species complex (KoSC) are emerging human pathogens causing infections of increasing significance especially in healthcare settings. KoSC strains are affiliated with distinct phylogroups based on genetic variation at the beta-lactamase gene (blaOXY) and it has been proposed that each major phylogroup represents a unique species. However, since the typing methods applied in clinical settings cannot differentiate every species within the complex, existing clinical, epidemiological and DNA sequence data is frequently misclassified. Here we systematically examined the phylogenetic relationship of KoSC strains to evaluate robustness of existing typing methods and to provide a simple typing strategy for KoSC members that cannot be differentiated biochemically. Initial analysis of a collection of K. oxytoca, K. michiganensis, K. pasteurii, and K. grimontii strains of environmental origin showed robust correlation of core phylogeny and blaOXY grouping. Moreover, we identified species-specific accessory gene loci for these strains. Extension of species correlation using database entries initially failed. However, assessment of average nucleotide identities (ANI) and phylogenetic validations showed that nearly one third of isolates in public databases have been misidentified. Reclassification resulted in a robust reference strain set for reliable species identification of new isolates or for retyping of strains previously analyzed by multi-locus sequence typing (MLST). Finally, we show convergence of ANI, core gene phylogeny, and accessory gene content for available KoSC genomes. We conclude that also the monophyletic members K. oxytoca, K. michiganensis, K. pasteurii and K. grimontii can be simply differentiated by a PCR strategy targeting blaOXY and accessory genes defined here.

Multiplex Visualized Closed-Tube PCR with Hamming Distance 2 Code for 15 HPV Subtype Typing

Abstract: Cervical cancer is the fourth leading cause of death in women, especially in developing countries. Specific and economic methodologies for HPV typing are crucial in cancer diagnosis and further disease control. However, routine assays based on real-time polymerase chain reaction (qPCR) or DNA-chip hybridization are either incapable of offering detailed subtype information or involve tedious open-tube operations with the risk of cross-contamination from PCR amplicons. Herein, we proposed a multiplex visualized closed-tube PCR (Multi-Vision) for HPV typing. Using gold nanoparticle probes (AuNPs) as a color change indicator combined with a Hamming distance 2 coding scheme, 13 high-risk HPVs and two subtypes associated with high-incidence benign lesions were successfully typed by performing six closed-tube PCRs. The assay demonstrates high specificity with no cross-reaction among different subtypes under several artificial sample concentrations (from 100 to 103 copies per reaction) and enables highly sensitive detection of as low as 0.5 copies/μL. Further, 105 clinical samples were successfully analyzed using our method with a high concordance rate of 99.05% (104/105) compared to a HPV typing kit. The inconsistent sample was confirmed by sequencing to be consistent with the typing results determined by our method, indicating that Multi-Vision could be a useful tool for HPV detection, especially in resource-limited regions.

Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes.

Abstract: Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasizing the need to investigate their survival strategy in greater depth.

Antimicrobial Resistance and CRISPR Typing Among Salmonella Isolates From Poultry Farms in China.

Abstract: Although knowledge of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been applied in many research areas, comprehensive studies of this system in Salmonella, particularly in analysis of antibiotic resistance, have not been reported. In this work, 75 Salmonella isolates obtained from broilers or broilers products were characterized to determine their antimicrobial susceptibilities, antibiotic resistance gene profiles, and CRISPR array diversities, and genotyping was explored. In total, 80.00% (60/75) of the strains were multidrug resistant, and the main pattern observed in the isolates was CN-AZM-AMP-AMC-CAZ-CIP-ATM-TE-SXT-FOS-C. The resistance genes of streptomycin (aadA), phenicol (floR-like and catB3-like), β-lactams (bla TEM, bla OXA, and bla CTX), tetracycline [tet(A)-like], and sulfonamides (sul1 and sul2) appeared at higher frequencies among the corresponding resistant isolates. Subsequently, we analyzed the CRISPR arrays and found 517 unique spacer sequences and 31 unique direct repeat sequences. Based on the CRISPR spacer sequences, we developed a novel typing method, CRISPR locus three spacer sequences typing (CLTSST), to help identify sources of Salmonella outbreaks especially correlated with epidemiological data. Compared with multi-locus sequence typing (MLST), conventional CRISPR typing (CCT), and CRISPR locus spacer pair typing (CLSPT), discrimination using CLTSST was weaker than that using CCT but stronger than that using MLST and CLSPT. In addition, we also found that there were no close correlations between CRISPR loci and antibiotics but had close correlations between CRISPR loci and antibiotic resistance genes in Salmonella isolates.

Antibiotic Resistance, spa Typing and Clonal Analysis of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Blood of Patients Hospitalized in the Czech Republic.

Abstract: Staphylococcus aureus is one of the major causes of bloodstream infections. The aim of our study was to characterize methicillin-resistant Staphylococcus aureus (MRSA) isolates from blood of patients hospitalized in the Czech Republic between 2016 and 2018. All MRSA strains were tested for antibiotic susceptibility, analyzed by spa typing and clustered using a Based Upon Repeat Pattern (BURP) algorithm. The representative isolates of the four most common spa types and representative isolates of all spa clonal complexes were further typed by multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. The majority of MRSA strains were resistant to ciprofloxacin (94%), erythromycin (95.5%) and clindamycin (95.6%). Among the 618 strains analyzed, 52 different spa types were detected. BURP analysis divided them into six different clusters. The most common spa types were t003, t586, t014 and t002, all belonging to the CC5 (clonal complex). CC5 was the most abundant MLST CC of our study, comprising of 91.7% (n = 565) of spa-typeable isolates. Other CCs present in our study were CC398, CC22, CC8, CC45 and CC97. To our knowledge, this is the biggest nationwide study aimed at typing MRSA blood isolates from the Czech Republic.

Carriage of Carbapenem-Resistant Enterobacterales in Adult Patients Admitted to a University Hospital in Italy.

Abstract: The emerging spread of carbapenemase-producing Enterobacterales (CPE) strains, in particular, Klebsiella pneumoniae and Escherichia coli, has become a significant threat to hospitalized patients. Carbapenemase genes are frequently located on plasmids than can be exchanged among clonal strains, increasing the antibiotic resistance rate. The aim of this study was to determine the prevalence of CPE in patients upon their admission and to analyze selected associated factors. An investigation of the antibiotic resistance and genetic features of circulating CPE was carried out. Phenotypic tests and molecular typing were performed on 48 carbapenemase-producing strains of K. pneumoniae and E. coli collected from rectal swabs of adult patients. Carbapenem-resistance was confirmed by PCR detection of resistance genes. All strains were analyzed by PCR-based replicon typing (PBRT) and multilocus sequence typing (MLST) was performed on a representative isolate of each PBRT profile. More than 50% of the strains were found to be multidrug-resistant, and the blaKPC gene was detected in all the isolates with the exception of an E. coli strain. A multireplicon status was observed, and the most prevalent profile was FIIK, FIB KQ (33%). MLST analysis revealed the prevalence of sequence type 512 (ST512). This study highlights the importance of screening patients upon their admission to limit the spread of CRE in hospitals.

Small-Scale Comparative Genomic Analysis of Listeria monocytogenes Isolated from Environments of Salmon Processing Plants and Human Cases in Norway

Abstract: Listeria monocytogenes is a food-borne bacterium that give rise to the potentially life-threatening disease listeriosis. Listeriosis has been mandatorily notifiable in Norway since 1991. All clinical L. monocytogenes isolates are sent to the Norwegian Institute of Public Health (NIPH) for typing. Since 2005 Multi-Locus Variable number tandem repeats Analysis (MLVA) has been used for typing but was recently replaced by whole genome sequencing using core genome Multi-Locus Sequence Typing (cgMLST). In the present study, L. monocytogenes isolates collected at salmon processing plants in Norway in 2007 (n = 12) and 2015 (n = 14) were first subject to MLVA. Twelve clinical L. monocytogenes isolates with matching MLVA profile and sampling time were selected from the strain collection at NIPH. Twenty-one isolates from the salmon processing plants and all clinical isolates (n = 12) were whole genome sequenced and compared using cgMLST and in silico detection of virulence genes. cgMLST revealed four pairs of environmental–human isolates with ≤10 allelic differences over 1708 genes, indicating that they may be assigned as clonal, with the implication that they are descended from the same recent ancestor. No relevant difference in carriage of virulence genes was found between environmental or human isolates. The present study shows that L. monocytogenes strains that genetically resemble contemporary isolates from human listeriosis circulate in Norwegian salmon slaughterhouses, and carry the same virulence genes.

Identification of a Depolymerase Specific for K64-Serotype Klebsiella pneumoniae: Potential Applications in Capsular Typing and Treatment.

Abstract: Carbapenem-resistant Klebsiella pneumoniae (CRKP), one of the major nosocomial pathogens, is increasingly becoming a serious threat to global public health There is an urgent need to develop effective therapeutic and preventive approaches to combat the pathogen Here, we identified and characterized a novel capsule depolymerase (K64-ORF41) derived from Klebsiella phage SH-KP152410, which showed specific activities for K pneumoniae K64-serotype We showed that this depolymerase could be used in the identification of K64 serotypes based on the capsular typing, and the results agreed well with those from the conventional serotyping method using antisera From this study, we also identified K64 mutant strains, which showed typing discrepancy between wzi-sequencing based genotyping and depolymerase-based or antiserum-based typing methods Further investigation indicated that the mutant strain has an insertion sequence (IS) in wcaJ, which led to the alteration of the capsular serotype structure We further demonstrated that K64-ORF41 depolymerase could sensitize the bacteria to serum or neutrophil killing by degrading the capsular polysaccharide In summary, the identified K64 depolymerase proves to be an accurate and reliable tool for capsular typing, which will facilitate the preventive intervention such as vaccine development In addition, the polymerase may represent a potential and promising therapeutic biologics against CRKP-K64 infections

Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance.

Abstract: Objectives:Burkholderia pseudomallei causes the severe disease melioidosis. Whole genome-sequencing (WGS) based typing methods currently offer the highest resolution for molecular investigations of this genetically diverse pathogen. Still, its routine application in diagnostic laboratories is limited by the need for high computing power, bioinformatic skills and variable bioinformatic approaches, the latter affecting the results. We therefore aimed to establish and validate a WGS-based core genome multilocus sequence typing (cgMLST) scheme, applicable in routine diagnostic settings. Methods: A soft defined core genome was obtained by challenging the B. pseudomallei reference genome K96243 with 469 environmental and clinical genomes, resulting in 4,221 core and 1,359 accessory targets. The scheme was validated with 320 WGS datasets. We compared our novel typing scheme with single nucleotide polymorphism based-approaches investigating closely and distantly related strains. Finally, we applied our scheme for tracking the environmental source of a recent infection. Results: The validation of the scheme detected >95% good cgMLST target genes in 98.4% of the genomes. Comparison with existing typing methods revealed very good concordance. Our scheme proved to be applicable to investigate not only closely related strains, but also the global B. pseudomallei population structure. We successfully utilized our scheme to identify a sugar cane field as the presumable source of a recent melioidosis case. Conclusion: We developed a robust cgMLST typing scheme which integrates high resolution, maximized standardization and fast analysis for the non-bioinformatician. Our typing scheme has the potential to serve as a routinely applicable classification system in B. pseudomallei molecular epidemiology.

Differential effects of chromosome and plasmid bla CTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection

Abstract: Objectives To compare antibiotic susceptibilities between chromosomal and plasmid blaCTX-M-15 locations in urinary tract infection-causing extended-spectrum β-lactamases-producing Escherichia coli blaCTX-M-15 isolated in Indonesia. Methods A total of 84 strains identified as extended-spectrum β-lactamases-producing E. coli were isolated from patients with urinary tract infection in Indonesia in 2015. Antimicrobial susceptibility tests were performed on these strains using 18 antibiotics, and extended-spectrum β-lactamase bla genes were detected by polymerase chain reaction. Gene localization of blaCTX-M-15 -positive strains was confirmed by Southern blot hybridization, and epidemiological typing was conducted using multilocus sequence typing. Results Of 54 strains harboring the blaCTX-M-15 gene, 27 showed localization on chromosome, 20 on plasmid, and seven on chromosome and plasmid. Most multilocus sequence typing sequence types of the 27 strains with chromosomal blaCTX-M-15 were ST405 (25.9%) and ST131 (22.2%) strains, whereas the 20 strains with plasmid-blaCTX-M-15 were mostly ST410 (55.0%). Conclusions Extended-spectrum β-lactamases-producing E. coli blaCTX-M-15 with plasmid genes show significantly higher resistant rates against piperacillin-tazobactam but lower resistant rates against chloramphenicol compared to chromosomal strains in Indonesian patients with urinary tract infection. Mechanistic investigations will be necessary to advance our knowledge of antimicrobial resistance in urinary tract infection.

Evaluation of a Combined Multilocus Sequence Typing and Whole-Genome Sequencing Two-Step Algorithm for Routine Typing of Clostridioides difficile.

Abstract: Multilocus sequence typing (MLST) is a low-resolution but rapid genotyping method for Clostridioides difficile. Whole-genome sequencing (WGS) has emerged as the new gold standard for C. difficile typing, but cost and lack of standardization still limit broad utilization. In this study, we evaluated the potential to combine the portability of MLST with the increased resolution of WGS for a cost-saving approach to routine C. difficile typing. C. difficile strains from two New York City hospitals (hospital A and hospital B) were selected. WGS single-nucleotide polymorphism (wgSNP) was performed using established methods. Sequence types (ST) were determined using PubMLST, while wgSNP analysis was performed using the Bionumerics software. An additional analysis of a subset of data (hospital A) was made comparing the Bionumerics software to the CosmosID pipeline. Cost and turnaround time to results were compared for the algorithmic approach of MLST followed by wgSNP versus direct wgSNP. Among the 202 C. difficile isolates typed, 91% (n = 185/203) clustered within the representative ST, showing a high agreement between MLST and wgSNP. While clustering was similar between the Bionumerics and CosmosID pipelines, large differences in the overall number of SNPs were noted. A two-step algorithm for routine typing results in significantly lower cost than routine use of WGS. Our results suggest that using MLST as a first step in routine typing of C. difficile followed by WGS for MLST concordant strains is a less technically demanding, cost-saving approach for performing C. difficile typing than WGS alone without loss of discriminatory power.

HLA‐A, ‐B, ‐C, ‐DRB1 allele and haplotype frequencies of the Korean population and performance characteristics of HLA typing by next‐generation sequencing

Abstract: INTRODUCTION Human leukocyte antigen (HLA) identification at the allelic level is important for haematopoietic stem cell transplantation (HSCT). Next-generation sequencing (NGS) resolves ambiguous alleles by determining the phase of the polymorphisms. The aim of this study was to validate the software for HLA-SBT (sequence-based typing), assess Korean allele frequency, and characterise the performance of NGS-HLA typing. METHODS From the 2009 to 2016 registry, 1293 unrelated healthy donors with a complete dataset of previously characterised HLA-A, -B, -C, and -DRB1 loci were selected and assessed for frequency, haplotype inference, and relative linkage disequilibrium. For performance characteristics of NGS-HLA, alleles included in 1293 cases and ambiguous or alleles assigned as new by SBT-HLA software, or unassigned alleles were included. A total of 91 and 41 quality control samples resulted in 1056 alleles (132 samples × 4 loci × 2 diploid) for analysis. The GenDx NGSgo kit was used for NGS-HLA typing using the Illumina MiSeq platform. RESULTS A panel of 132 samples covered 231 alleles, including 53 HLA-A, 80 HLA-B, 43 HLA-C, and 55 HLA-DRB1 by HLA-SBT typing. Comparison of SBT-HLA and NGS-HLA typing showed 99.7% (1053/1056) concordance and discrepant cases were resolved by manual evaluation. Typing by NGS resulted in 67 HLA-A, 112 HLA-B, 71 HLA-C, and 72 HLA-DRB1 alleles. A total of 132 ambiguous, 4 new, and 1 unassigned alleles by HLA-SBT were resolved by NGS-HLA typing. CONCLUSIONS NGS-HLA typing provided robust and conclusive results without ambiguities, and its implementation could support HSCT in clinical settings.

Differences in biofilm formation of Salmonella serovars on two surfaces under two temperature conditions

Abstract: Aims Salmonella is extremely diverse, with >2500 serovars that are genetically and phenotypically diverse. The aim of this study was to build a collection of Salmonella isolates that are genetically diverse and to evaluate their ability to form biofilm under different conditions relevant to a processing environment. Methods and results Twenty Salmonella isolates representative of 10 serovars were subtyped using Clustered regularly interspaced short palindromic repeats (CRISPR)-typing to assess the genetic diversity between isolates of each serovar. Biofilm formation of the isolates on both plastic and stainless-steel surfaces at 25°C and 15°C was assessed. At 25°C, 8/20 isolates each produced strong and moderate biofilm on plastic surface compared to stainless-steel (3/20 and 13/20, respectively). At 15°C, 5/20 produced strong biofilm on plastic surface and none on stainless-steel. Several isolates produced weak biofilm on plastic (11/20) and stainless-steel (16/20) surfaces. Serovar Schwarzengrund consistently produced strong biofilm while serovars Heidelberg and Newport produced weak biofilm. Conclusion These results suggest that Salmonellae differ in their attachment depending on the surface and temperature conditions encountered, which may influence persistence in the processing environment. Significance and impact of study These differences in biofilm formation could provide useful information for mitigation of Salmonella in processing environments.

Evidence of Common Isolates of Streptococcus agalactiae in Bovines and Humans in Emilia Romagna Region (Northern Italy).

Abstract: Streptococcus agalactiae (group B Streptococcus, GBS) is one of the most important agents of bovine mastitis and causes remarkable direct and indirect economic losses to the livestock sector. Moreover, this species can cause severe human diseases in susceptible individuals. To investigate the zoonotic potential of S. agalactiae, 203 sympatric isolates from both humans and cattle, isolated in the same time frame (2018) and in the same geographic area (Emilia Romagna region, Northern Italy), were characterized by molecular capsular typing (MCT), pilus island typing (PI), and multi-locus sequence typing (MLST). In addition, antibiotic-resistant phenotypes were investigated. The distribution of the allelic profiles obtained by combining the three genotyping methods (MCT-PI-MLST) resulted in 64 possible genotypes, with greater genetic variability among the human compared to the bovine isolates. Although the combined methods had a high discriminatory power (>96,2%), five genotypes were observed in both species (20,9% of the total isolates). Furthermore, some of these strains shared the same antibiotic resistance profiles. The finding of human and bovine isolates with common genotypes and antibiotic resistance profiles supports the hypothesis of interspecies transmission of S. agalactiae between bovines and humans.

Typing of Neisseria Gonorrhoeae isolates in Shenzhen, China from 2014-2018 reveals the shift of genotypes associated with antimicrobial resistance

Abstract: The growing antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a serious global threat to gonococcal therapy. Molecular typing is an ideal tool to reveal the association between specific genotype and resistance phenotype that provides effective data for tracking the transmission of resistant clones of N. gonorrhoeae In our study, we aimed to describe the molecular epidemiology of AMR and the distribution of resistance-associated genotypes in Shenzhen during 2014-2018. In total, 909 isolates were collected from Shenzhen from 2014-2018. Two typing schemes, multilocus sequence typing (MLST) and N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), were performed for all isolates. The distribution of resistance-associated genotypes was described using goeBURST analysis combined with data of logistic regression. Among 909 isolates, ST8123, ST7363, ST1901, ST7365, and ST7360 were most the common MLST sequence types (STs), and ST348, ST2473, ST497, and ST199 were the most prevalent NG-STAR STs. The logistic regression analysis showed that NG-STARST497, MLSTST7365, and MLSTST7360 were typically associated with decreased susceptibility to ceftriaxone. Furthermore, the internationally spreading ESC-resistant clone MLSTST1901 has been prevalent at least in 2014 in Shenzhen and showed a significant increase during 2014-2018. Additionally, MLSTST7363 owns the potential to become the next internationally spreading ceftriaxone-resistant ST. In conclusions, we performed a comprehensive epidemiological study to explore the correlation between AMR and specific STs, which provided important data for future studies of the molecular epidemiology of AMR in N. gonorrhoeae Besides, these findings provide insight for adjusting surveillance strategies and therapy management in Shenzhen.

Characterization of the novel HLA-DPB1*896:01 allele by sequencing-based typing.

Abstract: HLA-DPB1*896:01 differs from HLA-DPB1*15:01:01:01 by nucleotide substitutions at codons 183 and 215 (exons 3-4).

Whole Genome Sequencing of Pediatric Klebsiella pneumoniae Strains Reveals Important Insights Into Their Virulence-Associated Traits

Abstract: Klebsiella pneumoniae is recognized as a common cause of nosocomial infections and outbreaks causing pneumonia, septicemia, and urinary tract infections. This opportunistic bacterium shows an increasing acquisition of antibiotic-resistance genes, which complicates treatment of infections. Hence, fast reliable strain typing methods are paramount for the study of this opportunistic pathogen's multi-drug resistance genetic profiles. In this study, thirty-eight strains of K. pneumoniae isolated from the blood of pediatric patients were characterized by whole-genome sequencing and genomic clustering methods. Genes encoding β-lactamase were found in all the bacterial isolates, among which the blaSHV variant was the most prevalent (53%). Moreover, genes encoding virulence factors such as fimbriae, capsule, outer membrane proteins, T4SS and siderophores were investigated. Additionally, a multi-locus sequence typing (MLST) analysis revealed 24 distinct sequence types identified within the isolates, among which the most frequently represented were ST76 (16%) and ST70 (11%). Based on LPS structure, serotypes O1 and O3 were the most prevalent, accounting for approximately 63% of all infections. The virulence capsular types K10, K136, and K2 were present in 16, 13, and 8% of the isolates, respectively. Phylogenomic analysis based on virtual genome fingerprints correlated with the MLST data. The phylogenomic reconstruction also denoted association between strains with a higher abundance of virulence genes and virulent serotypes compared to strains that do not possess these traits. This study highlights the value of whole-genomic sequencing in the surveillance of virulence attributes among clinical K. pneumoniae strains.