Showing papers on "Typing published in 2014"
TL;DR: A real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli found whole-genome sequencing typing is a superior alternative to conventional typing strategies and may also be applied to typing and Surveillance of other pathogens.
Abstract: Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.
977 citations
TL;DR: The findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods, and suggest that cepinghalosporain resistance genes are mainly disseminated in animals and humans via distinct plasmids.
Abstract: Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.
269 citations
TL;DR: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing and “neXtype” for streamlined data analysis and HLA allele assignment was developed.
Abstract: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed “neXtype” for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.
232 citations
TL;DR: In this paper, a whole-genome sequencing (WGS) was applied retrospectively to an unusual spike in MRSA cases in two intensive care units (ICUs) over the course of four weeks.
Abstract: Multidrug-resistant nosocomial pathogens present a major burden for hospitals. Rapid cluster identification and pathogen profiling, i.e., of antibiotic resistance and virulence genes, are crucial for effective infection control. Methicillin-resistant Staphylococcus aureus (MRSA), in particular, is now one of the leading causes of nosocomial infections. In this study, whole-genome sequencing (WGS) was applied retrospectively to an unusual spike in MRSA cases in two intensive care units (ICUs) over the course of 4 weeks. While the epidemiological investigation concluded that there were two separate clusters, each associated with one ICU, S. aureus protein A gene (spa) typing data suggested that they belonged to single clonal cluster (all cases shared spa type t001). Standardized gene sets were used to extract an allele-based profile for typing and an antibiotic resistance and toxin gene profile. The WGS results produced high-resolution allelic profiles, which were used to discriminate the MRSA clusters, corroborating the epidemiological investigation and identifying previously unsuspected transmission events. The antibiotic resistance profile was in agreement with the original clinical laboratory susceptibility profile, and the toxin profile provided additional, previously unknown information. WGS coupled with allelic profiling provided a high-resolution method that can be implemented as regular screening for effective infection control.
216 citations
TL;DR: For S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS.
Abstract: Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly ‘real-time’ monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE reveling that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.
214 citations
TL;DR: A single strain type (USA300/t008/IV) predominates in all regions and infection sites and is now more common than USA 100 as a cause of MRSA BSI and nosocomial infections.
Abstract: Background The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is changing, with USA300 emerging first in community and then in healthcare settings. We performed nationwide surveillance to assess recent trends in the molecular epidemiology of MRSA. Methods One hundred consecutive unique clinically significant S. aureus isolates were recovered from patients at each of 43 US centers between July 1, 2011, and December 31, 2011. Susceptibility testing, pulsed-field gel electrophoresis (PFGE), staphylococcal protein A gene (spa) and staphylococcal cassette chromosome mec typing, and Panton-Valentine leukocidin detection were performed on all MRSA isolates. Results Of 4,131 isolates collected, 2,093 (51%) were MRSA. Specimen sources of MRSA isolates included wound or abscess (54%), blood (24%), lower respiratory tract (11%), and other sterile site (10%). Thirty percent were isolated more than 48 hours after hospital admission (ie, were associated with nosocomial acquisition of infection). USA300 was the most common PFGE type (1,269 isolates; 61%), overall and in all regions, followed by USA100 (368 isolates; 18%). Among 173 spa types found, the most common were t008 (51%) and t002 (18%); no other spa type accounted for more than 2% of isolates. One strain type (USA300/t008/IV) constituted almost half of all MRSA isolates (1,005 isolates; 48%) and was the most common at all body sites, causing 37% of MRSA bloodstream infections (BSIs) and 38% of nosocomial MRSA infections. Multidrug-resistant phenotypes were found among 34 USA300 isolates (3%) from 18 states. Conclusions The USA300 PFGE type continues to advance nationwide. A single strain type (USA300/t008/IV) predominates in all regions and infection sites and is now more common than USA100 as a cause of MRSA BSI and nosocomial infections. Although most USA300 retain typical susceptibility profiles, multidrug-resistant phenotypes are emerging.
139 citations
TL;DR: The coexistence of both TR₃₄/L98H and TR⁄₆/Y121F/T289A resistance mechanisms in azole-resistant A. fumigatus isolates originating from Tanzania is reported.
Abstract: OBJECTIVES: Azole resistance in Aspergillus fumigatus isolates has been increasingly reported with variable prevalence worldwide and is challenging the effective management of aspergillosis. Here we report the coexistence of both TR(3)(4)/L98H and TR(4)(6)/Y121F/T289A resistance mechanisms in azole-resistant A. fumigatus (ARAF) isolates originating from Tanzania, Africa. METHODS: A total of 30 soil and woody debris samples from the surroundings of Kilimanjaro Christian Medical Centre, Moshi, Tanzania, were processed for detection of ARAF isolates and were investigated for susceptibility to itraconazole, voriconazole, posaconazole and isavuconazole. All ARAF isolates were subjected to a real-time PCR assay for detection of mutations and were genotyped by microsatellite typing. RESULTS: Of the 30 samples, 29 yielded 108 A. fumigatus isolates. Overall, 15 ARAF isolates were obtained, which included 4 ARAF harbouring the TR(4)(6)/Y121F/T289A mutation and 11 isolates carrying TR(3)(4)/L98H. All four TR(4)(6)/Y121F/T289A A. fumigatus isolates showed high MICs of voriconazole (>16 mg/L) and isavuconazole (8 mg/L). In contrast, the 11 TR(3)(4)/L98H A. fumigatus isolates were pan-azole resistant. The isolates were cross-resistant to azole fungicides. Notably, 20% of environmental samples harboured ARAF and the TR(4)(6)/Y121F/T289A resistance mechanism was found in 5.5% of the soil samples, where it coexisted with TR(3)(4)/L98H. The Tanzanian TR(4)(6)/Y121F/T289A strains had a genotype identical to Dutch clinical TR(4)(6)/Y121F/T289A isolates. CONCLUSIONS: The present study reports the isolation of resistant A. fumigatus strains harbouring the TR(4)(6)/Y121F/T289A mutation from Africa. Recovery of TR(4)(6)/Y121F/T289A from the environment is worrisome and we must strive for effective surveillance of clinical and environmental sources to detect azole resistance in A. fumigatus.
123 citations
TL;DR: Identification of cross-reacting anti-CPS mAbs encourages their development as adjunctive therapy and indicates variability of virulence-associated traits in CR-Kp ST258 strains.
Abstract: Background. Novel therapies are urgently needed to treat carbapenem-resistant Klebsiella pneumoniae (CR-Kp)-mediated infection, which constitute a major health threat in the United States. In order to assess if it is feasible to develop anticapsular antibodies as a potential novel therapy, it is crucial to first systematically characterize capsular polysaccharide (CPS) and virulence traits in these strains.
Methods. Forty CR-Kp were genotyped by pulsed field gel electrophoresis, multilocus sequence typing (MLST), and molecular capsule typing (C-patterns and wzi sequencing). Their biofilm formation, serum resistance, macrophage-mediated killing, and virulence in Galleria mellonella were compared. MAb (1C9) was generated by co-immunization with 2 CPSs, and cross-reactivity was investigated.
Results. MLST assigned 80% of CR-Kp isolates to the ST258-clone. Molecular capsule typing identified new C-patterns, including C200/wzi-154, which was widely represented and associated with blaKPC-3-bearing strains. Heterogeneity was detected in biofilm formation and macrophage-mediated killing. Differences in serum resistance correlated with virulence in G. mellonella. ST258 strains carrying blaKPC-3 were less virulent than those with blaKPC-2. MAb 1C9 cross-reacted with 58% of CR-Kp CPSs.
Conclusions. CR-Kp ST258 strains exhibit variability of virulence-associated traits. Differences were associated with the type of KPC gene and CPS. Identification of cross-reacting anti-CPS mAbs encourages their development as adjunctive therapy.
112 citations
TL;DR: MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli in 2011 northern Germany and could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.
Abstract: Background
In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.
Methods
Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak.
Results
Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.
Conclusions
MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.
87 citations
TL;DR: The risk of zoonotic infection emanating from ruminants even in high prevalence areas is negligible and integration of sequence typing data with information on geographic origins of samples allows parasite sub-population tracing using current typing tools.
Abstract: Background: Giardia duodenalis is a common flagellated protozoan parasite that infects the small intestine of a wide range of vertebrate hosts. This study aimed to determine whether tracing of G. duodenalis isolates by current genetic typing tools is possible using an exemplary set of samples from infected cattle, buffalo and children from the Ismailia province, Egypt. Method: A total of 804 fecal samples from ruminant animals was collected from 191 herds and 165 samples from diarrheal children below the age of 10 years. Parasites were detected in these samples using the copro-antigen RIDA®QUICK test and by real-time PCR. Samples were then genetically characterized based on the triosephosphate isomerase, glutamate dehydrogenase and β-giardin genes. Results: The prevalence of G. duodenalis was 53% in ruminants and 21% in symptomatic children and infection was not positively correlated with diarrheal symptoms. Sequence typing analysis confirmed predominance of B-type sequences (>67%) in humans and E-type sequences (>81%) in ruminants over A-type sequences. For 39 samples the complete sequence information of the three marker gene fragments could be derived. Integration of the concatenated sequence information of the three marker gene fragments with the spatial data of the respective sample revealed that identical or near identical (only up to 1 out of 1358 bp different) concatenated sequencing types were spatially related in 4 out of 5 cases. Conclusion: The risk of zoonotic infection emanating from ruminants even in high prevalence areas is negligible. Genetic characterization indicated a predominant anthropogenic cycle of infection within the pediatric population studied. Integration of sequence typing data with information on geographic origins of samples allows parasite sub-population tracing using current typing tools.
81 citations
TL;DR: Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5′ untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which is observed occasionally in clinical samples.
Abstract: Human rhinoviruses (RVs), comprising three species (A, B, and C) of the genus Enterovirus, are responsible for the majority of upper respiratory tract infections and are associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types necessitate regular updating of the diagnostic assays for the accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5′ untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which are observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes for seminested PCR assays provided higher specificity and sensitivity for virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignments for RV-C isolates, based on phylogenetic analysis of partial VP4/VP2 coding sequences. The improved assay was evaluated by typing RVs in >3,800 clinical samples. RVs were successfully detected and typed in 99% of the samples that were RV positive in multiplex diagnostic assays.
TL;DR: The objective was to determine the location of blaCTX-M-1 and blaCMY-2 genes in 33 Escherichia coli isolates previously obtained from healthy humans, pets, and food-producing animals in Tunisia, and to characterize the genetic lineages of isolates.
Abstract: The objective was to determine the location of blaCTX-M-1 and blaCMY-2 genes in 33 Escherichia coli isolates previously obtained from healthy humans, pets, and food-producing animals in Tunisia, and to characterize the genetic lineages of isolates. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE)-XbaI and multilocus sequence typing (MLST). Plasmids were analyzed by S1-PFGE, polymerase chain reaction-based replicon typing, and plasmid MLST. Conjugation experiments were performed. The blaCTX-M-1 and blaCMY-2 genes were studied by I-Ceu1-PFGE and S1-PFGE, and subsequent hybridization with specific probes. Eighteen different sequence types (STs) were identified among the 30 CTX-M-1-producing isolates, 5 of them being detected in 17 isolates (ST/phylogroup): ST57/D, ST155/B1, ST58/B1, ST10/A, and ST398/A. Most of the blaCTX-M-1-positive isolates had different PFGE profiles, with the exception of four human and pet isolates of lineage ST57 with related PFGE profiles (>80% identity). Thr...
TL;DR: The results indicate that the Bordetella pertussis population causing this epidemic was diverse, with a few molecular types predominating, and the PFGE, MLVA, and MLST profiles were consistent with the predominate types circulating in the United States and other countries.
Abstract: Although pertussis disease is vaccine preventable, Washington State experienced a substantial rise in pertussis incidence beginning in 2011. By June 2012, the reported cases reached 2,520 (37.5 cases per 100,000 residents), a 1,300% increase compared with the same period in 2011. We assessed the molecular epidemiology of this statewide epidemic using 240 isolates collected from case patients reported from 19 of 39 Washington counties during 2012 to 2013. The typing methods included pulsed-field gel electrophoresis (PFGE), multilocus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), and pertactin gene (prn) mutational analysis. Using the scheme PFGE-MLVA-MLST-prn mutations-Prn deficiency, the 240 isolates comprised 65 distinct typing profiles. Thirty-one PFGE types were found, with the most common types, CDC013 (n = 51), CDC237 (n = 44), and CDC002 (n = 42), accounting for 57% of them. Eleven MLVA types were observed, mainly comprising type 27 (n = 183, 76%). Seven MLST types were identified, with the majority of the isolates typing as prn2-ptxP3-ptxA1-fim3-1 (n = 157, 65%). Four different prn mutations accounted for the 76% of isolates exhibiting pertactin deficiency. PFGE provided the highest discriminatory power (D = 0.87) and was found to be a more powerful typing method than MLVA and MLST combined (D = 0.67). This study provides evidence for the continued predominance of MLVA 27 and prn2-ptxP3-ptxA1 alleles, along with the reemergence of the fim3-1 allele. Our results indicate that the Bordetella pertussis population causing this epidemic was diverse, with a few molecular types predominating. The PFGE, MLVA, and MLST profiles were consistent with the predominate types circulating in the United States and other countries. For prn, several mutations were present in multiple molecular types.
TL;DR: C. striatum is an opportunistic nosocomial pathogen in long-term hospitalized patients and can be at the origin of major outbreaks and could be used as an easy and rapid epidemiological typing tool for outbreak investigation.
TL;DR: For long typing sessions or when typing productivity is at a premium, conventional keyboards with tactile feedback may be more suitable interface.
TL;DR: A method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency and comparison with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method.
Abstract: Unambiguous human leukocyte antigen (HLA) typing is important in transplant matching and disease association studies. High-resolution HLA typing that is not restricted to the peptide-binding region can decrease HLA allele ambiguities. Cost and technology constraints have hampered high-throughput and efficient high resolution unambiguous HLA typing. We have developed a method for HLA genotyping that preserves the very high-resolution that can be obtained by next-generation sequencing (NGS) but also achieves substantially increased efficiency. Unambiguous HLA-A, B, C and DRB1 genotypes can be determined for 96 individuals in a single run of the Illumina MiSeq. Long-range amplification of full-length HLA genes from four loci was performed in separate polymerase chain reactions (PCR) using primers and PCR conditions that were optimized to reduce co-amplification of other HLA loci. Amplicons from the four HLA loci of each individual were then pooled and subjected to enzymatic library generation. All four loci of an individual were then tagged with one unique index combination. This multi-locus individual tagging (MIT) method combined with NGS enabled the four loci of 96 individuals to be analyzed in a single 500 cycle sequencing paired-end run of the Illumina-MiSeq. The MIT-NGS method generated sequence reads from the four loci were then discriminated using commercially available NGS HLA typing software. Comparison of the MIT-NGS with Sanger sequence-based HLA typing methods showed that all the ambiguities and discordances between the two methods were due to the accuracy of the MIT-NGS method. The MIT-NGS method enabled accurate, robust and cost effective simultaneous analyses of four HLA loci per sample and produced 6 or 8-digit high-resolution unambiguous phased HLA typing data from 96 individuals in a single NGS run.
TL;DR: The low-cost and convenient bla OXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of the collection and could be effectively applied to type rapidly A. baumannii strains.
Abstract: Single-locus blaOXA-51-like sequence-based typing (SBT) was evaluated for its ability to determine correctly sequence types (STs) in Acinetobacter baumannii clinical isolates, in comparison with the Pasteur's multilocus sequence typing (MLST) reference method and 3-locus sequence typing (3-LST). The comparative study was performed in 585 multidrug-resistant (MDR) A. baumannii clinical isolates recovered from 21 hospitals located throughout Greece, Italy, Lebanon, and Turkey. The isolates belonged to nine clonal complexes (CCs) that correspond to 12 distinct sequence types (STs) and to one singleton ST. These clonal lineages predominate worldwide among nosocomial MDR A. baumannii strains. The most common clone was CC2 (ST2 and ST45; n = 278 isolates) followed by CC1 (ST1 and ST20; n = 155), CC25 (n = 65), ST78 (n = 62), CC15 (ST15 and ST84; n = 9), CC10 (n = 4), CC3 (n = 4), CC6 (n = 3), CC54 (n = 3), and CC83 (n = 2). Using the blaOXA-51-like SBT method, all 585 isolates of the study were typed and assigned correctly to the nine CCs and the singleton ST78. The 3-LST method was not able to classify isolates belonging to CC6, CC10, CC54, and CC83, which are not yet characterized in its database. The low-cost and convenient blaOXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of our collection and could be effectively applied to type rapidly A. baumannii strains.
TL;DR: This study confirms the high disease burden of GAS infection in New Caledonia and supports the added value of the emm-cluster typing system to analyze GAS epidemiology and to help inform global GAS vaccine formulation.
Abstract: Background. Group A Streptococcus (GAS)–related disease is responsible for high mortality and morbidity in the Pacific region. The high diversity of circulating strains in this region has hindered vaccine development due to apparently low vaccine coverage of type-specific vaccines. Method. Prospective passive surveillance of all GAS isolates in New Caledonia was undertaken in 2012 using emm typing and emm-cluster typing. Molecular data were compared with the results from a prior study undertaken in the same country and with data from 2 other Pacific countries, Fiji and Australia. Results. A high incidence of invasive infection was demonstrated at 43 cases per 100 000 inhabitants (95% confidence interval, 35–52 cases per 100 000 inhabitants). Three hundred eighteen GAS isolates belonging to 47 different emm types were collected. In Noumea, only 30% of the isolates recovered in 2012 belonged to an emm type that was present in the same city in 2006, whereas 69% of the isolates collected in 2012 belonged to an emm cluster present in 2006. When comparing New Caledonian, Australian, and Fijian data, very few common emm types were found, but 79%–86% of the isolates from each country belonged to an emm cluster present in all 3 countries. Avaccine that could protect against the 10 most frequent emm clusters in the Pacific region would potentially provide coverage ranging from 83% to 92%. Conclusions. This study confirms the high disease burden of GAS infection in New Caledonia and supports the added value of the emm-cluster typing system to analyze GAS epidemiology and to help inform global GAS vaccine formulation.
TL;DR: An overview of the genetic diversity of Campylobacter strains from Portugal is provided, showing a worrying antibiotic multiresistance rate and the emergence of campylobacteria strains resistant to antibiotics of human use.
TL;DR: The genetic diversity of class D oxacillinases, including OXA-23, -24 (-40), -58 and, particularly, the intrinsic OxA-51-like genes, among multidrug-resistant Acinetobacter baumannii strains from inpatients at a tertiary care hospital in Riyadh, Saudi Arabia is explored.
Abstract: We explore the genetic diversity of class D oxacillinases, including OXA-23, -24 (-40), -58 and, particularly, the intrinsic OXA-51-like genes, among multidrug-resistant (MDR) Acinetobacter baumannii strains from inpatients at a tertiary care hospital in Riyadh, Saudi Arabia. Sequence-based typing (SBT) of the OXA-51-like gene was carried out on 253 isolates. Selected isolates (n = 66) were subjected to multilocus sequence typing (MLST). The polymerase chain reaction (PCR) typing results showed that all isolates (n = 253) contained the OXA-51-like and OXA-23 genes. However, the OXA-58 gene was detected in five isolates. Further, none of the isolates had the OXA-40 (identical to the OXA-24) gene. SBT revealed a high OXA-51-like genotypic diversity and showed that all isolates were clustered into four main groups: OXA-66 (62.3 %), followed by OXA-69 (19.1 %), OXA-132 (7.6 %) and other OXA-51-like genes (10.3 %), including OXA-79, -82, -92, -131 and -197. MLST revealed four main sequence types (STs), 2, 19, 20 and 25, among the isolates, in addition to six isolates with newly designated ST194–ST197 singletons. Further, a high prevalence (81.4 %) of OXA-66 and OXA-69-like genes in A. baumannii was identified. More studies are essential in order to explore the molecular mechanisms that confer carbapenem-resistant phenotypes for A. baumannii isolates and to investigate the genetic diversity of other OXA-D genes.
TL;DR: This study is the first to link ftsI alleles to STs in H. influenzae and indicates that horizontal gene transfer contributes to the emergence of rPBP3 by phylogeny restricted transformation.
Abstract: Beta-lactam resistance in Haemophilus influenzae due to ftsI mutations causing altered penicillin-binding protein 3 (PBP3) is increasing worldwide. Low-level resistant isolates with the N526K substitution (group II low-rPBP3) predominate in most geographical regions, while high-level resistant isolates with the additional S385T substitution (group III high-rPBP3) are common in Japan and South Korea. Knowledge about the molecular epidemiology of rPBP3 strains is limited. We combined multilocus sequence typing (MLST) and ftsI/PBP3 typing to study the emergence and spread of rPBP3 in nontypeable H. influenzae (NTHi) in Norway. The prevalence of rPBP3 in a population of 795 eye, ear and respiratory isolates (99% NTHi) from 2007 was 15%. The prevalence of clinical PBP3-mediated resistance to ampicillin was 9%, compared to 2.5% three years earlier. Group II low-rPBP3 predominated (96%), with significant proportions of isolates non-susceptible to cefotaxime (6%) and meropenem (20%). Group III high-rPBP3 was identified for the first time in Northern Europe. Four MLST sequence types (ST) with characteristic, highly diverging ftsI alleles accounted for 61% of the rPBP3 isolates. The most prevalent substitution pattern (PBP3 type A) was present in 41% of rPBP3 isolates, mainly carried by ST367 and ST14. Several unrelated STs possessed identical copies of the ftsI allele encoding PBP3 type A. Infection sites, age groups, hospitalization rates and rPBP3 frequencies differed between STs and phylogenetic groups. This study is the first to link ftsI alleles to STs in H. influenzae. The results indicate that horizontal gene transfer contributes to the emergence of rPBP3 by phylogeny restricted transformation. Clonally related virulent rPBP3 strains are widely disseminated and high-level resistant isolates emerge in new geographical regions, threatening current empiric antibiotic treatment. The need of continuous monitoring of beta-lactam susceptibility and a global system for molecular surveillance of rPBP3 strains is underlined. Combining MLST and ftsI/PBP3 typing is a powerful tool for this purpose.
31 Oct 2014
TL;DR: This chapter describes the recently developed SBT method for HLA-A, -B, -C, and -DQB1, that is based on full-length hemizygous Sanger sequencing of the alleles, separated by group-specific amplification using the low-resolution typing result as reference starting point.
Abstract: Matching for HLA at the allele level is crucial for stem cell transplantation. The golden standard approach for allele definition of full gene polymorphism, the so-called high-resolution HLA typing, is sequence-based typing (SBT). Although the majority of the polymorphism for class I is located in exons 2 and 3 and for class II in exon 2, for allele definition it is necessary to unravel the complete coding and intron sequences leading to an ultrahigh HLA typing resolution at the allele level, i.e., a full-length gene polymorphism identification.This chapter describes our recently developed SBT method for HLA-A, -B, -C, and -DQB1, that is based on full-length hemizygous Sanger sequencing of the alleles, separated by group-specific amplification using the low-resolution typing result as reference starting point. Group-specific amplification has already been established for DRB. This method enables a cost-efficient, user-friendly SBT approach resulting in a timely unambiguous HLA typing to an ultrahigh resolution level with minimal hands-on time.
TL;DR: It is suggested the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease, is recommended.
Abstract: Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.
TL;DR: This study identifies plasmid lineages that are contributing to the dissemination of blaCTX-M-1 genes in the food chain, the environment, and humans.
Abstract: Objectives: The aim of this work was to determine the plasmid replicon profiles of a collection of blaCTX-M-1-positive enterobacterial strains The isolates originated from chicken in the production pyramid, healthy food-producing animals at slaughter (chicken, calves and pigs), chicken retail meat, environmental isolates originating from water bodies, and isolates from humans A selection of IncI and IncN plasmids were characterized by multilocus sequence typing in order to determine their epidemiological relatedness Methods: Transconjugants of 74 blaCTX-M-1-positive isolates were analysed by PCR-based replicon typing and by PCR-based plasmid multilocus sequence typing Results: The incompatibility groups detected among the blaCTX-M-1-harboring plasmids included IncI1, IncN, IncHI1B, IncF, IncFIIS, IncFIB and IncB/O, with plasmid lineage IncI1/ST3 predominating in isolates from chicken and from humans Lineage IncN/ST1 was detected mainly in isolates from pigs For the first time, blaCTX-M-1 genes encoded on IncHI1 plasmids were detected in isolates from cattle and from water bodies Conclusions: This study identifies plasmid lineages that are contributing to the dissemination of blaCTX-M-1 genes in the food chain, the environment, and humans
TL;DR: Evaluated the performance of a new NGS assay during an outbreak of sequence type 131 (ST131) Escherichia coli infections in a nursing home in The Netherlands, which showed that all isolates of this outbreak cluster were strongly related.
Abstract: Next-generation sequencing (NGS) has the potential to provide typing results and detect resistance genes in a single assay, thus guiding timely treatment decisions and allowing rapid tracking of transmission of resistant clones. We evaluated the performance of a new NGS assay (Hospital Acquired Infection BioDetection System; Pathogenica) during an outbreak of sequence type 131 (ST131) Escherichia coli infections in a nursing home in The Netherlands. The assay was performed on 56 extended-spectrum-beta-lactamase (ESBL) E. coli isolates collected during 2 prevalence surveys (March and May 2013). Typing results were compared to those of amplified fragment length polymorphism (AFLP), whereby we visually assessed the agreement of the BioDetection phylogenetic tree with clusters defined by AFLP. A microarray was considered the gold standard for detection of resistance genes. AFLP identified a large cluster of 31 indistinguishable isolates on adjacent departments, indicating clonal spread. The BioDetection phylogenetic tree showed that all isolates of this outbreak cluster were strongly related, while the further arrangement of the tree also largely agreed with other clusters defined by AFLP. The BioDetection assay detected ESBL genes in all but 1 isolate (sensitivity, 98%) but was unable to discriminate between ESBL and non-ESBL TEM and SHV beta-lactamases or to specify CTX-M genes by group. The performance of the hospital-acquired infection (HAI) BioDetection System for typing of E. coli isolates compared well with the results of AFLP. Its performance with larger collections from different locations, and for typing of other species, was not evaluated and needs further study.
TL;DR: The study findings indicate that virtual keyboards with a key size less than 16 mm may be too small for touch typing given the slower typing speed, higher static shoulder muscle activity, greater wrist extension, and lowest subjective preferences.
Abstract: Objective:We investigated whether different virtual keyboard key sizes affected typing force exposures, muscle activity, wrist posture, comfort, and typing productivity.Background:Virtual keyboard use is increasing and the physical exposures associated with virtual keyboard key sizes are not well documented.Method:Typing forces, forearm/shoulder muscle activity, wrist posture, subjective comfort, and typing productivity were measured from 21 subjects while they were typing on four different virtual keyboards with square key sizes, which were 13, 16, 19, and 22 mm on each side with 2-mm between-key spacing.Results:The results showed that virtual keyboard key size had little effect on typing force, forearm muscle activity, and ulnar/radial deviation. However, the virtual keyboard with the 13-mm keys had a 15% slower typing speed (p < .0001), slightly higher static (10th percentile) shoulder muscle activity (2% maximum voluntary contractions, p = .01), slightly greater wrist extension in both hands (2° to 3°...
TL;DR: In China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.
Abstract: Legionella pneumophila serogroup 1 causes Legionnaires' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.
TL;DR: The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs.
Abstract: Clostridium difficile infections (CDI) are a growing concern in North America, because of their increasing incidence and severity. Using integrated approaches, we correlated pathogen genotypes and host clinical characteristics for 46 C. difficile infections in a tertiary care medical center during a 6-month interval from January to June 2010. Multilocus sequence typing (MLST) demonstrated 21 known and 2 novel sequence types (STs), suggesting that the institution's C. difficile strains are genetically diverse. ST-1 (which corresponds to pulsed-field gel electrophoresis strain type NAP1/ribotype 027) was the most prevalent (32.6%); 43.5% of the isolates were binary toxin gene positive, of which 75% were ST-1. All strains were ciprofloxacin resistant and metronidazole susceptible, and 8.3% and 13.0% of the isolates were resistant to clindamycin and tetracycline, respectively. The corresponding resistance loci, including potential novel mutations, were identified from the whole-genome sequencing (WGS) of the resistant strains. Core genome single nucleotide polymorphisms (SNPs) determining the phylogenetic relatedness of the 46 strains recapitulated MLST types and provided greater interstrain differentiation. The disease severity was greatest in patients infected with ST-1 and/or binary gene-positive strains, but genome-wide SNP analysis failed to provide additional associations with CDI severity within the same STs. We conclude that MLST and core genome SNP typing result in the same phylogenetic grouping of the 46 C. difficile strains collected in a single hospital. WGS also has the capacity to differentiate those strains within STs and allows the comparison of strains at the individual gene level and at the whole-genome level.
TL;DR: This scheme combines the best loci from the most widely used MLST schemes for Leptospira spp.