Classical Serotypes Research Articles

Genome-based Salmonella serotyping as the new gold standard

Salmonella enterica is the second most reported bacterial cause of food-borne infections in Europe. Therefore molecular surveillance activities based on pathogen subtyping are an important measure of controlling Salmonellosis by public health agencies. In Germany, at the federal level, this work is carried out by the National Reference Center for Salmonella and other Bacterial Enteric Pathogens (NRC). With rise of next generation sequencing techniques, the NRC has introduced whole-genome-based typing methods for S. enterica in 2016. In this study we report on the feasibility of genome-based in silico serotyping in the German setting using raw sequence reads. We found that SeqSero and seven gene MLST showed 98% and 95% concordance, respectively, with classical serotyping for the here evaluated serotypes, including the most common German serotypes S. Enteritidis and S. Typhimurium as well as less frequently found serotypes. The level of concordance increased to >99% when the results of both in silico methods were combined. However, both tools exhibited misidentification of monophasic variants, in particular monophasic S. Typhimurium and therefore need to be fine-tuned for reliable detection of this epidemiologically important variant. We conclude that with adjustments Salmonella genome-based serotyping might become the new gold standard.

Differential Biophysical Behaviors of Closely Related Strains of Salmonella.

Salmonella is an important pathogen and is a world-wide threat to food safety and public health. Surveillance of serotypes and fundamental biological and biochemical studies are supported by a wide variety of established and emerging bioanalytical techniques. These include classic serotyping based on the Kauffmann–White nomenclature and the emerging whole genome sequencing strategy. Another emerging strategy is native whole cell biophysical characterization which has yet to be applied to Salmonella. However, this technique has been shown to provide high resolution differentiation of serotypes with several other paired strains of other microbes and pathogens. To demonstrate that biophysical characterization might be useful for Salmonella serotyping, the closely related strains sv. Cubana and sv. Poona were chosen for study. These two serovars were subjected to biophysical measurements on a dielectrophoresis-based microfluidic device that generated full differentiation of the unlabeled and native cells. They were differentiated by the ratio of electrophoretic (EP) to dielectrophoretic (DEP) mobilities. This differentiation factor is 2.7 ± 0.3 × 1010 V/m2 for sv. Cubana, versus 2.2 ± 0.3 × 1010 V/m2 for sv. Poona. This work shows for the first time the differentiation, concentration, and characterization of the Salmonella serotypes by exploiting their biophysical properties. It may lead to a less expensive and more decentralized new tool and method for microbiologists, complimenting and working in parallel with other characterization methods.

Novel Native and Engineered Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs), produced by Clostridia and other bacteria, are the most potent toxins known. Their cleavage of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins in neurons prevents the release of neurotransmitters, thus resulting in the muscle paralysis that is characteristic of botulism. This mechanism of action has been exploited for a variety of therapeutic and cosmetic applications of BoNTs. This chapter provides an overview of the native BoNTs, including the classical serotypes and their clinical use, mosaic BoNTs, and novel BoNTs that have been recently identified in clostridial and non-clostridial strains. In addition, the modular structure of native BoNTs, which are composed of a light chain and a heavy chain, is amenable to a multitude of novel fusions and mutations using molecular biology techniques. These novel recombinant BoNTs have been used or are being developed to further characterize the biology of toxins, to assist in vaccine production, to serve as delivery vehicles to neurons, and to be utilized as novel therapeutics for both neuronal and non-neuronal cells.

SeqSero2: Rapid and Improved Salmonella Serotype Determination Using Whole-Genome Sequencing Data.

SeqSero, launched in 2015, is a software tool for Salmonella serotype determination from whole-genome sequencing (WGS) data. Despite its routine use in public health and food safety laboratories in the United States and other countries, the original SeqSero pipeline is relatively slow (minutes per genome using sequencing reads), is not optimized for draft genome assemblies, and may assign multiple serotypes for a strain. Here, we present SeqSero2 (github.com/denglab/SeqSero2; denglab.info/SeqSero2), an algorithmic transformation and functional update of the original SeqSero. Major improvements include (i) additional sequence markers for identification of Salmonella species and subspecies and certain serotypes, (ii) a k-mer based algorithm for rapid serotype prediction from raw reads (seconds per genome) and improved serotype prediction from assemblies, and (iii) a targeted assembly approach for specific retrieval of serotype determinants from WGS for serotype prediction, new allele discovery, and prediction troubleshooting. Evaluated using 5,794 genomes representing 364 common U.S. serotypes, including 2,280 human isolates of 117 serotypes from the National Antimicrobial Resistance Monitoring System, SeqSero2 is up to 50 times faster than the original SeqSero while maintaining equivalent accuracy for raw reads and substantially improving accuracy for assemblies. SeqSero2 further suggested that 3% of the tested genomes contained reads from multiple serotypes, indicating a use for contamination detection. In addition to short reads, SeqSero2 demonstrated potential for accurate and rapid serotype prediction directly from long nanopore reads despite base call errors. Testing of 40 nanopore-sequenced genomes of 17 serotypes yielded a single H antigen misidentification.IMPORTANCE Serotyping is the basis of public health surveillance of Salmonella It remains a first-line subtyping method even as surveillance continues to be transformed by whole-genome sequencing. SeqSero allows the integration of Salmonella serotyping into a whole-genome-sequencing-based laboratory workflow while maintaining continuity with the classic serotyping scheme. SeqSero2, informed by extensive testing and application of SeqSero in the United States and other countries, incorporates important improvements and updates that further strengthen its application in routine and large-scale surveillance of Salmonella by whole-genome sequencing.

Rapid Detection of Staphylococcal Enterotoxin-B by Lateral Flow Assay.

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose–response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.

Molecular Evolution of Classic Human Astrovirus, as Revealed by the Analysis of the Capsid Protein Gene.

Classic human astroviruses (HAstV) are major global viral agents for gastroenteritis, but the molecular characteristics of classic HAstVs are not well understood. Here, we presented the molecular evolution of all classic HAstV serotypes by the analysis of the capsid protein sequences. Our results show that classic HAstVs can be divided into four groups with the most recent common ancestor (TMRCA) of 749. The overall evolutionary rate of classic HAstVs on the capsid gene was 4.509 × 10−4 substitutions/site/year, and most of the serotypes present a clock-like evolution with an amino acid accumulation of mutations over time. The mean effective population size of classic HAstVs is in a downward trend, and some positive and more than 500 negative selection sites were determined. Taken together, these results reveal that classic HAstVs evolve at the intra-serotype level with high genetic heterogeneity and are driven by strong purifying selection. Long-term surveillance of classic HAstVs are needed to enrich the genomic data for further analysis.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics

ABSTRACTThis study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Assessment and Comparison of Molecular Subtyping and Characterization Methods for Salmonella.

The food industry is facing a major transition regarding methods for confirmation, characterization, and subtyping of Salmonella. Whole-genome sequencing (WGS) is rapidly becoming both the method of choice and the gold standard for Salmonella subtyping; however, routine use of WGS by the food industry is often not feasible due to cost constraints or the need for rapid results. To facilitate selection of subtyping methods by the food industry, we present: (i) a comparison between classical serotyping and selected widely used molecular-based subtyping methods including pulsed-field gel electrophoresis, multilocus sequence typing, and WGS (including WGS-based serovar prediction) and (ii) a scoring system to evaluate and compare Salmonella subtyping assays. This literature-based assessment supports the superior discriminatory power of WGS for source tracking and root cause elimination in food safety incident; however, circumstances in which use of other subtyping methods may be warranted were also identified. This review provides practical guidance for the food industry and presents a starting point for further comparative evaluation of Salmonella characterization and subtyping methods.

Replication kinetics and cellular tropism of emerging reoviruses in sheep and swine respiratory ex vivo organ cultures

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.

Whole genome sequencing used in an industrial context reveals a Salmonella laboratory cross-contamination

In 2013, during a routine laboratory analysis performed on food samples, one finished product from a European factory was tested positive for Salmonella Hadar. At the same period, one environmental isolate in the same laboratory was serotyped Salmonella Hadar. Prior to this event, the laboratory performed a proficiency testing involving a sample spiked with NCTC 9877 Salmonella Hadar. The concomitance of Salmonella Hadar detection led to the suspicion of a laboratory cross-contamination between the Salmonella Hadar isolate used in the laboratory proficiency testing and the Salmonella Hadar isolate found on the finished product by the same laboratory. Since the classical phenotypic serotyping method is able to attribute a serotype to Salmonella isolates with a common antigenic formula, but cannot differentiate strains of the same serotype within the subspecies, whole genome sequencing was used to test the laboratory cross-contamination hypothesis. Additionally, 12 Salmonella Hadar from public databases, available until the time of the event, were included in the whole genome sequencing analysis to better understand the genomic diversity of this serotype in Europe. The outcome of the analysis showed a maximum of ten single nucleotide polymorphisms (SNPs) between the isolates coming from the laboratory and the finished product, and thus confirmed the laboratory cross-contamination. These results combined with all additional investigations done at the factory, allowed to release finished product batches produced and thus circumvented unnecessary food waste and economic losses for the factory.

Molecular Characterization of Enterotoxigenic Escherichia coli: Effect on Intestinal Nitric Oxide in Diarrheal Disease

This study was aimed to investigate the effect of enterotoxigenic E. coli (ETEC)-induced diarrhea on fecal nitric oxide (NO) and intestinal inducible nitric oxide synthase (iNOS) expression in rats. E. coli isolates were gotten from infants diarrheal samples. Multiplex polymerase chain reaction (PCR) was used to detect the presence of eltA gene for heat-stable (ST) enterotoxigenic E. coli and eltB for heat-labile (LT) enterotoxigenic E. coli. Disk diffusion method was used to determine the antibiotic susceptibility of the organisms. Biofilm formation was detected by thiazoylblue tetrazolium bromide dye in a 96-well plate. Fecal NO was measured using standard griess reaction system. Reverse transcription PCR was used to investigate the expression of iNOS expression. Although none of the ETECs isolated in this study belonged to the classic serotype, serogroup O6 and O8 were found to be associated with ETECs. Among the three ETECs, two were found to be multidrug resistant. The biofilm production abilities of all the ETECs were found to exist between weak and moderate biofilm producers. Fecal NO was found to be elevated in both LT and ST-induced diarrheal groups but there was no corresponding intestinal iNOS expression. This suggests that the elevated NO could be as a result of up-regulation of constitutive NOS rather than iNOS.

Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods.

In this study, different molecular typing tools were applied to characterize 95 Salmonella enterica blood isolates collected between 2008 and 2013 from patients at nine public hospitals in Lima, Peru. Combined results of multiplex PCR serotyping, two- and seven-loci multilocus sequence typing (MLST) schemes, serotyping, IS200 amplification and RAPD fingerprints, showed that these infections were caused by eight different serovars: Enteritidis, Typhimurium, Typhi, Choleraesuis, Dublin, Paratyphi A, Paratyphi B and Infantis. Among these, Enteritidis, Typhimurium and Typhi were the most prevalent, representing 45, 36 and 11% of the isolates, respectively. Most isolates (74%) were not resistant to ten primarily used antimicrobial drugs; however, 37% of the strains showed intermediate susceptibility to ciprofloxacin (ISC). Antimicrobial resistance integrons were carried by one Dublin (dfra1 and aadA1) and two Infantis (aadA1) isolates. The two Infantis isolates were multidrug resistant and harbored a large megaplasmid. Amplification of spvC and spvRA regions showed that all Enteritidis (n = 42), Typhimurium (n = 34), Choleraesuis (n = 3) and Dublin (n = 1) isolates carried the Salmonella virulence plasmid (pSV). We conclude that the classic serotyping method can be substituted by the multiplex PCR and, when necessary, sequencing of only one or two loci of the MLST scheme is a valuable tool to confirm the results. The effectiveness and feasibility of different typing tools is discussed.

Global distributions and strain diversity of avian infectious bronchitis virus: a review.

The poultry industry faces challenge amidst global food security crisis. Infectious bronchitis is one of the most important viral infections that cause huge economic loss to the poultry industry worldwide. The causative agent, infectious bronchitis virus (IBV) is an RNA virus with great ability for mutation and recombination; thus, capable of generating new virus strains that are difficult to control. There are many IBV strains found worldwide, including the Massachusetts, 4/91, D274, and QX-like strains that can be grouped under the classic or variant serotypes. Currently, information on the epidemiology, strain diversity, and global distribution of IBV has not been comprehensively reported. This review is an update of current knowledge on the distribution, genetic relationship, and diversity of the IBV strains found worldwide.

Phenotypic and Genotypic Characterization of Clinical Enterotoxigenic Escherichia coli Isolates from Shenzhen, China.

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of infectious diarrhea in developing countries. This study aimed to characterize the prevalence and phenotypic and genotypic features of ETEC isolates from Shenzhen, China. ETEC isolates were obtained from acute diarrheal patients and evaluated for enterotoxin, classical colonization factors (CFs), serotypes, antimicrobial susceptibility, and multilocus sequencing typing (MLST). A total of 168 (1.3%) ETEC strains were isolated from 13,324 diarrheal outpatients during 2009 and 2014. A vast majority of ETEC-infected patients (82.1%) belonged to the age ranging 20-59 years and only six patients were children aged <5 years. Heat-stable toxin (ST) was most frequently detected (81.5%), followed by heat-labile toxin (LT) (13.1%). One or multiple colonization factors (CFs) were identified in 91 ETEC strains (54.2%). The most frequently detected CF was CS6 (with or without other CFs) (84/91), followed by CS21 (14/91). The most common serotype was O159:H34 (n = 36), followed by O148:H28 (n = 25) and O27:H7 (n = 17). High resistant rate was observed to nalidixic acid (77.4%), cephalothin (41.7%), ampicillin (34.5%), and tetracycline (21.4%). Antimicrobial resistance profiles differed among different serogroups. Sequence type (ST) 10 complex, integrated with connected ST218, ST48, ST4, and ST1312 subgroups, covered 73 (43.5%) isolates. ETEC isolates in Shenzhen of China appeared highly diverse, yet some isolates belonged to well-defined clonal groups sharing a unique set of virulence factors, serotypes, and MLST sequence types. Facing the challenge of ETEC antigenic diversity and geographic variation, novel molecules and/or classical antigens designed by novel strategies might contribute to ETEC vaccine development.

Pneumococcal 23B Molecular Subtype Identified Using Whole Genome Sequencing.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae and the target of all currently licensed pneumococcal vaccines. At present, there are 92 serologically distinct pneumococcal serotypes. Structural and antigenic variation of capsular types is the result of genetic variation within the capsular polysaccharide synthesis (CPS) locus; however, genetic variation may not always result in phenotypic differences which produce novel serotypes. With the introduction of high throughput whole genome sequencing, discovery of novel genotypic variants is not unexpected and this study describes a novel variant of the serotype 23B CPS operon. This novel variant was characterized as a novel genotypic subtype (23B1) with ∼70% homology to the published 23B CPS sequence. High sequence variability was determined in eight cps genes involved in sugar biosynthesis. However, there was no distinction between the classic 23B serotype and 23B1 serologically or in terms of polysaccharide structure. Phylogenetic and eBURST analysis revealed a distinct lineage for 23B1 with multiple clones (UK, Thailand, and USA) that arose at different points during pneumococcal evolution. Analysis of the UK S. pneumoniae isolates (n = 121) revealed an upsurge of 23B1 ST2372 in 2011, after which this previously unseen ST increased to reach 50% proportion of the 23B sequenced isolates from 2013 and remained prevalent within our sequenced isolates from later years. Therefore, although the 23B1 variant appears to have no phenotypic impact and cannot be considered as novel serotype, it appears to have led to a genetic restructuring of the UK serotype 23B population.

Analysis of antigenicity and pathogenicity reveals major differences among QX-like infectious bronchitis viruses and other serotypes

Avian coronavirus infectious bronchitis virus (IBV) causes considerable damage to the poultry industry worldwide and the proportion of QX-like genotype isolates have increased over time. Here, to better understand the antigenicity and pathogenicity of this genotype, we conducted sequence analyses, cross neutralization tests, and also examined the pathogenicity of two strains, SD and SZ. Sequence analyses revealed that SD and SZ isolates belong to the QX-like IBV genotype and share high homology in their full-length genomes. Cross neutralization tests showed high cross neutralization between SD and SZ, but distant relationships with other representative strains of the classical IBV serotypes. Virus infection experiments showed that SD caused high mortality with strong respiratory and renal pathogenicity in chickens, whereas SZ caused milder lesions by comparison. This study highlights the big discrepancy in antigenicity that exists between QX-like strains and other serotypes. Collectively, these findings provide important information about the epidemiology and pathogenicity of IBV, which may benefit the control of IB in the poultry industry.

Utilization of a molecular serotyping method for Salmonella enterica in a routine laboratory in Alberta Canada

Salmonella is one of the most common enteric pathogens related to foodborne illness. Alberta's Provincial Laboratory for Public Health (ProvLab) provides Outbreak and Surveillance support by performing serotyping. The Check&Trace Salmonella™ (CTS) assay (Check-Points, Netherlands), a commercial DNA microarray system, can determine the serotype designation of a Salmonella isolate with automated interpretation. Here we evaluate 1028 Salmonella isolates of human clinical or environmental sources in Alberta, Canada with the CTS assay. CTS was able to assign a serovar to 98.7% of the most frequently occurring human clinical strains in Alberta (82.5% overall), and 71.7% of isolates which were inconclusive by conventional methods. There was 99.7% concordance in environmental isolates. The CTS database has potential to expand to identify rare serovars. With the anticipated shift to molecular methods for identification, CTS provides an easy transition and demonstrates ease-of-use and reduces the turn-around-time of a reported result significantly compared to classical serotyping.

Novel and classical human astroviruses in stool and cerebrospinal fluid: comprehensive screening in a tertiary care hospital, Switzerland

Classical human astroviruses (HAstV) are the third most common cause of non-bacterial acute gastroenteritis. Due to the lack of routine molecular assays, novel HAstV are underdiagnosed and the magnitude of their contribution to clinical disease remains unknown. To better understand their prevalence and the susceptible patient profile, we conducted a comprehensive screening of novel and classical HAstV in stool and cerebrospinal fluid (CSF) samples collected for clinical care in a tertiary care hospital using a specially designed rRT-PCR panel for the detection of novel (MLB1-3 and VA1-4) and classical HAstV. Of the 654 stool samples, 20 were positive for HAstV, and the novel (n=10; 3 MLB1, 4 MLB2; 3 VA2) and classical (n=10) serotypes were equally prevalent. None of the 105 CSF samples were positive. Investigating the patient profile, we found a higher prevalence (P=0.0002) of both novel and classical HAstV in pediatric stool samples (3.4% and 3%, respectively) compared with adult stool samples (0.5% and 0.7%, respectively). Furthermore, all novel and classical HAstV-positive pediatric subjects were ≤four years old, demonstrating similar susceptible populations. Forty-five percent of positive patients were immunocompromised (novel: 40%, classical: 50%). A comparison of novel and classical HAstV-positive cases showed a lower viral load for novel HAstV (P=0.0007) with significantly more upper respiratory symptoms (70% of subjects; P=0.02); this observation may suggest a unique pathogenic pathway. This study confirms the clinical and epidemiological relevance of novel HAstV and identifies a target population in which routine screening may yield clinically valuable information.Emerging Microbes & Infections (2017) 6, e84; doi:10.1038/emi.2017.71; published online 20 September 2017

Serogenotyping and antimicrobial susceptibility testing of Salmonella spp. isolated from retail meat samples in Lagos, Nigeria

Microarray-based serogenotyping, antimicrobial susceptibility tests and the detection of relevant resistance genes were performed on isolates of Salmonella spp. from retail meat samples obtained in Lagos, Nigeria.Out of 151 meat samples, 33 Salmonella isolates were obtained. Nine different Salmonella serovars (S. Amoutive, S. Bargny, S. Drac, S. Ealing, S. Urbana, S. Hadar, S. Nyborg, S. Anatum and S. Havana) were identified by microarray-based serogenotyping and confirmed afterwards using classical serotyping. Antibiotic susceptibility tests with 17 antibiotics showed that almost all isolates were fully susceptible to this panel.The results of this study indicated a high prevalence of Salmonella in retail meat, the presence of some previously rather rarely described Serovars in retail meat samples from Lagos, and a need to monitor for Salmonella and their antibiotic resistance determinants. The microarray-based system used herein proved to be perfectly suited as epidemiological tool to replace classical serotyping.

Updated Campylobacter jejuni Capsule PCR Multiplex Typing System and Its Application to Clinical Isolates from South and Southeast Asia.

Campylobacter jejuni produces a polysaccharide capsule that is the major determinant of the Penner serotyping scheme. This passive slide agglutination typing system was developed in the early 1980’s and was recognized for over two decades as the gold standard for C. jejuni typing. A preliminary multiplex PCR technique covering 17 serotypes was previously developed in order to replace this classic serotyping scheme. Here we report the completion of the multiplex PCR technology that is able to identify all the 47 Penner serotypes types known for C. jejuni. The number of capsule types represented within the 47 serotypes is 35. We have applied this method to a collection of 996 clinical isolates from Thailand, Cambodia and Nepal and were able to successfully determine capsule types of 98% of these.