Dataset Information

Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan.

ABSTRACT:

Background

Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis strains can exist in single animals. Typing techniques are required for identifying G. parasuis isolates. Different strains within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence-associated trimeric autotransporters (vtaA) gene is an indicator of virulence. The aim of this study was to characterize Taiwanese G. parasuis isolates via molecular serotyping, vtaA PCR and ERIC-PCR.

Methods

One hundred and forty-five strains were collected between November 2013 and March 2017 in Taiwan and further examined by molecular serotyping, vtaA PCR and ERIC-PCR.

Results

The dendrogram revealed heterogeneous genetic diversity within many clusters. Partial correlation between the ERIC-PCR clusters of different strains, serovars and lesion patterns was observed. Twelve herds (8.3%) infected with more than one strain. Group 1 vtaA positive rate reached 98.6%.

Discussion

This study showed the high genetic diversity of G. parasuis in Taiwan by a high discriminatory capability of ERIC-PCR. Group 1 vtaA commonly exists in G. parasuis isolates and may play important roles in the pathogenesis of Taiwanese G. parasuis isolates.

SUBMITTER: Lin WH

PROVIDER: S-EPMC6526895 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan.

Lin Wei-Hao WH Shih Hsing-Chun HC Lin Chuen-Fu CF Yang Cheng-Yao CY Lin Chao-Nan CN Chiou Ming-Tang MT

PeerJ 20190517

<h4>Background</h4>Glaesserella (Haemophilus) parasuis (G. parasuis) causes severe economic losses in the swine industry. Multiple G. parasuis strains can exist in single animals. Typing techniques are required for identifying G. parasuis isolates. Different strains within a serovar display varying virulence. Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) can assess the heterogeneity. The group 1 virulence-associated ...[more]

PMID: 31149406

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Project description:BackgroundGlaesserella parasuis (G. parasuis) belongs to the normal microbiota of the upper respiratory tract in the swine, but virulent strains can cause systemic infections commonly known as Glässer's disease that leads to significant economic loss in the swine industry. Fifteen serotypes of G. parasuis have been classified by gel immunodiffusion test while the molecular serotyping based on variation within the capsule loci have further improved the serotype determination of unidentified field strains. Serovar has been commonly used as an indicator of virulence; however, virulence can be significantly differ in the field isolates with the same serotype. To date, investigations of G. parasuis isolated in Taiwan regarding antimicrobial resistance, serotypes, genotypes and virulence factors remain unclear.MethodsA total of 276 G.parasuis field isolates were collected from 263 diseased pigs at the Animal Disease Diagnostic Center of National Chiayi University in Taiwan from January 2013 to July 2021. Putative virulence factors and serotypes of the isolates were identified by polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed by microbroth dilution assay. Additionally, the epidemiology of G. parasuis was characterized by multilocus sequence typing (MLST).ResultsSerotype 4 (33.3%) and 5 (21.4%) were the most prevalent, followed by nontypable isolates (15.9%), serotype 13 (9.4%), 12 (6.5%), 14 (6.2%), 7 (3.3%), 1 (1.8%), 9 (1.1%), 11 (0.7%) and 6 (0.4%). Nine out of 10 putative virulence factors showed high positive rates, including group 1 vtaA (100%), fhuA (80.4%), hhdA (98.6%), hhdB (96.0%), sclB7 (99.6%), sclB11 (94.9%), nhaC (98.2%), HAPS_0254 (85.9%), and cirA (99.3%). According to the results of antimicrobial susceptibility testing, ceftiofur and florfenicol were highly susceptible (>90%). Notably, 68.8% isolates showed multidrug resistance. MLST revealed 16 new alleles and 67 new sequence types (STs). STs of these isolated G. parasuis strains were classified into three clonal complexes and 45 singletons by Based Upon Related Sequence Types (BURST) analysis. All the G. parasuis strains in PubMLST database, including strains from the diseased pigs in the study, were defined into two main clusters by Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Most isolates in this study and virulent isolates from the database were mainly located in cluster 2, while cluster 1 included a high percentage of nasal isolates from asymptomatic carriers. In conclusion, this study provides current prevalence and antimicrobial susceptibility of G. parasuis in Taiwan, which can be used in clinical diagnosis and treatment of Glässer's disease.

Project description:BackgroundGlaesserella parasuis (G. parasuis) is a respiratory pathogen of swine and the etiological agent of Glässer's disease. The structural organization of genetic information, antibiotic resistance genes, potential pathogenicity, and evolutionary relationships among global G. parasuis strains remain unclear. The aim of this study was to better understand patterns of genetic variation, antibiotic resistance factors, and virulence mechanisms of this pathogen.MethodsThe whole-genome sequence of a ST328 isolate from diseased swine in China was determined using Pacbio RS II and Illumina MiSeq platforms and compared with 54 isolates from China sequenced in this study and 39 strains from China and eigtht other countries sequenced by previously. Patterns of genetic variation, antibiotic resistance, and virulence mechanisms were investigated in relation to the phylogeny of the isolates. Electrotransformation experiments were performed to confirm the ability of pYL1-a plasmid observed in ST328-to confer antibiotic resistance.ResultsThe ST328 genome contained a novel Tn6678 transposon harbouring a unique resistance determinant. It also contained a small broad-host-range plasmid pYL1 carrying aac(6')-Ie-aph(2")-Ia and bla ROB-1; when transferred to Staphylococcus aureus RN4220 by electroporation, this plasmid was highly stable under kanamycin selection. Most (85.13-91.74%) of the genetic variation between G. parasuis isolates was observed in the accessory genomes. Phylogenetic analysis revealed two major subgroups distinguished by country of origin, serotype, and multilocus sequence type (MLST). Novel virulence factors (gigP, malQ, and gmhA) and drug resistance genes (norA, bacA, ksgA, and bcr) in G. parasuis were identified. Resistance determinants (sul2, aph(3")-Ib, norA, bacA, ksgA, and bcr) were widespread across isolates, regardless of serovar, isolation source, or geographical location.ConclusionsOur comparative genomic analysis of worldwide G. parasuis isolates provides valuable insight into the emergence and transmission of G. parasuis in the swine industry. The result suggests the importance of transposon-related and/or plasmid-related gene variations in the evolution of G. parasuis.

Project description:Glaesserella parasuis is a commensal bacterial organism found in the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease, which causes severe economic losses in the swine industry. This study aimed to better understand the epidemiological characteristics of this opportunistic pathogen. We investigated the prevalence and distribution of sequence types (STs), serovars, antimicrobial resistance genes (ARGs), and potential virulence factors (VFs) in 764 G. parasuis isolates collected from diseased and healthy pigs from 19 countries, including China. Multilocus sequence typing showed a high degree of variation with 334 STs, of which 93 were not previously recognized. Phylogenetic analysis revealed two major clades distinguished by isolation year, source, country, and serovar. The dominant serovars of G. parasuis were serovars 4 (19.50%), 7 (15.97%), 5/12 (13.87%), and 13 (12.30%). Serovar 7 gradually became one of the dominant serovars in G. parasuis with more VFs and fewer ARGs. Serovars 4 and 5/12 were the most frequent serovars in diseased pigs, whereas serovars 2, 8, and 11 were predominant in healthy pigs. Serovars 7 and 13 possessed more VFs than the other serovars. This study provides novel insights into the global prevalence and epidemiology of G. parasuis and valuable clues for further investigation into the pathogenicity of G. parasuis, which will facilitate the development of effective vaccines.IMPORTANCEGlaesserella parasuis is a clinically important gram-negative opportunistic pathogen, which causes serious financial losses in swine industry on a global scale. No vaccine is known that provides cross-protection against all 15 serovars; furthermore, the correlation between serovar and virulence is largely unknown. This study provides a large number of sequenced strains in 19 countries and compares the genomic diversity of G. parasuis between diseased and healthy pigs. We found a slight change in the dominant serovar of G. parasuis in the world, with serovar 7 gradually emerging as one of the predominant serovars. The observed higher average number of VFs in this particular serovar strain challenges the previously held notion that serovar 7 is non-virulent, indicating a more complex virulence landscape than previously understood. Our analysis indicating that six ARGs [tet(B), sul2, aph(3')-Ia, aph (6)-Id, blaROB-1, and aph(3'')-Ib] are likely to be transmitted horizontally in their entirety. By analyzing VFs, we provided an improved understanding of the virulence of G. parasuis, and these key findings suggest that vaccine development will be challenging.

Project description:BackgroundGlaesserella parasuis is the causative agent of Glässer's disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted.ResultsIn silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency; blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%). CONCLUSION: This study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.

Project description:BackgroundGlässer's disease, caused by Glaesserella parasuis (G. parasuis), is a widespread bacterial infection in swine that leads to significant economic losses. G. parasuis, a member of the normal microbiota within the Pasteurellaceae family, exhibits horizontal resistance gene exchange and intracellular invasion capabilities, increasing the risk of developing resistant isolates. Accurate antimicrobial therapy is essential for controlling Glässer's disease. The production systems for exotic crossbred pigs and Taiwan black pigs differ considerably. To inform Glässer disease control and monitor antimicrobial resistance, we assessed the antimicrobial susceptibilities of G. parasuis isolates, analyzed them using normalized resistance interpretation (NRI), and compared findings between the two production systems.ResultsA total of 154 G. parasuis isolates from 106 exotic crossbred pig herds and 48 Taiwan black pig herds were tested against 16 antimicrobial agents between 2015 and 2020. Due to the absence of specific breakpoints for G. parasuis, NRI was utilized to define non-wild-type (non-WT) populations based on minimum inhibitory concentration (MIC) distributions. Non-WT subpopulations of isolates for amoxicillin, ampicillin, ceftiofur, gentamicin, kanamycin, and tiamulin were observed. The highest MIC90 (the concentration at which 90% of isolates were inhibited) was > 256 µg/mL for several antimicrobials, including gentamicin, kanamycin, lincomycin, lincospectin, spectinomycin, and tylosin. In contrast, the lowest MIC90 was observed for ceftiofur (0.5 µg/mL). The MIC values for cephalothin were significantly higher in exotic crossbred pigs than in Taiwan black pigs (p = 0.0016). Conversely, MIC values for florfenicol were significantly higher in Taiwan black pigs than in exotic crossbred pigs (p = 0.003).ConclusionsThis study provides the susceptibility profile of G. parasuis isolates for both exotic crossbred pigs and Taiwan black pigs in Taiwan and highlights potential antimicrobial resistance for aminocyclitol, aminoglycosides, beta-lactams, lincosamides, macrolides, and pleuromulin. Ceftiofur, cephalothin, doxycycline, and florfenicol could be most suitable for treating early-stage Glässer's disease. Nonetheless, increased attention should be paid to the responsible use of antimicrobials in light of the growing threat of antimicrobial resistance.

Dataset Information

Genotypic analyses and virulence characterization of Glaesserella parasuis isolates from Taiwan.

Background

Methods

Results

Discussion

Publications

Genotypic analyses and virulence characterization of <i>Glaesserella parasuis</i> isolates from Taiwan.

Similar Datasets

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