Antimicrobial Resistance and Molecular Characterization of Escherichia coli from Healthy Chickens in Shandong, China from 2009 to 2014.

Antimicrobial resistance (AMR) is a great threat to animal and public health. Here, we conducted a surveillance of Escherichia coli isolated from healthy chickens during 2009-2014 to identify the characteristics of AMR. A total of 351 (95.64%) E. coli isolates were obtained from 367 healthy chicken fecal samples collected from 6 farms located in Shandong Province, China. The susceptibility to 10 antimicrobials, the prevalence of antibiotic resistance genes (ARGs), phylogenetic clustering, and multilocus sequence typing were evaluated. The isolates exhibited high resistant rates (>95%) to ampicillin, cefotaxime, ciprofloxacin, ceftiofur, and enrofloxacin. The most prevalent ARGs were blaCTX-M (36.36%), aac(6')-Ib-cr (30.79%), qnrS (29.62%), oqxAB (27%), mcr-1 (15.83%), blaTEM (9.09%), qnrC (3.52%), qnrD (0.88%), and qepA (0.29%). Phylogenetic clustering analysis indicated that the most prevalent group was group D (37.89%), followed by group B1 (34.76%), A (24.22%), and B2 (3.13%). Fifty-seven sequence types (STs) were identified among the 124 blaCTX-M-positive strains, and the dominant STs were ST354 (13.71%), ST117 (5.65%), ST155, ST2309, and ST2505 (4.84% each). There was a significant association between 17 pairs of AMR phenotypes, 14 pairs of ARGs, and 11 pairs of AMR-ARGs. The strongest association was found between ST602 and qnrC (odds ratios: 22.2). This study implied that E. coli isolated from healthy chickens could potentially serve as a reservoir of AMR and ARGs, and significant associations exist among AMR, ARGs, phylogenetic groups, and STs. Our study highlighted the need for routine surveillance of AMR in healthy chickens, and promoting appropriate antibiotic use and implementing regular monitoring of resistance in broilers are crucial for fostering the development of the poultry industry and safeguarding public health.

Contribution of nadR to the cell growth and virulence of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) has been reported to be a highly invasive pathogen in swine and a zoonotic agent for humans. Although many bacterial virulence factors have been identified, our an insightful understanding of SS2 pathogenicity is lacking. The gene nadR, encoding nicotinamide-nucleotide adenylyltransferase, was first reported as a regulator and transporter of the intracellular NAD synthesis pathway in Salmonella typhimurium. In this study, we constructed a mutant strain of nadR (ΔnadR) to test the phenotypic and virulence variations between the deletion mutant and the wild-type strain ZY05719. The phenotypic experimental results showed that ΔnadR obviously inhibited cell growth and exhibited shorter chains than WT. The growth defect of ΔnadR was caused by the loss of the function of nadR for transporting the substrates nicotinamide mononucleotide and nicotinamide riboside in the intracellular NAD synthesis pathway. In the process of interaction with the host, ΔnadR participated in adherence and invasion to the host cells, and it was more easily cleared by RAW264.7 macrophages. More importantly, both zebrafish and BALB/c mice in vivo virulence experimental results showed that ΔnadR dramatically attenuated the virulence of SS2, and the ability of ΔnadR to colonize tissues was notably reduced in comparison with that of WT in the BALB/c mice infection model. To the best of our knowledge, this is the first time to demonstrate that nadR not only plays an important role in bacterial growth, but also in connection with the virulence of SS2 as a global transcriptional regulator.

Promising potential of machine learning-assisted MALDI-TOF MS as an effective detector for Streptococcus suis serotype 2 and virulence thereof.

IMPORTANCE: To the best of our knowledge, this study reveals a strong correlation between mass spectra pattern and virulence phenotype among S. suis for the first time. In order to make the findings applicable and to excavate the intrinsic information in the spectra, the classifiers based on the machine learning algorithms were established, and RF (Random Forest)-based models have achieved an accuracy of over 90%. Overall, this study will pave the way for virulent SS2 (Streptococcus suis serotype 2) rapid detection, and the important findings on the association between genotype and mass spectrum may provide a new idea for the genotype-dependent detection of specific pathogens.

Molecular characterization of invasive Streptococcus pneumoniae clinical isolates from a tertiary children's hospital in eastern China.

Streptococcus pneumoniae is a common opportunistic pathogen that causes invasive pneumococcal disease (IPD), especially in children. This study aimed to determine the prevalence and molecular characteristics of S. pneumoniae isolated from children with IPD. A total of 78 S. pneumoniae isolates from aseptic body fluids of 70 IPD patients were collected at the Children's Hospital of Nanjing Medical University (Jiangsu Province, China) during 2017-2021. Whole-genome sequencing technology was used to analyze the serotype, sequence type (ST), virulence, and antibiotic resistance of the 78 invasive S. pneumoniae clinical isolates. Our results showed that the pneumococcal infection rate declined after the COVID-19 outbreak in 2019. Serotypes 19F, 14, 6A, 23F, 19A, and 6B were the most common strains. The pneumococcal conjugate vaccine (PCV) 13 serotype coverage rate was 87.1%. All isolates were classified by multi-locus sequence typing (MLST) analysis into 27 different STs, including 3 novel STs (ST17941, ST17942, and ST17944) and 1 novel allele [recP (558)]. The most predominant ST was ST271, followed by ST320 and ST876. All isolates carried the following virulence genes: cbpG, lytB, lytC, pce (cbpE), pavA, slrA, plr (gapA), hysA, nanA, eno, piuA, psaA, cppA, iga, htrA (degP), tig (ropA), zmpB, and ply. All isolates were multidrug resistant and had high levels of resistance to macrolides, tetracyclines, and sulfonamides. Taken together, this study revealed extensive genetic diversity among S. pneumoniae isolates from a single Chinese hospital. Wearing masks, universal infant vaccination with PCV13, and the launch of recombinant protein vaccine development programs could reduce the burden of IPD in children. IMPORTANCE Invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae in children remains a global burden and should be given more attention due to the fact that the pneumococcal vaccine is not fully covered globally. The molecular epidemiological characteristics of S. pneumoniae are not so clear, especially in these years of COVID-19. In this study, we collected S. pneumoniae isolates from the aseptic body fluid of children with IPD from 2017 to 2021 in a tertiary children's hospital in China and revealed the extensive genetic diversity of these isolates. Most importantly, we first found that the rate of pneumococcal infection has declined since the COVID-19 outbreak in 2019, which means that wearing masks could reduce the transmission of S. pneumoniae. In addition, it was shown that universal infant vaccination with PCV13 seems essential for reducing the burden of IPD in children.

Identification of Subunits for Novel Universal Vaccines against Three Predominant Serogroups and the Emerging O145 among Avian Pathogenic Escherichia coli by Pan-RV Pipeline.

Avian pathogenic Escherichia coli, a causative agent of avian colibacillosis, has been causing serious economic losses in the poultry industry. The increase in multidrug-resistant isolates and the complexity of the serotypes of this pathogen, especially the recently reported emergence of a newly predominant serogroup of O145, make the control of this disease difficult. To address this challenge, a high-throughput screening approach, called Pan-RV (Reverse vaccinology based on pangenome analysis), is proposed to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened from the core genome of 127 Avian pathogenic Escherichia coli (APEC) genomes, and six were verified by Western blots using antisera. Overall, our research will provide a foundation for the development of an APEC subunit vaccine against avian colibacillosis. Given the exponential growth of whole-genome sequencing (WGS) data, our Pan-RV pipeline will make screening of bacterial vaccine candidates inexpensive, rapid, and efficient. IMPORTANCE With the emergence of drug resistance and the newly predominant serogroup O145, the control of Avian pathogenic Escherichia coli is facing a serious challenge; an efficient immunological method is urgently needed. Here, for the first time, we propose a high-throughput screening approach to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Importantly, using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened, and three were shown to be immunoreactive with all antisera (covering the four serogroups), thereby providing a foundation for the development of APEC subunit vaccines against avian colibacillosis. Further, our Pan-RV pipeline will provide immunological control strategies for pathogens with complex and variable genetic backgrounds such as Escherichia coli and will make screening of bacterial vaccine candidates more inexpensive, rapid, and efficient.

Combing Immunoinformatics with Pangenome Analysis To Design a Multiepitope Subunit Vaccine against Klebsiella pneumoniae K1, K2, K47, and K64.

Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a leading causative agent of nosocomial infections, mainly infecting patients with immunosuppressive diseases. Capsular (K) serotypes K1, K2, K47, and K64 are commonly associated with higher virulence (hypervirulent Klebsiella pneumoniae), and more threateningly, isolates belonging to the last two K serotypes are also frequently associated with resistance to carbapenem (hypervirulent carbapenem-resistant Klebsiella pneumoniae). The prevalence of these isolates has posed significant threats to human health, and there are no appropriate therapies available against them. Therefore, in this study, a method combining immunoinformatics and pangenome analysis was applied for contriving a multiepitope subunit vaccine against these four threatening serotypes. To obtain cross-protection, 12 predicted conserved antigens were screened from the core genome of 274 complete Klebsiella pneumoniae genomes (KL1, KL2, KL47, and KL64), from which the epitopes of T and B cells were extracted for vaccine construction. In addition, the immunological properties, the interaction with Toll-like receptors, and the stability in a simulative humoral environment were evaluated by immunoinformatics methods, molecular docking, and molecular dynamics simulation. All of these evaluations indicated the potency of this constructed vaccine to be an effective therapeutic agent. Lastly, the cDNA of the designed vaccine was optimized and ligated to pET-28a(+) for expression vector construction. Overall, our research provides a newly cross-protective control strategy against these troublesome pathogens and paves the way for the development of a safe and effective vaccine. IMPORTANCE Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a leading causative agent of nosocomial infections. Among the numerous capsular serotypes, K1, K2, K47, and K64 are commonly associated with higher virulence (hypervirulent K. pneumoniae). More threateningly, the last two serotypes are frequently associated with resistance to carbapenem (hypervirulent carbapenem-resistant K. pneumoniae). However, there is currently no therapeutic agent or vaccine specifically against these isolates. Therefore, development of a vaccine against these pathogens is very essential. In this study, for the first time, a method combining pangenome analysis, reverse vaccinology, and immunoinformatics was applied for contriving a multiepitope subunit vaccine against K. pneumoniae isolates of K1, K2, K47, and K64. Also, the immunological properties of the constructed vaccine were evaluated and its high potency was revealed. Overall, our research will pave the way for the vaccine development against these four threatening capsular serotypes of K. pneumoniae.

Whole-genome analyses of APEC carrying mcr-1 in some coastal areas of China from 2019 to 2020.

OBJECTIVES: Polymyxin is considered as one of the 'last lines of defense' for the treatment of multidrug resistant bacteria. Increased use of polymyxin during recent years poses a risk to public health. The purpose of this study was to investigate the carrying situation of the mcr-1 drug-resistance gene in waterfowl in some coastal areas of China from 2019 to 2020. METHODS: Fifty-seven isolated avian pathogenic Escherichia coli strains were selected from 493 APEC isolates for whole-genome sequencing. The 24 mcr-1-positive APEC strains were tested for conjugation and genome-wide analysis, including sequence type (ST) analysis, serotype analysis, and drug-resistance gene analysis. Numerous mcr-1-positive E. coli were downloaded from the National Center for Biotechnology Information (NCBI) for comparative genomic analysis. RESULTS: Antimicrobial susceptibility test results showed that 57 APEC isolates were highly resistant to gentamicin, cefotaxime, and ofloxacin, and 24 mcr-1-positive APEC isolates were resistant to polymyxin. Fourteen isolates of mcr-1-positive APEC plasmids were successfully conjugated to EC600. Both ST156 and ST10 were found in high proportions in human and avian sources through genome-wide analysis; it is worth noting that these two isolates of APEC were detected to contain the blaNDM-1 and blaNDM-4 genes, respectively. CONCLUSION: In this study, the epidemiological investigation of the mcr-1 gene was carried out on APEC in some coastal areas of China from 2019 to 2020, and our results have enriched the data on the transmission of APEC isolates carrying the mcr-1 gene in waterfowl.

Duck sewage source coliphage P762 can lyse STEC and APEC.

Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. P762, a coliphage isolated from duck farm sewage, was demonstrated to cloud lyse Shiga toxin-producing Escherichia Coli serotypes O157 and non-O157 (17/39), Avian pathogenic E. coli covered serotype O78, O83, and O9 (5/19), and other pathogenic Escherichia coli (5/17). Additional fundamental biological characteristics analysis revealed that P762 is stable at pH 3 ~ 11 and temperature between 4 °C and 60 °C, and its optimum multiplicity of infection (MOI) is 0.1. The one-step curve of P762 exhibited three bursts of growth stage: two rapid and one slow stage. Furthermore, the first rapid burst size is 80 CFU/PFU, the burst size of the slow stage is 10 CFU/PFU, and the second rapid burst size is about 990 CFU/PFU. In addition, P762 can form a "halo" on a double agar plate, implying that the phage secretes depolymerase. With 95.14% identity and 90% query coverage, genome sequence analysis revealed that P762 is most closely related to Escherichia phage DY1, which belongs to the genus Kayfunavirus. After screening using RAST and VFDB, no virulence factors were discovered in P762. In vitro antibacterial tests revealed that P762 has high bactericidal activity in lettuce leaves contaminated with STEC. In conclusion, phage P762 might be employed in the future to prevent and control pathogenic Escherichia coli.

Genomic characterization of Streptococcus parasuis, a close relative of Streptococcus suis and also a potential opportunistic zoonotic pathogen.

Streptococcus parasuis (S. parasuis) is a close relative of Streptococcus suis (S. suis), composed of former members of S. suis serotypes 20, 22 and 26. S. parasuis could infect pigs and cows, and recently, human infection cases have been reported, making S. parasuis a potential opportunistic zoonotic pathogen. In this study, we analysed the genomic characteristics of S. parasuis, using pan-genome analysis, and compare some phenotypic determinants such as capsular polysaccharide, integrative conjugative elements, CRISPR-Cas system and pili, and predicted the potential virulence genes by associated analysis of the clinical condition of isolated source animals and genotypes. Furthermore, to discuss the relationship with S. suis, we compared these characteristics of S. parasuis with those of S. suis. We found that the characteristics of S. parasuis are similar to those of S. suis, both of them have "open" pan-genome, their antimicrobial resistance gene profiles are similar and a srtF pilus cluster of S. suis was identified in S. parasuis genome. But S. parasuis still have its unique characteristics, two novel pilus clusters are and three different type CRISPR-Cas system were found. Therefore, this study provides novel insights into the interspecific and intraspecific genetic characteristics of S. parasuis, which can be useful for further study of this opportunistic pathogen, such as serotyping, diagnostics, vaccine development, and study of the pathogenesis mechanism.

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice.

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 µg/mouse) of depolymerase intraperitoneal injection protected 90%-100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.

O145 may be emerging as a predominant serogroup of Avian pathogenic Escherichia coli (APEC) in China.

Among the numerous serotypes of Avian pathogenic Escherichia coli (APEC), O1, O2 and O78 have long been considered the predominant serogroups. O145, a pivotal serogroup in non-O157 Shiga toxin-producing Escherichia coli, has never been considered an important serogroup among APEC. The prevalence of APEC O145 was determined from the results of molecular serogrouping based on 42 sequenced isolates from Jiangsu and Guangxi Provinces in China. After realizing the potential importance of O145, 224 APEC isolates isolated from Jiangsu, Guangxi, Anhui, Shandong, Henan, Yunnan and Fujian provinces were screened using PCR amplification. The results showed that the proportion of O145 detected was 37.9 % (85/224), which was higher than those of the three traditional APEC serogroups. The virulence evaluation experiment showed that this serogroup may have stronger pathogenicity. Here, we report for the first time that O145 may be emerging as a predominant serogroup of APEC in China. The possible reasons for its prevalence and oversight were analyzed through genomic analysis. Furthermore, pangenome analysis with STEC O145 was performed to assess the potential threat to humans. The discovery of the ubiquity of O145 may not be coincidental, which may also account for the failure of vaccines that target the three major serogroups. Therefore, this newly predominant serogroup should be paid more attention and the focus should not be limited to the so-called three major APEC serogroups.

Combining pangenome analysis to identify potential cross-protective antigens against avian pathogenic Escherichia coli.

RESEARCH HIGHLIGHTSPan-RV analysis was used for the first time in the discovery of APEC-protective proteins.A total of 53 potential protective proteins were screened out.Four proteins were verified as potential vaccine candidates using western blotting.

An Escherichia coli isolate from hospital sewage carries blaNDM-1 and blaoxa-10.

Carbapenems, as the "last line of defense" against Gram-negative bacteria, are increasingly being challenged by drug-resistant bacteria, especially Enterobacteriaceae. In this study, a carbapenem-resistant Gram-negative bacterium, named AH001, was isolated from hospital sewage, and a modified Hodge test confirmed that this bacterium can produce carbapenemase. Further analysis revealed that this bacterium exhibits multidrug resistance against an additional seven antibiotics. Whole-genome sequencing and analysis showed that AH001 could not be classified by existing MLST, and its serotype could not be distinguished among O9, O89 or O168 according to O antigen prediction. More attention should be given to the role of environmental sources of Escherichia coli in the development and transfer of drug resistance in the hospital environment.

Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect.

The increasing prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to global health. Phages and phage-derived enzymes gained increasing attention for controling CRKP infections. In this study, a lytic phage P510 infecting KL64 type K. pneumoniae was isolated and characterized. Whole genome analysis and electron microscopy analysis showed that phage P510 belonged to genus Przondovirus, family Autographiviridae, the order Caudovirales. The tail fiber protein of the phage was predicted to encode capsule depolymerase. Further analysis demonstrated that recombinant depolymerase P510dep had polysaccharide-degrading activity against KL64-types capsule of K. pneumoniae, and its lysis spectrum matched to host range of phage P510. We also demonstrated that the recombinant depolymerase was able to significantly inhibit biofilm formation. The discovery of the phage-derived depolymerase lays the foundation for controlling the spread of CRKPs.

The biological role of MutT in the pathogenesis of the zoonotic pathogen Streptococcus suis serotype 2.

Streptococcus suis (S. suis) is an important rising pathogen that causes serious diseases in humans and pigs. Although some putative virulence factors of S. suis have been identified, its pathogenic mechanisms are largely unclear. Here, we identified a putative virulence-associated factor MutT, which is unique to S. suis serotype 2 (SS2) virulent strains. To investigate the biological roles of MutT in the SS2 virulent strain ZY05719, the mutT knockout mutant (ΔmutT) was generated and used to explore the phenotypic and virulent variations between the parental and ΔmutT strains. We found that the mutT mutation significantly inhibited cell growth ability, shortened the chain length, and displayed a high susceptibility to H2O2-induced oxidative stress. Moreover, this study revealed that MutT induced the adhesion and invasion of SS2 to host cells. Deletion of mutT increased microbial clearance in host tissues of the infected mice. Sequence alignment results suggested that mutT was encoded in a strain-specific manner, in which the detection was strongly linked to bacterial pathogenicity. In both zebrafish and mice infection models, the virulence of ΔmutT was largely reduced compared with that of ZY05719. Overall, this study provides compelling evidence that MutT is indispensable for the virulence of SS2 and highlights the biological role of MutT in bacteria pathogenesis during infection.

Genome-wide association study identifies the virulence-associated marker in Streptococcus suis serotype 2.

Streptococcus suis (S. suis) has been reported to be a highly invasive pathogen in swine, which causes severe infections like meningitis, arthritis and septicemia, and also a zoonotic agent for humans. Although many putative virulence factors (VFs) have been identified, the exact and wildly accepted virulence associated marker and pathogenesis mechanism of S. suis are still unclear. To establish connection of the genotypes with virulence phenotypes, we performed an "internal standard" method based on the zebrafish model to assess the virulence phenotypes of S. suis and did the genome-wide association study (GWAS) based on the genomes of 68 S. suis isolates. Through GWAS, a total number of 172 genes were identified. Among these genes, 143 of them distribute in virulent isolates. Further VFs interaction network analysis based on protein-protein interaction database found that 71 genes identified in this study could interact with known VFs and some of them even played an important role as the bridge between known VFs or formed important hub. In addition, 12 genes were found conserved in virulent isolates and 3 genes were conserved in avirulent isolates, 8 genes of the virulent conserved genes were belonging to a srtBCD pili cluster. Considering that sbp2', a member of the srtBCD pili cluster has been reported as a virulence-associated factor, we predict that sbp2' could be a fitness virulence-associated marker of virulent isolates. Taken together, our findings contribute to the insights in S. suis pathogenesis, enhance the knowledge of the genomic evolution of S. suis and provide several novel virulence-associated candidates.

Evaluation of the immunogenicity and protective ability of a pili subunit, SBP2', of Streptococcus suis serotype 2.

Streptococcus suis is an important zoonotic pathogen that leads to huge economic losses in the swine industry. Because of the enormous genetic and phenotypic diversity within S. suis, it is necessary to develop effective vaccines to control this zoonotic pathogen. SBP2' is a major pili subunit in S. suis that belongs to an srtBCD pili cluster and has already been reported to be associated with the pathogenesis of this bacterium. In this study, we aimed to evaluate the immunogenicity and protective ability of SBP2'. The rSBP2' protein was expressed by an Escherichia coli expression system and emulsified with Montanide ISA 201 adjuvant to prepare the subunit vaccine. Through active immune assays, the results showed that rSBP2' exhibited good immunogenicity and could protect mice from a lethal dose challenge. Additionally, the qRT-PCR data showed that the transcription levels of cytokines associated with systemic symptoms caused by S. suis were decreased, indicating that immunization with rSBP2' could protect the host from cytokine storms caused by S. suis. Furthermore, the passive immune assay showed that the humoral immunity induced by rSBP2' played an important role against S. suis infection. Taken together, SBP2' could provide proper immune protection against S. suis challenge and could be a candidate for S. suis subunit vaccine. The results of this study could provide new ideas for the development of effective vaccines against S. suis.

Pan-genome analysis of Streptococcus suis serotype 2 revealed genomic diversity among strains of different virulence.

Streptococcus suis (SS) is an emerging zoonotic pathogen that causes severe infections in swine and humans. Among the 33 known serotypes, serotype 2 is most frequently associated with infections in pigs and humans. To better understand the virulence characterization of S. suis serotype 2 (SS2) and discriminate the difference between virulent and avirulent strains in SS2, characterization of the genomic features of strains with different virulence is required. The result showed that Streptococcus suis have an open pan-genome. The pan-genome shared by the 19 S. suis serotype 2 strains was composed of 1,239 core genes and 2,436 accessory genes. COG analysis indicated that core genes are involved in the basic physiological function, but accessory genes related to tachytely evolution. Comparative analysis between core genomes of virulent strains and 9 avirulent strains suggested that srtBCD pilus cluster was a significant discrepancy between virulent and avirulent strains. Analysis between high virulent and group B low virulent strains showed 53 and 58 genes specific to each other. Moreover, genomes of avirulent strains tend to be larger than virulent strains; avirulent strains tend to possess more prophages sequences than virulent strains. Our findings could be contributed to a better understanding of the genomics of S. suis serotype 2.

Identification of novel fibronectin-binding proteins by 2D-far Western blot in atypical enteropathogenic Escherichia coli serotype O55:H7.

Atypical enteropathogenic Escherichia coli (aEPEC) is a subgroup of EPEC, which is one of the major pathogens responsible for fatal diarrhoea in children. Compared with typical EPEC (tEPEC), aEPEC lack an EAF (EPEC adherence factor) plasmid (pEAF), which encodes a series of virulence-associated genes. The extracellular matrix (ECM) component of human cells has been reported to be an important element in the interaction between host and bacterial pathogens. In this research, a 2D-Far Western blot method was performed to identifiy the bacterial proteins that could bind to fibronectin, one of the most common constituents of ECM. A total of 17 protein spots were identified, including 4 outer membrane proteins (OMPs), namely, OmpC, OmpD, OmpX and LamB. In vitro studies were used to determine whether these OMPs were involved in the adherence process. Through indirect immunofluorescence assays, four OMPs could be observed on the surfaces of host cells. After incubating the cells with the recombinant proteins, the adhesion rate of the O55:H7 isolate was decreased. Furthermore, the deletion of OmpX and LamB can also decrease the adhesion rate of WT. Taken together, a high-throughput screening method for host ECM-binding proteins based on 2D Far-Western blot was established, and four outer membrane proteins identified by this method were found to be involved in the adherence process.

ICEHpsaHPS7, a Novel Multiple Drug Resistance Integrative Conjugative Element in Glaesserella parasuis.

Integrative conjugative elements (ICEs) are a kind of novel self-transmissible mobile genetic element. In this study, a novel ICE was identified in Glaesserella (Haemophilus) parasuis We confirmed that it could mediate the migration of antimicrobial resistance genes in G. parasuis and found that there may have been a transferring potential between different serovar strains of G. parasuis These findings demonstrate that the ICE is crucial to the horizontal transfer of antimicrobial resistance among G. parasuis strains.