Objective To explore the relationship between hs-CRP and the etiology of children with hand-foot-and-mouth disease(HFMD). Methods A total of 1 156 laboratory-confirmed children diagnosed with HFMD by pathogenic detection(detection methods include EV71-CA16 IgM antibody detection and universal real-time fluorescent quantitative RT-PCR detection of enterovirus nucleic acid EV71/CA16/EV) in the Children′s Hospital of Hangzhou were involved in the research from September 2014 to July 2016.The hs-CRP levels in the early days(≤5 days) were recorded, and all data were analyzed with SPSS16.0. Results Of all the 1 156 cases, there were 642 cases with hs-CRP level more than 10mg/dL, of whom 37 cases were infected by EV71(5.8%), 552 cases were infected by EV(86.0%), 53 cases were infected by CA16(8.2%). In 514 cases with hs-CRP level less than 10mg/dL, of whom 298 cases were infected by EV71(58.0%), 152 cases were infected by EV(29.6%), 64 cases were infected by CA16(12.4%). Of cases with hs-CRP>10mg/dL, EV universal type got a significantly higher rate, with statistically significant difference(P<0.05). Of cases with hs-CRP<10mg/dL, EV71 got a significantly higher rate, with statistically significant difference(P<0.05). Conclusion The higher the hs-CRP level of the HFMD, the higher infection rate of EV.The lower the hs-CRP level of the HFMD, the higher infection rate of EV71. Key words: Enterovirus infections; Fluorescent antibody technique; Virulence factors; C-reactive protein; Hand-foot-and-mouth disease; Etiology detection

Objetivo: Detectar genes asociados a factores de virulencia de Escherichia coli aisladas de muestras diarreicas de ninos menores de cinco anos con el empleo de PCR Multiplex. Materiales y metodos: Se realizo un trabajo descriptivo, transversal, de una sola cohorte de muestras diarreicas de ninos menores de cinco anos colectadas desde enero 2014 a marzo 2015. Se usaron primers especificos para genes de los seis patotipos causantes de diarreas infantiles: gen daaD (Escherichia coli difusamente adherente - DAEC), gen aggR (Escherichia coli enteroagregativa - EAEC), gen eaeA(Escherichia coli enteropatogena - EPEC), gen stx (Escherichia coli productora de toxina Shiga - STEC), gen ipaH (Escherichia coli enteroinvasiva - EIEC)y gen st (Escherichia coli enterotoxigenica - ETEC). Resultados: Se aislaron 106 cepas de Escherichia coli en las que se encontraron genes de virulencia analizados en el 37,74 % (40/106) de las mismas. El grupo etario mas afectado con la presencia de estos genes fue el comprendido entre 1-2 anos (48,6 %). Conclusiones: El gen daaD del patotipo DAEC presento la mayor distribucion en un 16,98 %; asi mismo la deteccion de los genes de virulencia especificos podria ayudar a tratar de manera adecuada y oportuna un episodio de diarrea aguda infantil.

Objective To learn the distribution, epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli (DEC) isolated from patients with acute diarrhea among children less than 5 years old. Methods Totally 684 stool samples collected from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between August 1, 2015 and September 30, 2016 were tested by culture and identified the common pathogens. PCR was applied to detect the virulence genes of DEC. Meanwhile, serotyping of enteropathogenic E. coli (EPEC) was performed by slide agglutination tests for all the isolates of EPEC. An antimicrobial sensitivity test was performed using the agar dilution method. Results A total of 149 (21.7%) enteric bacteria pathogens were isolated from 684 specimens. DEC was found in 54 cases, ranked 2nd among the pathogenic bacteria. DEC tended to occur in summer/autumn periods. EPEC was the most frequent DEC genotype, accounted for 50% (27/54). Among EPEC, atypical EPEC was dominant, accounted for 77.8% (21/27) and typical EPEC only accounted for 22.2% (6/27). Followed by enteroaggregative E.coli 20.4% (11/54), enterotoxigenic E.coli 14.8% (8/54), enteroinvasive E. coli and Shiga toxin-producing E.coli 3.7% (2/54), 7.4% (4/54) cases were co-infected with more than one DEC genotypes. About 17/18 of suspicious DEC isolates can get the same genotypes by commercial multiplex PCR kit and single PCR test. Among the 27 EPEC strains, only 11(40.7%) strains can be detected by the slide agglutination serotyping method. More than 50% (27/54) of DEC isolates were resistant to conventional first-line antibiotics (ampicillin, trimethoprim-sulfamethoxazole) and cefazolin, cefuroxime, cefotaxime, but relatively low resistance to cefoxitin, amikacin, piperacillin/tazobactam, imipenem and meropenem. However, there was still a 9.2% (5/54) resistance rate to carbapenems. Conclusions DEC strains exhibited a high frequency of resistance to many antibiotics. Empirical antimicrobial therapy of severe DEC infection faces the challenge from the high resistance to ampicillin, trimethoprim-sulfamethoxazole. Even worse, some strains were resistant to relatively efficient drugs imipenem and meropenem. It is necessary to strengthen the epidemiological survey and antimicrobial resistance of DEC. Key words: Diarrhea; Escherichia coli; Virulence; Escherichia coli infections; Drug resistance, bacterial; Epidemiologic studies

Lactobacillus rhamnosus versus Staphylococcus aureus: influence on growth and expression of virulence factors

Background: The current study was conducted to investigate the frequency of resistance in the bacteria isolated from various sources, in Shiraz, Iran. Acquisition of new resistance genes is an important factor in the increasing incidence of resistant strains. A critical feature of resistance gene transfer is their stability to adapt rapidly to a new host and make serious consequences. Methods: A total of 520 samples were chosen from human and animal sources in order to investigate the frequency of antibiotics resistance mobile genes using PCR assay. Results: The rates of 70%, 52%, 16.5%, 8.5%, 8%, 4%, 9.2% and 6.8% were confirmed for several genes including tetO, tetA, tetB, tetM, tetR, gyrA, blaz, and blaSHV, respectively. Our results have revealed a pool of mobile genetic elements in the bacteria isolated from various sources in Iran. Conclusion: Our findings indicated un-regulated use of antibiotics in the food production chains which require more investigation.

Objective To sequence the 3′UTR of enterovirus 71 strains, investigate its foundation and impact in virulence by constructing a 3′UTR-replaced recombinant cDNA infectious clone. Methods Viral RNA of EV-A71 isolated viruses were extracted, and the nucleotide analysis was performed after sequencing. The 3′UTR of a full-length infectious clone of SDLY107 strain was replaced by its corresponding part of SDLY1 strain, and then the recombinant virus was constructed and identified. Results The nine isolated strains were classified into sub-genotype C4a of enterovirus (EV)-A71 by analysis, and nucleotide sequence homology for 3′UTR were 94%-100%. 3′UTR of EV-A71 strains may be associated with its pathogenicity. Identification of the rescued virus by sequencing and indirect immunofluorescence confirmed the successful construction of infectious recombinant virus. Conclusions Sequence analysis indicated that the 3′UTR may be involved in the pathogenicity of EV-A71. The recombinant virus SDLY107(1-3′UTR) was rescued successfully. Our study may provide evidence for further research on the influence of 3′UTR on the virulence of enterovirus 71. Key words: Enterovirus 71; Untranslated region; Recombinant virus

Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity. In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis, particularly in people with pre-disposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. To investigate the virulence of multiple isolates, a variation of the Bar-seq (barcode sequencing) method was employed. A conserved chromosomal site was identified for subsequent insertion of a barcode identifier, unique for each strain. Barcode insertion did not affect growth in vitro or in a rabbit model of endocarditis. Pooling of these strains and inoculation into rabbits demonstrated that all strains were capable of causing disease; however, virulence varied widely among strains. Genomic comparisons of the more virulent strains versus less virulent strains failed to conclusively identify any single gene responsible for virulence. Given this result, we continued our examination of the manganese transport system SsaACB, which is present in every strain of S. sanguinis examined. Although its contribution to virulence has not been confirmed in any strain other than SK36, it has been shown to be required for virulence in multiple species of streptococci, making it a candidate for emerging targeted therapies. In S. sanguinis strain SK36, previous studies have confirmed that loss of the manganese transport protein SsaB is tantamount to loss of virulence. Moreover, ssaB-deficient mutants are deficient for serum growth—a phenotype we have previously found to be associated with virulence. Our in vitro studies of manganese transporter-deficient strain SK36 supported this, but also revealed the emergence of suppressor mutants. In each suppressor mutant that was isolated, mutations were identified that mapped to a common gene, SSA_0696. Deletion of SSA_0696 resulted in restored in vitro growth in the ssaACB-deficient background, unearthing a novel mechanism for bacterial growth under manganese limitation. Fortunately, the suppressor mutant phenotype was not maintained in vivo; however, the combined results of these experiments suggest the efficacy of future therapeutics may require consideration of virulence at the species level and the incorporation of multiple targets.

Bacterial wilt (Ralstonia solanacearum) is one of the major potato diseases in Rwanda. An in vitro study was carried out to identify and characterize the pathogen isolated from three potato cultivars (Kinigi, Kirundo and Gikungu) in Rwanda. This was achieved by cultural and morphological tests on triphenyl tetrazolium chloride (TTC) and casamino peptone glucose (CPG) agar as well as biochemical tests through Gram staining and biovar test. An in vivo experiment was also performed to assess the pathogenicity of those isolates on potatoes. All isolates showed typical morphological traits of virulent R. solanacearum on TTC and CPG media. The test isolates were Gram-negative bacteria. Biovar test confirmed that all the isolates belonged to race 1 biovar 3 of the pathogen. Furthermore, the highest disease severity (DS=100%) and disease incidence (DI=100%) were observed in Gikungu isolate followed by Kinigi (DS=97.33% and DI=98.25) and Kirundo (DS=94.67% and DI=92.61%). From this study, all three isolates were typical R. solanaceraum belonging to race 1 biovar 3 and were all pathogenic to potato plants. Gikungu and Kinigi isolates were highly virulent than Kirundo isolate. Therefore, Gikungu or Kinigi isolates can be used for further studies in plant protection in management of the disease. Key words: Biovar test, Gram-negative, Gram-positive, pathogenicity test.

Clostridium difficile causes antibiotic-associated diarrhoea and pseudomembranous colitis. The main virulence factors of C. difficile are the toxins A (TcdA) and B (TcdB). A third toxin, binary toxin (CDT), which pathogenetic role had been remained largely overlooked until few years ago, nowadays have been detected in 5%-23% of strains. C. difficile has spread around world. Clostridium difficile infection (CDI) is one of the most common health-care associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with disease. It is usually based on a clinical history of recent antimicrobial usage and diarrhoea in combination with laboratory tests. Although the conventional methods are crucial for the diagnosis and the subsequent treatment of CDI, a timely laboratory diagnosis is essential for the detection of toxigenic strains providing either to an effective and immediately treatment or to the prevention of further disease transmission. In this review we provide general recommendations for testing of samples collected from patients with suspected CDI.

Objective To analyze the biological characteristics of Yersinia pestis strains in Haixi Prefecture, Qinghai Province, in order to provide a scientific basis for plague prevention and control in future. Methods Totally 181 strains were separated from variety kinds of host in Haixi Prefecture, Qinghai Province from 1957 to 2011, and these strains were conducted biochemical test, virulence factor evaluation, plasmid analysis, different region (DFR) genotyping, drug and disinfectant resistant genes detection; 79 of the 181 strains were examined by toxicity test and classified according to the criteria (minimum lethal dose: MLD≤10 000 was velogenic strain, 10 000 < MLD < 100 000 was moderate virulence strain, MLD≥100 000 was hypovirulent strain). Results According to six biochemical typing about gelatin candy, rhamnose, maltose, melibiose, glycerin and denitrification, the 181 strains of Yersinia pestis were antique biovar and Qing-Tibet Plateau ecotype. Aproportion of 81.22% (147/181) of Yersinia pestis strains contained all the four virulence factors (F1, PstⅠ, VW, Pgm). Totally 63.54% (115/181) of the strains contained 3 kinds of plasmid - 6 × 106, 45 × 106, and 52 × 106; 31.49% (57/181) of the strains contained 3 kinds of plasmid - 6 × 106, 45 × 106, and 65 × 106. The strains had 8 genomovars, and were given priority to genomovar 8 (109 strains), secondly, genomovar 32 (33 strains), genomovar 5 (20 strains), genomovar 1b (14 strains), genomovar 44 (2 strains), genomovar 7 (1 strain), genomovar 37 (1 strain), and genomovar 49 (1 strain). Among the 181 Yersinia pestis strains, strains with genes related to streptomycin resistance, sulfanilamide resistance, beta lactam resistance and disinfectant resistance were not found; and 75 of 79 strains were velogenic strains by toxicity test (MLD≤10 000), accounted for 94.94% (75/79). Conclusion The strains separated in Haixi Prefecture, Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity; all strains don't have the characteristics of drug and disinfectant resistance genes. Key words: Yersinia pestis; Etiology; Drug and disinfectant resistance genes

Transcriptional silencing and anti-silencing affect many aspects of bacterial physiology, including virulence in bacterial pathogens. In Shigella species, a group of gram-negative pathogens that cause bacillary dysentery in humans, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences virulence genes found on the large virulence plasmid while VirB anti-silences these genes. However, the mechanistic details of their interplay are not fully understood. To elucidate their regulatory mechanisms, I use the icsP virulence locus, which shares a long intergenic region with the divergently transcribed ospZ gene (1535 bp from TSS to TSS). Prior to this work, two discrete H-NS binding regions had been identified, suggesting H-NS-mediated bridging of these two regions as the mechanism of silencing. However, I show that changes to the spacing and helical phasing designed to disrupt the potential bridging were tolerated, suggesting an alternate mechanism of silencing is at play. In addition to H-NS, two other H-NS homologs found in S. flexneri, StpA and Sfh, can also silence the icsP promoter. Interestingly, VirB counters transcriptional silencing mediated by these other H-NS homologs. The site required for VirB-dependent anti-silencing of the icsP promoter is located over 1 kb upstream of the TSS, and nearly 500 bp upstream of the ospZ promoter, but exactly how VirB accomplishes this long-range regulation is not known. I show that VirB docks to this recognition site in vitro and has a high specificity for this site in vivo. Using a combination of 1D and 2D chloroquine-based agarose gel electrophoresis, I demonstrate that, upon docking to its recognition site, VirB triggers a loss of negative supercoiling of our VirB-dependent PicsP-lacZ reporter; importantly, this phenomenon occurs with native VirB levels in S. flexneri. Because H-NS is sensitive to DNA topology at some promoters, it is tantalizing to envision that VirBmediated changes in supercoiling alleviate H-NS-mediated silencing of virulence genes in Shigella. Although anti-silencing proteins in other bacteria, including related pathogens, bear little sequence homology to VirB, the possibility that changes to DNA supercoiling mechanistically unite this group of proteins requires further consideration when studying transcriptional silencing and anti-silencing processes in bacteria.

Infections with Pseudomonas aeruginosa can be extremely difficult to treat due to its virulence, its intrinsic and rapidly acquired antimicrobial

Fire blight disease, caused by the bacterium Erwinia amylovora, has developed to an economical important disease in cultivation of pome fruits in many regions of the world. The bacterial cysteine protease AvrRpt2EA was identified as a central molecule in the host-pathogen interaction and it is important for pathogen recognition in the fire blight resistant crabapple Malus ×robusta 5. However, little is known about its role as virulence factor in susceptible apples. To investigate its function in planta, transgenic lines of the fire blight-susceptible cultivar ‘Pinova’ were generated, which contain an plant-optimized version of AvrRpt2EA driven by a heat shock-inducible promoter. After induced expression of AvrRpt2EA, the transgenic lines showed symptoms similar to natural fire blight infections, such as shoot necrosis and browning of older leaves. Furthermore, an increase of the expression of the PR-1 gene was shown, which was used as molecular marker for salicylic acid (SA) dependent systemic acquired resistance (SAR). Additional analysis reveal that the levels of SA and its derivatives were increased after AvrRpt2EA expression, too, with diverse kinetics in leaves of different ages. In contrast, no induction of the expression level of VSP2 paralogs was found, which were used as marker genes for the activation of the jasmonic acid (JA)-dependent defense pathway. This was also confirmed by metabolic profiling of JA and its derivatives. In conclusion, the results of this study show that AvrRpt2EA alone acts as virulence factor causing fire blight disease symptoms in susceptible apple plants and induces the formation of SA and SA-dependent SAR.

Pathogenesis and virulence.

The two host-adapted varieties of the smut fungus Sporisorium reilianum (namely S. reilianum f. sp. zeae, SRZ, and S. reilianum f. sp. reilianum, SRS) produce spores either on maize or on sorghum. For plant infection, mating compatible haploid sporidia need to fuse and form infectious dikaryotic filaments that infect the plant at seedling stage and cause disease phenotype in the form of spore development in male and female inflorescences of the plant. Despite both formae speciales being very closely related on the genomic level why they differ in terms of host selection remains elusive. To know the host determining factors we generated sexual spores by crossing the compatible SRZ strain SRZ1_5-2 (mating type a1b1) with the SRS strain SRS2_H2-7 (mating type a2b6). Meiotic progeny (SRSZ) with the mating type a1b1 were selected and tested for virulence on sorghum after mating with SRS_H2-7. Virulence assays showed that the progeny were either non-virulent or showed various degrees of disease phenotype. We selected 110 non-virulent offspring, 50 offspring with full virulence and 29 offspring with intermediate virulence on sorghum for genotype analysis. Genomic DNA of 191 strains (189 offspring and 2 parental strains) was isolated and sequenced using Illumina technology with a read length of 125 nt in paired end. Mapping of the reads against the two assembled sister parental genomes showed that parental origin of chromosomal regions could be un-ambiguously assigned for all SRSZ strains. Interestingly, few strains contained partially duplicated genomic regions, i.e. carrying the same chromosomal fragments from both parents. Correlation of phenotype with parental origin of genomic loci revealed a region of 35 genes in the left arm of chromosome 7 potentially associated with the virulence phenotype on sorghum. Analysis of this region revealed a cluster of 9 genes that were expressed in planta, showed low sequence conservation and coded for proteins carrying predicted secretion signal peptides. Deletion of this gene cluster in forma specialis SRS and subsequent virulence analysis showed that this cluster is essential for the spore forming capacity of SRS in sorghum. These results indicate the power of classical genetics combined with genotype to phenotype association analysis using Next generation sequencing approaches for the discovery of genes involved in determining host specificity in S. reilianum.

Climate change and anthropogenic activity are currently driving large changes in nutritional availability across ecosystems, with consequences for infectious disease. An increase in host nutrition could lead to more resources for hosts to expend on the immune system or for pathogens to exploit. In this paper, we conducted a meta-analysis of studies on host–pathogen systems across the tree of life, to examine the impact of host nutritional quality and quantity on pathogen virulence. We did not find broad support across studies for a one-way effect of nutrient availability on pathogen virulence. We thus discuss a hypothesis that there is a balance between the effect of host nutrition on the immune system and on pathogen resources, with the pivot point of the balance differing for vertebrate and invertebrate hosts. Our results suggest that variation in nutrition, caused by natural or anthropogenic factors, can have diverse effects on infectious disease outcomes across species.

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

S1BioRXivCode makes Pairwise Invasibility Plot, S2BioRXivCode generates dynamical simulation of the evolution of virulence (The model is presented in the paper Parasite-induced shifts in host movement may explain the transient coexistence of high- and low-pathogenic disease strains) , and S3BioRXivCode makes video of Pairwise Invasibility Plots for varying parameters.

Group B Streptococcus (GBS) is an important pathogen which may result in miscarriage, intrauterine infection and puerperal infection. Neonatal GBS infection may lead to septicemia, pneumonia and meningitis. GBS can be divided into a variety of serotypes according to the antigenic structure of capsular polysaccharide, and different serotypes of GBS vary in ethnicity, geographical distribution, pathogen virulence and pathogenic species. The distribution and identification of GBS serotypes, and their relationships with genotypes, drug resistance, virulence factors and vaccine preparation of GBS were reviewed. Key words: Peripartum period; Streptococcus agalactiae; Streptococcal infections; Serotyping