Analysis Of Virulence Genes Research Articles

Investigation of a Salmonellosis Outbreak Caused by Multidrug Resistant Salmonella Typhimurium in China.

The rapid emergence of multidrug resistant Salmonella is a global public-health concern as outbreaks in recent years have mostly been caused by multidrug resistant strains. Here, we evaluated an outbreak in China caused by multidrug resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) by employing an epidemiological and laboratory investigation using conventional methods and whole genome sequencing (WGS). Eleven of the 12 people who participated in a banquet showed gastrointestinal symptoms, and 8S. Typhimurium strains were recovered. Isolated outbreak strains showed multidrug resistance (MDR), and decreased susceptibility to ciprofloxacin, a first-line drug recommended by WHO for clinical treatment of intestinal infections. Antimicrobial resistance (AMR) gene analysis indicated that the MDR phenotype of these outbreak strains may be due to the presence of a number of AMR genes, including the blaOXA-1 and blaTEM-1 β-lactamase genes, which are often plasmid-borne and easily transferred. Further virulence gene analysis indicated that these outbreak strains also carried a large number of virulence genes, including 2 types of Salmonella pathogenicity islands (SPI-1 and SPI-2) and many adhesion-related virulence genes. Cluster analysis based on pulse-field gel electrophoresis data and phylogenetic analysis based on WGS revealed that the outbreak clone was closely related to and thus probably derived from local strains. This outbreak caused by multidrug resistant S. Typhimurium highlights the need for government improved strategies for the prevention and control of Salmonella infections.

A rare carbapenem-resistant hypervirulent K1/ST1265 Klebsiella pneumoniae with an untypeable blaKPC-harboured conjugative plasmid

ObjectiveWe investigated the pathogenesis of a patient with severe acute pancreatitis by comprehensively analysing a rare carbapenem-resistant hypervirulent K1/ST1265 Klebsiella pneumoniae (CR-HvKP) strain. MethodsWe conducted virulence and multidrug-resistance phenotypic characterization and identified a CR-HvKP strain from the patient. It was subjected to Pacbio sequencing, and subsequent analysis of virulence, resistance genes and mobile genetic elements. ResultsWe described the phenotype and genotype of a rare CR-HvKP strain with an untypeable blaKPC-harboured conjugative plasmid and a pLVPK-like virulent plasmid. Resistance gene analysis showed that the untypeable blaKPC-2-harboured plasmid was formed by IS26-mediated recombination of blaKPC-embedded transposon Tn6500 into pCN061p4 from Escherichia coli. Interestingly, it had an R-M system that might protect plasmid from cleavage. This may facilitate the stabilization of plasmids in bacteria in the event of missing CR-plasmid during transmission. Virulence gene analysis indicated 78 virulence genes on the genome, including 67 on the chromosome (37 in high-pathogenic island) and 11 on the pLVPK-like virulence plasmid (harbouring rmpA/rmpA2). Further phylogenetic analysis revealed that the CR-HvKP evolved from HvKP through acquiring an antimicrobial-resistance plasmid. ConclusionOur research, to our knowledge, first reported a ST1265/K1 CR-HvKP strain with an untypeable blaKPC-harboured plasmid. The trend that HvKP could evolve into CR-HvKP by obtaining stabilized/conjugative blaKPC-carrying plasmids is a considerable threat to public health and should be closely supervised.

Genomic characteristics of clinically important ST11 Klebsiella pneumoniae strains worldwide

ObjectivesST11 Klebsiella pneumoniae is among the most important clinical pathogens in China, and KL47 and KL64 are the dominant K types of these strains. Understanding the genomic characteristics of these strains would be critical to their anti-infection treatment. MethodsThere were 364 genome sequences of ST11 K. pneumoniae strains isolated and collected from 13 countries from 2003 to 2018. These genome sequences included 338 downloaded from the National Center for Biotechnology Information (NCBI) database and 26 newly sequenced. Phylogenetic analyses of pan-genome and unique genes, and resistance and virulence gene analyses, were carried out to elucidate the molecular characteristics of these strains. ResultsA total of 19 732 genes were identified from the 364 ST11 strains, and the pan-genome was open, indicating the genetic diversity of ST11 K. pneumoniae. These strains were clustered into three clades. Clade 1 contained the most various K types (14/15, 93.3%) and unique genes. KL47 and KL64 were the dominant K types of clades 2 and 3, accounting for 100% and 99.4% of strains in each clade, respectively. KL64 strains contained the most virulence genes, including iucA and rmpA, and the two genes tend to coexist. In addition, strains in clade 1 were isolated from all 13 countries; the strains in clades 2 and 3 were isolated mainly from China. ConclusionsThe ST11 K. pneumoniae strain of KL64 is a newly emerging superbug, with more resistance and virulence genes in China; this was significantly different from other countries, and we should be alert to the dissemination of this subclone.

Molecular characterization of virulence and drug resistance genes-producing Escherichia coli isolated from chicken meat: Metal oxide nanoparticles as novel antibacterial agents

Escherichia coli is a major global foodborne pathogen, infecting a wide range of animals and contaminating their meat products. E. coli, can lead to high morbidity and mortality with a huge economic loss especially if foodborne diseases are associated with multidrug resistant (MDR)- and multivirulent-producing pathogens. Due to the increased resistance to common antimicrobials used to treat livestock animals and human infections, the discovery of new and innovative nanomaterials are in high demand. Recently, metal oxides can be considered as effective inorganic agents with antimicrobial features. Hence, this study might be the first to evaluate the efficiency of metal oxide nanoparticles (MO-NPs) as novel antibacterial agents against MDR/multivirulent E. coli pathogens isolated from chicken meat. The occurrence of pathogenic E. coli was determined in fresh warm chicken meat parts (breast, thigh, liver and gizzard). Ninety-one of 132 (69%) chicken meat parts were Escherichia -positive with E. coli as the only species isolated. Out of identified 240 E. coli strains, 72.5% (174/240) were classified as MDR E. coli strains. Fifty-five profile patterns were obtained. From each pattern, one strain was randomly selected for further analysis of virulence and resistance genes. Extracted DNA was assessed for the presence of antibiotic resistance genes (blaIMP-7, blaIMP-25, blaTEM, blaSHV, blaOXA-2, tetA, aadA, and aac(3)-IV) and virulence genes (stx1, stx2, hlyA, eaeA, aggR, eltB, estIb, papA, afa and hlyD). Clustering analyses revealed that 10 E. coli harboring the highest number of virulence and resistance genes were shifted together into one cluster designated as cluster X. The average activities of zinc peroxide nanoparticles (ZnO2-NPs) were higher than that of zinc oxide nanoparticles (ZnO-NPs) and titanium dioxide nanoparticles (TiO2-NPs) by 20% and 29%, respectively. The anti-inflammatory activity of ZnO2-NPs in comparison with aspirin was assessed using membrane stabilization, albumin denaturation, and proteinase inhibition methods. Significant anti-inflammatory activity of ZnO2-NPs was achieved at concentration levels of 500–1000 μg/ml. It seems that MO-NPs are effective alternative agents, since they exhibited a competitive antibacterial capability against MDR/multivirulent-producing E. coli pathogens isolated from chicken meat. Hence, ZnO2-NPs are a promising nanoparticles-based material for controlling foodborne pathogens, thereby valued for food safety applications.

Large-scale profiling of the proteome and dual transcriptome in Nile tilapia (Oreochromis niloticus) challenged with low- and high-virulence strains of Streptococcus agalactiae

Streptococcus agalactiae is a common pathogen in aquatic animals, especially tilapia, that hinders aquaculture development and leads to serious economic losses. Previously, a S. agalactiae strain named HN016 was identified from infected tilapia, and the attenuated strain YM001 was subsequently obtained by continuous passaging in Tryptic Soy Broth (TSB) medium. YM001 has been demonstrated as a safe vaccine for S. agalactiae infection in tilapia. To understand the molecular bases of the virulence of these two strains, we performed proteomic and transcriptomic analysis to reveal the protein and gene expression changes in the liver and intestine during the infection process. HN016 significantly decreased the contents of white blood cells (WBCs), neutrophils (NEUs), red blood cells (RBCs) and hematocrit (HCT) and increased the levels of total protein (TP), albumin (ALB) and globulin (GLO), while no such significant differences were observed when comparing the control with YM001. During the infection process, pathogenic peptidoglycan hydrolase, CSPA and membrane proteins were significantly differentially expressed between YM001 and HN016. Furthermore, both proteome and transcriptome data showed that the complement and coagulation cascades pathway and the antigen processing and presentation pathway were stimulated in the liver and intestine, respectively, by YM001 infection compared to HN016 infection. The interaction network analysis of key virulence genes from pathogens suggested that CSPA, as a key node, affects the expression of DOLPP1, MIPEP, PA2G4, OCIAD1, G3BP1 and CLIC5 with a positive correlation. The present evidence suggests that during the infection process, CSPA was the key genes contributing to low virulence in YM001.

Pet dogs potential transmitters of pathogenic Escherichia coli with resistance to antimicrobials

Escherichia coli strains are part of the normal biota of humans and animals; however, several clinical reports have implicated E. coli as the etiological agent of diarrhea in humans and companion animals. Thus, the aim of the present study was to know if companion dogs in the city of San Luis Potosi are colonized with virulent potentially harmful E. coli strains. Rectal swabs from 30 dogs, 13 with and 17 without diarrhea were analyzed. Phylogenetic and virulence genes analysis was performed to the E. coli isolates. Additionally, the Kirby-Bauer test was used to analyze the sensitivity to 32 different antimicrobials from 14 families. Eighty-five isolates were identified as E. coli and detected in 97% of healthy and diarrheic dog samples. E. coli isolates from healthy dogs carried several virulence genes, in contrast with those from diarrheic animals that presented only eaeA. In healthy dogs, phylogenetic analysis showed that 57% and 43% of E. coli isolates belonged to commensal (A and B1) and virulent (B2 and D) groups respectively. Meanwhile, diarrheic dogs showed that 69% of the isolates were identified as virulent B2 and D phylogroups. Moreover, E. coli resistant to β-lactams, aminoglycosides, tetracycline, quinolones, and folate inhibitors were detected in both groups of dogs. The presence of E. coli with eaeA virulence gene in diarrheic dogs, suggest that these strains are associated with the animal´s condition. Finally, major attention must be drawn to the careful handling of dogs because of their capability to harbor and disseminate virulent E. coli strains.

Virulence and resistance determinants in Pseudomonas aeruginosa isolated from pericarditis in diseased broiler chickens in Egypt.

Objective:This study was performed to probe the antimicrobial resistance and virulence genes profiling in Pseudomonas aeruginosa recovered from the cases of pericarditis in broiler chickens.Materials and methods:The samples (n = 250) collected from the cases of pericarditis in broiler chickens were bacteriologically examined. Antimicrobial susceptibility was tested by disc diffusion technique. The isolates were genotypically studied for the presence of antimicrobial resistance and virulence gene traits. Finally, the nucleotide sequence of representative resistance gene (mexR gene) and virulence genes (toxA and lasI genes) was analyzed.Results:P. aeruginosa was isolated from 45 samples (18%). Antimicrobial susceptibility testing revealed multidrug resistance in most of the recovered P. aeruginosa isolates, whereas colistin and imipenem were the furthermost in vitro-sensitive antibiotics. Antimicrobial resistance genes, such as blaCTX, fox, and mexR, were prevalent in 100%, 80%, and 100% of the isolates, respectively. PCR confirmed virulence genes such as toxA, exoY, lasB, and lasI in 100%, 60%, 80%, and 80% of the isolates, respectively. Nucleotide sequence analysis of representative resistance gene (mexR gene) and virulence genes (toxA and lasI genes) revealed a high correlation between P. aeruginosa recovered from pericarditis in broiler chickens in the present study with PAO1 (reference strain) and with other sequences published on the GenBank representing different localities worldwide.Conclusion:It could be concluded that P. aeruginosa recovered from pericarditis in broiler chickens in the current study is highly virulent bacteria, resisting most of the therapeutic agents which not only bear hazards for poultry industry but also represent a public health concern.

Identification and pathogenicity analysis of <i>Streptococcus equinus</i> FMD1, a beta-hemolytic strain isolated from forest musk deer lung

Streptococcus spp. cause a wide range of diseases in animals and humans. A Streptococcus strain (FMD1) was isolated from forest musk deer lung. To identify the bacterium at the species level and investigate its pathogenicity, whole genome sequencing and experimental infections of mice were performed. The genome had 97.63% average nucleotide identity with the S. equinus strain. Through virulence gene analysis, a beta-hemolysin/cytolysin genome island was found in the FMD1 genome, which contained 12 beta-hemolysin/cytolysin-related genes. Hemolytic reaction and histopathological analysis established the strain’s pathogenicity in mice. This is the first report of a beta-hemolytic S. equinus strain in forest musk deer identified based on phenotypic and genotypic analyzes; this strategy could be useful for analyzing pathogens affecting rare animals.

Retrospective Study of Listeria Monocytogenes Isolated in the Territory of Inner Eurasia from 1947 to 1999.

Listeriosis is one of the most significant humans and animals foodborne infectious diseases. Here, we characterized 48 Listeria monocytogenes strains isolated in the territory of inner Eurasia during the second half of the 20th century. A total of 23 strains (52.3%) were susceptible to the nine antibiotics tested, 30.43%, 15.22%, and 8.7% were resistant penicillin G, ampicillin, and enrofloxacin, respectively. We applied the multilocus sequence typing (MLST) scheme to determine the phylogenetic positions of the strains. All but one strain belonged to the II phylogenetic lineage, and the majority of the strains belonged to one of the previously described clonal complexes (CCs). More than 60% of the strains belonged to the clonal complex CC7 that prevailed among all sources, including cattle (58%), small ruminants (64%), rodents (71%), and humans (50%). Further, CC7, CC101, and CC124 were found among human isolates. The MLST scheme was supplemented with virulence gene analysis. In total, eight inlA, six inlB, and six inlC allelic variants were found, and all but one strain carried one of the two inlE alleles. Most strains (62.5%) belonged to the same multivirulence locus sequence typing (MvLST) type, which includes CC7, inlA allele 4, inlB allele 14, inlC allele 6, and inlE allele 8.

Morphological characteristics, race distribution, and virulence gene analysis of Pyricularia oryzae isolates (teleomorph: Magnaporthe oryzae)

Rice blast disease caused by fungus Pyricularia oryzae (Teleomorph: Magnaporthe oryzae) has caused significant losses of rice yields in many world’s rice-producing countries. The planting of resistant varieties was initially effective in suppressing blast disease, however, in many cases, as the time goes by, the resistance becomes ineffective due to the emergence of a new race. Study on the distribution of race and genetic diversity of P. oryzae isolates can be useful in the development of resistant varieties. The aim of this research was to determine the distribution of P. oryzae races and genetic variations of the isolates collected from Pinrang District. Race testing was based on the reaction of a set of an Indonesian differential variety. The severity of blast disease was observed seven days after inoculation using the Standard Evaluation System for Rice (IRRI). The pathogen’s genetic diversity was determined using specific primers, coding the fungus virulence genes Pwl2, Erg2 and Cut1. The banding patterns of DNA amplification product contained in each primer were scored with a value of 1 (there is a DNA band) and 0 (no DNA bands). A total of 22 isolates of P. oryzae obtained from five sub-districts in Pinrang belonged to 10 different races. Race 001 is the dominant race identified in 4 isolate source sites. Molecular analysis of the 10 isolates of P. oryzae with different races was found haplotype F-110 (8 isolates) and G-100 (2 isolates).

Safety Evaluation of Oral Care Probiotics Weissella cibaria CMU and CMS1 by Phenotypic |and Genotypic Analysis.

Weissella cibaria CMU and CMS1 are known to exert beneficial effects on the oral cavity but have not yet been determined to be generally recognized as safe (GRAS), although they are used as commercial strains in Korea. We aimed to verify the safety of W. cibaria CMU and CMS1 strains through phenotypic and genotypic analyses. Their safety was evaluated by a minimum inhibitory concentration assay for 14 antibiotics, DNA analysis for 28 antibiotic resistance genes (ARGs) and one conjugative element, antibiotic resistance gene transferability, virulence gene analysis, hemolysis, mucin degradation, toxic metabolite production, and platelet aggregation reaction. W. cibaria CMU showed higher kanamycin resistance than the European Food Safety Authority (EFSA) cut-off, but this resistance was not transferred to the recipient strain. W. cibaria CMU and CMS1 lacked ARGs in chromosomes and plasmids, and genetic analysis confirmed that antibiotic resistance of kanamycin was an intrinsic characteristic of W. cibaria. Additionally, these strains did not harbor virulence genes associated with pathogenic bacteria and lacked toxic metabolite production, β-hemolysis, mucin degradation, bile salt deconjugation, β-glucuronidase, nitroreductase activity, gelatin liquefaction, phenylalanine degradation, and platelet aggregation. Our findings demonstrate that W. cibaria CMU and CMS1 can achieve the GRAS status in future.

Transcriptional analysis of flagellar and putative virulence genes of Arcobacter butzleri as an endocytobiont of Acanthamoeba castellanii.

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

Molecular analysis of accessory gene regulator functionality and virulence genes in Staphylococcus aureus derived from pediatric wound infections

Staphylococcus aureus is a major human pathogen causing infections with high morbidity and mortality in both healthcare and community settings. The accessory gene regulator (Agr) is a key genetic element controlling the expression of numerous virulence factors in S. aureus. The significance of a functional Agr system in clinical S. aureus isolates derived from pediatric wound infections is still unclear. Therefore, the present study was conducted to identify virulence genes and determine Agr functionality from this cohort of patients. A total of 48 S. aureus wound isolates were collected from patients referred to Tehran Children's Medical Center Hospital from April 2017 to April 2018. In addition, in vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion and E-test methods. Conventional PCR was performed for the detection of toxins (tsst-1, hla, hlb, hld, eta, etb, etd, edin-A, edin-B, edin-C) and Agr typing (agrI, agrII, agrIII, agrIV). Agr functionality was assessed by quantitative reverse transcriptase real-time PCR (qRT-PCR). All S. aureus isolates were found to be susceptible to linezolid and vancomycin. The most frequently detected toxin gene was eta (100%), and the most prevalent Agr type was agrIII (56.3%). Importantly, qRT-PCR revealed that Agr was functional in 28 (58%) of wound isolates. Consequently, our data suggests that a functional Agr system may not be required for the development of S. aureus wound infections.

Genomic comparison of serogroups O159 and O170 with other Vibrio cholerae serogroups

BackgroundOf the hundreds of Vibrio cholerae serogroups, O1 and O139 are the main epidemic-causing ones. Although non-O1/non-O139 serogroups rarely cause epidemics, the possibility exists for strains within them to have pathogenic potential.ResultsWe selected 25 representative strains within 16 V. cholerae serogroups and examined their genomic and functional characteristics. We tentatively constructed a gene pool containing 405 homologous gene clusters, which is well organized and functions in O-antigen polysaccharide (O-PS) synthesis. Our network analysis indicate that great diversity exists in O-PS among the serogroups, and several serogroup pairs share a high number of homologous genes (e.g., O115 and O37; O170 and O139; O12 and O39). The phylogenetic analysis results suggest that a close relationship exists between serogroups O170, O89 and O144, based on neighbor-joining (NJ) and gene trees, although serogroup O159 showed an inconsistent phylogenetic relationship between the NJ tree and the gene tree, indicating that it may have undergone extensive recombination and horizontal gene transfer. Different phylogenetic structures were observed between the core genes, pan genes, and O-PS genes. The virulence gene analysis indicated that the virulence genes from all the representative strains may have their sources from four particular bacteria (Pseudomonas aeruginosa, V. vulnificus, Haemophilus somnus and H. influenzae), which suggests that V. cholerae may have exchanged virulence genes with other bacterial genera or species in certain environments. The mobile genetic element analysis indicated that O159 carries nearly complete VSP-II and partial VPI-1 and VPI-2, O170 carries partial VPI-1 and VPI-2, and several non-O1/non-O139 strains contain full or partial VPI-1 and VPI-2. Several genes showing evidence of positive selection are involved in chemotaxis, Na + resistance, or cell wall synthesis, suggestive of environmental adaptation.ConclusionsThis study reports on the newly sequenced O159 and O170 genomes and their comparisons with other V. cholerae serogroups. The complicated O-PS network of constituent genes highlights the detailed recombination mechanisms that have acted on the serogroups’ genomes. The serogroups have different virulence-related gene profiles, and there is evidence of positive selection acting on other genes, possibly during adaptation to different environments and hosts.

Dissemination and characterisation of Escherichia coli producing extended-spectrum β-lactamases, AmpC β-lactamases and metallo-β-lactamases from livestock and poultry in Northeast India: A molecular surveillance approach.

The aim of this study was to identify and characterise probable extended-spectrum β-lactamase (ESBL)-, AmpC lactamase- and/or metallo-β-lactamase (MBL)-producing Escherichia coli variants circulating in the livestock and poultry environment to establish their epidemiological significance, genetic diversity, antimicrobial resistance (AMR) trends and virulence. The culture method and E. coli-specific multiplex PCR identified 78 E. coli strains from faecal samples of healthy livestock and poultry. The antibiogram was determined by the disk diffusion and minimum inhibitory concentration (MIC) methods. Antimicrobial-resistant E. coli isolates were screened for the presence of ESBL, AmpC and MBL genes. Isolates were further characterised by plasmid replicon typing, integron assay and virulence gene analysis. Genetic diversity was assessed by random amplification of polymorphic DNA (RAPD) analysis and multilocus sequence typing (MLST). ESBL (CTX-M group 1, CTX-M group 4, TEM), AmpC (EBC, FOX, CMY, DHA) and MBL (IMP, SIM) resistance determinants were identified in 75%, 19% and 6% of isolates, respectively. Nine plasmid replicon types were distributed among resistant E. coli strains, with the most common plasmid replicon types being L/M and Y. Integrons were detected in 19% of E. coli isolates. RAPD analysis categorised the E. coli isolates into three clusters. MLST revealed seven different sequence types (STs), with ST10 being the most common. This study demonstrated a high prevalence of animals carrying potential ESBL- and AmpC-producing E. coli and emphasises the need for rigorous surveillance in the animal sector to identify critical control points conducive to prevent the rapid dissemination of AMR.

Molecular characterization of Streptococcus pneumoniae in children living in southwest China and assessment of a potential protein vaccine, rPfbA

BackgroundFew children living in southwest China are vaccinated against Streptococcus pneumoniae (S. pneumoniae), which is an important pathogen in causing high morbidity and high mortality. This study aimed the molecular characterization of S. pneumoniae strains isolated from children and a new vaccine strategy based on a potential protein antigen. MethodsMolecular characterizations, including serotype, virulence gene and pilus analyses, were performed using PCR. Seven housekeeping genes were sequenced to identify the sequence types (STs), and antibiotic resistance was analysed using the microdilution broth method. In addition, we evaluated the protective effects of recombinant plasmin- and fibronectin-binding protein A (rPfbA) in murine pneumococcal infection models by challenge and passive transfer analyses, and assessed cytokine changes after immunization. ResultsThe prevalent serotypes were 19F (31.4%), 19A (21.6%), 6B (13.7%), 14 (11.8%) and 23F (9.8%), and the coverage rates of the 13-valent pneumococcal conjugate vaccine (PCV13) were high in 93.3% of the isolates. The predominant STs were ST271 (23.5%), ST320 (21.6%) and ST876 (11.8%). Most of the S. pneumoniae isolates were resistant to erythromycin (95.1%) and clindamycin (90.2%). The molecular distributions and antibiotic resistance rates of the S. pneumoniae isolates differed between the plateau and the basin regions. More than 93% of the S. pneumoniae isolates carried ply, cbpA, phtD and nanA, and over half of the isolates carried pilus-1, pilus-2 and pfbA. Mucosal immunization with rPfbA induced pneumococcal specific antibody responses which provided to eliminate colonization in lung and nasopharynx, and protection against pneumococcal challenge. ConclusionVaccine strategies based on epidemiological surveillance can be more adaptive to specific areas, reduce costs and protect against changing antigenic sites. We advise that children currently living in southwest China be vaccinated with PCV13.

Multiplex PCR analysis of virulence genes and their influence on antibiotic resistance in Enterococcus spp. isolated from broiler chicken.

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.

Molecular Study of Enterotoxins Genes of Staphylococcus aureus Isolated from Patients in Intensive Care Units

Introduction:Staphylococcus aureusis responsible for healthcare-associated sepsis. Various virulence genes may be responsible for the severity of the outcome of this infection.Aims & objectives:Therefore, the aim of the present study was to investigate the prevalence of enterotoxin genes inS .aureusassociated with HCA sepsis affecting adult's patients in Intensive Care Units (ICU).Materials & Methods:The study is a cross-sectional observational study included 432 patients admitted to intensive care units with Healthcare-Associated Infections (HCAIs). The patients were subjected to blood culture and identification of the isolated bacteria according to the standard microbiological method. Isolates identified asS. aureuswere subjected to molecular studies for the detection of meca gene and enterotoxins genes by multiplex Polymerase Chain Reaction (PCR).Results & Discussion:Blood culture was positive in 170 of the patients (39.3%). 47 of the blood culture yieldS. aureu.Regarding studies of virulence genes in isolatedS. aureus, the most frequent genes were seg (61.7%), mecA (46.8%) and sea (42.6%). The less frequent genes were tst and eta 6.4% for each. Analysis of demographic, clinical and virulence genes of isolatedS. aureusto HAIs outcome reveals that the virulence genes namely mec, sei and tst ofS. aureuswere the only significant factors associated with death The distribution of enterotoxigenic genes among MRSA and MSSA shows a significant association between seg and sei genes in MRSA isolates.Conclusion:The present study highlights the prevalence of enterotoxin genes in isolated S.aureus from patients with hospital-acquired infections in intensive care units in a tertiary hospital. There was high prevalence as about two-thirds of the isolates harbor seg and sea genes in addition to methicillin resistance gene mecA. The fatal outcome of sepsis in those patients was associated with the presence of enterotoxin genes sei and tst. There is a need for screening patients admitted to intensive care units forS. aureusharboring these genes for proper management and application of isolation and contact guidelines of infection control

Molecular analysis of virulence genes of Salmonella Infantis isolated from chickens and turkeys

In this study, virulence genes of S. Infantis strains, which are commonly isolated from chickens and turkeys in Turkey, were analyzed, and the virulence genes of S. Infantis and other common serovars aside from S. Infantis were compared.In this study, 200 S. Infantis strains isolated from litter, powder, environmental sources, rodent samples and broiler chicken carcasses from a chicken slaughterhouse obtained from chickens (broiler chickens, breeders, laying hens) and 24 S. Infantis strains isolated and identified from litter, powder, environmental and rodent samples obtained from turkeys were analyzed. A total of 40 strains, comprising 10 strains from each Salmonella serovar (S. Typhimurium, S. Enteritidis, S. Kentucky and S. Hadar) (chicken-origin) were also selected from the collection of strains for comparison with S. Infantis strains. The virulence genes of 264, comprising 224 S. Infantis strains and 40 strains from common serovars other than S. Infantis were analyzed. A conventional polymerase chain reaction (PCR) was used to analyze the 11 genes associated with the virulence (sipA, sipD, sopD, sopB, sopE, sopE2, sitC, ssaR, sifA, spvC and pefA) in these strains.SipA, ssaR and sopE genes were found in the 209 S. Infantis strains (93.3%), sipD in 208 (92.85%), sopB in 207 (92.41%), sitC in 206 (91.96%), sifA in 203 (90.62%), sopD in 198 (88.39%), sopE2 in 166 (74.1%), spvC in 20 (8.92%) and the pefA virulence gene in one strain (0.44%). It was found that 74.55% of S. Infantis strains were distributed in gene patterns 1 and 2. In this study, the sopE2 virulence gene in S. Infantis strains was analyzed for the first time. The involvement of the dominant gene patterns of the S. Infantis strains isolated from broiler chicken and broiler chicken carcasses, and the fact that none of S. Infantis strains belonging to the breeders and laying hens were included in these patterns, indicated that S. Infantis entered the broilers not through the breeders, but through environmental factors. The presence of sipA, sipD, sopD, ssaR, sopB, sopE, sifA and sitC virulence genes in S. Infantis strains were found to be similar, but remarkable differences were found compared to the S. Typhimurium and S. Enteritidis strains in the presence of sopE2, spvC and pefA virulence genes.This study examining the virulence genes of S. Infantis strains provides detailed information aimed at providing an understanding of the pathogenesis and epidemiology of Salmonella in poultry.

Pathogenicity, genotyping and antibacterial susceptibility of the Listeria spp. recovered from stray dogs

The present study aimed to determine the prevalence of Listeria spp. in stray dogs in the Kayseri province of Turkey. In addition, serotyping, genotyping and virulence gene analysis of the isolated Listeria spp. were performed and their pathogenicity and antibacterial susceptibility were investigated.The study included 80 rectal swaps taken from 80 stray dogs of different ages and gender that were sheltered in the Kayseri Municipal Dog Shelter. Listeria selective broth and Listeria selective agar were used for the isolation of Listeria spp. and the isolates were identified using a Microbact 12L (Oxoid, England) identification test kit. 16S rDNA sequencing and species-specific polymerase chain reaction (PCR) were performed for molecular identification of the isolates, multiplex PCR and a serological test were performed for serotyping, and PCR was used for virulence gene analysis. For determining the pathogenicity of L. monocytogenes and L. innocua isolates, a total of 100 mice (50 pregnant and 50 non-pregnant) were used. The mice were infected intraperitoneally; the inoculation dose was 1 × 109 CFU/mL and 0.2 mL was used for each animal. Tissue samples obtained from infected mice were processed for the re-isolation of the Listeria spp. and then stained with hematoxylin eosin and Brown-Brenn Gram stain. The antibiotic susceptibilities of the isolates were determined by the disk diffusion method.Listeria spp. were isolated from 5 (6.25%) of the 80 fecal samples. While 1 of the isolates was identified as L. monocytogenes, 4 of them were identified as L. innocua. Serotyping by serological and molecular methods revealed the isolate of L. monocytogenes to be serotype 1/2a. According to the phylogenetic trees, L. innocua and L. monocytogenes strains were clustered in different groups. The L. monocytogenes isolate was positive for all virulence genes tested. All L. innocua isolates were positive for the plcB gene. While all L. innocua isolates were negative for the lin1068 gene, 3 L. innocua isolates were found to be positive for the lin0558 gene. In mice infected with L. monocytogenes, pathological findings were observed in the uterus, intestines, pancreas, and heart. In mice infected with L. innocua, pathological findings were observed in the stomach, intestines and spleen. L. monocytogenes- or L. innocua-related infections or other inflammatory reactions were not observed in the brains of infected animals. On histopathological examination with Gram stain, an abundance of Listeria spp. was observed in the lesions of the liver, spleen, uterus, and kidney. Moreover, while abortion was observed in all animals infected with L. monocytogenes, it was not observed in any of the animals infected with L. innocua. Antibiotic susceptibility testing revealed that all 5 isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, erythromycin, gentamicin, penicillin G, and trimethoprim-sulfamethoxazole and were resistant to nalidixic acid, streptomycin, and cefuroxime sodium.Considering also the pathogenicity of the isolated microorganisms, it can be suggested that stray dogs as carriers of Listeria spp. are a significant risk to public health. As L. innocua isolates, which are considered apathogenic, led to the occurrence of lesions similar to those caused by L. monocytogenes, detailed studies on the pathogenesis of L. innocua infections caused by L. innocua isolates recovered from various sources are required.