Characterization Of Virulence Genes Research Articles

A15 LEVERAGING CULTURE-DEPENDENT AND -INDEPENDENT METAGENOMICS TO IDENTIFY ENTEROBACTERIACEAE GENES ASSOCIATED WITH ACTIVE ULCERATIVE COLITIS

Abstract Background Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) that is restricted to the large intestine and is characterized by mucosal inflammation. There is evidence that pathogenic bacteria contribute to UC pathogenesis in at least some patients and the bacterial family Enterobacteriaceae are prime suspects. Some strains of Enterobacteriaceae carry virulence genes important for colonizing the gut (e.g. fimH) and disrupting the intestinal epithelium (e.g. hemolysins). We hypothesize that key virulence genes carried by strains of many Enterobacteriaceae species contribute to disease activity in some UC patients. As we predict virulence genes are strain-specific, the detection of the species alone (e.g. Escherichia coli) is insufficient to predict pathogenic potential and this has confounded previous studies to identify infectious agents in UC. Aims We aim to use culture-dependent and -independent sequencing approaches to identify Enterobacteriaceae genes in the UC gut microbiome associated with active disease. Methods UC patient stool samples collected throughout enrolment in randomized control trials of fecal microbiota transplantation (FMT) for adult UC (n=10) and microbiome studies in early-onset pediatric UC (n=25) were cultured on MacConkey (MAC) agar to enrich for Enterobacteriaceae. Strains were isolated from baseline stool samples cultured on MAC agar for whole genome sequencing and virulence gene characterization. Baseline and post-treatment stool samples cultured on MAC agar were sent for metagenomic sequencing. Using a subset of 11 pediatric patients, I developed a metagenomic pipeline to identify Enterobacteriaceae genes enriched during active UC compared to periods of remission or milder disease. Results Known virulence genes, including fimH and hemolysins, were present in some strains across multiple Enterobacteriaceae species. Using an unbiased metagenomic approach with the pediatric cohort, we identified 42 genes enriched at baseline with the criteria they must be elevated in at least three of the 11 patients. The majority of the 42 genes were distributed into six gene clusters, including a small plasmid. Conclusions Our approach allows us to identify genes enriched in active UC that have not been previously described in the literature and our analysis will be repeated with our adult cohort. Genes that are enriched in active UC in the pediatric and/or adult cohort will be validated in publicly available metagenomic datasets that consist of both IBD patients and healthy controls. Furthermore, culture-dependent approaches allow us to test mechanisms in vivo that are informed by our bioinformatics. Leveraging microbiome and clinical data provides a unique window to guide future diagnostics and treatments to improve outcomes for UC patients. Funding Agencies CIHROGS

Identification and virulence gene characterization of pathogenic bacteria from diseased <i>Labeo rohita</i> (Hamilton, 1822): Insight into aquatic animal health management in Indian aquaculture

<p>Aquaculture is one of the major economic activities in India, providing livelihoods and nutritional security to millions of people. In recent times, fish diseases have come to the limelight resulting in significant economic losses. We aimed to identify pathogenicity and virulence profiling of thirty-six pathogenic bacterial strains isolated from diseased <italic>Labeo rohita</italic> in the district of Hooghly, West Bengal, India. The bacterial strains were characterized through a comprehensive approach involving the examination of morphological features, biochemical properties, amplification, and sequencing of the 16S rRNA, species-specific genes, and virulence genes. Considering the prevalence frequency, virulence potential, and statistical significance <italic>Aeromonas hydrophila</italic> and <italic>Pseudomonas aeruginosa</italic> were selected for a survival assay followed by the examination of histopathological features to elucidate their effects. The identified bacterial isolates were arranged based on their predominance frequency, i.e., <italic>Aeromonas hydrophila</italic> (25%), <italic>Aeromonas veronii</italic> (22%), <italic>Pseudomonas aeruginosa</italic> (22%), <italic>Enterococcus faecalis</italic> (14%), <italic>Klebsiella pneumoniae</italic> (6%), <italic>Staphylococcus aureus</italic> (6%) and <italic>Escherichia coli</italic> (5%). Sixteen virulence-associated genes related to pathogenicity were amplified across the thirty-six isolates; <italic>aer, alt, lip</italic> and <italic>hlyA</italic> for <italic>A. hydrophila</italic>; <italic>exoS, lasB</italic>, <italic>toxA, oprL</italic> and <italic>phzM</italic> for <italic>P. aeruginosa</italic>; <italic>entB, fimH</italic> and <italic>uge</italic> in <italic>K. pneumoniae</italic>; <italic>aer</italic> in <italic>A. veronii</italic>; <italic>hlyA</italic> in <italic>E. coli</italic>; <italic>hlb</italic> in <italic>S. aureus</italic> and <italic>gelE</italic> for <italic>E. faecalis</italic>. The log-probit analysis revealed that <italic>A</italic>. <italic>hydrophila</italic> was notably more pathogenic than <italic>P. aeruginosa</italic>, as indicated by its lower lethal dose of 1.5×10<sup>4</sup> CFU/mL. Additionally, histological examination revealed notable pathological changes, including tissue degeneration, inflammatory cell infiltration and vacuolation observed in the liver, kidney, gill and intestine of the challenged fish. We highlighted several potent aquatic microbial pathogens in order to manage and prevent such aquacultural maladies.</p>

Genomic Diversity and Virulence Genes Characterization of Bacillus cereus sensu lato Isolated from Processing Equipment of an Algerian Dairy Plant

Bacillus cereus is a ubiquitous spore-forming bacterium causing food spoilage. In this study, seventeen B. cereus were isolated from dairy processing equipment in Algeria and characterized. The pathogenic potential genes encoding hemolysin, nonhaemolytic enterotoxin, cytotoxin K and emetic toxin were investigated by PCR. Phylogeny and genetic markers for antimicrobial resistance and virulence were analyzed using whole-genome sequencing data using the Illumina MiSeq® technology. The identity of the isolated strains was confirmed as B. cereus sensu lato and the sequencing of the panC gene clustered 64.7% of them in phylogenetic group III and 35.3% in group IV. The results of PFGE showed that 8 (47.1%), 4 (23.5%), 3 (17.6%) and 2 (11.8%) were differentiated into four groups A, B, C and D, respectively. Each of the two panC phylogenetic groups contains two different types of isolates corresponding to PFGE group (A and C) and (B and D) for phylogenetic groups III and IV, respectively. None of the 14 isolates carried the emetic toxin (ces) gene, whereas other toxin genes were variably detected. Genotyping assigned all isolates to three different sequence types (ST 2226, ST1018 and ST1431) while one clonal complex (CC-142) was identified in three isolates. Hierarchical clustering with a threshold of 500 core genome single nucleotide polymorphism (cgSNPs) differences grouped the strains into three clusters while two strains were less than 20 SNPs different. Three B. cereus (25%) contained three enterotoxic HBL complex-encoding genes hblA/C/D. Enterotoxic NHE complex-encoding genes (nheA/B/C) and the ctyK1 gene were detected in all isolates. The overall results highlight the risk due to toxins produced by the B. cereus group in the food safety and dairy industries in Algeria. This study emphasized the potential of whole-genome sequencing for genotyping and predicting the virulence-associated genes and antimicrobial resistance. The present study will help to better assess the health and spoilage risk associated with B. cereus in dairy processing plants and to incorporate adequate preventive measures.

Characterization of Virulence Genes Associated with Type III Secretion System and Biofilm Formation in Pseudomonas aeruginosa Clinical Isolates.

Pseudomonas aeruginosa is a common pathogen with an increasing multidrug resistance (MDR) phenotype. Its virulence determinants include many factors such as antimicrobial resistance, biofilm formation, and type III secretion system (T3SS) which correlate with disease severity. There are no reports regarding the virulence features of P. aeruginosa in Cyprus. The aim of this study was to investigate the frequency and distribution of selected virulence-encoding genes and evaluate the biofilm formation potential as well as antibiotic resistance rates of isolates in the region. One hundred clinical P. aeruginosa isolates were obtained from clinical specimens and were identified using standard microbiological techniques. Antimicrobial susceptibility was assessed using the VITEK-2 system and biofilm quantification was performed by the microtiter plate assay with crystal violet staining. The presence of algD, exoU, exoT, and exoS was evaluated using polymerase chain reaction(PCR). Among all isolates, 35% were strong biofilm former, 28% were moderate biofilm former, 19% were weak biofilm former, and 18% were non-biofilm former. The rates of MDR and extensive drug resistance (XDR) were 26% and 1%. PCR analysis indicated that 93% of the isolates were algD positive. T3SS genes exoT, exoS, and exoU were detected in 91%, 63%, and 32% of the isolates, respectively. There was a high frequency of exoT + /exoS + genotype (61%), whereas exoT + /exoU + (32%) and exoS + /exoU + (2%) genotypes were relatively uncommon. This study reports the first dataset on the molecular profile of P. aeruginosa in Cyprus. Our results demonstrated that most strains have the biofilm-forming capacity with an algD-positive genotype and the majority carry exoT and exoS with a high frequency of exoT + /exoS + genotype.

Molecular Characterization of Virulence genes in Pseudomonas Species Isolated from Bulk tank Milk in El Menofia Governorate

Molecular Characterization of Virulence genes in Pseudomonas Species Isolated from Bulk tank Milk in El Menofia Governorate

Reduction of Rhizoctonia cerealis Infection on Wheat Through Host- and Spray-Induced Gene Silencing of an Orphan Secreted Gene.

Rhizoctonia cerealis is a soilborne fungus that can cause sharp eyespot in wheat, resulting in massive yield losses found in many countries. Due to the lack of resistant cultivars, fungicides have been widely used to control this pathogen. However, chemical control is not environmentally friendly and is costly. Meanwhile, the lack of genetic transformation tools has hindered the functional characterization of virulence genes. In this study, we attempted to characterize the function of virulence genes by two transient methods, host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), which use RNA interference to suppress the pathogenic development. We identified ten secretory orphan genes from the genome. After silencing these ten genes, only the RcOSP1 knocked-down plant significantly inhibited the growth of R. cerealis. We then described RcOSP1 as an effector that could impair wheat biological processes and suppress pathogen-associated molecular pattern-triggered immunity in the infection process. These findings confirm that HIGS and SIGS can be practical tools for researching R. cerealis virulence genes. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Investigation of Virulence Genes of the Predominant Bacteria Associated with Renal Stones and their Correlation with Postoperative Septic Complications.

PurposeNephrolithiasis is a worldwide disease, and 4.7% of the patients may develop postoperative sepsis. Characterization of virulence genes of bacteria associated with renal stones is still lacking in the literature. The study aimed to investigate the virulence genes of the predominant stone bacterial isolate and their association with postoperative septic complications in patients treated with percutaneous nephrolithotomy (PCNL).MethodsStone and midstream urine samples were collected from 200 nephrolithiasis patients who underwent PCNL. Microbiological examination and virulence profile were studied for the common bacteria isolated from the stones.ResultsMicrobiological analysis revealed that Staphylococcus aureus was the predominant organism in stone samples (42.8%), while Escherichia coli (56.6%) was the dominant pathogen in midstream urine. Eight patients (4%) developed septic complications; stone culture was positive for S. aureus in seven and E. coli in one patient, while all but one had negative midstream urine. The patient with positive midstream urine culture had also S. aureus infection. Detection of virulence genes in S. aureus isolated from stones showed a high positivity of the hemolysine gene hla (93.3%) and adhesion gene fnbA (73.3%), whereas enterotoxin genes (sec and sea) were negative in all S. aureus stone cultures. Moreover, the adhesion genes (fnbB and can), hemolysine gene (hlb), panton-valentine leukocidin (pvl) gene and the enterotoxin gene (seb) were significantly higher in septic patients compared to the non-septic ones (p< 0.05). Interestingly, there was a significant relation between the existence of virulence genes and the resistance of antibiotics (p < 0.05).ConclusionThere has been a notable shift toward gram-positive organisms (S. aureus) in the stone culture. Moreover, S. aureus virulence genes were significantly attributed to the resistance of some antibiotics and postoperative septic complications, suggesting that the stone culture could be more informative than urine culture, especially in predicting the risk of postoperative sepsis.

Evaluation of the microbiological and chemical aspects of autochthonous wild snails in Sardinia.

This study was conducted to acquire knowledge on the epidemiology and ecology of some zoonotic agents in snails. Chemical and microbiological analysis was carried out on 46 samples of snails belonging to the species of Helix aspersa and Helix (Eobania) vermiculata. The association between heavy metals and wild snails, a native consumer product in the Region of Sardinia, was determined.The molecular characterisation of Listeria monocytogenes virulence genes has shown a genetic profile that deserves more attention for the improvement of surveillance and risk prevention. Specimens of H. vermiculata showed higher concentrations of cadmium (M=0.80±0.56 mg/kg) than H. aspersa (M=0.61±0.17 mg /kg).A further objective was to determine whether the samples showed significant differences from the point of view of secretion characterisation, in terms of protein content, and to identify species-specific correlations and possible relationships with the environment. The presence of Salmonella enterica sub.sp houtenae (6,14: z4, z23) and Salmonella enterica subsp diarizonae (47: k: e, n, z15) (1 sample), Listeria monocytogenes (2 samples) with Molecular characterization of virulence genes together with the measurement of heavy metals in samples of wild snails has shown a health and hygiene profile that would deserve greater attention for the improvement of the surveillance and prevention of microbiological and chemical risk in such products which currently show a tendency towards increase in consumption.

Isolation, identification and virulence gene characterization of Aeromonasdhakensis isolated from sea lion (Zalophus californianus).

Species of Aeromonas are ubiquitous pathogens of fish and aquatic animals and can infect humans and other animals through the food chain. However, there are few reports of marine mammalian infections. In 2020, a sea lion (Zalophus californianus) died acutely at an aquarium in Harbin, Heilongjiang Province, China. In order to explore the cause of death, we dissected the animal and observed pathological changes. Ogans were aseptically collected and used for bacterial isolation and culture. This revealed that the sea lion had died of sepsis caused by a bacterial infection. Isolated bacteria were investigated by morphology, biochemical phenotype and molecular identification, and this determined the pathogen as A. dhakensis. The isolate contained six virulence genes, hlyA, aerA, act, lafA, ela, fla, and was susceptible to most antibiotics. This is the first report of A. dhakensis associated with septicaemia in pinnipeds and a description of its virulence and resistance profiles. Its presence in aquatic environments poses a potential threat to marine mammals.

Isolation and identification of Biofilm-producing E. coli from drinking water.

Background: The increasing rate of water-borne diseases in Karachi demands the characterization of associated pathogens. Nowadays, water-borne infections become more resistant to treatment due to biofilm formation and excessive use of antibiotics. Biofilm formation in water deteriorates the quality of water. This study identified and characterized biofilm-producing E. coli in drinking water. Methodology: Water samples were analyzed for heterotrophic plate count and total coliforms, fecal coliforms, and E. coli. The Kirby disk bar diffusion method was used to investigate antibiotic susceptibility in biofilm-producing E. coli. The Congo red and tube ring methods were used to identify biofilm producers. The effect of biofilm formation on the hydrophobicity of E. coli was performed by the BATH method. The soft agar method determines the colony spreading ability of biofilm and non-biofilm producers. Molecular characterization of virulence genes of E. coli was performed by a PCR. Scanning electron microscopy for biofilm construction was conducted. Results: The total 120 water samples were tested for heterotrophic plate count and total coliforms, fecal coliforms, and E. coli. 78% were unfit in this study, and 21.66% were fit. 38 E. coli strains were found in water samples. According to findings, the hydrophobicity of biofilm-producing isolates increased with the incubation period. Colony-forming unit drops one logarithm in the biofilm state compared to the planktonic stage. Biofilm producers were more resistant to antibiotics. The virulence genes, pet, lt, and stx2, were used for the molecular characterization. Conclusion: The presence of biofilm-producing E. coli in drinking water is alarming, and it indicates inappropriate treatment of the water supply system. To prevent rapid water-borne diseases, adequate actions are required to control drinking-water biofilm producers.

Stenotrophomonas maltophilia: Genotypic Characterization of Virulence Genes and The Effect of Ascorbic Acid on Biofilm Formation

Stenotrophomonas maltophilia is an environmental bacterium that has gained a lot of attention, as a nosocomial pathogen associated with significant mortality rates. Biofilm formation is considered the corner stone for establishing infections in many bacteria including S. maltophilia. The aim of this study was the genotypic characterization of the different virulence-associated genes and the investigation of the effect of ascorbic acid on S. maltophilia biofilm formation. A total of 20 S. maltophilia isolates from different sources were included in this study. Genes encoding different virulence factors were investigated genotypically. These included stmPr1, stmPr2, smlt3773 locus, smf-1, rpfF, rmlA and spgM. Biofilm formation was investigated phenotypically. The effect of ascorbic acid on biofilm formation was investigated using MIC as well as sub-inhibitory concentrations. Many of the isolates harbored both serine proteases genes stmPr-1 and stmPr-2. Fourteen (70%) of the 20 isolates carried stmPr-1 and 15 (75%) had stmPr-2. Most of the isolates (95%) possessed smlt-3773 locus. Genes linked to biofilm formation such as smf-1, rpfF, rmlA and spgM, were found in (90%), (45%), (85%) and (30%) of the isolates, respectively. Phenotypically, all S. maltophilia isolates (100%) were biofilm producers. Fifteen (75%) were strong biofilm producers and 5 (25%) were moderate biofilm producers. In attempts to seek a non-chemotherapeutic alternative that can hinder biofilm formation without provoking antimicrobial resistance, the results, herein, showed that ascorbic acid inhibits biofilm formation in a dose-dependent manner.

Clostridioides difficile from Brazilian hospitals: characterization of virulence genes by whole genome sequencing

Clostridioides difficile (CD) is the most frequent cause of healthcare related diarrhea and its severity has increased in the last decade by the spread of hypervirulent strains. Most important CD virulence factor is toxin production; however, not only toxins are responsible for Clostridioides virulence. We sequenced 38 strains and analyzed the presence and integrity of 24 virulence (including toxin) genes. We identified 28 toxigenic strains, six also presented the cdt genes. Only six strains didn't present all others genes searched. All absent genes were adhesion related. Understand others CD virulence factors can lead to a best understanding on this matter.

Distinction between Antimicrobial Resistance and Putative Virulence Genes Characterization in Plesiomonas shigelloides Isolated from Different Sources.

Plesiomonas shigelloides are gram-negative, thermotolerant, motile, and pleomorphic microorganisms that are only distantly related to those of the Enterobacteriaceae and Vibrionaceae families. One of the most common sources of P. shigelloides contamination is human stool, but it may also be found in a wide range of other animals, plants, and aquatic habitats. Antimicrobial resistance in P. shigelloides from seawater and shellfish was investigated, and pathogenicity involved genes were characterized as part of this study. Out of 384 samples of shellfish, 5.7% included P. shigelloides. The presence of P. shigelloides was also discovered in 5% of the seawater sampled. The antimicrobial resistance of 23 P. shigelloides isolates derived from those samples was investigated. All isolates were sensitive to nalidixic acid, carbenicillin, cephalothin, erythromycin, kanamycin, tetracycline, and ciprofloxacin in the study. Several strains isolated from diseased shellfish were tested for virulence in shellfish by intraperitoneal injections. The LD50 values ranged from 12 × 108 to 3 × 1012 cfu/shellfish. When looking for possible virulence factors that may play a significant role in bacterial infection in the current study, we found that all of these genes were present in these strains. These include genes such as elastase, lipase, flagellin, enterotoxin, and DNases. According to these findings, shellfish may serve as a reservoir for multi-resistant P. shigelloides and help spread virulence genes across the environment.

Virulence Gene Characterization and Serotyping of Major Bacterial Pathogens Isolated from Bovine Respiratory Disease in Ethiopia

Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (n = 107) and pneumonic lung tissue (n = 59). Laboratory assay confirmed isolation of M. haemolytica 37 (22.29%), P. multocida 25 (15.06%), B. trehalosi 12 (7.23%), and H. somni 15 (9.04%). PCR assay of P. multocida capsular typing revealed 21 (84.0%) cap A (hyaD-hyaC) and 4 (16.0%) cap D (dcbF) strains. M. haemolytica serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. P. multocida biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, respectively. M. haemolytica harbored more than 60% ssa gene, and 90.91% sodA while FbpA, TbpA, and lktC genes were found in all isolates. Likewise, all P. multocida exhibited toxA, FbpA, TbpA, and pmSLP genes. The current finding showed that M. haemolytica serotype A: 1 is frequently associated with BRD followed by P. multocida biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, virulence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak surveillance and molecular approaches are indispensable in designing efficient prevention strategies.

First Report of Antimicrobial Susceptibility and Virulence Gene Characterization Associated with Staphylococcus aureus Carriage in Healthy Camels from Tunisia

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin; lukE–lukD, encoding bicomponent leukotoxin LukE–LukD; the clfB gene, encoding clumping factor B; the laminin gene, encoding laminin-binding protein; and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Antibiotic Susceptibility Pattern, Molecular Characterization of Virulence Genes among Pseudomonas Aeruginosa Isolated from Burn Patients

Abstract&#x0D; In this research a total of 150 samples were obtained from burn and wound patients admitted to the West Erbil Emergence Hospital during period from September 2020 to January 2021. Through cultural, morphological features, biochemical testing and Vitek’s 2 compact systems, 40 isolates of P. aeruginosa have been identified. P. aeruginosa produced various pigments, including blue / green, and yellow / green. The iso1ates of P. aeruginosa were subjected to 14 different antibiotics. Impenim was the most effective antimicrobial agents against all P. aerugionsa isolates, and most of isolates showed high resistance degree to Ampicillin 100%, Chloramphenicol 100%, amoxicillin-clavulanic acid 100%, Cefotaxime 100% and Penicillin 100% while for Aztreonam 32.5%, Meropenem 42.5%, Tobramycin 45%, Gentamycin 45%, Amikacin 45%, Ciprofloxacillin 62.5%, ceftazidime 67.5, % Tetracycline 80%. All Psudomonas aeruginosa isolates were screened using Multiplex polymerase chain reaction (PCR) to check for the presence of (Pvda, LasB, Protease, exoA, exoT, exoU and plch) on its genomic DNA. The findings have shown that (Pvda was 55%, LasB 75%, Protease 65%, exoA 60%, exoT 75%, exoU 60% and, plch 55%) of isolates harbored these genes as a virulence genes.

Multidrug-, methicillin-, and vancomycin-resistant Staphylococcus aureus isolated from ready-to-eat meat sandwiches: An ongoing food and public health concern

Methicillin-resistant S. aureus (MRSA) and their antimicrobial resistance pose exacerbating global health threats and endangering everyone. Thus, the prevalence, molecular characterization of virulence genes, and antimicrobial resistance patterns of strains isolated from 225 beef burger and hot dog sandwiches vended in Mansoura city, Egypt were determined. 83.1% of the sandwiches tested were contaminated with coagulase-positive S. aureus, with a mean count of 4 × 103 CFU/g. Genes encoding mecA, α-hemolysin, staphylococcal enterotoxins, and toxic shock syndrome toxin-1 were detected in 22.6%, 96.3%, 61.1%, and 0% of the strains isolated, respectively. Of the 190 coagulase-positive strains, 43 (22.6%) were confirmed as MRSA. Among them, 4 strains (2.1%) were vancomycin-resistant S. aureus (VRSA) and resistant to all antimicrobials tested. Interestingly, all isolates were resistant to at least one of the antimicrobials tested, with 75.2% being multi-drug resistant (MDR) and an average multiple antimicrobial resistance (MAR) index of 0.503. Not less important, 100%, 96.3%, 90.5%, 79.5%, 73.7%, 62.6%, and 48.9% of isolates were resistant to Kanamycin, Nalidixic acid, Cefotaxime, Sulphamethoxazole-Trimethoprim, Penicillin G, Tetracycline, and Cephalothin, respectively. The potential hazard of MDR-, MRSA-, and VRSA-contaminated sandwiches may be an indication of the presence of what is more dangerous. Hence, strict hygienic measures and good standards of food handler's personal hygiene to prevent transmission of these pathogens to consumers are imperative.

Biofilm formation in uropathogenic Escherichia coli strains; Relationship with virulence factors and antimicrobial resistance

Background: Urinary tract infection (UTI) is a clinical condition with characteristic symptoms, colonization and multiplication of bacteria in significant numbers. At this juncture, antimicrobial resistance in uropathogens has become one of the major concerns globally. Therefore, the present study aimed to demonstrate the biofilm formation in uropathogenic Escherichia coli strains; characterize the phenotypic & genotypic virulence factors and their relationship with antimicrobial resistance. Methods and materials: In this study, 1000 suspected UTI cases were included. Urine samples were processed for culture and antimicrobial drug susceptibility testing. Only Escherichia coli isolates were further studied for production of biofilm, ESBL and haemolysin enzymes by phenotypic methods. Virulence genes, papEF, traT & PAI were detected by multiplex PCR. Molecular confirmation & characterization of virulence genes was done by Sanger sequencing. Results: From the 1000 urine samples, 395 E. coli were isolated. Higher resistance was observed with antibiotics - ampicillin (82.53%), cefuroxime (72.41%), amoxicillin-clavulanic acid (71.90%), ceftriaxone (66.58%), ciprofloxacin (65.82%) and cefepime (57.47%). In-vitro biofilm was formed by 71.39% isolates. Biofilm-forming strains developed higher resistance towards ampicillin, cefuroxime, amoxicillin-clavulanic acid, ciprofloxacin, cefepime and ceftriaxone. Phenotypic methods detected 62.3% isolates as ESBL producers and 40.2% were β-haemolytic. Virulence gene characterization revealed presence of traT in 73.2% strains, PAI & papEF among 62.9% and 33.5% strains. ESBL & Hemolysin producing UPEC exhibited higher resistant to ampicillin, amoxicillin-clavulanic acid, aztreonam, cefriaxone, cefuroxime, cefepime, ciprofloxacin, chloramphenicol & gentamicin. Statistically proved association was seen with gene RPAI and traT with antibiotics nitrofurantoin and amikacin. All the isolates were sensitive to nitrofurantoin, piperacillin-tazobactam, and imipenem. Strains showed 99% sequence identity and no mutations were detected. Conclusion: Increase in trend of resistance was observed with antibiotics which were routinely used to treat UTI. Results indicated significant correlation between phenotypic and genotypic virulence factors and antibiotic resistance. Antibiotics nitrofurantoin, piperacillin - tazobactam, and imipenem can be effective for severe UTIs. This study concludes that expression of virulence factors by UPEC strains is responsible for increased antibiotic resistance. Hence, characterizing the UPEC strains help clinicians to reach a better therapeutic outcomes and treatment regimens in this region.

Serotyping, Genotyping and Virulence Genes Characterization of Pasteurella multocida and Mannheimia haemolytica Isolates Recovered from Pneumonic Cattle Calves in North Upper Egypt.

Pasteurella (P.) multocida and Mannheimia (M.) haemolytica are the most two common pathogenic bacterial agents causing pneumonia in calves. Both bacteria are associated with significant economic losses in the cattle industry due to high morbidity and mortality rates, especially in the case of severe infections. The objectives of the present study were to perform serotyping and genotyping, as well as characterization of the virulence-associated genes in 48 bacterial isolates; 33 P. multocida and 15 M. haemolytica. All strains were isolated from pneumonic cattle calves showing respiratory manifestations such as fever, nasal discharges, and rapid breathing in North Upper Egypt governorates (Beni-Suef and El-Fayoum). PCR was applied as a confirmatory test using a specific universal gene, kmt1, and rpt2 for P. multocida and M. haemolytica, respectively. The results show that 29 (87.9%) P. multocida and 15 (100%) M. haemolytica isolates were positive for the corresponding universal gene. The results of serotyping indicate that 86.2% of P. multocida isolates belonged to serotype B:2, while 13.8% were untyped. Meanwhile, 60% and 40% of M. haemolytica isolates belonged to serotype 2 and serotype 1, respectively. Investigation of virulence-associated genes showed that all the tested P. multocida isolates harbored nanB, omp87, and toxA genes. Four M. haemolytica isolates harbored both gcp and lktC genes and of these, three isolates harbored the ssa gene. Sequencing of toxA gene of P. multocida and lktC gene of M. haemolytica in the current strains indicated a great homology with strains uploaded in gene banks from different hosts and localities worldwide.

Prevalence and characterization of virulence genes among methicillin-resistant Staphylococcus aureus isolated from Sudanese patients in Khartoum state.

Staphylococcus aureus is a versatile pathogen that can cause a variety of diseases, ranging from mild to fatal infection. This study aimed to detect the virulence genes (cna, ica, hlg and sdrE) in S. aureus isolated from different types of infections in Sudanese patients admitted to different hospital in Khartoum state. This is a descriptive cross-sectional study conducted over a period of 4 months from 1 April to 30 July 2017 in Khartoum. Overall, 65 S. aureus isolates were identified using standard biochemical and microbiologic tests. Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Nucleic acid was extracted using the guanidine hydrochloride method, and all the genes except for sdrE were detected by multiplex PCR. The ica gene was the predominant one, found in 73.85% of the isolates, with sdrE found in 38.46%, cna in 29.25% and hlg in 7.69%. The relationship between the virulence genes and resistance to antibiotics showed that the highest resistance was observed in isolates with ica and sdrE, followed by cna and hlg. There were significant relationships between methicillin resistance and the presence of sdrE and ica genes (p 0.01 for both) and between ciprofloxacin resistance and the presence of sdrE gene (p 0.03).