hlyA Gene Research Articles

Construction and characterization of different hemolysin gene deletion strains in Vibrio parahaemolyticus (ΔhlyA, ΔhlyIII) and evaluation of their virulence

Vibrio parahaemolyticus, a halophilic food-borne pathogen, possesses an arsenal of virulence factors. The pathogenicity of V. parahaemolyticus results from a combination of various virulence factors. HlyA and hlyIII genes are presumed to function in hemolysis, in addition to tdh and trh in V. parahaemolyticus. To confirm the hemolytic function of genes hlyA and hlyIII, ΔhlyA and ΔhlyIII strains of V. parahaemolyticus were separately constructed via homologous recombination. The cytotoxicity and pathogenicity of the ΔhlyA and ΔhlyIII strains were evaluated using a Tetrahymena-Vibrio co-culture model and an immersion challenge in Litopenaeus vannamei. Results indicated that the hemolytic activity of the ΔhlyA and ΔhlyIII strains decreased by approximately 31.4 % and 24.9 % respectively, compared to the WT strain. Both ΔhlyA and ΔhlyIII exhibited reduced cytotoxicity towards Tetrahymena. Then shrimp infection experiments showed LD50 values for ΔhlyA and ΔhlyIII of 3.06 × 108 CFU/mL and 1.23 × 108 CFU/mL, respectively, both higher than the WT strain’s value of 2.57 × 107 CFU/mL. Histopathological observations revealed that hepatopancreas from shrimps challenged with ΔhlyA and ΔhlyIII exhibited mild symptoms, whereas those challenged with the WT strain displayed severe AHPND. These findings indicate that the ΔhlyA and ΔhlyIII strains are significantly less virulent than the WT strain. In conclusion, both hlyA and hlyIII are vital virulence genes involved in hemolytic and cytotoxic of V. parahaemolyticus.

Genetic diversity and antibiotic resistance patterns of Escherichia coli isolates causing septicemia: A phylogenetic typing and PFGE analysis

IntroductionThis study aims to analyze clinical isolates of E. coli causing septicemia across various phylogroups utilizing the PFGE method. Materials and methodsA total of 100 clinical isolates were collected. The presence of CTX-M, TEM, SHV, KPC, MBL and OXA-48 genes was detected by PCR. Additionally, phylotyping, serotyping, and virulence-typing assay were done by PCR and PFGE methods to investigate the genetic diversity of the isolates. ResultsThe O1 serotype and the HlyA gene were the most prevalent serotype and virulence gene, respectively. Notably, 34% of the isolates harbored SHV, TEM, and CTX-M-1 β-lactamase genes. All isolates showed resistance to amoxicillin and tetracycline, but no resistance to fosfomycin was seen. The most and least common phylotypes, according to PFGE analysis, belonged to phylogroups B2 and B1, respectively. ConclusionThe data offers valuable insights into the genetic diversity and antibiotic resistance patterns of E. coli isolates responsible for septicemia.

The Impact of Subinhibitory Concentrations of Ɛ-polylysine, Hydrogen Peroxide, and Lauric Arginate on Listeria monocytogenes Virulence

Recent studies on the use of plant-derived and other bioactive compounds and antimicrobials in food have challenged the idea that exposure to antimicrobials at sublethal or subinhibitory concentrations (SICs) increases the virulence potential of bacterial pathogens including Listeria monocytogenes. The objective of this study was to determine the effect of exposure to SICs of Ɛ-polylysine (EPL), hydrogen peroxide (HP), and lauric arginate (LAE) on L. monocytogenes virulence. For all assays, L. monocytogenes strains Scott A and 2014L-6025 were grown to mid-log phase in the presence of SICs of EPL, HP, or LAE. Motility was determined by spot inoculating cultures on soft brain heart infusion agar (0.3% agar). Cultures grown in SICs of antimicrobials were also inoculated onto Caco-2 cells (10:1 MOI) to determine the effects on subsequent adhesion and invasion. Last, the relative expression of key virulence genes (prfA, plcB, hlyA, actA, inlA, inlB, sigB, and virR) following growth in SICs was determined by RT-qPCR. Results indicate that L. monocytogenes growth in the presence of SICs of EPL, HP, or LAE did not affect the motility, adhesion, or invasion capacity of either strain. Changes in gene expression were observed for both L. monocytogenes strains. More specifically, SICs of EPL and LAE reduced hlyA expression in Scott A, whereas SICs of EPL and HP increased the expression of virR. The upregulation of sigB and actA in the presence of EPL and LAE, respectively, was observed in strain 2014L-6025. These findings indicate that exposure to SICs of these antimicrobials has varying effects on L. monocytogenes that differ by strain. Although no phenotypic effects were observed in terms of motility, adhesion, and invasion, the observed changes in virulence gene expression warrant further investigation.

Suppression of virulence factors of uropathogenic Escherichia coli by Trans-resveratrol and design of nanoemulgel

BackgroundDevelopment of multidrug resistance in Uropathogenic Escherichia coli (UPEC) makes treatment of Urinary Tract Infections (UTIs) a major challenge. This study was conducted to investigate the effect of trans-resveratrol (t-RSV) at a subinhibitory concentration (sub-MIC-t-RSV) on phenotypic and genotypic expression of virulence factors of clinical isolates of UPEC and develop a nanoformulation of t-RSV. Fifty-five clinical UPEC strains were investigated for the presence of virulence factors by phenotypic methods and PCR detection of virulence genes. The effect of sub-MIC-t-RSV was studied on the phenotypic and genotypic expression of virulence factors. t-RSV-loaded nanoemulgel formulation was prepared and characterized.ResultsOut of the 55 tested isolates, 50.9% were biofilm producers, 23.6% showed both mannose-sensitive and mannose-resistant hemagglutination, 21.8% were serum-resistant, 18.2% were hemolysin producers, while 36.4% showed cytotoxic effect on HEp-2 cells. A total of 25.5% of the isolates harbor one or more of hly-A, cnf-1 and papC genes, while 54.5% were positive for one or more of fimH, iss and BssS genes. A concentration of 100 µg/mL of t-RSV effectively downregulates the phenotypic and genotypic expression of the virulence factors in positive isolates. A stable t-RSV-nanaoemulgel with droplet size of 180.3 nm and Zetapotential of -46.9 mV was obtained.ConclusionThe study proves the effective role of t-RSV as an antivirulence agent against clinical UPEC isolates in vitro and develops a stable t-RSV-nanoemulgel formulation to be assessed in vivo. The promising antibacterial and antivirulence properties of t-RSV place this natural compound to be a better alternative in the treatment of persistent UTIs.

Molecular diagnosis of E.coliO157:H7 Which Isolated from Children with Diarrhea by using Multiplex PCR

A total of 96 stool samples were collected from children with bloody diarrhea from two hospitals in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identifed by using morphological diagnostic methods, Samples were cultured on liquid enrichment medium, incubated at 37C? for 24 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 32 non-sorbitol fermenting bacterial isolates were obtained of which 11 were identified as Escherichia coli by using traditional biochemical tests and API20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates . Four special biochemical tests were done for serotype O157:H7 differentiation from other NSF bacteria. Only 3 isolates belonging to the serotype O157:H7 were obtained . Latex agglutination test for O157 and H7 showed that the 3 isolates gave positive results with both tests. The Bacterial isolates were identifed by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated By using specific primers in MPCR . The result showed that one E. coli O157:H7 isolates contain all 4 genes , other isolates contain 3 genes: Stx2, hlyA & eaeA.

Detection Of Escherichia Coli Virulence Genes ( Pap , Afa , Iha Ipr2 And Hly ) Isolated From Patients With Urinary Tract Infections In Babylon Province, Iraq

The current study included a collection of 110 urine samples of both sexes of different ages for patients with urinary tract infections, who consulted Al-Hilla Teaching Hospital and the Children and Maternity Hospital in Al-Hilla city, through the period from May 2020 to December 2020, to investigate some virulence factors associated with Escherichia coli bacterium. The results of the biochemical tests revealed that 74 (67.3%) isolates were belonging to E. coli that was isolated from the urine samples. The results also indicated that the females were more infected 47 (63.5 %) with urinary tract infections than males 27 (36.4 %). Some virulence factor genes have also been investigated using the polymerase chain reaction (PCR) technique, which are (pap, afa, iha, ipr2 and hly genes). The PCR results for E.coli genes revealed that, 11 isolates contain pap gene (7 ( 14.8 %) and 4 ( 14.8 %)), while 9 isolates possesses afa gene ( 6 ( 12.7 %) and 3 ( 11.1 %)), whereas 5 isolates have gene iha (3 ( 6.3 %) and 2 ( 7.4 % )) , In addition to 30 isolates have irp2 gene (21 ( 44.6 % ) and 9 ( 33.3 % )) and 27 isolates contain hly gene ( 19 ( 40.4 %) and 8 ( 29.6 %)). These percentages are for females and males respectively. Conclusions: E.coli is the bacterium that dominant the UTIs. Females are more likely to develop UTIs than males. The irp2 gene responsible for taking iron is the most genetically studied genome that causes UTIs, followed by the gene hly responsible for producing α-hemolysin enzyme second among the gene of the virulence factors under study.

Molecular Identification and Virulence Genes Detection of Cronobacter Spp Isolated from Clinical Cases in Infants Under Two Years in Mosul City, Iraq

Cronobacter spp. are facultative-anaerobic non-spore forming Gram-negative bacteria, that are part of the Enterobacteriaceae family. They are opportunistic organisms that occasionally cause severe infections such as meningitis, necrotizing enterocolitis, and bloodstream infections in newborns and young children. There is a limited amount of information available about the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources. This study was conducted to isolate and identify Cronobacter spp from different clinical cases in children under two years in Mosul city and determine some virulence genes which are indicators for their pathogenicity. One hundred and fifty clinical specimens (diarrhea, blood and cerebrospinal fluid) were collected and cultured on Enterobacter sakazakii agar (HiCrome) and trypton soy agar. Eight Cronobacter spp were isolated and presumptively identified according to their morphological characteristics on selective media and confirmed by 16S rRNA gene sequencing and direct sequencing of ropB gene. PCR was used to amplify five potential virulence genes (cpa, hly, sip, zpx, and ompA) in order to determine their presence in our strains. The results revealed that all strains exhibited ompA and zpx, however, only four C. sakazakii strains contained cpa and hly genes. The sip gene was found in only two C. malonaticus strains. Lastly, ropB gene were detected in all Cronobacter spp isolated. Identification of Cronobacter from clinical specimens helps in understanding the sources and risks associated with these pathogens. By determining virulence genes, the study provides insights into how these bacteria cause disease which is crucial for developing targeted interventions and treatments. understanding the presence and characteristics of Cronobacter spp. in clinical cases informs diagnostic and treatment strategies, potentially leading to better patient outcomes.

Impact of liposomal hesperetin in broilers: prospects for improving performance, antioxidant potential, immunity, and resistance against Listeria monocytogenes

ABSTRACT Liposomal encapsulated phytogenics, such as liposomal hesperetin, are considered novel substitutes for antibiotics in the broiler industry owing to their improved nutritional and therapeutic properties. Therefore, our key goal was to investigate liposomal hesperetin impact on broiler growth performance, health, antioxidant status, tight junction proteins (TJP), and resistance against Listeria monocytogenes. Four broiler groups were fed 0, 150, 250, or 400 mg/kg of liposomal hesperetin-supplemented diets and experimentally infected with L. monocytogenes strain. Herein, liposomal hesperetin, especially at higher concentrations, augmented broilers FCR with upregulation of genes encoding TJP (occludin, JAM-2, MUC-2), and antioxidant attributes (GPX-1, SOD-1, CAT, HO-1, NQO1, COX2), which reflect enhancing health and welfare of broilers. Muscle antioxidant biomarkers were enhanced; meanwhile, muscle MDA, ROS, and H2O2 levels were reduced in response to 400 mg/kg of liposomal hesperetin. Liposomal hesperetin fortification reduced L. monocytogenes loads and expression levels of its virulence-related genes (flaA, hlyA, and ami). Remarkably, histopathological alterations in intestinal and brain tissues of L. monocytogenes-infected broilers were restored post-inclusion at higher levels of liposomal hesperetin, which reflects increase of the birds’ resistance to L. monocytogenes infection. Transcription levels of genes encoding cytokines/chemokines (MyD88, AVBD6, CCL20, IL-1β, IL-18), and autophagy (Bcl-2, LC3, AMPK, AKT, CHOP, Bip, p62, XBP1) were ameliorated following dietary liposomal hesperetin fortification, which suggests enhancement of the birds’ immunity and health. Collectively, our research recommends liposomal hesperetin application in broiler diets owing to its promoting impact on growth performance, antioxidant status, immunity, health, and welfare besides its antibacterial, and antivirulence characteristics to fight against L. monocytogenes.

A new exploration of the amplification mechanism of saltatory rolling circle amplification (SRCA) for food safety detection

A new property of Bst DNA polymerase was discovered by our team, which led to the establishment of a new isothermal amplification method for SRCA nucleic acid, which was applied to food safety detection, investigated the amplification mechanism, and the SRCA version 1.0 amplification mechanism was proposed. This study proposes a new SRCA amplification mechanism (version 2.0) by using the autosynthetic sequences CDG2040 and CDG2068, as well as the Listeria monocytogenes hlyA gene and Vibrio parahaemolyticus toxR gene as target sequences to further investigate the amplification mechanism. The initiation of the SRCA reaction depends primarily on the spatial structure of the non-closed circle formed by the discontinuous complementation of the bases on both sides of the target sequence. The DNA fragments that connect the target sequences in the SRCA reaction are the sum of the complementary sequences of adjacent partial sequences on both sides of the target sequence.

Sanitizer Resistance and Persistence of Listeria monocytogenes Isolates in Tree Fruit Packing Facilities

The foodborne pathogen Listeria monocytogenes can persist in produce processing environments, which increases the risk for food contamination. Increased resistance to antimicrobials commonly used in cleaning and sanitizing procedures may contribute to L. monocytogenes’ persistence in these environments. This study aimed to evaluate sanitizer resistance in L. monocytogenes isolates collected from three tree fruit packing facilities (F1, F2, and F3) during packing seasons 2020–2021 (Y1) and 2021–2022 (Y2), and to assess evidence of persistence based on the genomic similarity of isolates to historical isolates collected in previous years. L. monocytogenes isolates collected in 2020–2022 (n = 44) were tested for resistance to peroxyacetic acid (PAA) and a proprietary biofilm−removing agent using a broth microdilution assay. Further, L. monocytogenes isolates were whole genome sequenced and screened for the presence of antimicrobial resistance and virulence genes, as well as to assess the genomic similarity of isolates using the CFSAN SNP bioinformatic pipeline. Over half (57%) of the tested isolates had a PAA minimum inhibitory concentration (MIC) of 250 ppm, which was similar to the applied concentration of the PAA sanitizer in the three facilities (230 ppm). In contrast, 80% of tested isolates had a biofilm remover MIC of 0.13 ppm, which was substantially below the concentration applied in the facilities (137 ppm). Genomes of all tested isolates carried antimicrobial resistance (fosX, lin, mdrL, mprF, and norB) and virulence (inlA, inlB, plcA, plcB, prfA, hly, mpl, and iap) genes. L. monocytogenes isolates collected between 2020 and 2022 belonged to three distinct lineages, with 22 multilocus sequence types (MLSTs) belonging to 22 different clonal complexes. Genomic similarity analysis with historical isolates collected from the same facilities in 2016–2017 demonstrated a 5-year persistence of the genotypes ST 1003 and ST 554 in F2, which were no longer detected in 2022. Overall, our results highlight the need to re-evaluate sanitizer concentrations to effectively control persistent L. monocytogenes strains in tree fruit packing facilities.

Genetic Diversity, Virulence Factors and Antibiotic Resistance of Listeria monocytogenes from Food and Clinical Samples in Southern Poland.

Listeriosis is one of the most serious foodborne diseases under surveillance, with an overall mortality rate in the EU currently being high at 18.1%. Therefore, this study aims to investigate Listeria monocytogenes strains isolated from clinical and food samples for susceptibility to antimicrobials, presence of virulence factors, and genetic diversity. Species were identified using the MALDI-TOF, resistance to 11 antibiotics was determined according to EUCAST guidelines, and multiplex PCR was used for serotyping and detecting virulence genes. Strains were genotyped using the PFGE method. Clinical strains showed full sensitivity to all tested antibiotics. In total, 33.3% of strains from food products were found to be resistant to ciprofloxacin and 4.2% to tetracycline. Most of the tested isolates (79.2%) belonged to serotype 1/2a-3a, and the rest (20.8%) belonged to serotype 4ab-4b,4d-4e. Five virulence genes (prfA, hlyA, plcB, inlA, and lmo2672) were detected in all strains studied. The llsX gene was the least common, in 37.5% of clinical strains and 18.75% of strains isolated from food products. Among the analyzed strains, 13 strains displayed unique PFGE profiles. The other 11 strains belong to 3 clusters of pulsotypes: cluster 1 (2 strains), cluster 2 (6 strains), and cluster 3 (2 strains). The percentage of hospitalizations and deaths of Polish patients with listeriosis indicates the seriousness of this disease, especially in an aging society, while the molecular testing of clinical strains has been rarely performed, which makes it difficult to determine the source of infection.

LAMP combined with Pyrococcus furiosus Argonaute for the ultrasensitive and highly specific point-of-care test platform for Listeria monocytogenes detection

Listeria monocytogenes is an important foodborne pathogen that causes serious infectious diseases in humans and animals. Rapid, accurate, and sensitive detection in the field is critical for the timely implementation of control measures and ensuring food safety. The study established a method that combines loop-mediated isothermal amplification (LAMP) technology and Pyrococcus furiosus Argonaute (PfAgo) reporter system, which can detect L. monocytogenes hly gene levels as low as 1.8 × 101 copies. To overcome the demands on instrumentation and equipment, lateral flow strip (LFS) technology was combined for the first time in a LAMP-PfAgo-based system to detect L. monocytogenes. The method was highly sensitive, with a limit of detection of 1.8 × 102copies, and specificity results showed no cross-reactivity with other pathogens. The performance of the method was comparable to that of real-time fluorescence quantitative PCR (qPCR) in the detection of 18 food origin samples. Moreover, the LAMP-PfAgo reaction was equipped a low-cost thin metal bath as a simple amplification device, allowing it to detect L. monocytogenes samples in shorter time, highlighting its potential as a portable, rapid, accurate, and visual nucleic acid assay. The method can facilitate the early detection of pathogenic infections and promoting food safety detection technologies.

Reaction kinetics and mechanism of UVC-LED265–285nm and UVC-LED265–285nm/hydrogen peroxide toward foodborne pathogenic Listeria monocytogenes cells and its chromosomally encoded virulent hly genes

To investigate the principle-based inactivation kinetics and mechanisms of the foodborne pathogen Listeria monocytogenes (Lm), along with its representative virulence gene (hly) encoded in both intracellular and extracellular genomic DNA, we applied disinfection treatments using UVC-LED265–285nm and UVC-LED265–285nm combined with hydrogen peroxide (10 mg/L). These treatments were conducted under controlled model reaction matrices (phosphate-buffered solutions at pH 6.2 and 7.9) and/or actual agricultural water, depending on filtration pre-treatment. Comprehensive analytical methods, such as plate count, flow cytometry, and quantitative real-time PCR, were adopted for the qualitative/quantitative determination of bacterial or gene inactivation parameters. According to the experimentally determined fluence-based first-order rate constants (kUVC-LED and kUVC-LED/H2O2), the k value of Lm viability and hly genes was 1.14–2.19 cm2/mJ and 2.18 × 10−2–2.42 × 10−1 cm2/mJ, respectively. Damage to the longer amplicon or the extracellular form of the hly gene was higher than that to the shorter amplicon or the intracellular form by a factor of 2∼10 fold. Increasing pH in reaction matrices were marginally affected to the rates of inactivation kinetics. Dose-dependent inactivation levels tested in agriculture water matrices were comparable with the kinetics from the PB solutions of both UVC-LED265–285nm and UVC-LED265–285nm/hydrogen peroxide owing to OH radical scavenging by the water matrix components. These results will be valuable for optimizing UVC-LED and UVC-LED-based advanced oxidation process disinfection systems, specifically tailored to the unique challenges of bacterial or gene inactivation in the (waste)water at food processing and agricultural sectors.

Investigating the Effect of Melittin Peptide in Preventing Biofilm Formation, Adhesion and Expression of Virulence Genes in Listeria monocytogenes.

Listeria monocytogenes is a notable food-borne pathogen that has the ability to create biofilms on different food processing surfaces, making it more resilient to disinfectants and posing a greater risk to human health. This study assessed melittin peptide's anti-biofilm and anti-pathogenicity effects on L. monocytogenes ATCC 19115. Melittin showed minimum inhibitory concenteration (MIC) of 100μg/mL against this strain and scanning electron microscopy images confirmed its antimicrobial efficacy. The OD measurement demonstrated that melittin exhibited a strong proficiency in inhibiting biofilms and disrupting pre-formed biofilms at concentrations ranging from 1/8MIC to 2MIC and this amount was 92.59 ± 1.01% to 7.17 ± 0.31% and 100% to 11.50 ± 0.53%, respectively. Peptide also reduced hydrophobicity and self-aggregation of L. monocytogenes by 35.25% and 14.38% at MIC. Melittin also significantly reduced adhesion to HT-29 and Caco-2 cells by 61.33% and 59%, and inhibited invasion of HT-29 and Caco-2 cells by 49.33% and 40.66% for L. monocytogenes at the MIC value. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed melittin's impact on gene expression, notably decreasing inlB (44%) and agrA (45%) gene expression in L. monocytogenes. flaA and hly genes also exhibited reduced expression. Also, significant changes were observed in sigB and prfA gene expression. These results underscore melittin's potential in combating bacterial infections and biofilm-related challenges in the food industry.

Antibiotic Resistance and Inflammatory Response in Urology Infections Caused by E. coli: A Molecular and Clinical Investigation

Background: Urinary tract infections (UTIs) are a common condition resulting from bacterial invasion of the urinary system, which includes the kidneys, ureters, bladder, and urethra. These infections are particularly prevalent among females due to anatomical differences and hormonal factors. UTIs can range from uncomplicated cases, where there are no structural abnormalities, to more severe infections that may ascend to the kidneys, causing pyelonephritis. Methods: This study aimed to isolate and diagnose Escherichia coli (E. coli) bacteria in patients with UTIs, identify specific virulence genes, and assess the bacteria's resistance to various antibiotics. A total of 100 clinical samples were collected from patients with UTIs at the General Hospital of Homadiyah and Ahmed Maher from October 2021 to December 2022. Bacterial identification was performed using biochemical tests and polymerase chain reaction (PCR) techniques, while antibiotic sensitivity was assessed using the Kirby-Bauer disk diffusion method. Additionally, inflammatory markers (TNF-α, IL-6, and IL-8) were measured using ELISA. Results: E. coli was isolated in 56% of the samples, with the highest prevalence in patients aged 31-40 years. The antibiotic sensitivity test revealed varying levels of resistance, with the highest sensitivity observed to third-generation cephalosporins, particularly ceftriaxone (32.2% resistance). PCR analysis identified the presence of several virulence genes, including the Hly gene in 96.6% of isolates and the iha gene in 10% of isolates. Elevated levels of inflammatory markers were observed in patients with UTIs, indicating a strong immune response to bacterial infection. Conclusion: E. coli is a significant pathogen in UTIs, exhibiting considerable antibiotic resistance, particularly to first-generation cephalosporins. The presence of specific virulence genes correlates with the severity of infection. These findings underscore the need for targeted antibiotic therapy and the development of strategies to manage antibiotic resistance in UTIs.

First report of white muscle disease caused by Photobacterium damselae subsp. damselae in Kuruma shrimp (Penaeus japonicus)

In August and September 2022, two disease outbreaks were observed in Kuruma shrimp (Penaeus japonicus) farms in Okinawa, Japan. Diseased animals exhibited clinical signs of whitened musculature extending from the abdominal to the distal region. Histopathology revealed degeneration of fibers due to liquified muscle necrosis and haemocytic infiltration. Analysis of nonhost contigs from the assembled unmapped RNA and DNA shotgun sequence reads showed significant taxonomic alignment to bacterial species. This led to the investigation of a bacterium as a possible causative agent by characterizing its population in the diseased shrimp muscle. The majority of the 16S rRNA sequence recombinant clones had their highest homology with Photobacterium sp. (> 99%). The three bacterial isolates from the whitened muscle tissue were identified as Photobacterium damselae subsp. damselae (WMD-P1, WMD-P2 and WMD-P3) by biochemical and molecular assays and were further characterized. The Pdd genomes consisted of two circular chromosomes with varying numbers of plasmid. Its size ranges from 4.47 Mb to 4.60 Mb with an average GC content of 40.8%, with predicted number of coding sequences (CDs) ranging from 3816 to 4031. hlyA and pldA genes encoding for leukocidin pore-forming toxin and phospholipase were identified. Putative virulence genes are involved in adherence, antiphagocytosis, chemotaxis and motility, iron uptake, quorum sensing, secretion system, and immune evasion. The presence of prophages, genomic islands and antimicrobial resistant genes in the Pdd genomes suggests episodes of horizontal gene transfer. Average nucleotide identity (ANI) and pangenome analyses revealed a high genetic relationship of the Pdd strains (>98%) to isolates from other sources. PCR assays validated the presence of two bacterial virulence genes encoding the pore-forming toxin and phospholipase respectively in all isolates. Also, the strains had chitinase, phospholipase, and hemolytic activities. Intramuscular injection at 1 × 108 CFU/ml bacterial concentration produced pathological signs similar to those in naturally infected shrimp after 24 hpi. Lower concentration of 1 × 103 CFU/ml resulted in morbidity after 10 dpi. These results show that P. damselae subsp. damselae (Pdd) is associated with the occurrence of white muscle disease in Kuruma shrimp.

Virulence genes and pulsed-field gel electrophoresis profiles of Shiga toxin-producing Escherichia coli isolated from different food samples and patients with acute diarrhea.

Escherichia coli O157: H7 is one of the most important causes of hemorrhagic colitis, and hemolytic uremic syndrome. The present study aimed to isolate E. coli O157: H7 from foods and patients with hemorrhagic colitis, and identify Shiga toxin genes, phylogenetic comparison, and antibiotic resistance of the isolates. In total 400 samples, including patients stool and food were taken in Isfahan-Iran province. Phenotypic tests and PCR were performed to identify Shiga toxin-producing E. coli. The isolated strains were compared phylogenetically by PFGE. Agar disk diffusion was performed to identify the antibiotic resistance of the isolates. Totally, 5 isolates of fecal samples were E. coli O157, but only 2 isolates carried H7 gene. Also, 9 isolates of E. coli O157 were isolated from food samples that 3 isolates were E. coli O157: H7. The isolates carried stx1, stx2, hlyA and eaeA genes. Also, E. coli non-O157: H7 identified from samples that contained stx1, stx2, hlyA genes. The highest susceptibility to imipenem and the highest resistance to ampicillin and ciprofloxacin were observed. There was a similarity of 100% between the E. coli O157: H7 strains isolated from patients and raw milk and minced beef samples. Serotypes other than the O157 of E. coli are more prevalent in patients and food. The E. coli O157: H7 isolates from patients had 100% genetic similarity with minced meat and cow milk isolates, which indicates cattle are the most important reservoir of this bacterium in Iran.

Prevalence of multidrug-resistant Escherichia coli isolates and virulence gene expression in poultry farms in Jos, Nigeria.

Antimicrobial resistance is increasingly becoming a global health concern. This study aimed to investigate and report MDR Escherichia coli (E. coli) prevalence, resistance, and virulence genes from poultry in Jos, Plateau State, Nigeria. The samples were analyzed using microbiological standard methods and polymerase chain reactions (PCRs). A total of 179 cloacal swabs were collected from bothlocal and exotic poultry breeds, of which 99.4% (178/179) tested positive for E. coli. Among these culturally identified samples, 99.4% (177/178) were furtherconfirmed Escherichia coli with a molecular weight of 401 bp. Multidrugresistance of 45% (80/178) was observed from the confirmed isolates. PCR assays were conducted to detect genes associated with resistance to antibiotics, specifically, tetracycline (tetA gene), sulfonamide (sul1 gene), ampicillin (ampC gene), and quinolone (gyrA gene). Antimicrobial susceptibility test (AST) results revealed substantial antibiotic resistance, with 81.9% (145/177) of the isolates being resistant to tetracycline, 80.2% (142/177) to quinolone, 69.5% (123/177) to sulfonamide, and 66.1% (117/177) to ampicillin. Further analysis on 18 isolates that showed resistance to up to four different antibiotics was carried out using multiplex PCR to detect eae, hlyA, rfbE, fliC, and fstx virulence genes. The study found that 44.4% (15/18) of the isolates were positive for the eae gene, 27.7% (5/18) for stx, 22.2% (4/18) for rfbe gene, and 5.5% (1) for hlya gene, and none tested positive for fliC gene. These results showed high antibiotic resistance, virulent genes, and significant levels of MDR in E. coli from poultry. This study highlights the urgent need for antimicrobial stewardship practices within the poultry industry due to their profound implications for food safety and public health. This issue is particularly critical in Nigeria, where poultry farming constitutes a significant portion of smallholder farming practices.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

Molecular Assessment of Resistance and Virulence Potential of Vibrio Species Isolated From Dumpsites in Port Harcourt, Nigeria

Background: Dumpsites have the potential to serve as reservoirs for various medically important bacteria and their virulence and resistance gene markers. For Vibrio spp., numerous genes associated with virulence have been identified in environmental strains. Due to the specific growth requirements of Vibrio spp., such strains can often be overlooked. This study aimed to assess the potential of Vibrio spp. isolated from two dumpsites in Port Harcourt, Nigeria, to serve as reservoirs of virulence and resistance genes. Methods: The soil samples were evaluated for the presence of Vibrio spp. following enrichment, using standard microbiological and biochemical test methods. DNA from Vibrio spp. was extracted using the boiling method, and isolates were tested for the presence of four resistance (sxt, strB, BlaTEM, and dfrA1) and four virulence (ctxA, hlyA, tcpA, and toxR) genes. Results: The study found a 40% occurrence of resistance genes and a 10% occurrence of virulence genes, with the strB streptomycin resistance gene being the most commonly detected (42%). Two of the virulence genes (ctxA and tcpA) were not detected. Seven of the test isolates exhibited multiple gene markers, with four gene markers present in each of two isolates. Conclusion: Overall, the study revealed a generally low potential for Vibrio sp. isolated from the dumpsites in Port Harcourt, Nigeria, to act as reservoirs of virulence and resistance genes. Additionally, the study reported an absence of major virulence markers associated with V. cholerae. A concerning finding was the high occurrence (42%) of the strB gene among these environmental isolates.