Toxin Genes Research Articles

Prevalence and risk factors of toxigenic Clostridioides difficile asymptomatic carriage in 11 French hospitals.

Clostridioides difficile infection (CDI) incidence has increased over the last 20 years. Studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We conducted a point prevalence study to estimate the toxigenic C. difficile asymptomatic carriage rate and the associated risk factors in patients >3 years old. Between September 16, 2019 and January 15, 2020, all patients hospitalized in 11 healthcare facilities in the Paris urban area were included in the study. They were screened on the day of the survey for toxigenic C. difficile carriage by rectal swab and interviewed. Isolates were characterized by PCR ribotyping and multiplex PCR targeting toxin genes. A logistic regression model was used to determine the risk factors associated with toxigenic C. difficile asymptomatic carriage using uni- and multivariate analysis in the subpopulation of patients >3 years old. During the study period, 2,389 patients were included and screened. The median age was 62 years (interquartile range 35-78 years) and 1,153 were male (48.3%). Nineteen patients had a previous CDI (0.9%). Overall, 185/2389 patients were positive for C. difficile (7.7%), including 93 toxigenic strains (3.9%): 77 (82.8%) were asymptomatic (prevalence 3.2%) whereas 12 (12.9%) were diarrheic. Prevalences of toxigenic C. difficile were 3.5% in patients >3 years old and 7.0% in ≤3 years old subjects, respectively. Toxigenic strains mainly belonged to PCR ribotypes 106 (n = 14, 15.0%), 014 (n = 12, 12.9%), and 020 (n = 10, 10.8%). Among toxigenic strains, 6 (6.4%) produced the binary toxin. In multivariate analysis, two factors were positively associated with toxigenic C. difficile asymptomatic carriage in patients >3 years old: multidrug-resistant organisms co-carriage [adjusted Odd Ratio (aOR) 2.3, CI 95% 1.2-4.7, p = 0.02] and previous CDI (aOR 5.8, CI 95% 1.2-28.6, p = 0.03). Conversely, consumption of raw milk products were associated with reduced risk of toxigenic C. difficile colonization (aOR 0.5, CI 95% 0.2-0.9, p = 0.01). We showed that there was a low prevalence of asymptomatic toxigenic C. difficile carriage in hospitalized patients. Consumption of raw milk prevents toxigenic C. difficile colonization, probably due to the barrier effect of milk-associated bacteria.

Genome characteristics of the optrA-positive Clostridium perfringens strain QHY-2 carrying a novel plasmid type.

Clostridium perfringens is a bacterial species of importance to both public and animal health. The gene optrA is the first gene that confers resistance to the tedizolid, a last-resort antimicrobial agent in human medicine. Herein, we whole-genome sequenced and analyzed one optrA-positive C. perfringens strain QHY-2 from Tibetan sheep in Qinghai province and identified one optrA plasmid pQHY-2. The plasmid shared similar structure with the optrA-positive plasmids p2C45 and p21-D-5b previously identified in C. perfringens, demonstrating the potential horizontal transmission of the optrA plasmids among C. perfringens strains. Annotation of the optrA-positive plasmids showed optrA and erm(A) located on a segment flanked by IS element IS1216E, and fexA, optrA, and erm(A) located on a segment flanked by IS element ISVlu1, which revealed the possible dissemination mechanism. Additionally, a Tn6218-like transposon carrying aac(6')-aph(2″) and erm(B) was also detected on pQHY-2, demonstrating the transposition of Tn6218 and spread of antibiotic resistance among Clostridium bacteria. Molecular analysis indicated the optrA-positive plasmids belonged to a plasmid type distinct from the pCW3-like plasmids, pCP13-like plasmids, or pIP404-like plasmids. Further structure analysis showed they might be formed by inserting segments into plasmid pCPCPI53k-r1_1, which coexist with two pCW3-like plasmids and one pCP13-like plasmid in C. perfringens strain CPI 53k-r1 isolated from a healthy human in Finland. IMPORTANCE Antimicrobial resistance is now a global concern posing threats to food safety and public health. The pCW3-like plasmids can encode several main toxin genes and three antibiotic resistance genes (ARGs), including tetA(P), tetB(P), and erm(B), which used to be considered as the main carrier of ARGs in Clostridium perfringens. In this study, we found the optrA plasmids, which belonged to a novel plasmid type, could also harbor many other ARGs, indicating this type of plasmid might be the potential repository of ARGs in C. perfringens. Additionally, this type of plasmid could coexist with the pCW3-like plasmids and pCP13-like plasmids that encoded toxin genes associated with gastrointestinal diseases, which showed the potential threat to public health.

Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella

The bacterial genus Kingella includes two pathogenic species, namely Kingella kingae and Kingella negevensis, as well as strictly commensal species. Both K. kingae and K. negevensis secrete a toxin called RtxA that is absent in the commensal species. Here we present a phylogenomic study of the genus Kingella, including new genomic sequences for 88 clinical isolates, genotyping of another 131 global isolates, and analysis of 52 available genomes. The phylogenetic evidence supports that the toxin-encoding operon rtxCA was acquired by a common ancestor of the pathogenic Kingella species, and that a preexisting type-I secretion system was co-opted for toxin export. Subsequent genomic reorganization distributed the toxin machinery across two loci, with 30-35% of K. kingae strains containing two copies of the rtxA toxin gene. The rtxA duplication is largely clonal and is associated with invasive disease. Assays with isogenic strains show that a single copy of rtxA is associated with reduced cytotoxicity in vitro. Thus, our study identifies key steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer, co-option of an existing secretion system, and gene duplication.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO–K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

Evaluation of DNA extraction methods for the detection of Shiga toxin producing Escherichia coli in food by polymerase chain reaction

Reported food-borne outbreaks of Shiga toxin producing Escherichia coli (STEC) have involved a very diverse range of foods. Contemporary analytical methods for the detection of STEC in foods typically include PCR screening of enrichment media. However, PCR inhibitors present in food enrichments can produce false negative results when screening for DNA sequences associated with the pathogen. To avoid false negative results in enrichment screening, it is advantageous to have DNA extraction methods that are effective at removing PCR inhibitors from a wide range of foods. The standard Canadian STEC method MFLP-52 uses Bio-Rad Instagene Matrix for DNA extraction. In this study, three DNA extraction protocols using commercial kits (Instagene Matrix with Beckman Coulter Ampure XP Beads; Qiagen Gentra Puregene Yeast/Bact. Kit; Qiagen DNeasy Blood & Tissue) were assessed as alternative DNA extraction methods for the detection of the Shiga toxin gene by PCR in enrichments from sixteen different foods inoculated with STEC O157. The inoculated foods were bean sprouts, blackberries, blue cheese, cilantro, cocoa powder, coleslaw, cream of mushroom dried soup mix, cream of vegetable dried soup mix, flaxseed, guacamole, peanut butter, soft cheese, soy butter, spinach, walnut, and wheat flour. Two of the protocols, Instagene Matrix with Ampure XP Beads, and Gentra Puregene Yeast/Bact, produced no false-negative or false positive results in the analysis of triplicate enrichment samples from sixteen inoculated foods.

A microcystin synthesis mcyE/ndaF gene assay enables early detection of microcystin production in a tropical wastewater pond

Cyanobacteria can dominate the algal community in wastewater ponds, which can lead to the production of cyanotoxins and their release into the environment. We applied traditional and molecular techniques to identify cyanotoxin hazards and high-risk periods in a tropical wastewater treatment system. Potentially toxic cyanobacteria were identified by microscopy and amplicon sequencing over the course of a year. Toxin gene levels were monitored and compared to toxin production to identify likely toxin producing species and high-risk periods. Cyanobacteria were persistent in the effluent year-round, with Planktothrix and Microcystis the most abundant genera; Microcystis could not be resolved beyond genus using amplicon sequencing, but M. flos-aquae was identified as a dominant species by microscopy. Microcystin toxin was detected for the first time in treated effluent at the beginning of the wet season (December 2018), which correlated with an increase in Microcystis amplicon sequence abundance and elevated microcystin toxin gene (mcyE/ndaF) levels. Concomitantly, microscopy data showed an increase in M. flos-aquae but not M. aeruginosa. These data informed a refined sampling campaign in 2019 and results showed a strong correlation between mcyE/ndaF gene abundance, microcystin toxin levels and Microcystis amplicon sequence abundance. Microscopy data showed that in addition to M. flos-aquae, M. aeruginosa was also abundant in February and March 2019, with highest levels coinciding with toxin detection and toxin gene levels. M. aeruginosa was the most abundant Microcystis species detected in selected treated effluent samples by metagenomics analysis, and elevated levels coincided with toxin production. All microcystin genes in the biosynthesis pathway were detected, but microcystin genes from Planktothrix agardhii were not detected.Gene toxin assays were successfully used to predict microcystin production in this wastewater system. Changes in amplicon sequence relative abundance were a useful indicator of changes in the cyanobacterial community. We found that metagenomics was useful not just for identifying the most abundant Microcystis species, but the detection of microcystin biosynthesis genes helped confirm this genus as the most likely toxin producer in this system. We recommend toxin gene testing for the early detection of potential toxin producing cyanobacteria to manage the risk of toxicity and allow the implementation of risk management strategies.

Multidrug-resistant phenotypes among biofilm-producing staphylococcal isolates from farm-raised fish: a molecular scrutiny.

To describe the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from farm-raised fish. Fish samples (n=180) collected from aquaculture farms in three major districts of Kerala, yielded 45 methicillin-resistant staphylococci (MRS) (25%) isolates. All of the isolates were resistant to tested beta-lactams, and 19 (42.22%) were resistant to flouroquinolones. According to the resistance profile of each isolate, 33 of 45 (73.33%) are identified as multidrug-resistant (MDR) isolates, with an average multiple antibiotic resistance (MAR) index of 0.41±0.09. Notably, virulence determinants such as classical enterotoxin (sea & seb), and SE-like toxin (seg & sei) genes co-existed with Antimicrobial Resistance Genes (ARGs) in isolates studied. Regardless of the isolates being strong biofilm producers, only 23 (51.1%) harbored icaA and icaD genes. The heterogeneity of MRSA (n=17) clones were evident as the species belonged to three major spa types (t345, t2526, and t1309) and sequence types (STs), viz., ST772, ST88, and ST672. The current study on molecular characterization of the MRS isolates sheds light on the preventive measures that need to be implemented to limit the spread of AMR pathogens in aquaculture.

Characterization of an Escherichia coli Isolate Coharboring the Virulence Gene astA and Tigecycline Resistance Gene tet(X4) from a Dead Piglet.

tet(X4) is the critical resistance gene for tigecycline degradation that has been continually reported in recent years. In particular, pathogenic bacteria carrying tet(X4) are a severe threat to human health. However, information describing Escherichia coli coharboring tet(X4) with virulence genes is limited. Here, we isolated an E. coli strain coharboring tet(X4) and the heat-stable toxin gene astA from a dead piglet. The strain named 812A1-131 belongs to ST10. The genome was sequenced using the Nanopore and Illumina platforms. The virulence genes astA and tet(X4) are located on the chromosome and in the IncHI1-type plasmid p812A1-tetX4-193K, respectively. The plasmid could be conjugatively transferred to recipient E. coli J53 with high frequency. In vivo experiments showed that strain 812A1-131 is pathogenic to Galleria mellonella and could colonize the intestines of mice. In summary, pathogenic E. coli could receive a plasmid harboring the tet(X4) gene, which can increase the difficulty of treatment. The prevalence and transmission mechanisms of pathogenic bacteria coharboring the tet(X4) gene need more attention.

Escherichia albertii as a Potential Enteropathogen in the Light of Epidemiological and Genomic Studies.

Escherichia albertii is a new enteropathogen of humans and animals. The aim of the study was to assess the prevalence and pathogenicity of E. albertii strains isolated in northeastern Poland using epidemiological and genomic studies. In 2015-2018, a total of 1154 fecal samples from children and adults, 497 bird droppings, 212 food samples, 92 water samples, and 500 lactose-negative E. coli strains were tested. A total of 42 E. albertii strains were isolated. The PCR method was suitable for their rapid identification. In total, 33.3% of E. albertii isolates were resistant to one antibiotic, and 16.7% to two. Isolates were sensitive to cefepime, imipenem, levofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and did not produce ESBL β-lactamases. High genetic variability of E. albertii has been demonstrated. In the PFGE method, 90.5% of the strains had distinct pulsotypes. In MLST typing, 85.7% of strains were assigned distinct sequence types (STs), of which 64% were novel ST types. Cytolethal distending toxin (CDT) and Paa toxin genes were found in 100% of E. albertii isolates. Genes encoding toxins, IbeA, CdtB type 2, Tsh and Shiga (Stx2f), were found in 26.2%, 9.7%, 1.7%, and 0.4% of E. albertii isolates, respectively. The chromosome size of the tested strains ranged from 4,573,338 to 5,141,010 bp (average 4,784,003 bp), and at least one plasmid was present in all strains. The study contributes to a more accurate assessment of the genetic diversity of E. albertii and the potential threat it poses to public health.

Antimicrobial resistance and virulence profiles of staphylococci isolated from clinical bovine mastitis.

Staphylococci, mainly including Staphylococcus aureus and coagulase-negative staphylococci (CNS), are one of the most common pathogens causing bovine mastitis worldwide. In this study, we investigated the antimicrobial resistance and virulence profiles of staphylococci from clinical bovine mastitis in Ningxia Hui Autonomous Region of China. Antimicrobial resistance was determined by disc diffusion combined with E-test method. Genes of antimicrobial resistance and virulence factors were determined by PCR. A total of 332 staphylococcal isolates were confirmed from 1,519 mastitic milk samples, including 172 S. aureus and 160 CNS isolates. Fifteen CNS species were identified, with S. chromogenes being the most frequent found (49.4%), followed by S. equorum (13.8%). Noticeably, 2 S. agnetis isolates were found among the CNS isolates. To our knowledge, this is the first report documenting the presence of S. agnetis from bovine mastitis in China. The S. aureus and CNS isolates showed high resistance against penicillin, followed by erythromycin and tetracycline. Multidrug resistance was found in 11.6 and 16.3% of the S. aureus and CNS isolates, respectively. Resistance to penicillin was attributed to the presence of blaZ, erythromycin resistance to ermC (alone or combined with ermB) and tetracycline resistance to tetK (alone or combined with tetM). Notably, one S. equorum isolate and one S. saprophyticus isolate were both methicillin-resistant and mecA positive. Additionally, all S. aureus isolates carried the adhesin genes fnbpA, clfA, clfB, and sdrC, and most of them contained cna and sdrE. Conversely, only a few of the CNS isolates carried clfA, cna, and fnbA. Regarding toxin genes, all S. aureus isolates harbored hlb, and most of them were hlg positive. The lukE-lukD, lukM, sec, sed, sei, sen, seo, tst, seg, seh, and sej were also detected with low frequencies. However, no toxin genes were observed in CNS isolates. This study reveals high species diversity of staphylococci from clinical bovine mastitis in Ningxia Hui Autonomous Region of China. The findings for the genetic determinants of antimicrobial resistance and virulence factor provide valuable information for control and prevention of staphylococcal bovine mastitis.

Establishment of a Novel Detection Platform for Clostridioides difficile Toxin Genes Based on Orthogonal CRISPR.

Clostridioides difficile is one of the leading pathogens causing nosocomial infection. The infection can range from mild to severe, and rapid identification is pivotal for early clinical diagnosis and appropriate treatment. Here, a genetic testing platform for toxins, referred to as OC-MAB (orthogonal CRISPR system combined with multiple recombinase polymerase amplification [RPA]), was developed to detect the C. difficile toxin genes tcdA and tcdB. While recognizing the amplified products of the tcdA gene and the tcdB gene, Cas13a and Cas12a could activate their cleavage activities to cut labeled RNA and DNA probes, respectively. The cleaved products were subsequently identified by dual-channel fluorescence using a quantitative PCR (qPCR) instrument. Finally, they could also be combined with labeled antibodies on immunochromatographic test strips to achieve visual detection. The OC-MAB platform exhibited ultrahigh sensitivity in detecting the tcdA and tcdB genes at levels of as low as 102 to 101 copies/mL. When testing 72 clinical stool samples, the sensitivity (95% confidence interval [CI], 0.90, 1) and specificity (95% CI, 0.84, 1) of the single-tube method based on the fluorescence readout was 100%, with a positive predictive value (PPA) value of 100% (95% CI, 0.90, 1) and a negative predictive value (NPA) value of 100% (95% CI, 0.84, 1), compared to the results of qPCR. Likewise, the sensitivity of the 2-step method based on the test strip readout was 100% (95% CI, 0.90, 1), while the specificity was 96.3% (95% CI, 0.79, 0.99), with a PPA of 98% (95% CI, 0.87, 0.99) and an NPA of 100% (95% CI, 0.90, 1). In short, orthogonal CRISPR technology is a promising tool for the detection of C. difficile toxin genes. IMPORTANCE C. difficile is currently the primary causative agent of hospital-acquired antibiotic-induced diarrhea, and timely and accurate diagnosis is crucial for hospital-acquired infection control and epidemiological investigation. Here, a new method for the identification of C. difficile was developed based on the recently popular CRISPR technology, and an orthogonal CRISPR dual system was utilized for the simultaneous detection of toxin genes A and B. It also uses a currently rare CRISPR dual-target lateral flow strip with powerful color-changing capabilities, which is appropriate for point-of-care testing (POCT).

Regulation of Clostridial Toxin Gene Expression: A Pasteurian Tradition.

The alarming symptoms attributed to several potent clostridial toxins enabled the early identification of the causative agent of tetanus, botulism, and gas gangrene diseases, which belongs to the most famous species of pathogenic clostridia. Although Clostridioides difficile was identified early in the 20th century as producing important toxins, it was identified only 40 years later as the causative agent of important nosocomial diseases upon the advent of antibiotic therapies in hospital settings. Today, C. difficile is a leading public health issue, as it is the major cause of antibiotic-associated diarrhea in adults. In particular, severe symptoms within the spectrum of C. difficile infections are directly related to the levels of toxins produced in the host. This highlights the importance of understanding the regulation of toxin synthesis in the pathogenicity process of C. difficile, whose regulatory factors in response to the gut environment were first identified at the Institut Pasteur. Subsequently, the work of other groups in the field contributed to further deciphering the complex mechanisms controlling toxin production triggered by the intestinal dysbiosis states during infection. This review summarizes the Pasteurian contribution to clostridial toxin regulation studies.

Genome engineering of Stx1-and Stx2-converting bacteriophages unveils the virulence of the dairy isolate Escherichia coli O174:H2 strain UC4224.

The past decade witnessed the emergence in Shiga toxin-producing Escherichia coli (STEC) infections linked to the consumption of unpasteurized milk and raw milk cheese. The virulence of STEC is primarily attributed to the presence of Shiga toxin genes (stx1 and stx2) carried by Stx-converting bacteriophages, along with the intimin gene eae. Most of the available information pertains to the "Top 7" serotypes associated with STEC infections. The objectives of this study were to characterize and investigate the pathogenicity potential of E. coli UC4224, a STEC O174:H2 strain isolated from semi-hard raw milk cheese and to develop surrogate strains with reduced virulence for use in food-related studies. Complete genome sequence analysis of E. coli UC4224 unveiled the presence of a Stx1a bacteriophage, a Stx2a bacteriophage, the Locus of Adhesion and Autoaggregation (LAA) pathogenicity island, plasmid-encoded virulence genes, and other colonization facilitators. In the Galleria mellonella animal model, E. coli UC4224 demonstrated high pathogenicity potential with an LD50 of 6 CFU/10 μL. Upon engineering E. coli UC4224 to generate single and double mutant derivatives by inactivating stx1a and/or stx2a genes, the LD50 increased by approximately 1 Log-dose in the single mutants and 2 Log-doses in the double mutants. However, infectivity was not completely abolished, suggesting the involvement of other virulence factors contributing to the pathogenicity of STEC O174:H2. Considering the possibility of raw milk cheese serving as a reservoir for STEC, cheesemaking model was developed to evaluate the survival of UC4224 and the adequacy of the respective mutants as reduced-virulence surrogates. All tested strains exhibited the ability to survive the curd cooking step at 48°C and multiplied (3.4 Log CFU) in cheese within the subsequent 24 h. These findings indicate that genomic engineering did not exert any unintended effect on the double stx1-stx2 mutant behaviour, making it as a suitable less-virulent surrogate for conducting studies during food processing.

A local-scale One Health genomic surveillance of Clostridioides difficile demonstrates highly related strains from humans, canines, and the environment.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation (tcdB, tcdA), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB- isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes (n=136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile. This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.

Supplementary Data to Koludarov et al Domain loss enabled evolution of novel functions in the snake three-finger toxin gene superfamily

Supplementary Data to Koludarov et al Domain loss enabled evolution of novel functions in the snake three-finger toxin gene superfamily

Stress Resistance and Virulence Gene Profiles Associated with Phylogeny and Phenotypes of Escherichia coli from Cattle

Seven serogroups of E. coli (Top seven E. coli) are frequently implicated in foodborne outbreaks in North America, largely due to their carriage of Shiga toxin genes (stx). This study aimed to profile resistance genes and virulence factors (VF), and their potential association with phylogeny and phenotypes of Top seven E. coli originating from cattle in Canada. 155 Top seven E. coli isolates previously characterized for heat and acid resistance and biofilm-forming ability were whole-genome sequenced and analyzed for phylogeny, VF, and stress resistance genes. The 155 E. coli strains belonged to six phylogroups: A (n = 32), B1 (n = 93), C (n = 3), D (n = 11), E (n = 15), and G (n = 1). Different phylogroups were clearly separated on the core genome tree, with strains of the same serotype closely clustered. The carriage of stx and the transmissible locus of stress tolerance (tLST), the extreme heat resistance marker, was mutually exclusive, in 33 and 15 genomes, respectively. A novel O84:H2 strain carrying stx1a was also identified. In total, 70, 41, and 32 VF, stress resistance genes and antibiotic resistance genes were identified. The stress resistance genes included those for metal (n = 29), biocides/acid (n = 4), and heat (n = 8) resistance. All heat resistance genes and most metal-resistance genes that were differentially distributed among the phylogroups were exclusively in phylogroup A. VF were least and most present in phylogroups A and D, respectively. No specific genes associated with acid resistance or biofilm formation phenotypes were identified. VF were more abundant (P < 0.05) in the non–biofilm-forming population and acid-resistant population.

Tea Polyphenols Inhibit the Activity and Toxicity of Staphylococcus aureus by Destroying Cell Membranes and Accumulating Reactive Oxygen Species.

Staphylococcus aureus can cause bacterial food intoxication and seriously affect human health. Tea polyphenols (TP) are a kind of natural, safe, and broad-spectrum bacteriostatic substances, with a wide range of bacteriostatic effects. In the study, we explored the possible bacteriostatic mode of TP. The minimum inhibitory concentration of TP against S. aureus was 64 μg/mL. Protein, DNA, and K+ leak experiments, fluorescence microscopy, and transmission electron microscopy suggested that TP disrupt cell membranes, leading to intracellular component loss. By studying the effect of TP on the toxicity of S. aureus, it was found that the expression levels of two toxin genes, coa and spa, were downregulated by 2.37 and 32.6, respectively. Furthermore, after treatment with TP, a large number of reactive oxygen species (ROS) were propagated and released, leading to oxidative stress in cells. We speculated that the bacteriostatic mechanism of TP may be through the destruction of the cell membrane and ROS-mediated oxidative stress. Meanwhile, the hemolysis activity proved the safety of TP. Our results suggested that TP may be a potential antimicrobial agent for food.

Investigation of virulence factors of shiga toxin-producing Escherichia coli strains isolated from sheep

All over the world, Escherichia coli (STEC), which produces Shiga toxin, is recognized as an important zoonotic pathogen. Ruminants, particularly cattle, are their main reservoirs, but the role of small ruminants in the epidemiology of human infections has not been extensively evaluated in many countries. In this study, it was aimed to determine the Shiga toxin subtypes of Stx1, Stx2, eae and ehxa genes of Shiga toxin producing isolates within Escherichia coli strains in sheep and to determine the comparative effect of variables such as race and sex on them. In this study, virulence genes of STEC strains and Shiga toxin subtypes were identified in 215 E. coli isolates isolated from 272 rectal swab from 12 different sheep herds in and around Ankara. Of the 215 isolates, only 19 (8.84%) Stx1, 3 (1.4%) Stx2, 5 (2.33%) eae and 9 (4.19%) ehxa genes, 13 (6%, 05) Stx1-Stx2-ehxa, 5 (2.33%) Stx1-eae -ehxa, 3 (1.4%) Stx2-eae-ehxa together and 1 (0.47%) Stx1- eae, 30 (13.95%) Stx1- ehxa, 1 (0.47%) Stx2- eae, 15 (6.98%) Stx2-ehxa and 10 (4.65%) eae – ehxa were detected together. Of the 101 (46.98%) isolates, none of the Stx1, Stx2, eae and ehxa genes were detected. In stx1 subtyping in sheep, a high rate (82.35%) of stx1c was isolated. In stx2 subtyping stx2f (%62,86), stx2c (%54,29), stx2b (%45,71), stx2d (%22,86), stx2h (%22,86) subtypes are more common in different combinations. Detected at a high rate. These data indicate the presence of STEC in sheep and their potential to cause serious infection in humans

Status of Bacterial Entomopathogens Used for Microbial Control of Arthropod Pests in Iran and Turkey

Bacterial entomopathogens, especially Bacillus thuringiensis (Bt), are the most popular entomopathogen in bacteria worldwide, thanks to their toxin genes and high virulence. In this context, to develop an alternative to chemical pesticides used to control agricultural and forest pest insects, Bt has been isolated from soils and many insects, and products in the form of formulations have been prepared. It is also known that Bt-based biopesticides are generally more specific and harmless to other organisms. The results demonstrate that countries have different conditions for releasing these organic products to the market and it is often difficult for scientists to obtain a license. This study discusses the current status of bacterial entomopathogens study in Iran and Turkey.

Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)-the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot-apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from -20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.