Detection Of Virulence Genes Research Articles

Prevalence, antimicrobial resistance and detection of virulence genes of Escherichia coli and Salmonella spp. isolated from white-lipped peccaries and collared peccaries.

Salmonella spp. and Escherichia coli are implicated in human and animal infections and require antimicrobial treatment in many situations. Faecal samples of healthy white-lipped peccaries (Pecari tajacu) (n = 30) and collared peccaries (Tayassu pecari ) (n = 60) obtained in three farms located in the Midwest Brazil. The antimicrobial profiles of commensal E. coli from P. tajacu and T. pecari from commercial herds in Brazil were isolated and analyzed and virulence genes were detected. Among 90 healthy animals, no Salmonella spp. were isolated. However, 30 samples (27%) tested positive for E. coli, with 18 isolates from P. tajacu and 12 from T. pecari, representing frequencies of 58.0% and 38.7%, respectively. Additionally, other Enterobacteriaceae family bacteria were detected but not included in this analysis. However, individual samples from 30 animals tested positive for E. coli, of which 16 were isolated from P. tajacu presenting multidrug resistance and six were isolated from T. pecari presenting a similar pattern. The E. coli virulence genes detected were papC (pilus-associated pyelonephritis) in five isolates, tsh (temperature-sensitive hemagglutinin) in one isolate, and eae (enteric attachment and effacement) in one isolate. The serum resistance gene, iss (increased serum survival), was detected in four isolates. An association between these genes and the presence of hemolysin was also observed in one isolate. Thus, T. pecari and P. tajacu are potential reservoirs of pathogenic and multidrug-resistant and E. coli. Faecal E. coli of healthy P. tajacu and T. pecari could act as a possible reservoir of antimicrobial resistance genes in environment.

Experimental vaccination against Aeromonas hydrophila in Colossoma macropomum: Bacterial characterization, lethal doses, and mortality

Aeromonas hydrophila is one of the most important pathogens affecting Amazonian fish. In recent years, outbreaks in native aquaculture species displaying hemorrhages, epithelial ulcers, and damaged fins, which often lead to fatalities, have been reported. In the current study, we characterized A. hydrophila isolated from an outbreak on a commercial farm of Colossoma macropomum (gamitana), one of the main native species farmed in Peru and South America. Gram-negative, motile, oxidase-, and catalase-positive bacteria were retrieved from diseased C. macropomum. Aeromonas hydrophila was confirmed by biochemical and molecular characterization. Virulence genes detection showed that A. hydrophila (FO129–26) harbors cytotoxic enterotoxin (act), hemolysin (hly), serine protease (ser), lipase (lip), flagellin (fla), and elastase (ahyB) virulence genes. Then, lethal dose tests (LD10, 50, 90, 99) were performed and stablished at 4.6 × 106; 6.4 × 106; 8.9 × 106; and 1.11 × 107 CFU mL−1 confirming the virulence of the bacterial isolate. Finally, there were used three methods of bacteria inactivation to vaccine gamitanas against this bacterium. 180 gamitanas (C: control; FO: formaldehyde-inactivated group; PH: peroxide-inactivated group; CF: chloroform-inactivated group) were challenged with the pathogen. FO and PH groups had better survival rates (∼ 40 %) than the control group. There were no significant differences (p ≥ 0.05) between the FO and PH groups. Fish from CF presented early mortality after vaccination, probably due to irritation caused by chloroform residues. Finally, this study shows the virulence characteristics of A. hydrophila FO129–26 in an important farmed native fish in Peru and broadens the geographic range of the presence of this pathogen worldwide. Vaccines are a good strategy to improve the resistance of C. macropomum against A. hydrophila. However, further research is necessary to expand our comprehension of the efficacy of this protective technique in Amazonian fish.

The presence of virulence factor genes downregulates uterine AQP3 and alters glutathione peroxidase activity and uterine histopathology in canine pyometra.

Present study was designed to evaluate the role of virulence factor genes (papG, cnf1 and hylA) in the pathogenesis of canine pyometra. Antimicrobial susceptibility test and detection of virulence genes were performed Escherichia coli (E. coli) detected in uterine swab samples. Animals were divided into two groups based on the presence (VF+, n:14) or absence (VF-, n:7) of the virulence factor genes papG, cnf1 and hylA. Blood and tissue glutathione peroxidase activity, uterine histopathologic analysis and AQP3, ESR1, PGR, OXTR gene expressions were determined in both groups. Statistical analyses were performed using Stata version 15.1. All E. coli isolates were susceptible to amikacin, whereas resistant to ampicillin, amoxicillin/clavulanic acid and lincomycin. None of the isolates were susceptible to cefotaxime. E. coli isolates had at least one virulence gene. The most prevalent gene was fimH (100%), followed by fyuA (95.8%), usp (83.3%), sfa (75%), cnf1 and hlyA (70.8%) genes. Blood GPx activity was greater in VF+ animals. On the other hand, uterine tissue GPx activity was lower in VF+ group compared to the control group. Expression levels of AQP3 were upregulated more than fivefold in VF-dogs compared to the control group. In addition, AQP3 expression levels were found approximately threefold higher in VF (-) than VF (+) group (p < .05). Varying degree of inflammation noted for all animals with pyometra, but the presence of bacteria noted only in VF+ animals. In conclusion, the presence of virulence factor genes does not play a role in the histopathological degree of inflammation, the presence of bacteria was found to vary. Serum GPx activity increased in VF+ animals. While the hormone receptor expressions were similar, AQP expression was upregulated in the absence of virulence factor genes.

OCCURRENCE OF VIRULENCE GENES OF Staphylococcus aureus AND Escherichia coli IN VENDED FOODS IN SELECTED MOTOR-PARKS IN OBIO/AKPOR, RIVERS STATE

The study aimed at evaluating the microbiological safety of vended foods in selected motor parks in Obio/Akpor Local Government Area, Rivers State, Nigeria. Forty-eight samples obtained from four motor parks (Choba, Rumuokoro, Eliozu and Oil-mill Park) were analyzed using standard microbiological methods for total heterotrophic bacteria count, virulent and antibiotic resistant profiles of Staphylococcus aureus and Escherichia coli. Total heterotrophic bacteria count ranged from 4.60 to 8.39 log10CFU/g. A total of 10 (20.83%) samples were positive for S. aureus while 14 (29.17%) were positive for E. coli. Total staphylococcal and Escherichia coli counts ranged from 4.89 to 7.01 and 2.70 to 5.31 log10cfu/g, respectively. Both bacteria were 100% resistant to augmentin. The confirmed E. coli showed varying resistant to cefixime (50%), ceftazidime (42.86%), cefuroxim (14.29%) and ofloxacin (7.14%); while S. aureus also showed varying resistant to erythromycin (10.00%), ceftriaxone (90.00%), cloxacillin (100.00%) and gentamycin (0.00%) The confirmed E. coli produced the expected bands for the E. coli attaching and effusing (eaeA), aggregative adherence fimbriae (aggR) and antimicrobial sensitivity testing (astA) virulence genes while S. aureus did not produce the expected bands with staphylococcal enterotoxin A (sea) and staphylococcal enterotoxin B (seb) genes. The detection of virulence gene bearing E. coli and multiple antibiotic resistant E. coli and S. aureus portends danger for commuters who patronizes these foods, hence the need for urgent public health interventions.

Serotype distribution, virulence and antibiotic resistance of Streptococcus agalactiae isolated from cultured tilapia Oreochromis niloticus in Lake Volta, Ghana.

Streptococcus agalactiae infection is one of the major factors limiting the expansion of tilapia farming globally. In this study, we investigated the serotype distribution, virulence and antimicrobial resistance of S. agalactiae isolates from tilapia farmed in Lake Volta, Ghana. Isolates from 300 moribund fish were characterised by Gram staining, MALDI-TOF/MS and 16S rRNA sequencing. Serotype identification was based on multiplex polymerase chain reaction (PCR) amplification of the capsular polysaccharide genes. Detection of virulence genes (cfb, fbsA and cspA) and histopathology were used to infer the pathogenicity of the isolates. The susceptibility of isolates to antibiotics was tested using the Kirby-Bauer disk diffusion assay. All 32 isolates identified as S. agalactiae were of serotype Ia. This was notably different from isolates previously collected from the farms in 2017, which belonged to serotype Ib, suggesting a possible serotype replacement. The prevalence of the pathogen was related to the scale of farm operation, with large-scale farms showing higher S. agalactiae positivity. Data from histopathological analysis and PCR amplification of targeted virulence genes confirmed the virulence potential and ability of the isolates to cause systemic infection in tilapia. Except for gentamicin, the majority of the isolates were less resistant to the tested antibiotics. All isolates were fully sensitive to oxytetracycline, erythromycin, florfenicol, enrofloxacin, ampicillin and amoxicillin. This study has improved our understanding of the specific S. agalactiae serotypes circulating in Lake Volta and demonstrates the need for continuous monitoring to guide the use of antimicrobials and vaccines against streptococcal infections in Ghanaian aquaculture systems.

Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.

The Use of Nanomaterials to Detect Virulence Genes in Staphylococcus Aureus Isolated from Bullous Impetigo, Impetigo and Ecthyma Skin Infections

The Use of Nanomaterials to Detect Virulence Genes in Staphylococcus Aureus Isolated from Bullous Impetigo, Impetigo and Ecthyma Skin Infections

Antimicrobial Resistance Profiles of Enterococcus Faecalis and Enterococcus Faecium Isolated from Clinical Specimens at Kitwe Teaching Hospital in Zambia

This research examined the occurrence of Enterococcus faecalis and Enterococcus faecium in clinical samples processed at Kitwe Teaching Hospital Laboratory, along with their resistance to antimicrobials and presence of virulence determinants. A total of 230 clinical specimens were analyzed to identify suspected Enterococcus strains. Standard laboratory techniques were employed to isolate, characterize, and confirm the species of Enterococcus, followed by testing their susceptibility to antimicrobials and detection of resistance and virulence genes using polymerase chain reaction. Among the 230 cultured specimens, 89 were stool samples, 3 were swabs, and 138 were urine specimens. Out of these, 71 isolates were confirmed Enterococcus by PCR using genus specific primers. The resulting prevalence rate of 30.9% was thus obtained. The most prevalent species was Enterococcus faecalis, accounting for 56.3% of the 71 Enterococcus isolates. The prevalence of E. faecium was 14.1%. All tested E. faecalis isolates displayed resistance to ciprofloxacin and erythromycin. Furthermore, high rates of resistance were observed among E. faecalis isolates for chloramphenicol and tetracycline (97.5%), vancomycin (90.0%), nitrofurantoin (75.0%), penicillin (75.0%) and ampicillin (67.5%). Only two E. faecium isolates exhibited susceptibility to tetracycline and vancomycin, while the remaining isolates were 100% resistant to all other tested antimicrobials. Multidrug resistance was detected in all E. faecium isolates. In addition, various antibiotic resistance genes (aac(6′)-Ie-aph(2″)-LA, ermA, ermB, tetK, tetL, tetM, tetX, vanA) were identified in both E. faecalis and E. faecium isolates. A positive association between phenotype and genotype was found for tetracycline and erythromycin.

Molecular characterization of pathogenic Aeromonas jandaei bacteria isolated from cultured walking catfish (Clarias sp.)

Abstract. Mulia DS, Dwi NR, Suwarsito, Muslimin B. 2024. Molecular characterization of pathogenic Aeromonas jandaei bacteria isolated from cultured walking catfish (Clarias sp.). Biodiversitas 25: 1185-1193. Aeromonas spp. is pathogenic bacteria capable of causing fish disease, including walking catfish (Clarias sp.). In bacteria, virulence genes play a significant role in determining pathogenicity, while resistance genes influence resistivity. Therefore, this study aimed to determine molecular characterization of pathogenic Aeromonas jandaei bacteria isolated from walking catfish. The purposive sampling method was used to obtain diseased walking catfish from aquaculture ponds in Banyumas District, Central Java Province, Indonesia. The identification of bacteria was molecularly carried out based on 16S rDNA, followed by the detection of virulence genes on aer/haem, alt, ast, flaA, lafA, and fstA, as well as resistance genes on tet-A, strA-strB, and qnrA. The results of molecular analysis showed that three isolates from Singasari, namely BmSL-02, BmSL-04, and BmSL-07, were closely related to A. jandaei strain CDC0787-80 with a similarity rate and query coverage of 99.86% and 99%, respectively. Meanwhile, isolates from Cikawung, including BmCL-02, BmCL-05, and BmCL-07 were closely related to A. jandaei strain ATCC 49568. A total of six A. jandaei isolates had aer/haem virulence genes while alt, ast, and fstA were not found. BmSL-04 and BmCL-07 isolates had flaA gene, while four isolates including BmSL-02, BmSL-04, BmSL-07, and BmCL-07 had lafA gene. Additionally, BmCL-05 had tetA resistance gene, while the strA-strB and qnrA genes were not detected in all isolates. A. jandaei isolates were resistant to bacitracin, tetracycline, and gentamycin, but BmSL-04, BmSL-07, BmCL-02, and BmCL-05 were sensitive to chloramphenicol. This study provided valuable information on molecular characteristics of A. jandaei, serving as guidance in effective control and monitoring of isolates from Indonesia, specifically Banyumas, Central Java.

Exploring multi-antibiotic resistance in Arcobacter butzleri isolates from a poultry processing plant in northern Italy: An in-depth inquiry

Arcobacter butzleri is a foodborne pathogen that has been identified in various animal-derived foods, notably in poultry meat. The prevalence of this bacterium in poultry underscores the need for comprehensive investigations into its dissemination within poultry meat processing plants. Such assessments are critical due to the potential cross-contamination that may occur, impacting the safety of the final food product. This study endeavours to evaluate the genomic similarity of 56 A. butzleri isolates obtained from chicken carcasses (specifically neck skins and caecum, post-slaughter) and equipment surfaces following cleaning and sanitizing procedures. All samples originate from a poultry slaughterhouse in Northern Italy. The genomic analysis includes single nucleotide polymorphism, average nucleotide identity, and core genome multi-locus sequence typing analysis, aiming to discern potential cross-contamination between carcasses and equipment. The study evaluates antibiotic resistance, biofilm production, and host cell colonization of A. butzleri strains through in vitro assays. Genomic de-replication reveals the presence of 31 distinct strains. Results elucidate that a portion of these strains is evident in multiple sources, indicative of cross-contamination even post-cleaning and sanitization. The in vitro tests underline multi-antibiotic resistance of A. butzleri with genes associated with antibiotic resistance, among them mexAB-oprM, showing a correlation to the observed resistance. This study establishes the colonization capability of A. butzleri strains on a human gut mucus-secreting cell model, suggesting a potential virulence factor. Detection of various putative virulence genes further supports the hypothesis of strains virulence potential. This study holds importance of A. butzleri as it unveils the antibiotic resistance and pathogenic capabilities inherent in this species, identifying associated genetic traits. Through comprehensive genomic analysis, it was conclusively validated instances of cross-contamination between various sources and equipment surfaces, underscoring the pervasive dissemination of this bacterium within a food processing plant and its persistence. This research serves as an exploration into the intricate dynamics of A. butzleri within poultry processing environments. It accentuates the imperative for stringent hygiene protocols and ongoing surveillance to curtail the risk of cross-contamination, thereby ensuring the safety of poultry products for discerning consumers.

Rabbit meat as a potential source of multidrug-resistant and enterotoxigenic &lt;i&gt;Staphylococcus aureus&lt;/i&gt; strains

Staphylococcus aureus in rabbit meat is a consequence of insufficient hygienic handling and improper processing posing a major health hazard. This study was conducted to assess rabbit meat as a potential source of Staphylococcus species, particularly Staphylococcus aureus (S. aureus). Furthermore, the identified S. aureus isolates were tested for the detection of the mecA virulence gene of methicillin-resistant Staphylococcus aureus (MRSA) and enterotoxin encoding genes (Sea, Seb, Sec, and Sed). A total of 80 samples of different rabbit meat cuts represented by shoulder, ribs, loin, and thigh (20 of each) were collected from various markets of different sanitation levels. The results obtained revealed that the mean counts of Staphylococcus species were 7.40×105, 7.58×105, 7.60×105 and 8.29×105 CFU/g in the examined shoulder, ribs, loin and thigh samples, respectively. Out of 17 identified S. aureus isolates, 5 (29.4%) strains were characterized by the presence of the mecA gene. A large proportion of the isolates obtained were resistant to at least three antibiotics. Enterotoxins were evaluated by ELISA. The results showed that three strains isolated from shoulder produced Sea, Seb, and Sec enterotoxins, the strains isolated from ribs failed to produce enterotoxins, while two strains isolated from loin and thigh produced Sea enterotoxin. The presence of S. aureus, especially MRSA strains, in the examined rabbit meat indicates the necessity of enforced application of strict hygienic measurements.

Molecular detection of virulence genes for multi drug resistance Klebseilla pneumoniae in patients with urinary tract infections, Thi-Qar Provenance, Iraq

An agent that contributes to hospital acquired illnesses is Klebsiella pneumoniae. In community-acquired infections and illnesses of the urinary tract, it is the primary and most frequent causal agent. The strains were detected by using Analytical Profile Index (Api-20 E) and 16s rRNA techniques, fifteen trains of K. pneumoniae were obtained from eighteen urine samples collected from patients in Al-Hussein Hospital, Thi-Qar Governorate, Iraq. This research sought to identify the antibiotic resistance of K. pneumoniae to Aztreonam, Ceftriaxone, Trimethoprim, Amikacin, Ciprofloxacin, Meropenem, and Piperacillin, the resistance was high and ranged between (86.6-100)%. The multidrug resistance percentage was 100%, in addition the investigation aimed to detect some virulence genes, (i.e. HtrA and MrkA gene) in K. pneumoniae. The HtrA and MrkA genes were found using the PCR method, which revealed that 6 (40%) of the isolates had the HtrA gene and that all strains under study lacked the MrkA gene.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Multiple-Drug Resistant Shiga Toxin-Producing E. coli in Raw Milk of Dairy Bovine.

Raw milk may contain pathogenic microorganisms harmful to humans, e.g., multidrug-resistant Escherichia coli non-O157:H7, which can cause severe colitis, hemolytic uremia, and meningitis in children. No studies are available on the prevalence of Shiga toxin-producing E. coli (STEC O157:H7) in sick or healthy dairy animals in the Khyber Pakhtunkhwa Province of Pakistan. This study aimed to isolate, characterize, and detect antibiotic resistance in STEC non-O157:H7 from unpasteurized milk of dairy bovines in this province. We collected raw milk samples (n = 800) from dairy farms, street vendors, and milk shops from different parts of the Khyber Pakhtunkhwa Province. E. coli was isolated from these samples followed by latex agglutination tests for serotyping. The detection of STEC was conducted phenotypically and confirmed by the detection of virulence genes genotypically. An antibiogram of STEC isolates was performed against 12 antibiotics using the disc diffusion method. A total of 321 (40.12%) samples were found to be positive for E. coli in this study. These samples were processed for the presence of four virulence genes (Stx1, Stx2, ehxA, eae). Forty samples (5.0%) were STEC-positive. Of these, 38%, 25%, 19%, and 18% were positive for Stx1, Stx2, ehxA, and eae, respectively. Genotypically, we found that 1.37% of STEC isolates produced extended-spectrum beta-lactamase (ESBL) and contained the blaCTX M gene. Resistance to various antibiotics ranged from 18% to 77%. This study highlights the risk of virulent and multidrug-resistant STEC non-O157:H7 in raw milk and the need for proper quality surveillance and assurance plans to mitigate the potential public health threat.

Determination of Virulence Factors and Antibiotic Resistance Profile of E. coli O157:H7 in some Marine and Freshwater Fish from Samsun, Türkiye

In this study, Escherichia coli O157:H7 contamination was investigated in Engraulis encrasicolus, Merlangius euxmus, Trachurus mediterraneus, Mullus barbutus, Tinca tinca, Cyprinus carpio and Oncorchynhus mykiss (total of 300 samples). The presence of certain virulence genes such as shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eaeA), and hemolysin (hlyA) was determined. The E-test method was employed to determine the antibiotic resistance profile and minimum inhibitory concentration (MIC) values of the isolates. Out of a total 141 freshwater fish samples, E. coli O157:H7 was detected in 5 of them (3.5%). In 159 marine fish, 2 of them (1.25%) were E. coli O157 positive. In terms of virulence characteristics, the E. coli O157:H7 isolate obtained from the Oncorhynchus mykiss sample had the stx1, eaeA, and hlyA genes. Both eaeA and hlyA genes were determined as positive in the E. coli O157 isolate identified from the Merlangius euxmus sample. Resistance to tetracycline, cefotaxime, cefepime, ciprofloxacin, gentamicin, ertapenem and cefoxitin was demonstrated in both samples. In this study, the presence of E. coli O157 in fish, detection of virulence genes and antibiotic resistance profile indicate the potential risk of infection from contaminated seafood.

Detection of Pathogenic Serogroups and Virulence Genes in Listeria monocytogenes Strains Isolated from Beef and Beef Products Retailed in Gauteng Province, South Africa, Using Phenotypic and Polymerase Chain Reaction (PCR)-Based Methods.

South Africa recently (2017-18) experienced the largest outbreak of human listeriosis in the world caused by L. monocytogenes following the consumption of "polony," a ready-to-eat meat product. Most (59%) cases originated from Gauteng province, South Africa. As a follow-up study to the outbreak, we used standard bacteriological and molecular methods to determine the prevalence of pathogenic and virulent serogroups of L. monocytogenes in various beef and beef products retailed in Gauteng province, South Africa. The overall prevalence of Listeria spp. was 28% (112/400), comprising Listeria monocytogenes (9.3%), Listeria innocua (16.3%), and Listeria welshimeri (2.5%) (p < 0.001). It is crucial to have detected that the region (p=0.036), type of product (p=0.032), and temperature at storage (p=0.011) significantly affected the occurrence of L. monocytogenes in beef products. It is alarming that pathogenic serogroups 4b-4d-4e (51.4%) and 1/2a-3a (43.2%) were detected among the isolates of L. monocytogenes. Importantly, they were all carriers of seven virulence-associated genes (hlyA, inlB, plcA, iap, inlA, inlC, and inlJ). Our study also demonstrated that 16.7% of "polony" samples investigated were contaminated with L. monocytogenes. Considering that pathogenic and virulent L. monocytogenes contaminated beef and beef products retailed in South Africa, the food safety risk posed to consumers remains and cannot be ignored. Therefore, it is imperative to reduce the contamination of these products with L. monocytogenes during beef production, processing, and retailing to avoid future outbreaks of human listeriosis in the country.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Exploring Aeromonas dhakensis in Aldabra giant tortoises: a debut report and genetic characterization

Aeromonas dhakensis (A. dhakensis) is becoming an emerging pathogen worldwide, with an increasingly significant role in animals and human health. It is a ubiquitous bacteria found in terrestrial and aquatic milieus. However, there have been few reports of reptile infections. In this study, a bacterial strain isolated from a dead Aldabra giant tortoise was identified as A. dhakensis HN-1 through clinical observation, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF/MS), and gene sequencing analysis. Subsequently, to evaluate its pathogenicity, the detection of virulence genes and mice infection experiments were performed. A. dhakensis HN-1 was found to contain seven virulence genes, including alt, ela, lip, act, aerA, fla, and hlyA. Mice infected with A. dhakensis HN-1 exhibited hemorrhage of varying degrees in multiple organs. The half-maximal lethal dose (LD50) value of A. dhakensis HN-1 for mice was estimated to be 2.05 × 107 colony forming units (CFU)/mL. The antimicrobial susceptibility test revealed that A. dhakensis HN-1 was resistant to amoxicillin, penicillin, ampicillin and erythromycin. This is the first report of A. dhakensis in Aldabra giant tortoises, expanding the currently known host spectrum. Our findings emphasize the need for One Health surveillance and extensive research to reduce the spread of A. dhakensis across the environment, humans, and animals.

Whole Genome Sequencing Reveals Antimicrobial Resistance and Virulence Genes of Both Pathogenic and Non-Pathogenic B. cereus Group Isolates from Foodstuffs in Thailand.

Members of the Bacillus cereus group are spore-forming Gram-positive bacilli that are commonly associated with diarrheal or emetic food poisoning. They are widespread in nature and frequently present in both raw and processed food products. Here, we genetically characterized 24 B. cereus group isolates from foodstuffs. Whole-genome sequencing (WGS) revealed that most of the isolates were closely related to B. cereus sensu stricto (12 isolates), followed by B. pacificus (5 isolates), B. paranthracis (5 isolates), B. tropicus (1 isolate), and "B. bingmayongensis" (1 isolate). The most detected virulence genes were BAS_RS06430, followed by bacillibactin biosynthesis genes (dhbA, dhbB, dhbC, dhbE, and dhbF), genes encoding the three-component non-hemolytic enterotoxin (nheA, nheB, and nheC), a gene encoding an iron-regulated leucine-rich surface protein (ilsA), and a gene encoding a metalloprotease (inhA). Various biofilm-associated genes were found, with high prevalences of tasA and sipW genes (matrix protein-encoding genes); purA, purC, and purL genes (eDNA synthesis genes); lytR and ugd genes (matrix polysaccharide synthesis genes); and abrB, codY, nprR, plcR, sinR, and spo0A genes (biofilm transcription regulator genes). Genes related to fosfomycin and beta-lactam resistance were identified in most of the isolates. We therefore demonstrated that WGS analysis represents a useful tool for rapidly identifying and characterizing B. cereus group strains. Determining the genetic epidemiology, the presence of virulence and antimicrobial resistance genes, and the pathogenic potential of each strain is crucial for improving the risk assessment of foodborne B. cereus group strains.

Phylogenomic and phenotypic analyses highlight the diversity of antibiotic resistance and virulence in both human and non-human Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that causes infections in the immunocompromised. With a high incidence of muti-drug resistance, carbapenem-resistant A. baumannii is designated as a priority 1 pathogen by the WHO. The current literature has expertly characterized clinical isolates of A. baumannii. As the challenge of these infections has recently been classified as a One Health issue, we set out to explore the diversity of isolates from human and non-clinical sources, such as agricultural surface water, urban streams, various effluents from wastewater treatment plants, and food (tank milk); and, importantly, these isolates came from a wide geographic distribution. Phylogenomic analysis considering almost 200 isolates showed that our diverse set is well-differentiated from the main international clones of A. baumannii. We discovered novel sequence types in both hospital and non-clinical settings and five strains that overexpress the resistance-nodulation-division efflux pump adeIJK without changes in susceptibility reflected by this overexpression. Furthermore, we detected a bla ADC-79 in a non-human isolate despite its sensitivity to all antibiotics. There was no significant differentiation between the virulence profiles of clinical and non-clinical isolates in the Galleria mellonella insect model of virulence, suggesting that virulence is neither dependent on geographic origin nor isolation source. The detection of antibiotic resistance and virulence genes in non-human strains suggests that these isolates may act as a genetic reservoir for clinical strains. This endorses the notion that in order to combat multi-drug-resistant infection caused by A. baumannii, a One Health approach is required, and a deeper understanding of non-clinical strains must be achieved.IMPORTANCEThe global crisis of antibiotic resistance is a silent one. More and more bacteria are becoming resistant to all antibiotics available for treatment, leaving no options remaining. This includes Acinetobacter baumannii. This Gram-negative, opportunistic pathogen shows a high frequency of multi-drug resistance, and many strains are resistant to the last-resort drugs carbapenem and colistin. Research has focused on strains of clinical origin, but there is a knowledge gap regarding virulence traits, particularly how A. baumannii became the notorious pathogen of today. Antibiotic resistance and virulence genes have been detected in strains from animals and environmental locations such as grass and soil. As such, A. baumannii is a One Health concern, which includes the health of humans, animals, and the environment. Thus, in order to truly combat the antibiotic resistance crisis, we need to understand the antibiotic resistance and virulence gene reservoirs of this pathogen under the One Health continuum.