Gallinarum Strains Research Articles

Evaluation of mechanical transmission of Enterococcus spp., resistant to antibiotics, having flies as vectors of dissemination

The objective of this work was to evaluate the mechanical transmission of Enterococcus spp., resistant to antibiotics, using flies as dissemination vectors. In this work, 50 flies were captured, 25 in “La Llanada” community, in Cumaná and 25 others in the surroundings of the “Juan Otaola Rogliani” outpatient clinic and the central cemetery of Carúpano. The main families of flies captured in the state of Sucre were Muscidae, Calliphoridae and Sarcophagidae. The flies captured in Cumaná had fewer strains of Enterococcus spp., on the surface than those from Carúpano, while those from Cumaná were more colonized in the intestine than those from Carúpano. A total of 14 strains of Enterococcus were isolated, among which were E. faecium (6%), E. gallinarum (36%) and E. casseliflavus (58%). The susceptibility profile of E. casseliflavus strains is resistance to linezolid, tetracycline, erythromycin, rifampicin and intermediate susceptibility to fluoroquinolones; E. gallinarum strains were sensitive to rifampicin and tetracycline, and resistant to the other antibiotics. The only strain of E. faecium isolated in Carúpano, presented low level of resistance to vancomycin. It did not amplify for the glycopeptide resistance ligase genes vanA, vanB, vanD, vanE, or vanG. Antibiotypes IA and IV of E. caseliflavus were detected in both Carúpano and Cumaná.

Recombinant Salmonella gallinarum (S. gallinarum) Vaccine Candidate Expressing Avian Pathogenic Escherichia coli Type I Fimbriae Provides Protections against APEC O78 and O161 Serogroups and S. gallinarum Infection.

Avian pathogenic Escherichia coli (APEC) is one of the leading pathogens that cause devastating economic losses to the poultry industry. Type I fimbriae are essential adhesion factors of APEC, which can be targeted and developed as a vaccine candidate against multiple APEC serogroups due to their excellent immunogenicity and high homology. In this study, the recombinant strain SG102 was developed by expressing the APEC type I fimbriae gene cluster (fim) on the cell surface of an avirulent Salmonella gallinarum (S. gallinarum) vector strain using a chromosome-plasmid-balanced lethal system. The expression of APEC type I fimbriae was verified by erythrocyte hemagglutination assays and antigen-antibody agglutination tests. In vitro, the level of the SG102 strain adhering to leghorn male hepatoma (LMH) cells was significantly higher than that of the empty plasmid control strain, SG101. At two weeks after oral immunization, the SG102 strain remained detectable in the livers, spleens, and ceca of SG102-immunized chickens, while the SG101 strain was eliminated in SG101-immunized chickens. At 14 days after the secondary immunization with 5 × 109 CFU of the SG102 strain orally, highly antigen-specific humoral and mucosal immune responses against APEC type I fimbriae protein were detected in SG102-immunized chickens, with IgG and secretory IgA (sIgA) concentrations of 221.50 μg/mL and 1.68 μg/mL, respectively. The survival rates of SG102-immunized chickens were 65% (13/20) and 60% (12/20) after challenge with 50 LD50 doses of APEC virulent strains O78 and O161 serogroups, respectively. By contrast, 95% (19/20) and 100% (20/20) of SG101-immunized chickens died in challenge studies involving APEC O78 and O161 infections, respectively. In addition, the SG102 strain effectively provided protection against lethal challenges from the virulent S. gallinarum strain. These results demonstrate that the SG102 strain, which expresses APEC type I fimbriae, is a promising vaccine candidate against APEC O78 and O161 serogroups as well as S. gallinarum infections.

Strain and serovar variants of Salmonella enterica exhibit diverse tolerance to food chain-related stress.

Non-Typhoidal Salmonella (NTS) continues to be a leading cause of foodborne illness worldwide. Food manufacturers implement hurdle technology by combining more than one approach to control food safety and quality, including preservatives such as organic acids, refrigeration, and heating. We assessed the variation in survival in stresses of genotypically diverse isolates of Salmonella enterica to identify genotypes with potential elevated risk to sub-optimal processing or cooking. Sub-lethal heat treatment, survival in desiccated conditions and growth in the presence of NaCl or organic acids were investigated. S. Gallinarum strain 287/91 was most sensitive to all stress conditions. While none of the strains replicated in a food matrix at 4°C, S. Infantis strain S1326/28 retained the greatest viability, and six strains exhibited a significantly reduced viability. A S. Kedougou strain exhibited the greatest resistance to incubation at 60°C in a food matrix that was significantly greater than S. Typhimurium U288, S Heidelberg, S. Kentucky, S. Schwarzengrund and S. Gallinarum strains. Two isolates of monophasic S. Typhimurium, S04698-09 and B54Col9 exhibited the greatest tolerance to desiccation that was significantly more than for the S. Kentucky and S. Typhimurium U288 strains. In general, the presence of 12mM acetic acid or 14mM citric acid resulted in a similar pattern of decreased growth in broth, but this was not observed for S. Enteritidis, and S. Typhimurium strains ST4/74 and U288 S01960-05. Acetic acid had a moderately greater effect on growth despite the lower concentration tested. A similar pattern of decreased growth was observed in the presence of 6% NaCl, with the notable exception that S. Typhimurium strain U288 S01960-05 exhibited enhanced growth in elevated NaCl concentrations.

Analysis of antibiotic resistance gene cassettes in a newly identified Salmonella enterica serovar Gallinarum strain in Korea

Antimicrobial resistant pathogens are a global health threat driven by the indiscriminate use of antimicrobials. Antimicrobial resistance can be acquired by resistance genes encoded by mobile genetic elements. In this study, we identified a strain of Salmonella enterica serovar Gallinarum (SG4021) from an infected chicken in Korea and characterized the presence of resistance genes in its plasmid by whole genome sequencing. The sequence was then compared with that of a plasmid (P2) from strain SG_07Q015, the only other strain of S. Gallinarum isolated in Korea for which a genome sequence is available. The results revealed that both strains harbored nearly identical DNA carrying antibiotic resistance gene cassettes inserted into integron In2 of the transposable element Tn21, namely an aadA1 resistance gene conferring resistance to aminoglycosides and a sul1 resistance gene conferring resistance to sulfonamide. Interestingly, despite the presence of sul1 in SG4021, an antibiotic sensitivity test revealed that it was sensitive to sulfonamides. Further analysis revealed that this disparity was due to the insertion of a ~ 5 kb ISCR16 sequence downstream of the promoter driving sul1 expression in SG4021. Using various mutants, we showed that the insertion of ISCR16 blocked the expression of the sul1 gene from the upstream promoter. Therefore, the functionality of antimicrobial resistance genes determines phenotypic antimicrobial resistance.

PCR based early detection and antibiotic resistance pattern of Salmonella Gallinarum isolates from Pakistan poultry

The poultry industry in developing countries is still combating mortality and economic loss due to Salmonella contamination. Salmonella Gallinarum is a common pathogen of poultry birds, being the etiologic agent of fowl typhoid, which specifically infects adult birds via the oral-fecal route. Timely detection of S. Gallinarum in poultry flocks can allow early treatment intervention leading to a decrease in economic losses. Detection of S. Gallinarum is challenging, while its PCR-based detection is a promising strategy, however, due to its high genomic similarity with other commonly existing Salmonella spp., identification of S. Gallinarum from poultry samples with high specificity is still a challenge. The current study was conducted to isolate S. Gallinarum from different districts of Pakistan, assess their antibiotic susceptibility profile, and develop a method for its early detection. A total of 20 strains were isolated using buffer peptone water, selenite cysteine broth, and Xylose Lysine Tergitol-4 (XLT-4) agar supplemented with tergitol and characterized by biochemical procedures. The antibiotic sensitivity profile highlighted the highest resistance of isolates towards novobiocin and nalidixic acid, commonly used antibiotics in Pakistan Poultry production. The primers designed to amplify a unique genomic region of S. Gallinarum, showed successful detection of twenty S. Gallinarum strains, while no amplification with genomic DNA from other common Salmonella spp. The reported method can be utilized to detect S. Gallinarum from tissue samples of infected birds in a short time leading to early diagnosis and timely treatment intervention.

Gut colonization with vancomicyn-resistant enterococci among patients with hematologic malignancies

BackgroundVancomycin-resistant enterococci (VRE) are well known agents that colonize the gastrointestinal tract of immunocompromised patients, especially those with hematologic malignancies. The aim of the current study was to determine the incidence of and risk factors for colonization with VRE among patients with hematologic malignancies.MaterialsFor a nine-month period, all patients admitted to the Hematology ward at University Hospital in Pleven, Bulgaria who had hematologic malignancy and duration of hospitalization of more than 48 h were screened for colonization with VRE. The data collected from patients and their medical records during the entire hospital stay included: demographic characteristics, clinical information and information about all antimicrobials used. A longitudinal study was used to assesses the risk factors and statistical analysis was performed using SPSS version 27.0.ResultsA total of 119 patients were enrolled in the study. Colonization with VRE was established in 18 of them. One patient carried two species, resulting in a total of 19 VRE: 12 Enterococcus gallinarum, 4 Enterococcus casseliflavus, 2 Enterococcus faecium and 1 Enterococcus faecalis. VanA phenotype, with high-level resistance of vancomycin (MIC ≥ 256 μg/ml) and teicoplanin (MIC = 96 μg/ml), was demonstrated by one E. faecium, which carried vanA. The other E. faecium and E. faecalis expressed low-level resistance to vancomycin (MICs: 8 μg/ml and 12 μg/ml), susceptibility to teicoplanin (MICs = 0.5 μg/ml) and vanB was detected. All E. gallinarum and E. casseliflavus showed low-level resistance to vancomycin and susceptibility to teicoplanin. E. gallinarum strains were positive for vanC1 and E. casseliflavus for vanC2. Only two patients were colonized with vanA or vanB enterococci and the rest 16 were positive for vanC. The univariate analysis revealed that patient’s age (70–79 years; p = 0.025) and multiple myeloma (p = 0.001) are risk factors for VRE acquisition among the investigated patients. In addition, the multivariate analysis confirmed that patient’s age (70–79 years) is an independent risk factor for VRE colonization.ConclusionsOur results showed that 15.1% of patients with hematologic malignancies were colonized with VRE. There was a distinct prevalence of vanC enterococci. Among the analyzed risk factors, advanced age and multiple myeloma contributed to VRE acquisition.

Characterization of two-component system CitB family in Salmonella enterica serovar Gallinarum biovar Gallinarum

Salmonella enterica serovar Gallinarum biovar Gallinarum is an avian-adapted pathogen causing fowl typhoid and leading to enormous economic loss in the global poultry industry. Two-component systems (TCSs) are crucial for bacteria survival, virulence, sensing and responding to the environment. 23 pairs of TCSs classified into five families were found in S. Gallinarum strain 287/91, of which the CitB family contains three pairs of TCSs, namely CitA/CitB, DcuS/DcuR and DpiB/DpiA, whose functions remained unaddressed. Thus, four mutants of S. Gallinarum strain U20, ΔcitAB (Δcit), ΔdcuSR (Δdcu), ΔdpiBA (Δdpi) and ΔcitABΔdcuSRΔdpiBA (Δ3), were constructed. The results suggested that the CitB family did not affect the growth or the metabolic capacities tested, while different TCSs exerted various effects on biofilm formation and antimicrobial resistance against multiple drug classes. Furthermore, the CitB family negatively impacted the tolerance of environmental stress, contributing to compromised virulence in chicken embryos and in vivo survival of S. Gallinarum. Collectively, this research provided new knowledge of how the CitB family is involved in the pathogenicity of S. Gallinarum.

Draft Genome Sequences of Enterococcus faecium, Enterococcus gallinarum, and Lactococcus lactis Strains Isolated from a Mastitis-Infected Camel in Isiolo County, Kenya

We report the draft genome sequences and annotation of Enterococcus faecium, Enterococcus gallinarum, and Lactococcus lactis isolates that were recovered from a mastitis-infected camel in Isiolo County, Kenya. Collectively, these data provide an invaluable repository for data mining to support the development of a potential multicomponent mastitis subunit vaccine.

Prevalence and Genetic Analysis of Resistance Mechanisms of Linezolid-Nonsusceptible Enterococci in a Tertiary Care Hospital Examined via Whole-Genome Sequencing.

(1) Background: Linezolid plays an important role in the treatment of invasive infections caused by vancomycin-resistant enterococci after its introduction to clinical practice. However, a detailed examination of linezolid-nonsusceptible enterococci (LNSE) is required. In this study, we attempted to analyze the mechanisms of LNSE strains isolated from a tertiary care hospital. (2) Methods: From 2019 to 2020, 18 Enterococcus faecalis, 14 E. faecium, and 2 E. gallinarum clinical isolates were collected at Severance Hospital. Agar dilution was performed to evaluate precise linezolid minimum inhibitory concentrations (MICs). Short-read whole-genome sequencing (WGS) was used to analyze resistance determinants. (3) Results: The presence of the optrA gene was likely the primary resistance mechanism in these three species, typically demonstrating a MIC value of 8 μg/mL. The co-existence of the cfr(D) and poxtA2 gene was the second major mechanism, primarily predicting a phenotype showing intermediate susceptibility to linezolid. G2576U mutation on 23S rRNA was only found in E. faecium; it mediated the most significant increase in linezolid MIC. (4) Conclusion: This is the first report examining poxtA2-cfr(D) co-harboring clinical enterococcal isolates in Korea and demonstrating the poxtA EF9F6-harboring clinical E. gallinarum strain worldwide. The comparison with resistance-gene-containing fragments in the isolates obtained from different countries and different sources demonstrated the spread of linezolid-resistance genes and suggested the possibility of foodborne transmission.

Whole-Genome Sequence of a Clinical Enterococcus gallinarum Strain in a Positive Hemoculture from Siriraj Hospital, Thailand.

Enterococcus gallinarum is a rare causative agent of hospital-acquired bacteremia. Here, we reported the complete genome of E. gallinarum strain WKB01, which was directly isolated from a positive hemoculture in Siriraj Hospital, Thailand. Comprehensive analysis demonstrated the clinically relevant tetM gene presenting in its genome.

Safety and protective effects of an avirulent Salmonella Gallinarum isolate as a vaccine candidate against Salmonella Gallinarum infections in young chickens

Fowl typhoid is an important disease of chickens and turkeys, which is caused by Salmonella Gallinarum (S. Gallinarum). Vaccines with high levels of protective effects against fowl typhoid need to be developed for the poultry industry. In this study, a S. Gallinarum strain, named SG01, was isolated from a poultry farm in Mashan region of Wuxi City, China, and identified through biochemical tests and specific PCR amplifications. Then, safety evaluations of the SG01 strain were performed in young chickens. No clinical symptom including depression and diarrhea and gross lesion involved in the cardiac nodule, hepatic necrotic lesion and splenic necrotic lesion, was determined on fifteen-day-old chickens after immunization with 1 × 1010 CFU of the SG01 strain through the oral route. However, diarrhea symptoms and hepatic lesions were identified from chickens immunized with the commercial vaccine strain SG9R by the same dose and route. At 14 days post inoculation, SG01 strain was eliminated in the liver and spleen from SG01-immunized chickens, while the SG9R strain still could be identified from SG9R-immunized chickens. After challenge with the virulent S. Gallinarum strain, significant reduction of the morbidity rate was found in the SG01 immunized group (20 %) compared to the challenge group (100 %) according to signs scoring systems for clinical symptoms and gross lesions. Additionally, immunization with the SG01 strain could provide more than 8 weeks of protection periods against fowl typhoid. These results demonstrate the SG01 strain is avirulent to young chickens and might be safer compared to the SG9R strain. In addition, SG01 strain is a potential vaccine candidate against fowl typhoid in young chickens.

Molecular screening the emergence of vancomycin and gentamycin resistant enterococcus species in burn' patients in basrah government, Iraq

Emergence of multidrug resistance bacteria (MDR) in burn infections including Enterococcus yet to be managed and highlighted in most of hospitals in our region. This study included 200 clinical samples collected from wound, urine, stool and blood of 75 burn patients in Al-Faiha Teaching Hospital in Basrah, Iraq, between 2020 -2021. Based on Enterococci chromo agar, biochemical test and PCR for 16S rRNA gene, 50 isolates were identified as Enterococcus spp., involving 20 (40%) E. faecium, 14 (28%) E. faecalis, 7 (14%) E. gallinarum, 6 (12%) E. gilvus, 2 (4%) E. casseliflavus and 1 (2%) E. avium. All detected Enterococci were reported worldly as pathogenic bacteria to human. Six new local strains of Enterococcus were recorded in NCBI and the Gene bank as follow; Enterococcus gallinarum strain IraqYaHa5 and IraqYaHa19, Enterococcus faecium strain IraqYaHa23, IraqYaHa48 and IraqYaHa60, Enterococcus gilvus strain IraqYaHa50. Phylogenetic tree was constructed to all isolated Enterococci. The clinical isolates showed resistance up to nine antibiotics. However, enterococci isolated from healthy people during this study were 100% sensitive to six of those antibiotics. Duplex PCR was applied to detect vancomycin and gentamicin resistance genes. All resistant Enterococcus to gentamicin had harboured aph (3’).

Characterization and Comparative Genomic Analysis of a Highly Colistin-Resistant Chryseobacterium gallinarum: a Rare, Uncommon Pathogen

For the first time, we describe the whole genome of a yellow-pigmented, capsule-producing, pathogenic, and colistin-resistant Chryseobacterium gallinarum strain MGC42 isolated from a patient with urinary tract infection in India. VITEK 2 automated system initially identified this isolate as C. indologenes. However, 16S rRNA gene sequencing revealed that MGC42 shared 99.67% sequence identity with C. gallinarum–type strain DSM 27622. The draft genome of the strain MGC42 was 4,455,926 bp long with 37.08% Guanine-Cytosine (GC) content and was devoid of any plasmid. Antibiotic resistance, virulence, and toxin genes were predicted by implementing a machine learning classifier. Potential homologs of 340 virulence genes including hemolysin secretion protein D, metalloprotease, catalase peroxidases and autotransporter adhesins, type VI secretion system (T6SS) spike proteins, and 27 toxin factors including a novel toxin domain Ntox23 were identified in the genome. Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs of 110 transporter proteins were predicted that were in agreement with moderate efflux activity. Twelve antibiotic resistance genes including two potentially novel putative β-lactamase genes sharing low similarity with known β-lactamase genes were also identified in the genome of this strain. The strain MGC42 was also resistant to several classes of antibiotics along with carbapenems and polymyxin. We also identified mutations in the orthologs of pmrB (M384T) and lpxD (I66V) that might be responsible for colistin resistance. The MGC42 strain shared 683 core genes with other environmental and clinical strains of Chryseobacterium species. Our findings suggest that the strain MGC42 is a multidrug-resistant, virulent pathogen and recommend 16S rRNA gene sequencing to identify clinical specimens of Chryseobacterium species.

CRISPR/Cas9-Based Deletion of SpvB Gene From Salmonella gallinarum Leads to Loss of Virulence in Chicken.

Salmonella Gallinarum causes fowl typhoid in poultry leading to a huge economic loss to the poultry industry. The large virulence plasmid of S. gallinarum has been associated with various systemic infections in poultry. A five-gene spanning region (spvRABCD) of 7.8 kb on the large plasmid mainly confers virulence to the bacteria. However, the exact role of these genes in virulence has not been elucidated yet. SpvB exhibits delayed cell death by preventing actin polymerization followed by apoptosis during intracellular infection. The specific role of SpvB in causing the disease is not known yet. In the current study, the SpvB gene was deleted through CRISPR/Cas9 method from a large virulent plasmid of locally isolated S. gallinarum strain (SG18). The homology-directed repair method was used for complete deletion of SpvB gene using the modified pCas9 plasmid. The SpvB-deleted S. gallinarum strain (ΔSpvB_SG18), when tested for its virulence in broiler chicken showed no diseases signs and mortality. In addition, the avirulent strain does not affect the bird’s weight and was rapidly cleared from the liver after infection. However, it cleared from the intestine only after 4–5 days, which suggests that the ΔSpvB_SG18 strain is unable to invade from the intestine to the liver. This is the first study to report a complete gene deletion from the S. gallinarum virulent plasmid and its effect. This method will be useful for the deletion of virulent genes from S. gallinarum, to study their role in pathogenesis, and to prepare an effective vaccine strain for controlling fowl typhoid in poultry.

Detection of virulence factors, biofilm formation, and biogenic amine production in vancomycin‐resistant lactic acid bacteria (VRLAB) isolated from foods of animal origin

The objective of this study was to determine phenotypic and genotypic characteristics of virulence factors, biofilm and biogenic amine production properties of vancomycin-resistant 26 Enterococcus, 18 Pediococcus, and three Weisella strains. The most common virulence genes were observed to be hyl (80.77%, 21/26), ccf (38.46%, 10/26), gelE (30.77%, 8/26), and acm (30.77%, 8/26) in Enterococcus isolates, cylM (44.44%, 8/18) and gelE (33.33%, 6/18) were detected in Pediococcus isolates. Biofilm production was apparent in 97.87% of the investigated isolates. The epaB biofilm formation gene was observed in four Enterococcus isolates, and epaE was identified in one Enterococcus isolate. Tyramine production was detected only in Enterococcus gallinarum DYE22 strain while the tdc gene was also identified in this strain. The results of this study showed that the detection of high-level of virulence genes in VRLAB strains from food of animal origin is a concern for consumer health. Novelty impact statement The most common virulence factor gene in VRLAB strains was found to be hyl. Biofilm production was found to be very common in VRLAB strains. Tyrosine decarboxylation and tdc gene was detected only in one Enterococcus gallinarum strain.

Biodiversity, antibiotic resistance and virulence traits of Enterococcus species in artisanal dairy products

The presence, biodiversity, and virulence traits of enterococci were investigated in 426 artisanal dairy products. Enterococci were detected in 62.91% of the samples, which consisted of 10 diverse species. Enterococcus faecalis, Enterococcus faecium, and Enterococcus casseliflavus isolates contained all 7 virulence genes tested. Of the tested virulence genes, esp with 63.55% and cylA with 13.41% showed the highest and the lowest frequency, respectively. The frequency for efaA, asa1, gelE, ace and hyl was estimated at 62.10%, 61.80%, 61.22%, 49.56% and 31.19%, respectively. Most of E. faecalis, E. faecium, Enterococcus durans, and Enterococcus gallinarum strains contained all antibiotic resistance genes tested, as well as Tn916-like transposons. In addition, 100% of E. durans, E. casseliflavus, and E. gallinarum were resistant to vancomycin. As part of this study, 75.55% of Enterococcus isolates produced biofilm. In conclusion, artisanal dairy products can be a vehicle in transmitting multidrug-resistant and virulent enterococci to humans.

Prevalence of trichomonads in the cloaca of wild wetland birds in the Netherlands

ABSTRACT Severe granulomatosis in productive layer chickens due to Tetratrichomonas gallinarum strain 13/16632 infection occurred in 2013 and 2017 on farms situated in a wetland area in the Netherlands. We hypothesized that wetland birds could be the source of the infection. Therefore, a prevalence study on trichomonads was performed by analysing cloaca swabs of 526 birds belonging to 13 species of wetland birds. The number of birds sampled ranged from 1 to 275 per species. Birds were sampled at 15 locations in the Netherlands. DNA extracted from the cloaca swabs was subjected to nested PCR using trichomonad-specific primers targeting the internal transcribed spacer 1 (ITS1)–5.8S rRNA–ITS2 region followed by cloning and sequencing. In nine bird species, trichomonads were detected; the overall prevalence was 9% (47/526), while the prevalence in the five species for which a substantial number of birds were examined (at least 39 per species) ranged from 4% to 24%. Three trichomonad species were found: T. gallinarum, Trichomonas tenax and Simplicimonas sp. of which T. gallinarum dominated. The virulent T. gallinarum strain 13/16632 was not detected, but closely related strains were. Phylogenetic analysis revealed that all T. gallinarum isolates belonged to two clusters within lineage 15 of Tetratrichomonas lineages. All T. tenax isolates were identical and clustered with reference strain H95, while Simplicimonas sp. isolates showed large genetic diversity. Some isolates may represent a new species of the genus Simplicimonas. We conclude that trichomonads are widespread amongst wetland birds, raising the question, amongst others, of their relevance for commercial poultry. RESEARCH HIGHLIGHTS Trichomonads occur among wild wetland birds in the Netherlands. T. gallinarum is the dominant trichomonad species in the cloaca of wetland birds. Some T. gallinarum isolates are closely related to a strain causing granulomas in layer chickens. Some isolates may represent a new species of the genus Simplicimonas.

Genomic-based characterization of Enterococcus spp.: an emerging pathogen isolated from human gut.

Enterococci are ubiquitous microorganisms having diverse ecological niches but most prominently in gastrointestinal tract of humans and animals. Production of enterocins makes them a good probiotic candidate. However, their role as probiotics has become ambiguous in the last few years because of the presence of virulence factors and antibiotic resistance genes. These virulence traits are known to be transferred genetically, which makes them opportunistic pathogens in the gastrointestinal tract leading to serious concerns about their being used as probiotics. In the present study, Enterococcusspp. isolated from the human gut were subjected to Whole-Genome Sequencing (WGS) to determine the presence of resistance and virulence genes. Four human origins Enterococcus spp. including Enterococcus faecalis, Enterococcus casseliflavus, and two Enterococcus gallinarum were isolated from human fecal samples and further cultured on blood agar. Sanger sequencing was done using Applied Biosystems 3730xl DNA Analyzer. These strains were further subjected to WGS using oxford nanopore technology MinION. Raw data were analyzed using the free online tool epi2me. The Comprehensive Antibiotic Resistance Database (CARD) and RAST (Rapid Annotation using Subsystem Technology) software were used to look for the presence of antibiotic resistance genes in these strains. Resistance determinants for clinically important antibiotics (vancomycin) and functional virulence factor genes were detected. G-view server was used for comparative genomics of all strains. The genomic sequencing of Enterococcus suggested that E. faecalis, E. casseliflavus, and E. gallinarum strains are opportunistic pathogens, having antibiotic resistance genes. All isolates had vancomycin resistance genes, which were expressed phenotypically. Genes related to bacteriocin resistance were also present in E. casseliflavus and E. gallinarum.

Complete Genomic Analysis of VRE From a Cattle Feedlot: Focus on 2 Antibiotic Resistance.

Practices in intensive animal farming such as the extensive use of antimicrobials have significant impacts on the genetic make-up of bacterial communities, especially on that of human/animal commensals. In this report, whole genome sequencing of two vancomycin-resistant enterococci (VRE) isolates from a cattle feedlot in the North West Province, South Africa, was used to highlight the threats that extensive antimicrobial usage in intensive animal rearing represents for environmental microbiomes and the food chain. The genomic DNA of the studied strains was extracted using a DNA extraction kit. Whole-genome sequencing was performed through next-generation sequencing. The genomes of Enterococcus durans strain NWUTAL1 and Enterococcus gallinarum strain S52016 consisted of 3,279,618 and 2,374,946 bp, respectively with G + C contents of 40.76 and 43.13%, respectively. Antibiotic resistance genes (ARG), plasmids and virulence factors (involved in biofilm formation, colonization and copper/silver efflux system), were detected in the genomes of both strains. The presence of these genetic determinants in the studied strains is a cause for concern as they may disseminate and find their way into the food chain via horizontal gene transfer amongst bacteria of the different ecological niches. Issues of this nature cannot be undermined and are relevant as far as food safety is concerned.

Whole genome sequence of Enterococcus gallinarum EG81, a porcine strain harbouring the oxazolidinone-phenicol resistance gene optrA with chromosomal and plasmid location

ObjectiveThe aim of this study was to characterise the whole genome sequence of linezolid-intermediate Enterococcus gallinarum strain EG81 of swine origin in China. MethodsWhole genome of EG81 was sequenced using Illumina MiSeq platform combined with the Nanopore PromethION platform, and assembled de novo using Canu v1.5. NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to annotate the genome of EG81. Antimicrobial resistance genes were identified using CGE ResFinder 3.2. ResultsThe genome of EG81 consists of one 3,433,237-bp chromosome and two plasmids, pEG81-1 (51,632 bp) and pEG81-2 (3425 bp). A total of 3285 coding sequences and 80 RNA genes were predicted by PGAP. The oxazolidinone-phenicol resistance gene optrA is located on both the chromosome and plasmid pEG81-1 associated with Tn554 and Tn558, respectively. In addition, EG81 harbours vanC1XY (vancomycin resistance), fexA (phenicol), dfrG (trimethoprim), aadD, ant(6)-Ia and ant(9)-Ia (aminoglycoside), erm(A) and erm(B) (macrolide), and tet(L) and tet(M) (tetracycline). ConclusionHere, we first report the oxazolidinone-phenicol gene optrA in E. gallinarum that is intrinsically resistant to vancomycin, which poses a great threat to public health. The genome sequence of E. gallinarum EG81 provides valuable information for the dissemination of optrA among vancomycin-resistant enterococci.