Global spread of the linezolid-resistant Enterococcus faecalis ST476 clonal lineage carrying optrA.

OBJECTIVES: To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage. CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.

Enterococcus cecorum lesion strains are less sensitive to the hostile environment of albumen and more resistant to lysozyme compared to cloaca strains.

RESEARCH HIGHLIGHTS: Egg albumen inhibits Enterococcus cecorum cloaca strains more than lesion strains.Enterococcus cecorum lesion strains are resistant to high concentrations of lysozyme.Lysozyme resistance could enhance survival in albumen and body fluids.

Membrane vesicles derived from Enterococcus faecalis promote the co-transfer of important antibiotic resistance genes located on both plasmids and chromosomes.

BACKGROUND: Bacterial membrane vesicles (BMVs) are novel vehicles of antibiotic resistance gene (ARG) transfer in Gram-negative bacteria, but their role in the spread of ARGs in Gram-positive bacteria has not been defined. The purpose of this study was to evaluate the role of MVs in the transmission of antimicrobial resistance in Gram-positive bacteria. METHODS: A linezolid-resistant Enterococcus faecalis CQ20 of swine origin was selected as the donor strain. Linezolid-susceptible E. faecalis SC032 of human origin, Enterococcus faecium BM4105 and Escherichia coli were selected as recipient strains. The presence of plasmids (pCQ20-1 and pCQ20-2) and an optrA-carrying transposon Tn6674 in CQ20, MVs and vesiculants was verified by WGS or PCR. MVs were isolated with density gradient centrifugation, and MV-mediated transformation was performed to assess the horizontal transferability of MVs. The MICs for CQ20 and its vesiculants were determined by the broth microdilution method. RESULTS: CQ20-derived MVs (CQ20-MV) were isolated, and PCR identified the presence of two plasmids and the optrA gene in the CQ20-MVs. MV-mediated transformation to E. faecalis SC032 and E. faecium BM4105 was successfully performed, and the WGS data also showed that both plasmids pCQ20-1 and pCQ20-2 and optrA-carrying transposon Tn6674 were transferred to E. faecalis SC032 and E. faecium BM4105, but failed for E. coli. Additionally, vesiculants that had acquired ARGs still had the ability to spread these genes via MVs. CONCLUSIONS: To our knowledge, this is the first report of MV-mediated co-transfer of ARG-carrying plasmids and transposons in the Gram-positive bacterium E. faecium.

Antimicrobial resistance and whole genome sequencing of novel sequence types of Enterococcus faecalis, Enterococcus faecium, and Enterococcus durans isolated from livestock.

The emergence of antimicrobial-resistant, livestock-associated Enterococcus faecalis represents a public health concern. Here, we report the isolation, molecular detection of virulence and antimicrobial resistance determinants, in addition to the phylogenetic analyses of 20 Enterococcus species using whole genome sequencing analysis of 15 Enterococcus faecalis strains including six strains of three novel sequence types, three Enterococcus faecium and two Enterococcus durans. All strains were isolated from food chain animals in South Africa. Enterococcus strains were isolated on bile aesculin azide agar, followed by identification using MALDI-TOF MS analysis. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method. The genomic DNA of the isolates was extracted and sequencing was performed using the Illumina MiSeq platform. Sequence reads were trimmed and de novo assembled. The assembled contigs were analyzed for antimicrobial resistance genes and chromosomal mutations, extra-chromosomal plasmids, and multi-locus sequence type (MLST). Multidrug antimicrobial resistance genes conferring resistance to aminoglycosides (ant(6)-Ia, aph(3')-IIIa, sat4, and spw), lincosamides (lnu(B), lsa(A), and lsa(E)), macrolides (erm(B)), trimethoprim (dfrG) and tetracyclines (tet(L) and tet(M)) were identified. Plasmid replicons were detected in seven E. faecalis and three E. faecium isolates. The sequence type (ST) of each isolate was determined using the Enterococcus PubMLST database. Ten STs were identified in the collection, three of which (ST1240, ST1241, and ST1242) have not been previously reported and are described in the present study for the first time. To compare the sequenced strains to other previously sequenced E. faecalis strains, assembled sequences of E. faecalis from livestock were downloaded from the PubMLST database. Core genome-based phylogenetic analysis was performed using ParSNP. The detection of multiple drug-resistance in Enterococcus including E. faecalis and E. faecium highlights the significance of genomic surveillance to monitor the spread of antimicrobial resistance in food chain animals. In addition, the genome sequences of Enterococcus strains reported in the present study will serve as a reference point for future molecular epidemiological studies of livestock-associated and antibiotic-resistant E. faecalis in Africa. In addition, this study enables the in-depth analysis of E. faecalis genomic structure, as well as provides valuable information on the phenotypic and genotypic antimicrobial resistance, and the pathogenesis of livestock-associated E. faecalis and E. faecium.

Molecular characterization of tetracycline and vancomycin-resistant Enterococcus faecium isolates from healthy dogs in Egypt: a public health threat.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are among the most common causative pathogens for nosocomial infections worldwide. Moreover, strains of VRE have been isolated from several domestic livestock in Egypt. METHODS: This study examined if healthy dogs are a potential source of VRE infection by isolating and characterizing Enterococcus faecium strains from stool samples on a morphological basis and biochemical activities. Subsequently, it was confirmed by genotypic characterization using polymerase chain reaction (PCR), followed by the detection of antibiotic resistance genes, virulence determinants, and genes contributing to enterocin production by PCR. Furthermore, the phylogenetic relationships among vanB and tetL genes were analyzed. RESULTS: All ten fecal samples were identified as E. faecium and confirmed by PCR. In addition, 90% of the isolates tested were positive for the virulence genes gelE and esp, and all the isolates tested were positive for the antibiotic resistance genes tetL and vanB. Only three of the five enterocin genes examined were detected. Ent As-48, bacteriocin 31, and Ent L50 were identified in 100%, 80%, and 60% of the samples, respectively. CONCLUSION: Dogs should be regarded as a reservoir of E. faecium that carries vancomycin resistance and virulence determinants that may affect public health in Egypt, considering a "One Health" task force approach to restrict their spread.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Characterization of an Emerging Enterococcus cecorum Outbreak Causing Severe Systemic Disease with Concurrent Leg Problems in a Broiler Integrator in the Southern United States.

Enterococcus cecorum has been associated mainly with osteomyelitis of the free thoracic vertebra in chickens. However, there are reports of E. cecorum producing septicemic lesions and having tropism for cartilages, resulting in the presentation of femoral head necrosis and synovitis. This paper discusses the presentation of E. cecorum as it relates to an outbreak in one vertical integrator where the main lesions were related to septicemia. Using a convenience sampling method, 100 broiler chicken cases received at the Poultry Research and Diagnostic Laboratory of Mississippi State University from April to December of 2021 were analyzed. The peak in cases was observed from June to August. The average age of broilers was 21 days with a range of 15-31 days. Most of these cases were related to systemic disease and leg problems, with gross lesions including characteristic pericarditis along with perihepatitis, osteomyelitis, and arthritis. In six of the 100 cases, E cecorum was isolated from the free thoracic vertebra, with the remaining being recovered from various other locations including liver, pericardium, hock/joint, femoral head, and bone marrow. Enterococcus cecorum identification was performed by using Vitek matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These results were then sent to the research-use only SARAMIS database for analysis. Once the spectra of the isolates were imported, the relative and absolute taxonomy were analyzed. Two super spectrums and three clusters by homology were identified. The minimal inhibitory concentrations obtained by antimicrobial sensitivity tests were analyzed using WHONET Microbiology Laboratory Database Software. No isolates were pan-susceptible, 80% of isolates were noted to be resistant to ≥3 classes of antibiotics and, in general, isolates exhibited a high degree of variability when examining antimicrobial resistance patterns.

Reporte de caso- Caracterización de un brote reciente de Enterococcus cecorum causante de una enfermedad sistémica grave simultáneamente con problemas en las patas en un integrador de pollo de engorde en el sur de los Estados Unidos. La bacteria Enterococcus cecorum se ha asociado principalmente con osteomielitis de la vértebra torácica móvil de los pollos. Sin embargo, existen reportes de E. cecorum produciendo lesiones septicémicas y presentando tropismo por los cartílagos, resultando en la presentación de necrosis de la cabeza femoral y sinovitis. Este artículo analiza la presentación de E. cecorum relacionada con un brote en un integrador vertical donde las principales lesiones estaban relacionadas con septicemia. Utilizando un método de muestreo de conveniencia, se analizaron 100 casos de pollos de engorde recibidos en el Laboratorio de Investigación y Diagnóstico Avícolas de la Universidad Estatal de Mississippi de abril a diciembre del 2021. El mayor número de casos se observó de junio a agosto. La edad promedio de los pollos de engorde fue de 21 días con un rango de 15 a 31 días. La mayoría de estos casos estaban relacionados con enfermedad sistémica y problemas en las patas, con lesiones macroscópicas que incluían pericarditis característica junto con perihepatitis, osteomielitis y artritis. En seis de los 100 casos, E cecorum se aisló de la vértebra torácica móvil, y el resto de los casos se recuperó de otros lugares, incluyendo el hígado, el pericardio, articulación del corvejón, la cabeza femoral y la médula ósea. La identificación de E. cecorum se realizó utilizando la plataforma Vitek de espectrometría de masas MALDI-TOF. Posteriormente, estos resultados se enviaron a la base de datos SARAMIS de uso exclusivo para investigación para su análisis. Una vez importados los espectros de los aislados, se analizó la taxonomía relativa y absoluta. Se identificaron dos superespectros y tres grupos mediante homología. Las concentraciones inhibitorias mínimas obtenidas mediante pruebas de sensibilidad antimicrobiana se analizaron utilizando el software de base de datos de laboratorio de microbiología de WHONET. Ningún aislamiento fue pan-susceptible, se observó que el 80% de los aislamientos eran resistentes a tres o más clases de antibióticos y en general, los aislamientos exhibieron un alto grado de variabilidad al examinar los patrones de resistencia a los antimicrobianos.

Genomic insights into linezolid-resistant Enterococci revealed its evolutionary diversity and poxtA copy number heterogeneity.

OBJECTIVES: This study aimed to determine the molecular mechanisms of linezolid-resistant enterococci (LRE) in swine slaughterhouses in China and apply the "One Health" perspective to analyse the evolutionary dynamics of poxtA-positive E. faecium in clinical and non-clinical settings worldwide. METHODS: The phenotypic and genomic characteristics of multiple LRE isolates were systematically investigated using antimicrobial susceptibility testing, transfer assays, evolutionary experiments, quantitative RT-PCR assays, whole-genome sequencing, and bioinformatics analyses. RESULTS: Swine faeces served as a significant reservoir for LRE isolates, and optrA and poxtA were the primary contributors to linezolid resistance. Co-occurrence network analysis revealed a significant interconnection between optrA and several other ARGs. The poxtA copy number heterogeneity and polymorphism were initially observed in E. faecium parental and evolved isolates. The poxtA-carrying tandem repeat region exhibits high mobility and has undergone extensive duplication owing to linezolid pressure. The poxtA copy number varies from four copies on the plasmid of E. faecium IC25 to 11 copies on the plasmid and six copies on the chromosome in the evolved isolate IC25-50_poxtA. Furthermore, phylogenetic analysis of 185 poxtA-positive E. faecium strains worldwide found that one isolate from a French patient in 2018 shared only two SNPs with CC17 E. faecium isolates IC25 and IC7-2 from this study, highlighting the potential global transmission of CC17 poxtA-positive E. faecium between humans and animals. CONCLUSION: This study identified amplification of poxtA as a response of E. faecium to linezolid pressure. Phylogenetic analysis shed light on the potential global transmission of hospital-associated CC17 poxtA-positive E. faecium in clinical and non-clinical settings.

Identification of plasmids co-carrying cfr(D)/optrA and cfr(D2)/poxtA linezolid resistance genes in two Enterococcus avium isolates from swine brain.

Oxazolidinones are critically important antibiotics to treat human infections caused by multidrug-resistant bacteria, therefore the occurrence of linezolid-resistant enterococci from food-producing animals poses a serious risk to human health. In this study, Enterococcus avium 38157 and 44917 strains, isolated from the brain of two unrelated piglets, were found to carry the linezolid resistance genes cfr(D)-optrA, and cfr(D2)-poxtA, respectively. Whole genome sequencing analysis of E. avium 38157 revealed that the genes were co-located on the 36.5-kb pEa_cfr(D)-optrA plasmid showing high identity with the pAT02-c of Enterococcus faecium AT02 from pet food. The optrA region, was 99% identical to the one of the pAv-optrA plasmid from a bovine Aerococcus viridans strain, whereas the cfr(D) genetic context was identical to that of the plasmid 2 of E. faecium 15-307.1. pEa_cfr(D)-optrA was not transferable to enterococcal recipients. In E. avium 44917 a cfr(D)-like gene, named cfr(D2), and the poxtA gene were co-located on the transferable 42.6-kb pEa-cfr(D2)-poxtA plasmid 97% identical to the Tn6349 transposon of the human MRSA AOUC-0915. The cfr(D2) genetic context, fully replaced the Tn6644 that in S. aureus AOUC-0915 harbor the cfr gene. In conclusion, this is, the best of our knowledge, the first report of the new cfr(D2) gene variant. The occurrence of plasmids co-carrying two linezolid resistance genes in enterococci from food-producing animals needs close surveillance to prevent their spread to human pathogens.

Assessment of antibiotic resistance and biofilm formation of Enterococcus species isolated from different pig farm environments in Poland.

BACKGROUND: Enteroccocus spp. are human opportunistic pathogens causing a variety of serious and life-threating infections in humans, including urinary tract infection, endocarditis, skin infection and bacteraemia. Farm animals and direct contact with them are important sources of Enterococcus faecalis (EFA) and Enterococcus faecium (EFM) infections among farmers, veterinarians and individuals working in breeding farms and abattoirs. The spread of antibiotic-resistant strains is one of the most serious public health concerns, as clinicians will be left without therapeutic options for the management of enterococcal infections. The aim of the study was to evaluate the occurrence and antimicrobial susceptibility of EFA and EFM strains isolated from a pig farm environment and to determine the biofilm formation ability of identified Enterococcus spp. strains. RESULTS: A total numer of 160 enterococcal isolates were obtained from 475 samples collected in total (33.7%). Among them, 110 of genetically different strains were identified and classified into EFA (82; 74.5%) and EFM (28; 25.5%). Genetic similarity analysis revealed the presence of 7 and 1 clusters among the EFA and EFM strains, respectively. The highest percentage of EFA strains (16; 19.5%) was resistant to high concentrations of gentamicin. Among the EFM strains, the most frequent strains were resistant to ampicillin and high concentrations of gentamicin (5 each; 17.9%). Six (7.3%) EFA and 4 (14.3%) EFM strains showed vancomycin resistance (VRE - Vancomycin-Resistant Enterococcus). Linezolid resistance was found in 2 strains of each species. The multiplex PCR analysis was performed to identify the vancomycin resistant enterococci. vanB, vanA and vanD genotypes were detected in 4, 1 and 1 EFA strains, respectively. Four EFA VRE-strains in total, 2 with the vanA and 2 with the vanB genotypes, were identified. The biofilm analysis revealed that all vancomycin-resistant E. faecalis and E. faecium strains demonstrated a higher biofilm-forming capacity, as compared to the susceptible strains. The lowest cell count (5.31 log CFU / cm2) was reisolated from the biofilm produced by the vancomycin-sensitive strain EFM 2. The highest level of re-isolated cells was observed for VRE EFA 25 and VRE EFM 7 strains, for which the number was 7 log CFU / cm2 and 6.75 log CFU / cm2, respectively. CONCLUSIONS: The irrational use of antibiotics in agriculture and veterinary practice is considered to be one of the key reasons for the rapid spread of antibiotic resistance among microorganisms. Owing to the fact that piggery environment can be a reservoir of antimicrobial resistance and transmission route of antimicrobial resistance genes from commensal zoonotic bacteria to clinical strains, it is of a great importance to public health to monitor trends in this biological phenomenon.

Immune profiling of rainbow trout (Oncorhynchus mykiss) exposed to Lactococcus garvieae: Evidence in asymptomatic versus symptomatic or vaccinated fish.

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤ .05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤ .05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤ .05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤ .05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection.

Virulence gene detection and antimicrobial resistance analysis of Enterococcus faecium in captive giant pandas (Ailuropoda melanoleuca) in China.

BACKGROUND: The emergence of multidrug resistance among enterococci makes effective treatment of enterococcal infections more challenging. Giant pandas (Ailuropoda melanoleuca) are vulnerable to oral trauma and lesions as they feast on bamboo. Enterococci may contaminate such oral lesions and cause infection necessitating treatment with antibiotics. However, few studies have focused on the virulence and drug resistance of oral-derived enterococci, including Enterococcus faecium, in giant pandas. In this study, we analyzed the prevalence of 8 virulence genes and 14 drug resistance genes in E. faecium isolates isolated from saliva samples of giant pandas held in captivity in China and examined the antimicrobial drug susceptibility patterns of the E. faecium isolates. RESULTS: Twenty-eight isolates of E. faecium were successfully isolated from the saliva samples. Four virulence genes were detected, with the acm gene showing the highest prevalence (89%). The cylA, cpd, esp, and hyl genes were not detected. The isolated E. faecium isolates possessed strong resistance to a variety of drugs; however, they were sensitive to high concentrations of aminoglycosides. The resistance rates to vancomycin, linezolid, and nitrofurantoin were higher than those previously revealed by similar studies in China and other countries. CONCLUSIONS: The findings of the present study indicate the drugs of choice for treatment of oral E. faecium infection in the giant panda.

Virulence genes and antimicrobial resistance in Enterococcus strains isolated from dogs and cats in Northeast China.

This study aimed to characterize the antimicrobial resistance and virulence of Enterococcus from dogs and cats in Northeast China and evaluate its zoonotic risk based on a total of 469 enterococci strains from 610 samples, including 238 strains of E. faecium and 128 strains of E. faecalis. The isolation rate from police dog samples was 93.79%, pet dog samples was 69.90% and pet cat samples was 76.67%. The differences in the prevalence of E. faecalis among different hosts were statistically significant (P<0.05). The assays showed that most of the virulence genes detected were existed in E. faecalis and police dogs carried the least number of virulence genes. The correlation between enterococcal surface protein (esp) and aggregation substance (asa1) was determined. Enterococci are most resistant to tetracycline and erythromycin, 68.92% of the isolates were classified as multiple drug resistant. Significant differences (P<0.01) were found between E. faecium and E. faecalis in the resistance rates of nine antimicrobials. Four positive and four negative correlations were found between virulence genes and antimicrobial resistance. The results show that Enterococcus colonization and excretion in dogs and cats were related to animal species and living environments. Some correlation between virulence factors and antimicrobial resistance was obtained. This study confirmed the presence of strains carrying multiple virulence factors and antimicrobial resistance at the same time, suggesting a public health risk for dogs and cats as reservoirs of enterococci.

High occurrence of Enterococcus faecalis, Enterococcus faecium, and Vagococcus lutrae harbouring oxazolidinone resistance genes in raw meat-based diets for companion animals - a public health issue, Switzerland, September 2018 to May 2020.

IntroductionEnterococci harbouring genes encoding resistance to florfenicol and the oxazolidinone antimicrobial linezolid have emerged among food-producing animals and meat thereof, but few studies have analysed their occurrence in raw meat-based diets (RMBDs) for pets.AimWe aimed to examine how far RMBDs may represent a source of bacteria with oxazolidinone resistance genes.MethodsFifty-nine samples of different types of RMBDs from 10 suppliers (three based in Germany, seven in Switzerland) were screened for florfenicol-resistant Gram-positive bacteria using a selective culture medium. Isolates were phenotypically and genotypically characterised.ResultsA total of 27 Enterococcus faecalis, Enterococcus faecium, and Vagococcus lutrae isolates were obtained from 24 of the 59 samples. The optrA, poxtA, and cfr genes were identified in 24/27, 6/27 and 5/27 isolates, respectively. Chloramphenicol and linezolid minimum inhibitory concentrations (MICs) ranged from 24.0 mg/L-256.0 mg/L, and 1.5 mg/L-8.0 mg/L, respectively. According to the Clinical and Laboratory Standards Institute (CLSI) breakpoints, 26 of 27 isolates were resistant to chloramphenicol (MICs ≥ 32 mg/L), and two were resistant to linezolid (MICs ≥ 8 mg/L). Multilocus sequence typing analysis of the 17 E. faecalis isolates identified 10 different sequence types (ST)s, with ST593 (n = 4 isolates) and ST207 (n = 2 isolates) occurring more than once, and two novel STs (n = 2 isolates). E. faecium isolates belonged to four different STs (168, 264, 822, and 1846).ConclusionThe high occurrence in our sample of Gram-positive bacteria harbouring genes encoding resistance to the critical antimicrobial linezolid is of concern since such bacteria may spread from companion animals to humans upon close contact between pets and their owners.

Virulence and antibiotic-resistance genes in Enterococcus faecalis associated with streptococcosis disease in fish.

Enterococcus faecalis is associated with streptococcosis like infection in fish. A whole-genome sequence study was conducted to investigate the virulence factor and antibiotic-resistance genes in three fish pathogenic E. faecalis. Genomic DNA was extracted from three strains of E. faecalis isolated from streptococcosis infected Nile tilapia (strains BF1B1 and BFFF11) and Thai sarpunti (strain BFPS6). The whole genome sequences of these three strains were performed using a MiSeq sequencer (Illumina, Inc.). All three strains conserved 69 virulence factor such as genes associated with protection against oxidative stress, bacterial cell wall synthesis, gelatinase toxin, multiple biofilm-associated genes and capsule producing genes. Moreover, 39 antibiotic-resistance genes against sixteen major groups of antibiotics were identified in the genome sequences of all three strains. The most commonly used antibiotic Tetracycline resistance genes were found only in BFPS6 strain, whereas, Bacteriocin synthesis genes were identified in both BFFF11 and BFPS6 strain. Phylogenetic analysis revealed that strains BF1B1 and BFFF1 form a different cluster than BFPS6. This is one of the first whole-genome sequence study of fish pathogenic E. faecalis, unfold new information on the virulence factor and Antibiotic resistance genes linked to pathogenicity in fish.

Genomic and proteomic aspects of p57 protein from Renibacterium salmoninarum: Characteristics in virulence patterns.

Renibacterium salmoninarum is one of the oldest known fish bacterial pathogens. This Gram-positive bacterium is the causative agent of Bacterial Kidney Disease (BKD), a chronic infection that primarily infects salmonids at low temperatures. Externally, infected fish may show exophthalmos, skin blisters, ulcerations, and hemorrhages at the base of the fins and along the lateral line. Internally, the kidney, heart, spleen, and liver may show signs of inflammation. The best characterized virulence factor of R. salmoninarum is p57, a 57 kDa protein located on the bacterial cell surface and secreted into surrounding fish tissue. The p57 protein in fish is the main mediator in suppressing the immune system, reducing antibody production, and intervening in cytokine activity. In this review, we will discuss aspects such as single nucleotide polymorphisms (SNPs) that modify the DNA sequence, variants in the number of copies of MSA genes, physical-chemical properties of the signal peptides, and the limited iron conditions that can modify p57 expression and increase the virulence of R. salmoninarum.

Comparison of injection and immersion challenges of Renibacterium salmoninarum strains in Rainbow Trout.

OBJECTIVE: Renibacterium salmoninarum is a pathogenic gram-positive bacterium and is the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in Rainbow Trout Oncorhynchus mykiss. METHODS: The CK-90 strain was intracoelomically injected (100 µL) at a high dose containing 4.80 × 106 CFU/g of fish (optical density at 525 nm [OD525 ] = 1.779) and a low dose containing 6.86 × 105 CFU/g of fish (OD525  = 1.077); alternatively, fish were immersed in a solution containing 4.5 × 107 CFU/mL of fish (OD525  = 0.886). The ATCC 33739 strain (originating from Brook Trout Salvelinus fontinalis) was also included and intracoelomically injected at 3.58 × 105 CFU/g of fish (OD525  = 1.431) to discern differences in virulence between the strains. RESULT: Clinical signs of BKD manifested at approximately 10 d postchallenge, and mortalities began at 19 days postchallenge. To confirm infection and quantify R. salmoninarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected after the challenge. Rainbow Trout that were challenged with CK-90 by injection (both high- and low-dose groups) exhibited significantly higher mortality than fish that were injected with ATCC 33739 or those that were exposed to CK-90 via immersion challenge. The R. salmoninarum p57 (57-kDa protein) antigen was confirmed via ELISA. Antigen load for fish injected with CK-90 (high dose: OD405  = 0.71; low dose: OD405  = 0.66) was significantly higher than that for fish injected with ATCC 33739 (OD405  = 0.34). The CK-90 strain (both high and low doses) was more virulent than ATCC 33739, which caused no mortalities over the 28-days trial. Although there were no mortalities among ATCC 33739 fish, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in those fish. CONCLUSION: The immersion challenge methodology for R. salmoninarum CK-90 was ineffective for inducing mortalities at the examined dose.

Various mobile genetic elements carrying optrA in Enterococcus faecium and Enterococcus faecalis isolates from swine within the same farm.

OBJECTIVES: In this study, the distribution of the oxazolidinone/phenicol resistance gene optrA and the mobile genetic elements involved in its dissemination were analysed among enterococcal isolates from a farrow-to-finish swine farm. METHODS: Enterococcus faecium and Enterococcus faecalis isolates were obtained from all pig production stages in the farm. The optrA-carrying E. faecium and E. faecalis isolates were subjected to PFGE and antimicrobial susceptibility testing. Complete sequences of the genetically unrelated optrA-carrying E. faecium and E. faecalis isolates were determined using Illumina HiSeq and MinION platforms. RESULTS: The optrA gene was present in 12.2% (23/188) of the E. faecium and E. faecalis isolates, most of which originated from nursery and finishing stages. The 23 optrA-positive Enterococcus isolates represented 15 PFGE types. WGS of representative isolates of the 15 PFGE types showed that optrA was carried by diverse genetic elements either located in the chromosomal DNA or on plasmids. A novel optrA-bearing genetic element was identified on two distinct multi-resistance plasmids from E. faecium. Two new hybrid plasmids carrying several resistance genes were found in two E. faecalis isolates. pC25-1-like plasmids and chromosomally integrated Tn6674 and Tn6823-like transposons were prevalent in the remaining Enterococcus isolates. CONCLUSIONS: The gene optrA was found in genetically unrelated E. faecium and E. faecalis isolates from the same farm. Analysis of the genetic contexts of optrA suggested that horizontal transfer including different plasmids and transposons played a key role in the dissemination of optrA in this farm.

Detection of optrA and poxtA genes in linezolid-resistant Enterococcus isolates from fur animals in China.

The emergence of linezolid-resistant (LR) enterococci found in food of animal origin arouses attention, but little is known about LR enterococci in fur animals. A total of 342 Enterococcus faecalis and 265 E. faecium strains isolated from fur animals in China from 2015 to 2017 were investigated to determine if LR enterococci (≥16 µg ml-1 ) are present. Overall, two E. faecalis and 12 E. faecium among these isolates were resistant to linezolid. In addition, all LR isolates were classified as multidrug-resistant isolates. We further explore the resistance genes of the LR enterococci, four E. faecalis and two E. faecium isolates contained optrA gene. Two of them co-harboured optrA and poxtA genes. We detected virulence genes in LR enterococci were the following: asa1, cylA, esp, gelE and hyl, among which the highest carrying rate gene was asa1. Besides, all of the LR enterococci we tested had the biofilm-forming ability. It is worth noting that we detected a novel ST type ST2010 from E. faecium 82-2. These data show LR enterococci exist in fur animals and have unique characteristics.