Asa1 Genes Research Articles

Prevalence and Molecular Characteristics of Enterococci Isolated from Clinical Bovine Mastitis Cases in Ningxia.

This study aimed to investigate the prevalence and genetic characterization of enterococcal isolates (Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae) isolated from clinical bovine mastitis cases in Ningxia, China. The enterococci were identified by 16S rRNA amplification and sequencing. Antimicrobial resistance was determined by disc diffusion method. Virulence and antimicrobial resistance genes were detected by PCR assays. Overall, 198 enterococcal isolates were identified from 2897 mastitis samples, including 137 (4.7%) E. faecalis, 50 (1.7%) E. faecium and 11 (0.4%) E. hirae. E. faecalis, E. faecium and E. hirae isolates showed high resistance to tetracycline (92.7%, 68.0%, 90.9%), followed by erythromycin (86.9%, 76.0%, 72.7%). The multidrug-resistant strains of E. faecalis and E. faecium were 29 (21.2%) and 13 (26.0%), respectively. The resistance of E. faecalis, E. faecium and E. hirae isolates to tetracycline is mainly attributed to the presence of tetL (alone or combined with tetM and/or tetK), the erythromycin resistance to ermB (alone or combined with ermC and/or ermA). Moreover, cpd (94.2%), gelE (77.4%), efaAfs (93.4%), and esp (79.6%) were the most common virulence genes in E. faecalis. In E. faecium, except for the gene efaAfs (82.0%), other virulence genes are rarely found. Only two strains of E. hirae carrying asa1 gene were detected. The results of this study can provide a reference for the prevention and treatment of bovine mastitis caused by enterococci.

Sociodemographic distributions and molecular characterization of colonized Enterococcus faecium isolates from locality hospitals in Khartoum, Sudan.

Enterococcus faecium is an opportunistic pathogen of humans with diverse hosts, encompassing animals as well as human beings. In the past twenty years, there has been a rise in the instances of nosocomial infections that are linked to antibiotic-resistant Enterococcus faecium. The acquisition of diverse antimicrobial resistance factors has driven the global development of robust and convergent adaptive mechanisms within the healthcare environment. The presence of microorganisms in hospitalized and non-hospitalized patient populations has been significantly aided by the facilitation of various perturbations within their respective microbiomes. This study aimed to determine the antimicrobial profile, demographic and clinical characteristics, along with the detection of virulence encoding genes, and to find out the clonal genetic relationship among colonized E. faecium strains. A hospital-based cross-sectional study was carried out between October 2018 and March 2020 at four Khartoum locality hospitals in Sudan. The study comprised a total of 108 strains of E. faecium isolated from patients admitted to four locality hospitals in Khartoum. A self-structured questionnaire was used to gather information on sociodemographic traits. Data were analyzed using chi-square test. In all cases, P value ≤ 0.05 with a corresponding 95% confidence interval was considered statistically significant. Moreover, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) was utilized to assess the prevalence of clonal relationships, and the gel was analyzed using CLIQS software. In this study, the isolation rate of colonized E. faecium strains was 108/170 (63.5%). The colonization of E. faecium and its association with various sociodemographic and clinical features was examined. 73 (67.6%) of patients had multidrug-resistant (MDR), and 22 (20.4%) had extensively drug-resistant (XDR), 73 (67.6%) of patients engaged in self-medication practices. Eighty patients (74.1%) were non-adherence to prescribed antibiotics, while 70 (64.8%) patients reported recent antibiotic usage within the 3 months. The present study suggests that demographic factors may not be significantly associated with the incidence of E. faecium infection except for patients who had a prior history of antibiotic use (P≤0.005). The analysis of virulence genes showed a high prevalence of asa1 gene (22.2%) among strains. In ERIC-PCR the genetic relatedness of E. faecium showed seven identical clusters (A-G) with 100% genetic similarity. This implies clonal propagation in hospitals and communities. This study found that the incidence of E. faecium isolated from locality hospitals in Khartoum was likely due to the spread of E. faecium clones, thereby highlighting the need for intensifying infection control measures to prevent the spreading of nosocomial infection.

The Emergence of Antimicrobial Resistance and Virulence Characteristics in Enterococcus Species Isolated from Bovine Milk.

Enterococcus spp., including E. faecalis and E. faecium, pose risks to dairy farms as opportunistic pathogens. The study evaluates antimicrobial resistance (AMR) and virulence characteristics of Enterococcus spp. isolated from bovine milk. Bile esculin agar was used to assess 1471 milk samples, followed by colony identification, gram staining, catalase tests, and 45 °C incubation. PCR analysis targeted E. faecalis and E. faecium in characteristic Enterococcus spp. colonies, with MALDI-TOF used for negative samples. Multiple tests, including disk diffusion, chromogenic VRE agar for vancomycin resistance, Vancomycin Etest® for MIC determination, and PCR for virulence factors (cylA, esp, efaA, ace, asa1, gelE, and hyl genes), were performed. Out of 100 identified strains, E. durans (30.66%), E. faecium (26.28%), and E. faecalis (18.25%) were predominant. AMR in Enterococcus spp. varied, with the highest rates against rifampicin (27%), tetracycline (20%), and erythromycin (18%). Linezolid (5%), vancomycin, ciprofloxacin, and teicoplanin (3% each) had lower prevalence. E. faecium and E. faecalis showed high AMR to rifampicin, erythromycin, and tetracycline. Thirty-two strains (18.98%) grew on VRE Chromoselect agar, while 4 (2 E. faecalis and 2 E. faecium) showed vancomycin resistance by MIC values. E. faecalis carried gelE (45.5%) and asa1 (36%), and E. gallinarum had 9.1% with the asa1 gene. Detecting resistant Enterococcus in bovine milk supports control strategies for enterococci on dairy farms, highlighting AMR concerns in the food chain.

Virulence Factors and Biofilm Formation in Vancomycin Resistant <i>Enterococcus faecalis</i> and <i>Enterococcus faecium</i> Isolates in Brazil

In this work, we evaluated biofilm formation of Vancomycin Resistant of E. faecalis and E. faecium (VRE) in different culture media and adhesion substrate, as well as cellular hydrophobicity and presence of virulence genes. For this, 35 isolates were collected from a public hospital in Recife, Pernambuco, Brazil and identified by the Matrix-Assisted Laser Desorption Ionization - Time-of-flight - Mass Spectrometry (MALDI-TOF-MS) technique. Biofilm formation was analyzed by the Crystal Violet (CV) method and fluorescence microscopy, cellular hydrophobicity by hydrocarbon interaction and the presence of gelE, esp and asa1 genes by Polymerase Chain Reaction (PCR). 12 isolates were identified as E. faecalis and 23 as E. faecium. Most were obtained in Coronary Units (40.0%) and Intensive Care Unit (31.4%). E. faecium isolates were more resistant to the antibiotics tested than E. faecalis; however, E. faecalis stood out as a biofilm producer. Regarding the presence and gene frequency, it was observed that gelE (54.3%) and esp (54.3%) were the most prevalent, followed by asa1 (22.9%). When comparing the gene frequency, it was observed that gelE and esp were predominant (48.6% for both species), while asa1 was more frequent in E. faecalis (20.0%). The data presented here are worrying, because they reveal the virulence potential of isolates VRE, which contributes to the dissemination and persistence of these pathogens in the hospital environment.

Effect of sub-inhibitory concentrations of antibiotics on biofilm formation and expression of virulence genes in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis

Biofilm formation is a key factor in the pathogenesis of enterococcal infections. Thus, the biofilm-forming ability and frequency of biofilm-related genes in penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) compared to penicillin- and ampicillin-susceptible E. faecalis (PSASEF) were assessed in the present study. In addition, the effect of sub-inhibitory concentrations (sub-MICs) of antibiotics on biofilm formation and expression of virulence genes was evaluated. Twenty PRASEF and 21 PSASEF clinical isolates were used to determine the effect of sub-MICs of antibiotics (ampicillin, penicillin, and gentamicin) on biofilm formation, and ten selected isolates were subjected to RT-qPCR to detect the transcript levels of virulence genes (efaA, asa1, esp, and ace). Antibiotic susceptibility was evaluated by the microdilution broth method. Biofilm formation assay was performed using the microtiter plate method. All PSASEF and PRASEF isolates produced biofilms in vitro. Most isolates had three or four virulence genes. Sub-MICs of ampicillin significantly decreased biofilm production and expression of ace and asa1 genes, although the transcript levels were significantly lower (−350% and −606.2%, respectively) among the PSASEF isolates only. Sub-MICs of gentamicin did not have any significant effect on biofilm formation, but slightly increased the transcript levels of efaA. In conclusion, this study showed that the biofilm-forming ability and frequency of the evaluated virulence genes were similar among the PRASEF and PSASEF isolates. Further, in vitro antibiotic sub-MICs were confirmed to interfere with the expression pattern of virulence genes and biofilm formation by E. faecalis. However, further studies are required to clarify the role of sublethal doses of antibiotics on enterococcal biofilms.

Genetic Diversity, Antimicrobial Resistance, and Virulence Factors of Enterococcus faecalis Isolates Obtained from Stool Samples of Hospitalized Patients

Background: Enterococcus faecalis rapidly develops resistance to different antibiotics, thereby resulting in serious nosocomial infections associated with high mortality rates and different problems in the healthcare systems. Objectives: This study aimed to analyze the genetic diversity, antimicrobial resistance, and virulence factors of E. faecalis isolates obtained from the stool samples of patients in a hospital in the center of Iran. Methods: In this cross-sectional descriptive-analytical study, 108 stool samples were collected from September 2019 to February 2020 from 108 patients hospitalized in a hospital in the center of Iran. Enterococcus faecalis isolates were detected using the ddlE gene detection technique, and antimicrobial resistance testing was performed using the disc agar diffusion method. Moreover, polymerase chain reaction was used to detect antimicrobial resistance genes and virulence factors. Genetic diversity was also analyzed by enterobacterial repetitive intergenic consensus using PCR. The BioNumerics software was used to construct a dendrogram. Results: Of 108 isolates, 50 samples were E. faecalis (46.2%). The prevalence of multidrug resistance among E. faecalis isolates was 62%, and most isolates were resistant to antibiotics tetracycline (70%), erythromycin (68%), and rifampin (60%). Among the E. faecalis isolates, the most prevalent antimicrobial resistance genes were ermB (96%), aph (2′′) Ia (66%), aac(6′)-Ie (40%), and ermC (30%), and the most prevalent virulence genes were gelE (78%), asa1 (74%), and esp (74%). The genetic diversity analysis showed 25 ERIC types in two major clusters (ie, clusters H and J) and eight minor clusters (ie, clusters A-G and I). There was no significant difference between clusters H and J in terms of antimicrobial resistance and resistance genes (P > 0.05). In contrast, the prevalence of the asa1 gene was significantly higher in cluster J than in cluster H (P < 0.05). Conclusions: This study showed the high prevalence of multidrug resistance, and high heterogeneity among the E. faecalis isolates obtained from the stool samples of hospitalized patients.

Molecular characterization of Streptococcus and Enterococcus species isolated from broiler chickens

Streptococcus and Enterococcus infections in chickens may result in significant negative effect on economy. In the current study, the prevalence of Streptococcus and Enterococcus species was planned in different broiler chickens farms in Beni-Suef Governorate. A total of 272 samples were collected from lesions (septicemic organs, enlarged organs, necrotic focci) of the affected organs including heart, lung, liver and kidney of diseased broiler chickens and freshly dead ones. Out of 272 samples a total of 49 isolates were recovered with incidence of 18% including 26Streptococcus spp. (53.1%) and 21 Enterococcus spp. (42.8%) meanwhile 2 isolates (4.1%) were unidentified. Streptococcus isolates were identified as S.gallinaceous (24.5%), S. dysgalactiae (16.3%) and S. durans (12.2%). Meanwhile all Enterococcus isolates were identified as E. faecalis. The in-vitro antibiotic sensitivity testing showed that all isolates were highly sensitive to amoxicillin (77.6%), sulfamethoxazole-trimethoprim (73.5%) and amoxicillin-clavulanic acid (65.3%). Meanwhile, all isolates were resistant to cephalexin, cefotaxime sodium, cefipime, cefotriaxone, tetracycline, kanamycin and apramycin while 87.8 and 63.2% of isolates showed resistance aganist gentamicin and enrofloxacin, respectively. Moreover, multidrug resistant were detected in all isolates. Polymerase chain reaction (PCR) was applied to identify 4 resistance-associated genes including (tetO, aac(6')aph(2''), blaZ and Pbp 1A) as well as 6 virulence-associated genes including (cylE, brp A, hyl, cylA, asa1 and gelE). The results indicated that tetO, aac(6')aph(2''), blaZ, Pbp 1A, cylE, brp A, cylA and asa1 genes were recovered from all the tested isolates (100%). Meanwhile, none of streptococcus isolates had hly gene also, gelE gene not detected in enterococcus isolates.

Determination of the biosafety of potential probiotic Enterococcus faecalis and Enterococcus faecium strains isolated from traditional white cheeses

The aims of this study were to characterize antibiotic resistance, determination of genotypic and phenotypic virulence characters; and to evaluate the safety of 12 Enterococcus faecalis and 8Enterococcus faecium strains isolated from Iranian and Turkish white cheeses. All strains were researched for production capability of cytolysin and gelatinase, biofilm formation and sensitivity against ten antibiotics. The presence of vancomycin resistance genes and virulence factors genes like cylA and cylB, esp, agg, asa1, EfaAfs, EfaAfm, cob, ccf, and hly were determined by polymerase chain reaction and sequence analysis. All the investigated strains were resistant to at least one class of antibiotic, and 40% of the strains were classified as multi drug resistant. E. faecalis RI 11, RI 17, RI 21 strains demonstrated both gelatinase and biofilm activities. Moreover, there was a significant correlation between gelatinase activity and the presence of gelE (P < 0.05). While ccf and asa1 genes were determined in all strains, efaAfm and clyB genes were not found in any strains. E. faecium RI 56, RI 58 and RI 59 strains were found to contain different molecular sizes of plasmid DNA. The phenotypic and genotypic characteristics of E. faecalis and E. faecium strains were determined in terms of functional and safety perspectives.

Identification, antimicrobial susceptibility and virulence factors of Enterococcus species isolated from clinical specimens at an Indian tertiary care hospital

Purpose: Enterococcus species have increasingly emerged as important pathogens in serious nosocomial and community acquired infections. The objective was to determine species identification, evaluate current susceptibility pattern and characterize various virulence determinants among clinical isolates of enterococci at an Indian hospital. Methods & Materials: Consecutive isolates from clinical specimens (April to September 2017) were included. Species identification was performed by conventional biochemical tests. Antimicrobial susceptibility testing was as per CLSI guidelines, including detection of high-level gentamicin resistance (HLGR), high-level streptomycin resistance (HLSR) and glycopeptide resistant enterococci (GRE). All screen-positive GRE isolates were investigated by PCR for species confirmation and presence of vanA/ vanB genes. Virulence genes asa1, esp, hyl, cyl, and gelE were investigated by molecular methods. Haemolysin and biofilm production were studied phenotypically. Results: Of 71 strains isolated, 57 (80.3%), 9 (12.7%) and 5 (7.0%) were from urine, pus and blood respectively consisting of E. faecalis (45, 63.4%), E. faecium (20, 28.2%), E. durans (3, 4.2%), and E. avium, E. hirae, E. mundtii (1, 1.4%). Six (8.4%) were GRE (5 E. faecium, 1 E. faecalis), all vanA phenotypically and genotypically. HLGR and HLSR was observed in 41 (57.7%) and 23 (32.4%) isolates respectively, higher in E. faecium compared to E. faecalis (85.0% and 40.0% vs 46.6% and 28.8%). Resistance to other antibiotics was also higher in E. faecium. All were susceptible to daptomycin. Hemolysin activity was significantly higher in E. faecalis compared to E. faecium (55.5% vs 0%). Biofilm production was observed in 23 (32.4%) isolates. Virulence genes detected were asa1 (9, 45%), gel (9, 45%), esp (7, 35%), cyt (6, 30%) and hyl (1, 5%). Frequency of asa, gel, and cyl was higher in E. faecalis than E. faecium (7, 7, 5 vs 2, 2, 1), whereas esp and hyl was more in E. faecium compared to E. faecalis (5, 1 vs 0, 0). Conclusion: Prevalence of a wide variety of Enterococcus species in clinical samples together with their variable antimicrobial susceptibility pattern and virulence traits emphasizes the need for their routine speciation and susceptibility testing which may help control the spread of multidrug resistant Enterococcus species.

Molecular characterization of antibiotic resistant and potentially virulent enterococci isolated from swine farms and feed mills

A total of 108 swine feed samples were collected from six feed mills and two farms and tested for enterococcal contamination. Nearly 43% of these samples were positive for enterococci. The mean ± SE concentration of enterococci in feed samples ranged from 2.0 × 101 to 7.3 × 103 CFU/g of feed. About 38% of processed feed mill samples were contaminated with enterococci compared to 59% of swine farm samples. A total of 208 enterococcal isolates were represented by Enterococcus casseliflavus (54.8% of total isolates), E. gallinarum (17.8%), E. faecium (17.8%), E. hirae (5.8%), and E. faecalis (3.8%). These isolates were phenotypically resistant to tetracycline (48.5%), erythromycin (14.4%), streptomycin (13.4%), kanamycin (11.5%), ciprofloxacin (10.0%), ampicillin (2.8%), and chloramphenicol (1.4%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM gene (52.8%), tetO (14.4%), tetK (1.0%), and tetS (0.5%), whereas ermB conferred erythromycin resistance in 10.6% of all isolates. Several isolates carried genes coding for virulence factors, including gelatinase (gelE; 18.2%), an enterococcal surface protein (esp; 2.4%), and cytolysin (cylA; 2.4%). Only E. faecalis was β-hemolytic (2.9%) and gelatinolytic (3.4%). The aggregation substance gene asa1 was detected in 5 out of 8 E. faecalis isolates, of which four were phenotypically positive. The transposon Tn916/1545 was detected in 11.5% of all isolates. Mating assays revealed that 7 out of 8 E. faecalis could transfer tetM gene, and rate of transfer ranged from 2.0 × 10−3 to 1.6 × 10−5. The presence of antibiotic resistant and potentially virulent enterococci in swine farm samples and feed mill samples, though in low prevalence, raises concern and emphasizes the need for improved hygiene and quality standards on farms and in feed mills.

Brazilian artisanal ripened cheeses as sources of proteolytic lactic acid bacteria capable of reducing cow milk allergy.

The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and β-caseins, α-lactalbumin and β-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that causedpartial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.

Vancomycin-resistant enterococci isolates colonizing and infecting haematology patients: clonality, and virulence and resistance profile

Vancomycin-resistant enterococci (VRE) are an important agent of colonization and infection in haematology patients. However, the role of virulence on VRE colonization and infection is controversial. To characterize the lineage, virulence and resistance profile of VRE infection and colonization isolates; as well as their impact on outcome of haematology patients using a regression logistic model. Eighty-six isolates (80 Enterococcus faecium and six E.faecalis) from 76 patients were evaluated. Polymerase chain reaction for resistance and virulence genes, and pulsed-field gel electrophoresis and whole genome sequencing of the major clusters, were performed. Bivariate and multivariate analyses were carried out to evaluate the role of virulence genes on outcome. All isolates harboured the vanA gene. Regarding the virulence genes, 96.5% of isolates were positive for esp, 69.8% for gelE and asa1 genes. VRE infection isolates were more virulent than colonization isolates and harboured more often the gelE gene (P=0.008). Infections caused by VRE carrying asa1 gene resulted more frequently in death (P=0.004), but only the predominant clone remained as protector in the multivariate model. The E.faecium strains were assigned to seven STs (ST78, ST412, ST478, ST792, ST896, ST987, ST963) that belonged to CC17. The E.faecalis sequenced belonged to ST9 (CC9). E.faecium was predominant, and infection isolates were more virulent than colonization isolates and harboured more often the gene gelE. Infections caused by VRE carrying the asa1 gene appeared to be associated with a fatal outcome.

Virulence Factor and Biofilm Formation in Clinical Enterococcal Isolates of the West of Iran

: Enterococcus spp., a part of the normal flora of the human intestine, possess several virulence factors that can develop biofilms to endure harsh environments. Their ability to cause nosocomial infections makes them as critical opportunistic pathogens in hospital settings. The current study aimed at determining the occurrence of 6 genes coding virulence factors and their ability to develop biofilms, and conducting phenotypical assessments of haemolysin and gelatinase in clinical enterococci isolated from the West of Iran. A total of 126 isolates were screened for harbouring the following genes: aggregation substance (asa1), cytolysin (cylABM), enterococcal surface protein (esp), and gelatinase (gelE). Isolates were tested for haemolysin and gelatinase expression phenotypically and for biofilm production quantitatively, using the microtiter method. Of the 126 tested isolates, 95 (73%) were Enterococcus faecalis and 28 (21%) were E. faecium. The total frequency of virulence gene was cylA 92 (73%), cylB 85 (67%), cylM 57 (45%), asa1 26 (21%), gelE 64 (51%), and esp 66 (53%); while 98 (75%) of the isolates were able to form biofilm. A total of 74 (58%) and 46 (35%) isolates could secret haemolysin and gelatinase. There was a significant difference between the frequency of virulence gene in E. faecalis and E. faecium. Enterococcus faecium isolates lacked the gelE and asa1 genes and the frequency of cylABM genes were lower than that of E. faecalis isolates. Enterococcus faecalis isolates were relatively rich in virulence factors; no association was observed between biofilm formation and the presence of specific virulence genes.

Evaluation of virulence factors in enterococcus species

Enterococci have recently become important due to their increased isolation rates in community-based and nosocomial infections and resistance to many antibiotics, including glycopeptides. The aim of this study was to evaluate the antimicrobial susceptible patterns and virulence factors of various clinical specimens; urine (n= 149), blood (n= 38), wound (n= 17), stool (n= 13), and other (n= 12) with a total of 229 enterococci including 138 E.faecalis and 91 E.faecium isolates. Aggregation factor (AF), enterococcus surface protein (esp), cytolysins and gelatinase encoding genes (asa1, esp, cylM, cylBcyl A, cylll, cylls, gelE, respectively) were investigated by molecular methods. Haemolysin production and gelatinase were studied phenotypically. A total of 30 isolates, 29 of E.faecium and one of E.faecalis isolates were resistant to vancomycin. High-level gentamicin and high-level streptomycin resistance in E.faecalis were 40.7% and 63.7% however, they were 47.1% and 55.8% in E.faecalis isolates. All strains were susceptible to linezolid. Ampicillin, penicillin and vancomycin resistance in E.faecium isolates were found to be higher than E.faecalis isolates (p= 0.001, p= 0.008 and p< 0.001). Asa1 (p< 0.001), cylll (p= 0.002) and cylls (p< 0.001) as well as gelatinase activity in isolates of E.faecalis were significantly higher than the isolates of E.faecium (p< 0.001). The most common virulence genes in our study were asa1 gene (45%), cyLs gene (33.2%) and esp gene (32.3%). Ciprofloxacin resistance in cylLL and cyLs gene positive isolates of E.faecalis were significantly higher compared to isolates that do not contain these genes (p= 0.035 and p= 0.047). Likewise, haemolysin producing E.faecium isolates were significantly more resistant to vancomycin compared to isolates that do not produce hemolysin (p< 0.001). When the virulence factors of vancomycin resistant and susceptible isolates were compared, the esp gene level in VRE E.faecium isolates was found to be 24.1%, while no esp gene was found in VRE E.faecalis isolates. The existence of asa1was negative in both VRE E.faecium and VRE E.faecalis isolates. The activity of hemolysin was found 42.3% for E.faecalis and 19.3% for E.faecium. In vancomycin-sensitive enterococcus (VSE) species, esp gene activity was 35.1% for E.faecalis, 29.4% for E.faecium, asa1 gene activity was 60.8% for E.faecalis and 47.1% for E.faecium, hemolysin activity was 52.8% for E.faecalis and 23.5% for E.faecium. In our study, it was found that VSE isolates have more virulence genes than VRE isolates. It should be kept in mind that VRE can causeinfections which are difficult-to-treat especially in hospitalized patients and VSE have significant virulence factors that can cause severe infections.

Genetic diversity of Enterococcus faecalis isolated from environmental, animal and clinical sources in Malaysia.

Enterococcus faecalis ranks as one of the leading causes of nosocomial infections. A strong epidemiological link has been reported between E. faecalis inhabiting animals and environmental sources. This study investigates the genetic diversity, antibiotic resistance and virulence determinants in E. faecalis from three sources in Malaysia. A total of 250 E. faecalis isolates were obtained consisting of 120 isolates from farm animals, 100 isolates from water sources and 30 isolates from hospitalized patients. Pulse-field gel electrophoresis-typing yielded 63 pulsotypes, with high diversity observed in all sources (D=≥0.901). No pulsotype was common to all the three sources. Each patient room had its own unique PFGE pattern which persisted after six months. Minimum inhibitory concentrations of Vancomycin, Gentamicin, Penicillin, Tetracycline, Nitrofurantoin, Levofloxacin, Ciprofloxacin and Fosfomycin were evaluated. Resistance to Tetracycline was most prevalent in isolates from farm animals (62%) and water sources (49%). Water isolates (86%) had a higher prevalence of the asa1 gene, which encodes for aggregation substance, whereas clinical (78%) and farm animal isolates (87%) had a higher prevalence of the esp gene, encoding a surface exposed protein. This study generates knowledge on the genetic diversity of E. faecalis with antibiotic resistance and virulence characteristics from various sources in Malaysia.

Virulence Genotyping of Enterococcus species isolated from meat and milk products

Enterococci have recently emerged as nosocomial pathogens. Their ubiquitous nature determines their frequent findingin foods as contaminants. As little is known about their virulence potential, this study aimed to investigate the frequencyof five potential virulence determinants in Enterococcus species isolated from various foodstuffs in Sharkia and DakahliaGovernorates, Egypt. A total of 59 enterococci isolates (59%) were recovered according to standard microbiologicalmethods, with milk and meat being most contaminated (76 and 60%, respectively). Species-specific PCR of tenenterococci isolates identified by 16S rDNA revealed the presence of E. faecalis, E. faecium and unidentified enterococciin 70, 20 and 10% of the isolates, respectively. PCR screening for esp (enterococcal surface protein), gelE (gelatinase),asa1 (aggregation substance), hyl (hyaluronidase) and ace (collagen binding antigen) virulence factors showed that allthe identified isolates were found to carry one or more virulence-encoding genes, with two or three being the mostcommon pattern. The esp and gelE were the predominant virulence traits among all investigated enterococci isolates (80%each), followed by ace, asa1 and hyl genes (50, 30 and 10%, respectively). Notably, E. faecalis and E. faecium isolatesshowed different patterns of virulence determinants; esp, gelE, ace and asa1 genes were more prevalent in E. faeciumthan E. faecalis. Simultaneous presence of virulence markers was observed among the analyzed isolates. Therefore, theresults of this study showed that food can play an important role in the spread of enterococci with virulence potentialthrough the food chain to the human population

Detection of virulence genes in resistant enterococci isolated from pediatric patients at high risk for nosocomial infections

BackgroundThe study of virulence genes carried by enterococci has become of greater relevance as nosocomial enterococcal infections have become more prevalent and possibly more severe. MethodsSurveillance swabs were performed on children less than 18 months of age in an intensive care unit in Iran in 2012–2013. Multiplex PCR and sequencing methods were used to detect gelE, esp and asa1 genes in enterococci with intermediate or full resistance to vancomycin. ResultsThe rate of carriage of the genes was gelE (91%), esp (79%) and asa1 (87%). ConclusionThe majority of enterococcal strains with resistance to vancomycin carry genes for all three potential virulence factors that were analyzed in this study. This might explain why enterococcal infections appear to be more virulent than in the past.

Prevalence of virulence and resistance to antibiotics in pathogenic enterococci isolated from mastitic cows.

The prevalence of enterococci was examined in 280 milk samples collected from dairy cattle diagnosed with mastitis in three provinces of western China. Sixty strains of enterococci were isolated, and the species were determined based on their biochemical characters and 16S rRNA sequences. Resistance to seven antibiotic agents, frequency of seven virulence genes and pathogenicity in Kunming mice were tested to evaluate biological risks. The correlation between the number of virulence genes and pathogenicity in Kunming mice was also evaluated. The 60 isolates were allocated to Enterococcus hirae (68.3%), E. faecium (25.0%), E. mundtii (3.3%) and E. durans (3.3%). A total of 83.3% of the isolates were resistant to penicillin, whereas 15.0% were resistant to ampicillin, 15.0% to vancomycin, 6.7% to tetracycline and 25.0% to ciprofloxacin. Moreover, isolates exhibited 50.0% and 21.7% resistance to high levels of gentamycin and streptomycin, respectively. The gene asa1 was detected in all enterococcal isolates, whereas 66.7% of strains harbored three or more virulence factors and 56.7% were asa1-ccf-gelE-positive. In pathogenicity tests, isolates harboring numerous virulence factors did not show greater invasiveness than isolates harboring fewer virulence traits against Kunming mice. In conclusion, the number of virulence factors does not appear to predict the risk of enterococcal infection. Isolates were commonly resistant to penicillin and sporadically to ampicillin and vancomycin. These results suggest that the use of gentamycin, streptomycin and ciprofloxacin against enterococci should be avoided in mastitic cows. Additionally, the results demonstrate that the majority of isolates are sensitive to tetracycline.

Evaluation of the proteolytic activity of Enterococcus faecalis FT132 and Lactobacillus paracasei FT700, isolated from dairy products in Brazil, using milk proteins as substrates

Lactic acid bacteria (LAB) have been used by mankind from immemorial times due to their technological properties and ability to improve sensorial properties of foods. Some LAB strains are also able to hydrolyze milk proteins, which increase their digestibility and contribute to the production of desirable flavors. Moreover, proteolytic activity of LAB on milk proteins may release bioactive peptides with different activities such as antihypertensive, antioxidant, antimicrobial, immunomodulatory and mineral-binding activities. This study aimed to evaluate the proteolytic activity of Enterococcus faecalis FT132 and Lactobacillus paracasei FT700, isolated from Brazilian dairy products. The proteolytic activity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography using milk proteins as substrates in different conditions. Both E. faecalis FT132 and L. paracasei FT700 were proteolytic at pH 6.5 in the range of 37–42 °C, and these activities were due to metalloproteases. L. paracasei FT700 could then be used as adjunct culture in fermented dairy products in order to increase their digestibility. On the contrary, E. faecalis FT132, which harbors three virulence genes asa1, ace and gelE and which is resistant to erythromycin and tetracycline, cannot be added to food products. However, the peptides produced after hydrolytic activity on milk proteins by both strains might be used as ingredients in purified formulas.

Prevalence of Enterococcus Species and Their Virulence Genes in Fresh Water Prior to and after Storm Events

Enterococcus spp. isolates (n = 286) collected from six surface water bodies in subtropical Brisbane, Australia, prior to and after storm events, were identified to species level and tested for the presence of seven clinically important virulence genes (VGs). Enterococcus faecalis (48%), Enterococcus faecium (14%), Enterococcus mundtii (13%), and Enterococcus casseliflavus (13%) were frequently detected at all sites. The frequency of E. faecium occurrence increased from 6% in the dry period to 18% after the wet period. The endocarditis antigen (efaA), gelatinase (gelE), collagen-binding protein (ace), and aggregation substance (asa1) were detected in 61%, 43%, 43%, and 23% of Enterococcus isolates, respectively. The chances of occurrence of ace, gelE, efaA, and asa1 genes in E. faecalis were found to be much higher compared to the other Enterococcus spp. The observed odds ratio of occurrence of ace and gelE genes in E. faecalis was much higher at 7.96 and 6.40 times, respectively. The hyl gene was 3.84 times more likely to be detected in E. casseliflavus. The presence of multiple VGs in most of the E. faecalis isolates underscores the importance of E. faecalis as a reservoir of VGs in the fresh water aquatic environment. Consequently, if contaminated surface water is to be used for production of potable and nonpotable water some degree of treatment depending upon intended use such as detention in basins prior to use or chlorination is required.