Esp Gene Research Articles

Analysis of virulence genes, drug resistance detection, and pathogenicity in Enterococcus from farm animals

This study aimed to investigate the presence of eight virulence genes (ace, asa1, esp, efaA, gelE, cylA, agg, fsr) in Enterococcus from a variety of animals and to explore the drug resistance and pathogenicity. This could provide a theoretical basis for clinical treatment of Enterococcus infections. Anal swabs from pigs, chickens, cattle, and dogs in farms and pet hospitals were collected for Enterococcus isolation and identification. Eight virulence genes were detected (PCR method), and drug resistance was assessed (drug-sensitive paper method). The strains containing different virulence genes were then divided into EV1, EV2, and EV3 groups. The LD50 and pathogenicity was examined by intra-peritoneal injection to infect mice. Differences were found in the detection rates of virulence genes in Enterococcus from the different animals. The highest overall detection rate was for the esp gene (78.0%), and the lowest for the cylA gene (15.5%). Eight genes were detected most frequently in Enterococcus from dogs and least frequently from cattle. Among the Enterococcus strains from four variety of animals, drug resistance was highest against sulfamethoxazole (100%), cefotaxime (>97%), and cefotaxitin (>93%). Drug resistance was lowest against vancomycin (0%), levofloxacin (<12%) and ciprofloxacin (<13%). The LD50 for each of the three groups was EV1LD50=8.71×109CFU， EV2LD50=2.34×1010CFU，and EV3LD50=9.33×1010CFU. The Enterococcus12LD50 dose group caused significant clinical symptoms in mice, with pathological effects on the heart, liver, lungs, and kidneys, and particularly on the urinary system. The abundance of Enterococcus virulence genes, drug resistance, and pathogenicity vary among different animal origins, and the pathology caused by Enterococcus requires effective treatment protocols based on species and regional characteristics.

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6′)Ie-aph(2″)Ia and aph(3′)-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6′)-Ie-aph(2″)-Ia or aph(3’)-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Antibiogram Pattern and Virulence Trait Characterization of Enterococcus Species Clinical Isolates in Eastern India: A Recent Analysis.

Objective We aimed to evaluate the current antimicrobial susceptibility pattern and characterize putative virulence traits among Enterococcus species isolates from various clinical specimens in view of their increased isolation rates in both community-related and serious nosocomial infections, as well as resistance to many antibiotics. Methods Study (April 2017–March 2018) included consecutive, nonrepeated, discrete, and clinically significant isolates of enterococci. Susceptibility testing included detection of high-level aminoglycoside-resistant (HLAR) and glycopeptide-resistant enterococci (GRE). All screen-positive GRE isolates were investigated by polymerase chain reaction for species confirmation and presence of vanA/vanB genes. Virulence genes ace , asa1 , cyt , efa , esp , gelE , and hyl were investigated by molecular methods. Hemolysin and biofilm production were studied using phenotypic methods. Results Of 111 isolates, 89 (80.1%), 16 (14.4%), and 6 (5.4%) were from urine, pus, and blood, respectively, consisting predominantly of E. faecalis (67, 60.4%) and E. faecium (32, 28.8%). E. hirae (5, 4.5%) was the predominant non- E. faecalis non- E. faecium isolate. Other species were E. durans (4, 3.6%), E. avium (2, 1.8%), and E. mundtii (1, 0.9%). Seven (6.3%) out of the 111 isolates were GRE, all vanA genotype. HLAR was observed in 70 (63.1%) isolates, significantly higher in E. faecium than E. faecalis (81.2 vs. 58.2%; p < 0.05). All were susceptible to daptomycin. Hemolysin activity and biofilm production were observed in 38 (34.2%) and 36 (32.4%) isolates. Most frequent virulence genes were efa (77, 69.4%), ace (71, 63.9%), asa1 (67, 60.3%), and gelE (66, 59.4%). There was a predominant association of esp and hyl genes with E. faecium and that of the other genes with E. faecalis . Conclusion The study will contribute to the existing limited data on virulence trait characterization of clinical E. spp. isolates in India. At the same time, it will help to serve as a guide in the choice of empirical therapy in enterococcal infections leading to favorable clinical outcomes by decreasing the clinical failure, microbiological persistence, and associated mortality, and will lead to future studies on controlling the spread of virulent and multiresistant isolates.

Prevalence and Virulence Characteristics of Enterococcus faecalis and Enterococcus faecium in Bovine Mastitis Milk Compared to Bovine Normal Raw Milk in South Korea.

Simple SummaryCharacterization of Enterococcus spp. among bovine mastitis pathogens is important in terms of the dairy industry and public health domain. The present study aimed to characterize virulence properties such as virulence genes (esp, asa1, gelE, and cylA), biofilm, gelatinase, hemolysis, and antimicrobial resistance and to compare them between bovine mastitis milk and bovine normal raw milk. Our results demonstrated that Enterococcus faecalis and Enterococcus faecium isolated from bovine mastitis milk exhibited higher virulence properties than those isolated from bovine normal raw milk. E. faecalis (90 isolates) was present at a significantly higher rate than E. faecium (32 isolates) and was more abundant in virulence genes. Furthermore, after analyzing the correlation between the virulence genes and the corresponding phenotype, we found that gelE and esp were involved in strong biofilm formation and the gelE was involved in gelatinase production. Taken together, E. faecalis in bovine mastitis milk should be monitored to control bovine mastitis and improve public health.Enterococcus spp. are pathogens that cause environmental mastitis and are difficult to eliminate owing to their resistance to antibiotics. To compare the virulence characteristics of isolates from bovine mastitis milk (BMM) and bovine normal raw milk (NRM), we isolated Enterococcus spp. from 39 dairy farms in South Korea from 2015–2020. A total of 122 Enterococcus spp. were identified, with Enterococcus faecalis (73.8%) accounting for the majority, followed by Enterococcus faecium (26.2%). E. faecalis isolated from BMM harbored gelE, asa1, esp, and cylA genes with a prevalence of 85.7, 71.4, 54.3, and 30.0%, respectively. These genes were significantly more abundant in BMM than in NRM, except for asa1 (p < 0.0001). Interestingly, strong biofilm and gelatinase formation was predominately observed for BMM isolates and this was significantly correlated to the presence of esp and gelE genes (p < 0.05). BMM isolates demonstrated higher resistance to tetracycline (59.3%), followed by chloramphenicol (21.0%), rifampicin (18.5%), doxycycline (4.9%), ciprofloxacin (1.2%), and nitrofurantoin (1.2%), than those from NRM. E. faecalis harboring esp, gelE, and cylA may be causative agents for bovine mastitis and act as a reservoir for the transmission of virulence factors to humans.

Association between virulence factors and biofilm formation in Enterococcus faecalis isolated from semen of infertile men.

Enterococcus faecalis is a common microbial semen contaminant. Although virulence factors and biofilm formation have often been analyzed in Enterococcus spp., there is little information about these features in isolates obtained from the genitourinary tract. This study was intended to characterize and determine the relationship between biofilm-forming ability and the presence of E. faecalis virulence factors isolated from human semen. A total of 32 patients diagnosed with primary infertility and 28 healthy men were included in the study. Semen analyses were performed according to the WHO guidelines. PCR reactions were applied for the detection of ace, esp, efeA, gelE, asa1, and cylA genes. Microtiter plate assay, via measurement of OD560, was used to measure the biofilm-forming ability of the isolates. Sixty E. faecalis isolates from semen of infertile and fertile men were characterized by phenotypic and genotypic methods. The prevalence of ace, esp, efeA, gelE, asa1 and cylA were reported to be 81.3%/100.0%, 81.3%/89.3%, 81.3%/85.7%, 71.9%/53.6%, 8.8%/75.0%, and 62.5%/67.9% in infertile/fertile groups; respectively. Strong, weak, and non-biofilm reactions were reported to be 50.0%/21.4%, 40.6%/64.3%, and 9.4%/14.3% in infertile and fertile groups; respectively. There was a significant relationship between fertility and weak biofilm reaction and also between biofilm formation and possession of the esp gene (P < .05). It could be speculated that colonization with E. faecalis with a strong ability for biofilm formation could become a potential threat to men's fertility.

Isolation of Enterococcus faecalis pathogenic genes isolated from oral infection by the effect of ionic liquids based on amino acid and its expression by real-time PCR.

Enterococcus faecalis is an important factor in nosocomial infections. The aim of this study was the isolation of the pathogenic genes of Enterococcus faecalis in mouth infection and the study of the expression of these genes by real-time PCR. In this study, 60 isolates of E. faecalis were isolated from oral infections. The presence and frequency of cyl, hyl, and esp genes and their expression by ionic liquid were evaluated using real-time PCR. MIC was determined by broth dilution method and biofilm production was measured, then biofilm inhibition ability and cytotoxicity test were performed by MTT method. The esp, cyl, and hyl genes were observed in 10, 11, and 2 isolates, respectively. cyl gene with the highest frequency of expression in the treated group was reduced by 1.14% under the influence of ionic liquid with methionine base. The results of MIC and Sub MIC concentrations were obtained with the effect of ionic liquid including 125 and 225µg/mL, respectively. Amino-acid-based ionic liquids can also reduce biofilm production at sub-MIC concentrations (P < 0.05), and changes in cytotoxicity at different concentrations and over time are significant (P-value < 0.001). E. faecalis strains are genetically diverse and this indicates the polyclonal prevalence of strains in clinical specimens. Combination treatment of ionic liquid with common antimicrobial drugs has good antibacterial effects against Enterococcus species, and ionic liquid with a minimum dose can be a good alternative to single-drug treatment of Enterococcus infections.

Molecular Detection of Some Vancomycin and Virulence Factor Genes in Enterococcus faecium

Multidrug-resistant (MDR) and pathogenic E. faecium is a crisis in healthcare settings. This survey aimed at antibiotic susceptibility profiling and virulence determinants of E. faecium. This study pooled 100 fecal E. faecium isolates identified by phenotypic and molecular tests. Antibiotic susceptibility and ampicillin MIC were determined according to clinical and laboratory standards institute (CLSI) 2017. Moreover, biofilm formation was assayed by a microtiter tissue plate assay. Virulence genes pattern was performed by polymerase chain reaction (PCR) method. Among 460 fecal samples, 100 isolates of E. faecium were identified, among which the highest resistance was related to penicillin (81%), cephalothin (78%), cefazolin (76%), tetracycline and cefepime (69%). In contrast, 83% of them were susceptible to vancomycin. Moreover, four vancomycin-resistant isolates had vancomycin MIC&gt;32µg/mL, and 11 isolates had MIC&gt;8µg/mL. Of 32 ampicillin-resistant isolates, 18 (56%) produced strong biofilm, and 14 isolates (44%) produced moderate biofilm. Among 17 vancomycin-resistant E. faecium (VREfa), 15 (88.23%) isolates produced strong biofilms, and two of them produced moderate-level biofilms, which was significantly higher than susceptible isolates (p=0.0144). The vanA (vancomycin MIC: 16-64µg/mL) and vanB (vancomycin MIC: 8-64µg/mL) genes were detected in twelve and five isolates, respectively. The rate of adhesin genes ace, esp and ebp included 68%, 97% and 82%, respectively. All the VREfa harbored the ace, esp and ebp genes. The antibiotic resistance rate of E. faecium was low. Biofilm formation was significantly different between VREfa and vancomycin-susceptible isolates but not between k9ampicillin-resistant and ampicillin-susceptible isolates. All the VREfa harbored the ace, esp and ebp genes. The virulence adhesin genes were not significantly associated with biofilm formation. Further studies are essential to appreciate better the relation between biofilm formation, drug non-susceptibility and adhesin genes. Keywords: E. faecium, antibiotic resistance, biofilm formation, virulence genes

Biofilm forming Enterococci and their Status as Emerging Multidrug Resistant Bacteria

Urine specimens were collected aseptically and inoculated directly onto CLED and MacConkey agar medium and incubated overnight. Single target colonies were sub cultured, and identification was based on culture, morphology, and biochemical characteristics. Enterococci isolates were subjected to biofilm assay (Tube method), and resistance pattern was determined for both biofilm-formers and non-biofilm-formers. Genomic DNA was extracted using the Bioneer kit, and the gene for virulence was detected by PCR and agarose gel electrophoresis. 148 Patients having one or more urinary symptoms were the study population. The aim of the study was to determine the multidrug resistant ability of biofilm-forming Enterococci. A number of growths were encountered; Enterococci accounted for only 9.46% (14), the highest prevalence was seen in the age group 21-30 (35.71%), and females (64.29%) were more prone to enterococcal infection than their males (35.71) counterpart. There is a relationship between biofilm production and antibiotic resistance because multidrug resistant Enterococci isolate produced bands against esp gene with an average of 510kbp. Biofilm forming strains showed the highest resistance to gentamicin and penicillin (83.3%), and vancomycin can be considered a good alternative therapy in enterococcal UTIs because of its lower resistance (75%).

Selection of Beneficial Bacterial Strains With Potential as Oral Probiotic Candidates.

This study aimed to select beneficial strains from the oral cavity of healthy volunteers and to evaluate these as potential oral probiotic candidates. The selection process was based on the isolation, differentiation, identification, and safety assessment of LAB strains, followed by a series of experiments for the selection of appropriate candidates with beneficial properties. In the screening procedure, 8 isolates from the oral cavity of a Caucasian volunteers were identified as Streptococcus (Str.) salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lactiplantibacillus plantarum (Lb.) (Lactobacillus plantarum) ST63HK and ST66HK; Latilactobacillus sakei (Lb.) (Lactobacillus sakei) ST69HK; and Lactobacillus (Lb.) gasseri ST16HK based on 16S rRNA sequencing. Physiological and phenotypic tests did not show hemolytic, proteinase, or gelatinase activities, as well as production of biogenic amines. In addition, screening for the presence of efaA, cyt, IS16, esp, asa1, and hyl virulence genes and vancomycin-resistant genes confirmed safety of the studied strains. Moreover, cell-to-cell antagonism indicated that the strains were able to inhibit the growth of tested representatives from the genera Bacillus, Enterococcus, Streptococcus, and Staphylococcus in a strain-specific manner. Various beneficial genes were detected including gad gene, which codes for GABA production. Furthermore, cell surface hydrophobicity levels ranging between 1.58% and 85% were determined. The studied strains have also demonstrated high survivability in a broad range of pH (4.0-8.0). The interaction of the 8 putative probiotic candidates with drugs from different groups and oral hygiene products were evaluated for their MICs. This is to determine if the application of these drugs and hygiene products can negatively affect the oral probiotic candidates. Overall, antagonistic properties, safety assessment, and high rates of survival in the presence of these commonly used drugs and oral hygiene products indicate Str. salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lb. plantarum ST63HK and ST66HK; Lb. sakei ST69HK; and Lb. gasseri ST16HK as promising oral cavity probiotic candidates.

Inhibitory Effects of Carvacrol on Biofilm Formation and Expression of Biofilm Related Genes in Clinical Isolates of Enterococcus faecalis.

Nowadays, novel antimicrobial strategies are being developed which focus on debilitating, rather than killing the microorganisms. In this regard, anti-biofilm therapy is one of the important ways to combat bacterial infections. Therefore, the aim of the current study was to evaluate the anti-biofilm activity of Carvacrol against E. faecalis by means of its effects on biofilm formation as well as on the gene expression levels of the two biofilm related genes, Epa and Esp. A total of 40 clinical strains of E. faecalis were collected from three hospitals in Tehran, Iran during 2020. These isolates were confirmed by biochemical and genotypic methods. Antibacterial and anti-biofilm activity of Carvacrol essence were determined according the standard protocol. Finally, expression level of the biofilm related genes (Epa and Esp) were evaluated before and after the treatment with Carvacrol. A total of 14 isolates were considered as strong biofilm producers and were used for analysis. Carvacrol essence showed the best antibacterial activity at 2,500 μg/mL concentration against all the isolates, the biofilm formation capacity was decreased by Carvacrol essence, and it was statistically significant (p < 0.05). Expression levels of the Esp gene were decreased in 5 isolates while increased in 3 isolates following the Carvacrol treatment. Ex-pression levels of the EpaI gene was significantly decreased (p < 0.05) in 4 isolates following the Carvacrol treatment. In conclusion, the results presented in this study suggest that carvacrol extract exhibits significant antimicrobial and anti-biofilm properties against E. faecalis, even against vancomycin resistant isolates.

GENETIC DIVERSITY OF ENTEROCOCCUS FAECALIS ISOLATES FROM CHILDREN WITH URINARY TRACT INFECTION IN PRIMORSKY KRAI OF RUSSIA

Background: Escherichia coli is the most common cause of urinary tract infections (UTI). However, Enterococcus faecalis has been shown to be the most common causative agent of UTI among children and newborns in Primorsky Krai of Russia warranting further research. Aim: To study the occurrence of pathogenicity factor genes in the E. faecalis isolates from children with UTI in the Primorsky Krai of the Russian Far East. Methods: Forty-two E. faecalis clinical isolates from children under the age of 16 with UTI identified in 2013-2017 were studied. Phylogenetic diversity of the strains was assessed by the ultilocus sequence typing. Six genes, namely, cylA, aggA, efaA, eep, gelE, esp were tested in the isolates by polymerase chain reaction . Results: CylA, aggA, efaA, eep, gelE and esp genes occurred in 50.0 %, 80.95 %, 100 %, 100 %, 76.2 % and 71.4 % of the isolates, respectively. Eleven different gene variants were detected for the combination of pathogenicity factor genes. The most common gene variants were (aggA, cylA, efaA, eep, gelE, esp) and (aggA, efaA, eep, gelE). Among the uropathogenic E. faecalis. Fourteen sequence-types were identified (ST6, ST16, ST21, ST25, ST40, ST41, ST64, ST116, ST133, ST151, ST179, ST480, ST537, ST774), with ST179, ST774, ST6 being the most common. Conclusions: The identified diversity of sequence-types indicates the genetic heterogeneity of uropathogenic enterococci isolated in the Primorsky Krai. The detection of a large amount of pathogenicity factors and their combinations causes the predominance of E. faecalis in the region as a clinically relevant etiological agent of UTI among children. The identification of highly virulent sequence types such as E. faecalis ST6, ST179 and ST774 warrants further research to determine the population structure of enterococci.

Analysis of antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism

ABSTRACT This research was to explore antibiotic-induced drug resistance of Salmonella enteritidis and its biofilm formation mechanism. Kirby-Bauer (K-B) disk method recommended by Clinical and Laboratory Standards Institute (CLSI) was used to test drug sensitivity of Salmonella enteritidis to 16 kinds of antibiotics including ß-lactams, aminoglycosides, quinolones, sulfonamides, chloramphenicols, and tetracyclines. Polymerase chain reaction (PCR) was performed to detect carrying of drug resistance genes of 29 kinds of antibiotics including ß-lactams, aminoglycosides, quinolones, sulfonamides, chloramphenicols, and tetracyclines of Salmonella enteritidis. The expressions of esp, ebpA, ge1E, and fsrB genes in biofilm group and plankton group were detected when Salmonella was induced, and difference of gene expression was detected by FQ-PCR. The drug resistance rates of Salmonella enteritidis to nalidixic acid, ampicillin, streptomyces, and cefoperazone were high, which were 94.5%, 75%, 67%, and 52%, respectively. 94 strains of Salmonella enteritidis formed 22 kinds of drug resistance spectrum, the strains were generally resistant to 4-5 antibiotics, and some strains formed fixed drug resistance spectrum as follows: AMP-CFP-STR-NA-TE (22.6,21.7%), AMP-STR-NA-TE (17,16%), and AMP-CFP-STR-NA (11.1,10.6%). During biofilm formation, fsr can increase expression of ge1E and decrease expression of esp and ebpA. Consequently, Salmonella enteritidis was generally resistant to nalidixic acid, ampicillin, and streptomycin, and the multidrug resistance was severe. The drug resistance genes sul2, sul3, blaTEM-1-like, tet(A), and tet(G) were highly carried in Salmonella enteritidis. Esp, ebpA, ge1E, and fsrB genes were closely related to biofilm formation of Salmonella enteritidis.

Characterization of Enterococcus spp. isolated from a fish farming environment in southern Brazil.

The aim of present study is to characterize the resistance and virulence profile of enterococci isolated from aquaculture excavated ponds and masonry tanks (6 samples) in southern Brazil. Samples were cultured in selective medium, 10 colonies were randomly selected from each sample, which were identified by MALDI-TOF and tested against 13 antimicrobials. The presence of resistance (tetL, tetM, tetS, ermB and msrC) and virulence (ace, esp, agg, cylA and gelE) genes were determined by PCR. A total of 79 enterococci were identified, and Entecococcus faecalis (44.3%) and E. casseliflavus (36.7%) were the most prevalent species isolated. Sixty-five strains (82.3%) were resistant to at least one of the antimicrobials tested, whereas 27 (34.2%) strains were multiresistant. The overall percentages of antimicrobial resistant isolates were: 58.2% to rifampicin, 40.5% to fluoroquinolones, 36.7% to erythromycin and 30.4% to tetracycline. The tetL and tetM genes were found in 57.7% of the tetracycline-resistant strains; and msrC in 31.01% of erythromycin-resistant strains. The most frequently detected virulence factors were ace and gelE genes. Although limited to a single farm, these data suggest that aquaculture may be a reservoir of resistant and virulent enterococci. This study is the first step towards enhancing our understandingof distribution, resistance and virulence profile in enterococci isolated from fish farming environments in the south Brazil.

Survey on phenotypic resistance in Enterococcus faecalis: comparison between the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells.

Phenotypic resistance is considered as a serious therapeutic challenge for which a definitive remedy has not been discovered yet. Biofilm and persister cell formation are two well-studied phenotypic resistance phenomena, leading to the recalcitrance and relapse of different types of chronic infections. The presence of persister cells in biofilm structures seems to be one of the main factors contributing to the relapse of infections and treatment failure. Given the dormant and inert nature of persister cells, they can be easy targets for the immune system factors. Biofilm formation can be a survival strategy for the defenseless persister cells. Thus, this study was aimed to evaluate the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells. Vancomycin susceptibility and biofilm formation ability were investigated among 95 E. faecalis clinical isolates using microtiter broth dilution and microtiter plate assays, respectively. PCR was used to determine the presence of biofilm-related genes (gelE, esp, and agg) among the vancomycin-susceptible, biofilm producer E. faecalis isolates (91 isolates). Minimum bactericidal concentration for biofilms (MBCB) were determined for vancomycin using the MTP assay. Bacterial persister assay was performed using an enzymatic lysis assay. Finally, the expression of biofilm-related genes was compared between the persister and non-persister isolates of E. faecalis using real-time qPCR. E. faecalis isolates showed a high level of susceptibility (95.8%) to vancomycin (MIC < 1µg/mL). The gelE, esp, and agg genes were found in 91 (100%), 72 (79.12), and 74 (81.32) of the isolates, respectively. All the E. faecalis isolates were tolerant to vancomycin in the biofilm condition, showing a MBCB of > 2500µg/mL. Based on the enzymatic lysis assay, only 3 isolates, out of the 91, had the ability to form persister cells. The expression of biofilm-associated genes was higher among the persister compared to non-persister E. faecalis isolates. Biofilm-associated persister cells indicated a high vancomycin tolerance compared to non-persister cells. Moreover, persister isolates showed a higher tendency for biofilm formation and a higher expression level of the biofilm-associated genes, compared to non-persister isolates.

Nasal Colonization of Serine Protease esp-Positive Staphylococcus epidermidis Affecting Staphylococcus aureus

Background: Staphylococcus aureus is a common colonizer of the nasal vestibule found in approximately 20%–30% of healthy adults, while Staphylococcus epidermidis appears to be the most frequent colonizer in all regions of the upper respiratory tract. Esp, a serine protease of S. epidermidis, was reported to inhibit S. aureus colonization. This study was performed to examine the nasal colonization of S. aureus and S.epidermidis and the presence of esp determinants. Methods: Nasal swab specimens from 54 patients were cultured on blood agar plates (BAP) and selective media for S. aureus for bacteria isolation. After 48 hours of incubation with BAP, three or four colonies suspected of being coagulase-negative staphylococci (CNS) were identified by mass spectrometry. Polymerase chain reaction (PCR) for esp was performed on all CNS isolates identified as S. epidermidis. Results: Forty-three S.epidermidis strains were isolated from 18 (33.3%) of the 54 patients.Nine (50.0%) of the 18 patients carried S. aureus, while the other nine did not. Of the 36 S. epidermidis non-carriers, 13 (36.1%) were colonized by S. aureus. All S. epidermidis strains were confirmed by PCR to have esp determinants. Conclusion: S. epidermidis colonization did not affect S. aureus colonization in the nasal cavity. All S.epidermidis strains harbored the esp gene. There were no differences in the nasal colonization rates of S. aureus according to the presence of esp-positive S. epidermidis. Further research on the characterization of S.epidermidis in Korea is needed to understand the association between S. epidermidis and S. aureus colonization.

Effects of osmotic and high pressure stress on expression of virulence factors among Enterococcus spp. isolated from food of animal origin

The aim of study was to determine the occurrence of virulence factors and virulence-related genes among enterococci isolated from food of animal origin and effects of osmotic and high pressure stress on expression of virulence-related genes. The number of 78 isolates were analyzed. None of them showed a strong ability to form biofilm, 38.5% (n = 30) had the slime production ability, 41% (n = 32) had gelatinase activity, γ -type hemolysis was observed in 55% of isolates, and α-type hemolysis in 45%. All of the isolates carried 1–13 virulence-related genes. The most common genes were gelE (85.9%), sprE (78.2%) and asa1 (75.6%). There were also observed changes in the expression of the gelE, esp, asa1 and cylL genes in response to various NaCl concentration and high pressure processing. Results obtained in this study indicate that enterococci isolated from food may act as reservoirs of virulence genes. The presence of virulence factors among enterococci, especially the ability to biofilm formation is important for food safety and the protection of public health. The results presented in our work demonstrate that stress that can occur during food preservation and food processing can induce the changes in the virulence-related genes expression.

Identification of bacterial communities of fermented cereal beverage Boza by metagenomic analysis

Bacterial microbiota of directly studied and pre-enriched Boza samples were investigated by metagenomic analysis. Virulence gene contents, biofilm formation, antibiotic susceptibility and clonal relationships of enterococci present in pre-enriched Boza samples were determined. Chemical properties of the samples were also investigated. Although directly studied samples showed a dominance by Lactococcus, Lactobacillus, Leuconostoc, and Streptococcus. NGS upon pre-enrichment of the same Boza samples demonstrated a dominance by Lactococcus, Enterococcus, Escherichia/Shigella, Bacillus, and Lactobacillus. All enterococci were identified as Enterococcus faecium and none of them was positive for vanA, vanB, vanC1, vanD, vanE, vanG, agg, gelE, efaAfs, cylA, ace, hyl, cob, cylB, and cylM genes. However, efaAfm, ccf, cpd, and esp genes were detected in the strains. Only one strain formed biofilm and seven strains showed low adherence. E. faecium strains were resistant to rifampin and erythromycin. PFGE revealed 54–100% clonal relationships of E. faecium strains. Percent acidity of Boza samples were 0.14%–0.51%, pH was 3.00–4.07, protein content was 0.35–1.23 mg/100 mg, total sugar content was 9.64–19.21 mg/100 mg Boza, crude ash content was 0.05–0.18 mg/100 mg dry sample, total dry matter was 13.79–28.04 mg/100 mg. Our results indicate to importance of the dynamics nature of microbial communities involved in Boza fermentation and virulence properties of enterococci.

Enterococcus faecalis and Enterococcus faecium in pasteurized milk: Prevalence, genotyping, and characterization of virulence traits

To assess the prevalence of enterococci in pasteurized milk and characterize their virulence traits, 416 pasteurized milk samples were purchased from retail stores in the Northwest of Iran. Enterococci were isolated by conventional methods and confirmed by genus-specific PCR. Amongst the samples, 269 were identified as either Enterococcus faecalis or E. faecium, which were then subjected to genotypic and phenotypic analyses. Using enterobacterial repetitive intergenic consensus (ERIC)-PCR, E. faecalis and E. faecium strains were effectively grouped into 15 and 16 clusters, respectively. Nonetheless, ERIC-PCR was unable to accurately cluster E. faecalis and E. faecium strains according to their virulence or phenotypic characteristics. Based on the results, most of the isolates, especially E. faecalis strains, carried gelE, hyl, asa1, esp, cylA, ace, and efaA genes. Results also revealed that E. faecalis and E. faecium isolates were highly resistant to most of the tested antibiotics. The resistance to vancomycin for E. faecalis and E. faecium was estimated at 71.9% and 77.6%, respectively. Moreover, 81% and 69% of E. faecalis and E. faecium strains were able to produce biofilm. It was concluded that pasteurized milk could be an intermediate vehicle for the transmission of multidrug-resistant virulent enterococci strains to humans.

Resistance and virulence distribution in enterococci isolated from broilers reared in two farming systems

BackgroundThe impact of enterococci in human health has been growing for the last decades, mainly due to their resistance to several antimicrobial agents. Human consumption of contaminated meat, especially poultry, has been identified as a possible route of transmission. The aim of the present study was to evaluate and compare the antimicrobial resistance profiles and virulence genes of enterococci isolated from Portuguese conventional and free-range broiler farms.ResultsAntibiotic susceptibility testing showed high frequencies of resistance to tetracycline in both farming systems. Resistance to erythromycin and gentamicin were detected in about half of the isolates. Resistance to penicillin was the less frequently observed and no vancomycin resistant isolates were identified. The majority of the enterococcal isolates, from either farming systems, were resistant to more than one antibiotic, and no statistical associations were found, except for penicillin resistance which associated with the genetic clusters. No differences were found between farming systems regarding the prevalence of tet(M), erm(B), aac (6′)-Ie-aph (2″)-Ia and pbp5 genes, nevertheless pbp5 prevalence was associated with the different genetic clusters. Hemolytic activity was identified in 26.47% of all isolates and gelatinase activity in 50%. The gelE gene was identified in the majority of the isolates, whereas esp and agg genes were rarely detected. The cylA determinant was not detected in any of the isolates.ConclusionsOverall, results suggest that similar resistance patterns and virulence genes can be found in both farming systems, though enterococci in free-range conditions should be less prone to acquire further resistance genes.

D-Alanine metabolic pathway, a potential target for antibacterial drug designing in Enterococcus faecalis

Enterococcus faecalis (E. faecalis) is associated with persistent root canal infection because of its biofilm and various virulence factors. However, E. faecalis exhibits extensive drug resistance. d-Alanine (D-Ala) metabolism is essential for bacterial peptidoglycan biosynthesis. d-cycloserine (DCS), a second line drug used in the treatment of Mycobacterium tuberculosis infection, can inhibit two key enzymes in D-Ala metabolism: alanine racemase and d-alanine–d-alanine ligase. The aim of this study was to evaluate the effect of D-Ala metabolism on E. faecalis growth, cell wall integrity, biofilm formation and virulence gene expression by additional DCS with or without D-Ala. The results showed that DCS inhibited the planktonic growth and biofilm formation of E. faecalis in a dose-dependent manner. Both the minimum inhibitory concentration (MIC) and minimum biofilm inhibition concentration (MBIC) of DCS against E. faecalis were 200 μg/ml, whereas 50 μg/ml of DCS could inhibit planktonic growth and biofilm formation effectively. The addition of DCS also resulted in bacterial cell wall damage, biofilm surface roughness increase and biofilm adhesion force reduction. Moreover, the treatment of DCS downregulated the expression of asa1, esp, efaA, gelE, sprE, fsrB and ace genes. However, all of these inhibitory effects of DCS could be rescued by the addition of exogenous D-Ala. Meanwhile, DCS exhibited no toxicity to HGEs and HOKs. Therefore, D-Ala metabolic pathway in E. faecalis is a potential target for drug designing.