Aggregation Substance Research Articles

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.

Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages.

Enterococcus faecalis represents an important factor of hospital-associated infections (HAIs). The knowledge on its evolution from a commensal to an opportunistic pathogen is still limited; thus, we performed a study to characterise distribution of factors that may contribute to this adaptation. Using a collection obtained from various settings (hospitalised patients, community carriers, animals, fresh food, sewage, water), we investigated differences in antimicrobial susceptibility, distribution of antimicrobial resistance genes, virulence-associated determinants and phenotypes, and CRISPR loci in the context of the clonal relatedness of isolates. Bayesian Analysis of Population Structure revealed the presence of three major groups; two subgroups comprised almost exclusively HAI isolates, belonging to previously proposed enterococcal high-risk clonal complexes (HiRECCs) 6 and 28. Isolates of these two subgroups were significantly enriched in antimicrobial resistance genes, presumably produced a polysaccharide capsule and often carried the aggregation substance asa1; distribution of other virulence-associated genes, such as esp and cyl, formation of a biofilm and gelatinase production were more variable. Moreover, both subgroups showed a low prevalence of CRISPR-Cas 1 and 3 and presence of small CRISPR2 variants. Our study confirms the importance of HiRECCs in the population of E. faecalis and their confinement to the hospital settings.

Bacteriocin producing Enterococcus faecalis isolated from chicken gastrointestinal tract originating from Phitsanulok, Thailand: Isolation, screening, safety evaluation and probiotic properties

A total of 230 isolates of lactic acid bacteria were isolated from gastrointestinal tracts (GIT) of indigenous chickens purchased from local market in Bang Rakam district, Phitsanulok, Thailand. Among 230 isolates, only 7 isolates, named CM6CR07, CM6CR11, CF1GI15, CF1GI14, CF1GI17-1, CF1GI17-2 and CF1GI 19 showed an inhibition zone against indicator strains. Bacteriocin activity was completely inactivated when Neutralized Cell Free Supernatants (NCFS) of the isolates were treated with proteases, confirming the proteinaceous nature of the bacteriocin component. NCFS of all selected bacteriocin producing lactic acid bacteria (SBP-LAB) retained their bacteriocin activity after heating at 100 °C for 30 min. All of SBP-LAB were identified as Enterococcus faecalis. These Ent. faecalis isolates harbored the same virulence genes. All strains were positive for gelE (gelatinase), efaAfs (cell wall adhesins), hyl (hyaluronidase), ace (adhesin of collagen protein), cylLL (cytolysin structural subunit) and cylLS (cytolysin structural subunit) and were negative for asa1 (aggregation substance), agg (aggregation protein involved in adherence to eukaryotic cells), and esp (enterococcal surface protein). These Ent. faecalis showed a wide range of inhibitory spectrum against food-spoilage and food-borne pathogens when studied by agar spot test. The CFS and NCFS also inhibited some of food-spoilage and food-borne pathogenic indicator strains when checked by agar well diffusion assay. All of these Ent. faecalis survived simulated stomach conditions, whereas all of them could survive in conditions of simulated intestinal juice.

Safety of Lactobacillus plantarum ST8Sh and Its Bacteriocin.

Total DNA extracted from Lb. plantarum ST8Sh was screened for the presence of more than 50 genes related to production of biogenic amines (histidine decarboxylase, tyrosine decarboxylase, and ornithine decarboxylase), virulence factors (sex pheromones, gelatinase, cytolysin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors), and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). Lb. plantarum ST8Sh showed a low presence of virulence genes. Only 13 genes were detected (related to sex pheromones, aggregation substance, adhesion of collagen, tetracycline, gentamicin, chloramphenicol, erythromycin, but not to vancomycin, and bacitracin) and may be considered as indication of safety for application in fermented food products. In addition, interaction between Lb. plantarum ST8Sh and drugs from different groups were determined in order to establish possible application of the strain in combination with commercial drugs. Cytotoxicity of the semi-purified bacteriocins produced by Lb. plantarum ST8Sh was depended on applied concentration-highly cytotoxic when applied at 25μg/mL and no cytotoxicity at 5μg/mL.

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.

血小板凝集能検査法に関する検討 : 第三報 血小板放出物質との関連について

血小板凝集能検査法に関する検討 : 第三報 血小板放出物質との関連について

Genotypic characterization of Enterococcus species isolated from the oral cavity and their pattern of antibiotic susceptibility

Background: Enterococci comprise a small share of oral flora and occur as contaminants in food such as meat and cheese. They are commonly encountered in patients with infections of oral tissues such as necrotic pulp, root canals and periodontitis. Objectives: To characterize the genotypes of Enterococcus isolated from patients with oral infection and to determine their antibiotic susceptibility. Methods: We genotypically characterized a number of isolates of Enterococcus species derived from patients with various oral infections. All the isolates were tested for the presence of putative virulence genes; efaA (gene for endocarditis), gelE (gene for gelatinase), ace (gene for collagen binding antigen), asa (gene for aggregation substance), cylA (gene for cytolysin activator), and esp (gene for surface adhesin) of E. faecalis and E. faecium as described in previous studies. Results: E. faecalis dominated in oral infections compared with E. faecium. Isolates were susceptible to most antibiotics (only four were resistant to vancomycin). To our knowledge, the first report from this region. Conclusions: Treatment of enterococcal infections of the oral mucosal and deep oral areas necessitate microbiological diagnosis and identification of resistance strains. Keywords: Antibiotic susceptibility pattern, Enterococcus species, genotypic characterization, oral infections

Effects of sarang semut (Myrmecodia Pendens Merr. & Perry) extracts on Enterococcus faecalis sensitivity

Background: Enterococcus faecalis (E. faecalis) is a gram positive oral pathogen that reported at the main agent infection of endodontic treatment. Its activities are influenced by the virulence factors facilitating the interaction process between agents with host cells. Like aggregation substance, cytolysin, extracellular superoxide, gelatinase, hyaluronidase, sex pheromones, and surface adhesions molecules. Plant extracts are reported as the material antibacterial as well as E. faecalis in pathogenesis of endodontic infections. Purpose: Purpose of this study was to analyse of sarang semut extracts (Myrmecodia Pendens Merr. & Perry) towards sensitivity of E. faecalis. Method: This research used the methanol extract of sarang semut, E. faecalis ATCC 29212, and fosfomycin also chlorhexidine as the positive controls. Whereas, Bradford protein method was measured the concentration of the surface protein of E. faecalis and active component of the sarang semut extract. Result: Generally, the sarang semut extract possessed low sensitivity toward E. faecalis (≤ 13 mm), but on the concentrations of 100 µg/ml and 75 µg/ml better than inhibition of other concentrations, round 10.6-11.6 (mm). Specifically, on 100 µg/ml has indicator the minimal bactericidal concentration (MBC) on E. faecalis. Whereas minimal inhibition concentration (MIC) on the concentration of 3,125 µg/ml. Conclusion: Based on MBC and MIC assay, the extract of sarang semut has potential effects to adherence growth of E. faecalis, mainly on the highest concentration 100 µg/ml also MIC on 3,125 µg/ ml.

PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis.

Upon sensing of the peptide pheromone cCF10, Enterococcus faecalis cells carrying pCF10 produce three surface adhesins (PrgA, PrgB or Aggregation Substance, PrgC) and the Prg/Pcf type IV secretion system and, in turn, conjugatively transfer the plasmid at high frequencies to recipient cells. Here, we report that cCF10 induction is highly toxic to cells sustaining a deletion of prgU, a small orf located immediately downstream of prgB on pCF10. Upon pheromone exposure, these cells overproduce the Prg adhesins and display impaired envelope integrity, as evidenced by antibiotic susceptibility, misplaced division septa and cell lysis. Compensatory mutations in regulatory loci controlling expression of pCF10-encoded prg/pcf genes, or constitutive PrgU overproduction, block production of the Prg adhesins and render cells insensitive to pheromone. Cells engineered to overproduce PrgB, even independently of other pCF10-encoded proteins, have severely compromised cell envelopes and strong growth defects. PrgU has an RNA-binding fold, and prgB-prgU gene pairs are widely distributed among E. faecalis isolates and other enterococcal and staphylococcal species. Together, our findings support a model in which PrgU proteins represent a novel class of RNA-binding regulators that act to mitigate toxicity accompanying overproduction of PrgB-like adhesins in E.faecalis and other clinically-important Gram-positive species.

Occurrence of Antibiotic Resistance and Virulent Factors in Enterococcus faecalis Isolated from Bush Meat Roasted and Sold along Road Sides in Ekiti State

Background and Objectives: In Nigeria wild animals are hunted for meat mainly. Meanwhile, meat from wild animals are known to consist of pathogens apart from being contaminated by unhygienic environments (road sides) where they are mainly prepared and sold. The objective of this study was to investigate the occurrence of Enterococcus faecalis (E. faecalis) in samples of roasted bush meat prepared and sold along Ado-Ekiti-Ilesha road. Materials and Methods: A total of 182 roasted bush meat samples were collected in seven selected towns in Ekiti State, Nigeria between January and February, May and June, 2016 representing dry and rainy seasons, respectively. The samples were examined for the presence of enterococci within 2 h of collection. Standard methods were used to identify Enterococcus faecalis, determined its resistance to antibiotics and also determine the virulence factors in the sample. Results: A total of 91 (32.38%) out of 281 samples of roasted bush meat examined were contaminated with E. faecalis. The highest rate of contamination 61.22% was observed in samples collected from Igede-Ekiti while the least 6.70% was observed from samples collected in Ado-Ekiti. Rates of contamination among samples from other selected towns were, 40, 38.71 and 38.1% from Efon-Alaye-Ekiti, Iyin-Ekiti and Erio-Ekiti, respectively. Antibiotic susceptibility test results reveal that some of the isolates have acquired resistance to a number of antibiotics. High resistance rate was recorded against ampicillin 35.71%, followed by gentamicin 30.22%, ciprofloxacin 28.02% and ofloxacin 24.73%. The incidence of virulence factors was low in all the isolates with aggregation substance, haemolysin and gelatinase recording 7.69, 8.24 and 27.47%, respectively. Conclusion: The incidence of virulence factors in E. faecalis is an evidence of potential pathogenesis. The roasted bush meat screened from road sides in Ekiti State was contaminated with E. faecalis. There is need for strict monitoring and proper hygiene education for the food handlers in the study area.

The Determination of Virulence Factors among Fish Originated Enterococci

Enterococci are commensal organisms of human and animals, and may cause diseases in particular conditions. Several virulence factors are responsible in the production of diseases. The aim of this study was to isolate enterococci from fish and to determine virulence factors of the isolates. A total of 26 (13%) Enterococcus faecalis strains were isolated from live, moribund and dead fish collected from fish farms in Aegian Region. Cytolysin and gelatinase activities and aggregation substance production of these strains were examined.Cytolysin production was not detected in any of E. faecalis strains. Of 26 strains tested, 27% was found to produce aggregation substance. Gelatinase activity was found in 11.5% of strains. The presence of strains with important virulence factors in enterococci from fish was established. It was suggested that these strains have the potential of producing disease in human and animals.

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

Soil humic acid and aggregation as restoration indicators of a seasonally flooded riparian forest under buffer zone system

A degraded riparian forest that has lost vegetation and has suffered periodic flooding was rehabilitated using native species under buffer zone system which was stratified according to the distance from the river (Zones I, II, III). To restore the soil stabilisation and water flow we compared the soil aggregation, porosity and soil humic substances of the experimental site (ES) with those of a disturbed site (DS) and a preserved site (PS). Six years post-transplantation, soil aggregation and micro-porosity were improved in the ES relative to the DS, but only micro-porosity achieved a pattern similar to that of the PS in Zone III. Differences in soil aggregation between ES and PS were correlated with the negative effect promoted by a high aliphatic-humic acid composition in Zone I and by an undifferentiated distribution of fulvic acid among sites which were attributed to the flooding effect. Similarities among ES and PS were related to aromatic:vinylhumic acid ratio, allowing for the clustering of samples from Zones II and III of both the PS and the ES, via a principal component analysis. Samples from Zones I of ES and PS were separated driven by aliphatic and fulvic acid contributions, while all of the DS samples formed an isolated group under the influence of aliphatic species and macro-porosity. Based on these attributes, the ES is evolving towards the PS, and the rehabilitation process has attained an intermediate phase of restoration. The zones system adopted for riparian forest restoration allowed the use of soil humic substances, aggregates and micro-porosity as indicators of restoration.

Detection of Virulence Genes in Enterococci Isolated From the Human Normal Flora by Multiplex-Polymerase Chain Reaction

Background Enterococcus species recently evolved from a common avirulent normal flora into a multidrug–resistant, health care–associated pathogen causing difficult-to-treat diseases. The aim of the current study was the detection of aggregation substance (asa), hyaluronidase (hyl), cytolysin (cyl), enterococcal surface protein (esp), collagen-binding protein (acm), gelatinase (gelE), and Enterococcus faecalis endocarditis antigen genes in Enterococcus faecium isolated from healthy volunteers by the Multiplex-polymerase chain reaction (PCR) method. Methods Stool samples were obtained from 24 healthy volunteers. After biochemical and microbiological tests, all isolates were tested for the presence of the virulence genes by Multiplex-PCR. Results In total, 91 E. faecium strains were collected from 54 different REP-PCR patterns. The distribution of the virulence genes showed that 33 (36%), 14 (15%), 3 (3%), 1 (1.1%), and 0 isolates were positive for acm, asa, esp, cylA, hyl, and gelE genes, respectively. Conclusions Despite the fact that E. faecium isolated from normal human intestines usually shows low incidence of virulence genes, they may act as harbors of virulence factors, allowing the distribution of these genes to the human normal flora through the food chain.

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria

ObjectivesTo evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). MethodsAdhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. ResultsThe overall prevalence was: esp – 44.3%, agg/asa1 – 38.4%, ace/acm – 64.3%, efaA – 85.9%, eep – 69.4%, gelE – 64.3%, hyl – 25.1%, and cylA – 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. ConclusionMost E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study.

Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci.

Background:The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression.Materials and Methods:A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes.Results:Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only.Conclusion:Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their capacity to form biofilms.

The controversial nature of the Weissella genus: technological and functional aspects versus whole genome analysis-based pathogenic potential for their application in food and health.

Despite the use of several Weissella (W.) strains for biotechnological and probiotic purposes, certain species of this genus were found to act as opportunistic pathogens, while strains of W. ceti were recognized to be pathogenic for farmed rainbow trout. Herein, we investigated the pathogenic potential of weissellas based on in silico analyses of the 13 whole genome sequences available to date in the NCBI database. Our screening allowed us to find several virulence determinants such as collagen adhesins, aggregation substances, mucus-binding proteins, and hemolysins in some species. Moreover, we detected several antibiotic resistance-encoding genes, whose presence could increase the potential pathogenicity of some strains, but should not be regarded as an excluding trait for beneficial weissellas, as long as these genes are not present on mobile genetic elements. Thus, selection of weissellas intended to be used as starters or for biotechnological or probiotic purposes should be investigated regarding their safety aspects on a strain to strain basis, preferably also by genome sequencing, since nucleotide sequence heterogeneity in virulence and antibiotic resistance genes makes PCR-based screening unreliable for safety assessments. In this sense, the application of W. confusa and W. cibaria strains as starter cultures or as probiotics should be approached with caution, by carefully selecting strains that lack pathogenic potential.

Prevalence, antimicrobial resistance and virulence traits in enterococci from food of animal origin in Turkey

The objective of this work was to investigate the antibiotic susceptibility, the mechanisms implicated and the potential virulence genes (gelatinase [gelE], cytolysins [cylA, cylM, cylB], cell wall adhesins [efaAfs and efaAfm], enterococcal surface protein [esp], sex pheromones [cpd, cob, ccf], enhanced expression of pheromone [eep], aggregation substance [aggA]) in enterococci isolated from retail chicken and beef meat samples in Hatay, Turkey. Hundred-one (96%) isolates from chicken meat and sixty-three (63%) from minced meat isolates showed resistance to at least one of the 12 antimicrobial agents tested. The highest frequency of resistance was against tetracycline (89.5% and 53%), erythromycin (59% and 2%), ciprofloxacin (35.2% and 12%) and trimethoprim/sulfamethoxazole (34.3% and 7%) for isolates from chicken and beef samples, respectively. The ermB, tetM and tetL genes were predominantly detected by PCR. Five enterococci from chicken meat were found to be phenotypically resistant to vancomycin and carried the vanA gene. The presence of virulence genes including gelE, ccf, cpd, efaAfs, and aggA were frequently detected. The results of this study show that retail chicken and beef meat is source of concern for public health due to having high prevalence of antibiotic resistance and as well as harbouring virulence factors.

Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs

We documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes. We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. The staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes. These findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patient's, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.

The Intraperitoneal Transcriptome of the Opportunistic Pathogen Enterococcus faecalis in Mice.

Enterococcus faecalis is a Gram-positive lactic acid intestinal opportunistic bacterium with virulence potential. For a better understanding of the adapation of this bacterium to the host conditions, we performed a transcriptome analysis of bacteria isolated from an infection site (mouse peritonitis) by RNA-sequencing. We identified a total of 211 genes with significantly higher transcript levels and 157 repressed genes. Our in vivo gene expression database reflects well the infection process since genes encoding important virulence factors like cytolysin, gelatinase or aggregation substance as well as stress response proteins, are significantly induced. Genes encoding metabolic activities are the second most abundant in vivo induced genes demonstrating that the bacteria are metabolically active and adapt to the special nutrient conditions of the host. α- and β- glucosides seem to be important substrates for E. faecalis inside the host. Compared to laboratory conditions, the flux through the upper part of glycolysis seems to be reduced and more carbon may enter the pentose phosphate pathway. This may reflect the need of the bacteria under infection conditions to produce more reducing power for biosynthesis. Another important substrate is certainly glycerol since both pathways of glycerol catabolism are strongly induced. Strongly in vivo induced genes should be important for the infection process. This assumption has been verified in a virulence test using well characterized mutants affected in glycerol metabolism. This showed indeed that mutants unable to metabolize this sugar alcohol are affected in organ colonisation in a mouse model.