Molecular Assessments of Antimicrobial Protein Enterocins and Quorum Sensing Genes and Their Role in Virulence of the Genus Enterococcus.

Enterococcus has emerged as an opportunistic pathogen because of its antibiotic resistance and virulence profile, which makes it a causative agent of several diseases like endocarditis, surgical site, and urinary tract infections. Currently, species of this genus are the 2nd most frequently isolated microorganisms from hospital-acquired infections. Significant association with hospitals and unhygienic conditions of the environments has made them resistant to a wide range of antibiotics. On the brighter side, enterococci have the ability to produce antimicrobial proteins (i.e., enterocins) that exhibit wide antagonistic activity, thus making them useful microbes in the food and pharmaceutical industries. Enterocins are also involved in niche control in gut microbiota which is regulated by the quorum sensing (QS) system. A bacterial communication system that is controlled by the fsr operon in enterococci consists of FsrABDC, ef1097, and GelE/SprE genes. Hence, the present study was conducted for molecular assessment of enterocins and quorum sensing genes, inter-environmental correlation, and species prevalence of enterococci isolated from different environmental niches of Karachi, Pakistan. Obtained results revealed the highest prevalence of E. faecium and E. faecalis in all environments. Bacterial antagonism and enterocin genes were observed significantly high in poultry environments. The inter-environmental correlation indicated a strong positive correlation of freshwater with sewage and soil environments. Similarly, the fsr regulatory system was mostly identified in poultry-related environments, and a significant correlation between QS system and biofilm formation was established. In conclusion, this study confirmed the high prevalence of E. faecium in all tested sources, high enterocin production in non-clinical environments, and more fsr regulatory genes in poultry-related environments.

Molecular Assessment and Validation of the Selected Enterococcal Strains as Probiotics.

Probiotics are live microorganisms which confer health benefits to the host. Lactic acid bacteria (LAB) are used as probiotics since decades. Enterococci being the member of LAB have proven probiotic strains; therefore, this study was aimed at finding out the potential probiotic candidates from the pool of locally isolated strains. For initial screening, one hundred and twenty-two strains were selected and subjected to different confirmatory and phenotypic tests to choose the best strains that have potential probiotic criteria, i.e., no potential virulence traits, antibiotic resistance, and having tolerance properties. Keeping this criterion, only eleven strains (n = 11) were selected for further assessment. All virulence traits such as production of hemolysin, gelatinase, biofilm, and DNase were performed and not found in the tested strains. The molecular assessment indicates the presence of few virulence-associated genes in Enterococcus faecalis strains with variable frequency. The phenotypic and genotypic assessments of antibiotic resistance profile indicate that the selected strain was susceptible to ten commonly used antibiotics, and there were no transferrable antibiotic resistance genes. The presence of CRISPR-Cas genes also confirmed the absence of antibiotic resistance genes. Various enterocin-producing genes like EntP, EntB, EntA, and EntQ were also identified in the selected strains which make them promising probiotic lead strains. Different tolerance assays like acid, NaCl, and gastric juice tolerance that mimic host conditions was also evaluated by providing artificial conditions. Cellular adhesion and aggregation properties like auto- and co-aggregation were also checked and their results reflect all in the favor of lead probiotic strains.

The unravelled Enterococcus faecalis zoonotic superbugs: Emerging multiple resistant and virulent lineages isolated from poultry environment.

This study aimed to investigate the zoonotic potential by virtue of phylogenetic analysis, virulence and resistance gene profiles of Enterococcus faecalis originating from poultry environment. The ERIC, BOX and RAPD PCR analysis showed the clustering of E. faecalis strains (n = 74) into five groups (G1-G5) and fifteen sub-clusters (B1-B15), which share 50%-80% similarities with ATCC E. faecalis and clinical strains of human infection. E. faecalis strains harboured seven enterocins genes including ent1097 (85%), entB (84%), enterolysinA (51%), entSEK4 (51%), entL50 (31%), entA (25.7%) and ent1071 (14.9%). The highest prevalence of gelE-sprE (90%), lip-fl (90%) followed by cylL (62%), hyl (60%), katA (16%) and cylA (5.4%) was observed in poultry isolates. The fsr operon and gelE-sprE was co-associated in 66.2% strains. E. faecalis also harboured biofilm and endocarditis-associated genes, including efaAfs (97%), ebp-pilli (ebpABC and srtC 69.9%-80%), asa1 (71%), agg (55%), ace (54%) and esp-Tim (3%). Despite all found sensitive to vancomycin, 98.6% strains were multi-drug resistant to five to twelve tested antimicrobials. An increased-level of resistance (≥32 µg/ml) was observed to ampicillin (8.1%), meropenem (21.6%), chloramphenicol (73.4%), erythromycin (90.5%), tetracycline (100%) and high-level resistance to kanamycin (79.7%) and gentamicin (52.7%). The multi-drug resistant E. faecalis (MDRe.f) were carried pbp4 (90%), tetL (90%), tetM (70%), ermB (81%), cat (52.7%), acc6-aph2 (58.1%), aaph(3)-III (49.9%), gyrA (97%) and parC (98%) genes. Moreover, these MDRe.f were also harboured, hospital-associated marker IS16 (58%) and pheromone responsive genes, that is ccf (88%), cpd (74%), cob (62%) and eep (66%). Thus, regardless of the distinct phylogenetic background of E. faecalis of poultry origin, ATCC E. faecalis and clinical strains of human origin, we found major similarities in virulence, resistance gene profiles and mobile genetic elements (IS16 and pheromone responsive plasmids), supporting the zoonotic/reverse zoonotic risk associated with this organism.

Rheumatoid Arthritis Masquerading as Fibromyalgia.

Symptoms of inflammatory arthritis such as rheumatoid arthritis (RA) can overlap with fibromyalgia syndrome (FMS). Moreover, FMS and RAcan coexist. Hence, the diagnosis of low intensity, antibody-negative RAmay present a challenge. Here, we present the case of a middle-aged woman thought to have FMS, osteoarthritis and osteoporosis for 4 years prior to being diagnosed as seronegative RAon musculoskeletal ultrasound (MSKUS). Targeted therapy with etanercept led to a complete clinical response and normalization of previously elevated C-reactive protein. We conclude that MSKUS is a sensitive tool for distinguishing low-intensity inflammatory arthritis from FMS with important clinical consequences.

Prevalence and genetic profiling of tetracycline resistance (Tet-R) genes and transposable element (Tn916) in environmental Enterococcus species.

Resistance against antimicrobial agents in enterococci is a global concern that not only challenges infection therapy but also make them reservoir of antibiotic resistance in human and animal alike. This study was conducted to establish tetracycline resistance profiles, prevalence of tet genes and transposable element (Tn916) in enterococcal soil and clinical isolates. Enterococci (n = 1210) from different environmental niche were collected and subjected to molecular identification. In total, 361 isolates showed tetracycline resistance at the breakpoint of 32 µg ml-1. MICs (32-512 µg ml-1) were established by both agar and micro-broth dilution methods. Soil isolates (n = 76) were further investigated for Tet genes (tet-A, C, K, L, M, S, O) and Tn916. Major resistance was observed in E. faecium 67% followed by E. faecalis 22%, E. hirae 8% and E. casseliflavus 2.6%. Results revealed that tet(L) was more frequently found in E. faecium 74.5%, while tet(M) was in high prevalence in E. faecalis 82.3%. Tn916 was detected in both clinical and soil isolates (i.e. 43.3% and 19.7%, respectively). RAPD-PCR analysis showed high diversity among the investigated isolates. Cumulatively, our results revealed high-level tetracycline resistance and the presence of multiple Tet genes and transposable element Tn916 in enterococci.

Molecular assessment of virulence determinants, hospital associated marker (IS16gene) and prevalence of antibiotic resistance in soil borne Enterococcus species.

Enterococci, no more regarded as GRAS (Generally Recognized As Safe) organism, are emerging as an important source of nosocomial infections worldwide. The main contributors in pathogenesis of enterococci are the presence of various virulent factors and antibiotic resistance genes. We aimed to examine the prevalence, dissemination, antibiotic resistance and virulent factors associated with enterococci from bulk soil (BS). A total of 372 enterococci were isolated from 500 soil samples. PCR was used to identify the isolates up to species level and for carriage of 16 virulence genes including hospital associated marker (i.e. IS16). E. faecium (77%), E. faecalis (10%), E. hirae (4%) and E. casseliflavus (1%) were the major species isolated. The efaAfs was the most dominant gene (100%), followed by gelE (78.9%), sprE (76.3%) and esp (13%) in E. faecalis isolates. The E. faecium carried largely efaAfm (86.8%) and acm (50.3%) genes. Presence of entP (10%), entA (8.3%) and entB (6.9%) genes was detected mostly in E. faecium, while enlA (18%) and ef1097 (2.6%) was only detected in E. faecalis isolates. 50% E. faecalis and 2% E. faecium isolates harbored IS16, while five E. faecalis harbored both IS16 and espTIM genes providing strong evidence about the presence of espTIM gene on 64 Kb pathogenicity island. BOX and RAPD PCR analysis revealed high degree of genetic variation within the species. Degree of resistance against 12 major antibiotics showed chloramphenicol as the most effective and meropenom as the least effective antibiotic. Presence of multiple antibiotic resistant, virulent and hospital associated enterococci in bulk soil represents a potential source for further dissemination to humans and animals and poses potential impact on public health.