Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci.

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

NEW TEMPERATE ENTEROCOCCUS PHAGE VB_GEC_EFS_2 WITH POTENTIAL OF LYSOGENIC CONVERSION.

Bacteriophages represent the widest group of viruses, from which only virulent phages are used as antibacterial agent. But the picture in the case of temperate phage is absolutely different; many lysogenic phages express gene products that have subtle effects on the phenotype of the host cell. This process is called lysogenic conversion. In present study we characterized new temperate Enterococcus faecium phage vB_GEC_EFS_2, which was isolated from river Mtkvari. The phage is a member of Siphoviridae family. Whole genome of phage vB_GEC-EfS_2 was sequenced and analyzed. Total length of the genome of phage vB_GEC_EFS_2 is 38 508bp, The assembly contains 65 ORFs, among them - 3 lysis genes , genes coded 13 structural proteins, 1 DNA replication-associated gene, 1 gene coded integration, 3 - lysis-lysogenic cycle regulation, 43 hypothetical proteins. One holin gene contained "Haemolysin XhIA" domain which is surface associated haemolisyn. We isolated and purified holin gene and determine its haemolitic activity alongside with vB_GEC_EfS_2 phage lysate. We clarified the XhIA domain function and role in protein's haemolytic nature and described another kind of lysogenic conversion.

Formação de biofilmes monotípicos e heterotípicos por Enterococcus faecalis E Enterococcus faecium em dentina radicular / Formation of monotypic and heterotypic biofilm by Enterococcus faecalis E Enterococcus faecium in root dentin

E. faecalis e E. faecium possuem grande relevância nas infecções hospitalares por apresentarem facilidade em adquirir resistência aos antibióticos. E. faecalis também apresentam alta prevalência nas infecções endodônticas, entretanto a importância de E. faecium para a odontologia ainda precisa ser esclarecida. Assim, o objetivo desse estudo foi comparar cepas clínicas de E. faecium com as cepas de E. faecalis em relação a capacidade de formação de biofilme na dentina radicular e penetração nos túbulos dentinários. Além disso, foi avaliada a interação dessas espécies em biofilmes mistos. Para a realização desse estudo, foram utilizadas cepas clínicas, previamante, isoladas de canais radiculares com infecções endodõnticas e identificadas pelo PCR multiplex. Entre as cepas isoladas, foram selecionadas 4 cepas de E. faecalis e 2 cepas de E. faecium. Primeiramente, foi realizado a formação dos biofilmes monotípicos das cepas de E. faecalis e E. faecium sobre dentinas radiculares de dentes bovinos. Os biofilmes foram formados em placas de microtitulação por diferentes tempos: 2, 4, 6, 24, 48, 72, 96 e 120 horas. Os biofilmes formados foram, então, analisados pela contagem de células viáveis (UFC/mL) e quantificação da biomassa total (método do cristal violeta). Além disso, os biofilmes foram analisados por Microscopia Eletrônica de Varredura (MEV) procurando-se observar a penetração das células de E. faecalis e E. faecium nos túbulos dentinários. A seguir foram formados biofilmes heterotípicos de E. faecalis e E. faecium para estudo das interações ecológicas estabelecidas entre as espécies. A análise dos biofilmes heterotípicos foi feita pela quantificação da biomassa total (cristal violeta) procurando-se detectar a presença de relações sinérgicas ou antagônicas. Os resultados foram submetidos à Análise de Variância (ANOVA) e teste de Tukey, considerando-se nível de 5%. Os resultados obtidos na contagem de UFC/mL dos biofilmes monotípicos, revelaram que as 6 cepas testadas apresentam grande capacidade para formar biofilmes na dentina radicular, alcançando valores de UFC/mL entre 8 a 12 log de acordo com o tempo de observação. Em relação a análise das imagens de MEV, as cepas clínicas de E. faecalis e E. faecium demonstraram capacidade semelhante para formar biofilmes e penetrar nos túbulos dentinários. Na comparação da quantificação da biomassa dos biofilmes monotípicos e heterotípicos, observamos que a interação das cepas clínicas E. faecalis e E. faecium favoreceu a adesão e crescimento do biofilme. Assim, concluiuse que as cepas de E. faecalis e E. faecium apresentam a mesma capacidade de formar biofilmes sobre a superfície radicular. Além disso, em biofilmes mistos, essas duas espécies estabelecem relações ecológicas sinérgicas, aumentando significativamente a formação de biofilmes(AU)

E. faecalis and E. faecium have a high relevance in hospital infections because they are easy to acquire resistance to antibiotics. E. faecalis also present high prevalence in endodontic infections, however the importance of E. faecium for dentistry still needs to be clarified. Thus, the objective of this study was to compare clinical strains of E. faecium with strains of E. faecalis in relation to the capacity of biofilm formation in root dentin and penetration into the dentin tubules. In addition, the interaction of these species in mixed biofilms was evaluated. In order to perform this study, clinical strains were used, pre-determined, isolated from root canals with endodontic infections and identified by multiplex PCR. Among the isolated strains, 4 strains of E. faecalis and 2 strains of E. faecium were selected. Firstly, the formation of the monotypic biofilms of the strains of E. faecalis and E. faecium on root dentin of bovine teeth was carried out. The biofilms were formed in microtiter plates at different times: 2, 4, 6, 24, 48, 72, 96 and 120 hours. The biofilms formed were then analyzed by counting viable cells (CFU / mL) and quantification of total biomass (violet crystal method). In addition, the biofilms were analyzed by Scanning Electron Microscopy (SEM), aiming to observe the penetration of E. faecalis and E. faecium cells into the dentin tubules. Then, heterophilic biofilms of E. faecalis and E. faecium were formed to study the ecological interactions established between the species. The analysis of the heterotypic biofilms was made by quantifying the total biomass (violet crystal) in order to detect the presence of synergistic or antagonistic relationships The results were submitted to Analysis of Variance (ANOVA) and Tukey test, considering a level of 5%. The results obtained in the CFU / mL count of the monotypic biofilms revealed that the six strains tested had a great capacity to form biofilms in the root dentin, reaching values of CFU / mL between 8 and 12 log according to the time of observation. In relation to SEM images, the clinical strains of E. faecalis and E. faecium demonstrated similar capacity to form biofilms and to penetrate the dentinal tubules. In the comparison of the biomass quantification of the monotypic and heterotypic biofilms, we observed that the interaction of the clinical strains E. faecalis and E. faecium favored the adhesion and growth of the biofilm. Thus, it was concluded that strains of E. faecalis and E. faecium have the same ability to form biofilms on the root surface. In addition, in mixed biofilms, these two species establish synergistic ecological relationships, significantly increasing the formation of biofilms(AU)

Complete genome sequence of a novel, virulent Ahjdlikevirus bacteriophage that infects Enterococcus faecium.

A novel virulent bacteriophage named vB_EfaP_IME199 that specifically infects Enterococcus faecium was isolated and characterized. Its optimal multiplicity of infection was 0.01, and it had a 30 minute outbreak period. High-throughput sequencing revealed that the phage has a dsDNA genome of 18,838 bp with 22 open reading frames. The genome has very low homology to all other bacteriophage sequences in the GenBank database. Run-off sequencing experiments confirmed that vB_EfaP_IME199 has short inverted terminal repeats. Phylogenetic analysis indicated that vB_EfaP_IME199 can be taxonomically classified as a new member of the genus Ahjdlikevirus of family Podoviridae.

Preliminary survey of local bacteriophages with lytic activity against multi-drug resistant bacteria.

Bacteriophages (phages) represent a potential alternative for combating multi-drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE-group (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus. Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two-thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity.

Characterization of Enterococcus faecium bacteriophage IME-EFm5 and its endolysin LysEFm5.

Due to the worldwide prevalence of antibiotic resistant strains, phages therapy has been revitalized recently. In this study, an Enterococcus faecium phage named IME-EFm5 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that IME-EFm5 belong to the Siphoviridae family, and has a double-stranded genome of 42,265bp (with a 35.51% G+C content) which contains 70 putative coding sequences. LysEFm5, the endolysin of IME-EFm5, contains an amidase domain in its N-terminal and has a wider bactericidal spectrum than its parental phage IME-EFm5, including 7 strains of vancomycin-resistant E. faecium. The mutagenesis analysis revealed that the zinc ion binding residues (H27, H132, and C140), E90, and T138 are required for the catalysis of LysEFm5. However, the antibacterial activity of LysEFm5 is zinc ion independent, which is inconsistent with most of other amidase members. The phage lysin LysEFm5 might be an alternative treatment strategy for infections caused by multidrug-resistant E. faecium.

A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini.

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel E. faecalis and E. faecium phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the E. faecalis phage IME-EF4, E. faecium phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics. RESULTS: The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3' protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3' protruding cohesive ends are 5'-TCATCACCG-3' (IME-EF4) and 5'-GGGTCAGCG-3' (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing. CONCLUSION: Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging.

Characterization and complete genome sequence analysis of novel bacteriophage IME-EFm1 infecting Enterococcus faecium.

We isolated and characterized a novel virulent bacteriophage, IME-EFm1, specifically infecting multidrug-resistant Enterococcus faecium. IME-EFm1 is morphologically similar to members of the family Siphoviridae. It was found capable of lysing a wide range of our E. faecium collections, including two strains resistant to vancomycin. One-step growth tests revealed the host lysis activity of phage IME-EFm1, with a latent time of 30 min and a large burst size of 116 p.f.u. per cell. These biological characteristics suggested that IME-EFm1 has the potential to be used as a therapeutic agent. The complete genome of IME-EFm1 was 42 597 bp, and was linear, with terminally non-redundant dsDNA and a G+C content of 35.2 mol%. The termini of the phage genome were determined with next-generation sequencing and were further confirmed by nuclease digestion analysis. To our knowledge, this is the first report of a complete genome sequence of a bacteriophage infecting E. faecium. IME-EFm1 exhibited a low similarity to other phages in terms of genome organization and structural protein amino acid sequences. The coding region corresponded to 90.7 % of the genome; 70 putative ORFs were deduced and, of these, 29 could be functionally identified based on their homology to previously characterized proteins. A predicted metallo-ß-lactamase gene was detected in the genome sequence. The identification of an antibiotic resistance gene emphasizes the necessity for complete genome sequencing of a phage to ensure it is free of any undesirable genes before use as a therapeutic agent against bacterial pathogens.

Bacteriophage control of vancomycin-resistant enterococci in cattle compost.

AIMS: To isolate bacteriophage that infects vancomycin-resistant enterococci (VRE) and to investigate the ability of this phage to diminish VRE number in vitro and in experimentally VRE-inoculated compost. METHODS AND RESULTS: We sampled 106 solid or water samples, including 101 bovine faecal samples; lytic phage named Vrep-5 was isolated from one bovine faecal sample by plaque assay using the clinical VRE isolate FN1 (Enterococcus faecium). Vrep-5 generated clear plaques 1 mm in diameter and exhibited characteristics of the family Myoviridae A1, with a spherical head (122 ± 16 nm) and a contractile tail (152 ± 17 nm long). Vrep-5 lysed other bacterial strains, including Enterococcus faecalis. Inoculation of vrep-5 into 0.5 g unsterilized compost experimentally inoculated with FN1 at the multiplicity of infection of 1500 (8.8 × 10(4) CFU g(-1) VRE and 1.3 × 10(8) PFU g(-1) vrep-5) led to a decrease of >3 log(10) in VRE abundance compared with the untreated control after 24 h of incubation. CONCLUSIONS: The data show that bacteriophage vrep-5 is effective in the rapid reduction in VRE colonization in compost. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study gives valuable new knowledge in the fight against VRE in the animal production.

Isolation of bacteriophages to multi-drug resistant Enterococci obtained from diabetic foot: a novel antimicrobial agent waiting in the shelf?

INTRODUCTION: While foot infections in persons with diabetes are initially treated empirically, therapy directed at known causative organisms may improve the outcome. Many studies have reported on the bacteriology of diabetic foot infections (DFIs), but the results have varied and have often been contradictory. The purpose of the research work is to call attention to a frightening twist in the antibiotic-resistant Enterococci problem in diabetic foot that has not received adequate attention from the medical fraternity and also the pharmaceutical pipeline for new antibiotics is drying up. MATERIALS AND METHODS: Adult diabetic patients admitted for lower extremity infections from July 2008 to December 2009 in the medical wards and intensive care unit of medical teaching hospitals were included in the study. The extent of the lower extremity infection on admission was assessed based on Wagner's classification from grades I to V. Specimens were collected from the lesions upon admission prior to the initiation of antibiotic therapy or within the first 48 h of admission. RESULTS: During the 18-month prospective study, 32 strains of Enterococcus spp. (26 Enterococcus faecalis and 06 E. faecium) were recovered. Antibiotic sensitivity testing was done by Kirby-Bauer's disk diffusion method. Isolates were screened for high-level aminoglycoside resistance (HLAR). A total of 65.6% of Enterococcus species showed HLAR. Multidrug resistance and concomitant resistance of HLAR strains to other antibiotics were quite high. None of the Enterococcus species was resistant to vancomycin. CONCLUSION: Multidrug-resistant Enterococci are a real problem and continuous surveillance is necessary. Today, resistance has rendered most of the original antibiotics obsolete for many infections, mandating the development of alternative anti-infection modalities. One of such alternatives stemming up from an old idea is the bacteriophage therapy. In the present study, we could able to demonstrate the viable phages against MDR E. faecalis.

Bacteriophage-mediated transduction of antibiotic resistance in enterococci.

AIMS: Temperate bacteriophages are bacterial viruses that transfer genetic information between bacteria. This phenomenon is known as transduction, and it is important in acquisition of bacterial virulence genes and antimicrobial resistance determinants. The aim of this study was to demonstrate the role of bacteriophages in gene transfer (antibiotic resistance) in enterococci. METHODS AND RESULTS: Three bacteriophages from environmental samples isolated on pig host strains of Enterococcus gallinarum and Enterococcus faecalis were evaluated in transduction experiments. Antibiotic resistance was transferred from Ent. gallinarum to Ent. faecalis (tetracycline resistance) and from Ent. faecalis to Enterococcus faecium, Enterococcus hirae/durans and Enterococcus casseliflavus (gentamicin resistance). CONCLUSIONS: Bacteriophages play a role in transfer of antibiotic resistance determinants in enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms previous suggestions on transduction in enterococci, in particular on interspecies transduction. Interspecies transduction is significant because it widens the range of recipients involved in antimicrobial resistance transfer.

[Inhibition of herpes simplex virus type 1 reproduction by probiotic bacteria in vitro].

Light and immunofluorescence microscopies were used to study the cytopathic effect of herpes simplex virus type 1 (HSV-1) grown on the Vero cell cultures in the absence or presence of supernatants of Enterococcus faecium L3, Lactobacillus plantarum 8A-P3, and Escherichia coil M17. The effect of the probiotic strains was evaluated estimating the proportion of changed cells and the infective dose of the virus. The supernatants of the cultures of Lactobacillus sp. and Enterococcus sp., unlike those of E. coil, have antiviral activity. Inhibited viral replication was more evident when the supernatants were added until the cultured HSV-1 cells were infective. An enterococcal supernatant and its obtained peptide extract showed the maximum antiviral activity. This strain may be associated with the production of bacteriocins and bacteriocin-like substances.

Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island.

BACKGROUND: The Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients. RESULTS: We present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI), which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner. CONCLUSIONS: Genes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come.

Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium.

Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 10(9) CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 x 10(8) PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.