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Endolysin LysEF-P10 shows potential as an alternative treatment strategy for multidrug-resistant Enterococcus faecalis infections.

ABSTRACT: Phage-derived lysins can hydrolyse bacterial cell walls and show great potential for combating Gram-positive pathogens. In this study, the potential of LysEF-P10, a new lysin derived from a isolated Enterococcus faecalis phage EF-P10, as an alternative treatment for multidrug-resistant E. faecalis infections, was studied. LysEF-P10 shares only 61% amino acid identity with its closest homologues. Four proteins were expressed: LysEF-P10, the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain (LysEF-P10C), the putative binding domain (LysEF-P10B), and a fusion recombination protein (LysEF-P10B-green fluorescent protein). Only LysEF-P10 showed highly efficient, broad-spectrum bactericidal activity against E. faecalis. Several key functional residues, including the Cys-His-Asn triplet and the calcium-binding site, were confirmed using 3D structure prediction, BLAST and mutation analys. We also found that calcium can switch LysEF-P10 between its active and inactive states and that LysEF-P10B is responsible for binding E. faecalis cells. A single administration of LysEF-P10 (5??g) was sufficient to protect mice against lethal vancomycin-resistant Enterococcus faecalis (VREF) infection, and LysEF-P10-specific antibody did not affect its bactericidal activity or treatment effect. Moreover, LysEF-P10 reduced the number of Enterococcus colonies and alleviated the gut microbiota imbalance caused by VREF. These results indicate that LysEF-P10 might be an alternative treatment for multidrug-resistant E. faecalis infections.

SUBMITTER: Cheng M

PROVIDER: S-EPMC5579260 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Endolysin LysEF-P10 shows potential as an alternative treatment strategy for multidrug-resistant Enterococcus faecalis infections.

Cheng Mengjun M Zhang Yufeng Y Li Xinwei X Liang Jiaming J Hu Liyuan L Gong Pengjuan P Zhang Lei L Cai Ruopeng R Zhang Hao H Ge Jinli J Ji Yalu Y Guo Zhimin Z Feng Xin X Sun Changjiang C Yang Yongjun Y Lei Liancheng L Han Wenyu W Gu Jingmin J

Scientific reports 20170831 1

Phage-derived lysins can hydrolyse bacterial cell walls and show great potential for combating Gram-positive pathogens. In this study, the potential of LysEF-P10, a new lysin derived from a isolated Enterococcus faecalis phage EF-P10, as an alternative treatment for multidrug-resistant E. faecalis infections, was studied. LysEF-P10 shares only 61% amino acid identity with its closest homologues. Four proteins were expressed: LysEF-P10, the cysteine, histidine-dependent amidohydrolase/peptidase ( ...[more]

PMID: 28860505

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Project description:Background:Enterococcus faecalis and Enterococcus faecium are ubiquitous opportunistic pathogens found in the guts of humans and farmed animals. This study aimed to determine the occurrence, antimicrobial resistance, virulence, biofilm-forming ability and genotypes of E. faecalis and E. faecium from swine farms. Correlations between the genotypes, virulotypes, antibiotic resistance, and the environmental factors such as locality of farms and farm hygiene practice were explored. Methods:E. faecalis and E. faecium strains were isolated from the oral, rectal and fecal samples of 140 pigs; nasal, urine and fecal samples of 34 farmers working in the farms and 42 environmental samples collected from seven swine farms located in Peninsular Malaysia. Antibiotic susceptibility test was performed using the disk diffusion method, and the antibiotic resistance and virulence genes were detected by Polymerase Chain Reaction. Repetitive Extragenic Palindromic-Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis were performed to determine the clonality of the strains. Crosstab/Chi-square test and DistLM statistical analyses methods were used to determine the correlations between the genotypes, virulence factors, antibiotic resistance, and the environmental factors. Results:A total of 211 E. faecalis and 42 E. faecium were recovered from 140 pigs, 34 farmers and 42 environmental samples collected from seven swine farms in Peninsular Malaysia. Ninety-eight percent of the strains were multidrug-resistant (resistant to chloramphenicol, tetracycline, ciprofloxacin and erythromycin). Fifty-two percent of the strains formed biofilms. Virulence genes efa, asaI, gelE, esp, cyl and ace genes were detected. Virulence genes efa and asaI were most prevalent in E. faecalis (90%) and E. faecium (43%), respectively. Cluster analyses based on REP-PCR and PFGE showed the strains were genetically diverse. Overall, the strains isolated from pigs and farmers were distinct, except for three highly similar strains found in pigs and farmers. The strains were regional- and host-specific. Discussion:This study revealed alarming high frequencies of multidrug-resistant enterococci in pigs and swine farmers. The presence of resistance and virulence genes and the ability to form biofilm further enhance the persistence and pathogenicity of the strains. Although the overall clonality of the strains were regionals and host-specific, strains with high similarity were found in different hosts. This study reiterates a need of a more stringent regulation to ensure the proper use of antibiotics in swine husbandry to reduce the wide spread of multidrug-resistant strains.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Project description:BackgroundVancomycin-resistant enterococci (VRE) are important pathogens categorized as high-priority bacteria in the Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics published by the World Health Organization. The aim of this study was to determine the risk factors, resistance, virulence, mobilomes associated with multidrug-resistant and clonal lineages of Enterococcus faecium and faecalis circulating among hospitalized patients following the health system in South Africa, using whole genome sequencing (WGS).MethodsA cross-sectional study was conducted during a two-month periods among hospitalized patients in 2017. Rectal swabs were collected from patients admitted to medical and surgical wards in an urban tertiary hospital, and a rural district hospital in uMgungundlovu district, South Africa. Enterococci were screened for vancomycin resistance on bile esculin azide agar supplemented with 6 mg/L of vancomycin and confirmation of VRE was done using ROSCO kits. Conventional and real-time PCR methods were used to ascertain the presence of VanA, VanB, VanC-2/3 and VanC-1 genes. All six multidrug-resistant Enterococcus faecalis and faecium selected were identified using multiplexed paired-end libraries (2 × 300 bp) with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and genome sequencing was done using Illumina MiSeq instrument with 100× coverage at the National Institute of Communicable Diseases Sequencing Core Facility, South Africa. Antibiotic resistance genes, virulence factors, plasmids, integrons and CRISPR were characterized using RAST, ResFinder, VirulenceFinder, PlasmidFinder, PHAST and ISFinder respectively.ResultsSequencing analysis revealed that these strains harbouring numerous resistance genes to glycopeptides (vanC[100%], vex3[100%], vex2[83,33%] and vanG[16,66%]), macrolides, lincosamides, sterptogramine B (ermB[33,32%], Isa[16,66%], emeA[16,66%]) and tetracyclines (tetM[33,32%]) in both district and tertiary hospitals. Multidrug efflux pumps including MATE, MFS and pmrA conferring resistance to several classes of antibiotics were also identified. The main transposable elements observed were in the Tn3 family, specifically Tn1546. Four single sequence types (STs) were identified among E. faecium in the district hospital, namely ST822, ST636, ST97 along with a novel ST assigned ST1386, while one lineage, ST29 was detected in the tertiary hospital.ConclusionThe study reveals the genetic diversity and high pathogenicity of multidrug-resistant Enterococcus faecalis and faecium circulating among hospitalized patients. It underlines the necessity to implement routine screening of admitted patients coupled with infection control procedures, antimicrobial stewardship and awareness should be strengthened to prevent and/or contain the carriage and spread of multidrug resistant E. faecium and E. faecalis in hospitals and communities in South Africa.

Dataset Information

Endolysin LysEF-P10 shows potential as an alternative treatment strategy for multidrug-resistant Enterococcus faecalis infections.

Publications

Endolysin LysEF-P10 shows potential as an alternative treatment strategy for multidrug-resistant Enterococcus faecalis infections.

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