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A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

ABSTRACT: The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.

SUBMITTER: Kodali S

PROVIDER: S-EPMC4528116 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

Kodali Srinivas S Vinogradov Evgeny E Lin Fiona F Khoury Nancy N Hao Li L Pavliak Vilo V Jones C Hal CH Laverde Diana D Huebner Johannes J Jansen Kathrin U KU Anderson Annaliesa S AS Donald Robert G K RG

The Journal of biological chemistry 20150624 32

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic aci ...[more]

PMID: 26109072

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Project description:UnlabelledEnterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation.ImportanceEnterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections.

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:Regulatory RNAs (sRNAs) are now considered as major players in many physiological and adaptive responses in pathogenic bacteria. sRNAs have been extensively studied in Gram-negative bacteria, but less information is available in Gram-positive pathogens. There is a spread of multidrug-resistant (MDR) opportunistic organisms, grouped as “ESKAPE” pathogens, which comprise enterococci, a leading cause of hospital-acquired infections and outbreaks with emergence of MDR isolates, especially vancomycin-resistant Enterococcus faecium (VREF). Note that no information about sRNA expression is known in this major opportunistic pathogen. By transcriptomic and genomic analyses using E. faecium Aus0004 reference strain, 249 transcribed IGRs, including sRNA candidates, were detected and, using a series of cut-offs, this set was lowered down to 54 sRNAs while 7 that were predicted based on comparative sequence analysis. RNA-seq was performed with and without subinhibitory concentrations (SIC) of daptomycin, a cyclic lipopeptide antibiotic used for VREF infections. Under daptomycin SIC exposure, 260 genes (9.1% of the genome) had a significant alteration of expression including 80 upregulated genes and 180 downregulated genes. Among the repressed genes, a large proportion (55%) coded for proteins involved in carbohydrate and transport metabolism. Also, we focused on the 9 sRNAs exhibiting the highest expression, and all of them were confirmed as expressed along bacterial growth by Northern blots and qPCR. Out of these 9 sRNAs, four had significantly lower or higher expression in the presence of daptomycin SIC, and therefore responded to antibiotic exposure. Finally, we also tested the expression of these 9 sRNAs in a collection of isogenic Aus0004 mutants with increasing levels of daptomycin resistance, and we observed by qPCR that some sRNAs had a significantly modified expression in daptomycin resistance mutants. It highlights the significant implication of some of the E. faecium sRNAs in the early steps of the development of daptomycin resistance. This is the first experimental genome-wide sRNA identification in Gram-positive E. faecium, a leading cause of hospital acquired infections.

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

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

Publications

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium.

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