Johannes Huebner

Alanine Esters of Enterococcal Lipoteichoic Acid Play a Role in Biofilm Formation and Resistance to Antimicrobial Peptides

ABSTRACT Enterococcus faecalis is among the predominant causes of nosocomial infections. Surface molecules like d-alanine lipoteichoic acid (LTA) perform several functions in gram-positive bacteria, such as maintenance of cationic homeostasis and modulation of autolytic activities. The aim of the present study was to evaluate the effect of d-alanine esters of teichoic acids on biofilm production and adhesion, autolysis, antimicrobial peptide sensitivity, and opsonic killing. A deletion mutant of the dltA gene was created in a clinical E. faecalis isolate. The absence of d-alanine in the LTA of the dltA deletion mutant was confirmed by nuclear magnetic resonance spectroscopy. The wild-type strain and the deletion mutant did not show any significant differences in growth curve, morphology, or autolysis. However, the mutant produced significantly less biofilm when grown in the presence of 1% glucose (51.1% compared to that of the wild type); adhesion to eukaryotic cells was diminished. The mutant absorbed 71.1% of the opsonic antibodies, while absorption with the wild type resulted in a 93.2% reduction in killing. Sensitivity to several cationic antimicrobial peptides (polymyxin B, colistin, and nisin) was considerably increased in the mutant strain, confirming similar results from other studies of gram-positive bacteria. Our data suggest that the absence of d-alanine in LTA plays a role in environmental interactions, probably by modulating the net negative charge of the bacterial cell surface, and therefore it may be involved in the pathogenesis of this organism.

A Putative Sugar-Binding Transcriptional Regulator in a Novel Gene Locus in Enterococcus faecalis Contributes to Production of Biofilm and Prolonged Bacteremia in Mice

A biofilm-negative transposon mutant was created from an Enterococcus faecalis strain that produces a lot of biofilm. The transposon had been inserted in the second gene of a locus consisting of 4 open-reading frames, designated bop (biofilm on plastic surfaces). A nonpolar deletion of this gene and of parts of the 2 flanking genes was created; production of biofilm by this deletion mutant was significantly enhanced, compared with that by the wild-type strain. Expression of a downstream gene was significantly lower in the transposon mutant than in the wild-type strain and the biofilm-enhanced deletion mutant. Transformation of this gene into the transposon mutant partially restored production of biofilm. Mice challenged by intravenous injection with the biofilm-negative mutant strain showed significantly reduced numbers of colony-forming units in the blood, compared with mice challenged with the biofilm-enhanced deletion mutant and the wild-type. These results indicate that bop is involved in production of biofilm and probably regulates expression of biofilm in the E. faecalis strain tested.

Accumulation of Pharmaceuticals, Enterococcus, and Resistance Genes in Soils Irrigated with Wastewater for Zero to 100 Years in Central Mexico

Irrigation with wastewater releases pharmaceuticals, pathogenic bacteria, and resistance genes, but little is known about the accumulation of these contaminants in the environment when wastewater is applied for decades. We sampled a chronosequence of soils that were variously irrigated with wastewater from zero up to 100 years in the Mezquital Valley, Mexico, and investigated the accumulation of ciprofloxacin, enrofloxacin, sulfamethoxazole, trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen, diclofenac, as well as the occurrence of Enterococcus spp., and sul and qnr resistance genes. Total concentrations of ciprofloxacin, sulfamethoxazole, and carbamazepine increased with irrigation duration reaching 95% of their upper limit of 1.4 µg/kg (ciprofloxacin), 4.3 µg/kg (sulfamethoxazole), and 5.4 µg/kg (carbamazepine) in soils irrigated for 19–28 years. Accumulation was soil-type-specific, with largest accumulation rates in Leptosols and no time-trend in Vertisols. Acidic pharmaceuticals (diclofenac, naproxen, bezafibrate) were not retained and thus did not accumulate in soils. We did not detect qnrA genes, but qnrS and qnrB genes were found in two of the irrigated soils. Relative concentrations of sul1 genes in irrigated soils were two orders of magnitude larger (3.15×10−3±0.22×10−3 copies/16S rDNA) than in non-irrigated soils (4.35×10−5±1.00×10−5 copies/16S rDNA), while those of sul2 exceeded the ones in non-irrigated soils still by a factor of 22 (6.61×10–4±0.59×10−4 versus 2.99×10−5±0.26×10−5 copies/16S rDNA). Absolute numbers of sul genes continued to increase with prolonging irrigation together with Enterococcus spp. 23S rDNA and total 16S rDNA contents. Increasing total concentrations of antibiotics in soil are not accompanied by increasing relative abundances of resistance genes. Nevertheless, wastewater irrigation enlarges the absolute concentration of resistance genes in soils due to a long-term increase in total microbial biomass.

Control of multiply resistant cocci: do international comparisons help?

Antibiotic resistance has become a worldwide problem. However, the reasons for the uneven geographic distribution of antibiotic-resistant microorganisms are not fully understood. For instance, there are striking differences in the epidemiology of multiresistant gram-positive cocci between the USA and Germany. According to recent reports, the prevalence of high-level penicillin-resistant pneumococci (PRP), meticillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) in clinically relevant isolates of hospitalised patients in the USA and Germany are: PRP, 14% versus less than 1%; MRSA, 36% versus 15%; and VRE, 15% versus 1%. These disparities may be explained by several determinants: (1) diagnostic practice and laboratory recognition (all three pathogens); (2) clonal differences and pathogen transmissibility (VRE); (3) antibiotic prescribing practices (all three pathogens); (4) population characteristics, including extensive daycare exposure in the USA (PRP); (5) cultural factors (all three pathogens); (6) factors related to the health-care and legal system (all three pathogens); and (7) infection-control practices (MRSA and VRE). Understanding these determinants is important for preventing further spread of multiresistant cocci within the USA. A rational approach to national surveillance is urgently needed in Germany to preserve the favourable situation and decrease MRSA transmission. Finally, we suggest that a macro-level perspective on antibiotic resistance can broaden the understanding of this worldwide calamity, and help prevent further dissemination of multiply resistant microorganisms.

Opsonic Antibodies to Enterococcus faecalis Strain 12030 Are Directed against Lipoteichoic Acid

ABSTRACT A teichoic acid (TA)-like polysaccharide in Enterococcus faecalis has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from E. faecalis strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with d-Ala and kojibiose. We also detected a novel substituent at position C-2, [d-Ala→6]-α-d-Glcp-(1→2-[d-Ala→6]-α-d-Glcp-1→). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the E. faecalis strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to E. faecalis 12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.

Prophylactic and Therapeutic Efficacy of Antibodies to a Capsular Polysaccharide Shared among Vancomycin-Sensitive and -Resistant Enterococci

ABSTRACT Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics, including vancomycin. A recently described capsular polysaccharide (CP) isolated from Enterococcus faecalis 12030 was used to evaluate the potential efficacy of active or passive immunotherapy regimens as adjunctive treatments. Evaluation of protective efficacy was carried out in immunocompetent mice challenged intravenously (i.v.) with live enterococci. In nonimmune mice, i.v. inoculations resulted in high levels of bacteria in kidneys, spleens, and livers 5 days after challenge. Mice immunized with four 10-μg doses of CP antigen/mouse were protected against challenge with the homologous E. faecalis strain. High-titer opsonic immunoglobulin G was also induced by immunizing rabbits with the purified CP, and passive transfer of this antiserum to mice produced significantly lower bacterial counts in organs than did normal rabbit serum or sterile saline. Antibodies to the polysaccharide isolated from E. faecalis 12030 were protective againstEnterococcus faecalis OG1RF and against two serologically related, vancomycin-resistant Enterococcus faecium clinical isolates. Antibodies to this CP antigen were also effective as a therapeutic reagent in mice when passive therapy was initiated 48 h after live bacterial challenge. These data indicate that CP antigens from enterococci are potential targets of protective antibodies and that these antibodies may be useful for prophylaxis and treatment of enterococcal infections.

Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor

Neutrophils are the main proinflammatory cell type in chronically infected lungs of cystic fibrosis (CF) patients; however, they fail to effectively clear the colonizing pathogens. Here, we investigated the molecular composition of non-mucoid and mucoid Pseudomonas aeruginosa-induced neutrophil extracellular traps (NETs) in vitro and compared them to the DNA-protein complexes present in the CF sputum. The protein composition of P. aeruginosa-induced NET fragments revealed that irrespective of the inducing stimuli, NET fragments were decorated with a conserved set of proteins. The DNA-protein complexes derived from CF sputum were consistent with NETosis and shared a similar protein signature, suggesting that the majority of the extracellular DNA was NET derived. The ability of polymorphonuclear leukocytes to produce NETs in response to P. aeruginosa was driven by macrophage migration-inhibitory factor (MIF) by promoting mitogen-activated protein kinase. Analysis of 132 CF patient samples revealed that elevated MIF protein levels correlated with poorer lung function. We suggest that targeting MIF by small molecular inhibitors might reduce the presence of extracellular DNA and serve as an adjunct to the use of antimicrobial drugs that could ultimately reduce bacterial fitness in the lungs during the later stages of CF disease.

Glycolipids are involved in biofilm accumulation and prolonged bacteraemia in Enterococcus faecalis

Biofilm production is thought to be an important step in many enterococcal infections. In several Gram‐positive bacteria, membrane glycolipids have been implicated in biofilm formation. We constructed a non‐polar deletion mutant of a putative glucosyltransferase designated biofilm‐associated glycolipid synthesis A (bgsA) in Enterococcus faecalis 12030. Analysis of major extracted glycolipids by nuclear magnetic resonance spectroscopy revealed that the cell membrane of 12030ΔbgsA was devoid of diglucosyl–diacylglycerol (DGlcDAG), while monoglucosyl–diacylglycerol was overrepresented. The cell walls of 12030ΔbgsA contained longer lipoteichoic acid molecules and were less hydrophobic than wild‐type bacteria. Inactivation of bgsA in E. faecalis 12030 and E. faecalis V583 led to an almost complete arrest of biofilm formation on plastic surfaces. Overexpression of bgsA, on the other hand, resulted in increased biofilm production. While initial adherence was not affected, bgsA‐deficient bacteria did not accumulate in the growing biofilm. Also, adherence of E. faecalisΔbgsA to Caco‐2 cells was impaired. In a mouse bacteraemia model, E. faecalis 12030ΔbgsA was cleared more rapidly from the bloodstream than the wild‐type strain. In summary, BgsA is a glycosyltransferase synthetizing DGlcDAG, a glycolipid and lipoteichoic acid precursor involved in biofilm accumulation, adherence to host cells, and virulence in vivo.

Serological and Genetic Diversity of Capsular Polysaccharides in Enterococcus faecalis

ABSTRACT Enterococci possess capsular carbohydrate antigens that are targets of opsonic antibodies. These antigens may be used to develop alternative options for the treatment and prevention of enterococcal infections. The present study was done to analyze the diversity of capsular polysaccharides in Enterococcus faecalis. Four type-specific sera were used in an enzyme-linked immunosorbent assay format to detect polysaccharide antigen extracted from bacterial cell walls. A total of 55% of a collection of 29 E. faecalis strains could be grouped into one of four serogroups. Additional analysis of the strains by opsonophagocytic assays revealed agreement between the results of the two methods for 72% of the isolates. An additional four strains could be assigned to a serogroup on the basis of opsonic killing by sera with antibodies against the four prototypes strains, provisionally named CPS-A to CPS-D. The results of the two methods disagreed for one strain (4%). When the results of both methods were combined, 66% of the strains could be classified. One strain had to be assigned to two serogroups. The assignments to the four serogroups were confirmed by analysis of the genetic organization of the biosynthetic capsular polysaccharide (cps) locus. All strains grouped into serotypes CPS-A and CPS-B possessed only the cpsA and cpsB genes, while all strains grouped into serogroups CPS-C and CPS-D possessed an additional eight or nine genes. Our results suggest the existence of a limited number of E. faecalis capsule serotypes, and we provisionally propose four serotypes, named CPS-A to CPS-D, and the respective prototype strains for these families.

The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins

Background: Conjugative plasmid transfer is the prevalent means for spreading antibiotic resistance genes among bacteria. Results: Surface exposure of transfer protein TraM from the Gram-positive (G+) plasmid pIP501 was confirmed, and its crystal structure was solved. Conclusion: Structural relations to type IV secretion (T4S) proteins provide a novel classification scheme. Significance: The novel classification will help elucidate structure-function relationships in G+ T4S systems. Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins.