Synthesis of mosaic peptidoglycan cross-bridges by hybrid peptidoglycan assembly pathways in gram-positive bacteria

The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly(5), l-Ala(2), and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred beta-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs.     

Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC 9790 in Enterococcus faecium and Enterococcus faecalis

Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.     

Affinities of PBPs of enterococci to cefepime and ampicillin]

The beta-lactam resistance of genus Streptococcus has been explained by the low binding affinity of penicillin-binding proteins (PBPs) to the drug. This study was carried out to resolve the mechanisms of resistance to beta-lactam antibiotics in the species of genus Enterococcus by means of binding affinities of PBPs. Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium and Enterococcus avium were employed as assay microbes. Cefepime (CFPM) and ampicillin (ABPC) were used as representatives of cephems and penicillins, respectively. All the PBP fractions of S. pyogenes manifested high binding affinities to CFPM and ABPC, whereas PBPs 1 and 4 of E. faecalis showed high binding affinities to ABPC but not to CFPM. In E. faecium, PBPs of an exceptionally penicillin-susceptible strain manifested a high binding affinity to ABPC, but PBPs 5 and 6 showed low affinities to CFPM. beta-lactam resistant strains of E. faecium possessed PBPs 5 and 6 with low binding affinities to CFPM and ABPC. All the fractions of PBPs but PBP 1 in E. avium showed low binding affinities to CFPM. Although all the PBP fractions but PBPs 3 and 6 manifested high binding affinities to ABPC, PBPs 3 and 6 showed low binding affinities to ABPC. A strain of E. avium, which is susceptible to ABPC but moderately resistant to CFPM, lacked PBP 6. In conclusion, the resistance of E. avium to CFPM is based upon low binding affinities of the many fractions to this drug, and ABPC resistance is based upon PBPs 3 and 6 with low binding affinities to ABPC.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Molecular characterization of enterococci harboring genotype and phenotype incongruence related to glycopeptide resistance isolated in Brazilian hospitals

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.     

Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.     

A "retrocidal" plasmid in Enterococcus faecalis: passage and protection

Enterococcus faecalis MC4 harbors a 130 kb conjugative, pheromone (cCF10)-responding plasmid, pAMS1, conferring chloramphenicol, streptomycin and tetracycline resistances. A plasmid-borne class IIa bacteriocin (MC4-1) determinant and cognate immunity gene were present, but not expressed in MC4. However, pAMS1 transfer to E. faecalis JH2-2 (but not to the non-isogenic OG1SS) generated the surprising ability to express bacteriocin activity against the plasmid donor, MC4. The bacteriocin target spectrum includes E. faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, and Listeria monocytogenes. Those donors unable to express bacteriocin or immunity could protect themselves from the "retrocidal" behavior of transconjugants by a switch to bacteriocin resistance at a frequency of approximately 10(-3). Reversion to sensitivity occurred at a relatively high frequency, suggestive of involvement of a phase variation event. These observations concerning a conjugative plasmid with novel "retrocidal" properties, coupled with a defense mechanism independent of plasmid-borne immunity functions, may relate to phenomena exploiting regulatory features with broader ecological and evolutionary implications.     

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis

Abstract Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in β-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in β-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not stictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.     

Genome-wide identification of ampicillin resistance determinants in Enterococcus faecium

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism.     

Peptidoglycan synthesis in the absence of class A penicillin-binding proteins in Bacillus subtilis

Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.     

Determination of the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium.

Pulsed-field gel electrophoresis was used to determine the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium. The size determinations of OG1X, a strain of E. faecalis widely used in many laboratories for genetic studies, using Sma I, Not I, and Sfi I alone or in combination, ranged from 2,750 to 2,761 kb. Using the same enzymes as with OG1X, the size of HH-67, a plasmid-free clinical isolate of E. faecalis, was determined to be 2,170-2,288 kb and the size of JH2-2, an E. faecalis recipient strain, ranged from 2,008 to 2,135 kb. The size range generated for GE-1, a plasmid-free E. faecium strain, with the use of Sma I, Not I, and Apa I was 2,045-2,155 kb. Although OG1X differed in size from the other three enterococci, each individual enterococcal strain generated reproducible results in different experiments. However, for both E. faecalis OG1X and E. faecium GE-1, one of the enzymes used generated a considerably smaller molecular size than that generated by the other two enzymes. The discrepancy was due to visually undiscernible comigrating fragments, and serves to point out a potential source of error if fewer than two enzymes are used to size a genome. The size discrepancies were resolved by digesting individual fragments with a second enzyme. The molecular sizes of these enterococcal strains are larger than that recently reported for Campylobacter, smaller than that of Escherichia coli and Pseudomonas aeruginosa, and similar (OG1X) or smaller (JH2-2, HH67, and GE-1) than the 2,819-kb reported for Streptococcus mutans.     

Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

Gene vanXYC encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococci in which the C-terminal D-Ala residue has been replaced by D-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis of D-Ala-D-Ser has a polar effect on both D, D-dipeptidase and D,D-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[D-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP-MurNAc-pentapeptide[D-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.