Jean-Luc Mainardi

Comprehensive Diagnostic Strategy for Blood Culture-Negative Endocarditis: A Prospective Study of 819 New Cases

BACKGROUND. Blood culture-negative endocarditis (BCNE) may account for up to 31% of all cases of endocarditis. METHODS. We used a prospective, multimodal strategy incorporating serological, molecular, and histopathological assays to investigate specimens from 819 patients suspected of having BCNE. RESULTS. Diagnosis of endocarditis was first ruled out for 60 patients. Among 759 patients with BCNE, a causative microorganism was identified in 62.7%, and a noninfective etiology in 2.5%. Blood was the most useful specimen, providing a diagnosis for 47.7% of patients by serological analysis (mainly Q fever and Bartonella infections). Broad-range polymerase chain reaction (PCR) of blood and Bartonella-specific Western blot methods diagnosed 7 additional cases. PCR of valvular biopsies identified 109 more etiologies, mostly streptococci, Tropheryma whipplei, Bartonella species, and fungi. Primer extension enrichment reaction and autoimmunohistochemistry identified a microorganism in 5 additional patients. No virus or Chlamydia species were detected. A noninfective cause of endocarditis, particularly neoplasic or autoimmune disease, was determined by histological analysis or by searching for antinuclear antibodies in 19 (2.5%) of the patients. Our diagnostic strategy proved useful and sensitive for BCNE workup. CONCLUSIONS. We highlight the major role of zoonotic agents and the underestimated role of noninfective diseases in BCNE. We propose serological analysis for Coxiella burnetii and Bartonella species, detection of antinuclear antibodies and rheumatoid factor as first-line tests, followed by specific PCR assays for T. whipplei, Bartonella species, and fungi in blood. Broad-spectrum 16S and 18S ribosomal RNA PCR may be performed on valvular biopsies, when available.

A Novel Peptidoglycan Cross-linking Enzyme for a β-Lactam-resistant Transpeptidation Pathway

The β-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the β-lactam ring and the d-alanyl4-d-alanine5 extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in aβ-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldtfm, catalyzes in vitro the cross-linking of peptidoglycan subunits in a β-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldtfm for the l-lysyl3-d-alanine4 peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic β-lactams in contrast to d-alanyl4-d-alanine5 in the pentapeptide donors required for dd-transpeptidation. Ldtfm homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.

Evolution of peptidoglycan biosynthesis under the selective pressure of antibiotics in Gram‐positive bacteria

Acquisition of resistance to the two classes of antibiotics therapeutically used against Gram-positive bacteria, the glycopeptides and the beta-lactams, has revealed an unexpected flexibility in the peptidoglycan assembly pathway. Glycopeptides select for diversification of the fifth position of stem pentapeptides because replacement of D-Ala by D-lactate or D-Ser at this position prevents binding of the drugs to peptidoglycan precursors. The substitution is generally well tolerated by the classical D,D-transpeptidases belonging to the penicillin-binding protein family, except by low-affinity enzymes. Total elimination of the fifth residue by a D,D-carboxypeptidase requires a novel cross-linking enzyme able to process the resulting tetrapeptide stems. This enzyme, an L,D-transpeptidase, confers cross-resistance to beta-lactams and glycopeptides. Diversification of the side chain of the precursors, presumably in response to the selective pressure of peptidoglycan endopeptidases, is controlled by aminoacyl transferases of the Fem family that redirect specific aminoacyl-tRNAs from translation to peptidoglycan synthesis. Diversification of the side chains has been accompanied by a parallel divergent evolution of the substrate specificity of the L,D-transpeptidases, in contrast to the D,D-transpeptidases, which display an unexpected broad specificity. This review focuses on the role of antibiotics in selecting or counter-selecting diversification of the structure of peptidoglycan precursors and their mode of polymerization.

The impact of valve surgery on short- and long-term mortality in left-sided infective endocarditis: do differences in methodological approaches explain previous conflicting results?

AIMS The aim of this study was to evaluate the effect of valve surgery (VS) in infective endocarditis (IE) on 5-year mortality and to evaluate whether conflicting results reported by previous studies could be due to differences in their methodological approaches. METHODS AND RESULTS Four hundred and forty-nine patients with a definite left-sided IE were selected from a prospective, population-based study. Association between VS and 5-year mortality was examined with a Cox model. To determine the impact of different methodological approaches, we also analysed the relationship between VS and mortality in our database, according to each method used in the five previous studies. Valve surgery was performed in 240 patients (53%). It was associated with an increase in short-term mortality [within the first 14 post-operative days; adjusted hazard ratio (HR), 3.69; 95% confidence interval (CI), 2.17-6.25; P<0.0001] and a decrease in long-term mortality (adjusted HR, 0.55; 95% CI, 0.35-0.87; P=0.01). At least 188 days of follow-up were required for VS to provide an overall survival advantage. When applying each studys method to our database, we obtained results similar to those reported. CONCLUSION Previous conflicting results appear to be related to differences in statistical methods. When using appropriate models, we found that VS was significantly associated with reduced long-term mortality.

Synergistic effect of amoxicillin and cefotaxime against Enterococcus faecalis.

The antibacterial efficacy of the combination of amoxicillin and cefotaxime was assessed against 50 clinical strains of Enterococcus faecalis. For 48 of 50 strains, the MIC of amoxicillin that inhibited 50% of isolates tested decreased from 0.5 microgram/ml (range, 0.25 to 1 microgram/ml) to 0.06 microgram/ml (range, 0.01 to 0.25 microgram/ml) in the presence of only 4 micrograms of cefotaxime per ml. Alternatively, the MIC of cefotaxime that inhibited 50% of isolates tested decreased from 256 micrograms/ml (range, 8 to 512 micrograms/ml) to 1 micrograms/ml (range, 0.5 to 16 micrograms/ml) in the presence of only 0.06 microgram of amoxicillin per ml. For JH2-2, a reference strain of E. faecalis, the MICs of amoxicillin, cefotaxime, and amoxicillin in the presence of cefotaxime (4 micrograms/ml) were 0.5, 512, and 0.06 microgram/ml, respectively. By using a penicillin-binding protein (PBP) competition assay, it was shown that with cefotaxime, 50% saturation of PBPs 2 and 3 was obtained at very low concentrations (< 1 microgram/ml), while 50% saturation of PBPs 1, 4, and 5 was obtained with > or = 128 micrograms/ml. With amoxicillin, 50% saturation of PBPs 4 and 5 was obtained at 0.12 and 0.5 microgram/ml, respectively. Therefore, the partial saturation of PBPs 4 and 5 by amoxicillin combined with the total saturation of PBPs 2 and 3 by cefotaxime could be responsible for the observed synergy between these two compounds.

Identification of the l,d-Transpeptidases Responsible for Attachment of the Braun Lipoprotein to Escherichia coli Peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.

Comparative molecular and microbiologic diagnosis of bacterial endocarditis.

Sequencing of 16S rDNA, and of sodAint and rpoBint in some cases, was applied to DNA from heart valves of 46 patients (36 with definite and 10 with possible endocarditis). Sequence-based identifications were compared with those obtained with conventional methods. Among the 36 definite cases, 30 had positive blood cultures and 6 had negative cultures. Among the 30 positive cases, sequencing of 16S rDNA permitted identification of species (18), genus (8), or neither (4); sodAint and rpoBint sequencing was necessary for species identification in 8 cases. Species identifications were identical in only 61.5%, when conventional techniques and DNA sequencing were used. In five of the six blood culture–negative endocarditis cases, sequencing identified Bartonella quintana (3), B. henselae (1), and Streptococcus gallolyticus (1). Our results demonstrate a clear benefit of molecular identification, particularly in cases of blood culture–negative endocarditis and of possible endocarditis, to confirm or invalidate the diagnosis. Moreover, in 19.4% of the definite cases, the improvement in species identification by sequencing led to improved patient management.

Whole body [18F]fluorodeoxyglucose positron emission tomography imaging for the diagnosis of pacemaker or implantable cardioverter defibrillator infection: a preliminary prospective study

We studied the potential use of [(18) F]fluorodeoxyglucose ((18) F-FDG) whole body positron emission tomography (PET)-computed tomography for the diagnosis of device infection and extension of infection. Twenty-one patients with suspected device infection were prospectively included and compared with 14 controls free of infection. (18) F-FDG uptake on the box and on the leads was visually and quantitatively interpreted (using the maximal standard uptake value). The final diagnosis was obtained either from bacteriological data after device culture (n = 11) or by a 6-month follow-up according to modified Dukes criteria (n = 10). Ten patients finally showed infection on bacteriological study (n = 8) or during follow-up (n = 2). Sensitivity, specificity, positive predictive value and negative predictive value were, respectively, 80%, 100%, 100% and 84.6% on patient-based analysis (presence or absence of infection). They were 100%, 100%, 100% and 100% for boxes, but only 60%, 100%, 100% and 73% for leads. Quantitative analysis could be useful for boxes but not for leads, for which the presence of a mild hot spot was the best criterion of infection. The four false negatives on leads received antibiotics for longer than the six true positives (20 ± 7.2 vs. 3.2 ± 2.3 days, p <0.01). Although the study was not designed for this purpose, management could have been modified by PET results in six of 21 patients. (18) F-FDG PET imaging may be useful for the diagnosis of device infection, and could impact on clinical management. Interpretation of negative cases should be performed with caution if patients have received antibiotics.

Value of Microimmunofluorescence for Diagnosis and Follow-up of Bartonella Endocarditis

ABSTRACT Bartonella endocarditis is a disease of emerging importance that causes serious complications and high rates of mortality. Due to the fastidious nature of Bartonella species and their high degrees of antibiotic susceptibility, cultures of clinical samples most often remain sterile and valvular biopsy specimens, the best specimens for PCR amplification, are seldom available. Therefore, serology appears to be the easiest diagnostic tool. In order to determine the best cutoff value for serology and its predictive values for the detection of Bartonella endocarditis, we studied 48 patients with culture- and/or PCR-confirmed Bartonella endocarditis. We also applied these serological criteria to 156 patients with blood culture-negative endocarditis. Furthermore, we compared the kinetics of the antibody responses to Bartonella spp. in order to estimate the value of serology for prediction of the occurrence of relapses. A titer of ≥1:800 for immunoglobulin G antibodies to either Bartonella henselae or B. quintana has a positive predictive value of 0.810 for the detection of chronic Bartonella infections in the general population and a value of 0.955 for the detection of Bartonella infections among patients with endocarditis. When this cutoff was applied to 156 patients with blood culture-negative endocarditis, we were able to diagnose Bartonella infections in an additional 45 patients with definite endocarditis for whom a positive Bartonella serology was the only evidence of infection. On follow-up, the kinetics of the decrease in antibody titers were significantly delayed in two patients with relapses. In conclusion, we recommend the determination of antibodies to both B. quintana and B. henselae and the use of a cutoff value of 1:800 for the diagnosis of Bartonella endocarditis. We propose that this criterion, which may also help with the detection of late relapses, be included as a major criterion in the Duke criteria for the diagnosis of infective endocarditis.

Unexpected Inhibition of Peptidoglycan LD-Transpeptidase from Enterococcus faecium by the β-Lactam Imipenem

The β-lactam antibiotics mimic the d-alanyl4-d-alanine5 extremity of peptidoglycan precursors and act as “suicide” substrates of the dd-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the ld-transpeptidase of Enterococcus faecium (Ldtfm) leads to high level resistance to ampicillin. Ldtfm is specific for the l-lysyl3-d-alanine4 bond of peptidoglycan precursors containing a tetrapeptide stem lacking d-alanine5. This specificity was proposed to account for resistance, because the substrate of Ldtfm does not mimic β-lactams in contrast to the d-alanyl4-d-alanine5 extremity of pentapeptide stems used by the dd-transpeptidases. Here, we unexpectedly show that imipenem, a β-lactam of the carbapenem class, totally inhibited Ldtfm at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldtfm is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldtfm by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldtfm containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldtfm is likely to involve rupture of the β-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of β-lactams is not restricted to transpeptidases of the dd-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the ld- and dd-specificities.