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Resume Contexte La surveillance de l’evolution de la resistance aux antibiotiques et des serotypes des souches de Streptococcus pneumoniae en France repose sur un reseau alliant les observatoires du pneumocoque, le Centre national de reference du pneumocoque et l’Institut de veille sanitaire. Methodes Entre 2009 et 2013, les 23 observatoires regionaux du pneumocoque (ORP) ont recueilli et analyse 13 033 souches de pneumocoque dont 2953 isolees chez l’enfant et 10 080 chez l’adulte. Resultats Durant cette periode, le nombre de souches isolees a fortement diminue, avec une baisse de 42,1 %. La diminution a ete particulierement importante chez les enfants, a la fois pour les infections invasives (–59,7 %) et pour les pus d’otite moyenne aigue (–74,4 %). Chez les adultes, le nombre de souches isolees d’infections invasives a baisse de 31,0 %. Conjointement, il est a noter une baisse des souches de sensibilite diminuee a la penicilline G (34,2 % en 2009 vs 25,7 % en 2013), ainsi qu’une baisse de la resistance a l’amoxicilline (17,9 % en 2009 vs 11,6 % en 2013) et au cefotaxime (9,7 % en 2009 vs 6,0 % en 2013). Enfin, en 2009, la prevalence des souches appartenant aux serotypes presents dans le vaccin anti-pneumococcique PCV13 etait de 64,2 % et en 2013, de 35,6 %. En parallele, les serotypes 12F, 22F, 24F, 15A, 10A et 23B, non inclus dans le vaccin PCV13, etaient en augmentation. Conclusion La surveillance de la resistance et des serotypes reste primordiale car les infections a S. pneumoniae representent, de part leur frequence et leur gravite, un enjeu majeur de sante publique.

The production of β-lactamases Bla Mab and BlaC contributes to β-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis , respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥256 to 16 to 64 μg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations.

Imipenem and cefoxitin are used to treat Mycobacterium abscessus infections and have moderate activity against this fast-growing mycobacterium (MIC₅₀ of 16 and 32 mg/L, respectively). M. abscessus is highly resistant to most other β-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A β-lactamase (Bla(Mab)) and investigated its role in β-lactam resistance.Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various β-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of β-lactams were determined for M. abscessus CIP104536 and for Escherichia coli producing Bla(Mab) and BlaC.Bla(Mab) had a broad hydrolysis spectrum, similar to that of BlaC, but with overall higher catalytic efficiencies, except for cefoxitin. As expected from its in vivo efficacy, cefoxitin was very slowly hydrolysed by Bla(Mab) (k(cat)/K(m) = 6.7 M(-1) s(-1)). Bla(Mab) hydrolysed imipenem more efficiently (k(cat)/K(m) = 3.0 × 10(4) M(-1) s(-1)), indicating that the in vivo activity of this drug might be improved by combination with a β-lactamase inhibitor. β-Lactamase inhibitors clavulanate, tazobactam and sulbactam did not inhibit Bla(Mab). This enzyme efficiently hydrolysed clavulanate, in contrast to BlaC, which is irreversibly acylated by this inhibitor. Bla(Mab) and BlaC were functional in E. coli and the resistance profiles mediated by these enzymes were in agreement with the kinetic parameters.M. abscessus produces a clavulanate-insensitive broad-spectrum β-lactamase that limits the in vivo efficacy of β-lactams.

Overall, 2,337 rectal screening samples (RSSs) were seeded by using the Wasp instrument for automated microbiological processing with five media for detection of extended-spectrum β-lactamase (ESBL): CHROMagar, ChromID, Brilliance, BD Drigalski, and HEGP media. Of 354 RSSs harboring ESBL-producing isolates, 89.3% were found to be positive on all media. Sensitivity and specificity ranged from 95.5 to 98.3% and from 57.9 to 72.3%, respectively. No medium was perfectly ESBL selective, and non-ESBL-producing strains were mainly Enterobacteriaceae overproducing AmpC β-lactamase and nonfermenting Gram-negative bacilli, mostly Pseudomonas aeruginosa .

International audience; Evidence before this study We searched PubMed on June 30, 2016, for English-language cohort studies published since Jan 1, 1980, of patients with invasive listeriosis worldwide with the keywords " listeria " , " listeriosis " , " maternal " , and " neurolisteriosis ". Studies had to include epidemiological or clinical data on listeriosis. All clinical forms of infection were included (bacteraemia, neurolisteriosis, and maternal–neonatal infection). Host risk factors for listeriosis have been well identified, but the clinical features and prognostic factors of the disease are based on retrospective studies compiling heterogeneous data or random collected cases. Furthermore, no clinical trial has ever been done and medical management is not evidence based. Added value of the study Our study is the first prospective clinical study focusing on all forms of invasive listeriosis. The study is based on a national mandatory system that allowed the nearly complete capture of microbiologically proven cases. The study shows a higher burden of listeriosis than reported before: more than 80% of infected mothers experienced major fetal or neonatal complications (fetal loss, very high prematurity, early or late onset disease); only 39% of patients with neurolisteriosis survived and fully recovered. The study provides important new data to improve management and predict outcome in listeriosis, such as determination of the time window for fetal losses (

Implementation of 13-valent conjugate pneumococcal vaccine in France has led to an overall antibiotic resistance decrease among invasive pneumococcal isolates. However, among emerging non-PCV13 serotypes, 12F, 24F, 15A and 35B are associated to antibiotic resistance and should be further monitored., Background. In 2010, the pneumococcal 13-valent conjugate vaccine (PCV13), containing 6 additional serotypes including the multidrug-resistant 19A, replaced the PCV7 in France. This study aimed at analyzing trends in antibiotic resistance in invasive pneumococcal disease (IPD) isolates in France after PCV13 introduction. Methods. A total of 5243 pneumococci isolated from IPD in 2008–2009 (late PCV7 era) and 2011–2012 (PCV13 era) were studied according to their serotype and antibiotic resistance profile. Multilocus sequence typing analysis was performed on strains of the predominant serotypes (12F and 24F) isolated from young children. Results. Overall, the prevalence of antibiotic resistance decreased in France (−21.5% for penicillin from 2008–2009 to 2011–2012), mainly driven by the decline of the 19A serotype. Among non-PCV13 serotypes that concomitantly emerged, serotypes 12F, 24F, 15A, and 35B were consistently associated with resistance to 1 or more antibiotics. In children under 2 years, serotypes 15A, 35B, and 24F accounted together for 37.8% and 31.9% of penicillin-nonsusceptible and erythromycin-resistant isolates, respectively. Chloramphenicol and cotrimoxazole resistance were mainly associated with serotypes 12F and 24F, respectively. Genetic analysis showed that although emergence of serotype 12F pneumococci resulted from the expansion of various pre-existing lineages, increase in serotype 24F was related to the clonal expansion of the ST162 penicillin-susceptible cotrimoxazole-resistant lineage. Conclusions. We showed that decline of PCV13-related IPD was associated with a decline in antibiotic resistance in France, but that it likely favored the spread of several resistant nonvaccine serotypes. However, antibiotic resistance does not seem to be the only element that may drive this phenomenon.

Bypass of classical penicillin-binding proteins by the l , d -transpeptidase of Enterococcus faecium (Ldt fm ) leads to high-level ampicillin resistance in E. faecium mutants, whereas carbapenems remain the lone highly active β-lactams. Kinetics of Ldt fm inactivation was determined for four commercial carbapenems and a derivative obtained by introducing a minimal ethyl group at position 2. We show that the bulky side chains of commercial carbapenems have both positive and negative effects in preventing hydrolysis of the acyl enzyme and impairing drug binding.

The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l , d -transpeptidases that replace 4→3 cross-links formed by the d , d -transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l , d -transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis . Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l , d -transpeptidase Ldt Mt1 by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt Mt1 , which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt Mt1 , with a binding rate constant of 0.08 μM −1 min −1 . The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (−1 ) in comparison to the acylation rate constant (3.1 min −1 ). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10 −3 min −1 . Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt Mt1 inactivator. Cephalosporins also formed covalent adducts with Ldt Mt1 , although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis .

In clinical studies on bacteraemia, the negativity of blood cultures is an important endpoint for comparing the efficacy of different therapeutic regimens. In FAN ° anaerobic blood culture medium (BacT/ALERT system), daptomycin displayed increased MIC against Staphylococcus aureus and improved abolishment of its carryover effect in charcoal when compared with vancomycin. Differences between these two drugs can lead to a false interpretation of negative blood cultures. To compare different antibiotic regimens for the treatment of bacteraemia, preliminary studies are mandatory to ensure that ex vivo antibiotic behaviour is similar in the blood-culture system used.

Few therapeutic alternatives remain for the treatment of infections due to multiresistant Mycobacterium abscessus . Here we show that the peptidoglycans of the “rough” and “smooth” morphotypes contain predominantly 3→3 cross-links generated by l , d -transpeptidases, indicating that these enzymes are attractive targets for the development of efficient drugs.

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d,d and l,d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt(Cjk1), a member of the active site cysteine l,d-transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of beta-lactam, the PBPs and the l,d-transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt(Cjk1) and Pbp2C were not inhibited by ampicillin, the participation of the l,d-transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt(Cjk1) for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt(Cjk1) was produced by an ampicillin-sensitive d,d-carboxypeptidase (Pbp4(Cjk)). Acquisition and mutational alterations of pbp2C accounted for high-level beta-lactam resistance in C. jeikeium.

Antimicrobial, antioxidant, and antiviral activities of flower extracts of Retama raetam Forssk. Webb (Fabaceae) were screened both from standard and isolated Gram-positive and Gram-negative bacteria by solid medium technique. Oxacillin, Amoxicillin, Ticarcillin, Cefotaxim, and Amphotericin were used as the control agents. The antiviral activity was evaluated against human cytomegalovirus (HCMV) strain AD-169 (ATCC Ref. VR 538) and coxsackie B virus type 3 (CoxB-3) using a cytopathic effect (CPE) reduction assay. The antioxidant activity was evaluated using two tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical scavenging and the ammonium thiocyanate methods. All extracts were characterized quantitatively for the presence of polyphenols, flavonoids, and tannins. Of the extracts tested, butanol and ethyl acetate extracts showed important antibacterial activity against Gram-positive and Gram-negative bacteria but only moderate antifungal activity. Methanol extract exhibited moderate antiviral activity against HCMV with IC50 of 250 μg/ml. Ethyl acetate, chloroform, and methanol fractions were found to cause significant free-radical-scavenging effects in both assays. These results may suggest that R. raetam flowers could be used as a natural preservative ingredient in the food and/or pharmaceutical industries.

The plasmid-borne quinolone resistance gene qnrA1 is prevalent in multidrug-resistant Enterobacteriaceae . A chromosomally encoded homologue in Shewanella algae , qnrA3 , has been described. We isolated two qnrA3 -positive strains, one of Klebsiella pneumoniae (He96) and one of Kluyvera ascorbata (Kas96), from the feces of an immunocompromised outpatient. The qnrA3 allele was identical to that of S. algae except for 5 nucleotides and differed from qnrA1 by 29 nucleotides affecting three amino acids. The analysis of the qnrA3 genetic environment showed that qnrA3 was inserted downstream from an IS CR1 element at a recombination crossover site described for other resistance genes, including qnrA1 , and immediately upstream from IS 26 , a situation not described before. IS 26 preceded an incomplete class 1 integron which contained, among other genes, aac ( 6 ′)- Ib-cr , another transferable quinolone resistance gene, and the β-lactamase gene bla OXA-1/30 . The 10-kb fragment encompassing qnrA3 was compared to previously described qnrA1 -containing plasmids and multidrug-resistant plasmids; it shares identical sequences with pC15a, pHSH2, pQR1, pQKp311H, and pSAL-1 but with rearrangements, deletions, and mutations. Conjugal transfer of qnrA3 was highly efficient (10 −2 ) from K. pneumoniae He96 or K. ascorbata Kas96 to Escherichia coli J53 but less so (10 −5 ) from either donor to a clinical strain of Enterobacter cloacae . This first description of a plasmid-borne copy and of the in vitro transfer of qnrA3 is taken to illustrate its likely in vivo transfer from S. algae to the Enterobacteriaceae .

Using the BacT/Alert automated system, we conducted a 1-year retrospective study on blood cultures, focusing on the relevance of routine use of the anaerobic bottle. The rate of patients with positive blood cultures was 19.7%. Among these, 13.5% had a positive anaerobic bottle in the absence of any aerobic bottle, and 2/3 of these grew with nonobligate anaerobes. These patients were hospitalized in 20 out of 26 wards of the hospital group. For 65.4% of the monomicrobial-positive blood cultures growing Enterobacteriaceae , the anaerobic bottle detected growth earlier than the corresponding aerobic bottle. These data suggest that, in our institution, the use of an anaerobic bottle is still relevant.

Streptococcus pneumoniae is an important cause of acute otitis media (AOM). The aim of this study was to evaluate trends in antibiotic resistance and circulating serotypes of pneumococci isolated from middle ear fluid of French children with AOM during the period 2001–2011, before and after the introduction of the PCV-7 (2003) and PCV-13 (2010) vaccines. Between 2001 and 2011 the French pneumococcal surveillance network analysed the antibiotic susceptibility of 6683 S. pneumoniae isolated from children with AOM, of which 1569 were serotyped. We observed a significant overall increase in antibiotic susceptibility. Respective resistance (I+R) rates in 2001 and 2011 were 76.9% and 57.3% for penicillin, 43.0% and 29.8% for amoxicillin, and 28.6% and 13.0% for cefotaxime. We also found a marked reduction in vaccine serotypes after PCV-7 implementation, from 63.0% in 2001 to 13.2% in 2011, while the incidence of the additional six serotypes included in PCV-13 increased during the same period, with a particularly high proportion of 19A isolates. The proportion of some non-PCV-13 serotypes also increased between 2001 and 2011, especially 15A and 23A. Before PCV-7 implementation, most (70.8%) penicillin non-susceptible pneumococci belonged to PCV-7 serotypes, whereas in 2011, 56.8% of penicillin non-susceptible pneumococci belonged to serotype 19A. Between 2001 and 2011, antibiotic resistance among pneumococci responsible for AOM in France fell markedly, and PCV-7 serotypes were replaced by non-PCV-7 serotypes, especially 19A. We are continuing to assess the impact of PCV-13, introduced in France in 2010, on pneumococcal serotype circulation and antibiotic resistance.

Objectives Two β-lactams, cefoxitin and imipenem, are part of the reference treatment for pulmonary infections with Mycobacterium abscessus. M. abscessus has recently been shown to produce a broad-spectrum β-lactamase, BlaMab, indicating that the combination of β-lactams with a BlaMab inhibitor may improve treatment efficacy. The objectives of this study were to evaluate the impact of BlaMab production on the efficacy of β-lactams in vitro and to assess the benefit of BlaMab inhibition on the activity of β-lactams intracellularly and in an animal model. Methods We analysed the mechanism and kinetics of BlaMab inactivation by avibactam, a non-β-lactam β-lactamase inhibitor currently in Phase III of development, in combination with ceftazidime for the treatment of serious infections due to Gram-negative bacteria. We then deleted the gene encoding BlaMab to assess the extent of BlaMab inhibition by avibactam based on a comparison of the impact of chemical and genetic inactivation. Finally, the efficacy of amoxicillin in combination with avibactam was evaluated in cultured human macrophages and in a zebrafish model of M. abscessus infection. Results We showed that avibactam efficiently inactivated BlaMab via the reversible formation of a covalent adduct. An inhibition of BlaMab by avibactam was observed in both infected macrophages and zebrafish. Conclusions Our data identify avibactam as the first efficient inhibitor of BlaMab and strongly suggest that β-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections.

Three sets of mutants of Bacillus anthracis resistant to fluoroquinolones were selected on ciprofloxacin and moxifloxacin in a stepwise manner from a nalidixic acid-resistant but fluoroquinolone-susceptible plasmidless strain harboring a Ser85Leu GyrA mutation. A high level of resistance to fluoroquinolones could be obtained in four or five selection steps. In each case, ParC was the secondary target. However, in addition to the GyrA mutation, expression of high-level resistance required (i) in the first set of mutants, active drug efflux associated with a mutation in the QRDR of ParC; (ii) in the second set, two mutations in the QRDR of ParC associated with a mutation in GyrB; and (iii) in the third set, two QRDR mutations, one in ParC and one in GyrA. Interestingly, several selection steps occurred without obvious mutations in the QRDR of any topoisomerase, thereby implying the existence of other resistance mechanisms. Among the fluoroquinolones tested, garenoxacin showed the best activity.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis , class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 ( ponA , pbpF , and pbpZ ). Among mutants with single or double deletions, only JH2-2 Δ ponA Δ pbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus . Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis , and heterospecific complementation was observed with an ortholog from E. faecium . Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

Background/Aims : We herein focused on identifying biological factors possibly involved in non-parenteral transmission of hepatitis C virus (HCV), such as HCV excretion patterns and antibody-based immunity to the virus in saliva and/or cervicovaginal secretions (CVS). Methods : Paired blood, saliva and cervicovaginal lavage samples were obtained from HCV-RNA plasma-positive hemoglobin (Hb) antigen and HIV-seronegative, HCV-seropositive males ( n =13) and females ( n =21). HCV-specific antibodies were detected by ELISA in paired samples, and HCV-RNA was detected in cell-free and cell-associated body fluids. Results : Antibodies to E1 HCV surface glycoprotein of the IgG and IgA isotypes showed similar, but less pronounced, profiles as IgG and IgA to E2. HCV-specific IgG and IgA in mucosal fluids likely originated predominantly from the systemic compartment, because HCV-specific mucosal immunoglobulins involved primarily monomeric antibodies, including monomeric IgA, and because their specific activities for HCV antigens in corporeal fluids were similar to those in paired serum (Se). Viral shedding in saliva or CVS was restricted to cell-associated, non-replicating strand (+) HCV-RNA in 42% (12 out of 28) of saliva and in 19% (four out of 21) of cervicovaginal fluids. Conclusions : The association in body fluids of HCV-specific IgG, and to a lesser extent IgA, directed to E1/E2 surface glycoproteins (which may block critical steps of virus–cell interactions), of undetectable free viral RNA, and of occasional non-replicating cell-associated HCV, suggests a resulting poor infectivity of saliva or cervicovaginal fluid in chronically HCV-infected individuals. Taken together, these observations provide the basis for the low risk of non-parenteral transmission of HCV infection.

AKlebsiella pneumoniaeisolate was identified that had reduced susceptibility to several expanded-spectrum cephalosporins and imipenem. That isolate produced a chromosome-encoded SHV-type β-lactamase, SHV-38, that had an alanine to valine substitution in position Ambler 146 compared to β-lactamase SHV-1. The kinetic parameters for purified β-lactamases SHV-38 and SHV-1 showed that the hydrolytic spectrum of SHV-38 included only ceftazidime and imipenem. This report is the first example of an SHV-type β-lactamase capable of hydrolyzing imipenem.

Streptococcus pneumoniae may be considered like multiresistant bacteria, responsible for non-nosocomial infections. In France during 1999, 44 % of the strains were non-susceptible to penicillin G (PNSP) (with 12 % of resistant strains), 26 % were non-susceptible to amoxicilline (with 1,8 % of resistant strains) and 17,1% were non-susceptible to cefotaxime (with 0,4 % of resistant strains). The percentage of resistant strains was 1.8 % for amoxicilline and 0.4 % for cefotaxime. The PNSP's percentage was variable with regions, from 32 % in the “Pays de Loire” to 60 % in “Brie”. It was higher for children (52.7 %) than for adults (39.8 %). It was also a function of sample's site and age (child/adult): bloodstream 41.8 % / 37.2 %, broncho-pulmonary sample 54.2 % / 41.4 %, and cerebrospinal fluid 45.5 % / 37.5 %. These PNSP were often poly-resistant : 30.1 % of penicillin susceptible pneumococci but 82.2 % of the PNSP were resistant to erythromycin's. In children, the percentage of erythromycin's resistant pneumococci was about 73.2 % for ears, 66 % for broncho-pulmonary, 50.2 % for bloodstream and 56.3 % in cerebrospinal fluid. The most common serotype for PNSP strains was the 23 serotype. Lastly, for anti-pneumococci fluoroquinolones, few resistant strains have been mentioned.

In gram-positive bacteria, the ɛ-amino group of l-lysine in the pentapeptide stem of peptidoglycan precursors is often replaced by amino acid chains of various lengths and compositions (19) (Fig. ​(Fig.1).1). These amino acids form cross bridges between l-Lys3 and d-Ala4 following cross-linking of the peptide stems from nascent chains of peptidoglycan by the d,d-transpeptidases. The ligases for the addition of glycine and l-amino acids to the ɛ-amino group of l-lysine use aminoacyl-tRNAs as substrates, whereas d-amino acids are added in a tRNA-independent reaction (15, 20). FIG. 1 Structure of cross-linked peptidoglycan. (A) Fragment of the primary structure of peptidoglycan. The position of the peptide bond formed by the d,d-transpeptidases (penicillin-binding proteins) between the acceptor and donor peptide stems is indicated ... In Staphylococcus aureus, the femA, femB, and fmhB genes were shown to be essential for incorporation of glycine into the side chain of peptidoglycan precursors (18, 21). The femAB locus was initially identified as a factor essential for methicillin resistance (fem) based on random insertional inactivation of chromosomal genes and a screen for reduced expression of resistance mediated by the penicillin binding protein 2A (PBP2A) (2, 5). Inactivation of femA or femB was subsequently reported to prevent incorporation of glycine residues at positions 2 to 5 or positions 4 to 5 of the penta-glycine cross bridge since muropeptides cross-linked by one or three glycine residues were detected in the corresponding mutants (4, 21). Inactivation of fmhB, formerly femX, is lethal, but the construction of a mutant conditionally expressing fmhB under the control of a xylose-inducible promoter showed that the gene was essential for synthesis of branched peptidoglycan precursors (18). These results indicated that the fem gene products were required for incorporation of glycine at positions 1 (FmhB), 2 and 3 (FemA), and 4 and 5 (FemB) of the cross bridge, although the catalytic activity of the proteins was not directly assessed (18). Similarly, inactivation of two fmhB homologues in Streptococcus pneumoniae, designated murM (fibA) and murN (fibB), reduced addition of l-Ala or l-Ser to the ɛ-amino group of l-Lys and subsequent addition of a second l-Ala residue, respectively (6, 7, 23). Overall, disruption of the murMN operon reduced the proportion of branched peptide stems in the peptidoglycan from 89 to 33% (6). In contrast to what occurs in S. aureus (18), direct cross-linking of l-Lys to d-Ala occurs in S. pneumoniae, and the murMN operon was accordingly reported to be unessential (6). However, production of branched peptidoglycan precursors was necessary for expression of penicillin resistance (6) and appeared to affect mainly the activity of mosaic PBP2x (23). In enterococci, streptococci, and staphylococci, the ligation reactions are catalyzed by membrane-associated enzymes acting exclusively or preferentially on late peptidoglycan precursors linked to the undecaprenyl-phosphate lipid carrier, namely undecaprenyl-PP-MurNAc-pentapeptide (lipid I) and undecaprenyl-PP-MurNAc(pentapeptide)-GlcNAc (lipid II) (14). This conclusion is supported by the detection of only small amounts (

For an in vitro mutant of Streptococcus pneumoniae selected on moxifloxacin four- to eightfold-increased MICs of new fluoroquinolones, only a twofold-increased MIC of ciprofloxacin, and a twofold-decreased MIC of novobiocin were observed. This phenotype was conferred by two mutations: Ser81Phe in GyrA and a novel undescribed His103Tyr mutation in ParE, outside the quinolone resistance-determining region, in the putative ATP-binding site of topoisomerase IV.

In Enterococcus gallinarum SC1, a low-level vancomycin-resistant strain, only monomeric muropentapeptides with a C-terminal d -alanine were detected after growth without vancomycin. In contrast, in SC1 induced by vancomycin, as well as in AIB39, a constitutive vancomycin-resistant strain, monomeric and dimeric muropentapeptides with a C-terminal d -serine were detected.

Salvage therapy with ritonavir (RTV) and saquinavir (SQV) failed to achieve virological and immunological improvement in 24 HIV-infected patients who discontinued triple therapy with RTV or indinavir (IDV) because of failure or intolerance to treatment. Changes in the HIV-1 protease gene sequence were analyzed prospectively in 14 patients. No primary protease mutation was found prior to the use of protease inhibitors. After 7 months of treatment with IDV or RTV, primary resistance mutations at codons pol 46 and/or pol 82 were observed in 11 of 13 patients. After 16 weeks on RTV-SQV, novel primary mutations related to SQV emerged in 7 of 13 patients, together with an increase in the number of secondary resistance mutations. Our observations indicate that the cumulative occurrence of resistance mutations in the protease gene was associated with failure of antiretroviral therapy. The presence of mutations to a first protease inhibitor may represent a risk factor for the failure of a subsequent treatment with a second line protease inhibitor.

Oropharyngeal samples from 60 hospitalized patients (30 fluoroquinolone [FQ]-treated and 30 non-FQ-treated patients) and 30 untreated nonhospitalized healthy control subjects yielded 20 isolates of viridans group streptococci with reduced susceptibility to FQ, mostly from the hospitalized patients. An efflux phenotype was commonly encountered, expressed either alone or with topoisomerase mutations. Interspecies transfer of the efflux phenotype was demonstrated via transformation of Streptococcus pneumoniae R6 with DNA from S. mitis and S. oralis .

Epidemiological relationships were investigated between 40 methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased glycopeptide susceptibility isolated from November 1998 to March 1999 from 39 patients (17 infected and 22 colonized patients) in nine wards of the Broussais Hospital, Paris, France. Reduced glycopeptide susceptibility was readily detected on brain heart infusion (BHI) agar containing 6 μg of teicoplanin per ml and on gradient plates, but not by the standard disk diffusion method. The MICs of vancomycin and teicoplanin, determined on BHI agar, were 4 and 8 to 32 μg/ml, respectively (standard antibiotic dilution), and 4 to 8 and 8 to 32 μg/ml, respectively (E-test). All strains were resistant to macrolides, aminoglycosides, tetracycline, rifampin, sulfonamides, and pefloxacin, showed reduced susceptibility to fusidic acid and fosfomycin, and were susceptible to trimethoprim and chloramphenicol. Pulsed-field gel electrophoresis and lysotyping revealed that a multidrug-resistant MRSA clone with decreased susceptibility to glycopeptides has been discretely endemic since at least 1996 in our institution, where it was responsible for an outbreak in November and December 1998.

We investigated human immunodeficiency virus (HIV) type 1 RNA, proviral DNA, and antiretroviral drug-resistant variants in cervicovaginal secretions of HIV-1-infected women receiving antiretroviral therapy. The prevalence of detectable HIV-1 RNA in genital secretions was inversely related to the number of antiretroviral drugs taken by the patients. Proviral DNA was detected in approximately half of all samples of cervicovaginal secretions from HIV-1-infected women, regardless of the presence or absence of HIV-1 RNA in cervicovaginal secretions and of the antiretroviral regimen. In cervicovaginal secretions of most women with persisting genital viral replication, HIV variants exhibiting mutations associated with drug resistance against protease and reverse-transcriptase pol genes were found. Our observations indicate that antiretroviral therapy is not effective in purging the female genital tract of cell-associated provirus and that antiretroviral drugs that penetrate the female genital tract at suboptimal concentrations exert a potent selective pressure on genital HIV variants when local replication of free HIV-1 RNA persists.

The peptidoglycan structure of in vitro selected ampicillin-resistant mutant Enterococcus faecium D344M512 and of the susceptible parental strain D344S was determined by reverse phase high performance liquid chromatography and mass spectrometry. The muropeptide monomers were almost identical in the two strains. The substantial majority (99.3%) of the oligomers from the susceptible strain D344S contained the usual d-alanyl --> d-asparaginyl (or d-aspartyl)-l-lysyl cross-link (d-Ala --> d-Asx-l-Lys) generated by beta-lactam-sensitive DD-transpeptidation. The remaining oligomers (0.7%) were produced by beta-lactam-insensitive LD-transpeptidation, because they contained l-Lys --> d-Asx-l-Lys cross-links. The muropeptide oligomers of the ampicillin-resistant mutant D344M512 contained only these l-Lys --> d-Asx-l-Lys cross-links indicating that resistance was due to the bypass of the beta-lactam-sensitive DD-transpeptidation reaction. The discovery of this novel resistance mechanism indicates that DD-transpeptidases cannot be considered anymore as the sole essential transpeptidase enzymes.

Objective To study the efficacy and safety of high-concentration antibiotic locks in association with systemic antibiotherapy in Staphylococcus epidermidis infections of totally implantable venous access devices (TIVADs) in patients with AIDS. Methods Thirty-one episodes of S. epidermidis TIVAD infection were observed in nine patients. Locks consisted of high concentrations of aminoglycosides or vancomycin according to antibiogram results (susceptibility results obtained with the disk diffusion technique). Genotyping of bacterial strains was performed by pulsed-field gel electrophoresis (PFGE) in 26 of the 31 infectious episodes. Results The infections occurred within a median period of 62 catheter-days. The median duration of systemic antibiotic therapy was 17.5 days in association with a median of three antibiotic locks. Failure as defined by the occurrence of a novel episode within 2 months was observed in 17 of the 31 infections (58%). According to the PFGE results, relapse with the same strain of S. epidermidis or reinfection with a different strain of S. epidermidis could be assigned to 10 episodes which failed to respond to therapy. Relapse was observed in six of 10 episodes. Four episodes were followed by the occurrence of a novel infection with a different S. epidermidis strain. In one patient, a relapse occurred despite TIVAD removal. Conclusion: Our observations further document the poor efficacy of associating antibiotic locks with systemic antibiotic therapy for the treatment of TIVAD infections in patients with AIDS.

To evaluate the role of known topoisomerase IV and gyrase mutations in the fluoroquinolone (FQ) resistance of Streptococcus pneumoniae , we transformed susceptible strain R6 with PCR-generated fragments encompassing the quinolone resistance-determining regions (QRDRs) of parC or gyrA from different recently characterized FQ-resistant mutants. Considering the MICs of FQs and the GyrA and/or ParC mutations of the individual transformants, we found three levels of resistance. The first level was obtained when a single target, ParC or GyrA, depending on the FQ, was modified. An additional mutation(s) in a second target, GyrA or ParC, led to the second level. The highest increases in resistance levels were seen for Bay y3118 and moxifloxacin with the transformant harboring a double mutation in both ParC and GyrA. When a single modified target was considered, only the ParC mutation(s) led to an increase in the MICs of pefloxacin and trovafloxacin. In contrast, the GyrA or ParC mutation(s) could lead to increases in the MICs of ciprofloxacin, sparfloxacin, grepafloxacin, Bay y3118, and moxifloxacin. These results suggest that the preferential target of trovafloxacin and pefloxacin is ParC, whereas either ParC or GyrA may both be initial targets for the remaining FQs tested. The contribution of the ParC and GyrA mutations to efflux-mediated FQ resistance was also examined. Active efflux was responsible for two- to fourfold increases in the MICs of ciprofloxacin for the transformants, regardless of the initial FQ resistance levels of the recipients.

SUMMARY: The influence of NaCl on the susceptibility of Enterococcus faecalis to cefotaxime was tested with JH2-2, a laboratory strain, and 20 clinical strains grown on tryptic soy agar supplemented with 5% horse blood. Growth with 3% NaCl in the medium resulted in an increase in cefotaxime resistance and the appearance of a heterogeneous resistance phenotype: for the majority of the strains, the MlCs of cefotaxime increased from 4 to 512 pg m1-Y By a competition assay using cefotaxime and [3H]benzylpenicillin, it was shown for strain JH2-2 that at the MIC penicillin-binding protein (PBP) 2 and PBP3 were the apparent essential PBPs in medium without NaCI, whilst the low-affinity PBPs 4 and 1 were the apparent essential PBPs for cell growth in medium containing 3% NaCl. Analysis of JH2-2 peptidoglycan by HPLC and MS after growth in the presence of 3% NaCl showed a relative increase in unsubstituted monomers and a relative decrease in alanine- and dialanine-substituted monomers. It is therefore hypothesized that modification of the number of alanine-substituted precursors in the presence of NaCl could interfere with the functions of the different PBPs and thus play a role in cefotaxime resistance in E. faecalis.

Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in β-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of β-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzylpenicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 μg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental S. pneumoniae strain. The results stress the potential for the acquisition by S. pneumoniae of high-level β-lactam resistance by interspecies gene transfer.

The accumulation of fluoroquinolones (FQs) was studied in a FQ-susceptible laboratory strain of Streptococcus pneumoniae (strain R6). Uptake of FQs was not saturable, was rapidly reversible, and appeared to occur by passive diffusion. In the presence of glucose, which energizes bacteria, the uptake of FQs decreased. Inhibitors of the proton motive force and ATP synthesis increased the uptake of FQs in previously energized bacteria. Similar results were observed with the various FQs tested and may be explained to be a consequence simply of the pH gradient that exists across the cytoplasmic membrane. From a clinical susceptible strain (strain SPn5907) we isolated in vitro on ciprofloxacin an FQ-resistant mutant (strain SPn5929) for which the MICs of hydrophilic molecules were greater than those of hydrophobic molecules, and the mutant was resistant to acriflavine, cetrimide, and ethidium bromide. Strain SPn5929 showed a significantly decreased uptake of ciprofloxacin, and its determinant of resistance to ciprofloxacin was transferred by transformation to susceptible laboratory strain R6 (strain R6tr5929). No mutations in the quinolone resistance-determining regions of the gyrA and parC genes were found. In the presence of arsenate or carbonyl cyanide m-chlorophenylhydrazone, the levels of uptake of ciprofloxacin by the two resistant strains, SPn5929 and R6tr5929, reached the levels of uptake of their susceptible parents. These results suggest an active efflux of ciprofloxacin in strain SPn5929.

Antibiotic-resistant enterococci have become an increasingly important cause of nosocomial infections, frequently involved in hospital outbreaks. Acquired antibiotic resistance among enterococci include high-level aminoglycosides, β -lactams and glycopeptide resistance. Vancomycin-resistant enterococci (VRE) have had a major impact in the USA and are now widely distributed among different European countries. The treatment of VRE infections is usually difficult and relatively inefficient, leading to failure of antibiotic therapy and surgery for uncontrolled sepsis. Since there is often no therapeutic agent available of established efficacy for patients infected by VRE, in vitro studies and animal models represent useful tools to test new antibiotic molecules and to evaluate the efficacy of new antibiotics given alone or in association.

Resume Les effets des beta-lactamines vis-a-vis du pneumocoque, a l'exception de ceux de la penicilline G, sont peu connus. Nous avons compare l'effet lytique et bactericide de la penicilline G, de l'amoxicilline et du cefotaxime vis-a-vis de 4 souches de pneumocoque de sensibilite differente a la penicilline G. L'amoxicilline et la penicilline G ont un effet lytique comparable et les souches ainsi lysees sont rapidement tuees. Les effets du cefotaxime sont differents. Sur 3 des 4 souches etudiees, l'effet lytique du cefotaxime est inferieur a celui de la penicilline G ou de l'amoxicilline. Son activite bactericide est variable selon les souches independemment de leur sensibilite aux beta-lactamines, et peut s'observer en absence d'effet lytique. L'activite bactericide des penicillines resulte de la mise en jeu de systemes complexes qui ne dependent pas seulement du syteme autolytique. Les cephalosporines, qui n'inhibent pas les memes proteines de liaison aux penicillines, favoriseraient l'elaboration d'un peptidoglycane immature et pourraient impliquer ces systemes de facon differente.

A spontaneous Escherichia coli mutant, named Q3, resistant to nalidixic acid was obtained from a previously described clinical isolate of E. coli, Q2, resistant to fluoroquinolones but susceptible to nalidixic acid (E. Cambau, F. Bordon, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 37:1247-1252, 1993). Q3 harbored the mutation Asp82Gly in addition to the Gly81Asp mutation of Q2. The different mutations leading to Gly81Asp, Asp82Gly, and Gly81AspAsp82Gly were introduced into the gyrA gene harbored on plasmid pJSW102, and the resulting plasmids were introduced into E. coli KNK453 (gyrAts) by transformation. The presence of Asp82Gly or Gly81Asp alone led to a low-level resistance to fluoroquinolones but not to nalidixic acid resistance. When both mutations were present, resistance to both nalidixic acid and fluoroquinolones was expressed. Purified gyrases of the different mutants showed similar rates of supercoiling. Dominance of the various gyrA mutant alleles harbored on plasmids was examined. The susceptibility to quinolones associated with wild-type gyrA was always dominant. The susceptibility to nalidixic acid expressed by the Gly81Asp mutant was dominant, while that expressed by the Asp82Gly mutant was recessive. From these results, we hypothesize that some amino acids within the quinolone resistance-determining region of gyrase A are more important for the association of subunits rather than for the activity of the holoenzyme.

Forty-seven bronchoalveolar lavages (BAL) were obtained from 41 patients with acute pneumonia attending an intensive care unit. By molecular diagnosis, 30% of total BAL and 63% of bacteria-negative BAL were positive for respiratory viruses. Molecular detection allows for high-rate detection of respiratory viral infections in adult patients suffering from severe pneumonia.

International audience; Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt(fm)) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt(fm) does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt(fm) by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt(fm) inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt(fm) acylation was followed by rupture of the C(5)-C(6) bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt(fm) blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.

Pneumococcus is an important human pathogen, highly antibiotic resistant and a major cause of bacterial meningitis worldwide. Better prevention requires understanding the drivers of pneumococcal infection incidence and antibiotic susceptibility. Although respiratory viruses (including influenza) have been suggested to influence pneumococcal infections, the underlying mechanisms are still unknown, and viruses are rarely considered when studying pneumococcus epidemiology. Here, we propose a novel mathematical model to examine hypothetical relationships between Streptococcus pneumoniae meningitis incidence (SPMI), acute viral respiratory infections (AVRIs) and antibiotic exposure. French time series of SPMI, AVRI and penicillin consumption over 2001–2004 are analysed and used to assess four distinct virus–bacteria interaction submodels, ascribing the interaction on pneumococcus transmissibility and/or pathogenicity. The statistical analysis reveals strong associations between time series: SPMI increases shortly after AVRI incidence and decreases overall as the antibiotic-prescription rate rises. Model simulations require a combined impact of AVRI on both pneumococcal transmissibility (up to 1.3-fold increase at the population level) and pathogenicity (up to threefold increase) to reproduce the data accurately, along with diminished epidemic fitness of resistant pneumococcal strains causing meningitis (0.97 (0.96–0.97)). Overall, our findings suggest that AVRI and antibiotics strongly influence SPMI trends. Consequently, vaccination protecting against respiratory virus could have unexpected benefits to limit invasive pneumococcal infections.

A 78-year-old woman developed fatal endocarditis of her prosthetic aortic valve, caused by Bacteroides fragilis fragilis , and associated with ovarian carcinoma. The strain showed multiple antibiotic resistance, including resistance to beta-lactam agents and combinations with beta-lactamase inhibitors. Seventeen previously described cases of endocarditis caused by Bacteroides spp. have been found in the literature. The mean age of the 18 patients was 50.3 years, the gastro-intestinal tract was the most common site of associated disease, embolism occured in ten cases and eight patients died. Previous isolates showed the antibiotic susceptibility customarily associated with the B. fragilis group.

Bacteriocin susceptibilities indicate that during cloacin DF13 uptake the F porin of Enterobacter cloacae plays a similar role to that reported for the OmpF porin of Escherichia coli during colicin A entry. The translocatory activities of these two porins during the bacteriocin uptake can be substituted by the porins D and OmpC, respectively, under conditions not requiring the receptor binding step. Using anti-peptide antibodies, a peptide located in the internal loop L3 of the Escherichia coli OmpF porin was identified in the D and F porins of Enterobacter cloacae. The results demonstrated the existence of a close relationship between porins in terms of both antigenic determinants and bacteriocin susceptibilities.

Bacteriocin susceptibilities indicate that during cloacin DF13 uptake the F porin of Enterobacter cloacae plays a similar role to that reported for the OmpF porin of Escherichia coli during colicin A entry. The translocatory activities of these two porins during the bacteriocin uptake can be substituted by the porins D and OmpC, respectively, under conditions not requiring the receptor binding step. Using anti-peptide antibodies, a peptide located in the internal loop L3 of the Escherichia coli OmpF porin was identified in the D and F porins of Enterobacter cloacae. The results demonstrated the existence of a close relationship between porins in terms of both antigenic determinants and bacteriocin susceptibilities.

To identify risk factors for and prognostic indicators of the nosocomial acquisition of multiresistant Acinetobacter baumannii in an intensive care unit, we prospectively studied 40 patients: 13 who were infected with this organism and 27 who were colonized. Isolates were identified by pulsed-field gel electrophoresis; the infected/colonized patients were compared with 348 noninfected, noncolonized patients by logistic regression analysis and with matched historical controls in a cohort study. The severity of illness (evaluated by the APACHE II score; P < .05) and previous infection (P < .001) were retained as independent risk factors for acquiring A. baumannii. Logistic regression analysis selected a high APACHE II score (P < .01) and the acquisition of A. baumannii (P < .01) as factors independently associated with death. The acquisition of A. baumannii was associated not only with high mortality but also with a length of stay on the intensive care unit in excess of that due to the underlying disease alone; specifically, the attributable mortality was 25%, with a risk ratio for death of 2.0 (95% confidence interval, 1.11-3.62), and the duration of stay for infected/colonized patients was 10.3 days longer than that for controls (P < .001).

A synergistic bactericidal effect between penicillin, vancomycin, and gentamicin has been described against enterococci highly resistant to beta-lactams and glycopeptides. Since such a synergy was also observed in vitro between ceftriaxone, teicoplanin, and gentamicin against Enterococcus faecium 70/90, the efficacy of different combinations including penicillin or ceftriaxone, vancomycin or teicoplanin, and gentamicin was compared in vivo in experimental endocarditis. In vitro, all four triple combinations provided a bactericidal effect. In rabbits, after a 5-day treatment, the ceftriaxone-vancomycin-gentamicin combination was the most effective, both in reducing the total bacterial titers and in eradicating the subpopulation resistant to the synergy. Compared with the 5-day regimen, 10- and 20-day regimens of ceftriaxone-vancomycin-gentamicin, each followed by a 3-day antibiotic-free period, increased the number of sterilized animals but failed to avoid the emergence of resistant bacteria, which occurred in 10%-20% of animals.

Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet (M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 μg/ml) were found to harbor truncated tet (M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet (M) promoter region, among which four displayed increased Tc MICs (16 to 64 μg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet (M). Expression in Enterococcus faecalis of a tet (M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet (M) and by alterations of the upstream transcriptional attenuator.