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1.  Molecular Basis for Different Levels of tet(M) Expression in Streptococcus pneumoniae Clinical Isolates 
Antimicrobial Agents and Chemotherapy  2012;56(10):5040-5045.
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.
doi:10.1128/AAC.00939-12
PMCID: PMC3457367  PMID: 22802249
3.  Plasmidic qnrA3 Enhances Escherichia coli Fitness in Absence of Antibiotic Exposure 
PLoS ONE  2011;6(9):e24552.
The widespread presence of plasmid-mediated quinolone resistance determinants, particularly qnr genes, has become a current issue. By protecting DNA-gyrase from quinolones, Qnr proteins confer a low level quinolone resistance that is not sufficient to explain their emergence. Since Qnr proteins were hypothesized to act as DNA-binding protein regulators, qnr genes could have emerged by providing a selective advantage other than antibiotic resistance. We investigated host fitness of Escherichia coli isogenic strains after acquisition of the qnrA3 gene, inserted either alone onto a small plasmid (pBR322), or harbored on a large conjugative native plasmid, pHe96(qnrA3) found in a clinical isolate. The isogenic strains were derived from the susceptible E. coli CFT073, a virulent B2 group strain known to infect bladder and kidneys in a mouse model of pyelonephritis. In vitro experiments included growth analysis by automatic spectrophotometry and flow cytometry, and competitions with CFU enumeration. In vivo experiments included infection with each strain and pairwise competitions in absence of antimicrobial exposure. As controls for our experiments we used mutations known to reduce fitness (rpsL K42N mutation) or to enhance fitness (tetA deletion in pBR322). E. coli CFT073 transformed with pBRAM(PBR322-qnrA3) had significantly higher maximal OD than E. coli CFT073 transformed with pBR322 or pBR322ΔtetA, and in vivo competitions were more often won by the qnrA3 carrying strain (24 victories vs. 9 loss among 42 competitions, p = 0.001). In contrast, when pHe96(qnrA3) was introduced by conjugation in E. coli CFT073, it exerted a fitness cost shown by an impaired growth observed in vitro and in vivo and a majority of lost competitions (33/35, p<0.0001). In conclusion, qnrA3 acquisition enhanced bacterial fitness, which may explain qnr emergence and suggests a regulation role of qnr. However, fitness was reduced when qnrA3 was inserted onto multidrug-resistant plasmids and this can slow down its dissemination without antibiotic exposure.
doi:10.1371/journal.pone.0024552
PMCID: PMC3168526  PMID: 21915350
4.  Nocardia pseudobrasiliensis as an Emerging Cause of Opportunistic Infection after Allogeneic Hematopoietic Stem Cell Transplantation▿  
Journal of Clinical Microbiology  2009;48(2):656-659.
We report the case of a 55-year-old man who exhibited a nodular pneumonia 4 months after an allogeneic hematopoietic stem cell transplantation. Culture of the bronchoalveolar lavage fluid revealed Nocardia pseudobrasiliensis. This recently described carbapenem-resistant species should be included in the differential diagnosis of fungal infection in this setting.
doi:10.1128/JCM.01244-09
PMCID: PMC2815612  PMID: 19940053
5.  Postoperative Mediastinitis Due to Finegoldia magna with Negative Blood Cultures▿  
Journal of Clinical Microbiology  2009;47(12):4180-4182.
We report a case of Finegoldia magna (formerly known as Peptostreptococcus magnus) mediastinitis following coronary artery bypass in a 50-year-old patient. Even if staphylococci remain the main causative organism of postoperative mediastinitis, the responsibility of anaerobic bacteria must be considered in cases of fever and sternal drainage with negative blood cultures.
doi:10.1128/JCM.01192-09
PMCID: PMC2786666  PMID: 19812272
6.  First Case of Streptococcus oligofermentans Endocarditis Determined Based on sodA Gene Sequences after Amplification Directly from Valvular Samples▿  
Journal of Clinical Microbiology  2008;47(3):855-856.
We report the first case of infection due to Streptococcus oligofermentans, which is a recently described oral Streptococcus species. It was responsible for the endocarditis and left forearm abscess of a 43-year-old woman. Identification was made using molecular techniques performed directly from valvular and surgical samples.
doi:10.1128/JCM.01436-08
PMCID: PMC2650917  PMID: 19116351
7.  Case of Indolent Endocarditis Due to Pseudomonas stutzeri with Genetic Evidence of Relapse after 4 Years▿  
Journal of Clinical Microbiology  2008;47(2):503-504.
Pseudomonas stutzeri, a gram-negative bacterium, is a common inhabitant of soil and water. We report an unusual case of a relapse of infective endocarditis due to P. stutzeri 4 years after the initial episode. The identity of the strains was proven by genomic analysis.
doi:10.1128/JCM.00707-08
PMCID: PMC2643672  PMID: 19052175
8.  A Plasmid-Borne Shewanella algae Gene, qnrA3, and Its Possible Transfer In Vivo between Kluyvera ascorbata and Klebsiella pneumoniae▿  
Journal of Bacteriology  2008;190(15):5217-5223.
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 ISCR1 element at a recombination crossover site described for other resistance genes, including qnrA1, and immediately upstream from IS26, a situation not described before. IS26 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 blaOXA-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.
doi:10.1128/JB.00243-08
PMCID: PMC2493261  PMID: 18515416
10.  Early Diagnosis of Disseminated Mycobacterium genavense Infection 
Emerging Infectious Diseases  2008;14(2):346-347.
doi:10.3201/eid1401.070901
PMCID: PMC2600216  PMID: 18258141
RNA; ribosomal; 16S; Mycobacterium infections; heart; transplant; letter
11.  Dolosigranulum pigrum Causing Nosocomial Pneumonia and Septicemia▿  
Journal of Clinical Microbiology  2007;45(10):3474-3475.
We report a case of non-ventilator-associated nosocomial pneumonia and septicemia due to Dolosigranulum pigrum, a rare gram-positive opportunistic pathogen. The organism was isolated from bronchoalveolar lavage fluid and blood of a debilitated patient. D. pigrum was identified after 16S rRNA gene sequencing.
doi:10.1128/JCM.01373-07
PMCID: PMC2045320  PMID: 17687015
12.  Molecular Diagnosis of Kingella kingae Pericarditis by Amplification and Sequencing of the 16S rRNA Gene▿  
Journal of Clinical Microbiology  2007;45(9):3133-3134.
Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.
doi:10.1128/JCM.00809-07
PMCID: PMC2045294  PMID: 17634294
13.  Modes and Modulations of Antibiotic Resistance Gene Expression 
Clinical Microbiology Reviews  2007;20(1):79-114.
Since antibiotic resistance usually affords a gain of function, there is an associated biological cost resulting in a loss of fitness of the bacterial host. Considering that antibiotic resistance is most often only transiently advantageous to bacteria, an efficient and elegant way for them to escape the lethal action of drugs is the alteration of resistance gene expression. It appears that expression of bacterial resistance to antibiotics is frequently regulated, which indicates that modulation of gene expression probably reflects a good compromise between energy saving and adjustment to a rapidly evolving environment. Modulation of gene expression can occur at the transcriptional or translational level following mutations or the movement of mobile genetic elements and may involve induction by the antibiotic. In the latter case, the antibiotic can have a triple activity: as an antibacterial agent, as an inducer of resistance to itself, and as an inducer of the dissemination of resistance determinants. We will review certain mechanisms, all reversible, that bacteria have elaborated to achieve antibiotic resistance by the fine-tuning of the expression of genetic information.
doi:10.1128/CMR.00015-06
PMCID: PMC1797629  PMID: 17223624
14.  Relevance of Routine Use of the Anaerobic Blood Culture Bottle▿  
Journal of Clinical Microbiology  2007;45(8):2711-2715.
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.
doi:10.1128/JCM.00059-07
PMCID: PMC1951263  PMID: 17581942
15.  Hip Prosthesis Infection Due to Mycobacterium wolinskyi 
Journal of Clinical Microbiology  2006;44(9):3463-3464.
Mycobacterium wolinskyi, first described in 1999, is a rapidly growing mycobacterium related to the Mycobacterium smegmatis group. Only eight cases of infection due to this microorganism have been reported, including three cases of bone infection. Here, we present the first case of a joint prosthesis infection cured with the combination of surgery and prolonged antibiotic therapy. The microorganism was identified by biochemical tests and 16S rRNA and Hsp65 gene sequence analysis.
doi:10.1128/JCM.02685-05
PMCID: PMC1594673  PMID: 16954303
16.  Activities of Different Fluoroquinolones against Bacillus anthracis Mutants Selected In Vitro and Harboring Topoisomerase Mutations 
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.
doi:10.1128/AAC.48.8.3024-3027.2004
PMCID: PMC478509  PMID: 15273116
18.  Comparative Molecular and Microbiologic Diagnosis of Bacterial Endocarditis 
Emerging Infectious Diseases  2003;9(12):1543-1547.
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.
doi:10.3201/eid0912.030229
PMCID: PMC3034331  PMID: 14720393
Endocarditis; negative blood culture; molecular diagnosis; PCR; 16S rDNA; rpoB and sodA genes
19.  Emergence in Klebsiella pneumoniae of a Chromosome-Encoded SHV β-Lactamase That Compromises the Efficacy of Imipenem 
A Klebsiella pneumoniae isolate 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.
doi:10.1128/AAC.47.2.755-758.2003
PMCID: PMC151740  PMID: 12543688
20.  Salmonella enterica Serovar Typhimurium blaPER-1-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-N-Acetyltransferase of Type Ib 
We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 β-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6′)-Ib11, was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the β-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6′)-Ib11 translation. AAC(6′)-Ib11 had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6′)-Ib11 and two other acetyltransferase gene variants, aac(6′)-Ib7 and -Ib8, which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119.
doi:10.1128/AAC.47.2.697-703.2003
PMCID: PMC151738  PMID: 12543680
21.  Fluoroquinolone Resistance Associated with Target Mutations and Active Efflux in Oropharyngeal Colonizing Isolates of Viridans Group Streptococci 
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.
PMCID: PMC90040  PMID: 10898702
22.  Outbreak of Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Glycopeptides in a Parisian Hospital 
Journal of Clinical Microbiology  2000;38(8):2985-2988.
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.
PMCID: PMC87166  PMID: 10921964
23.  Overexpression of the marA or soxS Regulatory Gene in Clinical Topoisomerase Mutants of Escherichia coli 
The contribution of regulatory genes to fluoroquinolone resistance was studied with clinical Escherichia coli strains bearing mutations in gyrA and parC and with different levels of fluoroquinolone resistance. Expression of marA and soxS was evaluated by Northern blot analysis of isolates that demonstrated increased organic solvent tolerance, a phenotype that has been linked to overexpression of marA, soxS, and rob. Among 25 cyclohexane-tolerant strains detected by a screen for increased organic solvent tolerance (M. Oethinger, W. V. Kern, J. D. Goldman, and S. B. Levy, J. Antimicrob. Chemother. 41:111–114, 1998), we found 5 Mar mutants and 4 Sox mutants. A further Mar mutant was detected among 11 fluoroquinolone-resistant, cyclohexane-susceptible E. coli strains used as controls. Comparison of the marOR sequences of clinical Mar mutants with that of E. coli K-12 (GenBank accession no. M96235) revealed point mutations in marR in all mutants which correlated with loss of repressor function as detected in a marO::lacZ transcriptional assay. We found four other amino acid changes in MarR that did not lead to loss of function. Two of these changes, present in 20 of the 35 sequenced marOR fragments, identified a variant genotype of marOR. Isolates with the same gyrA and parC mutations showed increased fluoroquinolone resistance when the mutations were accompanied by overexpression of marA or soxS. These data support the hypothesis that high-level fluoroquinolone resistance involves mutations at several chromosomal loci, comprising structural and regulatory genes.
PMCID: PMC105868  PMID: 9687412
24.  Multidrug Resistance following Expression of the Escherichia coli marA Gene in Mycobacterium smegmatis 
Journal of Bacteriology  1998;180(11):2995-2998.
Expression of the Escherichia coli multiple antibiotic resistance marA gene cloned in Mycobacterium smegmatis produced increased resistance to multiple antimicrobial agents, including rifampin, isoniazid, ethambutol, tetracycline, and chloramphenicol. Cloned marR or marA cloned in the antisense direction had no effect. Resistance changes were lost with spontaneous loss of the plasmid bearing marA. A MarA mutant protein, having an insertional mutation within either of its two alpha-helices of the first putative helix-turn-helix domain, failed to produce the multiresistance phenotype in E. coli and M. smegmatis, indicating that this region is critical for MarA function. These results strongly suggest that E. coli marA functions in M. smegmatis and that a mar-like regulatory system exists in this organism.
PMCID: PMC107271  PMID: 9603894
25.  Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii 
Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Both compounds, however, as well as isepamicin, retained good bactericidal activity against the three strains. The aac genes were borne by conjugative plasmids (pLMM562 and pLMM564 of ca. 100 kb and pLMM563 of ca. 20 kb). By PCR mapping and nucleotide sequence analysis, an aac(6′)-Ib gene was found in each strain upstream of an ant(3")-I gene in a sulI-type integron. The size of the AAC(6′)-Ib variant encoded by pLMM562 and pLMM564, AAC(6′)-Ib7, was deduced to be 184 (or 177) amino acids long, whereas in pLMM563 a 21-bp duplication allowing the recruitment of a start codon resulted in the translation of a variant, AAC(6′)-Ib8, of 196 amino acids, in agreement with size estimates obtained by Western blot analysis. Both variants had at position 119 a serine instead of the leucine typical for the AAC(6′)-Ib variants conferring resistance to amikacin. By using methods that predict the secondary structure, these two amino acids appear to condition an α-helical structure within a putative aminoglycoside binding domain of AAC(6′)-Ib variants.
PMCID: PMC105389  PMID: 9527761

Results 1-25 (25)