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1.
BMC Pulm Med ; 14: 137, 2014 Aug 12.
Article in English | MEDLINE | ID: mdl-25115239

ABSTRACT

BACKGROUND: Fiberoptic bronchoscopy (FOB) is a useful diagnosis tool in low-burden countries for patients with suspected pulmonary tuberculosis (TB) who are smear-negative or sputum-scarce. This study sought to determine the accuracy of the Xpert® MTB/RIF (XP) assay using FOB samples. METHODS: We retrospectively reviewed clinical, radiological, and microbiological characteristics of 175 TB-suspected patients requiring diagnostic FOB (bronchial aspirate or bronchoalveolar lavage) with XP assay. Polymerase chain reaction (PCR) and smear microscopy (SM) performances were first compared to culture, then to the final diagnosis, established based on clinical or radiological evolution when cultures were negative. RESULTS: Of the total 162 included patients, 30 (18.5%) had a final diagnosis of pulmonary TB, with positive cultures reported in 23. As compared to culture, sensitivity and specificity values were 80.0% and 98.6% for the XP assay, and 25.0% and 95.8% for SM, respectively. As compared to final diagnosis, the corresponding performance values were 60.0% and 100.0% for the XP assay, and 16.7% and 95.5% for SM, respectively. The sensitivity of the XP assay was significantly higher than that of SM in both cases (p=0.003 and p=0.001). Concerning the final diagnosis, both XP assay and culture sensitivities were similar (60% vs. 66.7%). PCR assay enabled pulmonary TB to be diagnosed earlier in 13 more cases, compared to SM. CONCLUSION: Our study has confirmed the clinical benefits provided by XP assay compared to SM for the early diagnosis of suspected pulmonary TB cases requiring FOB, on per procedure samples, especially in a low TB-burden country.


Subject(s)
Bronchoalveolar Lavage Fluid/microbiology , Bronchoscopy , Molecular Diagnostic Techniques , Real-Time Polymerase Chain Reaction , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Pulmonary/diagnosis , Adult , Aged , Early Diagnosis , Female , Humans , Male , Microscopy , Middle Aged , Predictive Value of Tests , Retrospective Studies , Sputum/microbiology , Tuberculosis, Multidrug-Resistant/microbiology , Tuberculosis, Pulmonary/microbiology
2.
Antimicrob Agents Chemother ; 57(9): 4463-9, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23836170

ABSTRACT

As opposed to Enterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A), Enterococcus faecium is naturally susceptible. Since this phenotype may be selected for in vivo by quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance in E. faecium. Six LSAP-resistant in vitro mutants of E. faecium HM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of an in vitro mutant (E. faecium UCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in all in vitro and in vivo resistant strains but was not present in susceptible strains. The wild-type allele was named eat(A) (for Enterococcus ABC transporter), and its mutated allelic variant was named eat(A)v. The introduction of eat(A)v from UCN90B into HM1070 conferred the LSAP phenotype, whereas that of eat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance in E. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Enterococcus faecium/genetics , Lincosamides/pharmacology , Streptogramin A/pharmacology , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Diterpenes/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Enterococcus faecium/drug effects , Enterococcus faecium/metabolism , Genetic Complementation Test , Genotype , Molecular Sequence Data , Phenotype , Point Mutation , Polycyclic Compounds , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Pleuromutilins
3.
PLoS One ; 7(8): e43520, 2012.
Article in English | MEDLINE | ID: mdl-22937059

ABSTRACT

BACKGROUND: QuantiFERON®-TB Gold in-Tube (QFT) assay is a recently developed test to assess latent tuberculosis infection in contagious tuberculosis (TB) contact subjects. To assess the QFT assay in recently exposed contacts of active tuberculosis patients in a French area with low TB incidence but high Bacille Calmette-Guerin coverage, and evaluate progression rates to TB disease. METHODOLOGY/PRINCIPAL FINDINGS: Between January 2007 and December 2009, 687 contacts of culture-confirmed tuberculosis cases underwent the QFT assay, with tuberculin skin test (TST) in 473, and a 34 months mean follow-up. Of 687 contacts, 148 were QFT positive, while 526 were negative and 13 indeterminate. QFT was positive in 35% of individuals with TST ≥ 10 mm, 47.5% with TST ≥ 15 mm or phlyctenular, but in 21% of cases in which two-step TST (M0 and M3) remained negative. Conversely, QFT was negative in 69% of cases with two-step TST showing conversion from negative to positive. All indeterminate QFT were associated with TST induration <10 mm in diameter. For 29 QFT-positive subjects, no chemoprophylaxis was given due to medical contraindications. Of the remaining 119 QFT-positive contacts, 97 accepted chemoprophylaxis (81.5%), and 79 (81.4%) completed the treatment. Two contacts progressed to TB disease: one subject was QFT positive and had declined chemoprophylaxis, while the other one was QFT negative. QFT positive predictive value for progression to TB was 1.96% (1/51) with a 99.8% (525/526) negative predictive value. CONCLUSIONS/SIGNIFICANCE: Our results confirm the safety of the QFT-based strategy for assessing the TB chemoprophylaxis indication, as only one contact developed TB disease out of 526 QFT-negative subjects.


Subject(s)
Tuberculin Test/methods , Tuberculosis/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Incidence , Infant , Infant, Newborn , Male , Middle Aged , Prospective Studies , Tuberculosis/epidemiology , Young Adult
4.
Antimicrob Agents Chemother ; 55(4): 1470-4, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21245447

ABSTRACT

Streptococcus agalactiae UCN70, isolated from a vaginal swab obtained in New Zealand, is resistant to lincosamides and streptogramins A (LS(A) phenotype) and also to tiamulin (a pleuromutilin). By whole-genome sequencing, we identified a 5,224-bp chromosomal extra-element that comprised a 1,479-bp open reading frame coding for an ABC protein (492 amino acids) 45% identical to Lsa(A), a protein related to intrinsic LS(A) resistance in Enterococcus faecalis. Expression of this novel gene, named lsa(C), in S. agalactiae BM132 after cloning led to an increase in MICs of lincomycin (0.06 to 4 µg/ml), clindamycin (0.03 to 2 µg/ml), dalfopristin (2 to >32 µg/ml), and tiamulin (0.12 to 32 µg/ml), whereas no change in MICs of erythromycin (0.06 µg/ml), azithromycin (0.03 µg/ml), spiramycin (0.25 µg/ml), telithromycin (0.03 µg/ml), and quinupristin (8 µg/ml) was observed. The phenotype was renamed the LS(A)P phenotype on the basis of cross-resistance to lincosamides, streptogramins A, and pleuromutilins. This gene was also identified in similar genetic environments in 17 other S. agalactiae clinical isolates from New Zealand exhibiting the same LS(A)P phenotype, whereas it was absent in susceptible S. agalactiae strains. Interestingly, this extra-element was bracketed by a 7-bp duplication of a target site (ATTAGAA), suggesting that this structure was likely a mobile genetic element. In conclusion, we identified a novel gene, lsa(C), responsible for the acquired LS(A)P resistance phenotype in S. agalactiae. Dissection of the biochemical basis of resistance, as well as demonstration of in vitro mobilization of lsa(C), remains to be performed.


Subject(s)
Anti-Bacterial Agents/pharmacology , Lincosamides/pharmacology , Streptococcus agalactiae/drug effects , Streptogramins/pharmacology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Diterpenes/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Microbial Sensitivity Tests , Molecular Sequence Data , Open Reading Frames/genetics , Polycyclic Compounds , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Streptococcus agalactiae/genetics , Pleuromutilins
5.
Antimicrob Agents Chemother ; 53(9): 4007-9, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19546366

ABSTRACT

We compared the propensities of Enterococcus faecalis JH2-2 and of the recombination-deficient JH2-2 recA strain to develop mutational resistance to linezolid. In both organisms, a mutation in a single rrl copy conferred resistance to linezolid. Delay in acquisition of the mutation by other rrl copies in JH2-2 recA showed that gene conversion contributed to the acquisition of resistance.


Subject(s)
Acetamides/pharmacology , Anti-Bacterial Agents/pharmacology , Enterococcus faecalis/drug effects , Enterococcus faecalis/genetics , Oxazolidinones/pharmacology , Drug Resistance, Bacterial/genetics , Linezolid , Microbial Sensitivity Tests , Mutation
6.
Int J Antimicrob Agents ; 34(3): 274-7, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19414240

ABSTRACT

The genetic basis of erythromycin resistance in Turicella otitidis, a coryneform bacteria associated with otitis, was studied in five macrolide-resistant clinical isolates. Macrolide resistance genes were searched for by polymerase chain reaction (PCR). Genes for domain V of 23S rRNA (rrl) as well as rplD (L4 protein) and rplV (L22 protein) genes were characterised, amplified by PCR from total genomic DNA and sequenced. In the resistant isolates, cross-resistance to macrolides and clindamycin was associated with mutations at positions 2058 and/or 2059 (Escherichia coli numbering). Three isolates displayed A2058 mutations, one isolate had an A2059G mutation whereas another one contained mutations at positions 2058 and 2059. Southern blot experiments revealed that T. otitidis had three copies of the rrl gene. In conclusion, resistance to macrolides in T. otitidis is due, at least in part, to mutations in the rrl gene.


Subject(s)
Amino Acid Substitution , Bifidobacteriales Infections/microbiology , Bifidobacterium/drug effects , Bifidobacterium/genetics , Drug Resistance, Multiple, Bacterial/genetics , Macrolides/pharmacology , RNA, Ribosomal, 23S/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bifidobacterium/isolation & purification , Clindamycin/pharmacology , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Gene Dosage , Humans , Ribosomal Proteins/genetics , Sequence Analysis, DNA
7.
Antimicrob Agents Chemother ; 52(2): 626-30, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18086844

ABSTRACT

Streptococcus uberis UCN 42, isolated from a case of bovine mastitis, was intermediately resistant to lincomycin (MIC = 2 microg/ml) while remaining susceptible to clindamycin (MIC = 0.06 microg/ml) and erythromycin. A 1.1-kb SacI fragment was cloned from S. uberis UCN 42 total DNA on plasmid pUC 18 and introduced into Escherichia coli AG100A, where it conferred resistance to both clindamycin and lincomycin. The sequence analysis of the fragment showed the presence of a new gene, named lnu(D), that encoded a 164-amino-acid protein with 53% identity with Lnu(C) previously reported to occur in Streptococcus agalactiae. Crude lysates of E. coli AG100A containing the cloned lnu(D) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl(2). Mass spectrometry experiments demonstrated that the lnu(D) enzyme catalyzed adenylylation of clindamycin. A domain conserved in deduced sequences of lincosamide O-nucleotidyltransferases Lnu(A), Lnu(C), LinA(N2), and Lin(D) and in the aminoglycoside nucleotidyltransferase ANT(2'') was identified.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Lincomycin/pharmacology , Mastitis, Bovine/microbiology , Nucleotidyltransferases/genetics , Streptococcus/drug effects , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Cattle , Female , Microbial Sensitivity Tests , Molecular Sequence Data , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Sequence Analysis, DNA , Streptococcus/enzymology , Streptococcus/genetics , Streptococcus/isolation & purification
8.
J Antimicrob Chemother ; 54(6): 1040-4, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15537693

ABSTRACT

OBJECTIVES: To characterize a new type of resistance to clindamycin in Streptococcus agalactiae. METHODS: Nineteen erythromycin-susceptible, clindamycin-resistant S. agalactiae isolates from New Zealand were studied. MICs of macrolide, lincosamide and streptogramin antibiotics were determined. Clindamycin and streptogramin resistance genes were searched for by PCR. Isolates were compared by serotyping and by DNA macrorestriction patterns determined by PFGE. Conjugative transfer of resistance traits to recipient strains of S. agalactiae and Enterococcus faecium was assayed. RESULTS: The 19 S. agalactiae isolates were intermediate or resistant to clindamycin (MIC range: 0.5-2 mg/L) and lincomycin (MIC range: 1-8 mg/L) and had high MICs of dalfopristin (4-32 mg/L), a streptogramin A-type antibiotic, compared with controls. By contrast, the strains were susceptible to macrolides and quinupristin, a streptogramin B-type antibiotic. This new phenotype was called LSA (lincosamide-streptogramin A). Clindamycin resistance could not be transferred to recipient strains. Thirteen isolates belonged to serotype III and to a single PFGE genotype A, and five isolates belonged to serotype I and to genotype B. One isolate was non-typeable and belonged to a distinct genotype C. CONCLUSIONS: We have characterized a new LSA phenotype in S. agalactiae. Analysis of restriction patterns of S. agalactiae chromosomal DNA showed that the resistance was spread in a minimum of three bacterial clones. The genetic and biochemical basis for the resistance remains unknown.


Subject(s)
Anti-Bacterial Agents/pharmacology , Clindamycin/pharmacology , Drug Resistance, Bacterial , Streptococcal Infections/epidemiology , Streptococcus agalactiae/drug effects , Adult , Aged , Aged, 80 and over , Drug Resistance, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Female , Humans , Infant, Newborn , Lincosamides , Macrolides/pharmacology , Male , Microbial Sensitivity Tests , Middle Aged , New Zealand/epidemiology , Phenotype , Streptococcal Infections/microbiology , Streptococcus agalactiae/classification , Streptococcus agalactiae/genetics , Streptogramin A/pharmacology
9.
J Antimicrob Chemother ; 54(1): 117-21, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15190021

ABSTRACT

OBJECTIVES: To determine the prevalence of macrolide and streptogramin resistance in Streptococcus mitis isolates from neutropenic patients and to identify mechanisms of macrolide and streptogramin resistance in resistant isolates. METHODS: MICs of erythromycin, spiramycin, lincomycin and pristinamycin were determined for S. mitis isolates. Macrolide-resistance genes were characterized by PCR and ribosomal mutations by sequencing. RESULTS: A total of 169 S. mitis isolates were recovered from 66 patients at the Tunisian Bone Marrow Transplant Centre. Of these, 120 (70%) were non-susceptible to erythromycin and one was resistant to pristinamycin; 48.5% of isolates had an MLSB phenotype with cross-resistance between erythromycin, spiramycin and lincomycin, 4% had a dissociated MLSB phenotype with resistance to erythromycin and spiramycin but apparent susceptibility to lincomycin and 47.5% displayed the M phenotype. Resistance determinants were characterized in 33 isolates. Ten of 14 isolates with the cross MLSB resistance contained an ermB-like gene and four a combination of ermB- and mefA-like genes. Four of the five isolates with a dissociated MLSB phenotype contained ermB-like and one a combination of ermB- and mefA-like genes. All the 14 isolates with an M phenotype contained mefA-like genes. The pristinamycin-resistant strain had G105 and A108 substitutions in the conserved C terminus of the L22 ribosomal protein. CONCLUSIONS: The prevalence of macrolide resistance is high in S. mitis from neutropenic patients and is due to the spread of ermB- or mefA-like genes alone or combined. Resistance to streptogramins is rare and in this case associated with ribosomal mutation.


Subject(s)
Anti-Bacterial Agents/pharmacology , Macrolides/pharmacology , Neutropenia/microbiology , Streptococcal Infections/microbiology , Streptococcus mitis/drug effects , Streptococcus mitis/genetics , Streptogramins/pharmacology , Amino Acid Sequence , Bone Marrow Transplantation , DNA Primers , Drug Resistance, Bacterial , Erythromycin/pharmacology , Genotype , Humans , Immunocompromised Host , Microbial Sensitivity Tests , Molecular Sequence Data , Phenotype , Pristinamycin/pharmacology , Reverse Transcriptase Polymerase Chain Reaction
10.
Crit Care Med ; 32(3): 708-13, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15090951

ABSTRACT

OBJECTIVE: To compare effectiveness in preventing central venous catheter colonization and infection of two protocols of cutaneous antisepsis using povidone-iodine solution in combination with ethanol or water. DESIGN: Randomized trial. SETTING: Medical intensive care department in a university hospital. PATIENTS: Consecutive patients requiring central venous catheter in two similar 11-bed units from January 1, 2001, to January 1, 2002. INTERVENTIONS: Alcoholic povidone-iodine solution protocol was randomly assigned to one of two units when the study began. Every 3 months the alcoholic protocol was switched from one unit to the other. Depending on the unit and the time the patient was admitted, catheters were inserted and cared for with 10% aqueous povidone-iodine solution or 5% povidone-iodine solution 70% ethanol-based combination. MEASUREMENTS AND MAIN RESULTS: Rates of catheter colonization, catheter-related bacteremia, and catheter-related infection were compared in the two protocols; 223 catheters were included in an intent-to-treat analysis. The incidence of catheter colonization was significantly lower in the alcoholic povidone-iodine solution protocol than in the aqueous povidone-iodine solution protocol (relative risk, 0.38; 95% confidence interval, 0.22-0.65, p <.001), and so was the incidence of catheter-related infection (relative risk, 0.34; 95% confidence interval, 0.13-0.91, p <.04). Catheter-related bacteremia were similar in both protocols. After adjusting for other risk factors, time to central venous catheter colonization was significantly longer in the alcoholic solution (adjusted hazards ratio, 0.3; 95% confidence interval, 0.2-0.6, p <.001). Based on a subgroup of 114 patients (57 in each protocol), analysis of 57 pairs of central venous catheters matched for age, duration, and site of insertion found similar results regarding the superiority of alcoholic povidone-iodine solution in preventing central venous catheter colonization and infection. CONCLUSIONS: The use of alcoholic povidone-iodine for skin disinfection reduced the incidence of catheter colonization and related infection compared with aqueous 10% povidone-iodine disinfection in an adult intensive care unit.


Subject(s)
Anti-Infective Agents, Local/chemistry , Catheterization, Central Venous/methods , Ethanol , Pharmaceutical Vehicles , Povidone-Iodine/chemistry , Water , Anti-Infective Agents, Local/therapeutic use , Catheterization, Central Venous/adverse effects , Catheters, Indwelling , Cross Infection/epidemiology , Cross Infection/etiology , Cross Infection/mortality , Cross Infection/prevention & control , Cross-Over Studies , Equipment Contamination/prevention & control , France/epidemiology , Humans , Incidence , Matched-Pair Analysis , Middle Aged , Multivariate Analysis , Povidone-Iodine/therapeutic use , Proportional Hazards Models , Skin/microbiology
11.
Antimicrob Agents Chemother ; 48(1): 297-304, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14693554

ABSTRACT

Mutations in genes coding for L4 (rplD) or L22 (rplV) ribosomal proteins or in 23S rRNA (rrl gene) are reported as a cause of macrolide resistance in streptococci and staphylococci. This study was aimed at evaluating a denaturing high-performance liquid chromatography (DHPLC) technique as a rapid mutation screening method. Portions of these genes were amplified by PCR from total DNA of 48 strains of Streptococcus pneumoniae (n = 22), Staphylococcus aureus (n = 16), Streptococcus pyogenes (n = 6), Streptococcus oralis (n = 2), and group G streptococcus (n = 2). Thirty-seven of these strains were resistant to macrolides and harbored one or several mutations in one or two of the target genes, and 11 were susceptible. PCR products were analyzed by DHPLC. All mutations were detected, except a point mutation in a pneumococcal rplD gene. The method detected one mutated rrl copy out of six in S. aureus. This automated method is promising for screening of mutations involved in macrolide resistance in gram-positive cocci.


Subject(s)
Gram-Positive Cocci/genetics , Macrolides/pharmacology , Mutation/genetics , Protein Denaturation/genetics , Ribosomes/genetics , Chromatography, High Pressure Liquid , Drug Resistance, Bacterial/genetics , Gene Deletion , Phospholipase D/genetics , RNA, Bacterial/genetics , Reverse Transcriptase Polymerase Chain Reaction , Staphylococcus aureus/genetics , Streptococcus/genetics
12.
Antimicrob Agents Chemother ; 47(7): 2307-9, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12821484

ABSTRACT

The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux. The lsa-like genes of two clinical isolates of E. faecalis susceptible to lincosamides and dalfopristin contained mutations that produced premature termination codons. Revertant mutants were obtained by selection on agar plates containing clindamycin.


Subject(s)
Anti-Bacterial Agents/pharmacology , Codon, Nonsense , Enterococcus faecalis/drug effects , Enterococcus faecalis/genetics , Macrolides , Streptogramin A/pharmacology , Amino Acid Sequence , Drug Resistance, Bacterial/genetics , Genes, Bacterial , Lincosamides , Molecular Sequence Data , Phenotype
13.
J Infect Dis ; 187(11): 1709-16, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12751028

ABSTRACT

Incidence of resistance to erythromycin at our institution reached 53% in 122 Staphylococcus aureus isolates obtained from patients with cystic fibrosis (CF) from 1997 to 1999. Macrolide-resistance genes were sought for in 20 erythromycin-resistant isolates from 9 patients with CF by use of polymerase chain reaction; 13 strains did not contain any known macrolide-resistance genes. Sequence of ribosomal genes rrl (23S rRNA), rplD (L4 protein), and rplV (L22 protein) revealed the presence of mutations in the target site of macrolides in 15 of the 20 isolates. A higher proportion of hypermutator strains was observed in a group of 89 CF staphylococcal isolates, compared with that in the 74 non-CF control isolates (13/89 vs. 1/74 with resistance to rifampin [P=.0045]; 9/89 vs. 1/74 with resistance to streptomycin [P=.04]). Various mutations or deletions of the mutator mutS gene were found not only in 5 of 11 hypermutable strains but also in 3 nonhypermutable strains harboring a large number of ribosomal mutations. The presence of a high proportion of hypermutable strains might explain the adaptation of certain strains in the patients, as well as the emergence of macrolide resistance as a result of antibiotic selective pressure in CF.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins , Cystic Fibrosis/microbiology , DNA-Binding Proteins , Drug Resistance, Multiple, Bacterial , Mutation/genetics , Staphylococcal Infections/drug therapy , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Adenosine Triphosphatases/genetics , Adult , DNA, Ribosomal/genetics , Escherichia coli Proteins/genetics , Female , Humans , Macrolides , Male , MutS DNA Mismatch-Binding Protein , Phenotype , Staphylococcus aureus/classification
14.
Antimicrob Agents Chemother ; 46(9): 3054-6, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12183270

ABSTRACT

Six strains of Staphylococcus aureus isolated from cystic fibrosis patients after treatment with azithromycin were cross-resistant to azithromycin and erythromycin. None of the isolates contained erm or msr(A) genes, but they all carried either A2058G/U or A2059G mutations within the rrl genes, with a majority of the rRNA copies bearing the mutation. One strain displayed an additional mutation in the rplV gene, encoding the L22 ribosomal protein.


Subject(s)
Anti-Bacterial Agents/pharmacology , Membrane Transport Proteins , Methyltransferases , Mutation/genetics , Ribosomes/genetics , Staphylococcal Infections/microbiology , Staphylococcus aureus/genetics , Azithromycin/pharmacology , Bacterial Proteins/genetics , Cystic Fibrosis/microbiology , DNA, Bacterial/genetics , Drug Resistance , Erythromycin/pharmacology , Genes, Bacterial/genetics , Humans , Microbial Sensitivity Tests , Oligonucleotide Probes , Reverse Transcriptase Polymerase Chain Reaction
15.
J Antimicrob Chemother ; 49(6): 935-9, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12039885

ABSTRACT

OBJECTIVE: Two clinical strains of Streptococcus pyogenes, 237 and 544, one isolated in Slovakia and the other in Croatia, that were resistant to azithromycin (MIC 8 and 2 mg/L, respectively) but susceptible to erythromycin (MIC 0.5 and 0.12 mg/L, respectively) did not contain any gene known to confer macrolide resistance by ribosomal modification (erm gene) or efflux [mef(A) and msr(A) genes]. The aim of the study was to determine the mechanisms of macrolide resistance in both strains. METHODS: Portions of genes encoding ribosomal proteins L22 and L4, and 23S rRNA (domains II and V) in the two macrolide-resistant strains and in control strains susceptible to macrolides, were analysed by PCR and single-strand conformational polymorphism, to screen for mutations. The DNA sequences of amplicons from resistant strains that differed from those of susceptible strains, in terms of their electrophoretic migration profiles, were determined. RESULTS: S. pyogenes 237 displayed a KG insertion after position 69 in ribosomal protein L4. S. pyogenes 544 contained a C2611U mutation in domain V of 23S rRNA. CONCLUSION: Mutations at a similar position in ribosomal protein L4 and 23S rRNA have been reported previously in macrolide-resistant pneumococci. This report shows that similar mutations can be found in macrolide-resistant S. pyogenes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Mutation/genetics , Ribosomal Proteins/genetics , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/genetics , Adult , Humans , Infant , Macrolides , Microbial Sensitivity Tests/methods , Microbial Sensitivity Tests/statistics & numerical data , Sequence Homology, Amino Acid , Streptococcal Infections/drug therapy , Streptococcal Infections/microbiology , Streptococcus pyogenes/isolation & purification
16.
Antimicrob Agents Chemother ; 46(7): 2200-7, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12069975

ABSTRACT

The mechanism of resistance to the streptogramin antibiotics quinupristin and dalfopristin was studied in a Staphylococcus aureus clinical isolate selected under quinupristin-dalfopristin therapy, in four derivatives of S. aureus RN4220 selected in vitro, and in a mutant selected in a model of rabbit aortic endocarditis. For all strains the MICs of erythromycin, quinupristin, and quinupristin-dalfopristin were higher than those for the parental strains but the MICs of dalfopristin and lincomycin were similar. Portions of genes for domains II and V of 23S rRNA and the genes for ribosomal proteins L4 and L22 were amplified and sequenced. All mutants contained insertions or deletions in a protruding beta hairpin that is part of the conserved C terminus of the L22 protein and that interacts with 23S rRNA. Susceptible S. aureus RN4220 was transformed with plasmid DNA encoding the L22 alteration, resulting in transformants that were erythromycin and quinupristin resistant. Synergistic ribosomal binding of streptogramins A and B, studied by analyzing the fluorescence kinetics of pristinamycin I(A)-ribosome complexes, was abolished in the mutant strain, providing an explanation for quinupristin-dalfopristin resistance.


Subject(s)
Bacterial Proteins/genetics , Drug Resistance, Microbial/genetics , Drug Therapy, Combination/pharmacology , Mutation , RNA-Binding Proteins/genetics , Ribosomal Proteins , Staphylococcus aureus/drug effects , Virginiamycin/pharmacology , Amino Acid Sequence , Humans , Molecular Sequence Data , RNA, Ribosomal, 23S/genetics , RNA-Binding Proteins/chemistry , Ribosomes/metabolism , Staphylococcus aureus/genetics
17.
Antimicrob Agents Chemother ; 46(5): 1199-203, 2002 May.
Article in English | MEDLINE | ID: mdl-11959545

ABSTRACT

The mechanisms of resistance to macrolides in seven group A streptococcal (Streptococcus pyogenes) isolates that were the cause of pharyngitis in children who were unsuccessfully treated with azithromycin (10 mg/kg of body weight/day for 3 days) were evaluated. All posttreatment strains were found to be genetically related to the pretreatment isolates by random amplified polymorphism DNA analysis and pulsed-field gel electrophoresis. Two isolates had acquired either a mef(A) or an erm(B) gene, responsible for macrolide efflux and ribosomal modification, respectively. Three isolates displayed mutations in the gene encoding the L4 ribosomal protein that is part of the exit tunnel within the 50S subunit of the bacterial ribosome. In the two remaining posttreatment strains, the mechanisms of macrolide resistance could not be elucidated.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Pharyngitis/microbiology , Streptococcal Infections/microbiology , Streptococcus pyogenes/drug effects , Child , Child, Preschool , DNA, Bacterial/analysis , Electrophoresis, Gel, Pulsed-Field , Humans , Macrolides , Microbial Sensitivity Tests , Random Amplified Polymorphic DNA Technique , Ribosomal Proteins/genetics , Streptococcus pyogenes/genetics
18.
Antimicrob Agents Chemother ; 46(1): 125-31, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11751122

ABSTRACT

Mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible Streptococcus pneumoniae by serial passage in various macrolides (T. A. Davies, B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum, Antimicrob. Agents Chemother., 44:414-417, 2000). Portions of genes encoding ribosomal proteins L22 and L4 and 23S rRNA (domains II and V) were amplified by PCR and analyzed by single-strand conformational polymorphism analysis to screen for mutations. The DNA sequences of amplicons from mutants that differed from those of parental strains by their electrophoretic migration profiles were determined. In six mutants, point mutations were detected in the L22 gene (G95D, P99Q, A93E, P91S, and G83E). The only mutant selected by telithromycin (for which the MIC increased from 0.008 to 0.25 microg/ml) contained a combination of three mutations in the L22 gene (A93E, P91S, and G83E). L22 mutations were combined with an L4 mutation (G71R) in one strain and with a 23S rRNA mutation (C2611A) in another strain. Nine other strains selected by various macrolides had A2058G (n = 1), A2058U (n = 2), A2059G (n = 1), C2610U (n = 1), and C2611U (n = 4) mutations (Escherichia coli numbering) in domain V of 23S rRNA. One mutant selected by clarithromycin and resistant to all macrolides tested (MIC, >32 microg/ml) and telithromycin (MIC, 4 microg/ml) had a single base deletion (A752) in domain II. In six remaining mutants, no mutations in L22, L4, or 23S rRNA could be detected.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance/genetics , Escherichia coli Proteins , Ketolides , Macrolides , Streptococcus pneumoniae/genetics , Amino Acid Sequence , Clindamycin/pharmacology , Genetic Testing , Genetic Variation , Humans , Molecular Sequence Data , Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , RNA, Ribosomal, 23S/genetics , RNA-Binding Proteins/genetics , Ribosomal Proteins/genetics , Streptococcus pneumoniae/drug effects , Streptogramins/pharmacology
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