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1.  Occurrence of the Campylobacter resistance gene tetO in Enterococcus and Streptococcus spp. 
Antimicrobial Agents and Chemotherapy  1988;32(12):1793-1796.
The distribution of nucleotide sequences related to tetK, tetL, tetM, and tetO was studied by dot blot hybridization in 178 strains of Streptococcus and Enterococcus spp. that were resistant to tetracycline. The tetO gene, which is responsible for tetracycline resistance in Campylobacter spp., was detected in six Streptococcus strains and two Enterococcus strains, in which it was borne by similar plasmids. This observation confirms our previous proposal that tetO originated in gram-positive cocci. tetM, the most prevalent resistance gene, was present alone in 109 strains and associated with tetL in 33 strains in which the two genes contributed cooperatively to high-level tetracycline resistance. tetL was present alone in five Enterococcus strains, and tetK was detected in a single Streptococcus strain. The existence of 22 strains that did not hybridize to the probes suggest that tetracycline resistance in streptococci and enterococci involves additional gene classes as well.
PMCID: PMC176020  PMID: 3245693
2.  Loss of intrinsic aminoglycoside resistance in Acinetobacter haemolyticus as a result of three distinct types of alterations in the aac(6')-Ig gene, including insertion of IS17. 
Antimicrobial Agents and Chemotherapy  1997;41(12):2646-2651.
The distribution of the aac(6')-Ig gene, encoding aminoglycoside 6'-N-acetyltransferase-Ig [AAC(6')-Ig], was studied in 96 Acinetobacter haemolyticus strains and 12 proteolytic Acinetobacter strains, including Acinetobacter genomospecies 6, 13, and 14 and 3 unnamed species assigned to this genomic group by DNA-DNA hybridization. This gene was detected by DNA-DNA hybridization in all 96 A. haemolyticus strains and by PCR in 95 strains but was not detected in strains of other species, indicating that it may be used to identify A. haemolyticus. Three A. haemolyticus strains were susceptible to tobramycin and did not produce an aminoglycoside 6'-N-acetylating activity, although they contained aac(6')-Ig-related sequences. An analysis of three susceptible A. haemolyticus strains indicated that aminoglycoside resistance was abolished by the following three distinct mechanisms: (i) a point mutation in aac(6')-Ig that led to a Met56-->Arg substitution, which was shown by analysis of a revertant to be responsible for the loss of resistance; (ii) a polythymine insertion that altered the reading frame; and (iii) insertion of IS17, a new member of the IS903 family. These observations indicated that AAC(6')-Ig is not essential for the viability of A. haemolyticus, although the aac(6')-Ig gene was detected in all members of this species.
PMCID: PMC164184  PMID: 9420034
3.  VanD-type glycopeptide-resistant Enterococcus faecium BM4339. 
Enterococcus faecium BM4339 was constitutively resistant to vancomycin (MIC, 64 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml). A 605-bp product obtained with the V1 and V2 primers for amplification of genes encoding D-Ala:D-Ala ligases and related glycopeptide resistance proteins was sequenced after cloning. The deduced amino acid sequence had 69% identity with VanA and VanB and 43% identity with VanC, consistent with the finding that BM4339 synthesized peptidoglycan precursors terminating in D-lactate. This new type of glycopeptide resistance phenotype was designated VanD.
PMCID: PMC164056  PMID: 9303405
4.  Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae. 
We report a mutation in the parE genes of two in vitro mutants of Streptococcus pneumoniae responsible for low-level resistance to fluoroquinolones. Sequential acquisition of mutations in parE and gyrA leads to higher levels of resistance. This confirms that topoisomerase IV is the primary target of fluoroquinolones in S. pneumoniae.
PMCID: PMC163872  PMID: 9145891
5.  Emergence of the trimethoprim resistance gene dfrD in Listeria monocytogenes BM4293. 
The sequence of the trimethoprim resistance gene of the 3.7-kb plasmid (pIP823) that confers high-level resistance (MIC, 1,024 microg/ml) to Listeria monocytogenes BM4293 was determined. The gene was identical to dfrD recently detected in Staphylococcus haemolyticus MUR313. The corresponding protein, S2DHFR, represents the second class of high-level trimethoprim-resistant dihydrofolate reductase identified in gram-positive bacteria. We propose that trimethoprim resistance in L. monocytogenes BM4293 could originate in staphylococci.
PMCID: PMC163863  PMID: 9145882
6.  Active efflux of antimicrobial agents in wild-type strains of enterococci. 
Enterococci are intrinsically resistant to numerous antimicrobial agents. We examined the energy-dependent efflux of radiolabeled drugs from four reference strains of Enterococcus faecalis and a strain of Enterococcus faecium and found that most strains pumped out norfloxacin and chloramphenicol. Efflux of tetracycline was detected only in certain strains.
PMCID: PMC163815  PMID: 9087510
7.  Activity of the new fluoroquinolone trovafloxacin (CP-99,219) against DNA gyrase and topoisomerase IV mutants of Streptococcus pneumoniae selected in vitro. 
Antimicrobial Agents and Chemotherapy  1996;40(12):2691-2697.
The MICs of trovafloxacin, ciprofloxacin, ofloxacin, and sparfloxacin at which 90% of isolates are inhibited for 55 isolates of pneumococci were 0.125, 1, 4, and 0.5 microgram/ml, respectively. Resistant mutants of two susceptible isolates were selected in a stepwise fashion on agar containing ciprofloxacin at 2 to 10 times the MIC. While no mutants were obtained at the highest concentration tested, mutants were obtained at four times the MIC of ciprofloxacin (4 micrograms/ml) at a frequency of 1.0 x 10(-9). Ciprofloxacin MICs for these first-step mutants ranged from 4 to 8 micrograms/ml, whereas trovafloxacin MICs were 0.25 to 0.5 microgram/ml. Amplification of the quinolone resistance-determining region of the grlA (parC; topoisomerase IV) and gyrA (DNA gyrase) genes of the parents and mutants revealed that changes of the serine at position 80 (Ser80) to Phe or Tyr (Staphylococcus aureus coordinates) in GrlA were associated with resistance to ciprofloxacin. Second-step mutants of these isolates were selected by plating the isolates on medium containing ciprofloxacin at 32 micrograms/ml. Mutants for which ciprofloxacin MICs were 32 to 256 micrograms/ml and trovafloxacin MICs were 4 to 16 micrograms/ml were obtained at a frequency of 1.0 x 10(-9). Second-step mutants also had a change in GyrA corresponding to a substitution in Ser84 to Tyr or Phe or in Glu88 to Lys. Trovafloxacin protected from infection mice whose lungs were inoculated with lethal doses of either the parent strain or the first-step mutant. These results indicate that resistance to fluoroquinolones in S. pneumoniae occurs in vitro at a low frequency, involving sequential mutations in topoisomerase IV and DNA gyrase. Trovafloxacin MICs for wild-type and first-step mutants are within clinically achievable levels in the blood and lungs of humans.
PMCID: PMC163605  PMID: 9124824
8.  Development of fluoroquinolone resistance in Enterococcus faecalis and role of mutations in the DNA gyrase gyrA gene. 
Antimicrobial Agents and Chemotherapy  1996;40(11):2558-2561.
We have analyzed the development of fluoroquinolone resistance between 1986 and 1993 among clinical isolates of Enterococcus faecalis from a French hospital. One hundred randomly selected isolates per year were screened for resistance to ciprofloxacin (MIC > 2 micrograms/ml) and for high-level resistance to gentamicin (MIC > 1,000 micrograms/ml). The percentages of ciprofloxacin-resistant strains for these years were as follows: 1986, 0; 1987, 1; 1988 to 1989, 2; 1990, 6; 1991, 16; 1992, 24; and 1993, 14. Eighty-three percent of the ciprofloxacin-resistant isolates were coresistant to high levels of gentamicin. Forty-eight high-level gentamicin-resistant E. faecalis strains, which were resistant (24 strains) or susceptible (24 strains) to ciprofloxacin, were examined by pulsed-field gel electrophoresis (PFGE) of SmaI-digested total DNA. Numerous PFGE types were observed among the ciprofloxacin-susceptible isolates, whereas one type was largely predominant among the ciprofloxacin-resistant strains, which suggests that the increase in fluoroquinolone resistance was due to the spread of a single clone. A 241-bp fragment of gyrA, corresponding to the quinolone resistance-determining region, was amplified and sequenced for seven ciprofloxacin-resistant isolates. Six strains had high levels of resistance (MICs, 32 to 64 micrograms/ml) and had a mutation at position 83 (Escherichia coli coordinates) from Ser to Arg (three strains) or to Ile (two strains) or at position 87 from Glu to Gly (one strain), whereas the low-level-resistant isolate (MIC, 8 micrograms/ml) had no mutations.
PMCID: PMC163575  PMID: 8913464
9.  Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro. 
Antimicrobial Agents and Chemotherapy  1996;40(11):2505-2510.
We have analyzed by gene amplification and sequencing mutations in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes of fluoroquinolone-resistant Streptococcus pneumoniae mutants obtained during therapy or in vitro. Mutations leading to substitutions in ParC were detected in the two mutants obtained in vivo, BM4203-R (substitution of a histidine for an aspartate at position 84 [Asp-84-->His]; Staphylococcus aureus coordinates) and BM4204-R (Ser-80-->Phe), and in two mutants obtained in vitro (Ser-80-->Tyr). An additional mutant obtained in vitro, BM4205-R3, displayed a higher level of fluoroquinolone resistance and had a mutation in gyrA leading to a Ser-84-->Phe change. We could not detect any mutation in the three remaining mutants obtained in vitro. Total DNA from BM4203-R, BM4204-R, and BM4205-R3 was used to transform S. pneumoniae CP1000 by selection on fluoroquinolones. For the parC mutants, transformants with phenotypes indistinguishable from those of the donors were obtained at frequencies (5 x 10(-3) to 8 x 10(-3)) compatible with monogenic transformation. By contrast, transformants were obtained at a low frequency (4 x 10(-5)), compatible with the transformation of two independent genes, for the gyrA mutant. Resistant transformants of CP1000 were also obtained with an amplified fragment of parC from BM4203-R and BM4204-R but not with a gyrA fragment from BM4205-R3. All transformants had mutations identical to those in the donors. These data strongly suggest that ParC is the primary target for fluoroquinolones in S. pneumoniae and that BM4205-R3 is resistant to higher levels of the drugs following the acquisition of two mutations, including one in gyrA.
PMCID: PMC163565  PMID: 8913454
10.  Specificity of induction of glycopeptide resistance genes in Enterococcus faecalis. 
Antimicrobial Agents and Chemotherapy  1996;40(10):2291-2295.
Regulation of VanA- and VanB-type glycopeptide resistance in enterococci is mediated by related two-component regulatory systems (VanR-VanS and VanRB-VanSB). The transglycosylase inhibitors vancomycin, teicoplanin, and moenomycin induced synthesis of the VanX D,D-dipeptidase in a VanA-type Enterococcus faecalis harboring transposon Tn1546. Inhibitors of reactions immediately preceding (ramoplanin) or following (penicillin G and bacitracin) transglycosylation were not inducers. These results identify accumulation of membrane-bound lipid intermediate II as a potential signal for induction of VanA-type resistance. In E.faecalis BM4281 harboring a wild vanB genetic element, D,D-dipeptidase synthesis was only inducible by vancomycin. Induction of the production of the VanB ligase by vancomycin was required for growth of a vancomycin-dependent derivative of BM4281, since introduction of a plasmid coding for constitutive synthesis of the VanA ligase eliminated the requirement of glycopeptide for growth. Both vancomycin and teicoplanin were able to induce D,D-dipeptidase synthesis in BM4281 derivatives that were vancomycin and teicoplanin resistant or vancomycin and teicoplanin dependent. Acquisition of teicoplanin resistance in the latter types of strains was due to alteration in induction specificity associated with an increase in the sensitivity of the regulatory system to vancomycin. Thus, the wild VanRB-VanSB system is unable or not sensitive enough to sense teicoplanin, although mutations can lead to recognition of this antibiotic.
PMCID: PMC163522  PMID: 8891132
11.  Efficacy of vancomycin and teicoplanin alone and in combination with streptomycin in experimental, low-level vancomycin-resistant, VanB-type Enterococcus faecalis endocarditis. 
The efficacy of vancomycin (VM) and teicoplanin (TE), alone and in combination with streptomycin (SM), against enterococci that express low-level VanB-type VM resistance was investigated in experimental endocarditis using isogenic strains of Enterococcus faecalis susceptible to glycopeptides and aminoglycosides or inducibly resistant to low levels of VM (MIC = 16 micrograms/ml). VM was significantly less active against the resistant strain than against the susceptible strain, establishing that low-level VanB-type VM resistance can influence therapeutic efficacy. By contrast, TE had equally good activity against both strains. VM or TE combined with SM was synergistic and bactericidal against the resistant strain in vitro. While both combinations were efficient in reducing bacterial density in vivo, TE plus SM was significantly superior to VM plus SM if valve sterilization was considered. These data suggest that despite the presence of low-level VanB-type resistance, combination therapy with a glycopeptide and SM (and presumably other aminoglycosides to which there is not high-level resistance) will nevertheless provide effective bactericidal activity.
PMCID: PMC163056  PMID: 8787879
12.  Detection of aac(6')-I genes in amikacin-resistant Acinetobacter spp. by PCR. 
Antimicrobial Agents and Chemotherapy  1994;38(12):2925-2928.
The distribution of aac(6')-I genes in 62 strains of Acinetobacter spp. resistant to amikacin, netilmicin, and tobramycin and susceptible to gentamicin, a phenotype compatible with synthesis of an AAC(6')-I enzyme, was studied by PCR and by DNA hybridization. Both methods gave similar results. Among the 51 Acinetobacter baumannii strains, aac(6')-Ib was found in 19 isolates and aac(6')-Ih was found in the remaining strains. The aac(6')-Ig gene was present in all 10 A. haemolyticus strains studied and was detected only in this species. A pair of degenerate oligonucleotides complementary to conserved regions of aac(6')-Ic, -Id, -If, -Ig, and -Ih enabled detection of these genes and also of aac(6')-Ij, recently recognized in Acinetobacter sp. strain 13.
PMCID: PMC188310  PMID: 7695286
13.  Characterization of the chromosomal aac(6')-Ij gene of Acinetobacter sp. 13 and the aac(6')-Ih plasmid gene of Acinetobacter baumannii. 
The amikacin resistance genes aac(6')-Ih of Acinetobacter baumannii BM2686 and aac(6')-Ij of Acinetobacter sp. 13 BM2689 encoding aminoglycoside 6'-N-acetyltransferases were characterized. The 441-bp coding sequences predict proteins with calculated masses of 16,698 and 16,677 Da, respectively. Analysis of the deduced amino acid sequences indicated that the proteins belonged to a subfamily of 6'-aminoglycoside acetyltransferase type I enzymes from gram-negative bacteria. The aac(6')-Ih gene of BM2686 was located on a 13.7-kb nonconjugative plasmid. The aac(6')-Ij gene from BM2689 was not transferable either by conjugation to Escherichia coli or A. baumannii or by transformation to Acinetobacter calcoaceticus. Plasmid DNA from BM2689 did not hybridize with an intragenic aac(6')-Ij probe. These results suggest a chromosomal location for this gene. The aac(6')-Ij gene was detected by DNA hybridization in all 28 strains of Acinetobacter sp. 13 tested but not in other Acinetobacter strains, including A. baumannii, proteolytic genospecies 4, 6, 14, 15, 16, and 17, and ungrouped strains. The aac(6')-Ih and -Ij probes did not hybridize in dot blot assays with DNA from members of the families Enterobacteriaceae and Pseudomonadaceae that produced 6'-N-acetyltransferases. These data suggest that the genes are confined to the Acinetobacter genus and that the aac(6')-Ij gene is species specific and may be used to identify Acinetobacter sp. 13.
PMCID: PMC284657  PMID: 7810994
14.  Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors. 
The vanR, vanS, vanH, vanA, and vanX genes of enterococcal transposon Tn1546 were introduced into the chromosome of Enterococcus faecalis JH2-2. Complementation of this portion of the van gene cluster by a plasmid encoding VanY D,D-carboxypeptidase led to a fourfold increase in the vancomycin MIC (from 16 to 64 micrograms/ml). Multicopy plasmids pAT80 (vanR vanS vanH vanA vanX) and pAT382 (vanR vanS vanH vanA vanX vanY) conferred similar levels of vancomycin resistance to JH2-2. The addition of D-alanine (100 mM) to the culture medium restored the vancomycin susceptibility of E. faecalis JH2-2/pAT80. The pentapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala partially replaced pentadepsipeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac when the strain was grown in the presence of D-alanine. In contrast, resistance mediated by pAT382 was almost unaffected by the addition of the amino acid. Expression of the vanY gene of pAT382 resulted in the formation of the tetrapeptide UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala, indicating that a portion of the cytoplasmic precursors had been hydrolyzed. These results show that VanY contributes to glycopeptide resistance in conditions in which pentapeptide is present in the cytoplasm above a threshold concentration. However, the contribution of the enzyme to high-level resistance mediated by Tn1546 appears to be moderate, probably because hydrolysis of D-alanyl-D-alanine by VanX efficiently prevents synthesis of the pentapeptide.
PMCID: PMC284659  PMID: 7810996
15.  Emergence of high-level resistance to glycopeptides in Enterococcus gallinarum and Enterococcus casseliflavus. 
Enterococcus gallinarum BM4231 and Enterococcus casseliflavus BM4232, isolated from the feces of a patient under oral therapy with vancomycin, were resistant to high levels of vancomycin (MICs of > 256 micrograms/ml) and teicoplanin (MICs of 128 and 64 micrograms/ml, respectively). This phenotype is new for these bacterial species that are naturally resistant to low levels of vancomycin and appears to be due to in vivo acquisition of plasmid pIP218 carrying the vanA gene cluster.
PMCID: PMC284616  PMID: 7979308
17.  In vivo selection during pefloxacin therapy of a mutant of Staphylococcus aureus with two mechanisms of fluoroquinolone resistance. 
Staphylococcus aureus BM4626 (ciprofloxacin MIC, 0.5 microgram/ml) and BM4627 (ciprofloxacin MIC, 32 microgram/ml) were isolated from the same patient before and during pefloxcin therapy for septic tibial nonunion, respectively. The two strains had similar serotypes and indistinguishable phage types and SmaI-generated restriction fragment length polymorphisms. Portions of the gyrA (codons 60 to 120) and the gyrB (codons 420 to 480) genes of each clinical isolate were amplified by PCR and sequenced. Strain BM4627 had a serine-to-leucine substitution resulting from a cytosine-to-thymidine mutation at codon 84 of gyrA relative to the sequence of the gyrA gene of BM4626. Norfloxacin accumulation, measured in a whole-cell uptake assay, was significantly lower in BM4627 than BM4626. These data indicate that double mutants can be selected in vivo under fluoroquinolone therapy.
PMCID: PMC188166  PMID: 7915098
18.  Characterization of Acinetobacter haemolyticus aac(6')-Ig gene encoding an aminoglycoside 6'-N-acetyltransferase which modifies amikacin. 
Antimicrobial Agents and Chemotherapy  1993;37(10):2093-2100.
The amikacin resistance gene acc(6')-Ig of Acinetobacter haemolyticus BM2685 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 438 bp corresponding to a protein with a calculated mass of 16,522 Da. Analysis of the deduced amino acid sequence suggested that it was the fourth member of a subfamily of aminoglycoside 6'-N-acetyltransferases. The resistance gene was not transferable either by conjugation to Escherichia coli or to Acinetobacter baumannii or by transformation into Acinetobacter calcoaceticus. Plasmid DNA from strain BM2685 did not hybridize with an intragenic aac(6')-Ig probe. These results suggest a chromosomal location for this gene. The gene was detected by DNA hybridization in all 20 strains of A. haemolyticus tested but not in 179 other Acinetobacter strains, including A. baumannii, A. lwoffii, A. junii, and A. johnsonii and genospecies 3, 6, 11, 13, 14, 15, 16, and 17, of which 162 were amikacin resistant. The probe did not hybridize in dot blot assays with DNAs purified from members of the families Enterobacteriaceae and Pseudomonadaceae that encode 6'-N-acetyltransferases. These data suggest that the aac(6')-Ig gene is species specific and may be used to identify A. haemolyticus.
PMCID: PMC192234  PMID: 8257129
20.  Vancomycin resistance gene vanC is specific to Enterococcus gallinarum. 
Nearly all strains of Enterococcus gallinarum are resistant to low levels of vancomycin. The glycopeptide resistance gene vanC from E. gallinarum BM4174 has recently been cloned and sequenced. A probe specific for vanC hybridized with a 2.7-kb EcoRI and a 4.5-kb HindIII fragment of total DNA from the 42 strains of E. gallinarum studied. No homology was detected with DNA of strains belonging to other species intrinsically resistant to vancomycin, including Enterococcus casseliflavus, a species that expresses a vancomycin resistance phenotype similar to that of E. gallinarum. No hybridization with DNA of enterococcal strains with acquired resistance to high or low levels of vancomycin was observed. The specificity of the vanC probe allowed us to distinguish E. gallinarum from 12 other species of enterococci, indicating that this probe is a useful tool for species identification within the genus Enterococcus.
PMCID: PMC192426  PMID: 1416893
21.  Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant enterococci. 
The VanA ligase encoded by the vancomycin resistance plasmid pIP816 of Enterococcus faecium BM4147 condenses D-alanine with various D-2-hydroxy and D-2-amino acids in vitro. D-Lactate added to the culture medium restored the vancomycin resistance of a strain that does not produce the VanH dehydrogenase and therefore appears to be a substrate of VanA in vivo.
PMCID: PMC189469  PMID: 1503450
22.  Nucleotide sequences specific for Tn1545-like conjugative transposons in pneumococci and staphylococci resistant to tetracycline. 
The distributions of tet(M) and conjugative transposons related to Tn1545 were studied by hybridization in 47 clinical isolates of Streptococcus pneumoniae resistant to tetracycline. Resistance to tetracycline was always associated with resistance to minocycline and was due to the presence of the tet(M) gene. Association of tet(M) with int-Tn, the gene encoding the protein required for the movements of Tn1545-like transposons, was found in all but one strain of S. pneumoniae. In contrast, int-Tn was detected in only 2 of 37 strains of Staphylococcus spp. harboring tet(M).
PMCID: PMC245236  PMID: 1656870

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