Biomedical subjects
Publications and source records attributed to J Tankovic.
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.
S. typhimurium AlhR, S. enteritidis OulR, and S. hadar GueR resistant to fluoroquinolones (QR), ciprofloxacin MICs, 0.25 to 1 microgram/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml; nalidixic acid MIC, 256 micrograms/ml were isolated from urinary tract infections (AlhR and OulR) during FQ therapy in immunocompromised patients infected by the parent FQ-susceptible strains (AlhS and OulS) (ciprofloxacin MICs, 0.032-0.063; norfloxacin MICs, 0.125-0.25; nalidixic acid MICs, 4-8) or from intestinal infection (GueR). Transformation of AlhR, OulR, and GueR by plasmid pJSW101 carrying the wild-type gyrA gene of Escherichia coli resulted in complementation (nalidixic acid MICs, 4 to 8), proving that these strains had a gyrA mutation. A 800-bp fragment of gyrA from the five strains was amplified by PCR. Direct DNA sequencing of 252-bp region of this fragment identified a single point mutation leading to a substitution Ser-83 to Tyr in AlhR and to a substitution Ser-83 to Phe in OulR and in GueR. These results emphasize the potential risk of selection of FQ-resistant Salmonella during FQ therapy in immunocompromised patients and suggest that these strains differ from the parent strains at least by one mutation in the gyrA gene. They also confirm the role of substitutions in position 83 of gyrA in FQ-resistant clinical isolates of Salmonella.
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.
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.
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.
OBJECTIVE: Infection is a major complication in vascular stents. Stents impregnated with gelatine and dipped in Rifampicin have been shown to resist methicillin-resistant Staphylococcus aureus in both animal experiments and in man. It has been suggested that all aorto-ilio-femoral stents should be treated. To evaluate this method, we reassessed all stent infections observed in our patients who had undergone revascularization of the lower limbs from January 1985 to 1994. We excluded stents implanted for ruptured aneurysms or implanted in patients with a past history of local infection on vascular stents. RESULTS: The rate of septic complications observed during the first year was 1% for all patients in the series, 0% for aorto-aortic and aorto-biiliac stents and 0.7% for aorto- bifemoral stents. These rates are similar to those reported in the multicentric study directed by Goeau Brissonière using antibiotic impregnated stents. The extra cost involved in using such stents for aorto-ilio-femoral revascularization was estimated in this series at 2,180,000 Francs. The costs resulting from the three infections was estimated at 960,000 Francs. CONCLUSION: Based on the findings in this series, antibiotic impregnated stents should be indicated only in selected patients due to the extra cost: past history of local infection, ruptured aneurysms, femoro-tibial stents, cross or axillo-femoral revascularization for which the rate of stent infection is 6.3 - 3.2 and 1.4%, immunodeficient patients, multiple reoperations, post-irradiation arteritis and situations known to involve major risk of infection.
Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.
We have constructed a gyrA trans-complementation plasmid, pAT512, by cloning the wild-type gyrA gene of Staphylococcus aureus into the expression vector pAT392. Introduction by electrotransformation of pAT512 into a high-level fluoroquinolone resistant mutant of S. aureus (ciprofloxacin MIC = 16 micrograms ml-1) having a gyrA mutation which results in a Ser-84 to Leu substitution, reduced the MICs of norfloxacin and ciprofloxacin for the host of four- and eight-fold, respectively.
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.
During a 13-month period, 31 patients hospitalized primarily in two intensive care units (ICUs) were either colonized or infected by imipenem-resistant Acinetobacter baumannii. Typing of the isolates by three methods (antibiotyping, biotyping, and pulsed-field gel electrophoresis) revealed that two distinct strains were involved in the first 9 cases of the outbreak and that one of these strains, which had acquired a higher level of imipenem resistance as well as resistance to all aminoglycosides, accounted for 21 of 22 cases in the second part of the outbreak. ICU environmental contamination was recognized as an important reservoir of this epidemic strain. The outbreak ceased only after the ICUs were closed for complete cleaning and disinfection.
We determined the MICs of 28 antimicrobial agents against 36 clinical strains of Pediococcus spp. (25 P. acidilactici, 9 P. pentosaceus, and 2 P. urinaeequi strains). Penicillin G, imipenem, gentamicin, netilmicin, erythromycin, clindamycin, rifampin, chloramphenicol, daptomycin, and ramoplanin were the most active. All strains of P. acidilactici were susceptible to novobiocin, whereas all isolates of P. pentosaceus were resistant. Novobiocin could therefore be helpful for differentiation of these two closely related species. P. acidilactici HM3020 was inducibly resistant to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics. Resistance was due to a determinant homologous to ermAM and carried by a nontransferable 46-kb plasmid, pVM20. This plasmid was structurally distinct from two enterococcal MLS resistance plasmids, pIP819 and pAM beta 1. The 34 strains of P. acidilactici and P. pentosaceus were resistant to tetracycline, and total DNA of these strains did not hybridize to probes specific for tetK, tetL, tetM, and tetO.
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