Search

A total of 198 nonrepetitive clinical strains of Clostridium difficile isolated from different French hospitals in 1991 (n = 100) and 1997 (n = 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 micro g of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA (eight mutations, amino acid changes Asp71-->Val in one, Thr82-->Ile in six, and Ala118-->Thr in one) or in gyrB (six mutations, amino acid changes Asp426-->Asn in five and Arg447-->Leu in one). These changes are similar to those already described in other species except for Asp71-->Val, which is novel, and Ala118-->Thr, which is exceptional. Attempts to detect the topoisomerase IV parC gene by PCR amplification with universal parC primers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 micro g/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficile is rare in France and is associated with the occurrence of a gyrA or gyrB mutation.

ABSTRACTA total of 198 nonrepetitive clinical strains of Clostridium difficileisolated from different French hospitals in 1991 (n= 100) and 1997 (n= 98) were screened for decreased susceptibility to fluoroquinolones by plating onto Wilkins-Chalgren agar containing 16 μg of ciprofloxacin per ml. The frequency of decreased susceptibility was 7% (14 of 198) and was identical for the years 1991 and 1997. Serogroups C, H, D, A9, and K accounted for five, four, two, one, and one of the resistant strains, respectively, one strain being nontypeable. Arbitrarily primed PCR typing showed that all resistant strains had unique patterns except two serotype C strains, which could not be clearly distinguished. All isolates with decreased susceptibility carried a mutation either in gyrA(eight mutations, amino acid changes Asp71→Val in one, Thr82→Ile in six, and Ala118→Thr in one) or in gyrB(six mutations, amino acid changes Asp426→Asn in five and Arg447→Leu in one). These changes are similar to those already described in other species except for Asp71→Val, which is novel, and Ala118→Thr, which is exceptional. Attempts to detect the topoisomerase IV parCgene by PCR amplification with universal parCprimers or DNA-DNA hybridization under low-stringency conditions were unsuccessful. The susceptibilities of all resistant strains to ciprofloxacin and ethidium bromide were not affected by the addition of reserpine at 20 μg/ml. In conclusion, decreased susceptibility to fluoroquinolones in C. difficileis rare in France and is associated with the occurrence of a gyrAor gyrBmutation.

Identification of stress proteins in Lactobacillus delbrueckii bulgaricus by two-dimensional electrophoresis. We studied Lactobacillus bulgaricus adaptation to an acid stress. In L. bulgaricus, adapted cells (30 min incubation at pH 4.75) were ?250 fold more tolerant to lethal acid stress (30 min at pH 3.6) than non adapted ones. In order to identify proteins induced during the adaptation, de novo protein synthesis was monitored by 35S methionine labeling of exponentially growing cultures, under standard (pH 6) and adaptive (pH 4.75) conditions. After 2 dimensional (2-D) electrophoresis separation, the protein patterns were compared. At least 30 protein spots showed an increased radioactivity level under acidic conditions. We determined the N-terminal amino acid sequence of 3 highly induced proteins. By comparison to databases, they were identified as the heat shock proteins GroES, GroEL and DnaK. A differential induction was observed for GroES and GroEL, suggesting a post-transcriptionnal regulation mechanism. In this report we established a method for the study of acid stress response in L. bulgaricus. This method will also be useful for further studies on the adaptation of this bacterium to environmental variations. Nous avons ?tudi? l'adaptation de la bact?rie lactique Lactobacillus bulgaricus au stress acide. Nous avons montr? que les bact?ries cultiv?es en milieu "neutre " (pH 6) pr?sentaient une forte l?talit? apr?s transfert dans un milieu acide (choc acide ? pH 3,6). En revanche, les bact?ries pr?alablement adapt?es dans un milieu acide non l?tal (pH 4,75) devenaient ?250 fois plus tol?rantes au choc acide. Apr?s marquage ? la m?thionine 35S et s?paration par ?lectrophor?se bidimensionnelle (2-D), les profils prot?iques obtenus en condition d'adaptation (pH 4,75) et en condition standard (pH 6) sont compar?s. On observe une augmentation significative (> 2) de l'intensit? d'environ 30 spots pendant l'adaptation. La d?termination des s?quences peptidiques N-terminales de 3 prot?ines fortement induites ? pH 4,75 permet d'identifier les prot?ines de choc thermique GroES, GroEL et DnaK. Nous observons des niveaux d'induction diff?rents pour GroES et GroEL, qui pourraient ?tre li?s ? une r?gulation post-transcriptionnelle. Au final, nous avons ?tabli une m?thode d'?tude de la r?ponse au stress acide chez L. bulgaricus. De fa?on plus g?n?rale, cette m?thode permettra d'?tudier l'adaptation de cette bact?rie aux variations environnementales.

Identification of stress proteins in Lactobacillus delbrueckii bulgaricus by two-dimensional electrophoresis. We studied Lactobacillus bulgaricus adaptation to an acid stress. In L. bulgaricus, adapted cells (30 min incubation at pH 4.75) were $\sim 250$ fold more tolerant to lethal acid stress (30 min at pH 3.6) than non adapted ones. In order to identify proteins induced during the adaptation, de novo protein synthesis was monitored by $^{35}$S methionine labeling of exponentially growing cultures, under standard (pH 6) and adaptive (pH 4.75) conditions. After 2 dimensional (2-D) electrophoresis separation, the protein patterns were compared. At least 30 protein spots showed an increased radioactivity level under acidic conditions. We determined the N-terminal amino acid sequence of 3 highly induced proteins. By comparison to databases, they were identified as the heat shock proteins GroES, GroEL and DnaK. A differential induction was observed for GroES and GroEL, suggesting a post-transcriptionnal regulation mechanism. In this report we established a method for the study of acid stress response in L. bulgaricus. This method will also be useful for further studies on the adaptation of this bacterium to environmental variations.