CTX-M-type extended-spectrum beta-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop.

Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniae ILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 beta-lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 beta-lactamase. The amino acid sequence of the CTX-M-18 beta-lactamase differed from that of the CTX-M-9 beta-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. The bla(CTX-M-18) and bla(CTX-M-19) genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.

In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy.

We report the emergence after 4 days of ciprofloxacin monotherapy of a double mutant of Pseudomonas aeruginosa overexpressing the multidrug efflux system MexAB-OprM and harbouring a mutation in the gyrB gene. Compared with its initial susceptible counterpart, this mutant exhibited a significant increase in resistance to most of the beta-lactam antibiotics tested (16 x MIC of ticarcillin) and to ciprofloxacin (128 x MIC). Combined ceftazidime and amikacin therapy finally eradicated the resistant isolate and cured the patient of his infection. This case illustrates how strains of P. aeruginosa may develop high levels of fluoroquinolone resistance by combining efflux mechanisms and target alterations.

Mechanisms of macrolide resistance in clinical group B streptococci isolated in France.

Macrolide susceptibility was investigated in clinical group B streptococci obtained from neonates or pregnant women in 2000 in France. Of 490 consecutive isolates, 18% were resistant to erythromycin. The erm(B), erm(A) subclass erm(TR), and mef(A) genes were harbored by 47, 45, and 6% of these strains, respectively. Two isolates did not harbor erm or mef genes.

Sequential colonization by Streptococcus pneumoniae of healthy children living in an orphanage.

A prospective study of nasopharyngeal colonization by Streptococcus pneumoniae in the exceptional conditions of a closed community of abandoned children was done over a 1-year period; 71 children (age <24 months) were studied monthly. S. pneumoniae was isolated from 58 (81.7%), and 94.5% of the 111 isolates were resistant to penicillin. The mean rate of carriage was estimated at 57.4%, ranging from 42.8% to 70.4%. Children were sequentially colonized by a mean of 3 different isolates. The mean duration of carriage for a given isolate was approximately 2.2 months. Serotyping and molecular typing by pulsed-field gel electrophoresis showed that children were colonized by a limited number of clones belonging to only 4 serotypes and 4 pulsotypes. These clones rapidly spread in the community and colonized the children in waves, with a rapid turnover of S. pneumoniae isolates, facilitated by close contact between children.

Resistance to macrolides in Streptococcus pyogenes in France in pediatric patients.

A total of 1,500 recent throat isolates of Streptococcus pyogenes collected between 1996 and 1999 from children throughout France were tested for their susceptibility to erythromycin, azithromycin, josamycin, clindamycin, and streptogramin B. The erythromycin-resistant isolates were further studied for their genetic mechanism of resistance, by means of PCR. The clonality of these strains was also investigated by means of serotyping and ribotyping. In all, 6.2% of the strains were erythromycin resistant, and 3.4 and 2.8% expressed the constitutive MLS(B) and M resistance phenotypes and harbored the ermB and mefA genes, respectively; ermTR was recovered from one isolate which also harbored the ermB gene. Ten serotypes and 8 ribotypes were identified, but we identified 17 strains by combining serotyping with ribotyping. Among the eight ribotypes, the mefA gene was recovered from six clusters, one being predominant, while the ermB gene was recovered from four clusters, of which two were predominant.

Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae.

Klebsiella pneumoniae ORI-1 was isolated in 1998 in France from a rectal swab of a 1-month-old girl who was previously hospitalized in Cayenne Hospital, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferable plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistance profile antagonized by the addition of clavulanic acid, tazobactam, or imipenem. The gene for GES-1 (Guiana extended-spectrum beta-lactamase) was cloned, and its protein was expressed in Escherichia coli DH10B, where this pI-5. 8 beta-lactamase of a ca. 31-kDa molecular mass conferred resistance to oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum beta-lactamases (ESBLs). The highest percentage of amino acid identity was obtained with the carbenicillinase GN79 from Proteus mirabilis; with YENT, a chromosome-borne penicillinase from Yersinia enterocolitica; and with L-2, a chromosome-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% amino acid identity each). However, a dendrogram analysis showed that GES-1 clustered within a class A ESBL subgroup together with ESBLs VEB-1 and PER-1. Sequencing of a 7,098-bp DNA fragment from plasmid pTK1 revealed that the GES-1 gene was located on a novel class 1 integron named In52 that was characterized by (i) a 5' conserved segment containing an intI1 gene possessing two putative promoters, P(1) and P(2), for coordinated expression of the downstream antibiotic resistance genes and an attI1 recombination site; (ii) five antibiotic gene cassettes, bla(GES-1), aac(6')Ib' (gentamicin resistance and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel chloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomycin resistance); and (iii) a 3' conserved segment consisting of qacEDelta1 and sulI. The bla(GES-1) and aadA2 gene cassettes were peculiar, since they lacked a typical 59-base element. This work identified the second class A ESBL gene of a non-TEM, non-SHV series which was located in the plasmid and integron, thus providing it additional means for its spread and its expression.

The NADH oxidase of Streptococcus pneumoniae: its involvement in competence and virulence.

A soluble flavoprotein that reoxidizes NADH and reduces molecular oxygen to water was purified from the facultative anaerobic human pathogen Streptococcus pneumoniae. The nucleotide sequence of nox, the gene which encodes it, has been determined and was characterized at the functional and physiological level. Several nox mutants were obtained by insertion, nonsense or missense mutation. In extracts from these strains, no NADH oxidase activity could be measured, suggesting that a single enzyme encoded by nox, having a C44 in its active site, was utilizing O2 to oxidize NADH in S. pneumoniae. The growth rate and yield of the NADH oxidase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development of competence for genetic transformation during growth was markedly altered. Conditions that triggered competence induction did not affect the amount of Nox, as measured using Western blotting, indicating that nox does not belong to the competence-regulated genetic network. The decrease in competence efficiency due to the nox mutations was similar to that due to the absence of oxygen in the nox+ strain, suggesting that input of oxygen into the metabolism via NADH oxidase was important for controlling competence development throughout growth. This was not related to regulation of nox expression by O2. Interestingly, the virulence and persistence in mice of a blood isolate was attenuated by a nox insertion mutation. Global cellular responses of S. pneumoniae, such as competence for genetic exchange or virulence in a mammalian host, could thus be modulated by oxygen via the NADH oxidase activity of the bacteria, although the bacterial energetic metabolism is essentially anaerobic. The enzymatic activity of the NADH oxidase coded by nox was probably involved in transducing the external signal, corresponding to O2 availability, to the cell metabolism and physiology; thus, this enzyme may function as an oxygen sensor. This work establishes, for the first time, the role of O2 in the regulation of pneumococcal transformability and virulence.

[Bacterial concentration in the cerebrospinal fluid in childhood meningitis]. / Concentrations bactériennes dans le liquide céphalo-rachidien au cours des méningites de l'enfant.

OBJECTIVES: The aim of this study was to analyze the epidemiological evolution of causal germs in meningitis in children aged 1 day to 15 years and determine the relationship between pretreatment concentrations of bacteria in cerebral spinal fluid (CSF), patient age, bacterial species and bacteriological eradication. PATIENTS AND METHODS: A quantitative analysis of germs was performed in 212 children with bacterial meningitis (mean age 19.8 months). RESULTS: Bacterial counts ranged from 2.10(1) to 4.10(9) CFU/ml in CSF. Among the 212 patients, 52 (24.5%) had counts 10(7)/ml. Infants had significantly higher counts than the other age groups. Mean counts for Hoemophilus influenzoe serotype B were not different from those for Streptococcus pneumoniae but were significantly higher than for Neisseria meningitidis. Compared with initial germ counts, 98.5% of the CSF specimens were sterile at 24 and 48 hours and 100% at 72 hours. CONCLUSION: Germ counts were higher in infants.

[Role of group B streptococcus serotype V in materno-fetal infections]. / Place du streptocoque B sérotype V dans les infections maternofoetales.

BACKGROUND: The classification of serogroup B streptococci in serotype is based on the structural differences of capsular polysaccharides and on presence or absence of a protein c antigen. They are classified as Ia, Ia/c, Ib/c, II, II/c, III, IV and V. The serotype V, unknown in 1970, seems emerging, and is placed in third position of frequency in some American studies. We have therefore decided to evaluate its frequency in Paris. POPULATION AND METHODS: In a population of 137 pregnant women and 60 neonates carrying streptococcus of serogroup B, the serotype was systematically determined using the test "Group B streptococcus serotyping test" (Dako, Danemark). RESULTS: In the pregnant women population, 12% of the isolated strains were of serotype V, 26% of serotype III, 15% of serotype II, 14% of serotype Ia, and 21% could not be typed. In neonates, it represented 15% of the isolates and took place after the serotype Ia (20%), the serotype III (18%) and the serotype II (15%). None of the neonates had early- or late-onset disease. They were only colonized. Only one mother exhibited, during the per-partum, a positive blood culture with a streptococcus group B of serotype V. CONCLUSION: These results confirm, in Paris, the importance of this serotype previously observed in foreign studies. It represents 11 to 15% of the isolated streptococcus group B in the neonates and can cause early or late-onset disease. However, larger studies are needed to evaluate the exact risk of pathology for the serotype V and its significance in neonatal infectious disease.