Detection of penicillin and extended-spectrum cephalosporin resistance among Streptococcus pneumoniae clinical isolates by use of the E test.

Increasing penicillin resistance and the initial recognition of resistance to extended-spectrum cephalosporins among Streptococcus pneumoniae isolates have placed greater emphasis on accurate methods for susceptibility testing of clinical isolates. This study has evaluated the use of the E test (AB Biodisk NA, Piscataway, N.J.) for the detection of penicillin and cefotaxime resistance among 147 pneumococcal clinical isolates in three geographically separate laboratories. These included 42 penicillin-resistant (MIC, > or = 2 micrograms/ml) and 14 cefotaxime-resistant (defined here as an MIC of > or = 2 micrograms/ml) isolates. E test strips were applied to the surface of Mueller-Hinton sheep blood agar plates and incubated at 35 degrees C in 5% CO2 for 20 to 24 h. E test MICs were compared with MICs determined with lysed horse blood-supplemented Mueller-Hinton broth in a microdilution format as recommended by the National Committee for Clinical Laboratory Standards. Penicillin MICs agreed within one log2 dilution for 136 of 147 (92.5%) isolates, and cefotaxime MICs agreed within one log2 dilution for 142 of 147 (96.6%) isolates. No very major or major interpretive errors occurred with either penicillin or cefotaxime E test MIC results. There were 9.5 and 5.4% minor interpretive category errors with penicillin and cefotaxime E test MICs, respectively. These data indicate that the E test represents a convenient and reliable method for the detection of penicillin or cephalosporin resistance in pneumococci.     

Relationship between penicillin and cephalosporin resistance of Streptococcus pneumoniae strains and its inflammatory activity in the experimental model of meningitis

Using a rabbit model of meningitis, we sought to compare the inflammatory activity induced by three pneumococcal strains with different susceptibilities to penicillin and cephalosporins, belonging to the serotypes 3, 6B and 23F at different inoculum sizes. These serotypes are prevalent in Western Europe and are believed to produce a moderate-to-severe cerebrospinal fluid (CSF) inflammatory response. Only minor differences were observed in the inflammatory activity evoked by the three strains in the subarachnoid space, and most were probably related to differences in bacterial counts. Infection by serotype 23F caused secondary bacteremia in all challenged animals. Our findings reinforce the concept that resistant pneumococci are not more virulent, a fact that should be taken into account when evaluating the efficacy of different anti-pneumococcal therapies. However, the frequent induction of secondary bacteremia by the resistant serotype 23F requires further study.     

Streptococcus pneumoniae meningitis after myelography and intrathecal infiltration

A 76 year old patient is admitted in an intensive care unit with severe acute pneumococcal meningitis. He dies 2 days after admission besides antibiotherapy by ampicillin. Interest of measures for preventing the infections after lumbar puncture is discussed.     

Streptococcus pneumoniae meningitis in children. Case records 1985 - 2003

Acute bacterial meningitis and sepsis are the most severe among invasive diseases due to Streptococcus pneumoniae, particularly in pediatric age, and present a high risk of mortality and neurologic sequelae. S. pneumoniae is a major worldwide pathogen in children. The widespread emergence of penicillin-resistant pneumococci, a new pneumococcal conjugate vaccine, and the epidemiological prevalence of some serotypes, have recently focused attention on S. pneumoniae disease. We reviewed the data on incidence, epidemiology, diagnosis, therapy in children hospitalized with acute bacterial meningitis in the Division of Infectious Diseases of the Bambino Gesu Children's Hospital, Rome, between 1985- 2003. S. pneumoniae was isolated in 16.3% of the children, progressively emerging as the prevalent pathogen. The highest incidence was found in children younger than 2 yrs. The disease still presents a high rate of long-term sequelae, especially hearing loss and neurological handicap. Penicillin and ampicillin resistant isolates were found in 2.3% of positive cultures; no strain was resistant to cephalosporins and vancomycin. Our data support the recommendations to consider administration of the 7-valent pneumococcal conjugate vaccine for children older than 2 months of age, with special consideration for selected groups. We recommend monitoring all invasive pneumococcal infections in children, the emergence of antibiotic-resistance and changes in prevalence of pathogen serotypes.     

Decreased virulence of a pneumolysin-deficient strain of Streptococcus pneumoniae in murine meningitis

Pneumolysin, neuraminidases A and B, and hyaluronidase are virulence factors of Streptococcus pneumoniae that appear to be involved in the pathogenesis of meningitis. In a murine model of meningitis after intracerebral infection using mutants of S. pneumoniae D39, only mice infected with a pneumolysin-deficient strain were healthier at 32 and 36 h, had lower bacterial titers in blood at 36 h, and survived longer than the D39 parent strain. Cerebellar and spleen bacterial titers, meningeal inflammation, and neuronal damage scores remained uninfluenced by the lack of any of the virulence factors.     

Streptococcus pneumoniae damages the ciliated ependyma of the brain during meningitis

Streptococcus pneumoniae meningitis remains a disease with a poor outcome for the patient. A region of the brain that has been neglected in the study of meningitis is the ependyma, which has been identified as a location of adult pluripotent cells. In this study we have used a rat model of meningitis to examine whether the ependymal layer is affected by S. pneumoniae. The effects included localized loss of cilia, a decrease of the overall ependymal ciliary beat frequency, and damage to the ependymal ultrastructure during meningitis. In conclusion, loss of ependymal cells and ciliary function exposes the underlying neuronal milieu to host and bacterial cytotoxins and this is likely to contribute to the neuropathology commonly observed in pneumococcal meningitis.     

Non-Penicillin-Binding protein mediated high-level penicillin and cephalosporin resistance in a Hungarian clone of Streptococcus pneumoniae

A clone of Hungarian pneumococcal strains has recently been isolated with notably high levels of beta-lactam resistance (penicillin MIC, 16 microg/mL; cefotaxime MIC, 4 microg/mL). The role that each penicillin-binding protein (PBP) plays in the development of resistance in these strains was investigated via transformation of susceptible strain R6 with pbp DNA from resistant strain 3191. Transformation of strain R6 with pbp2X DNA resulted in transformants with penicillin and cefotaxime MICs of 0.06 and 0.25 microg/mL, respectively. Further introduction of pbp2B and 1A DNA increased penicillin MICs to 0.25 and 4 microg/mL, respectively. Transformation of strain R6 with a combination of pbp2X and pbp1A DNA produced R63191/2X/1A strains with an unexpected low cefotaxime MIC of 0.5 microg/mL. This low-level of cefotaxime resistance was surprisingly increased from 0.5 to 2 microg/mL in R63191/2X/2B/1A strains. This suggests the involvement of altered PBP 2B in cefotaxime resistance. Therefore, within this particular setting of resistance, the environmental presence of selectors for altered PBP 2B (penicillin or piperacillin) are required for the development of high-level cefotaxime resistance. The MICs of R63191/X/2B/1A strains never reached the MICs of the donor strain. Full MICs of the donor were eventually reached by transforming R63191/2X/2B with chromosomal3191 DNA. Resultant transformants revealed the introduction of altered PBP 1A, while unaltered PBPs 1B, 2A, and 3 proved that these PBPs were not involved in resistance. A non-PBP resistance determinant has therefore made up the difference in resistance between R63191/2X/2B/1A and donor strain 3191. Beta-lactamase activity and efflux systems have so far been eliminated as causes of resistance. This resistance determinant represents a novel mechanism for beta-lactam resistance in clinical isolates of pneumococci, operates at the highest level of resistance, and remains to be identified.     

Calcium signaling in Streptococcus pneumoniae: implication of the kinetics of calcium transport

The kinetics and pharmacological characterization of a Na+/Ca2+ exchange system, essential for the growth of the extracellular pathogen Streptococcus pneumoniae in high-calcium media, demonstrated that calcium transport, in addition to its role in calcium homeostasis, is involved in the induction of autolysis and of competence for genetic transformation. These responses are expressed respectively in cultures entering the stationary phase and growing with exponential rates. Experimental virulence also appears to be modulated by the kinetics of calcium transport. Calcium transport in S. pneumoniae is electrogenic and shows sigmoidicity, indicating a cooperative mechanism with an inflexion point at 1 mM Ca2+. Mutant strains with Hill number values of 4 and 1, compared to 2 in the wild-type strain, were isolated. These changes were associated with altered regulation of competence and autolysis, and also with reduced experimental virulence. By contrast, they could not be related to a specific calcium requirement for growth. This indicates that the cooperativity of Ca2+ transport is not involved in vegetative growth, but rather regulates competence and autolysis. Competence and autolysis represent two growth-phase-dependent responses to an oligopeptide-activator exported to the medium, the competence-stimulating peptide. Addition of this activator to noncompetent cells, triggers net and transient 45Ca2+ influx. One effect of the activator might be to activate a calcium transporter by enhancing its cooperativity. In addition to an increase in intracellular calcium, a transient membrane depolarization induced by electrogenic calcium influx may be part of the signaling mechanism. The competence activator is a quorum-sensing molecule whose synthesis is autoregulated. This regulation might involve calcium-mediated signaling. As an extracellular pathogen, S. pneumoniae probably develops in niches with variable calcium concentration. Interestingly, virulence depends strongly upon the kinetics of Ca2+ transport. Regulation of calcium influx may represent a common mechanism of sensing the environment, if the Na+/Ca2+ exchanger is the target for external mediators including the competence activator.     

Clinical and microbiological epidemiology of Streptococcus pneumoniae bacteraemia

A survey of Streptococcus pneumoniae bacteraemia in the Grampian region of Scotland was carried out over a 2-year period. One hundred and four bacteraemic episodes were identified in 103 patients, an incidence of 9.8/100,000 population/year, and the mortality was 24%. Clinical information was abstracted from 92 sets of patient notes and 98 isolates of S. pneumoniae were available for further study. The incidence of S. pneumoniae bacteraemia was highest at the extremes of age and peaked at 78 cases/100,000 population/year in those over 80 years old. Many patients had predisposing conditions, of which chronic lung disease (23%), chronic alcohol abuse (10%) and malignant disease (10%) were the commonest. Age was the highest risk factor for mortality, with 20 of the 22 deaths in those over 65 years old. The commonest serotype of S. pneumoniae isolated was serotype 14 (23.5%). Only one isolate (serotype 6A) showed intermediate resistance to penicillin, but 12 isolates (12.2%) were resistant to erythromycin. Nine of these 12 isolates were of serotype 14 and had MICs clustered in the range 12-24 mg/L. Examination of all serotype 14 isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two distinct genetic clusters, with all the erythromycin-resistant isolates in the same cluster. These isolates had similar PFGE profiles to erythromycin-resistant serotype 14 strains isolated elsewhere in the UK and they were positive for the mefE gene by PCR, confirming that resistance was of the M phenotype. The recent increase in erythromycin resistance in S. pneumoniae may be due, at least in part, to the spread of a serotype 14 clone of the M phenotype which appears to be an important cause of invasive disease.     

Genome-wide identification of Streptococcus pneumoniae genes essential for bacterial replication during experimental meningitis

Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during infection. The results point to essential roles for capsular polysaccharides, nutrient uptake, and amino acid biosynthesis in bacterial replication during experimental meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive infection models of experimental rat meningitis. It appeared that adenylosuccinate synthetase, flavodoxin, and LivJ, the substrate binding protein of a branched-chain amino acid ABC transporter, are relevant as targets for future therapy and prevention of pneumococcal meningitis, since their mutants were attenuated in both models of infection as well as in competitive growth in human cerebrospinal fluid in vitro.     

Mixed bacterial meningitis due to Streptococcus pneumoniae and Neisseria meningitidis in an 18-month-old child

We report an unusual case of culture-proven pneumococcal and meningococcal mixed meningitis in an 18-month-old girl. The patient responded well to antimicrobial therapy and recovered completely without sequelae. No underlying condition could be demonstrated except a rhinitis of unknown etiology 2 days before the onset of the symptoms suggesting meningitis.     

Streptococcus pneumoniae meningitis in Amiens Hospital between 1990 and 2005. Bacteriological characteristics of strains isolated

Streptococcus pneumoniae is actually the first most likely organism to cause meningitis in children 2 months to 2 years old and in adults older than 65 years. From January 1990 to December 2005, 72 cases of S. pneumoniae-positive cerebrospinal fluid culture were indexed in our hospital. Among the 72 cases, 25 came from children, and 60% of these came from children under two years of age and 47 came from adults whose the mean age was 55 years. The first penicillin-resistant S. pneumoniae (PNSP) meningitis was identified in 1993. The susceptibility to penicillin of pneumococcal isolates causing meningitis varied according to time; until 1995, 25% of the strains were PNSP, then from 1996 to 2005, 50% of strains were PNSP. The overall prevalence of non-susceptible was 34.7% (25/72). Among the 25 PNSP, 21 were intermediate to penicillin G and four of them were resistant. Among children, seven PNSP meningitis were indexed and one of them was resistant. The antimicrobial MICs of amoxicillin and cefotaxim varied from 0.064 to 1 mg/l and from 0.016 to 0.5 mg/l respectively. Among adults, 18 PNSP meningitis were indexed. Three strains were penicillin-resistant. The antimicrobial MICs of amoxicillin varied from 0.064 to 2 mg/l. Nine strains of 18 PNSP had cefotaxim MIC>/=0.5 mg/l and, four of them had MIC 1 mg/l. None amoxicillin and cefotaxim-resistant strain was isolated. Serotyping of all strains was performed in the Reference Center. Serotypes 6B, 9V and 19 were the most frequent in child and serotypes 6B, 23F, 19, 9, 4 were the most frequent in adult. So, all serotypes were represented.     

Vancomycin-tolerant Streptococcus pneumoniae in Korea

A nationwide surveillance study was undertaken to monitor antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in Korea, with a special focus on vancomycin tolerance. For the 6-month period from March to August 2002, clinical isolates of S. pneumoniae were collected from 11 university hospitals and 1 reference laboratory. One-hundred eighty-eight isolates were measured for lysis rates after exposure to vancomycin for 4 h. Two vancomycin-tolerant S. pneumoniae (VTSP) strains, S3 and H8, were isolated from sputum cultures of two patients, who had stayed in intensive-care units of different hospitals with long-term antibiotic therapy and were not treated for pneumococcal pneumonia. The penicillin, cefotaxime, and vancomycin MICs for S3 were 8 microg/ml, >16 microg/ml, and 0.5 microg/ml, and those for H8 were 2 microg/ml, 2 microg/ml, and 0.5 microg/ml, respectively. While S3 belonged to serotype 23F and was autolysin defective, H8 belonged to serotype 13F and had intact autolysin. These strains were not clonally related as determined by pulsed-field gel electrophoresis of chromosomal DNA. In agreement with previous reports, both isolates showed pairing of TIGR4 vex2 with R6 pep27 and had two identical amino acid substitutions, Q441K in vncS and N25D in vex2. These findings indicate that two VTSP strains have emerged independently in Korea, suggesting a prevalence rate of 1.1%. The emergence of VTSP would be a serious threat in Korea, where there are significant rates of penicillin resistance in S. pneumoniae. Monitoring of the prevalence of VTSP and further investigation of the clinical relevance of VTSP are warranted.     

Macrolide Resistance Phenotypes and Genotypes in French Clinical Isolates of Streptococcus pneumoniae

 The aim of this study was to analyze the mechanisms of macrolide resistance in French clinical isolates of Streptococcus pneumoniae. A total of 838 strains of pneumococci were isolated in 1997 in Normandy, a region of western France, by 19 microbiology laboratories. Fifty-three percent had displayed diminished susceptibility to penicillin G and 50% were resistant to erythromycin. From this collection, 92 penicillin-intermediate or -resistant and 18 penicillin-susceptible strains resistant to erythromycin were studied. The presence of erm genes coding for ribosomal methylases and of mefE-like genes responsible for macrolide efflux was screened by a multiplex polymerase chain reaction and confirmed by DNA/DNA hybridization. Of the 110 strains studied, 108 were cross-resistant to erythromycin, spiramycin and clindamycin, including 105 strains containing ermB-related genes and three strains that contained a combination of ermB- and mefE-related genes. Two strains apparently susceptible to clindamycin but resistant to spiramycin also contained ermB-related genes. No strain was resistant to erythromycin alone or contained only a mef-like gene. Therefore, resistance to erythromycin is mostly related to ribosomal methylation in this region of France.     

Mixed bacterial meningitis in an adult caused by Haemophilus influenzae and Streptococcus pneumoniae. A case report

A case of mixed bacterial meningitis with Haemophilus influenzae and Streptococcus pneumoniae is reported in a 26-year-old woman without demonstrable predisposing conditions, who recovered after treatment with ampicillin, without sequelae.