Drawbacks of antibiotic susceptibility of Streptococcus pneumoniae

During 10 years penicillin resistance among clinical isolates of S. pneumoniae was more or less stable. The cumulative proportion of penicillin non susceptible S. pneumoniae (PNSP) was 13.0%; 7.2% when isolates with MIC = 2 mg/l were considered. Among PNSP only 2 strains were isolated from sterile body fluids, one having MIC >3.84 mg/l. Lack of susceptibility to cotrimoxazole was 38.4%. All isolates were susceptible to ceftriaxone, levofloxacin, moxifloxacin, and quinupristin-dalfopristin.     

Genetic relatedness of Streptococcus pneumoniae isolates from paired blood and respiratory specimens

To assess the relatedness of Streptococcus pneumoniae isolates recovered concurrently from blood and respiratory tract specimens from patients with pneumonia, we analyzed 24 paired isolates by pulsed-field gel electrophoresis (PFGE), serotyping, and antimicrobial susceptibility testing. PFGE, serotype, and/or susceptibility patterns were identical for 22 of 24 pairs. Susceptibility results for blood isolates should guide therapy.     

Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates

The increasing prevalence of penicillin-resistant Streptococuus pneumoniae urges for fast and accurate susceptibility testing methods. This study evaluated the comparability of three commonly used techniques; disk diffusion, E-test and agar dilution, to detect penicillin susceptibility in clinical isolates of S. pneumoniae. Fifty pneumococcal isolates, obtained from patients at the University of Malaya Medical Centre, were selected to include both penicillin-susceptible strains and those that had decreased susceptibility (resistant and intermediate) to penicillin. The minimum inhibitory concentration (MIC) values of penicillin to serve as the reference was determined by the agar dilution method in which, based on the MIC breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 27 strains had decreased susceptibility to penicillin with 17 strains resistant and 10 intermediate. Comparing to the agar dilution method, oxacillin disk diffusion test detected all strains with decreased penicillin susceptibility as such while E-test showed a close agreement of susceptibility (92%) of the isolates to penicillin. This confirmed that oxacillin is a good screening test for S. pneumoniae isolates with decreased susceptibility to penicillin while E-test is very reliable for rapid and accurate detection of penicillin susceptibility.     

Antimicrobial susceptibility of invasive isolates of Streptococcus pneumoniae in Ireland

Between January 1999 and June 2002, 646 invasive isolates of Streptococcus pneumoniae were collected in Ireland. MICs of penicillin, ciprofloxacin, cefotaxime, moxifloxacin and linezolid were determined by Etest methodology. Eighty-seven (13.5%) isolates showed intermediate resistance to penicillin, while seven (1.1%) showed high-level resistance. Eighty-seven (13.5%) isolates were resistant to erythromycin, but all isolates were susceptible to cefotaxime, moxifloxacin and linezolid. The prevalence of pneumococcal isolates non-susceptible to penicillin in Ireland is worryingly high, but currently there are alternative agents available to treat invasive infection.     

Methods for predicting susceptibility of Streptococcus pneumoniae to cefixime.

Among 698 Streptococcus pneumoniae isolates, 475 were penicillin susceptible and > 99% of those were susceptible to 0.5 microgram of cefixime per ml; other pneumococci were tentatively assumed to be resistant to cefixime. A 1-microgram oxacillin disk was more reliable than a 5-micrograms cefixime disk for predicting susceptibility to cefixime.     

Molecular epidemiology of macrolide-resistant isolates of Streptococcus pneumoniae collected from blood and respiratory specimens in Norway

Norway has a low prevalence of antimicrobial resistance, including macrolide-resistant Streptococcus pneumoniae (MRSP). In a nationwide surveillance program, a total of 2,200 S. pneumoniae isolates were collected from blood cultures and respiratory tract specimens. Macrolide resistance was detected in 2.7%. M-type macrolide resistance was found in 60% of resistant isolates, and these were mainly mef(A)-positive, serotype-14 invasive isolates. The erm(B)-encoded macrolide-lincosamide-streptogramin B (MLS(B)) type dominated among the noninvasive isolates. One strain had an A2058G mutation in the 23S rRNA gene. Coresistance to other antibiotics was seen in 96% of the MLS(B)-type isolates, whereas 92% of the M-type isolates were susceptible to other commonly used antimicrobial agents. Serotypes 14, 6B, and 19F accounted for 84% of the macrolide-resistant isolates, with serotype 14 alone accounting for 67% of the invasive isolates. A total of 29 different sequence types (STs) were detected by multilocus sequence typing. Twelve STs were previously reported international resistant clones, and 75% of the macrolide-resistant isolates had STs identical or closely related to these clones. Eleven isolates displayed 10 novel STs, and 7/11 of these "Norwegian strains" coexpressed MLS(B) and tetracycline resistance, indicating the presence of Tn1545. The invasive serotype-14 isolates were all classified as ST9 or single-locus variants of this clone. ST9 is a mef-positive M-type clone, commonly known as England(14)-9, reported from several European countries. These observations suggest that the import of major international MRSP clones and the local spread of Tn1545 are the major mechanisms involved in the evolution and dissemination of MRSP in Norway.     

Antimicrobial susceptibility of Streptococcus pneumoniae from children attending day-care centers in a central Italian city

Objective: To undertake a survey of nasopharyngeal carriage of Streptococcus pneumoniae , which reflects strains causing infection, in 100 children under 3 years of age attending day-care centers in Frosinone, a city near Rome.
Methods: Fifty-three unique isolates of S. pneumoniae , isolated from 41 of the children tested, were tested for antimicrobial susceptibility to penicillin, cefotaxime, erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole.
Results: Resistance rates were as follows: penicillin, 20.7% (15% intermediate; 5.7% resistant); trimethoprim-sulfamethoxazole, 64.2%; erythromycin, 64.2%; clindamycin, 30.2%; tetracycline, 32.1%; and chloramphenicol, 3.8%. Except for three intermediate strains, all strains were susceptible to cefotaxime. Only five strains were susceptible to all of the antibiotics tested. An unusual finding of this study was that 23 of the 34 erythromycin-resistant strains were penicillin susceptible, whereas erythromycin-resistant strains found in other countries are predominantly penicillin resistant as well. In addition, 18 of the 34 erythromycin-resistant strains were susceptible to clindamycin. Serogroups 6, 14, 19 and 23 accounted for 84.9% of the isolates.
Conclusions: These data show that carriage of antibiotic-resistant pneumococci in children under 3 years of age is high in Frosinone, Italy. Information on resistance rates in pneumococcal disease in different age groups and on prevalence of drug resistance in other parts of the country is urgently needed.     

Antimicrobial susceptibility testing of Streptococcus pneumoniae: quality assessment results.

Six strains of Streptococcus pneumoniae were distributed to 405 United Kingdom laboratories who were asked to test the susceptibility of the strains to penicillin, tetracycline, chloramphenicol and erythromycin and to provide details of methodology to test the standards of susceptibility testing. High error rates were seen only in failure to detect moderate resistance to penicillin (12%) and resistance to chloramphenicol (16%). Increased error rates were associated with several methods or practices. These included the use of certain culture media; failure to standardise the inoculum; inoculation by loop rather than by swab; failure to use control organisms; failure to measure zone sizes; the use of discs containing a high content of penicillin to test susceptibility to penicillin, and the use of high content discs for testing erythromycin, tetracycline, and chloramphenicol.     

Determination of Penicillin MICs for Streptococcus pneumoniae by Using a Two- or Three-Disk Diffusion Procedure

The potential for the use of the disk diffusion method to accurately predict penicillin MICs for Streptococcus pneumoniae was investigated with penicillin (6 μg), methicillin (5 μg), and oxacillin (1 μg) disks. A total of 183 S. pneumoniae isolates were tested by three MIC procedures (agar dilution, microdilution, and E-test). Regression analyses of the geometric mean of the three MIC results against (i) the sum of the zone diameters for methicillin, penicillin, and oxacillin disks; (ii) the sum of the zone diameters for methicillin and penicillin disks; and (iii) each of the three individual zone diameters were performed. Calculated MICs were determined from each of these regression analyses and compared to the mean reference MICs. A high level of correlation was obtained with both the two- and the three-disk procedures (r = 0.97), with essential agreement rates (±1 doubling dilution) between MICs calculated by the three-disk procedure and the two-disk procedure and the mean reference MICs of 98.4 and 98.9%, respectively. No major or very major errors were obtained with the two- or three-disk procedures. The accuracy of the disks used individually was lower (r = 0.84 to 0.93). However, oxacillin and methicillin disk testing remain excellent for screening strains, with all penicillin-susceptible strains having zones of >21 and >22 mm, respectively. The combination disk procedure, which involves the use of three disks (methicillin, oxacillin, and penicillin) or two disks (methicillin and penicillin) for testing S. pneumoniae, can provide accurate penicillin MICs and qualitative category results that are comparable to results obtained by the E-test, agar, and microdilution MIC methods.     

Detection of Penicillin Susceptibility in Streptococcus pneumoniae by pbp2b PCR-Restriction Fragment Length Polymorphism Analysis

A PCR-restriction fragment length polymorphism strategy directed against the pbp2b gene was evaluated for identification of penicillin susceptibility. A total of 106 United Kingdom (U.K.), 30 Danish, and 11 Papua New Guinean strains were tested. Of the U.K. strains, all the susceptible and all but one of the resistant isolates were correctly assigned. By using conventional definitions of “not resistant” and “not susceptible,” the sensitivities were 97.5 and 94.4%, the specificities were 100 and 98.9%, the positive predictive values were 100 and 94.4%, and the negative predictive values were 93.1 and 98.9%, respectively. This technique may allow susceptible (MIC, <0.1 mg/liter) and resistant (MIC, >1 mg/liter) isolates to be distinguished in a single PCR.     

Screening for cephalosporin-resistant Streptococcus pneumoniae with the Kirby-Bauer disk susceptibility test.

Kirby-Bauer disk susceptibility tests with five standard cephalosporin disks were performed on 23 penicillin-resistant Streptococcus pneumoniae isolates for which ceftriaxone MICs were 0.125 to 4 micrograms/ml. Cefuroxime disk inhibition zone diameters distinguished clearly isolates for which ceftriaxone MICs were > or = 2 micrograms/ml from more susceptible strains, whereas cephalothin, ceftizoxime, cefotaxime, and ceftriaxone disks distinguished these isolates less clearly than the cefuroxime disk did.     

In vitro susceptibility of six fluoroquinolones against invasive Streptococcus pneumoniae isolated from 1996 to 2001 in Taiwan

A total of 331 invasive nonduplicated Streptococcus pneumoniae isolates from three sampling periods during 1996 to 2001 were tested for susceptibility to recently developed fluoroquinolones. Five major serotypes, 23F, 6B, 14, 19F, and 3, were frequently encountered in this collection. Penicillin nonsusceptible isolates constituted 52.9% from 1996 to 1997, 61.6% from 1998 to 1999, and 60.0% from 2000 to 2001. Fifty-seven percent of the isolates were susceptible to cefotaxime, 56.5% to ceftriaxone, 54.1% to cefepime, and 52.6% to cefuroxime. Macrolide-susceptible isolates constituted less than 14% of the total sample, and no vancomycin-resistant isolates were detected. For fluoroquinolones, MIC90 was lowest for gemifloxacin (MIC90 = < or = 0.12 microg/ml), followed by moxifloxacin (MIC90 = 0.25 microg/ml), gatifloxacin (MIC90 = 0.5 microg/ml), sparfloxacin (MIC90 = 0.5 microg/ml), levofloxacin (MIC90 = 1 microg/ml), and ciprofloxacin (MIC90 = 2 microg/ml). All isolates were susceptible to sparfloxacin, levofloxacin, gatifloxacin, and gemifloxacin apart from one isolate (0.3%), which was simultaneously resistant to sparfloxacin, levofloxacin, and gatifloxacin. Mutations at the positions S81F of GyrA and D435N and I460V of ParC were detected for this multiple drug resistant isolate. The in vitro results suggest that recently developed fluoroquinolones are very effective against invasive S. pneumoniae isolates in Taiwan. Nevertheless, emerging fluoroquinolone resistance should be acknowledged and clinicians alerted. Surveillance should be carried out to monitor any changes in antibiotic resistance of S. pneumoniae.     

Detection of Streptococcus pneumoniae in lower respiratory tract specimens by anaerobic culture technique.

The relative efficacy of aerobic and anaerobic culture techniques for the recovery of Streptococcus pneumoniae from 1,173 lower respiratory tract specimens was determined. Unlike previous studies, this investigation found no significant difference between the two techniques.     

Determination of penicillin susceptibility of Streptococcus pneumoniae using the polymerase chain reaction.

AIM: To develop a polymerase chain reaction (PCR) based method to detect penicillin susceptibility in isolates of Streptococcus pneumoniae (SP). METHOD: PCR primers were designed to amplify differential nucleotide sequences of the penicillin-binding protein (PBP) genes 2b, 2x, and 1a in penicillin susceptible and resistant strains of SP. Primers derived from the PBP 2x and 2b genes were designed to amplify products from penicillin susceptible S pneumoniae (PSSP), whereas primers derived from the PBP 1a gene were designed to amplify gene sequences of penicillin resistant S pneumoniae (PRSP). RESULTS: Two hundred and thirty clinical isolates of SP from the USA, UK, Kenya, Romania, and the Kingdom of Saudi Arabia were tested. Of the isolates, 116 were penicillin susceptible, 65 were intermediately resistant, and 49 were highly resistant. PCR identified 108 (93%) of 116 of PSSP isolates, 55 (85%) of 65 intermediately resistant isolates, and all of the 49 highly resistant isolates of SP. The susceptibility of 16 (7%) isolates could not be determined using PCR. All of these 16 isolates had a minimum inhibitory concentration (MIC) of penicillin < 1 mg/l. None of the highly resistant isolates was identified as penicillin susceptible by PCR, although two of the isolates with intermediate resistance (MIC = 0.125 mg/l) were. CONCLUSION: Using primers that differentially identify the genotypes of susceptible and resistant strains of SP, PCR provides a rapid method for determining the penicillin susceptibility of SP isolates and could potentially be used for testing clinical samples.     

Large-Scale Identification of Virulence Genes from Streptococcus pneumoniae

Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Streptococcus pneumoniae is the major cause of community-acquired bacterial pneumonia, and it is also responsible for otitis media and meningitis (2). Capsular polysaccharides were the first virulence factors to be identified. The capsule is thought to protect the bacteria from the host immune system by preventing phagocytosis (17). Purified capsular extracts do not have an inflammatory or toxic effect (31, 32). Among the proteins considered to be virulence factors (17, 45) are pneumolysin (3, 7, 12), autolysin (4, 12, 56), hyaluronidase (5), pneumococcal surface protein A (PspA) (8), PsaA (6), neuraminidase (10), immunoglobulin A1 (IgA1) protease (46, 59), and pyruvate oxidase (55), although for some of them a role in virulence has not been demonstrated.Recent advances in the field of bacterial pathogenesis have allowed a large-scale identification of new virulence genes in different bacterial species. The methods developed are based on the concept that specific gene products are required for each stage of an infection process and that their expression is often regulated by the different environmental conditions encountered in the host. Mahan et al. (38) developed a system called IVET (in vivo expression technology) aimed at identifying bacterial genes that were preferentially expressed in the host during infection and were poorly transcribed under laboratory conditions. IVET was originally developed for use with Salmonella typhimurium (38) and then applied to Vibrio cholerae (11) and Pseudomonas aeruginosa (58). Hensel et al. (28) expanded the concept of tagging originally developed by Walsh and Cepko (57) to monitor the fate of clonally related neocortical cells during brain development, developing a strategy to identify virulence genes by negative selection. This system, called STM (signature-tagged mutagenesis), exploits a pool of transposons in which each transposon is tagged with a unique DNA sequence so that the resulting insertion mutants are marked with a different DNA sequence. This permits the identification of bacteria recovered from hosts infected with a mixed population of mutants.Tagged insertion mutants are combined into pools, which are used to infect the animals. At a defined time point, bacteria are recovered from the animals. Tag sequences are amplified from each pool by using a radioactive label before and after the infection. These two labeled tag probes are hybridized to filters containing spotted genomic DNA from all mutants of the corresponding pool. Mutants whose tags are positive for hybridization with the probe from the original pool and negative with the one from the recovered bacteria are considered to be virulence attenuated. This system was originally used to identify genes involved in virulence in S. typhimurium (28) and recently applied to Staphylococcus aureus and V. cholerae (15, 42).S. pneumoniae has been studied for many years, yet its virulence mechanisms are not fully understood (17). Therefore, we have modified the original STM methodology to discover novel virulence factors in S. pneumoniae. Initially, we attempted to use for this purpose the encapsulated type 3 strain GP119, obtained by transformation from the nonencapsulated avirulent laboratory strain Rx1. Surprisingly, when GP119 was tested for virulence in a mouse septicemia model, it was found to be not virulent (unpublished observations); this result indicates that in factors in addition to the capsule are required for virulence. We then selected the encapsulated type 19F strain G54, a recent clinical isolate. This strain was chosen because of its high transformation rate (47) induced by using the 17-residue competence-stimulating peptide (26) and its virulence in a mouse pneumonia model, as assessed in our laboratory. The following two major changes were introduced in the original STM protocol (28).(i) A library of tagged mutants was constructed by insertion-duplication mutagenesis using short random genomic DNA fragments inserted in a suicide plasmid vector bearing the molecular tag. The original transposon mutagenesis technique (28) was not applied to S. pneumoniae, since we did not expect transposons to insert randomly as shown for Streptococcus mutans (24) and Lactococcus lactis (49).(ii) While in the original method (28) the filters corresponding to each pool had spots of genomic DNA from each mutant obtained by transferring bacterial colonies to the filters (colony hybridization), we used filters containing amplified tags from each mutant. This modification was necessary since in our hands colony hybridization generated a high background giving rise to false positives.Using the modified STM technique, we identified 126 putative virulence genes from S. pneumoniae. Some of them corresponded to previously described pneumococcal virulence factors, while others showed homology to virulence genes found in other bacteria. In addition, we have identified genes not previously known to be involved in virulence.