Pbp2b Genes Research Articles

Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes. One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay. A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics. The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.

Molecular characteristics of serotype 3 Streptococcus pneumoniae isolates among community-acquired pneumonia patients in Japan

In order to understand the spread of the erythromycin-resistant serotype 3 Streptococcus pneumoniae clone in Japan, we have assessed the molecular characteristics of this clone. Among 156 S. pneumoniae isolates recovered from adults with community-acquired pneumonia between 2003 and 2005, 42 were serotype 3 and 40 were sequence type (ST) 180/Netherlands(3)-31 by multilocus sequence typing. Thirty-eight of the 40 ST 180 isolates had acquired resistance to erythromycin via the ermB gene. Although the ermB-positive ST180 clone isolates were more susceptible to penicillin and trimethoprim-sulfamethoxazole than ermB-positive non-ST180 isolates and contained a less mutated pbp1a or pbp2b gene, without a mefA gene, the ST180 clone was highly prevalent among ermB-positive isolates. Routine surveillance for the ST180 S. pneumoniae clone may soon become necessary.

Lack ofin vitrobiofilm formation does not attenuate the virulence ofStreptococcus gordoniiin experimental endocarditis

The ability to induce experimental endocarditis of biofilm-deficient mutants of Streptococcus gordonii was studied in an isogenic background. Strains were inactivated in either comD, fruK or pbp2b genes, which are involved in biofilm formation. These strains were clearly impaired (>75% reduction) in biofilm production in vitro. However, this did not result in a decreased severity of infection in vivo.

High Prevalence of Streptococcus pneumoniae with Mutations in pbp1a, pbp2x, and pbp2b Genes of Penicillin-Binding Proteins in the Nasopharynx in Children in Japan

Objective: To evaluate the resistances of Streptococcus pneumoniae to β-lactams developed by stepwise alterations in high-molecular-weight penicillin-binding proteins (PBPs) with a reduced binding affinity of β-lactams. Among the numerous mutations in pbp genes that alter the affinity for β-lactams, the decreased affinity of PBP1A, 2X and 2B is especially important in the development of resistances to β-lactams. Study Design: Retrospective review. Methods:In this study, we investigated the mutations in pbp1a, pbp2x, and pbp2b genes evaluated by polymerase chain reaction (PCR) in 866 pneumococcal isolates collected from the nasopharynx of Japanese children with acute otitis media. Results: 210 strains (24.3%) exhibited no mutations in the three pbp genes. 333 strains (38.5%) had mutations in the three pbp genes, 78 (9.0%) in two pbp genes, whereas 245 (28.3%) displayed mutations in only one pbp gene. Among the 656 strains with mutations in pbp genes, 620 (94.5%) strains had mutations in pbp2x. The annual prevalence of antimicrobial-resistant S. pneumoniae showed a gradual increase in strains with mutations in the three pbp genes and a parallel decrease in strains without mutations. Conclusions: PCR-based genotyping can characterize the antimicrobial resistances in pneumococci along with minimal inhibitory concentrations (MICs). Physicians should pay attention to the recent increase in antimicrobial-resistant S. pneumoniae when treating pediatric acute otitis media.

Incremental Increase in Fitness Cost with Increased β‐Lactam Resistance in Pneumococci Evaluated by Competition in an Infant Rat Nasal Colonization Model

We evaluated the impact of resistant penicillin-binding protein (PBP) allele acquisition on the ability of penicillin-resistant (PEN-R) pneumococcal strains to compete with penicillin-susceptible (PEN-S) ancestors for upper-respiratory-tract (URT) colonization. PEN-S serotype 2, 6B, and 9V strains were transformed into derivatives expressing an increasing number of PEN-R PBP forms (2X, 2X-1A, and 2X-1A-2B for serotype 2 and 2X, 2X-2B, and 2X-2B-1A for 6B and 9V). Infant rats were inoculated intranasally with a mix of a PEN-R and PEN-S strains. For consecutive days, samples were collected for assessment of the ratio of PEN-S to PEN-R cells colonizing the URT. The selective index (SI), defined as the change in the natural logarithm of the ratio of PEN-S to PEN-R strains from the inoculum to the nasal-wash samples, quantified differences in fitness. SIs significantly > 0 (indicating a cost of resistant allele acquisition) were observed 4-5 days after colonization in all but serotype 6B pbp2x transfomants. Additional replacements with low-affinity forms of pbp2b and pbp1a genes reduced further ability to compete in all strains. The cost of penicillin-resistance acquisition for the Streptococcus pneumoniae strain competing with its susceptible ancestor to colonize the URT increases with the number of resistant pbp alleles acquired.

Alterations of the penicillin-binding proteins and murM alleles of clinical Streptococcus pneumoniae isolates with high-level resistance to amoxicillin in Spain

The aim of this study was to analyse the nucleotide sequences of regions encoding the penicillin-binding domains of pbp1A, pbp2B and pbp2X genes and murM alleles from 14 selected amoxicillin-resistant Streptococcus pneumoniae isolates (MICs 8-16 mg/L) obtained in Spain. PFGE and dideoxynucleotide chain termination sequencing were used. Analysis of PFGE profiles showed that the amoxicillin-resistant S. pneumoniae strains belonged to six different PFGE patterns including the Spain23F-1, Spain6B-2, Spain9V-3 and Spain(14)-5 international clones; however, 8 of the 14 strains belonged to the Spain9V-3 clone. These strains showed the typical changes in penicillin-binding proteins (PBPs) 1A and 2X and had 10 unique changes in the 590-641 region of PBP2B as described previously. Transformation experiments tried to incorporate the transpeptidase domain of PBP2B including the 590-641 region associated with amoxicillin-resistant pneumococci. Sequencing of the pbp2B genes revealed that part of the 3' region of the pbp2B sequence encoding a region of the domain (around amino acid 514-538 to the C terminus of PBP2B) did not recombine with the R6 pbp2B gene. The murM sequence analysis showed that 6, 6 and 2 amoxicillin-resistant S. pneumoniae strains had murMA, murMB5 and murMB6 alleles, respectively. However, strains with murMB5 or murMB6 alleles showed a single mutation (N537D) in the 537-581 region of PBP2B, while strains with the murMA allele had 12 unique changes. Ten unique changes in the 590-641 region of PBP2B and no specific murM alleles were found in S. pneumoniae strains isolated in Spain with an amoxicillin MIC>or=8 mg/L (MICs from 6 to 12 mg/L by 1 mg/L step dilution). In addition, the presence of specific mutations in PBP2B seems to play a key role in the presence of different murM alleles in these amoxicillin-resistant pneumococcal strains.

Antibiotic susceptibility according to genotype of penicillin-binding protein and macrolide resistance genes, and serotype of Streptococcus pneumoniae isolates from community-acquired pneumonia in children

Antibiotic susceptibilities, genes mediating beta-lactam and macrolide resistance, and serotypes were analysed for strains of Streptococcus pneumoniae. A total of 392 strains of S. pneumoniae were isolated from paediatric patients with community-acquired pneumonia between May 2002 and 2004. All strains were classified into six genotype patterns according to the mutations found in the pbp1a, pbp2x and pbp2b genes identified by PCR. These results are represented by adding 'g', indicating genotypic identification. Thirty-nine per cent of the isolates showed mutations in either one or two PBP genes (gPISP, where PISP stands for penicillin-intermediate resistant S. pneumoniae) and 52.3% had mutations in three genes (gPRSP, where PRSP stands for penicillin-resistant S. pneumoniae). The majority of the strains had a macrolide resistance gene: mefA, (30.6%); ermB, (48.5%); or both mefA and ermB, (7.7%). The most frequent serotypes of these strains were: 6B (23.2%), 23F (17.6%), 19F (17.3%), 14 (10.5%) and 6A (8.2%). Serotypes of the seven-valent conjugate vaccine covered 70.9% of all isolates, and 89.8% of gPRSP. Serotypes of the strains with cefotaxime MICs of > or =2 mg/L were almost all of a vaccine type. The results suggest that introduction of conjugate vaccines into infants and children is necessary for the prevention of pneumococcal infections in Japan.

Novel Antibiotic-Resistant Pneumococcal Strains Recovered from the Upper Respiratory Tracts of HIV-Infected Adults and Their Children in Kisumu, Kenya

In a survey of genetic diversity within penicillin-nonsusceptible pneumococcal isolates in Kenya, we examined 162 upper respiratory isolates from 104 human immunodeficiency virus (HIV)-infected adults and 46 children in a cotrimoxazole prophylaxis study. Antibiotic resistance levels were high; 152 (94.4%) were cotrimoxazole nonsusceptible (134 fully resistant) and 124 (77%) were intermediately penicillin resistant. Isolates nonsusceptible to penicillin and cotrimoxazole (PNCNP) were found among 24 of the 29 serotypes encountered, 15 of which have rarely or never had documented nonsusceptibility to penicillin. These included serotypes 3, 4, 7C, 7F, 10A, 11A, 13, 15A, 15B, 16F, 17F, 19B, 21, 35A, and 35B. Segments of pbp2b genes from 9 PNCNP (serotypes 3, 13, 15A, 16F, 20, and 35A) were typical of resistance-conferring alleles in that they were highly divergent and contained two substitutions thought to be critical for resistance. Similarly, the dhfr genes from 3 PNCNP were divergent and contained a substitution required for cotrimoxazole resistance. Multilocus sequence typing (MLST) of 48 PNCNP revealed 33 sequence types (STs), none of which were previously recorded at http://www.mlst.net. Comparisons with all known STs revealed that 23 of these STs were unrelated to other known STs, whereas 10 STs were highly related to STs from internationally disseminated strains, including 2 of the 26 antibiotic-resistant clones recognized by the Pneumococcal Molecular Epidemiology Network. Based upon differing serotypes expressed by strains of identical or closely similar genotypes, there has been an extensive history of capsular switching within seven genetic clusters represented by these 10 STs and related STs described at http://www.mlst.net.

Comparison of activities of β-lactam antibiotics against Streptococcus pneumoniae with recombinant penicillin-binding protein genes from a penicillin-resistant strain

We investigated the antibacterial activities of 19 beta-lactams against three recombinant bacterial strains, in which three penicillin-binding protein genes, pbp2x, pbp1a, and pbp2b, from penicillin-resistant Streptococcus pneumoniae (PRSP), were transformed to a penicillin-susceptible strain. By the acquisition of the pbp2x gene from PRSP, the minimum inhibitory concentrations (MICs) of third-generation cephalosporins were increased more than eight fold. When the strain acquired the PRSP pbp1a gene in addition to pbp2x, the MICs of all tested beta-lactams increased 2- to 16-fold. When the strain acquired the PRSP pbp2b gene in addition to pbp2x and pbp1a, the MICs of penicillins and carbapenems increased 4- to 16-fold. However, two novel carbapenems, ME1036 and L-036, showed excellent antibacterial activities against these recombinant strains, as well as against the parent PRSP.

Genotyping of Streptococcus pneumoniae and Haemophilus influenzae Isolated from Paired Middle Ear Fluid and Nasopharynx by Pulsed-Field Gel Electrophoresis

Twenty-eight isolates of Streptococcus pneumoniae and 30 isolates of Haemophilus influenzae from paired nasopharynx and middle ear fluids of 21 children with acute otitis media (AOM) were evaluated to determine genotypes by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE). Among the 28 isolates of S. pneumonaie, 21 isolates (75.0%) possessed mutations in the pbp1a,pbp2x, and pbp2b genes, and 7 isolates (25%) had mutations in the pbp2x gene. Nineteen isolates (67.9%) expressed the mefE gene, and 5 isolates (17.9%) possessed the ermB gene. Among the 30 isolates of H. influenzae, 5 isolates (16.7%) had mutations in pbp3 genes, 3 isolates (10.0%) produced β-lactamase, and 2 (6.7%) isolates possessed mutations both in the pbp3 gene and the β-lactamase gene. Ten out of the 14 pairs (71.4%) of the restriction fragment patterns of S. pneumoniae from paired nasopharynx and middle ear fluids were indistinguishable following PFGE analysis. The same patterns were identified among 5 children of unrelated families. The restriction fragment patterns of H. influenzae isolated by PFGE were also indistinguishable in 13 out of the 15 pairs (86.7%) of nasopharynx and middle ear fluids. The genetic similarity between nasopharyngeal and middle ear isolates suggests that the causative bacteria migrate from the nasopharynx into the middle ear cavity via the Eustachian tube. Some resistant strains might be prevalent. In children with AOM, the nasopharynx could have been colonized by a virulent strain of bacteria that replaced the benign, commensal bacteria and then progressed to the middle ear, where they caused AOM.

Antimicrobial susceptibilities and analysis of genes related to penicillin or macrolide resistance in Streptococcus pneumoniae

One hundred and seventy-seven strains of Streptococcus pneumoniae derived from respiratory specimens between 1987 and 2001 were evaluated for their antimicrobial susceptibilities and distribution of genes related to penicillin and macrolide resistance. Resistance rates tended to be higher for the 1996–2001 isolates than for the 1987–1995 isolates for all β-lactams tested. For benzylpenicillin the MIC 90 value of the isolates derived between 1996 and 2001 was 1.56 mg/L, while that of strains isolated between 1987 and 1990 was 0.05 mg/L. Furthermore, the number of strains susceptible to macrolides also decreased, but only two strains isolated in 1993 were resistant to levofloxacin of the 177 S. pneumoniae strains tested. When of genes relating to penicillin resistance were analysed using PCR with primers specific to susceptible alleles, although more than 50% of strains from 1987 to 1990 and 1991 to 1995 revealed no mutations in the pbp 1a, 2x and 2b genes, only 30.0% of strains derived between 1996 and 2001 showed no mutations in the pbp gene. Strains having mutations in all three pbp genes ( 1a, 2x and 2b) by the PCR method increased from only 2.2% in the 1987–1990 derived strains to 27.5% in the 1996–2001 strains. Furthermore, 64.1 and 60.0% of the isolates from 1987 to 1990 and 1991 to 1995, respectively, did not possess either the mefA or ermB by PCR analysis. Conversely, 75.0% of isolates from 1996 to 2001 possessed mefA and/or ermB. These genetic changes may explain the increase in the number of penicillin and macrolide resistant strains. We believe that it is important to evaluate changes in MIC as well as genetic mutations in order to select the most appropriate therapy for S. pneumoniae infections.

Genomic analysis of penicillin-resistant Streptococcus pneumoniae in Southeastern Michigan.

The emergence of multidrug resistance within Streptococcus pneumoniae population was analysed, correlating penicillin resistance Pen(R) with secondary antibiotic resistance, capsular serotype, and genetic diversity among isolates. DNA fingerprinting, following macro-restriction enzyme digestion and pulse field gel electrophoresis (PFGE), and restriction fragment analysis of the PBP 2b gene, following PCR amplification, were performed on the Pen(R) S. pneumoniae, among 377 clinical isolates obtained from the clinical microbiology laboratory (University of Michigan Medical Center). Overall 35% of the isolates were Pen(R) of which 45% demonstrated high-level penicillin (Pen(R)-R, MIC>1). Respiratory isolates were more likely to be Pen(R) (p <0.001) than non-respiratory isolates and the rate of Pen(R)-R was significantly increased in children <10 years of age (59.6%, p <0.02). Secondary antibiotic resistance was more frequently associated with Pen(R)-R. Genomic DNA fingerprinting analysis and restriction fragment analysis of the PBP 2b gene demonstrated genomic divergence with discrete conserved pattern in the PBP 2b gene among the resistant isolates. The emergence of multidrug resistance in the S. pneumoniae population in SE Michigan is not due to expansion of a single or limited number of resistant clones, is occurring most frequently in the paediatric population and is associated with a decreased susceptibility to penicillin.

Application of a multiplex pbp2b and pbp2x PCR for prediction of penicillin resistance in Streptococcus pneumoniae

Sir, In Streptococcus pneumoniae, penicillin binding proteins (PBPs) 2b and 2x are the primary targets for penicillin. Hence, they are the first PBPs to be altered following mutations that lead to the development of penicillin resistance. Within the transpeptidase-encoding regions of the pbp2b and pbp2x genes, a few specific nucleotide changes have been shown to be common to penicillin-resistant pneumococci from many parts of the world. Polymerase chain reaction (PCR) primers have been developed to specifically amplify these mutant alleles. The primers are designed to be complementary to the mutant sequence, such that a PCR product is only produced if the mutation is present in the gene. 1,2 However, these previously developed PCR assays are limited to only detecting mutations in one of the pbp genes, and/or they require two or more separate PCR reactions to accommodate tests for all the resistance alleles. In this study, we report the evaluation of a one-tube, multiplex PCR reaction for detection of pbp2b and pbp2x mutations associated with penicillin resistance in S. pneumoniae. The primers used in the reaction are R1 to R4, P5 and P6, specific for pbp2b, and Pn-RX1 and Pn-RX2 for pbp2x. The sequences of these primers have been reported previously. 2,3 Possible PCR products have sizes (in bp) of 183 (primer pair Pn-RX1 and Pn-RX2), 214 (primer pair R4 and P6), 328 (primer pair R2 and P6), 331 (primer pair R1 and P6), 334 (primer pair R3 and P6) and 682 (primer pair P5 and P6—used as a PCR control). To test the multiplex PCR assay, a collection of 383 nasopharyngeal isolates of S. pneumoniae4 obtained from among 1978 children aged less than 6 years attending 79 day care centres and kindergartens in all areas of Hong Kong was tested. The penicillin MICs of the isolates were determined by the Etest method. The 40 µL optimal PCR reaction comprised 2.2 mM MgCl2, dNTP at 200 µM, 1 U of AmpliTaq Gold in 1× PCR buffer (Applied Biosystems, Foster City, CA, USA), primers (P5 and P6 at 1 µM; R1 to R4 at 0.1 µM; PnRx1 and PnRx2 at 0.2 µM) and 4 µL of DNA template (generated by rapid alkaline lysis of cells). PCR was carried out using the following conditions: an initial activation at 95°C for 8 min, then 10 cycles of

Identification of the anginosus group within the genus Streptococcus using polymerase chain reaction

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B ( pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin ( ily) gene of S. intermedius. In the case of Streptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.

Detection of penicillin-binding protein 2b gene alteration in Streptococcus mitis by polymerase chain reaction.

Three isolates of beta-lactam-resistant streptococci from the saliva of healthy adults were identified as Streptococcus mitis. Minimum inhibitory concentrations (MICs) were 2 to 4 micro g/ml for ampicillin (ABPC) and 64 to more than 128 micro g/ml for cefaclor (CCL). To determine the position of base alterations of the penicillin-binding protein 2b ( pbp2b) gene, upstream primers containing possible mutation points were designed, and used for polymerase chain reaction (PCR), together with a downstream primer. Alterations adjacent to the conserved motifs of the pbp2b gene were apparent. DNA sequencing data indicated replacements in deduced amino acid sequences in all resistant isolates: from threonine to alanine just after the serine-serine-asparagine (SSN) motif, and from alanine to glycine two residues downstream of the lysine-threonine-glycine (KTG) motif. These changes were the same as those in penicillin-resistant Streptococcus pneumoniae (PRSP), suggesting importance for the enzymatic activity of the protein. Thus, Beta-lactam susceptibility of S. mitis may be partially predicted by PCR using our primer set for pbp2b.

Penicillin susceptibility and molecular characteristics of clinical isolates of Streptococcus pneumoniae at the University of Malaya Medical Center, Kuala Lumpur, Malaysia

Objective: To determine the prevalence of penicillin resistance and molecular characteristics of pneumococcal isolates at the University of Malaya Medical Center. Methods: From March 1999 to July 2000, 100 clinical isolates of Streptococcus pneumoniae were obtained from 93 patients of various ages and from various body sites. The minimum inhibitory concentrations (MICs) for penicillin and ceftriaxone were determined by E test, and results were interpreted according to guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Fifty isolates were further serotyped, and analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) of the penicillin-binding protein ( pbp) 2b and 2x genes. Results: The majority of the isolates were from respiratory sites. Thirty-one isolates showed decreased susceptibility to penicillin (PRSP), and many of these also showed decreased susceptibility to ceftriaxone. Twelve serogroup/types (SGTs) were present, with 19F being the most common. PFGE analysis identified two dominant profiles, consisting mainly of PRSPs that had common serotypes (19F) and pbp gene patterns within their respective groups, although PCR-RFLP analysis showed different patterns of pbp genes among the PRSPs as compared to penicillin-susceptible strains, which had a uniform pattern. Conclusion: PRSPs were genetically related as shown by PFGE and serotype. The consistency of pbp gene patterns, observed among many of the PRSPs within their respective PFGE profiles, supported their relatedness as established by PFGE.

Phenotypic and genotypic characterization of two penicillin-susceptible serotype 6B Streptococcus pneumoniae clones circulating in Italy.

Twenty-nine penicillin-susceptible serotype 6B strains isolated from patients with invasive diseases and from healthy carriers were examined by different genotyping methods. Ten groups were identified on the basis of the pulsed-field gel electrophoresis (PFGE) profiles, and two of these contained multiple isolates and were analyzed further. PFGE group 1 comprised 12 isolates, the majority of which had a multiresistant phenotype (resistance to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole), corresponding to that of a clone previously described in the Mediterranean area and related to penicillin-resistant clone Spain(6B)-2. The pbp2b, pbp2x, dhf, and pspA genes of the isolates had identical restriction profiles; and the partial sequence of pspA was identical to that of clone Spain(6B)-2. In all isolates the resistance determinants erm(B) and tet(M) were inserted in a Tn1545-like element; 11 isolates carried cat as part of the integrated plasmid pC194. Multilocus sequence typing (MLST) performed with two isolates confirmed that their profiles corresponded to that of the Mediterranean clone. PFGE group 2 comprised nine strains, of which the majority showed no antibiotic resistance. Their pspA profiles were different, and the partial sequences obtained for two representative isolates indicated the presence of PspA proteins of different clades. The MLST profile of one strain was identical to that of a serotype 6B strain from the United Kingdom, while two other isolates were novel one-allele variants. This clone appears to be related (five of seven identical alleles) to two other internationally disseminated clones, Hungary(19A)-6 and Poland(23F)-16, both of which are penicillin resistant. The presence of antibiotic-susceptible isolates of this clone suggests that traits other than antibiotic resistance can make a clone successful.

Two major Spanish clones of penicillin-resistant Streptococcus pneumoniae in Portuguese isolates of clinical origin.

We studied the genetic relatedness of 47 Portuguese penicillin-resistant 9V and 23F Streptococcus pneumoniae of clinical origin, using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism analysis. PFGE fingerprinting showed that 24 isolates of serotype 9V and 23 isolates of serotype 23F were variants of the Spain(9V)-3 and Spain(23F)-1 clones, respectively. Fingerprinting of pbp1a, pbp2b and pbp2x genes showed that all the penicillin-resistant clones gave similar HinfI restriction patterns. In this study, serotypes 9V and 23F have different clonal origins and identical PBP genotypes, suggesting a horizontal transfer of resistance. Visual and computer-assisted analysis of PFGE patterns correlated well (r = 0.983).

In vitro activities of new ketolide, telithromycin, and eight other macrolide antibiotics against Streptococcus pneumoniae having mefA and ermB genes that mediate macrolide resistance

The comparative in vitro activity of a new ketolide, telithromycin (TEL), and eight other macrolide-lincosamide antibiotics (MLS) against 215 strains, of Streptococcus pneumoniae including penicillin-resistant isolates (PRSP), was determined by the agar dilution method. These strains were isolated from patients with pneumonia, otitis media, and purulent meningitis between 1995 and 1997. Two genes, mefA and ermB, that encode MLS resistance in the strains were identified by polymerase chain reaction (PCR). Of the strains, 30.2% (n = 65) had the mefA gene, 37.7% (n = 81) had the ermB gene, and 1.4% (n = 3) had both resistant genes. The minimum inhibitory concentration (MIC90s) of TEL and 16-membered ring MLS for strains having the mefA gene were 0.063-0.25 microg/ml, which were the same level as those for MLS-susceptible strains. On the other hand, the strains with the mefA gene showed low-level resistance to 14- and 15-membered ring MLS, with MIC90s ranging from 1 to 4 microg/ml. Only the MIC90 of TEL at 0.5 microg/ml, for strains having the ermB gene was superior to that of the 14-, 15-, and 16-membered ring MLS (MIC90, > or =64 microg/ml). TEL also showed excellent activity against PRSP having abnormal pbp1a, pbp2x, and pbp2b genes. Most strains having the mefA and ermB genes were serotyped to 3, 6, 14, 19, and 23. These results suggest that TEL may be a useful chemotherapeutic agent for respiratory tract infections caused by S. pneumoniae.

Culture-Negative Childhood Empyema Is Usually Due to Penicillin-Sensitive Streptococcus pneumoniae Capsular Serotype 1

Two studies in the United States have shown that isolates of Streptococcus pneumoniae from children with empyema are more likely to be penicillin sensitive and of capsular serotype 1 than those from uncomplicated pneumococcal pneumonia (1, 4). In the United Kingdom, childhood empyema is becoming more common (S. D. Playfor, A. R. Smyth, and R. J. Stewart, Letter, Thorax 52:932, 1977; J. H. M. Rees, D. A. Spencer, D. Parikh, and P. Weller, Letter, Lancet 349:402, 1997). Many cases have no definitive etiology, despite attempts at noncultural diagnosis by antigen detection, and are most likely due to previous antibiotic therapy. It is uncertain, therefore, if these culture-negative cases of childhood empyema are from the same population as the S. pneumoniae culture-positive ones or whether they are a separate and different problem. We have examined pleural aspirates from 43 children (aged 6 months to 17 years; mean age, 6.2 years; median age, 6 years; 30 of whom were male) presenting with empyema thoracis over a 4-year period to the Freeman Hospital in Newcastle upon Tyne, a tertiary referral center for the North East of England. Routine culture and a latex agglutination test for pneumococcal antigen (SLIDEX; BioMerieux, Marcy l'Etoile, France) were performed with these specimens, after which the remaining material was digested with dithiothreitol. A 1-ml aliquot of the digest was centrifuged at 10,000 × g for 10 min, and the pellet was washed twice in phosphate-buffered saline (PBS). The pellet was resuspended in 12.5% Chelex-100 resin (Bio-Rad Laboratories Ltd.) and heated at 56°C for 30 min and then at 100°C for 15 min. The supernatant was then tested for the presence of S. pneumoniae DNA by a previously described real-time PCR assay targeting the pneumolysin gene (ply) (A. M. Kearns, R. Freeman, O. M. Murphy, P. R. Seiders, M. Steward, and J. Wheeler, Letter, J. Clin. Microbiol. 37:3434, 1999). Positive samples underwent a second real-time PCR assay targeting a conserved sequence of the PBP2B gene (pbp2b) (3) found in all strains of penicillin-susceptible S. pneumoniae (PSSP) for which the MIC of penicillin was <0.06 mg/liter. Finally, whenever sample volumes permitted, 250 μl of the digested sample was mixed with 750 μl of PBS and heated to 100°C for 10 min and then centrifuged for 2 min (21,000 × g). The supernatant was used in solid-phase indirect sandwich enzyme-linked immunosorbent assays (ELISAs) for the detection of type-specific pneumococcal capsular polysaccharides, with monoclonal antibodies to types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F donated by Wyeth Vaccines Research, West Henrietta, N.Y. Two samples were culture positive. One of them, for which all tests for S. pneumoniae were negative, yielded Staphylococcus aureus. The other grew scanty Staphylococcus epidermidis, which was regarded as a skin contaminant. All pbp2b assays indicated “penicillin sensitivity.” Other results are found in Table ​Table11. TABLE 1. Results of real-time PCR assays for pneumococcal DNA and ELISAs for 13 pneumococcal capsular serotype antigens on 43 pleural aspirates from children with empyema We found evidence of S. pneumoniae infection in over 70% of culture-negative empyema fluids. The infecting strains were likely to have been highly penicillin sensitive, and at least 60% were of capsular serotype 1, which, in the United Kingdom, as in the United States, is not the commonest serotype in invasive pneumococcal disease (2). We conclude that cases of culture-negative childhood empyema presenting at tertiary referral centers are not an etiologically distinct group, that childhood pneumococcal vaccines must include capsular serotype 1 antigen, and that early detection of S. pneumoniae capsular serotype 1 antigen in cases of childhood pneumonia may aid management and predict complications, especially empyema.