Genetic diversity of penicillin-binding protein 2B and 2X genes from Streptococcus pneumoniae in South Africa.

Streptococcus pneumoniae (the pneumococcus) is believed to have developed resistance to penicillin by the production of altered forms of penicillin-binding proteins (PBPs) that have decreased affinity for penicillin. Sixty-eight clinical isolates of serogroup 6 and 19 pneumococci (MICs, < 0.015 to 8 micrograms/ml) were randomly selected from hospitals across South Africa which are at substantial geographic distance from each other. The polymerase chain reaction was used to isolate the penicillin-binding domain of PBPs 2B and 2X from the chromosomal DNAs of the bacteria; the purified PBP DNA was digested with restriction enzymes, the fragments were end-labelled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. Fingerprint analysis revealed that at least 19 PBP 2B gene variants occur in the serogroup 6 and 19 pneumococci. The PBP 2B gene revealed a uniform profile among penicillin-susceptible isolates, with variation from this profile occurring only in isolates for which MICs were > or = 0.06 micrograms/ml. Analysis of the PBP 2X gene revealed a greater diversity in the population with 26 variant genes, including some diversity among susceptible isolates. Discrete profiles of both genes were found only within narrow bands of the penicillin MIC, so that the gene pattern predicted the MIC. PBP 2X gene variation and the lack of variability among PBP 2B genes in pneumococci inhibited at low MICs confirm that PBP 2X alteration may be responsible for low-level penicillin resistance, while alterations in both PBP 2B and PBP 2X are required for high-level resistance. The extensive diversity of PBP genes in South African serogroup 6 and 19 strains suggests that altered PBP genes have arisen frequently in this population.     

In Vitro Activity of Sanfetrinem and Affinity for the Penicillin-Binding Proteins of Streptococcus pneumoniae

Against penicillin-susceptible pneumococci, the activity of sanfetrinem was similar to those of penicillin, amoxicillin, cefotaxime, imipenem, and meropenem, while against penicillin-resistant strains, sanfetrinem and the carbapenems exhibited superior activity (MICs at which 90% of strains are inhibited, ≤1 μg/ml). PBP 1a in the penicillin-susceptible strain and PBP 1a and PBP 2b in the more resistant isolates seemed to be the essential penicillin-binding proteins for imipenem and sanfetrinem.     

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

AIMS: 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. METHODS: PFGE and dideoxynucleotide chain termination sequencing were used. RESULTS: 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. CONCLUSIONS: 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.     

Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the beta-lactam binding site

There are several major differences between the penicillin-binding proteins (PBPs) of highly penicillin-resistant and -susceptible strains of pneumococci. The highest-molecular-size PBP 1a (98 kilodaltons [kDa]) of susceptible pneumococci is not detectable in resistant bacteria. Instead, resistant strains contain a PBP of smaller size: 92 and 94 kDa in South African strains 8249 and A95210, respectively, and 96 kDa in New Guinea strain 2955 (S. Zighelboim and A. Tomasz, Antimicrob. Agents Chemother. 17:434-442, 1980). Using antibodies prepared against PBP 1a of penicillin-susceptible pneumococci, we demonstrated that these anomalous-sized proteins in the resistant strains are immunologically related to PBP 1a of penicillin-susceptible bacteria. A second difference between the PBP patterns of strain 8249 and the susceptible pneumococci is that the 78-kDa PBP 2b is not detectable by the radioactive penicillin binding assay in the resistant strain. Using antibodies prepared against PBP 2b of susceptible cells, we demonstrated the presence of PBP 2b in membrane preparations from strain 8249 cells. Thus, the poor detection of this PBP appears to be related to its greatly decreased affinity for the antibiotic molecule. We also compared the patterns of penicillin-labeled peptides derived from PBPs of resistant and susceptible cells during partial proteolysis by V8 protease. Several changes were observable in small peptides carrying the beta-lactam binding site generated from the high Mr (PBP 1a-related) binding proteins. In contrast, no differences in the pattern of penicillin-labeled peptides were seen when the pattern of PBP 2a of susceptible pneumococci was compared with the peptide pattern of PBP 2a from resistant strains. One of the resistant isolates (strain 2955) also had a PBP 3 with a higher-than-normal molecular weight. This protein gave strong positive reaction with antibodies against PBP 3 of susceptible cells. Examination of the pattern of penicilloyl peptides generated from the susceptible and resistant PBP 3s during partial proteolysis revealed only differences which seem to reside distant from the beta-lactam binding site.     

Mutations in ponA, the gene encoding penicillin-binding protein 1, and a novel locus, penC, are required for high-level chromosomally mediated penicillin resistance in Neisseria gonorrhoeae

Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were > or = 1 microg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (k2/K') than wild-type PBP 1 with a variety of beta-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene (ponA1) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC, which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 microg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC, is required along with ponA1 to achieve high-level penicillin resistance.     

First molecular characterization of group B streptococci with reduced penicillin susceptibility

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 microg/ml, 2 to 8 microg/ml, and 4 to 128 microg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of beta-lactam-insusceptible GBS from a phenotypic standpoint.     

Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotypes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillin-susceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotype 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone of S. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.     

Antipneumococcal Activity of Ertapenem (MK-0826) Compared to Those of Other Agents

The activities of ertapenem (MK-0826) and eight other agents against a range of penicillin-susceptible and -resistant pneumococci were tested by determination of MICs by the microdilution method and by the time-kill methodology. For 125 penicillin-susceptible, 74 penicillin-intermediate, and 86 penicillin-resistant pneumococci, the MICs at which 50% of isolates are inhibited (MIC(50)s) and MIC(90)s, as determined by the microdilution method, were as follows: for ertapenem, 0.016 and 0.03, 0.125 and 0.5, and 0.5 and 1.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; for amoxicillin, < or =0.016 and 0.03, 0.25 and 1.0, and 2.0 and 2.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; for cefprozil, 0.125 and 0.25, 1.0 and 8.0, and 16.0 and 16.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; for cefepime, < or =0.016 and 0.06, 0.5 and 1.0, and 1.0 and 2.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; for ceftriaxone, < or =0.016 and 0.06, 0.25 and 1.0, and 1.0 and 2.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; for imipenem, < or =0.008 and < or =0.008, 0.03 and 0.25, and 0.25 and 0.25 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; for meropenem, < or =0.008 and 0.016, 0.125 and 0.5, and 0.5 and 1.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively; and for clarithromycin, 1.0 and >32.0, 1.0 and >32.0, and >32.0 and >32.0 microg/ml for penicillin-susceptible, penicillin-intermediate, and penicillin-resistant pneumococci, respectively. For 64 strains for which quinolone MICs were increased (ciprofloxacin MICs, > or =4.0 microg/ml), the MIC(90) of ertapenem was 1.0 microg/ml and the MIC(90)s of the other beta-lactams tested and clarithromycin were >32.0 microg/ml. Against four penicillin-susceptible, four penicillin-intermediate, and four penicillin-resistant strains, testing by the time-kill methodology showed that ertapenem at two times the MIC was bacteriostatic (99% killing) after 12 h and bactericidal (99.9% killing) against all strains by 24 h, with 90% killing of all strains at two times the MIC after 6 h. At the MIC, ertapenem was bacteriostatic against all strains tested after 24 h. Although the bactericidal activity of imipenem at the MIC after 24 h was significantly greater than that of ertapenem, the kinetics of the two drugs at two times the MIC were similar after 24 h. The killing kinetics of clarithromycin were slower than those of ertapenem and other agents, with clarithromycin at two times the MIC having bactericidal activity against seven of eight macrolide-susceptible strains after 24 h.     

Effects of amino acid alterations in penicillin-binding proteins (PBPs) 1a, 2b, and 2x on PBP affinities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor in 18 clinical isolates of penicillin-susceptible, -intermediate, and -resistant pneumococci

Amino acid alterations in or flanking conserved motifs making up the active binding sites of penicillin-binding proteins (PBPs) 1a, 2b, and 2x of pneumococci were correlated with changes in affinities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor for these PBPs. Four penicillin-susceptible (PSSP), eight penicillin-intermediate (PISP), and six penicillin-resistant (PRSP) pneumococci were studied by DNA sequencing of the penicillin-binding sites of the pbp1a, -2x, and -2b genes of strains and by determining 50% inhibitory concentrations of the seven agents for PBP1a, -2x, and -2b. Two PSSP strains had alterations in PBP2x (L(546)-->V) (one strain) or PBP2b (T(445)-->A) (one strain). All eight PISP strains had at least two alterations--T(338)-->P or A or H(394)-->Y in PBP2X and T(445)-->A in BPB2b. All PRSP strains had the same changes seen in PISP strains, as well as T(371)-->A or S substitutions in PBP1a. The two most resistant PRSP strains had a second change in PBP2x (M(339)-->F) in a conserved motif. The affinities of penicillin and ampicillin for all three PBPs were decreased for PRSP and most PISP strains. The affinity of amoxicillin for PBP1a and -2x was decreased only for PRSP. Cefaclor and cefprozil showed decreased affinity of PRSP but not PISP for all three PBPs. Cefuroxime showed decreased affinity of PISP and PRSP for PBP1a and -2x but no change for PBP2b. Cefditoren showed no difference in PBP affinity based on penicillin or cefditoren MICs, indicating a different PBP target for this agent. Overall, the MICs for and PBP affinities of the strains correlated with the changes found in the PBP active binding sites.     

In Vitro Activity of Cephalosporin RWJ-54428 (MC-02479) against Multidrug-Resistant Gram-Positive Cocci

RWJ-54428 (MC-02479) is a novel cephalosporin that binds to penicillin-binding protein (PBP) PBP 2' (PBP 2a) of methicillin-resistant staphylococci. Its in vitro activity was assessed against 472 gram-positive cocci, largely selected as epidemiologically unrelated isolates with multidrug resistance. The MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90) of RWJ-54428 for methicillin-resistant Staphylococcus aureus (MRSA) were 1 and 2 microg/ml, respectively, whereas they were 0.5 and 0.5 microg/ml, respectively, for methicillin-susceptible S. aureus. The MIC(50) and MIC(90) were 1 and 4 microg/ml, respectively, for methicillin-resistant coagulase-negative staphylococci (MRCoNS), whereas they were 0.25 and 1 microg/ml, respectively, for methicillin-susceptible isolates. The highest MICs for MRSA and MRCoNS isolates were 2 and 4 microg/ml, respectively. The MIC(50) and MIC(90) of RWJ-54428 for Enterococcus faecalis were 0.5 and 1 microg/ml, respectively, but they were 4 and 8 microg/ml, respectively, for Enterococcus faecium. For penicillin-susceptible, -intermediate, and -resistant pneumococci, the MIC(90)s of RWJ-54428 were 0.03, 0.25, and 0.5 microg/ml, respectively, with the highest MIC for a pneumococcus being 1 microg/ml, recorded for a strain for which penicillin and cefotaxime MICs were 8 and 4 microg/ml. MICs for Lancefield group A, B, C, and G streptococci were < or =0.008 microg/ml; those for viridans group streptococci, including isolates not susceptible to penicillin, were from 0.015 to 0.5 microg/ml. RWJ-54428 did not select resistant mutants of MRSA or enterococci in challenge experiments and has the potential to be useful for the treatment of infections caused by gram-positive cocci.     

In vitro activity of cefditoren against clinical isolates of penicillin-susceptible and penicillin-intermediate strains of Streptococcus pneumoniae isolated in Germany, 1992-1998

This study investigates the susceptibility to cefditoren of penicillin-susceptible strains of invasive Streptococcus pneumoniae (n = 312) and of penicillin-intermediate strains of S. pneumoniae (n = 30) isolated mainly from patients with respiratory tract infections. The MIC(90)s of penicillin-susceptible and -intermediate isolates were as follows: cefditoren, < or =0.06 and 1 mg/L; penicillin G, < or =0.06 and 0.5 mg/L. Cefditoren showed the highest activity against the penicillin-intermediate strains investigated compared with the other beta-lactam antibiotics and is therefore considered to be a promising agent for the treatment of infections caused by pneumococci with reduced penicillin susceptibility.     

MIC and time-kill studies of antipneumococcal activity of GV 118819X (sanfetrinem) compared with those of other agents.

Agar dilution MIC methodology was used to test the activities of GV 118819X (sanfetrinem), ampicillin, amoxicillin, amoxicillin-clavulanate, cefpodoxime, loracarbef, levofloxacin, clarithromycin, ceftriaxone, imipenem, and vancomycin against 53 penicillin-susceptible, 84 penicillin-intermediate and 74 penicillin-resistant pneumococci isolated in the United States. GV 118819X was the most active oral beta-lactam, with MIC at which 50% of the isolates were inhibited (MIC50)/MIC90 values of 0.008/0.03, 0.06/0.5, and 0.5/1.0 micrograms/ml against penicillin-susceptible, -intermediate, and -resistant stains, respectively. Amoxicillin and amoxicillin in the presence of clavulanate (2:1) were the second most-active oral beta-lactams, followed by ampicillin and cefpodoxime; loracarbef was not active against penicillin-intermediate and -resistant strains. Clarithromycin was most active against penicillin-susceptible strains but was less active against intermediate and resistant stains. All pneumococcal stains were inhibited by ceftriaxone and imipenem at MICs of < or = 4.0 and < or = 1.0 micrograms/ml, respectively. The activities of levofloxacin and vancomycin were unaffected by penicillin susceptibility. Time-kill studies of three penicillin-susceptible, three penicillin-intermediate, and three penicillin-resistant pneumococci showed that all compounds, at the broth microdilution MIC, yielded 99.9% killing of all strains after 24 h. Kinetic patterns of all oral beta-lactams, ceftriaxone, and vancomycin were similar relative to the MIC, with 90% killing of all strains first observed after 12 h. However, killing by amoxicillin-clavulanate, imipenem, and levofloxacin was slightly faster and that by clarithromycin was slower than that by the above-described drugs. At 2 x the MIC, more strains were killed earlier than was the case at the MIC, but the pattern seen at the MIC prevailed. When MICs and kill kinetics were combined, sanfetrinem was the most active oral antipneumococcal agent in this study.     

Emergence and spread of Neisseria gonorrhoeae clinical isolates harboring mosaic-like structure of penicillin-binding protein 2 in Central Japan

Of 150 clinical isolates of Neisseria gonorrhoeae recovered in 2001, we examined 55 clinical isolates of N. gonorrhoeae for which cefixime MICs were > or=0.125 microg/ml and randomly selected 15 isolates for which cefixime MICs were < or =0.06 microg/ml for analysis of alterations in the penicillin-binding protein 2 (PBP 2) gene. We found insertion of an extra codon (Asp-345a) in the transpeptidase domain of PBP 2, and this insertion occurred alone or in conjunction with other amino acid substitutions. We also found a mosaic PBP 2 that was composed of fragments of the PBP 2 proteins from Neisseria cinera and Neisseria perflava. This mosaic PBP 2 was significantly associated with decreased susceptibilities to penicillin and cephalosporins, especially oral cephalosporins. For most of the isolates with a mosaic PBP 2, the cefixime MICs were > or =0.5 microg/ml and the cefdinir MICs were > or =1 microg/ml. Analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis revealed that most isolates with the mosaic PBP 2 were genetically similar. The recombination events that generated the mosaic PBP 2 would likely have contributed to the decreased sensitivities to cephalosporins. Isolates with the mosaic PBP 2 appear to threaten the efficacy of the currently recommended regimen with cefixime. The emergence of such strains may be the result of the in vivo generation of clones in which interspecies recombination occurred between the penA genes of N. gonorrhoeae and commensal Neisseria species.     

Susceptibilities of 228 penicillin- and erythromycin-susceptible and -resistant pneumococci to RU 64004, a new ketolide, compared with susceptibilities to 16 other agents.

The susceptibilities of 228 penicillin- and erythromycin-susceptible and -resistant pneumococci to RU 64004, a new ketolide, were tested by agar dilution, and the results were compared with those for penicillin G, erythromycin, azithromycin, clarithromycin, rokitamycin, clindamycin, pristinamycin, ciprofloxacin, sparfloxacin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, cefuroxime, ceftriaxone, imipenem, and vancomycin. RU 64004 was very active against all strains tested, with MICs at which 90% of the isolates are inhibited (MIC90s) of 0.016 microg/ml for erythromycin-susceptible strains (MIC, < or = 0.25 microg/ml) and 0.25 microg/ml for erythromycin-resistant strains (MIC, > or = 0.5 microg/ml). All other macrolides had MIC90s of 0.03 to 0.25 and > or = 128 microg/ml for erythromycin-susceptible and -resistant strains, respectively. Among erythromycin-resistant strains, clindamycin MICs for 28 of 91 (30.7%) were < or = 0.125 microg/ml. Pristinamycin MICs for all strains were < or = 1.0 microg/ml. MIC90s of ciprofloxacin and sparfloxacin were 4.0 and 0.25 microg/ml, respectively, and were unaffected by susceptibility to penicillin or erythromycin. Vancomycin and imipenem inhibited all strains at < or = 0.5 and < or = 0.25 microg/ml, respectively. MICs of cefuroxime and cefotaxime rose with those of penicillin G. MICs of trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol were variable but were generally higher for penicillin- and erythromycin-resistant strains. RU 64004 is the first member of the macrolide group which has low MICs for erythromycin-resistant pneumococci.     

MIC and time-kill study of activities of DU-6859a, ciprofloxacin, levofloxacin, sparfloxacin, cefotaxime, imipenem, and vancomycin against nine penicillin-susceptible and -resistant pneumococci.

MIC and time-kill methods were used to test the activities of DU-6859a, ciprofloxacin, levofloxacin, sparfloxacin, cefotaxime, imipenem, and vancomycin against nine penicillin-susceptible, -intermediate, and -resistant pneumococci. The MIC of penicillin for penicillin-susceptible strains was 0.016 micrograms/ml, those for intermediate strains were 0.25 to 1.0 microgram/ml, and those for resistant strains were 2.0 to 4.0 micrograms/ml. Of the four quinolones tested, DU-6859a had the lowest MIC (0.064 micrograms/ml), followed by sparfloxacin (0.25 to 0.5 micrograms/ml) and levofloxacin and ciprofloxacin (both 1.0 to 4.0 micrograms/ml). Vancomycin inhibited all strains at MICs of 0.25 to 0.5 micrograms/ml. The MICs of imipenem and cefotaxime for penicillin-susceptible, -intermediate, and -resistant strains were 0.004 to 0.008, 0.008 to 0.032, and 0.25 micrograms/ml and 0.016, 0.125 to 0.5, and 2.0 micrograms/ml, respectively. DU-6859a was bactericidal at eight times the MICs (0.5 micrograms/ml) for seven of the nine strains after 4 h and bactericidal for all nine strains after 6 h at eight times the MICs and after 12 h at two times the MICs. By comparison, sparfloxacin, the next most active quinolone, was uniformly bactericidal at two times the MICs only after 24 h, with little activity after 2 h. Levofloxacin and ciprofloxacin were bactericidal against all strains after 12 h at eight times the MICs and against all strains at 24 h at four times the MICs. Imipenem was bactericidal against all strains, at concentrations exceeding the MICs, after 24 h. Cefotaxime was also uniformly bactericidal only after 24 h of incubation at two times the MICs. Vancomycin, despite having uniformly low MICs for all strains irrespective of their penicillin susceptibility, was uniformly bactericidal only at two times the MICs after 24 h.     

Molecular characteristics of pbp1a and pbp2b in clinical Streptococcus pneumoniae isolates in Quebec, Canada

OBJECTIVES: To investigate the nature of the amino acid motifs found in penicillin-binding protein (PBP) 2b and PBP1a of penicillin-resistant Streptococcus pneumoniae isolates across Quebec (Canada), and to obtain preliminary information regarding the prevalence of these alterations. METHODS: DNA sequences of pbp2b (codons 210-675) and pbp1a (codons 310-682) transpeptidase domains were determined and compared in 48 clinical isolates comprising 17 penicillin-susceptible (PSSP), 19 penicillin-intermediate (PISP) and 12 penicillin-resistant (PRSP) pneumococci. RESULTS: The degree of diversity within PBP1a and PBP2b correlated with increased resistance to beta-lactam antibiotics. There were an average of 0.6 +/- 0.4 and 2.9 +/- 0.2 mutations in PSSP, 16.8 +/- 1.4 and 36.3 +/- 5.2 in PISP, and 18.7 +/- 2.5 and 51.4 +/- 1.3 in PRSP isolates compared with control penicillin-susceptible R6-PBP2b and R6-PBP1a sequences, respectively. At least seven PBP2b and six PBP1a distinct amino acid profiles were identified among intermediate or resistant strains isolated in Quebec. The pattern of distribution of the PBPs' altered amino acids differs from that of other countries, with pneumococci isolates from Quebec showing a unique genetic signature. CONCLUSION: This study will serve as a basis for future monitoring of genetic changes associated with the emergence and spread of beta-lactam resistance in Quebec, Canada.     

An internationally spread clone of Streptococcus pneumoniae evolves from low-level to higher-level penicillin resistance by uptake of penicillin-binding protein gene fragments from nonencapsulated pneumococci

Low-level penicillin resistance in an international Streptococcus pneumoniae serotype 19F clone emerging in Switzerland was characterized by mutations in the penicillin-binding protein PBP2x. Some isolates of this clone had evolved to higher resistance levels (penicillin MICs of 0.094 and 1 microg/ml), probably by acquisition of pbp2x fragments from local nonencapsulated pneumococci.     

Antimicrobial susceptibility and penicillin-binding protein 1 and 2 mutations in Neisseria gonorrhoeae isolated from male urethritis in Sapporo, Japan

The spread of antimicrobial-resistant Neisseria gonorrhoeae worldwide is a critical issue in the control of sexually transmitted infections. The purpose of this study was to clarify recent trends in the susceptibility of N. gonorrhoeae to various antimicrobial agents and to compare these data with our previous data. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined in N. gonorrhoeae strains clinically isolated from male gonococcal urethritis. In addition, amino acid sequencing of penicillin-binding protein (PBP) 2, encoded by the penA gene, was analyzed so that genetic analysis of mosaic PBP 2 could clarify the susceptibility of the strains to cefixime and other cephalosporins. The susceptibility rate for ceftriaxone, cefodizime, and spectinomycin, agents whose use is recommended by the guideline of the Japanese Society of Sexually Transmitted Infections (JSSTI), was 100 %. The susceptibility rates of the strains to penicillin G and ciprofloxacin were lower than those in previous reports. Mosaic PBP 2 structures were detected in 51.9 % of the strains and the MICs of the strains with the mosaic PBP 2 to cefixime were much higher than those of the strains without the mosaic PBP 2. In the clinical situation, the treatment regimen recommended by the JSSTI remains appropriate; however, the susceptibility to cephalosporins should be intensively surveyed because strains with mosaic PBP 2 were commonly detected.     

Study of comparative antipneumococcal activities of penicillin G, RP 59500, erythromycin, sparfloxacin, ciprofloxacin, and vancomycin by using time-kill methodology.

Time-kill studies were used to examine the in vitro activities of penicillin G, RP 59500, erythromycin, ciprofloxacin, sparfloxacin, and vancomycin against 10 pneumococci expressing various degrees of susceptibility to penicillin and erythromycin. RP 59500 MICs for all strains were 0.5 to 2.0 micrograms/ml, while erythromycin MICs were 0.008 to 0.06 microgram/ml for erythromycin-susceptible strains and 32.0 to 64.0 micrograms/ml for erythromycin-resistant strains. Strains were more susceptible to sparfloxacin (0.125 to 0.5 microgram/ml) than to ciprofloxacin (0.5 to 4.0 micrograms/ml), and all were inhibited by vancomycin at MICs of 0.25 to 0.5 microgram/ml. Time-kill studies showed that antibiotic concentrations greater than the MIC were bactericidal for each strain, with the following exceptions. Erythromycin was bactericidal for one penicillin-resistant strain at 6 h, with regrowth after 12 and 24 h. Three penicillin-susceptible strains were bacteriostatically inhibited by erythromycin at concentrations greater than or equal to the MIC by 6 h. One penicillin-susceptible strain (penicillin MIC, 0.06 microgram/ml) was bacteriostatically inhibited by penicillin G at 24 h at the MIC or at one-half the MIC; a bactericidal effect was found only with penicillin G at concentrations of > or = 0.25 microgram/ml. At 10 min after inoculation a 1- to 3-log10-unit reduction (90 to 99.9%) in the original inoculum was seen for 6 of 10 strains with RP 59500 at concentrations greater than or equal to the MIC. This effect was not found with any of the other compounds tested. A bactericidal effect was found at > or = 6 h with RP 59500 at concentrations of one-half to one-quarter the MIC in 7 of 10 strains, and a bacteriostatic effect was found in 3 or 10 strains, with regrowth at 24 h. One penicillin-resistant strain was examined by the time-kill methodology at 0, 1, 2, and 3 h. RP 59500 at a concentration equal to the MIC was bactericidal within 1 h, and at a concentration of one-half the MIC it was bactericidal within 3 h. This phenomenon was not seen with the other antimicrobial agents tested. Regrowth of strains at ciprofloxacin concentrations equal to the MIC or at a one-half to one-quarter the MIC was found. For sparfloxacin, three of the four penicillin-susceptible strains and two of four penicillin-resistant strains were bacteriostatically inhibited by 6 h. Bactericidal effects were found at 6, 12, and 24 h with both intermediate-resistant, one penicillin-susceptible, and two penicillin-resistant strains. Complete killing was observed with vancomycin at concentrations greater than MIC. Of the new compounds tested, RP 59500 and sparfloxacin show promise for the treatment of infections caused by penicillin-susceptible and -resistant pneumococci. The clinical significance of rapid killing by RP 59500 remains to be determined.