Clinical Pneumococcal Isolates Research Articles

Clinical isolates of Streptococcus pneumoniae that exhibit tolerance of vancomycin.

The ability of Streptococcus pneumoniae to escape lysis and killing by vancomycin, a property termed "tolerance," has recently been noted in a laboratory strain of the species. Vancomycin tolerance in clinical isolates represents a potential new health risk. We determined the prevalence of vancomycin and penicillin tolerance among 116 clinical isolates of pneumococci by monitoring lysis and viability after exposure to the respective antibiotic for 4 hours. Eight percent of the strains were tolerant to penicillin and 3% were tolerant to vancomycin. The 3 vancomycin-tolerant isolates also had a high ratio of minimum bactericidal concentration to minimum inhibitory concentration, in contrast to nontolerant strains. They were of serotype 9V and had reduced susceptibility to penicillin. Only 1 was also tolerant to penicillin. Growth rate and ability to divide were not affected in the 3 vancomycin-tolerant strains, and they all lysed with deoxycholate, which indicates autolysin production. Vancomycin tolerance among clinical isolates of pneumococci will necessitate tracking to determine the magnitude of the evolving health risk, since tolerance may contribute to treatment failure (in particular, cases of meningitis, in which bactericidal activity is critical for eradication) and since it may also be a favored background for acquisition of resistance of vancomycin.

In vitro Activity of Gemifloxacin against Streptococcus pneumoniae Isolates in Germany

The MICs (minimal inhibitory concentrations) of gemifloxacin, moxifloxacin, ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, penicillin G, tetracycline and erythromycin were determined for 200 clinical pneumococcal isolates using the microbroth dilution method. MIC₅₀ and MIC₉₀ values were as follows (mg/l): 0.016 and 0.016 (gemifloxacin, penicillin-intermediate strains), 0.03 and 0.06 (gemifloxacin, penicillin-susceptible strains); 0.125–0.5 and 0.25 (moxifloxcacin); 1–2 and 4 (ciprofloxacin and ofloxacin); 1 and 2 (levofloxacin), and 0.25–0.5 and 0.5 (sparfloxacin). MIC₅₀ and MIC₉₀ values for the fluoroquinolones were nearly identical for individual antimicrobial agents in the penicillin-susceptible and penicillin-intermediate groups of strains. Erythromycin and tetracycline were less active against penicillin-intermediate pneumococci isolates (erythromycin: MIC₉₀ 8 mg/l; MIC range 0.06–≧64 mg/l; tetracycline: MIC₉₀ ≧64 mg/l, MIC range 0.25– ≧64 mg/l) as compared to penicillin-susceptible isolates (erythromycin: MIC₉₀ 0.5 mg/l, MIC range 0.06–≧64 mg/l; tetracycline: MIC₉₀ 8 mg/l, MIC range 0.06–≧64 mg/l).

Mechanisms of macrolide resistance in clinical pneumococcal isolates in France.

The genetic basis of macrolide resistance was investigated in a collection of 48 genotypically unrelated clinical isolates of Streptococcus pneumoniae obtained between 1987 and 1997 in France. All strains were resistant to erythromycin, clindamycin, and streptogramin B, exhibiting a macrolide-lincosamide-streptogramin B resistance phenotype, and harbored the erm(B) gene. None of the strains carried the mef(A) or erm(A) subclass erm(TR) gene.

MM1, a temperate bacteriophage of the type 23F Spanish/USA multiresistant epidemic clone of Streptococcus pneumoniae: structural analysis of the site-specific integration system.

We have characterized a temperate phage (MM1) from a clinical isolate of the multiply antibiotic-resistant Spanish/American 23F Streptococcus pneumoniae clone (Spain(23F)-1 strain). The 40-kb double-stranded genome of MM1 has been isolated as a DNA-protein complex. The use of MM1 DNA as a probe revealed that the phage genome is integrated in the host chromosome. The host and phage attachment sites, attB and attP, respectively, have been determined. Nucleotide sequencing of the attachment sites identified a 15-bp core site (5'-TTATAATTCATCCGC-3') that has not been found in any bacterial genome described so far. Sequence information revealed the presence of an integrase gene (int), which represents the first identification of an integrase in the pneumococcal system. A 1.5-kb DNA fragment embracing attP and the int gene contained all of the genetic information needed for stable integration of a nonreplicative plasmid into the attB site of a pneumococcal strain. This vector will facilitate the introduction of foreign genes into the pneumococcal chromosome. Interestingly, DNAs highly similar to that of MM1 have been detected in several clinical pneumococcal isolates of different capsular types, suggesting a widespread distribution of these phages in relevant pathogenic strains.

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

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

Novel purification scheme and functions for a C3-binding protein from Streptococcus pneumoniae.

To isolate microbial proteins capable of binding the third component of complement (C3), we coupled the free sulfhydryl group of methylamine-inactivated C3 to a thiolSepharose matrix. This simple technique facilitated the purification of the first C3-binding protein isolated from a bacterium (Streptococcus pneumoniae). Both metastable (native) and thioester-disrupted C3 were recognized by this protein; binding of C3 was noncovalent, independent of thioester conformation, and preferential for the C3 alpha-chain. Sequencing of amino-terminal and internal peptides from the C3-binding protein disclosed a proline-rich region spanning approximately 20 amino acids and a signal peptide that had not been previously reported. The gene was isolated from a library of genomic DNA from laboratory strain CP1200 by screening with a 1200 bp PCR product amplified from degenerate oligonucleotides encoding the amino terminal sequence and the internal proline-rich sequence. The open reading frame spanned 1692 bp; all peptide sequences were identified in the translated gene product, which also contained at least three choline-binding repeats at the carboxy-terminus. The gene was conserved, and the translated protein was functionally active in pneumococcal clinical isolates of serotypes 1, 3, 4, 14, and 19F. Serum from a patient recovering from acute pneumococcal infection contained IgG antibodies specific for this protein by immunoblot. Wide conservation among clinical isolates, saturable binding of C3, and the ability to stimulate the human immune response have not previously been reported for this choline-binding protein. A similar biochemical approach should enable the identification of other C3-binding proteins in microorganisms able to elude complement-mediated host defense.

Assessment of the Susceptibility of Streptococcus pneumoniae to Cefaclor and Loracarbef in 13 Countries

Between July 1998 and July 1999, 2,644 clinical isolates of Streptococcus pneumoniae were collected from 27 study centers in 13 countries and their susceptibilities to penicillin, cefaclor and loracarbef were determined by E-test” (AB BIODISK, Solna, Sweden). Overall, 96.3% of isolates were penicillin-susceptible (79.8%) or -intermediate (16.6%) (MIC, <1 μg/ml). Rates of penicillin-resistant S. pneumoniae isolation varied widely and were highest in the study centers tested in New Zealand (10.9%), Canada (10.0%), Mexico (9.1%) and the United States (5.1%). Low rates of penicillin-resistance were found in the study centers tested in Russia (0%), Turkey (0%), Brazil (0.5%), Germany (0.6%), Philippines (1.6%), Italy (2.1%), United Kingdom (2.3%), Australia (3.0%) and Poland (3.1%). Using recently published NCCLS interpretative breakpoints (M100-S10, 2000), 87.2% (median) of all isolates tested were cefaclor-susceptible and 87.8% (median) of all isolates tested were loracarbef-susceptible. Of the penicillin-susceptible S. pneumoniae isolates, 99.5% were susceptible to both cefaclor and loracarbef. Susceptibility to cefaclor and loracarbef was also retained by 30.8% and 32.9% of penicillin-intermediate isolates, respectively. These findings are in contrast to recent publications reporting lower cefaclor and loracarbef activities using non-validated interpretative criteria. In conclusion, rates of penicillin resistance among recent clinical isolates of pneumococci remain low in many centers worldwide. Cefaclor and loracarbef demonstrated excellent in vitro activity against recent clinical isolates of penicillin-susceptible and many isolates of penicillin-intermediate S. pneumoniae.

Prevalence of resistance to MLS antibiotics in 20 European university hospitals participating in the European SENTRY surveillance programme. Sentry Participants Group.

Macrolide, lincosamide and streptogramin (MLS) antibiotics are chemically distinct inhibitors of bacterial protein synthesis. Resistance to MLS antibiotics may be constitutive or inducible. The purpose of this study is to update our understanding of the prevalence of different forms of MLS resistance in Europe. The analysis of 3653 clinical pneumococcal, staphylococcal and enterococcal isolates exhibited an average percentage of 21.3% and 6.2% intermediate and high-level penicillin-resistant Streptococcus pneumoniae, 21.8% methicillin-resistant Staphylococcus aureus and 11% vancomycin-resistant Enterococcus faecium. Geographical differences in erythromycin and clindamycin resistance in isolates of S. pneumoniae and S. aureus strongly reflect geographical variations in susceptibility to penicillin and methicillin, respectively. A very narrow range of MICs was obtained with quinupristin/dalfopristin, with no S. pneumoniae, S. aureus and E. faecium isolate having an MIC of > 4 mg/L, indicating a possible role of quinupristin/dalfopristin in the treatment of infections by multi-resistant Gram-positive bacteria.

Sparfloxacin resistance in clinical isolates of Streptococcus pneumoniae: involvement of multiple mutations in gyrA and parC genes.

Antimicrobial susceptibility testing revealed among 150 clinical isolates of Streptococcus pneumoniae 4 pneumococcal isolates with resistance to fluoroquinolones (MIC of ciprofloxacin, >/=32 microgram/ml; MIC of sparfloxacin, >/=16 microgram/ml). Gene amplification and sequencing analysis of gyrA and parC revealed nucleotide changes leading to amino acid substitutions in both GyrA and ParC of all four fluoroquinolone-resistant isolates. In the case of strains 182 and 674 for which sparfloxacin MICs were 16 and 64 microgram/ml, respectively, nucleotide changes were detected at codon 81 in gyrA and codon 79 in parC; these changes led to an Ser-->Phe substitution in GyrA and an Ser-->Phe substitution in ParC. Strains 354 and 252, for which sparfloxacin MICs were 128 microgram/ml, revealed multiple mutations in both gyrA and parC. These strains exhibited nucleotide changes at codon 85 leading to a Glu-->Lys substitution in GyrA, in addition to Ser-79-->Tyr and Lys-137-->Asn substitutions in ParC. Moreover, strain 252 showed additional nucleotide changes at codon 93, which led to a Trp-->Arg substitution in GyrA. These results suggest that sparfloxacin resistance could be due to the multiple mutations in GyrA and ParC. However, it is possible that other yet unidentified mutations may also be involved in the high-level resistance to fluoroquinolones in S. pneumoniae.

Killing kinetics of meropenem against penicillin-resistant pneumococci.

The killing kinetics of meropenem against sixteen clinical isolates of pneumococci (12 penicillin-resistant, three penicillin-intermediate and one penicillin-sensitive) were studied. Meropenem was tested at 1x, 2x and 4x the MIC for each individual isolate. The results showed a good bactericidal activity with rapid killing of pneumococci (killing > 3 log10 cfu/mL was obtained for nine strains). This strategy needs clinical assessment and might prove to be a suitable alternative to penicillin and cephalosporins for the treatment of mixed infections involving multiresistant pneumococci.

Development of interpretive criteria and quality control limits for broth microdilution and disk diffusion antimicrobial susceptibility testing of Streptococcus pneumoniae.

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.

Carboxy-terminal deletion analysis of the major pneumococcal autolysin.

Autolysins are endogenous enzymes that specifically degrade the covalent bonds of the cell walls and eventually can induce bacterial lysis. One of the best-characterized autolysins, the major pneumococcal LytA amidase, has evolved by the fusion of two domains, the N-terminal catalytic domain and the C-terminal domain responsible for the binding to cell walls. The precise biochemical role played by the six repeat units that form the C-terminal domain of the LytA amidase has been investigated by producing serial deletions. Biochemical analyses of the truncated mutants revealed that the LytA amidase must contain at least four units to efficiently recognize the choline residues of pneumococcal cell walls. The loss of an additional unit dramatically reduces its hydrolytic activity as well as the binding affinity, suggesting that the catalytic efficiency of this enzyme can be considerably improved by keeping the protein attached to the cell wall substrate. Truncated proteins lacking one or two repeat units were more sensitive to the inhibition by free choline than the wild-type enzyme, whereas the N-terminal catalytic domain was insensitive to this inhibition. In addition, the truncated proteins were inhibited by deoxycholate (DOC), and the expression of a LytA amidase lacking the last 11 amino acids in Streptococcus pneumoniae M31, a strain having a deletion in the lytA gene, conferred to the cells an atypical phenotype (Lyt+ DOC-) (cells autolysed at the end of the stationary phase but were not sensitive to lysis induced by DOC), which has been previously observed in some clinical isolates of pneumococci. Our results are in agreement with the existence of several choline-binding sites and suggest that the stepwise acquisition of the repeat units and the tail could be considered an evolutionary advantage for the enzyme, since the presence of these motifs increases its hydrolytic activity.

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

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

Infections due to Penicillin-Resistant Pneumococci

Pneumococcal infection remains a common cause of serious morbidity and mortality throughout the world. Until recently, clinical isolates of pneumococci that were penicillin resistant were rare. However, 4% to 5% of the clinical isolates in the United States were recently found to be either intermediately resistant or highly resistant to penicillin. Clinicians in every field of medicine must therefore be better informed regarding penicillin-resistant pneumococcal infections to minimize their attendant morbidity and mortality and increase compliance with preventive measures. We reviewed the molecular, genetic, and epidemiologic aspects of penicillin-resistant pneumococcal infections, with emphasis on their microbiologic and clinical features.

Penicillin-binding protein families: evidence for the clonal nature of penicillin resistance in clinical isolates of pneumococci.

In view of the worldwide emergence of penicillin-resistant pneumococci among clinical isolates it was of importance to examine a large number of strains to test the uniformity of the resistance mechanism. Among 160 clinical isolates of pneumococci (minimum inhibitory concentration [MIC], 0.005-16 micrograms/mL), susceptible strains showed a common pattern of five penicillin-binding proteins (PBPs) with high penicillin affinities (PBP 3 greater than 1A greater than or equal to 2A greater than 1B greater than 2B). PBPs 1A, 2A, and 2B (but not PBP 3) each showed distinct stepwise decreases in penicillin affinities parallel with increasing levels of antibiotic resistance. The number and molecular sizes of PBPs became variable in strains with MIC values greater than 1.0 microgram/mL; among 39 strains with a MIC of greater than or equal to 1.0 microgram/mL, 11 distinct and stable PBP patterns could be identified. Using PBP profiles, serotypes, and antibiotic resistance patterns, as well as data on isolation dates and sites, we identified at least three groups of resistant strains that showed clear indication of clonal origin.

Penicillin resistance and defective lysis in clinical isolates of pneumococci: evidence for two kinds of antibiotic pressure operating in the clinical environment.

Seventy percent of clinical isolates of penicillin-resistant pneumococci also exhibit defective lysis when treated with penicillin exceeding the minimal inhibitory concentration (MIC). To provide a possible explanation for the frequent association of these two traits, we exposed penicillin-susceptible pneumococci to two kinds of antibiotic pressures in the laboratory. Treatment of cultures with cycles of high concentrations of penicillin (20 X MIC) followed by growth of the survivors in drug-free medium selected for lysis-defective mutants that died only slowly during antibiotic treatment but had unchanged MICs. Exposure to sustained, low levels of penicillin produced resistant mutants, with elevated MICs, that lysed normally with penicillin. We suggest that the cyclic antibiotic exposure generally used in the clinical setting may select primarily for enhanced survival. From these survivors a second type of antibiotic exposure--sustained antibiotic concentrations just above the MIC (concentrations that may be restricted to the tail-end trough of a dosing interval)--selects for penicillin-resistant mutants.

Alterations in penicillin-binding proteins of clinical and laboratory isolates of pathogenic Streptococcus pneumoniae with low levels of penicillin resistance.

Several recent surveys of clinical isolates have indicated that substantial fractions of naturally occurring populations of Streptococcus pneumoniae have undergone a distinct upward move in the required minimal inhibitory concentration (MIC) of benzylpenicillin (from a range of 0.006-0.008 to 0.03-0.05 microgram/ml). Evidence is presented that in clinical pneumococcal isolates, penicillin-binding proteins (PBPs) groups 1 and 2 have a decreased affinity for radioactive benzylpenicillin as compared with penicillin-sensitive isolates from the same locale. Exposure of a penicillin-sensitive type 2 strain (MIC, 0.006 microgram/ml) to sequentially increasing concentrations of penicillin allowed the isolation of spontaneous resistant mutants with stepwise increases in the MIC of penicillin required (0.01-0.02, 0.025-0.05, and 0.1 microgram/ml), and in these laboratory isolates too, PBP groups 1 and 2 showed decreased affinity for labeled benzylpenicillin. DNA from the low-level resistant clinical or laboratory isolates could be used to transform the appropriate levels of penicillin resistance into penicillin-sensitive laboratory isolates. These findings suggest that significant fractions of natural pneumococcal populations may have acquired one or two of the low-level penicillin resistance genes.