Levels Of Penicillin Resistance Research Articles

Nasopharyngeal carriage and antimicrobial resistance in isolates of Streptococcus pneumoniae and Haemophilus influenzae type b in children under 5 years of age in Botswana

Objectives: A prospective survey was conducted to determine the prevalence of asymptomatic nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae type b in children under 5 years of age in Botswana and to determine the antibiotic resistance patterns of these organisms to commonly used antimicrobial agents. Methods: Children 2 months to 5 years of age (n = 249) were recruited from outpatient clinics in Gaborone and Francistown, and 29 were sampled from the pediatric wards at Princess Marina (Gaborone) and Nyangabgwe (Francistown) Hospitals. Nasopharyngeal specimens were collected and the carriage and antibiotic resistance of S. pneumoniae and H. influenzae type b were determined. Analyses of risk factors associated with carriage and resistance were performed. Results: Streptococcus pneumoniae was isolated from 69% of the outpatient children in Gaborone and 85% of the children in Francistown; the carriage rate in hospitalized children was 36% and 33% in Gaborone and Francistown, respectively. Approximately half of the isolates at both sites were resistant to at least one antibiotic, the most common being cotrimoxazole and penicillin. Resistance to three or more antibiotics (multiple resistance) was found in less than 10% of the isolates. Most penicillin resistance at both sites was at the intermediate level; however, almost 20% of the isolates demonstrated high-level resistance to cotrimoxazole. The most prevalent serogroups or serotypes of antibiotic-resistant isolates were 14, 19F, 19A, 6A, 6B, and 4. No risk factors for antibiotic resistance were identified. Haemophilus influenzae type b was isolated from 8% of the children in Gaborone and from 3% of the children in Francistown. Almost a third of the isolates were resistant to ampicillin. Conclusions: The high levels of antibiotic resistance in pneumococci isolated from children in Botswana suggest that the clinical management of meningitis and ottis media with a β-lactam antibiotic may fail in a significant proportion of cases and that empiric first-line use of cefotaxime or ceftriaxone for meningitis and higher dose amoxicillin (90 mg/kg/day) for otitis media is recommended. The levels of penicillin resistance in this study would not impact on the management of pneumonia with amoxicillin.

Epidemiology, Laboratory Detection, and Therapy of Penicillin-resistant Streptococcal Infections

Streptococci cause a wide range of infections in humans including respiratory tract infections, endocarditis, meningitis, bacteremias, and skin and soft tissue lesions. Mutations in the penicillin binding proteins target sites in these organisms have recently caused resistance to penicillins and cephalosporins. The passage of resistant genetic material from one streptococcal species to another has been recognized as one of the mechanisms by which this resistance has occurred and spread. Such resistance has been a particular problem in Streptococcus pneumoniae and viridans group streptococci with penicillin resistance levels in excess of 25%, now common in both groups of organisms worldwide. Fourth-generation cephalosporins, with their enhanced antibacterial activity against Gram-positive organisms (cefpirome > cefepime) and their increased stability to the β-lactamases produced by many bacterial species, offer a new option for the treatment of potentially life-threatening infections such as pneumococcal pneumonia and meningitis with or without bacteremia. Clinical trials are currently in place to evaluate the role of these agents in these, and other, indications of Gram-positive infections. Prior studies of cefpirome therapy for infections caused by Streptococcus spp. were successful, and recent expanded in vitro investigations profess a future for expanded use of cefpirome to treat infections produced by several Gram-positive species.

Epidemiology of penicillin resistance in Streptococcus pneumoniae isolates in eastern France

OBJECTIVE: To assess the rates of intermediate and high-level resistance to penicillin among Streptococcus pneumoniae isolates and to identify clonal relationship of isolates within the different serotypes by means of pulsed-field gel electrophoresis. METHODS: We studied all clinical isolates obtained between April 1995 and March 1996 from patients admitted to 10 hospitals in eastern France. Antibiotic susceptibility testing and serotyping were performed on all isolates. The genetic polymorphism of isolates susceptible, intermediately resistant and highly resistant to penicillin was studied by using pulsed-field gel electrophoresis with ApaI and SmaI endonucleases. RESULTS: The prevalence of intermediate and high-level resistance was respectively 30.3% and 9.7%. Diminished sensitivity to penicillin was mainly encountered in serotypes 6, 9V, 14 and 23F. The 9V isolates from the different hospitals were genetically closely related, unlike the 23F isolates. Different levels of resistance (MICs from 0.5 to 2 mg/L) were expressed by closely related isolates. Three 9V isolates, three capsular-type 14 isolates and one non-typeable isolate were genetically closely related in studies with the two endonucleases. CONCLUSIONS: The capsular type was not a good indicator of genetic relatedness. The level of penicillin resistance was independent of the clonal classification. Horizontal gene transfer may be the main factor determining the degree of resistance.

Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries: an overview. PAHO/Rockefeller University Workshop. Pan American Health Organization.

The Pan American Health Organization (PAHO) has conducted a study of Streptococcus pneumoniae in six Latin-American countries: Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay. Sterile site isolates from children aged < or =5 years showing clinical symptoms of pneumonia (as defined by the clinical criteria of WHO), meningitis, sepsis or bacteremia (without infectious foci), arthritis, and peritonitis were the source of most of the invasive pneumococcal isolates collected between the end of 1993 and 1996 in the six participating countries. Partial characterization of these isolates (antibiotic resistance and serotyping) have already been described (Microbial Drug Resistance 3:(2):131-163, 1997). In the next phase of the study, 326 S. pneumoniae isolates with reduced penicillin susceptibility were transferred to the Laboratory of Microbiology at The Rockefeller University for molecular characterization, and a summary and overview of the findings is described in this article. Some of the most interesting findings were as follows: (1) There was a surprisingly high representation of two internationally spread clones, which made up >80% of the strains with penicillin MIC of 1 microg/ml or higher; most of these isolates were recovered in large cities, supporting the likelihood that the source of these clones is through international travel. (2) The frequency of resistance to trimethoprim/sulfamethoxazole was extremely high (present in 85% of all isolates with decreased penicillin susceptibility). (3) None of these isolates was resistant to ofloxacin, and macrolide resistance was rare (present in 6.4% of the isolates). (4) There was an apparent inverse relationship between level of penicillin resistance and genetic diversity. (5) There were striking differences in the "microbiologic profiles" of the six different Latin-American countries.

Comparative activity of twelve beta-lactam drugs tested against penicillin-resistant Streptococcus pneumoniae from five medical centers: Effects of serum protein and capsular material on potency and spectrum as measured by reference tests

A total of 152 strains of Streptococcus pneumoniae from diverse geographic areas in the United States and with different levels of penicillin resistance were tested against five broad-spectrum cephalosporins, ampicillin, piperacillin, ticarcillin, and three beta-lactamase inhibitor combinations. Also, the effect of human serum proteins on the activity of selected “third-generation” cephalosporins was examined. The overall rank order of activity among the cephalosporins against penicillin-susceptible strains was: ceftriaxone (MIC 90, 0.03 μg/mL) > cefotaxime > ceftizoxime = cefuroxime > ceftazidime (MIC 90, 0.5 μg/mL). Only cefotaxime and ceftriaxone exhibited significant activity against penicillin-intermediate or -resistant isolates. Ampicillin, piperacillin, and penicillin were generally eight- to 16-fold more potent than ticarcillin and no increase in the effectiveness of these agents was observed with the addition of the beta-lactamase inhibitors (clavulanate, sulbactam, tazobactam). Ceftriaxone potency was significantly decreased (⩾four-fold) by the modest addition of 25% pooled human serum proteins and this change modified the rank order of potency against nonpenicillin-susceptible pneumococci to favor cefotaxime (41% resistant versus 71% for ceftriaxone; MICs at ⩾2 μg/mL). Induced high-level capsular production had no measurable effect on the MIC results of tested agents. These results confirm the continued activity advantages of cefotaxime and ceftriaxone against various populations of pneumococci compared to other alternative beta-lactams. The predictive value, however, of the utilized breakpoint concentrations of the cephalosporins, remains in question for pneumococcal infections other than those in the central nervous system and at unaltered, “usual” dosing.

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae

Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 μg benzylpenicillin ml −1. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae.

Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 micrograms benzylpenicillin ml-1. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.

Molecular epidemiology of penicillin-resistant pneumococci isolated in Nairobi, Kenya.

A total of 26% of the pneumococci isolated from an outpatient clinic in Nairobi, Kenya, during 1991 to 1992 had intermediate levels of penicillin resistance. Gene fingerprinting and DNA sequencing were used to distinguish the penicillin-binding protein (PBP) 1A, 2B, and 2X genes in 23 resistant isolates. Isolates were grouped into those that had identical forms of each of the three PBP genes (fingerprint groups) and those that had identical rRNA gene restriction patterns (ribotypes). Both methods divided the isolates into 11 groups. In a few cases, horizontal gene transfer appeared to have distributed an identical altered PBP gene into different pneumococcal lineages. Eight isolates were indistinguishable by ribotyping or multilocus enzyme electrophoresis and contained identical PBP 1A genes. Although these isolates were therefore members of the same clone, they were divided into two fingerprint groups which contained different PBP 2X and 2B genes. Presumably, members of this clone have acquired different altered PBP 2X and 2B genes on two separate occasions. One of these fingerprint groups contained isolates of serotype 14, whereas the other contained isolates of both serotypes 14 and 7. The identification of isolates in the latter group that are identical by all criteria, except serotype, implies the occurrence of a change in serotype. The predominant serotypes of the penicillin-resistant pneumococci from Nairobi were serotypes 14 and 19. In both cases, isolates of the same serotype which required the same MIC of penicillin were not members of a single clone, indicating that identity of serotype and MIC are not sufficient criteria for defining clones of resistant pneumococci even when the bacteria are isolated from a single clinic.

Epidemiological studies of penicillin resistance in Danish Staphylococcus aureus strains in the period 1977-1990.

During the period 1977-1990 the frequency of penicillin resistance increased from 78.7 to 87.5% among a total of 278,193 Danish Staphylococcus aureus strains from hospitalized patients. By combining these data with phage-type patterns the increase was shown to be caused by 1) the introduction and spread of mainly resistant strains of type 95 and the 94,96 complex; 2) an increased occurrence of strains of group II which during the observation period became more frequently penicillin resistant; and 3) a gradual disappearance of strains of group III and the 83A complex which showed a decreasing degree of penicillin resistance. During the observation period community acquired strains reached nearly the same level of penicillin resistance as the hospital-acquired strains, and the frequency of penicillin resistance did not increase during hospitalization among the predominant strains of type 95, group II and the 94,96 complex. S. aureus isolated from airways had a higher frequency of penicillin resistance mainly caused by increasing amount of penicillin resistant strains of group II. Strains from urine had a lower frequency of penicillin resistance. The total antibiotic consumption and the usage of beta-lactam antibiotics remained nearly unchanged during the observation period. The frequent use of beta-lactam antibiotics for airway infections might explain the possible selection of penicillin resistant strains of group II.

Insertion of an extra amino acid is the main cause of the low affinity of penicillin‐binding protein 2 in penicillin‐resistant strains of Neisseria gonorrhoeae

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin-binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp-345A) that is absent from PBP2 of penicillin-sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp-345A, compared to PBP2 of penicillin-sensitive strains. Site-directed mutagenesis has been used to insert the Asp-345A codon into the penA gene from a penicillin-sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a penicillin-sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp-345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin-sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp-345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin-sensitive strains only in the insertion of Asp-345A have been identified.

Variation in penicillin-binding protein patterns of penicillin-resistant clinical isolates of pneumococci.

A large number of pneumococcal isolates (over 80 strains) from a variety of geographic locales and representing a spectrum of resistance levels from a penicillin MIC of 0.003 microgram/ml up to an MIC of 16 micrograms/ml were analyzed for their penicillin-binding protein (PBP) patterns. With a few exceptions, the great majority of strains with penicillin MICs up to about 0.05 microgram/ml contained the same set of five PBPs with molecular sizes typical of those of susceptible pneumococci. In strains with penicillin MICs of about 0.1 microgram/ml and up, virtually all isolates showed two common features: (i) all isolates showed loss of PBP 1A (98 kilodaltons) with or without a parallel appearance of a "new" PBP that ranged in molecular size between 96 and 97 kilodaltons; and (ii) in strains with penicillin MICs of 0.5 microgram/ml or more, PBP 2B could not be detected on the fluorograms even with very high concentrations of radioactive penicillin. Beyond these two common features, resistant strains with similar penicillin MICs showed a surprising variety of PBP profiles (i.e., in the number and molecular sizes of PBPs), each characteristic of a given isolate. We suggest that in pneumococci remodeling of critical PBPs in more than one way may result in comparable levels of penicillin resistance.

Altered peptidoglycan structure in a pneumococcal transformant resistant to penicillin.

A series of isogenic pneumococcal transformants differing in their levels of penicillin resistance and containing altered penicillin-binding proteins were compared for their cell wall structures by using a recently developed technique that can resolve the peptidoglycan stem peptides of Pneumococcus strains to over 40 components (J. F. Garcia-Bustos, B. T. Chait, and A. Tomasz, J. Biol. Chem. 32:15400-15405). The stem peptides from the highly resistant transformants differed strikingly from those of the susceptible recipient strain, and the peptide patterns were almost identical to that of the DNA donor. Four peptides representing the major components in the walls of susceptible cells were replaced by six new peptides that were only minor components of susceptible cell walls. A remarkable common feature of these new species was their high alanine content. Amino acid analysis, sequencing, and mass spectrometry allowed the assignment of the extra alanine residues to dialanine or alanylserine cross bridges in the six new stem peptides. The common feature of the four peptide species that were present as major components in the susceptible walls, but became minor species in the resistant cells, was the absence of a cross bridge in at least one of the stem peptide components. We suggest that the extensive remodelling of cell wall synthetic enzymes that accompanies acquisition of penicillin resistance eventually also alters the reactivity of these proteins towards their natural substrates in cell wall synthesis. As a result, highly penicillin-resistant pneumococci will shift from the use of wall precursors with linear stem peptides to a preferential use of precursors containing the more-hydrophobic peptides carrying dialanyl or alanylserine cross bridges.

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.

Multiple antibiotic resistance in South African strains of Streptococcus pneumoniae: mechanism of resistance to beta-lactam antibiotics.

Multiply antibiotic-resistant strains of Streptococcus pneumoniae appeared in South Africa in 1977. In these organisms resistance to chloramphenicol is caused by an inducible, drug-inactivating enzyme; however, the basis for resistance to other antibiotics is unknown. Pneumococci with increased resistance to beta-lactam antibiotics do not produce beta-lactamases, a finding indicating the presence of intrinsic resistance to these drugs. One approach to the understanding of the mechanism of this resistance was to study the pattern of penicillin-binding proteins (PBPs) in the South African pneumococci. With the use of highly radioactive penicillin to label PBPs in vivo, it was found that the South African pneumococci have a PBP pattern that differs from that of the sensitive laboratory strain R6 in several respects. Differences include (1) a lack of PBPs la and lb; (2) the presence of a new, faster moving protein (lower molecular weight), named PBP lc; (3) an apparent decrease in the affinity of PBP 2a for [3H]penicillin; and (4) a lack of PBP 2b. Taking advantage of the fact that penicillin resistance is a property acquired in a stepwise process, a series of isogenic and progressively more resistant transformants was constructed. DNA from the resistant South African strain 8249 of S. pneumoniae was used as the donor in a series of transformations for which the recipients were either strain R6 or transformants of organisms with lower levels of resistance. In vivo labeling of the PBPs of these transformants revealed a gradual shift from a pattern similar to that of the sensitive strain (in the transformants of lower resistance) to a pattern resembling that of the highly resistant donor strain (in the transformants of higher resistance) as the level of penicillin resistance increased.

Quantitative correlation between penicillin resistance and beta-lactamase activity specified by the R plasmids R1, R1 bla-45, and RP1 in Escherichia coli K-12.

A mutant of the R plasmid R1 which synthesizes a beta-lactamase with altered kinetic characteristics was isolated. The level of penicillin resistance specified by this plasmid was correctly predicted from the properties of the wild-type R1 according to a simple theoretical model published by Zimmermann and Rosselet (Antimicrob. Agents Chemother. 12:368--372, 1977). The model also accounts for the high level of penicillin resistance specified by the R plasmid RP1.

Penicillin-binding proteins of penicillin-susceptible and intrinsically resistant Neisseria gonorrhoeae.

The penicillin-binding proteins (PBPs) of Neisseria gonorrhoeae were investigated by using [3H]benzylpenicillin of high specific activity. This made it possible to label the PBPs both in cytoplasmic membranes and in the membranes of actively growing cells (in vivo labeling). A total of 20 strains isolated from different geographic locales showed the same pattern of three major PBPs, which had molecular weights of approximately 90,000, 63,000, and 48,000. Five clinical isolates of intrinsically penicillin-resistant gonococci each exhibited reduced penicillin binding of PBPs 1 and 2. The construction of an isogenic set of transformants with increasing levels of penicillin resistance indicated that the penA gene was associated with a decrease in penicillin binding fo PBP 2. Decreased binding to PBP 1 is likely to accompany the newly reported pem and tem genes, which govern to highest level of penicillin resistance.

Distribution of levels of penicillin resistance among freshly isolated strains of N. gonorrhoeae. Application of a novel sensitivity assay.

A novel diffusion zone method of quantitative assay of the antibiotic sensitivity of bacterial strains was tested on freshly isolated gonococci. Smoothly variable estimates of the minimum inhibitory concentration of penicillin for these strains was obtained with sufficient accuracy and precision (coefficient of variation c. 10 per cent.) by means of a simple graphical analysis and without replication. Such estimates were free from the chief sources of error associated with the commonly applied 'incorporation' and 'diffusion' methods. The method revealed that 816 isolates of gonococci obtained in the Bristol area during a 6-month period fell into a large 'sensitive' group (MIC c. 0.02 unit per ml.) and three smaller more resistant groups, and that this pattern occurred in three widely spaced centres within the area. It is suggested that the method is capable of revealing details of distribution that may be masked by the usual techniques and that it is of wide applicability.