Increasing Penicillin Resistance Research Articles

Presence of the blaTEM Gene in Commensal Neisseria spp.: A Possible Cause for the Acquired Drug Resistance Among Pathogenic Respiratory Bacteria.

Background The oral microbiome consists of various bacterial genera, withNeisseriaspp. being a prominent part of this niche. WhileNeisseria gonorrhoeaeandNeisseriameningitidisare human-restricted pathogens, non-pathogenicNeisseriaspecies likeNeisseria sicca,Neisseria perflava, etc.,are primarily commensals that can also behave as opportunistic pathogens. With increasing penicillin resistance in commensalNeisseria, there is a concern that these bacteria might harbor resistance genes that can be transferred to other pathogens. This study aimed to characterize the blaTEM gene (encodes for the plasmid-mediated β-lactamase enzyme that hydrolyzes the β-lactam ring) of commensalNeisseriaspp. isolated from respiratory samples. Methodology The research was conducted in the Department of Clinical Microbiology at Sri Ramachandra University, Chennai. The specimens used were sputum and throat swabs, which were subjected to a series of phenotypic methods and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) for speciation. The antibiogram was determined using the Kirby-Bauer disk diffusion method, and a PCR assay was utilized to identify theblaTEMgene responsible for β-lactamase production. Results Out of 274 processed samples, 65 unique commensalNeisseriaspp. were identified. The study highlighted the presence of theblaTEMgene in 93.9% (61) of the isolates, which is responsible for β-lactamase production. All isolates exhibitedresistance to penicillin. MostblaTEM-positive commensalNeisseria spp. were susceptible to cefuroxime (83.6%), ceftriaxone (85.2%), and cefotaxime (85.2%). The high prevalence of theblaTEMgene in commensalNeisseriais alarming. The gene, found on plasmids, could potentially transfer to other related species likeNeisseria gonorrhoeaeandNeisseriameningitidis, as well as other Gram-negative bacilli. Conclusion The presence of resistance genes in commensal bacteria is of concern, as they might be reservoirs for resistance transfer to pathogenic strains. The study emphasizes the importance of continuous monitoring and deeper investigations into commensal bacteria, emphasizing the need for a broader community screening approach to understand resistance mechanisms in the normal microbiome.

Analysis of Amino Acid Sequences of Penicillin-Binding Proteins 1a, 2b, and 2x in Invasive Streptococcus pneumoniae Nonsusceptible to Penicillin Isolated from Children in India.

Penicillin-binding proteins are the primary targets for beta lactam drugs, which are main stay of treatment for Streptococcus pneumoniae. The emergence of increased penicillin resistance in meningeal isolates of S. pneumoniae in India is alarming. With this background, we aimed to analyze the pbp gene mutations of penicillin nonsusceptible pneumococcal (PNSP) isolates from within India and their association with international clones. A total of 32 PNSP invasive isolates with a penicillin minimal inhibitory concentrations (MIC) of ≥0.12 μg/mL were subjected to PCR and sequencing for multilocus sequence typing and the pbp genes (pbp2b, pbp2x, and pbp1a). The S. pneumoniae R6 susceptible strain was used as the reference for the comparison analyses. In the majority of the present study isolates, amino acid substitutions were only seen in one of the three active sites of one of the three pbp genes. Thus, pbp genes in the absence of the major substitutions usually associated with penicillin resistance combined with mosaicism in pbp1a resulted in a slight increase in the penicillin MIC to between 0.06 and 2.0 μg/mL, which according to meningeal break point denote resistance. Clonal analyses revealed that the emergence of PNSP in India is due to the gradual expansion of the resistant clones CC320, CC230, and CC63.

Illusions regarding Helicobacter pylori clinical trials and treatment guidelines

Identification of reliable Helicobacter pylori eradication therapy has proved difficult, in part because brief exposure of H. pylori to commonly used antimicrobials such as macrolides, nitroimidazoles or quinolones often results...

Increasing Incidence of Penicillin- and Cefotaxime-resistant Streptococcus pneumoniae Causing Meningitis in India: Time for Revision of Treatment Guidelines?

Purpose: Pneumococcal meningitis is a life-threatening infection, requiring prompt diagnosis and effective treatment. Penicillin resistance in pneumococcal infections is a concern. Here, we present the antibiotic susceptibility profile of pneumococcal meningeal isolates from January 2008 to August 2016 to elucidate treatment guidelines for pneumococcal meningitis. Materialsand Methods: Invasive pneumococcal isolates from all age groups, were included in this study. Minimum inhibitory concentrations for the isolates were identified by agar dilution technique and VITEK System 2. Serotyping of isolates was done by co-agglutination technique. Results: Out of 830 invasive pneumococcal isolates, 167 (20.1%) isolates were from meningeal infections. Cumulative penicillin resistance in pneumococcal meningitis was 43.7% and cefotaxime non-susceptibility was 14.9%. Penicillin resistance amongst meningeal isolates in those younger than 5 years, 5–16 years of age and those aged 16 years and older was 59.7%, 50% and 27.3%, respectively, with non-susceptibility to cefotaxime in the same age groups being 18%, 22.2% and 10.4%. Penicillin resistance amongst pneumococcal meningeal isolates increased from 9.5% in 2008 to 42.8% in 2016, whereas cefotaxime non-susceptibility increased from 4.7% in 2008 to 28.5% in 2016. Serotypes 14, 19F, 6B, 6A, 23F, 9V and 5 were the most common serotypes causing meningitis, with the first five accounting for over 75% of resistant isolates. Conclusions: The present study reports increasing penicillin resistance and cefotaxime non-susceptibility to pneumococcal meningitis in our setting. This highlights the need for empiric therapy with third-generation cephalosporins and vancomycin for all patients with meningitis while awaiting results of culture and susceptibility testing.

Increasing penicillin resistance in pneumococci isolated from cerebrospinal fluid samples: Fifteen-year experience from a teaching hospital

Objective: There have been prominent changes in evaluation of resistance patterns of pneumococci and breakpoint values in recent years. We aimed to investigate the penicillin sensitivity of pneumococcal strains isolated from the cesrebrospinal fluid specimens between the years 1997-2011 in our hospital and determine the MIC values under the light of these changes. Methods: Identification of pneumococci was made with conventional methods in patients with meningitis. MIC values for penicillin G were investigated by E test method in all strains. MIC values of pneumococci strains were evaluated acscording to values determined by CLSI for meningitis isolates in 2008. Results: A total of 57 strains were investigated in this study. The MIC range for penicillin was 0.016 - 0.75 mg/ml in S. pneumoniae strains. Seventy five percent of the strains were sensitive and eight strains (14%) had resistance. MIC50 value was sensitive, and a MIC90 value was resistant. The first resistant strain was detected in 2000, and three strains were resistant in the last year. When examined over the years increased resistance was 5.3% in the first five-year period seems to be 28.6% in the last five-year period. Conclusions: Knowledge on penicillin resistance patterns and surveillance is very important in the empirical treatment in pneumococcal meningitis.

Susceptibility patterns of Streptococcus pneumoniae isolates in North America (2002–2003): contemporary in vitro activities of amoxicillin/clavulanate and 15 other antimicrobial agents

A contemporary (2002–2003) national collection of 2100 strains of Streptococcus pneumoniae obtained from 30 sites in the nine United States (US) census regions were tested to determine the comparative antimicrobial properties of amoxicillin/clavulanate and 15 other antimicrobials. The rank order of antimicrobials with the lowest susceptibility rates was: penicillin (67.9%) < trimethoprim/sulphamethoxazole (69.5%) < macrolides (≤73.3%) < orally administered cephalosporins (≤78.8%). Bloodstream infection isolates were more susceptible than strains isolated from community-acquired respiratory tract infections; penicillin (11.0% difference), erythromycin (9.8%) and trimethoprim/sulphamethoxazole (6.5%). The resistance rates of nearly all tested antimicrobials increased with increasing penicillin resistance: levofloxacin (98.0%), ceftriaxone (87.4%) and clindamycin (70.7%) being the most active agents against penicillin-resistant strains. Highest resistance rates were noted for the <2 year-old age group for the macrolides (≥41.1%), trimethoprim/sulphamethoxazole (38.9%), tetracyclines (22.2%) and clindamycin (10.0%). Geographical variation in the susceptibility patterns among US census zones was present with lowest penicillin and erythromycin susceptibility noted for West South Central and West North Central zones (≤58.7% penicillin; ≤65.5% erythromycin). New formulations of amoxicillin/clavulanate (Augmentin XR and ES-600) had a higher percentage of potentially susceptible S. pneumoniae isolates compared with amoxicillin/clavulanate for the entire organism collection (≥+0.9%), sinus isolates (+2.7%), middle ear fluid isolates (+5.5%), penicillin-resistant strains (≥+5.8%) and strains from patients <2 years of age (≥+2.4%). Local and global surveillance studies of common respiratory pathogens such as S. pneumoniae remain instrumental to guide clinicians in appropriate empirical treatments and to emphasize the need for prudent antimicrobial use.

Epidemiology of invasive Streptococcus pneumoniae infections in children in an area of Barcelona, Spain.

Streptococcus pneumoniae is the most common cause of bacteraemia, pneumonia, sinusitis and acute otitis media. With the advent of conjugate vaccines, there is now the possibility of preventing disease caused by this organism. However, little is known about the epidemiology of invasive pneumococcal disease in children in Spain. The aim of this study was to determine the incidence and the clinical and microbiologic characteristics of invasive pneumococcal disease in Sabadell, an industrial area in the province of Barcelona, Spain. From January 1990 to December 2000, the case records of children with pneumococcal invasive disease at Sabadell Hospital were retrospectively (1990-1996) reviewed and prospectively (1997-2000) collected. The hospital serves a population of 61,143 children under 15 y of age, 18,073 children under 4 y of age and 7300 children under 2 y of age. A total of 112 children (54% under 24 mo of age and 93% under 6 y of age) with invasive pneumococcal disease were diagnosed during a period of 11 y. The incidence of invasive pneumococcal disease was 76 per 100,000 for children aged 0 to 24 mo, 45 for children aged 0-48 mo and 16.6 for children aged 0-14 y. Occult bacteraemia was the most common manifestation of invasive pneumococcal disease (66 cases), pneumonia was the second form (34 cases) and meningitis (10 cases) and arthritis (2 cases) were the other clinical manifestations. Of the 105 strains tested, 8.6% were highly penicillin resistant, 37.1% were intermediately penicillin resistant. 16.2% were intermediately cefotaxime resistant and 32.4% were erythromycin resistant. Pneumococci of serogroups 6, 14, 18, 19, 1, 5, 4, 9, 23 and 33 were the most frequently isolated groups (92%) but only 6, 9, 14, 19 and 23 were resistant to penicillin, cefotaxime, or erythromycin. In this study the incidence of invasive pneumococcal disease was found to be greater than that reported elsewhere in Spain and Europe. Penicillin resistance levels are high but the trend towards increasing penicillin resistance may have ended over the past few years. The currently licensed seven-valent (7-V) pneumococcal conjugate vaccine would cover 78% of cases of invasive pneumococcal in children aged 0-14 y, 80% in children aged 0-24 mo and 100% of cases of penicillin- or cefotaxime-resistant invasive pneumococcal disease.

Epidemiology of invasive Streptococcus pneumoniae infections in children in an area of Barcelona, Spain

Aim: Streptococcus pneumoniae is the most common cause of bacteraemia, pneumonia, sinusitis and acute otitis media. With the advent of conjugate vaccines, there is now the possibility of preventing disease caused by this organism. However, little is known about the epidemiology of invasive pneumococcal disease in children in Spain. The aim of this study was to determine the incidence and the clinical and microbiologic characteristics of invasive pneumococcal disease in Sabadell, an industrial area in the province of Barcelona, Spain. Methods: From January 1990 to December 2000, the case records of children with pneumococcal invasive disease at Sabadell Hospital were retrospectively (1990–1996) reviewed and prospectively (1997–2000) collected. The hospital serves a population of 61 143 children under 15 y of age, 18 073 children under 4y of age and 7300 children under 2 y of age. Results: A total of 112 children (54% under 24 mo of age and 93% under 6 y of age) with invasive pneumococcal disease were diagnosed during a period of 11 y. The incidence of invasive pneumococcal disease was 76 per 100000 for children aged 0 to 24 mo, 45 for children aged 0–48 mo and 16.6 for children aged 0‐14 y. Occult bacteraemia was the most common manifestation of invasive pneumococcal disease (66 cases), pneumonia was the second form (34 cases) and meningitis (10 cases) and arthritis (2 cases) were the other clinical manifestations. Of the 105 strains tested, 8.6% were highly penicillin resistant, 37.1% were intermediately penicillin resistant, 16.2% were intermediately cefotaxime resistant and 32.4% were erythromycin resistant. Pneumococci of serogroups 6, 14, 18, 19, 1, 5, 4, 9, 23 and 33 were the most frequently isolated groups (92%) but only 6, 9, 14, 19 and 23 were resistant to penicillin, cefotaxime, or erythromycin. Conclusions. In this study the incidence of invasive pneumococcal disease was found to be greater than that reported elsewhere in Spain and Europe. Penicillin resistance levels are high but the trend towards increasing penicillin resistance may have ended over the past few years. The currently licensed seven‐valent (7‐V) pneumococcal conjugate vaccine would cover 78% of cases of invasive pneumococcal in children aged 0–14 y, 80% in children aged 0–24 mo and 100% of cases of penicillin‐ or cefotaxime‐resistant invasive pneumococcal disease.

Susceptibility of Canadian isolates of Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae to oral antimicrobial agents

We measured the susceptibility of Canadian isolates of three respiratory tract pathogens ( Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae) to several currently approved antimicrobial agents by two different methods. We also measured the susceptibility of isolates to seven fluoroquinolones. Beta-lactamase was produced by 123/566 (21.7%) of H. influenzae isolates compared with 178/200 (89%) of M. catarrhalis isolates. For S. pneumoniae 83/374 (22.2%) isolates were penicillin resistant and of these 2.1% (8/374) showed high level resistance (MIC≥2 mg/l). Regardless of methodology, all fluoroquinolones were highly active against H. influenzae (MIC 90 ≤0.031 mg/l) and M. catarrhalis (MIC 90 ≤0.064 mg/l) isolates. Susceptibility of H. influenzae to cefuroxime and amoxycillin/clavulanic acid was 99–100% whereas 84–85.5% were susceptible to cefaclor and cefprozil. Azithromycin susceptibility ranged from 82.6 to 99.2% depending on the method. M. catarrhalis isolates were uniformly susceptible to all agents tested except amoxycillin. Cross-resistance in S. pneumoniae to all non-quinolone agents was concurrent with increasing penicillin resistance as shown by increasing MIC 90 values. For the fluoroquinolones tested, the rank order of potency based on MIC 90 values was as follows: gemifloxacin (0.031–0.063 mg/l), trovafloxacin (0.125 mg/l), moxifloxacin (0.125–0.25 mg/l), grepafloxacin (0.125–0.25 mg/l), gatifloxacin (0.5 mg/l), levofloxacin (1 mg/l) and ciprofloxacin (2 mg/l). Our study confirms either a high or increasing prevalence of antimicrobial resistant respiratory pathogens in Canada and also compares the new and old fluoroquinolones and their potential role as therapy for community-acquired infections. The prevalence of β-lactamase positive H. influenzae may have decreased from levels reported in previous studies.

Antibiotic use and pneumococcal resistance to penicillin: the French experience

Antibiotic use and pneumococcal resistance to penicillin: the French experience

New faces of an old pathogen: emergence and spread of multidrug-resistant Streptococcus pneumoniae

The image of Streptococcus pneumoniae is split between the world of infectious diseases and the world of the science laboratory. In the eyes of medical practitioners and clinical microbiologists, this bacterium is a dangerous pathogen capable of causing potentially life-threatening community-acquired diseases. In the eyes of the microbiologist, S. pneumoniae is the microbe that has provided some of the most important insights into molecular biology. It was laboratory studies with S. pneumoniae that led to the identification of the first polysaccharide antigen, the unique recombinational mechanism of genetic transformation, and identification of DNA as the genetic material; the first bacterial quorum-sensing polypeptide, a microbial hormone inducing and controlling the physiological state of competence, and the first bacterial autolysin, were also first identified in pneumococci. The novel feature of our era is that these two images of the pneumococcus, the disease-causing pathogen and the source of unique insights into molecular biology, are becoming fused through the introduction of molecular fingerprinting techniques that allow one to observe molecular-level interactions among these bacteria in their natural habitat, which is the human host. There is little doubt that the appearance and massive geographic spread of penicillin-resistant and multidrug-resistant pneumococcus among clinical isolates in the 1980s and 1990s were major motives for the development and application of the molecular techniques that began to play an increasingly important role in the identification of the novel genetic elements, new physiologic, biochemical, and epidemiologic features that constitute the new face of S. pneumoniae in the 1990s. Analysis of penicillin-resistant strains of S. pneumoniae isolated from the clinical environment revealed a remarkable number of changes in these bacteria: Genetic determinants of cell wall synthetic enzymes were shown to contain patches of nucleotide sequence mosaics, indicating the acquisition of extraspecies DNA; penicillin-resistant bacteria contained membrane proteins of altered catalytic properties and altered molecular size; the chemical composition of the cell wall peptidoglycan was changed; the control of autolytic enzymes was changed; and clear-cut evidence for the change of capsular type through the replacement of large pieces of DNA was obtained. Directly or indirectly, most, if not all, of these changes were related to the appearance of antibiotic-resistant pneumococcus. The first report describing a penicillin-resistant S. pneumoniae isolate that caught the attention of the world of infectious diseases was a capsular type 4 S. pneumoniae isolated from the throat of a healthy 3-year-old boy in the village of Anguganak, New Guinea (population 507) on April 15, 1969. The isolate had an increased minimum inhibitory concentration (MIC) for penicillin (0.5 mg/ mL) but was susceptible to chloramphenicol, tetracycline, erythromycin, and sulfa drugs. The putative selective pressure in operation in this part of New Guinea was the extensive use of prophylactic procaine penicillin. The publication of this report prompted an editorial in The New England Journal of Medicine in 1970, which recognized the implication: the specter of a potential problem in the treatment of serious pneumococcal infections. However, the editorial continued with the statement that the particular epidemiologic circumstances and the special properties of the strain involved made the prospect of spread of the resistant pneumococcus extremely remote. This, of course, turned out to be more wishful thinking than a correct prediction of things to come. The next report with major impact came in 1977, from South Africa, with an epidemic pneumococcal disease caused by strains with greatly increased penicillin MIC values and carrying resistance traits to tetracycline, macrolides, and chloramphenicol as well. A comparison of the antibiotic susceptibilities of a prepenicillin era S. pneumoniae and one of the multidrug-resistant South African strains illustrates the magnitude of change (Table 1). In between and after these two dramatic reports, detection of penicillin-resistant pneumococci among clinical isolates began to be reported with increasing frequency in the clinical and microbiologic literature. Careful surveillance in Spain showed the increasing geographic spread and also the increasing penicillin resistance level of the pneumococcal strains from a mean MIC of 0.1– 0.2 mg/mL in the 1970s to a mean of 1–2 mg/mL in the 1980s. In most parts of the world where surveillance for resistant pneumococci was performed at several time inFrom the Laboratory of Microbiology, The Rockefeller University, New York, New York. This article was written during the tenure of a grant from the National Institutes of Health (RO1 AI37275). Requests for reprints should be addressed to Alexander Tomasz, Laboratory of Microbiology, The Rockefeller University, 1230 York Avenue, New York, New York 10021.

Treatment of pneumococcal infections in the era of increasing penicillin resistance

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Gonococcal arthritis in an era of increasing penicillin resistance. Presentations and outcomes in 41 recent cases (1985-1991).

To assess the impact of recent reports of disseminated gonococcal infection caused by penicillin-resistant organisms, we reviewed the presenting features, clinical course, and outcomes of a group of patients with gonococcal arthritis treated in recent years. We reviewed the records of all cases of acute arthritis associated with a culture positive for Neisseria gonorrhoeae at our institution from July 1985 through December 1991. Forty-one cases were identified. Patients included 34 women and 38 blacks; the mean age was 22.6 years. Duration of symptoms averaged 4.8 days at presentation. Other features included migratory arthralgias (n = 27), urogenital symptoms or signs (n = 26), fever (n = 21), and skin lesions (n = 16). Comorbid conditions included intravenous drug use (n = 8) and systemic lupus erythematosus (n = 3). The knee was the most commonly affected joint. Positive culture results were obtained from 32 urogenital samples (86%), 14 synovial fluid samples (44%), seven rectal samples (39%), four blood samples (12%), and two throat samples (7%). All synovial fluid samples with positive culture results had white blood cell counts higher than 20.0 x 10(9)/L. Response to therapy with penicillin and/or ceftriaxone was prompt, and mean duration of hospitalization was 5.8 days. Patients who required longer hospitalization had a higher mean erythrocyte sedimentation rate and higher frequencies of positive synovial fluid culture results and comorbid conditions. Penicillin sensitivity could be determined in 30 patients on the basis of clinical response or in vitro testing. Among these patients, two cases of penicillin-resistant organisms were identified, one beta-lactamase positive and one beta-lactamase negative. The clinical features of patients with gonococcal arthritis have changed very little since the last large reported series over a decade ago. Underlying conditions appear to be more common, but response to antibiotic therapy and eventual outcome remain excellent. The finding of penicillin-resistant organisms in at least 5% of patients reinforces recent recommendations that third-generation cephalosporin agents be used as initial therapy for disseminated gonococcal infections until drug susceptibilities are known.

Activity of oral antibiotics in middle ear and sinus infections caused by penicillin-resistant Streptococcus pneumoniae: implications for treatment.

The increasing prevalence of intermediately and highly penicillin-resistant strains of Streptococcus pneumoniae is a problem worldwide. However, optimal management of patients with middle ear and sinus infections caused by resistant pneumococci has not been established. We performed agar dilution susceptibility studies on 71 strains of penicillin-resistant pneumococci (minimum inhibitory concentration (MIC), > or = 0.1 microgram/ml) recovered from middle ear and sinus cultures of Houston children against 13 oral antibiotics with the use of both established and newly proposed National Committee for Clinical Laboratory Standards susceptibility criteria. Of the 62 middle ear isolates 35 (56%) were intermediately resistant and 27 (44%) were highly resistant to penicillin. Of the 9 sinus isolates tested, 5 (56%) were intermediately resistant (MIC between 0.1 and 1 micrograms/ml) and 4 (44%) were highly resistant (MIC > or = 2 micrograms/ml) to penicillin. The MIC90 increased with increasing penicillin resistance for the antibiotics tested except for rifampin, ciprofloxacin, loracarbef, clindamycin and trimethoprim-sulfamethoxazole. None of the highly penicillin-resistant isolates was susceptible to loracarbef or trimethoprim-sulfamethoxazole. The MIC90 values for clindamycin and rifampin were similar for the intermediately and highly penicillin-resistant groups, and the number of susceptible isolates in each group remained greater than 90% for both antibiotics. Thirty-five isolates were resistant to erythromycin but susceptible to clindamycin, a susceptibility pattern distinctly different from that seen in South Africa and Europe, where clindamycin resistance parallels erythromycin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Increased rate of isolation of penicillin-resistant Streptococcus pneumoniae in a children's hospital and in vitro susceptibilities to antibiotics of potential therapeutic use.

The isolation of Streptococcus pneumoniae with both high and intermediate resistance to penicillin has increased in our institution since 1989 to an average of 12.1% of all isolates. We determined the susceptibilities of 95 isolates (34 susceptible to penicillin, 42 intermediate in resistance to penicillin, and 19 resistant to penicillin) to 16 antimicrobial agents of potential use in the treatment of disease caused by S. pneumoniae. Susceptibility to penicillin was determined by broth macrodilution with Mueller-Hinton broth supplemented with 5% lysed horse blood. Isolates were classified as highly resistant when the MIC was greater than or equal to 2.0 micrograms/ml, intermediate in resistance when the MIC was between 0.1 and 1.0 microgram/ml, and susceptible when the MIC was less than 0.1 microgram/ml. Fifteen of 19 isolates found to be highly resistant to penicillin were recovered from the middle ear of children. None of the isolates recovered from cerebrospinal fluid was highly resistant to penicillin. Fifteen of these isolates highly resistant to penicillin were found to be serogroup 6. Susceptibilities to other antibiotics were determined by the agar dilution method with Mueller-Hinton agar containing 5% lysed horse blood and an inoculum of 10(4) CFU per spot delivered by a replicator device. The MIC for 90% of isolates increased with increasing penicillin resistance for all antibiotics tested, except chloramphenicol, ciprofloxacin, rifampin, and vancomycin. Regardless of the classification of penicillin resistance, all isolates were classified as susceptible to cefotaxime, cefpirome, cefpodoxime, clarithromycin, imipenem, rifampin, and vancomycin on the basis of National Committee for Clinical Laboratory Standards interpretive guidelines. Interpretation of susceptibilities on the basis of currently available guidelines is difficult in that susceptibility guidelines applicable specifically to S. pneumoniae are not available.

Antimicrobial susceptibility changes in Enterococcus faecalis following various penicillin exposure regimens.

Penicillin-"virgin" strains of Enterococcus faecalis collected from a population of individuals with no previous antibiotic exposure were subjected in vitro to penicillin delivered as repeated pulses, stepwise increasing concentrations, or sustained levels of a single concentration. Changes in resistance to penicillin were assessed by determination of MICs, and time-kill studies were performed to evaluate changes in tolerance to the bactericidal effects of penicillin. Isogenic clones, derived from various exposure regimens, which exhibited changes in either resistance or tolerance were further examined for changes in penicillin-binding proteins. Exposure to repeated pulses of penicillin resulted in the development of tolerance to penicillin without changes in the level of resistance. Clones derived from a regimen of stepwise increases in the penicillin concentration acquired both increased penicillin resistance and tolerance. Clones selected after prolonged continuous exposure to a fixed concentration of penicillin displayed minimally increased resistance to penicillin, but they retained the lytic, nontolerant response to the bactericidal effect of penicillin. Clones which acquired tolerance to the bactericidal effect of penicillin without changes in penicillin resistance exhibited a penicillin-binding protein pattern identical to that of the parental strain. Increased labeling of several penicillin-binding proteins accompanied the development of increased penicillin resistance in both penicillin-tolerant and nontolerant strains. Exposure of E. faecalis to penicillin in repeated pulses of brief duration, for prolonged periods at a constant concentration, or in stepwise graded concentrations can result in the selection of clones with increased resistance to the inhibitory or bactericidal effects of penicillin, or both. These observations may be relevant to the selection of dosing regimes for penicillin in the treatment of enterococcal infections, when bactericidal synergism cannot be achieved with penicillin-aminoglycoside combinations.

Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution

To identify any change in the antibiotic resistance of Enterococcus faecium, we examined the antibiotic susceptibilities of clinical strains (n = 84) isolated at one institution during the 22 years since 1968. A significant increase in resistance to penicillin was observed during the study period: the MICs of penicillin for 50 and 90% of isolates tested were 16 and 64 micrograms/ml, respectively, from 1969 to 1988 (n = 48; geometric mean MIC, 14 micrograms/ml) , whereas they were 256 and 512 micrograms/ml, respectively, from 1989 to 1990 (n = 36; geometric mean MIC, 123 micrograms/ml) (P less than 0.001). A comparable increase in resistance to ampicillin was also noted (P less than 0.001). No strains produced detectable beta-lactamase. In contrast, susceptibilities to vancomycin, teicoplanin, and ciprofloxacin remained stable. High-level resistance to gentamicin was observed in none of 48 isolates from 1969 to 1988, but was present in 22 of 36 strains (61%) from 1989 to 1990 (P less than 0.001) and was significantly associated with resistance (MIC, greater than or equal to 128 micrograms/ml) to penicillin (P less than 0.001). To assess the potential evolution of antibiotic resistance in this species, clinical isolates (n = 24) were compared with strains isolated in 1968 from a human population in the Solomon Islands that was never exposed to antibiotics. Solomon Island isolates were significantly more susceptible than all clinical strains to penicillin, ampicillin, and vancomycin (P less than 0.001 for each), but they exhibited no differences in susceptibility to teicoplanin or ciprofloxacin. The penicillin-binding affinity of penicillin-binding protein 5 (PBP 5) in penicillin-resistant clinical strains (MIC, 512 micrograms/ml) was notably lower than that in strains with more typical susceptibilities, suggesting an alteration in this PBP as a possible mechanism for increased penicillin resistance. Solomon Island strains most susceptible to penicillin demonstrated a prominent PBP 5* and the absence of PBP 5. These changes in the antibiotic resistance of E. faecium emphasize the importance of identifying this species in patients with serious enterococcal infections and the necessity of assessing its susceptibility to both beta-lactams and aminoglycosides if effective therapy is to be identified.

Comparison of Enterococcus raffinosus with Enterococcus avium on the basis of penicillin susceptibility, penicillin-binding protein analysis, and high-level aminoglycoside resistance

We reidentified our laboratories' collections of 57 enterococcal isolates previously classified as Enterococcus avium by the API Rapid Strep identification system (Analytab Products, Plainview, N.Y.) with the identification criteria recommended by Facklam and Collins (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27: 731-734, 1989). Thirty isolates were identified as true E. avium, 25 isolates were identified as E. raffinosus, and 2 isolates were identified as E. pseudoavium. E. raffinosus could be differentiated from E. avium on the basis of penicillin susceptibility, as follows: MIC for 50% of E. raffinosus isolates tested (MIC50), 32 micrograms/ml; MIC90, 64 micrograms/ml (range, 4 to 64 micrograms/ml); E. avium MIC50, 1 microgram/ml; MIC90, 2 micrograms/ml (range, 0.5 to 2 micrograms/ml). No strains produced detectable beta-lactamase. Penicillin-binding protein (PBP) analysis of all E. raffinosus isolates demonstrated the unique pattern reported previously (M. D. Collins, R. R. Facklam, J. A. E. Farrow, and R. Williamson, FEMS Microbiol. Lett. 57:283-288, 1989); however, a number of newly identified PBPs were noted. Of 25 isolates, 13 had an additional PBP of 77 kDa (designated PBP 6*), while all isolates possessed a 52-kDa PBP (PBP 7) and a 46-kDa PBP (PBP 8). The presence or absence of PBP 6* did not correlate with penicillin susceptibility; however, PBP 7 demonstrated many features suggestive of low penicillin-binding affinity and may represent a possible mechanism for the relative resistance of this species to penicillin, although this hypothesis remains speculative since attempts to develop a penicillin-hypersusceptible E. raffinosus mutant were unsuccessful. E. raffinosus isolates were significantly more likely to exhibit high-level resistance to kanamycin than E. avium strains were (P < 0.001; chi-square); however, no strains demonstrated high-level resistance to gentamicin. No trend toward increasing penicillin resistance was noted among this collection of E. avium and E. raffinosus isolates collected over the past 35 and 14 years, respectively. Relative resistance to penicillin may be a helpful differentiating feature between E. avium and E. raffinosus when assessment of raffinose metabolism is not possible or is indeterminant.

Intra- and inter-specific transformation of Streptococcus pneumoniae to penicillin resistance

Transformation studies were carried out with a penicillin susceptible (MIC 0.006 mg/l) laboratory strain of Streptococcus pneumoniae as recipient. Donor DNA was prepared from two clinical isolates of S. pneumoniae, four isolates of S. mitior and five isolates of S. sanguis. DNA from both isolates of S. pneumoniae generated penicillin-resistant transformants (MICs 0.03-2.0 mg/l). In addition, one isolate each of S. mitior and S. sanguis transformed the recipient to increased penicillin resistance, with MICs of 0.125 mg/l. DNA from the S. sanguis strain generated a transformant which showed a reduction in the penicillin-binding affinity of penicillin-binding protein (PBP) 2B. DNA from a second S. mitior strain generated intermediately resistant (MIC 0.5 mg/l) transformants in a single transformation round. The PBP profiles of these transformants showed an apparent molecular size alteration of PBP 2B, corresponding to a PBP found in the donor strain, and PBP 1B was no longer detected. Isolates of S. pneumoniae may apparently develop penicillin resistance either intrinsically, by intragenetic transfer, or by transfer of penicillin resistance determinants from viridans streptococci.