A global gene pool for high-level cephalosporin resistance in commensal Streptococcus species and Streptococcus pneumoniae

Highly penicillin- and cephalosporin-resistant Streptococcus mitis and Streptococcus oralis were isolated in Spain, Hungary, and Berlin. With chromosomal DNA of these strains, resistant transformants of Streptococcus pneumoniae were obtained that expressed low-affinity variants of penicillin-binding proteins (PBPs) 2x, 1a, 2a, and 2b in different combinations, depending on the selective conditions. The transformants had cefotaxime MICs of up to 6 microg/mL, and those with a low-affinity PBP 2b were highly deficient in penicillin-induced lysis. Sequence analysis of the pbp2x genes confirmed the presence of a global gene pool of penicillin resistance determinants shared by commensal and pathogenic streptococci.     

Detection of Streptococcus pneumoniae in clinical specimens, its antimicrobial susceptibility and serotypes

The purpose of our investigation was the detection of S. pneumoniae and the isolation frequency of PRSP/PISP from the patients of the area. 457 strains of S. pneumoniae were isolated from the clinical specimens which had been requested for cultivation from the individual general practitioners in the city. Screening of the PRSP/PISP in 457 strains was carried out with the oxacillin (MPIPC) disk method. The MIC (benezylpenicillin: PCG) was measured at the same time in 73 strains, and compared with the disk method. The results were as follows: 1) Regarding the detection rate of S. pneumoniae, nose discharge was the highest (11.4%) in all the specimens. 2) There was a greater number of isolation of S. pneumoniae in one year from November to March. Then it was scarce in August and September. This change in seasons was noted. 3) There were many 9-year olds and over-60 year olds in the age group of the patients in whom the S. pneumoniae was isolated. 4) All 30 strains where a 6 mm diameter was found when a MIC was compared with the MPIPC disk method were resistant (PRSP/PISP). All 20 strains with a > or = 20 mm diameter were sensitive (PSSP). But in the 23 strains which had a 7 mm -19 mm, 7 strains (30%) were PSSP, and 16 strains (70%) in the intermediate (PISP). 5) A diameter of 6 mm was found in 194 strains (42.5%) with the MPIPC disk method, among the 457 strains isolated from the clinical specimens. 141 strains (30.9%) were found with a diameter of 7 mm -19 mm and 122 strains (26.7%) with a diameter of > or = 20 mm. 6) More than 70% in PRSP/PISP were multiple antimicrobiotics resistant strains to PCG, EM and MINO. 7) The distribution of the serotype in PRSP/PISP was in the order of 19 type (41.3%), 23 type (21.7%), 6 type (13.0%). Moreover, it was in the order of 3 type (18.5%), 6 type (11.1%), 19 type (11.1%), and there were few 23 type (3.7%) for PSSP. The distribution of the serotype was different for PRSP/PISP and PSSP.     

Comparison of Etest to broth microdilution method for testing Streptococcus pneumoniae susceptibility to levofloxacin and three macrolides

When the Etest was compared to broth microdilution for susceptibility testing of Streptococcus pneumoniae, levofloxacin, erythromycin, and penicillin results correlated for both methods; azithromycin and clarithromycin showed discrepancies of > or = 2 dilutions for 95.8% and 31.5% of the isolates, respectively. Levofloxacin was active against 141 of 142 isolates (< or = 2.0 micrograms/ml), making it a potentially useful new fluoroquinolone.     

Comparison of E test with standard broth microdilution for determining antibiotic susceptibilities of penicillin-resistant strains of Streptococcus pneumoniae

We compared the E test (AB Biodisk North America, Inc., Culver City, Calif.) with the National Committee for Clinical Laboratory Standards broth microdilution method for the determination of MICs of penicillin and cefotaxime for 108 isolates of Streptococcus pneumoniae. The E test was performed following manufacturer's recommendations with Mueller-Hinton blood agar, and the broth microdilution procedure was performed with lysed horse blood-supplemented Mueller-Hinton broth. The microdilution method classified 26 isolates as highly penicillin resistant (MIC, > or = 2 micrograms/ml), 33 as intermediately resistant to penicillin (MIC, > or = 0.1 < 2.0 micrograms/ml), and 49 as susceptible to penicillin (MIC, < 0.1 micrograms/ml). Discordant results obtained with the E test for penicillin susceptibility testing compared with broth microdilution occurred for 19 of the 108 isolates tested. Cefotaxime MICs for 90% of isolates found highly resistant, intermediately resistant, and susceptible to penicillin by broth microdilution were 2.0, 0.5, and 0.06 micrograms/ml, respectively. There were 16 susceptibility category changes when the E test was used to determine cefotaxime MICs. All of the discrepancies in the penicillin and cefotaxime MICs determined by the E test occurred at the susceptibility category breakpoints, and all represented differences of only one twofold dilution factor. Properly performed and controlled, the E test should be a reliable quantitative procedure for more accurately predicting the susceptibility of S. pneumoniae to several antibiotics.     

Distribution of Streptococcus pneumoniae resistant to penicillin in the USA and in-vitro susceptibility to selected oral antibiotics

Surveillance by 33 laboratories in 19 states during a 4 1/2 month period between December 1993 and April 1994 found that 263 of 1627 (16.2%) isolates of Streptococcus pneumoniae were resistant to penicillin. One hundred and seventy (10.4%) isolates were determined to be intermediately resistant to penicillin (MICs 0.1-1.0 mg/L and 93 (5.7%) were found to be highly resistant to penicillin (MICs > 2.0 mg/L). MIC90s for intermediately penicillin resistant strains were: amoxycillin/clavulanate 2.0 mg/L, cefaclor 64 mg/L, cefixime 32 mg/L, cefprozil 8 mg/L and loracarbef 128 mg/L. MIC90s for highly penicillin resistant strains were: amoxycillin/clavulanate 4.0 mg/L, cefaclor > or = 128 mg/L cefixime 64 mg/L, cefprozil 32 mg/L and loracarbef > or = 128 mg/L.     

Mechanisms of the spread of penicillin resistance in Streptococcus pneumoniae strains causing meningitis in children in France

The molecular epidemiology of penicillin-resistant Streptococcus pneumoniae strains causing meningitis in children was studied in France. Typing procedures included analysis of total DNA polymorphism by random amplification of polymorphic DNA, restriction fragment length polymorphism (RFLP) analysis of the ribosomal RNA gene regions, and pulsed-field gel electrophoresis. Penicillin-binding protein (PBP) genes pbp2b and pbp2x were studied by RFLP analysis and DNA sequencing in selected cases. Statistical analysis of the data by factorial analysis of correspondence established that the emergence of penicillin-resistant pneumococci in this pathology is the result of the spread of two highly resistant closely related clusters and a cluster of serotype 23 strains with an intermediate level of resistance, the spread of genes confering high resistance to penicillin between the two highly resistant clusters, and complex genetic events involving the pbp genes in a heterogeneous population of strains leading to an intermediate level of resistance.     

Comparison of agar dilution, broth dilution, disk diffusion, and the E-test for susceptibility testing of penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae

An evaluation to determine the optimal methods for the in vitro susceptibility testing of 41 clinical isolates and the ATCC 49619 strain of Streptococcus pneumoniae to penicillin was undertaken. No very major or major interpretive errors were observed with the following test methods and media: agar dilution using either Mueller-Hinton medium with lysed horse blood or Haemophilus test medium; broth dilution using cation-adjusted Mueller-Hinton medium with lysed horse blood, Haemophilus test medium, or Todd-Hewitt medium; and the epsilo-meter test (E-test) using agar containing Mueller-Hinton medium and 5% sheep blood. The disk diffusion method using agar containing Mueller-Hinton medium and 5% sheep blood agar was an effective screening method, requiring confirmation by a dilution susceptibility test method.     

Prevalence of antimicrobial resistance among respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY antimicrobial surveillance program

Rabies is a widespread zoonosis that recently reached epidemic proportions in gray foxes (Urocyon cinereoargenteus) in central Texas. The objectives of this study were to determine bait and attractant preferences among captive gray foxes, to determine the behavioral responses of gray foxes to selected bait-attractant combinations, and to evaluate baits as a delivery mechanism of oral rabies vaccines. Trials were conducted to determine bait preferences of captive gray foxes to selected baits and attractants. Tested baits consisted of a polymer-bound cube made of either dog food meal or fish meal, a polymer-bound cylinder made of dog food meal, and a wax-lard cake that was enhanced with marshmallow or chicken flavoring. Attractants were additives to baits that exuded sweet, sulfurous, fruity, fatty, cheesy, honey, and fishy odors and flavors. Captive gray foxes (n = 31) exhibited a preference for marshmallow wax cakes and polymer dog food baits with a lard interior and granulated sugar exterior. However, gray foxes exhibited chewing behaviors consistent with ingesting an oral vaccine only with the wax cake baits.     

Unreliability of disc diffusion test for screening for reduced penicillin susceptibility in Neisseria meningitidis

The 2-U penicillin and microgram oxacillin discs proposed for screening meningococci for susceptibility to penicillin were evaluated by using MICs measured by the E test. The discs yielded unacceptably high frequencies of misclassification of susceptibility category and should be abandoned in favor of MIC estimations. An agreed breakpoint for reduced penicillin susceptibility in meningococci is needed for the E test.     

Penicillin resistance in laboratory isolates of Streptococcus pneumoniae, in Western Australia, 1990-1994

Increasing frequency of penicillin resistance in Streptococcus pneumoniae has been reported worldwide. We report on clinical isolates of penicillin-resistant pneumococci (PRP) in Western Australia (WA) from 1990-1994. A retrospective survey of laboratories performing susceptibility testing, or receiving isolates referred from rural areas found resistant on oxacillin disc screening, was undertaken. Four of 11 laboratories could provide data for the five year time period inclusive. Information was provided on susceptibility to penicillin, type of specimen, date of isolation and; age, sex and race of individuals with PRP. Penicillin resistance increased from 1.3% to 9.0% over the five year period. PRP were rarely invasive. Highest age specific rates per 100,000 were found in children < 5 years (19.4) and adults > or = 60 years (5.1). Aboriginal ethnicity was associated with resistance. The increasing frequency of PRP in WA indicates the need for surveillance systems for their detection.     

Evaluation of the E test for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from patients with long-term bladder catheterization

The E test was evaluated in comparison with reference agar methods (National Committee for Clinical Laboratory Standards) for the susceptibility testing of 248 Pseudomonas aeruginosa isolates from bladder-catheterized patients against nine antibiotics. The E-test MICs correlated well with those determined by the agar dilution and disk diffusion reference methods (88 and 92.5% within 1 log2 dilution step, respectively), confirming that the E test is a reliable method for the determination of MICs of antibiotics for catheterization-associated P. aeruginosa isolates.     

Serotype distribution and prevalence of resistance to benzylpenicillin in three representative populations of Streptococcus pneumoniae isolates from the coast of Kenya

As surveillance data from sub-Saharan Africa are few, three representative populations of Streptococcus pneumoniae isolates were examined in Kenya for serotype distribution and Etest minimum inhibitory concentrations (MICs) of benzylpenicillin: (1) 75 lung aspirate or blood culture isolates from 301 consecutive adult patients with pneumonia, (2) 112 invasive isolates from continuous pediatric inpatient surveillance over 4 years, and (3) 97 nasopharyngeal isolates from systematically selected sick children. The proportions with benzylpenicillin MICs of > or = 0.1 microgram/mL were 0.27, 0.29, and 0.47, respectively. Vaccine-related serotypes accounted for 96% of invasive isolates from children and 90% of those from human immunodeficiency virus (HIV)-seropositive adults. Serotype 1 accounted for 44% of pneumococci from HIV-seronegative patients but only 5% of those from HIV-seropositive patients (P = .0002). Of serotype 1 isolates, 98% were susceptible to benzylpenicillin, but serogroups 13, 14, 19, and 23 were strongly associated with an MIC of > or = 0.1 microgram/mL.     

Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae

An open reading frame (ORF) homologous to norA was insertionally inactivated with cat in a fluoroquinolone-resistant pneumococcus with an efflux phenotype; this inactivation caused reversion to drug sensitivity. The ORF product has 24% amino acid sequence identity each to NorA and Bmr, which suggests that it is a major facilitator system pump of the 12-transmembrane-segment class.     

In vitro selection of resistance to four beta-lactams and azithromycin in Streptococcus pneumoniae

Selection of resistance to amoxicillin (with or without clavulanate), cefaclor, cefuroxime, and azithromycin among six penicillin G- and azithromycin-susceptible pneumococcal strains and among four strains with intermediate penicillin sensitivities (azithromycin MICs, 0.125 to 4 microg/ml) was studied by performing 50 sequential subcultures in medium with sub-MICs of these antimicrobial agents. For only one of the six penicillin-susceptible strains did subculturing in medium with amoxicillin (with or without clavulanate) lead to an increased MIC, with the MIC rising from 0.008 to 0.125 microg/ml. Five of the six penicillin-susceptible strains showed increased azithromycin MICs (0.5 to >256.0 microg/ml) after 17 to 45 subcultures. Subculturing in medium with cefaclor did not affect the cefaclor MICs of three strains but and led to increased cefaclor MICs (from 0.5 to 2.0 to 4.0 microg/ml) for three of the six strains, with MICs of other beta-lactams rising 1 to 3 twofold dilutions. Subculturing in cefuroxime led to increased cefuroxime MICs (from 0.03 to 0.06 microg/ml to 0.125 to 0.5 microg/ml) for all six strains without significantly altering the MICs of other beta-lactams, except for one strain, which developed an increased cefaclor MIC. Subculturing in azithromycin did not affect beta-lactam MICs. Subculturing of the four strains with decreased penicillin susceptibility in amoxicillin (with or without clavulanate) or cefuroxime did not select for beta-lactam resistance. Subculturing of one strain in cefaclor led to an increase in MIC from 0.5 to 2.0 microg/ml after 19 passages. In contrast to strains that were initially azithromycin susceptible, which required >10 subcultures for resistance selection, three of four strains with azithromycin MICs of 0.125 to 4.0 microg/ml showed increased MICs after 7 to 13 passages, with the MICs increasing to 16 to 32 microg/ml. All azithromycin-resistant strains were clarithromycin resistant. With the exception of strains that contained mefE at the onset, no strains that developed resistance to azithromycin contained ermB or mefE, genes that have been found in macrolide-resistant pneumococci obtained from clinic patients.     

In Vitro antimicrobial susceptibilities of Streptococcus pneumoniae isolated from two teaching hospitals in Taiwan, 1989-1995

The susceptibility of 46 pneumococcal isolates collected during October 1989 to May 1995 from National Taiwan University Hospital and Taipei Municipal Yang Ming Hospital was studied. Among these isolates, the resistant rate of penicillin G was 21.7%; the penicillin G-resistant strains were more frequently resistant than the penicillin-sensitive strains to other beta-lactam antimicrobial drugs. The minimum bactericidal concentrations (MBCs) of penicillin G for all isolates were equal to, or one dilution higher than, minimum inhibitory concentrations (MICs). Three strains were false positive for penicillin resistance among isolates of Streptococcus pneumoniae screened with oxacillin. On the other hand, resistance to penicillin G was often independent of resistance to erythromycin. Vancomycin was the most active agent tested.     

Antimicrobial resistance and serotype distribution of Streptococcus pneumoniae strains causing childhood infections in Bangladesh, 1993 to 1997

Three hundred sixty-two Streptococcus pneumoniae strains were isolated from children under 5 years of age at Dhaka Shishu (Children) Hospital from 1993 to 1997. The strains were isolated from blood (n = 105), CSF (n = 164), ear swab (n = 61), eye swab (n = 20), and pus (n = 12). Of the 362 isolates, 42 (11.6%) showed intermediate resistance (MIC, <0.1 microgram/ml) and only 4 (1.1%) showed complete resistance (MIC, >2.0 microgram/ml) to penicillin. Penicillin resistance exhibited a strong relationship with serotype 14; 47.8% of the penicillin-resistant strains belonged to this type. A remarkably high (64.1%) resistance to co-trimoxazole was observed, along with a significant increase during the time period studied; there was no relationship to capsular type. By way of contrast, penicillin resistance did not show any significant change during the study period. Resistance to chloramphenicol (2.2%) and erythromycin (1.1%) was rare. The high resistance to co-trimoxazole and its increasing trend demand elucidation of the clinical impact of pneumonia treatment by this antimicrobial and reconsideration of the World Health Organization recommendation for co-trimoxazole administration to children with community-acquired pneumonia at the health care worker level in Bangladesh.     

Recent trends in incidence of respiratory tract pathogens and antimicrobial susceptibilities of Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis isolated in 1994 and 1995

The incidence of pathogenic bacteria in respiratory tract infections in 1994 and 1995 was investigated using quantitative cultures of sputa from patients with the infections in our department. Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were isolated at high rates (70.5% in 1994 and 73.8% in 1995) from the specimens of out-patients, and the incident rates were similar to the past data. The antimicrobial susceptibilities of these three pathogens were examined with the agar dilution method. The incidence of penicillin (Pc) resistant S. pneumoniae against which MIC of Pc-G was higher than 0.125 microgram/ml was markedly increased from 24% in 1994 to 34.9% in 1995. Most of the Pc resistant isolates were also resistant to other antibiotics including erythromycin, minocycline and tosufloxacin. Serotype of strains against which MIC of Pc-G was higher than 1.0 microgram/ml was 19. The ratios of beta-lactamase-producing strains among H. influenzae isolated in 1994 and 1995 were 20 and 15.8%, respectively, which were slightly higher than those in the past. One quinolone resistant strain was isolated in this study. Although the ratio of beta-lactamase-producing strains among M. catarrhalis was as high (96.7%) as in the past, no increased resistance against the drugs examined was observed.