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We evaluated the efficacy of disk diffusion methods for detection of low-level β-lactamase-negative ampicillin-resistant (low-BLNAR) Haemophilus influenzae. Four hundred and seventy unselected, recent clinical isolates were tested with ampicillin (10 μg), cefaclor (30 μg) and cefuroxime (30 μg) on iso-Sensitest agar enriched with nicotinamide adenine dinucleotide (NAD) and horse blood [ST agar; Swedish Reference Group for Antibiotics (SRGA) guidelines], and on chocolate agar (in-house guidelines). Selected isolates (n = 147) were subjected to partial sequencing of the ftsI gene. Forty-seven strains (10.0%) were genotypically identified as low-BLNAR, which was confirmed by determination of minimal inhibitory concentration (MIC) using microbroth dilution method: only low level resistance to ampicillin was detected [MIC ≤1 μg/mL; MIC(50) = 0.5 μg/mL, implying susceptibility by Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antibiotic Susceptibility Testing (EUCAST) interpretative criteria]. The MIC of cefuroxime varied between 1 and 4 μg/mL (MIC(50) = 2 μg/mL), indicating susceptibility to cefuroxime by CLSI but not by EUCAST guidelines. Disk diffusion methods were able to discriminate low-BLNAR H. influenzae from the wild-type population with sensitivities ranging from 87% to 98% and specificities from 96% to 99%. Cefaclor was found to be superior to cefuroxime and ampicillin. Cefaclor zone diameter breakpoints of 30/29 and 23/22 mm are suggested for ST agar and chocolate agar, respectively., (© 2011 The Authors. APMIS © 2011 APMIS.)

Plasmid pB1000 is a small replicon recently identified as bearing bla(ROB-1) in animal and human Pasteurellaceae in Spain. We identified pB1000 in 11 bla(ROB-1)-positive Australian and North American Haemophilus influenzae isolates, suggesting a wider role for pB1000 in disseminating bla(ROB-1). Native H. influenzae conjugative elements can mobilize plasmids similar to pB1000 at a low frequency of 10(-8), and this might account for the infrequency of bla(ROB-1) compared to the rate of occurrence of bla(TEM-1). Altered penicillin-binding protein 3 was associated with an increased cefaclor MIC in 3 isolates.

Currently, beta-lactamase-negative (BLN) ampicillin-resistant (AR) strains of Haemophilus influenzae are prevalent in Japan. BLNAR strains are defined by the presence of specific mutation(s) in the ftsI gene but are not phenotypically distinguishable by ampicillin (ABPC) susceptibility. In the present study, we showed that cephalexin (CEX), cefsulodin (CFS), and cefaclor (CCL) disk diffusion tests can be effectively used to identify BLNAR strains. A total of 169 clinical isolates of BLN H. influenzae, consisting of 113 of BLNAR and 56 of BLN, ampicillin-susceptible (AS), were included. All the isolates were genetically defined by detection of the TEM gene and partial sequencing of the ftsI gene. The Clinical and Laboratory Standards Institute (CLSI) standard broth microdilution and disk diffusion tests for ABPC provided 20% and 19% false susceptible rates, respectively. Alternatively, 34 cephem agents were tested using disk diffusion. Of the agents tested, CEX, CFS, and CCL disks could effectively discriminate between BLNAR and BLNAS isolates. All the BLNAS isolates showed visible growth inhibitory zones around CEX and CFS disks, but 108 (95.6%) and 106 (93.8%) BLNAR isolates did not. The results indicated 100% predictive values (PVs) for BLNAR and PVs for BLNAS were 91.8% for CEX and 88.9% for CFS. The CLSI-based interpretations for CCL (> or =20 mm) also highly correlated with BLNAR and BLNAS, PVs for BLNAR and for BLNAS being 100% and 93.3%, respectively. With simplicity and discriminability of the test method, we recommend a CEX disk diffusion test in combination with a rapid beta-lactamase test to identify BLNAR isolates in clinical laboratories.

Plasmid pB1000 is a mobilizable replicon bearing the bla(ROB-1) beta-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, > or =64 microg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

We previously reported that standard methods overestimate cefaclor minimum inhibitory concentrations (MICs) for Streptococcus pneumoniae due to in vitro chemical instability. This study aimed to ascertain if standard methods accurately measure cefaclor MICs to Haemophilus influenzae. Cefuroxime was used as a comparator. Standard NCCLS broth microdilution and E-Test MICs were determined for eight isolates of H. influenzae. Kill curves determined the "bacteriostatic" MIC, defined as the concentration showing no significant growth or kill over six hours taking into account cefaclor instability. On average, cefaclor and cefuroxime bacteriostatic MICs were 0.2 x MIC and 0.6 x MIC determined by NCCLS methodology respectively. The mean MIC determined by NCCLS methodology was 3.0 mg/L for cefaclor and 0.8 mg/L for cefuroxime. Cefaclor MICs by NCCLS methodology were overestimated due to chemical instability over 18-24 hours. The bacteriostatic MICs by kill curve were not significantly different from those of cefuroxime.

The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 beta-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or beta-lactams plus beta-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which bla(ROB-1) mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to beta-lactamase inhibitors, a plasmid harboring bla(ROB-1) was transformed into hypermutagenic strain Escherichia coli GB20 (DeltaampC mutS::Tn10), and this construct was used in place of H. influenzae bla(ROB-1). Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected beta-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the bla(ROB-1) gene were obtained during the passages in the presence of the different concentrations of the selective agents. Mutants resistant to extended-spectrum cephalosporins harbored either the Leu169-->Ser169 or the Arg164-->Trp164 substitution or the double amino acid change Arg164-->Trp164 and Ala237-->Thr237. ROB-1 mutants that were resistant to beta-lactams plus beta-lactamase inhibitors and that harbored the Arg244-->Cys244 or the Ser130-->Gly130 replacement were also obtained. The cefaclor-hydrolyzing efficiencies of the ROB-1 variants were strongly decreased in all mutants, suggesting that if bla(ROB-1) mutants were selected by cefaclor, this drug would prevent the further evolution of this beta-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or beta-lactamase inhibitors.

Epidemiological and microbiological studies were carried out using 575 strains of Haemophilus influenzae isolated from clinical specimens at Kitakyushu municipal medical center from January 1996 through December 1999. The strains isolated multiply were excluded. The strains of H. influenzae did not increase for 4 years, and were detected more in summer season, peaked in July, and less in winter season. Like the cases of Streptococcus pneumoniae, most (91.8%) of the strains was detected in the specimens from the respiratory tract, and also they were isolated mainly from infants under 4-years old (25.6%) and adults over 65-years old (24.2%) MICs of 7 antimicrobial agents, such as ampicillin (ABPC), sulbactam/ABPC, cefaclor, imipenem, panipenem, meropenem (MEPM), and levofloxacin (LVFX) were determined using broth microdilution methods. Among 575 strains of H. influenzae isolated from clinical specimens, 51 ABPC-resistant strains (8.9%) produced beta-lactamase, and 67 strains (11.6%) were beta-lactamase negative ampicillin resistant H. influenzae. The ABPC-resistant strains existed in 20.5%. Both of MEPM and LVFX showed excellent antimicrobial activity against H. influenzae including ABPC-resistant strains. Four cases of meningitis were reviewed. All of H. influenzae isolated possessed type b capsular antigen. All patients recovered by appropriate antimicrobial treatment. But one adult patient developed serious sequela.

Objective: To determine the antimicrobial activity of Cefaclor against common respiratory tract pathogens isolated from patients in Pakistan., Setting: Department of Microbiology, Liaquat National Postgraduate Medical Centre, Karachi., Method: A laboratory analysis was done on 466 isolates of respiratory tract pathogens collected from 13 laboratories from all over Pakistan. Antibiotic sensitivity test was done by Kirby Bauer disc diffusion method and MIC of Cefaclor was determined by 'E' test., Result: Of the 163 Streptococcus pneumoniae, 87 Moraxella catarrhalis and 216 Haemophilus influenzae > 95% isolates were susceptible to Cefaclor. The MIC 90 of all these pathogens were < 2 ug., Conclusion: Resistance of respiratory tract pathogens to the 2nd generation Cephalosporin, Cefaclor is very low. MIC 90 of Cefaclor against all three common respiratory tract pathogens is < 2 ug, which indicates that Cefaclor would be effective in more than 90% of cases infected with these bacteria.

In vitro cefditoren antimicrobial activity was tested by broth microdilution and disk diffusion methods against 300 Streptococcus pneumoniae and 299 Haemophilus influenzae isolates. MICs were also determined for three comparison drugs. The MICs of cefditoren were very comparable to those of cefotaxime against both species. If penicillin-resistant pneumococci are to be considered not susceptible to cefditoren, the tentative MIC breakpoints for cefditoren of < or = 0.25 microg/ml for susceptible and > or 1.0 microg/ml for resistant could be selected. With these breakpoints, all penicillin-susceptible pneumococci were cefditoren-susceptible, as were 85% of penicillin-intermediate strains. Provisional zone diameter breakpoints would be > or = 26 mm for susceptible and < or = 20 mm for resistant. If penicillin-resistant pneumococcal infections are shown to clinically respond to cefditoren therapy, then a susceptible MIC breakpoint of < or = 1.0 microg/ml would be appropriate, with a corresponding zone diameter breakpoint of > or = 21 mm. A susceptible MIC breakpoint of < or = 0.5 or < or = 1.0 microg/ml is appropriate for H. influenzae, but lack of correlation between cefditoren MICs and disk diffusion zone diameters when testing H. influenzae leads us to make no recommendations at this time regarding cefditoren disk tests for H. influenzae.

beta-Lactamase production in Canadian isolates of Haemophilus influenzae has remained relatively constant (25-35%) over the last decade despite increasing cefaclor resistance (MIC >/= 32 mg/L). TEM (294/324, 90.7%) and ROB-1 (30/324, 9.3%) prevalence rates among 324 isolates of H. influenzae obtained from across Canada in 1997-1998 were similar (P > 0.05) to previously published reports. However, 66. 7% (26/39) of cefaclor-resistant isolates were ROB-1-positive (P < 0. 001) and the remaining four ROB-1-positive isolates were cefaclor-intermediate (MIC 16 mg/L). Susceptibilities to loracarbef (P < 0.001) and cefprozil were also reduced in the presence of ROB-1 while the activities of cefuroxime, cefotaxime, cefixime and imipenem were similar in both TEM- and ROB-1-positive solates.

Cefditoren, a third generation orally administered aminothiazolyl cephalosporin, has demonstrated bactericidal activity against many Gram positive and negative bacterial pathogens and stability against clinically important beta-lactamases. Cefditoren was compared to cefaclor, cefixime, and penicillins against 1 435 recently isolated strains of streptococci (312 Streptococcus pneumoniae, 165 viridans group streptococci, 142 beta-haemolytic streptococci), Haemophilus influenzae (521 strains), and Moraxella catarrhalis (295 strains). Streptococcus pneumoniae and viridans group streptococci had penicillin nonsusceptible rates of 37.8 and 35.8%, respectively. Cefditoren (MIC(90) in microg/ml/% susceptible) activity against all tested H. influenzae (0.03/100) and M. catarrhalis (0.06-0.5/100) was comparable to cefixime and significantly greater than cefaclor. Cefditoren (MIC(90), 0.5 microg/ml) was 4- to 128-fold more active than comparison beta-lactams against the pneumoococci and was the most potent beta-lactam (including penicillin) versus beta-haemolytic streptococci. Cefditoren pharmacokinetics demonstrate a T(1/2) of 1.5-2 h and C(max) values of 2.8 and 4.6 microg/ml, respectively with 200 or 400 mg doses of cefditoren pivoxil; plasma concentrations exceed 1 microg/ml for 4 to 6 hours (33-50% of dosing interval). Consequently, a susceptible MIC of /= 18 and >/= 15 mm (5-microg disk) for all cited fastidious species tested. Categorical agreement between MIC and disk tests was 94.6 to 100% with a correlation coefficient (r) range of 0.50 to 0.90 for streptococci. H. influenzae intermethod comparison results using the same interpretive criteria were in complete agreement, but exhibited a low r = 0.39. Cefditoren clearly possesses the most potent activity among currently studied oral cephalosporins or penicillin against commonly isolated bacterial pathogens causing bronchitis, pneumonia, sinusitis, or pharyngitis and was active against nearly all penicillin-resistant streptococci at

Study Objectives: To determine the time above minimum inhibitory concentration (T > MIC) and serum bactericidal activity of five oral cephalosporins against two strains of Haemophilus influenzae., Design: Randomized, crossover study., Setting: University-associated research center., Subjects: Ten healthy volunteers., Interventions: Each subject received a single dose of cefpodoxime 200 mg, cefuroxime 500 mg, cefaclor 500 mg, cefprozil 500 mg, or loracarbef 400 mg each week for 5 weeks. Blood for serum levels was obtained at time zero and 1, 2, 3, 4, 6, 8, and 12 hours after each dose., Measurements and Main Results: Cefpodoxime produced serum concentrations above the MIC for more than 90% of the time for both beta-lactamase-negative and -positive strains of H. influenzae. Moreover, it had serum bactericidal activity for 12 hours against both isolates. Cefuroxime was the second most active cephalosporin, with serum concentrations above the MIC of both isolates for 60% of the time. Cefuroxime provided serum bactericidal activity for 12 hours against the beta-lactamase-negative strain and 6 hours against the beta-lactamase-positive strain of H. influenzae. Even though the T > MIC was less than 50% of the study period for the other cephalosporins, all but cefaclor provided serum bactericidal activity for 12 hours against the beta-lactamase-negative isolate. Cefaclor provided measurable serum bactericidal activity for only 3 hours. The duration of serum bactericidal activity of cefprozil, loracarbef, and cefaclor against the beta-lactamase-positive isolate was 4, 2, and 0 hours, respectively., Conclusion: Cefpodoxime was the most active cephalosporin studied based on T > MIC and serum bactericidal activity against isolates of H. influenzae.

One hundred eighty consecutive, unduplicate isolates of Haemophilus influenzae from clinical specimens collected from November 1994 through February 1995 in nine general hospitals throughout Belgium were examined for beta-lactamase production using a nitrocefin-based test, and for their in vitro susceptibilities to ampicillin, amoxycillin/clavulanate, cefaclor, cefuroxime, cefotaxime, clarithromycin and azithromycin by means of the NCCLS agar dilution test. The isolates were all from respiratory tract specimens. The prevalence of capsular type b was 1.1%, and the overall rate of beta-lactamase production 16.7%. Rates of beta-lactamase production were higher in isolates from children (22.0%) than in those from adults (15.3%), and in isolates from upper respiratory tract specimens (22.0%) than in those from the lower respiratory tract (15.1%). Beta-lactamase-negative ampicillin resistance amounted to 1.1%. Cefotaxime had the highest activity on a weight basis [MIC (minimal inhibitory concentration) for 50% of the isolates tested (MIC50) < or = 0.06 microgram/ml], followed by ampicillin (MIC50 of 0.25 microgram/ml), amoxycillin/clavulanate and cefuroxime (MIC50 of 0.5 microgram/ml), azithromycin (MIC50 of 2 micrograms/ml), cefaclor (MIC50 of 4 micrograms/ml), and clarithromycin (MIC50 of 8 micrograms/ml). Cefotaxime was also the most active drug in terms of susceptibility rates of the isolates (100.0%), followed by amoxycillin/clavulanate and azithromycin (98.9%), cefuroxime (97.2%), cefaclor (89.4%), clarithromycin (82.8%), and ampicillin (82.2%). In conclusion, amoxycillin/clavulanate and cefuroxime retain an excellent activity against H. influenzae, while cefaclor lost some of its activity. The rate of susceptibility to azithromycin was markedly higher than that to clarithromycin; however, its ability to accumulate intracellularly while concentrations in serum and interstitial fluid remain low, should be considered, as it may represent a major drawback to its use in H. influenzae infections.

The aim of the current five-center collaborative study was to reassess the interpretive criteria for cefaclor, loracarbef, cefprozil and cefixime previously adopted or proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for disk diffusion susceptibility tests with Haemophilus influenzae on Haemophilus Test Medium (HTM) agar. MICs and zones of inhibition were determined using NCCLS methods, HTM and two collections of strains of Haemophilus influenzae. One group of strains consisted of 118 stock organisms taken largely from various recent U.S. antibiotic resistance surveillance studies. The emphasis in this selected group of organisms was on strains that were beta-lactamase negative but ampicillin resistant (BLNAR) by some other mechanism. The second collection of test organisms consisted of 50 recent clinical isolates of Haemophilus influenzae obtained from each of the five participating study centers. This group was considered representative of the type of Haemophilus influenzae currently recovered from clinical sources in the USA. Frequency distribution assessment and error-rate bounded analysis of scattergram comparisons of MICs and zone sizes were used to develop the following zone diameters interpretive for disk diffusion susceptibility tests with Haemophilus influenzae on HTM agar: cefaclor, > or = 20 mm (susceptible, S) and < or = 16 mm (resistant, R); loracarbef, > or = 19 mm (S) and < or = 15 mm (R); and cefprozil, > 18 mm (S) and < 14 mm (R). The respective MIC correlates for all three antimicrobial agents were < or = 8 micrograms/ml (S) and 32 micrograms/ml (R).(ABSTRACT TRUNCATED AT 250 WORDS)

HTM agar was used for in vitro study of bêta-lactam antibiotics activity on H. influenzae. Tested strains belong to the three ampicillin phenotypes: sensitive, betalactamase production and ampicillin resistance without production of betalactamase. Using 2 micrograms ampicillin disk, diameters > or = 20 mm and < 20 mm separate ampicillin sensitive and resistant strains. The following zone-size breakpoints could be suggested: ampicillin (10 micrograms) > or = 25 and < 22 mm; amoxicillin (25 micrograms) > or = 26 mm and < 23 mm; ampicillin/sulbactam (10/10 micrograms) > or = 25 mm and < 22 mm; amoxicillin/clavulanic acid (20/10 micrograms) > or = 26 mm and < 23 mm; cefaclor (30 micrograms) > or = 25 mm and < 20 mm; cefuroxime (10 micrograms) > or = 22 mm and < 19 mm; cefixime (10 micrograms) and cefpodoxime (10 micrograms) > or = 26 mm; cefotaxime (30 micrograms) et ceftriaxone (30 micrograms) > or = 30 mm. The zone-size breakpoints concentrations, distribution of bacterial populations, mechanisms of resistance. In vitro study may screen for ampicillin resistance mechanisms.

Cefaclor sustained its inhibitory activity against a beta-lactamase-producing strain of Haemophilus influenzae. Although a relatively high permeability coefficient was calculated for ampicillin compared with that calculated for cefaclor, the resulting periplasmic concentration of cefaclor was 5.7 times that of ampicillin. The efficacy of cefaclor may be due to its higher beta-lactamase resistance, which allows it to achieve a greater periplasmic concentration and adequate binding to crucial penicillin-binding proteins.

The susceptibility of 2,212 Haemophilus influenzae isolates cultured in UK clinical laboratories in 1991 was determined for four orally-administered beta-lactam drugs. These isolates included 1,893 ampicillin-susceptible, 191 beta-lactamase-positive and 128 ampicillin-resistant, beta-lactamase-negative Haemophilus influenzae. While 150 (6.8%) isolates were resistant to cefaclor (MIC > or = 16 mg/l) and 85 (3.8%) to loracarbef, all were inhibited by < or = 2 mg/l cefetamet and < or = 1 mg/l cefixime and were therefore susceptible to these agents. Ranges and modes of inhibition zone diameters and MICs indicated that the susceptibility of a variable proportion of the 191 beta-lactamase-positive isolates to cefaclor, loracarbef and cefetamet was reduced compared with the fully susceptible population. In contrast, a major reduction in susceptibility to all four antimicrobial agents was seen among the 128 ampicillin-resistant (MIC 1-64 mg/l) beta-lactamase-negative isolates such that these accounted for 53% and 67% of the total number of organisms resistant to cefaclor and loracarbef respectively. In addition, 23 of 25 isolates inhibited only by > or = 1 mg/l cefetamet and all eight inhibited only by > or = 0.5 mg/l cefixime showed this type of resistance to ampicillin. Results indicate the importance of detecting non-beta-lactamase-mediated resistance to ampicillin and any concomitant diminished susceptibility to other beta-lactam drugs.

Inconsistent quality control results in disk diffusion testing of cefaclor, cefamandole, cefonicid, and cefuroxime with Haemophilus influenzae ATCC 49247 and Haemophilus test medium (HTM) prompted a search for an alternative control strain that would provide more reliable results. A five-laboratory study was conducted to evaluate two candidate H. influenzae strains as possible alternatives to the aforementioned strain. Repetitive testing of the candidate strains and H. influenzae ATCC 49247 over several days with a total of six different lots of HTM documented consistent performance of the two candidate strains and confirmed inconsistent results for some of the antibiotics with H. influenzae ATCC 49247. In particular, certain lots of HTM failed to yield cefaclor and cefamandole zone sizes within the quality control range advocated by the National Committee for Clinical Laboratory Standards. Because of the greater consistency offered by the new strains, one was selected (now designated H. influenzae ATCC 49766) to be recommended for routine quality control testing of cefaclor, cefamandole, cefonicid, cefuroxime, and the related carbacephem loracarbef. The new control strain and zone size ranges proposed here have been approved by the National Committee for Clinical Laboratory Standards in place of the previously recommended strain and zone size limits for testing of these five cephem antibiotics.

The relationship between the growth rate and the kill rate was used to evaluate and to compare the in vitro bactericidal activities of cefdinir, a new oral cephalosporin, and cefaclor against Streptococcus pneumoniae and beta-lactamase-producing strains of Haemophilus influenzae. These frequently encountered pathogens of community-acquired respiratory tract infections are usually susceptible to both drugs. The MIC ranges for cefdinir and cefaclor were, respectively, 0.03 to 0.06 and 0.25 to 0.5 micrograms/ml for S. pneumoniae and 0.25 and 4 to 8 micrograms/ml for H. influenzae. The colony counts (CFU per milliliter) measured after 6 h of exposure to a range of antibiotic concentrations in broth were plotted against the colony count of the control culture over the same period of time. Higher kill rates versus bacterial growth rates were noted for S. pneumoniae for both drugs (positive balance). Conversely, lower kill rates versus growth rates were noted for H. influenzae for both drugs (negative balance). In conclusion, the bactericidal activities of both drugs against S. pneumoniae and H. influenzae were similar when expressed by the relationship between the growth rate and the kill rate at 6 h, but cefdinir was more active at lower concentrations.

The in vitro activity of ceftibuten, a new orally administered cephalosporin, was assessed against clinical isolates of Haemophilus influenzae and Branhamella catarrhalis. The activity of ceftibuten was compared to that of ampicillin, amoxicillin-clavulanic acid, and three oral cephalosporins, cefaclor, cefuroxime, and cefixime. With the exception of rare beta-lactamase-negative ampicillin-resistant strains of H. influenzae, resistance to ceftibuten was not observed with any of the study isolates. Ceftibuten was more active than amoxicillin/clavulanic acid for beta-lactamase-positive and -negative strains of H. influenzae; it was less active than this combination for B. catarrhalis. Ceftibuten was essentially equivalent in activity to cefixime against both Haemophilus and Branhamella but more active than cefaclor and cefuroxime against these two organisms.

Between 40 and 43 Haemophilus influenzae isolates obtained from Greece, Lebanon, Israel and Morocco respectively (a total of 167 strains) were analysed for prevalence of resistance to six different drugs. Of these isolates 12.6% produced beta-lactamase and were resistant to ampicillin. All isolates, except a single strain from Greece, were susceptible to cefaclor. The frequency of resistance to chloramphenicol and tetracycline was below 2%. The frequency of isolates susceptible and resistant to erythromycin and cotrimoxazole varied from country to country.

We evaluated the incidence of Haemophilus influenzae resistance to selected antimicrobials used in Canada. From 1985 to 1987, 2503 H. influenzae isolates obtained in 14 hospitals across Canada were sent to the Centre hospitalier de l'université Laval (CHUL) for identification, serotyping, biotyping and testing for beta-lactamase production. Susceptibility tests were done with the use of 12 antibiotics. Of the strains 424 (16.9%) produced beta-lactamase; the proportion varied from 12.8%, in Newfoundland, to 19.6%, in Ontario. Of the strains 18.3% were type b; 19.4% of those produced beta-lactamase. Almost 82% of the strains were not type b. The proportion of beta-lactamase-producing strains varied according to the isolation site, from 15.3% in the respiratory tract to 25.6% in the blood. The overall level of resistance was 19.3% to ampicillin, 24.2% to erythromycin, 3.8% to trimethoprim-sulfamethoxazole, 1.7% to amoxicillin-potassium clavulanate, 1.4% to cefaclor, 1.3% to tetracycline, 1.0% to rifampin, 0.7% to cefuroxime and 0.1% to cefamandole. Disc diffusion susceptibility testing revealed 64 strains (2.6%) that did not produce beta-lactamase but were resistant to ampicillin and 9 (0.4%) that produced beta-lactamase but were susceptible to ampicillin. The results of beta-lactamase production tests were identical regardless of whether the tests were done by the CHUL or by the other hospitals, but there was a marked difference in the susceptibility test results between the CHUL and the other centres. Our results suggest that the level of resistance of H. influenzae to antibiotics is increasing in Canada and that the initial choice of drug therapy may have to be modified.

In the second European survey of the prevalence of antimicrobial resistance in Haemophilus influenzae, 2529 clinical isolates collected in 1988/89 from 78 laboratories in nine countries (Austria, Belgium, Federal Republic of Germany, France, Italy, The Netherlands, Spain, Switzerland, United Kingdom) were examined. Of these, 23.8% were type b strains. The overall rate of beta-lactamase production was 9.1%, being slightly higher in type b isolates (10.5%) compared to non-type b isolates (8.6%). The MICs of six antimicrobials (ampicillin, cefaclor, chloramphenicol, erythromycin, tetracycline, and cotrimoxazole) were determined by an agar dilution procedure at a single central laboratory. The proportion of isolates resistant to the antimicrobials varied considerably amongst the individual countries. The highest incidence of resistance to all six drugs was observed in strains collected in Spain, whereas resistance was rarely encountered among strains isolated in Austria and the FRG. Resistance to ampicillin (MIC greater than or equal to 4 mg/l) among strains that lacked beta-lactamase activity was uncommon (0.3%). Based on the NCCLS Haemophilus influenzae breakpoints, the rates of susceptibility and resistance to cefaclor, chloramphenicol, tetracycline, and cotrimoxazole were 96.4/1.5, 96.2/2.8, 92.4/4.7, and 87.9/7.3%, respectively. The rate of susceptibility and resistance to erythromycin was 2.3/19.9. Multiple resistance to ampicillin, chloramphenicol, tetracycline, and cotrimoxazole was observed in 15 isolates (0.6%), and resistance to three drugs simultaneously in 72 (1.5%). The incidence of beta-lactamase producing strains was similar to that seen in the first European study performed in 1986 (9.1% vs. 10.9%), and was half that observed in US isolates collected in 1986.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro activity of amoxicillin in the presence of clavulanic acid against clinical isolates of Haemophilus influenzae and Branhamella catarrhalis was assessed in comparison with ampicillin, amoxicillin, cefaclor and erythromycin. The isolates were selected so as to yield equal numbers of beta-lactamase producing and non-beta-lactamase producing strains of the two species. MICs obtained by agar dilution indicated that amoxicillin in the presence of clavulanic acid was the most active of the drugs tested. Clavulanic acid potentiated the activity of amoxicillin against beta-lactamase-producing strains of both Haemophilus influenzae and Branhamella catarrhalis. Further studies on a few strains of each species revealed that the beta-lactamase of Haemophilus influenzae (TEM-1) rapidly inactivated ampicillin and slowly inactivated cefaclor but not cefuroxime. The Branhamella catarrhalis enzyme rapidly inactivated cefaclor, ampicillin and to some extent cefuroxime. Clavulanic acid afforded protection against the beta-lactamase action of both species when beta-lactam antibiotics were added to bacterial cultures.

The zone sizes and MICs of ampicillin, cefaclor and cephalexin were determined for 2458 clinical isolates of Haemophilus influenzae collected in 1986 during a national survey of the prevalence of resistance in this species. Cefaclor showed greater in-vitro activity than cephalexin against all isolates, with a modal MIC of 4 mg/l compared with 8 mg/l. MIC50 values for the 2201 ampicillin-sensitive and 157 beta-lactamase-positive resistant strains were similar for both cephalosporins (4 mg/l for cefaclor, and 8 mg/l for cephalexin). A further 100 strains with a reduced zone (less than 20 mm) to a 2 micrograms ampicillin disc showed a definite (MIC greater than or equal to 4 mg/l) or intermediate (MIC 1 or 2 mg/l) degree of intrinsic resistance to ampicillin. Both cephalosporins showed a marked and significant rise in MIC values for this group. Cefaclor is the more active agent against ampicillin-sensitive and beta-lactamase-positive H. influenzae but strains with an intrinsic mechanism of resistance to ampicillin are markedly less sensitive to both cefaclor and cephalexin.

Cefaclor appeared to be an effective antibiotic against both beta-lactamase-positive and beta-lactamase-negative strains of Haemophilus influenzae type b when tested with 10(5) colony-forming units per ml. With inocula in excess of 10(6) colony-forming units per ml, these organisms were neither inhibited nor killed at concentrations of 400 micrograms/ml. This inoculum effect was also demonstrated in time-kill curve studies.

The activities of ampicillin, cefaclor, cephalothin, and cefuroxime against invasive clinical isolates of Haemophilus infleunzae were studied to determine the correlation between resistance and beta-lactamase production. Approaches to in vitro susceptibility testing of cephalosporins in the clinical laboratory were also assessed. Three hundred and eight isolates of H. influenzae were tested for ampicillin susceptibility, and those which required for inhibition greater than or equal to 1.0 micrograms/ml of ampicillin were tested for beta-lactamase production with a chromogenic cephalosporin. Twenty-two percent of isolates produced beta-lactamase and 85% were serotype b. All isolates considered resistant (MIC greater than or equal to 2.0 micrograms/ml) to ampicillin produced beta-lactamase. A single beta-lactamase-producing isolate was identified, but was inhibited by 1.0 microgram/ml of ampicillin, and a zone diameter of 20 mm was produced by disk diffusion testing. One hundred and ninety-seven isolates were tested for susceptibility to cefaclor, cefuroxime, and cephalothin. Chloramphenicol susceptibility testing by disk diffusion was also performed on these isolates. General agreement and interchangeable results were found among these three cephalosporins by both agar dilution and disk diffusion methods. We conclude that ampicillin-resistant H. influenzae not producing beta-lactamase is rare among these isolates of H. influenzae responsible for invasive disease. Susceptibility testing results of cephalothin, cefaclor, or cefuroxime appear to be interchangeable, although results of cephalothin testing would tend to underestimate the activities of cefaclor or cefuroxime.

A total of 2,811 clinical isolates of Haemophilus influenzae were obtained during 1986 from 30 medical centers and one nationwide private independent laboratory in the United States. Among these, 757 (26.9%) were type b strains. The overall rate of beta-lactamase-mediated ampicillin resistance was 20.0%. Type b strains were approximately twice as likely as non-type b strains to produce beta-lactamase (31.7 versus 15.6%). The MICs of 12 antimicrobial agents were determined for all isolates. Ampicillin resistance among strains that lacked beta-lactamase activity was extremely uncommon (0.1%). Percentages of study isolates susceptible to cefamandole, cefaclor, cephalothin, and cephalexin were 98.7, 94.5, 87.3, and 43.3%, respectively. For 14 strains (0.5% of the total), chloramphenicol MICs were greater than or equal to 8.0 micrograms, and thus the strains were considered resistant. All of these resistant strains produced chloramphenicol acetyltransferase. In addition, all 14 strains were resistant to tetracycline; 11 produced beta-lactamase. The percentage of isolates susceptible to tetracycline was 97.7%. In contrast, erythromycin and sulfisoxazole were relatively inactive. The combination of erythromycin-sulfisoxazole (1/64) was more active than erythromycin alone but essentially equivalent in activity to sulfisoxazole alone. Finally, small numbers of clinical isolates of H. influenzae were resistant to trimethoprim-sulfamethoxazole and rifampin.

The first European survey of the prevalence of antibiotic resistance in Haemophilus influenzae was conducted between February and October 1986. Eighty laboratories in nine countries participated (Austria, Belgium, France, FRG, The Netherlands, Spain, Sweden, Switzerland and the UK). A total of 1,961 clinical isolates was examined for type b encapsulation, beta-lactamase production and susceptibility to ampicillin, chloramphenicol, cefaclor, erythromycin and tetracycline, using a unique microdilution method. The proportion of isolates resistant to these antibiotics varied considerably between individual countries. The highest prevalence of ampicillin resistance was found in Spain (30.6%), and the lowest in the FRG (1.6%), with a mean value of 10% for all countries. Chloramphenicol resistance was highest in Spain (24.9%) and Belgium (10.9%) and lowest in The Netherlands (0.6%) and Austria (0.5%), with a mean value of 4.7%. Resistance to erythromycin ranged from 27% of the isolates in The Netherlands to 1.1% in Austria. For tetracycline, values ranged from 1.5% in the UK to 17.8% in Belgium and 25.4% in Spain. The lowest mean prevalence of resistance was observed for cefaclor (breakpoint 8 mg/l): 5% or less in all countries. These inter-country differences could only partially be explained by variations in the proportion of type b strains, the source of the isolates and the mode of collection.

The activity of phenoxymethylpenicillin (PcV), ampicillin, cefaclor, cefuroxime, chloramphenicol, co trimoxazole, doxycycline and erythromycin against clinical isolates of Branhamella catarrhalis, Haemophilus influenzae, pneumococci, group A streptococci and Staphylococcus aureus in 1983 was investigated with the MIC-method (plate-dilution technique). Forty-six percent of B. catarrhalis, 2% of H. influenzae and 78% of S. aureus were beta-lactamase producing and had high MIC-values for penicillin and ampicillin. Thus MIC for 90% of all strains of B. catarrhalis was 32 mg/l and 8 mg/l for penicillin and ampicillin while MIC for 90% of non beta-lactamase producing Branhamella strains was 2 mg/l and 0.25 mg/l respectively. This indicates a high susceptibility of penicillins to the action of Branhamella beta-lactamase. Almost all strains of B. catarrhalis, pneumococci, group A streptococci and S. aureus were inhibited at low concentrations of erythromycin. However, 4 mg/l was required to inhibit 90% of H. influenzae. Co-trimoxazole and doxycycline had good activity against all B. catarrhalis and H. influenzae strains while a few pneumococci, streptococci and staphylococci had intermediate sensitivity or were resistant. Essentially all strains were sensitive to cefuroxime and chloramphenicol.

The antibacterial activity of beta-lactams against H. influenzae is difficult to evaluate as a result of the osmotic-pressure-dependent formation of filaments and spheroplasts whose viability is still under debate. We compared growth curves obtained by optic density measurements and by UFC/ml counts for H. influenzae in the presence of amoxicillin, cefaclor and cefotaxime in various concentrations. Only the early response to antibiotics, observed during the first six hours of antibiotic-culture contact, was considered. Results showed, for each of the three antibiotics: an increase in optic density with formation of abnormal organisms that correlated poorly with antibiotic concentrations; a stable number of UFC/ml for more than 90 mn, followed by a slow fall reaching at the most 1.5 Log 10 UFC/ml at the sixth hour. We conclude that amoxicillin, cefaclor and cefotaxime in active concentrations rapidly produce the formation of abnormal organisms with an increase in biomass as a result. These abnormal organisms lose their viability slowly, even with concentrations greater than 50 time the MIC. Cefaclor's MIC and MBC are underestimated when results are read only after 24 hours.

Between October 1, 1980, and September 30, 1981, a prospective statewide study of Haemophilus influenzae infections identified 220 culture-proved systemic infections. Of these, 92% were caused by H. influenzae type b. Antimicrobial susceptibility testing of the type b isolates showed that 17% were resistant to ampicillin, but virtually all were susceptible to chloramphenicol, trimethoprim-sulfamethoxazole, cefaclor and rifampin. Ampicillin resistance is common in all parts of Tennessee and chloramphenicol remains the drug of choice for initial therapy of systemic H. influenzae infections.

A total of 126 strains of Haemophilus influenzae were examined for susceptibility to amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, cefaclor, and erythromycin by an agar dilution procedure. Fifty strains (eight type B, 42 non-type B), all with ampicillin minimal inhibitory concentrations (MIC) of greater than or equal to 6.2 micrograms/ml, produced beta-lactamase. The remaining 76 strains (18 type B, 59 non-type B) were beta-lactamase-negative. All of these strains had ampicillin MICs of less than or equal to 0.8 micrograms/ml. The combination of amoxicillin and clavulanic acid (2:1) was highly active against all strains tested. With the exception of two strains with amoxicillin/clavulanic acid MICs of 1.6/0.8 ug/ml, all strains were inhibited by concentrations of less than or equal to 0.8/0.4 ug/ml. Trimethoprim/sulfamethoxazole was also found to be highly active (MICs uniformly less than or equal to 0.1/1.9 ug/ml). Cefaclor and erythromycin were the least active of the agents tested. Fourteen strains (10.6%) had cefaclor MICs of greater than 32 ug/ml. Forty-seven strains (35.6%) had erythromycin MICs of greater than 8 micrograms/ml. With the exception of amoxicillin/clavulanic acid beta-lactamase production did not seem to influence the activity of any of the antimicrobials tested. Minimum inhibitory concentrations of amoxicillin/clavulanic acid, although still well within achievable serum levels, were approximately one twofold dilution higher with beta-lactamase-producing H. influenzae type B strains than with beta-lactamase-negative strains.

The results are presented of the first surveillance study in Greece on resistance in strains of Haemophilus influenzae (n = 61) and Haemophilus parainfluenzae (n = 96) to six antibiotics. Strains were isolated in a three-month period in 1987 in most cases from sputum specimens from adult patients with lung infections. The overall rate of resistance to ampicillin was 28.3%, to chloramphenicol 2.7%, to cefaclor 2.7%, to tetracycline 10.6%, to erythromycin 38.1% and to cotrimoxazole 5.3%. It is evident that the resistance rate of Haemophilus spp. is steadily increasing in Greece as in other European countries. Regular surveys of resistance patterns for both older and newer antimicrobial agents are therefore necessary.

The in vitro determination of the T log and the minimal bactericidal time of cefatrizine was evaluated against H. influenzae, Str. pyogenes A isolated from clinical specimens. and K. pneumonia ATCC 10031 and compared with that of amoxycillin, cefaclor, cefroxadine and miocamycin against the same bacterial strains. Cefatrizine demonstrate the shorter TMB against all bacterial strains and antibiotic tested with MBCs.

Altogether 403 Haemophilus spp. were isolated in seven hospital laboratories in Hong Kong during June 1986, mostly from sputum. Of these 73% were Haemophilus influenzae and 27% Haemophilus parainfluenzae. All the isolates of H. influenzae were non-capsulated; Haemophilus spp. were not isolated from blood or cerebrospinal fluid (CSF) during the period of the study. Antimicrobial resistance, including multiple resistance, was common. Of all the strains of H. influenzae, 20% were resistant to 1 mg/l ampicillin, (all except one by production of TEM-1 beta-lactamase), 65% were resistant to 0.5 mg/l erythromycin, 25% to 1 mg/l tetracycline, 14% to 1 mg/l chloramphenicol (mediated by the production of a chloramphenicol-destroying enzyme) and less than 1% to 8 mg/l cefaclor and 0.5 mg/l trimethoprim. All isolates were susceptible to cephamandole and cefuroxime. Haemophilus parainfluenzae showed similar susceptibilities, except that a greater proportion of strains was sensitive to erythromycin and chloramphenicol. Only 50% of the ampicillin-resistant strains of H. influenzae and H. parainfluenzae contained detectable plasmids of 2-55 Mdal arranged in six to nine different plasmid profiles. Resistance to ampicillin and chloramphenicol has increased markedly in isolates of H. influenzae in Hong Kong over the last 5 years. This resistance may be associated with transposable genes.

The antibacterial activity of cefaclor against 100 non-beta-lactamase producing and 11 beta-lactamase producing isolates of Haemophilus influenzae was compared with that of cephalexin, ampicillin, chloramphenicol, tetracycline and co-trimoxazole. A new standardized microtiter dilution technique was used. Cefaclor showed greater activity than did cephalexin and inhibited beta-lactamase producing H. influenzae isolates. Ampicillin was the most active compound against non-beta-lactamase producing isolates. One of our strains was resistant to chloramphenicol and one resistant to tetracycline.

The prevalence of antimicrobial resistance was assessed among a total of 3,356 clinical isolates of Haemophilus influenzae obtained from 22 medical centers distributed throughout the United States during the period July, 1983 through June, 1984. All strains were examined for beta-lactamase production with a rapid acidometric assay and for resistance to ampicillin, chloramphenicol, cephalothin, cefamandole, cefaclor, tetracycline, and erythromycin with a standardized disk diffusion procedure. The overall rate of beta-lactamase production was 15.2%, although results of disk diffusion tests suggested that the overall rate of ampicillin resistance was 19.5%. Twenty-one percent of encapsulated type b strains produced beta-lactamase; 12.1% of non-type b strains were beta-lactamase positive. Specific rates of beta-lactamase production obtained at individual study centers varied widely with no evidence of geographic clustering. The highest rates of beta-lactamase production were observed with isolates of H. influenzae recovered from infants and young children, and from blood and cerebrospinal fluid specimens. The overall rate of chloramphenicol resistance was 0.6%. The prevalence of cephalothin, cefamandole, cefaclor, tetracycline, and erythromycin resistance was 9.9%, 2.4%, 2.8%, 6.4%, and 64.2%, respectively. beta-Lactamase positive isolates of H. influenzae had higher rates of resistance to all of the cephalosporins than did strains that lacked beta-lactamase.

BMY-28100 is a new orally absorbed cephalosporin whose in vitro activity was compared with that of cefaclor. BMY-28100 was more active against Staphylococcus aureus and Haemophilus influenzae than was cefaclor. In addition, there was only a small rise in MIC50 and MIC90 values for BMY-28100 when the inoculum size was increased 100- or 10,000-fold. In contrast, MIC50 and MIC90 values increased significantly for cefaclor with similar increases in inoculum size. These data suggest that BMY-28100 may be a promising agent to test for oral administration in infections caused by S. aureus and H. influenzae.