Comparison of the serum-supplemented Todd-Hewitt and the new Haemophilus test media for broth microdilution susceptibility testing of Streptococcus pneumoniae.

Horse serum-supplemented Todd-Hewitt broth (STH) in use at Hôpital Ste-Justine for the last 12 years was compared to the recently proposed Haemophilus test medium (HTM), for broth microdilution susceptibility testing of Streptococcus pneumoniae. One hundred and twenty S. pneumoniae isolates from pediatric clinical specimens were used in this study. In general, the minimum inhibitory concentrations (MICs) in STH for 15 antimicrobial agents were quite comparable to those determined in HTM but tended to be higher. Drugs which generated MICs within +/- 1 log2 concentration differences in both media included penicillin, ampicillin, oxacillin, cefuroxime, cefotaxime, cefixime, clindamycin, chloramphenicol, trimethoprim-sulfamethoxazole, rifampin, ciprofloxacin and vancomycin. Cefaclor and tetracycline MICs tended to be > or = 2 log2 dilutions higher with STH for most of the isolates tested, while erythromycin MICs were often 2 log2 dilutions lower with STH than with HTM. Despite some differences in MICs noted above, few very major (0.4%), major (0.2%) and minor interpretive category errors (4.4%) were observed. The visual reading of the MICs for most of the 120 clinical isolates tested was generally easier in STH which was superior in supporting best the bacterial growth as detected by spectrophotometry. The risk of false susceptibility is thus decreased by using STH rather than HTM; furthermore, STH is free of the technical problems of the lysed horse blood Mueller-Hinton (LHB-MH) recommended by the NCCLS.

Routine susceptibility testing of four antibiotic combinations for improvement of laboratory guide to therapy of cystic fibrosis infections caused by Pseudomonas aeruginosa.

Previous studies have demonstrated synergy between an aminoglycoside and a beta-lactam for treating Pseudomonas aeruginosa infections. Cystic fibrosis patients are prone to infection by this bacterium, which becomes very resistant with recurrent antibiotic treatments. The purpose of this study was to evaluate the susceptibility patterns of 122 isolates of P. aeruginosa isolated from cystic fibrosis patients to five individual antibiotics (tobramycin, ceftazidime, piperacillin, ticarcillin, and imipenem) and to four antibiotic combinations (tobramycin associated with one of the other antibiotics). Strains were selected because of their resistance to individual antimicrobial agents, which ranged from 21.3% for imipenem to 56.5% for tobramycin. By using an automated broth microdilution method, we were able to demonstrate synergy against 39 strains (32%) with tobramycin-ticarcillin, against 38 strains (31%) with tobramycin-piperacillin, against 47 strains (39%) with tobramycin-ceftazidime, and against 23 strains (19%) with tobramycin-imipenem. Of the 122 isolates, 77 (63%) were rendered significantly susceptible to at least one of the four antibiotic combinations by synergy. These results suggest that when appropriate technology is available, susceptibility to antibiotic combinations greatly improves the guide to antibiotic therapy for infections due to P. aeruginosa in cystic fibrosis patients.

Effect on Pseudomonas aeruginosa alginate expression of direct plating and culture of fresh cystic fibrosis sputum on to pseudomonas isolation agar containing subinhibitory concentrations of roxithromycin and rifampicin.

The effects of subinhibitory concentrations of roxithromycin (16 mg/L) or rifampicin (16 mg/L) on alginate production by Pseudomanas aeruginosa were investigated. The weight of purified alginate from antibiotic-free cultures was significantly greater (52.5 +/- 24.0 mg, range 22.4-109.5), compared with alginate from cultures bacteria exposed to sub-MIC of roxithromycin (21.9 +/- 17.0, 0.0-42.1 (P < or = 0.037)) and to sub-MIC of rifampicin (28.6 +/- 15.0, 2.9-47.5 (P < or = 0.038)). Chromatographic analysis of hydrolysed and chemically transformed sub-units of alginate revealed that the presence and the molar ratio of D-mannuronic acid and L-guluronic acid were not affected in the remnant alginate exposed to sub-MIC of roxithromycin in contrast to that in the remnant alginate exposed to sub-MIC of rifampicin.

Pneumococcal resistance to antimicrobial agents in the province of Québec, Canada.

The serogroup/serotypes (SGTs) and antimicrobial susceptibilities to 10 antimicrobial agents of 110 clinical strains of Streptococcus pneumoniae were determined. Strains intermediately resistant or highly resistant to penicillin G (80 of 110) belonged predominantly to SGTs 23 (45.0%), 19 (13.7%), 6 (10.0%), 9 (6.2%), and 14 (3.7%). The MICs of all cephalosporins, tetracycline, trimethoprim-sulfamethoxazole, and chloramphenicol increased along with the MICs of penicillin G. However, erythromycin resistance and clindamycin resistance were observed more frequently among the intermediately penicillin-resistant strains. Multiple resistance was observed for 32 strains, of which 25 were highly resistant to penicillin G and belong to SGT 23F. All strains were susceptible to vancomycin.

The evolving pattern of pediatric endocarditis from 1960 to 1985.

A diagnosis of endocarditis was made in 37 patients (three days to 21 years old) on the basis of the following: histology in 11; at least two positive blood cultures in patients with underlying cardiac disease in 22; less than two positive blood cultures, vegetations seen at echocardiography and a suggestive clinical syndrome in four. Twenty-six patients had primary endocarditis (17 with pre-existing cardiopathy, nine with normal hearts). The 11 others developed secondary endocarditis following heart surgery (early onset in six, late onset in five). The mean delay before diagnosis was prolonged 35.8 days. The clinical and laboratory findings included weakness in 36 patients, fever in 35, new or modified heart murmur in 14, positive blood cultures in 30, anemia in 12, high white blood cell count in 15, increased sedimentation rate in 14, and positive echocardiogram in 11. Etiologic agents isolated were: streptococci in 17, staphylococci in seven, miscellaneous germs in eight, and aspergillus in two. Mortality was greater in patients less than one year old, infected with aspergillus or without underlying heart disease. The present study suggests that childhood endocarditis remains uncommon but presents a poor prognosis with a mortality of 27% and a morbidity of 85.7%.

Quantitative effect of roxithromycin and rifampicin on mucoid cultures from directly plated sputum of cystic fibrosis patients chronically colonized with Pseudomonas aeruginosa.

A previous study has shown that non-anti-Pseudomonas aeruginosa antibiotics can modify the mucoid character, a major virulence factor in cystic fibrosis P. aeruginosa. The purpose of the present study was to investigate further this phenomenon using a quantitative approach on fresh sputum. A total of 0.1 to 0.5 ml of sputum were homogenized, decimally diluted, plated on three series of Difco Pseudomonas isolation agar containing no antibiotics, roxithromycin (16.0 microgram/ml) or Rifampicin (16.0 microgram/ml) and incubated at 35 degrees C. In each of the three series, the cfus of PA by ml of sputum were evaluated after 24h, and the entire culture of plates with confluent growth was harvested after 96h and weighed on an analytical scale according to the correlation observed between the mucosity and the density of the culture. To date, 36 sputa from twenty-nine cystic fibrosis patients known to be chronically colonized by mucoid P. aeruginosa have been submitted to the protocol; the mean weight of control, roxithromycin and rifampicin cultures was respectively 3.12 +/- 1.62, 1.90 +/- 1.67 (p less than or equal to 0.0005) and 1.83 +/- 1.24 g (p less than or equal to 0.005) in parallel with mean cfu/ml of 2.24 +/- 6.45 x 10(8) (p less than 0.03) and 1.65 +/- 4.42 x 10(8) (p greater than 0.05); a more constant reduction (greater than or equal to 20%) of mucosity (expressed as the ratio of weight of antibiotic culture/control culture x 100) was observed with rifampicin (29 vs. 23/36 sputa), whereas a more drastic reduction (greater than or equal to 80%) was observed with roxithromycin (12 vs. 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating immune complexes, antibodies to Pseudomonas aeruginosa, and pulmonary status in cystic fibrosis.

Serum samples from 37 patients with cystic fibrosis (CF), whose lungs were colonized by Pseudomonas aeruginosa, were tested in a 1 yr prospective study to examine a possible relationship between levels of circulating immune complexes (CIC) and the following parameters: level of specific antibodies to P. aeruginosa; relative importance of P. aeruginosa mucoid and non-mucoid strains isolated from sputum; the forced expiratory volume (FEV1; percentage predicted); the chest X-Ray score (Brasfield system) and the clinical score (Shwachman system). Reactivity of CIC against P. aeruginosa, Staphylococcus aureus, Haemophilus influenzae and Escherichia coli antigens were also assayed. We found that the FEV1, the chest X-Ray and the clinical scores were significantly lower in patients with high levels of CIC than in those with normal levels of CIC (p less than 0.001 for each). We also found that the level of IgG antibodies against P. aeruginosa was significantly higher (p less than 0.001) in patients with high levels of CIC than in those with normal levels of CIC. 78% of patients with high levels of CIC had predominantly mucoid P. aeruginosa isolates whereas only 21% of patients with normal levels of CIC had also predominantly mucoid P. aeruginosa isolates. Specific antibodies to P. aeruginosa were detected in all CIC isolated by polyethylene glycol precipitations from CF patients exhibiting both high levels of CIC and inferior pulmonary status. Our findings support the hypothesis that a high level of CIC in association with an aggressive humoral response to P. aeruginosa correlates with defective pulmonary status in cystic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA probe technology for rapid detection of Haemophilus influenzae in clinical specimens.

In a previous study, we reported that a 5-kilobase Haemophilus influenzae DNA fragment involved in penicillin-binding protein expression could be used as a probe for specific detection of H. influenzae strains (F. Malouin and L. E. Bryan, Mol. Cell. Probes 1:221-232, 1987). Here, we report the ability of this probe to detect H. influenzae in clinical specimens. In a bacterial dot experiment, there was strong hybridization of the 32P-labeled probe to nonencapsulated and serotype a through f H. influenzae strains. The detection of H. influenzae in body fluids was then evaluated by using pooled human serum, urine, cerebrospinal fluid, and sputum as dilution media for H. influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, and Escherichia coli cells. At 65 degrees C, the probe hybridized to H. influenzae and H. aegyptius (greater than or equal to 10(5) cells) in all fluids. There was no hybridization with the E. coli negative control, and H. parainfluenzae hybridized when greater than or equal to 10(7) cells were used. Experiments performed at 73 and 80 degrees C permitted elimination of H. parainfluenzae hybridization. The detection of H. influenzae in 232 sputa from patients with respiratory tract infections was very specific (96 to 97%) and sensitive (74 to 100%) when the total time of the procedure was sufficient (6 to 24 h) and when the experiments were performed at 80 degrees C. In addition, the probe detected three of three and four of four H. influenzae-infected cerebrospinal fluids and blood cultures, respectively, and did not react with pneumococcus- or streptococcus-infected cerebrospinal fluids. Finally, by using a small-scale procedure, the probe rapidly detected H. influenzae in cerebrospinal fluid and sputum specimens (4 and 8 h, respectively). These results imply prompt diagnosis of H. influenzae infections caused by nonencapsulated and serotype a through f strains.

Mucoid and pigmentation characters can be suppressed by non-anti-Pseudomonas aeruginosa antibiotics in cystic fibrosis: a report of promising preliminary results.

In an effort to develop new strategies, the authors are exploring the hypothesis that non-anti-Pseudomonas aeruginosa antibiotics can affect some major virulence factors in cystic fibrosis P. aeruginosa. Recent samples (127) of P. aeruginosa isolated from the sputum of cystic fibrosis patients and manifesting a mucoid growth were stored at -70 degrees C until experimentation. The mucoid character was retested after thawing and one 24-h in-vitro passage at 37 degrees C onto 100-mm blood agar plates to prepare a bacterial suspension for inoculation (Steers or MIC-2000 plus replicator) and cultivation (48 h, 37 degrees C) onto different 100-mm or 150-mm Mueller-Hinton agar plates containing either no antibiotics or antibiotics alone, and antibiotic combinations without presumed anti-P. aeruginosa activity. Some isolates (49, 38.6%) of P. aeruginosa expressed again the mucoid character on antibiotic-free Mueller-Hinton agar and growth was prevented by antibiotics in a number of them: 7 with doxycycline (16.0 micrograms/ml), 2 with rifampicin (16.0 micrograms/ml), 6 with roxithromycin-sulfamethoxazole (16.0-304.0 micrograms/ml) and 23 with trimethoprim-sulfamethoxazole. In the remaining evaluable isolates, the mucoid was modified towards the rough character in 36 out of 42 with doxycyline, 36 out of 47 with rifampicin, 31 out of 43 with roxithromycin-sulfamethoxazole and 20 out of 26 with trimethoprim-sulfamethoxazole. In parallel, the pigmentation was lost in the presence of antibiotics in those P. aeruginosa isolates having manifested this character on Mueller-Hinton agar. These results suggest that non-anti-P. aeruginosa antibiotics can modify the in vitro markers of virulence in cystic fibrosis P. aeruginosa and that they merit further investigation.

Roxithromycin alone and in combination with sulphamethoxazole against Haemophilus influenzae.

Haemophilus influenzae is only moderately susceptible to erythromycin but previous studies in vitro and in vivo have shown that it is fully susceptible when erythromycin is combined with sulphonamides. The purpose of this study was to evaluate whether the activity of the new macrolide antibiotic roxithromycin was improved after combination with sulphamethoxazole. One hundred and eighty fresh clinical isolates of Haemophilus influenzae, comprising 74 ampicillin-susceptible, beta-lactamase negative, and 106 ampicillin-resistant, beta-lactamase positive, strains, were tested for their susceptibility to erythromycin, roxithromycin, sulphamethoxazole, erythromycin-sulphamethoxazole and roxithromycin-sulphamethoxazole in a fixed ratio of 1:19. Bacteriostatic and bactericidal synergy was found between roxithromycin and sulphamethoxazole more commonly than between erythromycin and sulphamethoxazole. Antagonism, was less common between roxithromycin and sulphamethoxazole than erythromycin and sulphamethoxazole. These results suggest that the in-vitro activity of roxithromycin is improved by combination with sulphamethoxazole, making this combination more attractive in the oral therapy of infections due to H. influenzae.

Antibiotic interaction of amoxycillin and clavulanic acid against 132 beta-lactamase positive Haemophilus isolates: a comparison with some other oral agents.

We studied the specific beta-lactamase inhibitory activity of clavulanic acid in association with amoxycillin against 132 beta-lactamase producing Haemophilus isolates. Inhibitory synergy between amoxycillin and clavulanic acid (ratio 2:1) was found in 131/132, partial synergy or antagonism in none; bactericidal synergy was found in 124/131, partial synergy in 4 and antagonism in 1. In comparison, inhibitory synergy between trimethoprim and sulphamethoxazole (ratio 1:19) was found in only 39/104 beta-lactamase positive strains, partial synergy in 42 and antagonism in 3 and bactericidal synergy in 18/104, partial synergy in 8 and antagonism in 3. The amoxycillin-clavulanic acid combination expressed significantly (P less than 0.001) more frequent synergy, at both inhibitory and bactericidal levels, than the trimethoprim-sulphamethoxazole combination. The synergy of amoxycillin-clavulanic acid resulted in a significant decrease of MIC90 (greater than or equal to 32.0-2.0 mg/l) and MBC90 (greater than or equal to 32.0-4.0 mg/l) of amoxycillin; the synergy of trimethoprim-sulphamethoxazole resulted in a significant decrease of MIC90 (8.0-2.0 mg/l) of trimethoprim but did not change MBC90. The amoxycillin-clavulanic acid combination was also more active than cefaclor or erythromycin alone against the 132 beta-lactamase producing strains.

In vitro comparison of ampicillin-chloramphenicol and ampicillin-cefotaxime against 284 Haemophilus isolates.

Since November 1982 at the Sainte-Justine Hospital in Montreal, ampicillin and cefotaxime were used in association as initial treatment (greater than or equal to 48 h) for childhood bacterial meningitis. In this report is described the in vitro interaction of the new regimen in comparison with that of the previous ampicillin-chloramphenicol combination against 284 Haemophilus isolates. Among the 156 ampicillin-susceptible, beta-lactamase-negative isolates, synergy was detected in 13 with ampicillin-cefotaxime, and antagonism was detected in only 1; in contrast, synergy was found in only 2 strains with ampicillin-chloramphenicol, and antagonism was found in 15. These differences were statistically significant (P less than 0.01). Such significant differences were not observed among the 128 ampicillin-resistant, beta-lactamase-positive Haemophilus isolates. The synergy of ampicillin-cefotaxime did not contribute to a decrease of the MIC of cefotaxime for 90% of isolates tested, whereas the antagonism of ampicillin-chloramphenicol did not contribute to increase the MIC of ampicillin for 90% of isolates tested.

A comparison of ampicillin-cefotaxime and ampicillin-chloramphenicol in childhood bacterial meningitis: an experience in 55 patients.

Ampicillin-cefotaxime was tested as initial therapy of presumptive bacterial meningitis in 55 children greater than or equal to 2 months of age at our hospital. During the first year of this ongoing trial, 11 patients, 10 whose CSF yielded ampicillin-resistant Haemophilus influenzae type b (MIC greater than 16 mg/l, beta-lactamase +) and one, indole-negative proteus (MIC 4 mg/l), were begun on ampicillin-cefotaxime and then continued on cefotaxime alone. All did well clinically except one who convulsed briefly but recovered without sequelae. The cefotaxime MICs/MBCs of the beta-lactamase-positive H. influenzae isolates (less than or equal to 0.007 to 0.03/less than or equal to 0.007 to 0.12) and the proteus isolate (0.03/0.12) were significantly lower than chloramphenicol MICs/MBCs (0.25 to 1.0/0.5 to 1.0 and 8/greater than 16). We followed 44 other children with meningitis due to ampicillin-sensitive organisms who were treated with ampicillin or penicillin after 1 or 2 days of ampicillin-cefotaxime. Aetiological agents included ampicillin-sensitive H. influenzae (25), pneumococci (9), meningococci (8), Strept. MG (1) and Listeria monocytogenes (1). 40/44 recovered uneventfully. There were 4 neurological complications: the streptococcal meningitis sustained a brain abscess and the three others were motor incoordination (sensitive haemophilus), hearing loss and subdural effusion (2 pneumococci). There were no deaths. 18/48 children managed initially with ampicillin-chloramphenicol during the same 12-month period one year earlier had significant neurological complications and/or sequelae and there was one death; aetiological agents included sensitive H. influenzae (30), pneumococci (9), ampicillin-resistant haemophilus (5), meningococci (3) and pneumococci plus strept. MG (1). The two groups were comparable except for the number of resistant haemophilus and meningococcal strains and underlying disease more frequent in the ampicillin-cefotaxime group. A significant reduction of neurological morbidity (5/55 or 9.1% vs. 18/48 or 37.5%:P less than 0.001) was therefore associated with the ampicillin-cefotaxime schedule in the initial treatment of proven bacterial meningitis. A prolonged hospitalization (greater than 15 days) was less frequent (P less than 0.01) in the ampicillin-cefotaxime group (3/55 or 5.5% vs. 13/48 or 27.1%). The results of the trial to date are considered to be very promising.

Growth retardation and prevention of Ehrlich solid tumor by Clostridium perfringens type A spores and culture supernatant.

When given by direct s.c. injection into the Ehrlich solid carcinoma 1 week after s.c. tumor transfer, viable crude spores of Clostridium perfringens type A (attenuated mutant strain LNG11 ATCC 29348) inhibited tumor growth and significantly prolonged the life span of male outbred Swiss mice. Under these conditions a concentrated sterile supernatant of a C. perfringens culture proved to be slightly more effective than were viable crude spores. In contrast viable crude spores were ineffective in the treatment of female Swiss mice, but the sterile supernatant retained significant activity. When given at the time and site of s.c. grafting of Ehrlich tumor cells, a concentrated sterile supernatant of a C. perfringens culture prevented tumor growth in 80% of male outbred Swiss mice. Under these conditions viable crude spores prevented tumor growth in 70% of mice and significantly prolonged the life span in the other 30%. When given by i.p. injection and before i.p. grafting of tumor cells, viable crude spores of C. perfringens prevented Ehrlich ascites tumor in 5 of 12 Swiss mice and prolonged life span in the other 7. In contrast concentrated sterile supernatant and viable purified spores were ineffective in the prevention or delay of the growth of Ehrlich ascites tumor. These data indicate that C. perfringens can be a potent antitumor agent without producing the harmful anaerobic infection of solid tumors (clostridial oncolysis.