In vitro development of resistance of Streptococcus pneumoniae to beta-lactam antibiotics.

In recent years, increasing numbers of Streptococcus pneumoniae strains displaying relative resistance to penicillin have been reported. Epidemiological studies have shown a correlation between aminopenicillin administration and resistance. We investigated the development of resistance in six strains (four sensitive and two intermediate-resistant to penicillin) by serial daily passages in subinhibitory concentrations of amoxicillin (AMX), amoxicillin + clavulanic acid (AMC), imipenem (IMP), cefixime (CFM), cefatrizine (CTZ), cefadroxil (CDX), and cefuroxime (CXM). MICs were determined by the macrodilution method in brain-heart broth for each daily passage. The number of daily passages needed to increase the MIC by a factor of 8 was achieved with AMX, AMC, and CFM for most of the strains after a mean of 24, 20, and 11 passages, respectively, and for one-third of the strains, with CDX, IMP, and CTZ after 11, 11, and 21 passages, respectively. Decreased susceptibility to breakpoints for intermediate-resistant S. pneumoniae populations was noted for all strains with CFM, AMX, and AMC after a mean of 10, 18, and 21 serial passages, respectively, and for four of five strains with IMP and CTZ after 12 and 13 passages. CTZ-, CDX-, and CXM-passaged variants had increased MIC values only for cephalosporins, while AMX-, AMC-, IMP-, and CFM-passaged variants exhibited increased MICs to all antibiotics tested. These in vitro data appear to be in agreement with epidemiological studies and warrant further exploration with respect to possible clinical implications.

Effect of beta-lactam antibiotics on the in vitro development of resistance in Pseudomonas aeruginosa.

OBJECTIVE: To investigate whether stepwise selection of resistance mutations may mirror the continued bacterial exposure to antibiotics that occurs in the clinical setting. METHODS: We examined the in vitro development of resistance to a number of commonly used antibiotics (cefepime, cefpirome, ceftazidime, cefotaxime, piperacillin and imipenem) in Pseudomonas aeruginosa, a significant nosocomial pathogen. Stepwise resistance was assessed by serial passage of colonies located nearest to the inhibition zone on antibiotic-containing gradient plates. RESULTS: The lowest frequencies of spontaneous resistance mutations were found with cefepime and imipenem; these drugs also resulted in the slowest appearance of resistance of spontaneous resistance mutations. In five wild-type P. aeruginosa strains, cefepime-selected isolates required a mean of 30 passages to reach resistance; resistance occurred more rapidly in strains selected with other cephalosporins. P. aeruginosa strains that produced beta-lactamase or non-enzymatic resistance generally developed resistance more rapidly than wild-type strains. For most strains, resistance to all antibiotics except imipenem correlated with increased levels of beta-lactamase activity. Cross-resistance of cephalosporin-selected resistant mutants to other cephalosporins was common. Cephalosporin-resistant strains retained susceptibility to imipenem and ciprofloxacin. CONCLUSIONS: From our in vitro study, we can conclude that the rate of development of resistance of P. aeruginosa is lower with cefepime compared with other cephalosporines.

Efficacy of subinhibitory concentration of pefloxacin in preventing experimental Staphylococcus aureus foreign body infection in mice.

Adhesion is the first step leading to colonization and infection of a foreign body (FBI). To assess the ability of a subinhibitory concentration (subMIC) of pefloxacin (P) to prevent such infection, an experimental model was developed in Swiss albino mice. Subcuts of polyurethane catheters (Vygon) were placed in the peritoneal cavity of animals and 24 hours later, different inocula of an adherent strain of Staphylococcus aureus (SA) (MIC of P:0.8 mg/l) were injected i.p. Unexposed SA served as controls. Two days later the removed catheters, blood and spleen specimens were quantitatively cultured for bacterial content and identity. Infection was defined as more than 10 CFU/ml of SA recovered. Significant protection of mice, with lower dissemination, was found with inoculum sizes of 10(5) and 10(6). These results suggest that subMICs of P may confer protection against foreign body infection.

Pharmacokinetic parameters and killing rates in serum of volunteers receiving amoxicillin, cefadroxil or cefixime alone or associated with niflumic acid or paracetamol.

Pharmacokinetic parameters and killing rates in serum of volunteers receiving amoxicillin, cefadroxil or cefixime alone or associated with niflumic acid or paracetamol were studied. Niflumic acid (250 mg) or analgesic and antipyretic drugs such as paracetamol (500 mg) are often combined with antibiotics to avoid inflammation and pain in acute ear, nose and throat diseases. Pharmacokinetic interactions between these two classes of drugs have been described in experimental models, and exceptionally in humans. The aim of the present investigation was to study the interactions of these two drugs with three antibiotics (amoxicillin 500 mg x 2, cefadroxil 500 mg x 2, cefixime 200 mg and one placebo capsule) on pharmacodynamic parameters and on rate of killing in the serum of six healthy volunteers receiving the antibiotic associated or not with the product in a randomized cross-over double-blind trial. The bacteria most often involved in sinusitis, bronchitis and otitis media (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus) three target diseases for oral cephalosporins and amoxicillin, were chosen for bacteriological study. Blood samples were obtained at 0.25, 0.50, 1, 1.5, 2, 4, 6 and 12 h after oral administration of antibiotics alone or associated with the drugs. There was a wash-out period of at least 1 week between the eleven sequences. Antibiotics were measured by two methods: bioassay and high performance liquid chromatography (HPLC). All serum samples obtained at peak level, 4 and 6 h were tested for killing rate. Area under the time kill curve was calculated by the trapezoidal rule method and relative bioactivity in percent was defined as follows: (AUC control - AUC test)/AUC control x 100. No pharmacokinetic interaction was found in the AUC and T1/2 of the plasma concentrations of the antibiotics or associated with the drugs, regardless of dose, as determined by HPLC or microbiological assay. For these beta-lactam antibiotics killing rate was found to be time-dependent. Bactericidal activity was improved on H. influenzae when cefixime was associated with niflumic acid and became concentration-dependent. A significant concentration relation was also found with niflumic acid or paracetamol associated with cefixime on Strep. pneumoniae.

[Infection prophylaxis in open leg fractures. Comparison of a dose of pefloxacin and 5 days of cefazolin-oxacillin. A randomized study of 616 cases]. / Prophylaxie des infections dans les fractures ouvertes de jambe. Comparaison entre une dose de péfloxacine et 5 jours cefazoline-oxacilline. Etude randomisée sur 616 cas.

THE PURPOSE OF THE STUDY: Was to compare the efficacy of a single 800 mg injection of Pefloxacin (PF) versus 2 days of cefazolin (1 gr.Q.6 H) followed by 3 days of oxacillin (1 gr.Q.8 H) in patients with an open tibial fracture and to examine the predictive factors for infection. A double-blind double dummy, multicentric, randomized trial was performed. 616 adults with an open tibial fracture requiring single-stage bone coverage were included. The end point was wound infection within 3 months. RESULTS: Within 3 months, 21/316 patients were infected in the PF group (6.6 p. 100) versus 24/300 in the CZ-OX group (8 p. 100), the difference was not significant (95 p. 100 Cl for difference: -4.8 p. 100 to 2.1 p. 100). Twenty one strains were isolated in 18 infected patients in the PF group, and 27 in 20 patients in the CZ-OX group. Negative gram bacteria were less frequent in the PF group (10 p. 100) than in the CZ-OX group (48 p. 100), and positive gram bacteria were more frequent in the PF group (90 p. 100) than in the CZ-OX group (52 p. 100). Independent risk factors for infection were severe contamination, widespread contusion, unstable fracture, positive sample in the emergency room and at the end of surgery. Resistant infecting bacteria rate was 24 p. 100 in infected cases. CONCLUSION: There was no difference in infection rates after surgery for open tibial fractures between a 800 mg injection of Pefloxacin and 2 days of pephazolin followed by 3 days of oxacillin. Infecting bacteria were mainly nosocomially acquired.

Effect of beta-lactam antibiotics on the in vitro development of resistance in Pseudomonas aeruginosa.

OBJECTIVE: To investigate whether stepwise selection of resistance mutations maz mirror the continued bacterial exposure to antibiotics that occurs in the clinical setting. METHODS: We examined the in vitro development of resistance to a number of commonly used antibiotics (cefepime, cefpirome, ceftazidime, cefataxime, piperacillin and imipenem) in Pseudomonas aeruginosa, a significant nosocomial pathogen. Stepwise resistance was assessed by serial passage of colonies located nearest to the inhibition zone on antibiotic-containing gradient plates. RESULTS: The lowest frequencies of spontaneous resistance mutations were found with cefepime and imipenem; these drugs also resulted in the slowest appearance of resistance of spontaneous resistance mutations. In five wild-type P. aeruginosa strains, cefepime-selected isolates required a mean of 30 passages to reach resistance; resistance occurred more rapidly in strains selected with other cephalosporins. P. aeruginosa strains that produced beta-lactamase or non-enzymatic resistance generally developed resistance more rapidly than wild-type strains. For most strains, resistance to all antibiotics except imipenem correlated with increased levels of beta-lactamase activity. Cross-resistance of cephalosporin-selected resistant mutants to other cephalosporins was common,. Cephalosporin-resistant retained susceptibility to imipenem and ciprofloxacin. CONCLUSIONS: From our in vitro study, we can conclude that the rate of development of resistance of P. aeruginosa is lower with cefepime compared with other cephalosporines.

Pneumococcal resistance patterns in Europe.

The emergence of Streptococcus pneumoniae strains with decreased susceptibility to penicillin has been reported worldwide over the past 20 years. However, there are striking discrepancies in penicillin susceptibility among various European countries, suggesting that local conditions may affect clonal propagation or de novo selection of resistant strains. In the present study, data on penicillin resistance patterns, antibiotic use and mode of administration, and treatment compliance in five European countries (France, Spain, Germany, Italy, and the UK) were compared. High prevalence rates of penicillin-resistant pneumococci have been reported in Spain and France, where antibiotics are widely prescribed, and overall in Europe, patient compliance with more than 50% of oral antimicrobial prescriptions is inadequate. The low prevalence of penicillin resistance in Germany and the UK coincides with lower antibiotic consumption and better treatment compliance in these countries. Recent attempts to raise public awareness and to restrict and improve indications for antimicrobial agents have resulted in decreased pneumococcal resistance in Hungary and Iceland, suggesting that pneumococcal resistance can be reversed.

Effect of subinhibitory concentrations of cefamandole and cefuroxime on adherence of Staphylococcus aureus and Staphylococcus epidermidis to polystyrene culture plates.

The ability of cefamandole and cefuroxime to inhibit adherence of staphylococci to polystyrene culture plates was tested in an in vitro assay using eight strains each of Staphylococcus aureus and Staphylococcus epidermidis. The results indicated that subinhibitory concentrations of cefamandole and cefuroxime altered the adherence ability of both staphylococcal species, inhibition of adherence being more marked in the presence of cefamandole. It may be important to consider antiadherence properties in association with bactericidal activity when selecting agents for antibiotic prophylaxis.

In-vitro development of resistance to beta-lactam antibiotics in Streptococcus pneumoniae.

Selection of resistant mutants was induced in broth by exposing pneumococci to serial sub-inhibitory concentrations of various beta-lactam antibiotics. Aminopenicillins selected for resistance to themselves and to cephalosporins although cephalosporins tended to select for resistance to their own class, with the exception of cefixime which seems to select cross-resistant organisms.

Effects of subinhibitory concentrations of vancomycin and teicoplanin on adherence of staphylococci to tissue culture plates.

Bacterial adhesion is the first step in infection of medical devices. Staphylococcus aureus and Staphylococcus epidermidis are the pathogens recovered most often. The effects of subinhibitory concentrations of vancomycin and teicoplanin on the adherence of eight clinical strains of S. aureus and eight strains of S. epidermidis to tissue culture plates in vitro were tested. The mean relative inhibitions of adherence at one-fourth and one-eighth the MIC were statistically different for teicoplanin and vancomycin. Slime production seemed not to be involved in adherence.

Efficacy and safety of vancomycin constant-rate infusion in the treatment of chronic Gram-positive bone and joint infections.

OBJECTIVE: To evaluate the efficacy and safety of vancomycin constant-rate infusion over 24 h in the treatment of Gram-positive bone infections, METHODS: Vancomycin (40 mg/kg/day) was administered without a loading dose to 15 patients (12 male, three female) aged 23--90 years, weighing 46--85 kg, with postoperative chronic bone and joint infections. The 24-h dose was adjusted to maintain plasma levels between 25 and 35 mg/L. Mean duration of therapy was 6.2 months (4--8.5) via a portable infusion pump. Sites of infection included hip and femur (10), tibia (three), patella (one) and vertebrae (one). Sequestrectomy (two), removal of material (7/8 prosthetic hips, 1/5 metal implants) and debridement (two) were performed at the beginning of the treatment. Involved bacteria included Staphylococcus aureus (eight, six methicillin resistant), S. epidermidis (four methicillin-resistant), Enterococcus faecalis (one), Enterococcus avium (one) and Streptococcus bovis (one). RESULTS: MIC of vancomycin ranged from 1 to 4 mg/L. The mean vancomycin bone concentration when available was 67.7plus minus38.9 microg/L. Based on a mean post-treatment follow-up of 14plus minus4 months (6--20.6), cure was achieved in 10 patients (66.6%). Failures were related to the inability to remove the infected prosthesis (one) or implants (three) and to the persistence of a deep wound abcess (one). Adverse events included pruritus (four cases), tinnitus (two), mild transient elevation of creatinine level (three) and transient neutropenia (two). Vancomycin was maintained in all the patients. CONCLUSIONS: Prolonged treatment with vancomycin constant-rate infusion is effective and safe for treatment of Gram-positive chronic bone and joint infections, providing that complete surgical débridement and prosthetic material removal are performed.

Epidemiology of bacterial infection during management of open leg fractures.

In a randomised double-blind trial conducted between 1990 and 1994, 616 patients from 43 centres, pefloxacin (group P, 316 patients) and a cefazolin-oxacillin combination (group C, 300 patients) were compared in the prophylaxis of bone infection after grade 1 and 2 open leg fractures. Samples were obtained at emergency, before and during surgery, and from drain aspirates. Antimicrobial susceptibility, slime production and adherence properties of the bacteria were tested. Cultures at emergency and before surgery showed similar distributions of gram-positive and gram-negative bacteria in both groups, while wound closure and infecting isolates showed prevailing gram-positive bacteria in group P and gram-negative bacteria in group C. Positive cultures at each stage were correlated with the occurrence of infection but were not predictive of the infecting species, which were nosocomial bacteria in most cases. Positive cultures at wound closure warn of a higher infection risk. Twenty-one of 316 (6.6%) patients in group P and 24 of 300 (8%) in group C were considered infected within 3 months. The difference is not significant (chi-square test = 0.42; P = 0.51). Infecting strains were isolated from 38 patients (group P, 18; group C, 20). Infecting species, although not predictable, appear to be those escaping the spectrum of the prescribed antimicrobial prophylaxis.

[Nasopharyngeal carriage of resistance pneumococci in day-care centers in the Alps-Maritimes region]. / Portage nasopharyngé de pneumocoques résistants dans les crèches des Alpes-Maritimes.

To provide ongoing information on regional trends of antibiotic resistance prevalence to pneumococci, a cross selectional survey was conducted on a large representative sample of children attending day-care centers. Children were analyzed in spring (n = 378) and autumn (n = 379) for nasopharyngeal carriage. Streptococcus pneumoniae was detected in 149 children (39.4%) in Spring and 204 (59.8%) in Autumn. Half of these isolated strains showed penicillin insensitivity or resistance. A high proportion of children (43.6% in spring and 47.5% in autumn) had been treated with antibiotics during the 3 months prior to sample collection; 21.6% of isolated strains were serotype 6B, 20.1% type 23F, 18.9% type 19A and 19F, 11.5% type 14. Reduced susceptibility was frequently noted in serotype 23F, 14 and 19F, representing 93%, 94% and 46% of identified serotypes, respectively. Acquisition of a strain of PRP was correlated with prescription of antibiotics during the previous three months (p < 0.05). This type of survey on children in day-care centers can contribute to the understanding of regional variations in antibiotic resistance and provide information for epidemiological surveillance.

Gradient plate method to induce Streptococcus pyogenes resistance.

In recent years, increasing numbers of Streptococcus pyogenes (GAS) strains displaying resistance to macrolides have been reported in Finland, Japan, Asia and Spain. Antibiotic use has been shown to be a risk factor for infection with and carriage of drug-resistant streptococci. The aim of this study was to compare in-vitro development of resistance of streptococci to beta-lactams (penicillin, amoxycillin, cefotiam and cefuroxime) and erythromycin by serial passages in subinhibitory concentrations of antibiotics (subMICs) by gradient plate method. Three clinical strains of GAS were tested. Two were susceptible to erythromycin (MIC = 0.015 mg/L and 0.013 mg/L) and one resistant. Serial passages were performed daily by gradient plate method until a four-fold increase of the MIC was achieved. GAS variants obtained after serial passages in beta-lactams had MICs increased at least four-fold. They remained susceptible to these antibiotics. With erythromycin, final MICs reached intermediate and resistant level. Results obtained in this study with erythromycin are in good correlation with clinical studies showing that prior exposure to macrolides may help to facilitate the emergence of drug-resistant strains of streptococci.

Possible prevention of in vitro selection of resistant Streptococcus pneumoniae by beta-lactamase inhibitors.

The development of resistance in vitro in five strains of Streptococcus pneumoniae (3 with full susceptibility and 2 with intermediate susceptibility to penicillin) was investigated by serial passages in the presence of subinhibitory concentrations of amoxicillin and ampicillin. At the end of passaging, MICs of antibiotics for all the strains increased by a factor of four or more, reaching at least intermediate levels. MICs of cephalosporins, ampicillin and amoxicillin increased for almost all variants obtained. Similar results were obtained with amoxicillin plus clavulanic acid at a ratio of 2:1 and at a constant concentration of 2 micrograms/ml, and with ampicillin plus sulbactam at a ratio 2:1. In contrast, no significant modification of MIC was seen with ampicillin plus sulbactam at a constant concentration of 4 micrograms/ml sulbactam. These results suggest interaction of sulbactam with penicillin binding proteins as described previously for other bacterial species, and merit further investigation.