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New colorimetric microdilution method for in vitro susceptibility testing of Borrelia burgdorferi against antimicrobial substances.

A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, < or = 0.125 microg/ml), ceftriaxone (MIC range, < or = 0.015-0.06 microg/ ml), and azithromycin (MIC range, < or = 0.015-0.06 microg/ml). Whereas tobramycin (MIC range, 8-64 microg/ml) exhibited little activity, spectinomycin (MIC range, 0.25-2 microg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P < 0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.

Anti-Bacterial Agents↗

Enhanced activity of beta-lactam antibiotics with amdinocillin in vitro and and in vivo.

An important advantage of antibiotic combinations which can be determined is the increase in spectrum of activity. Ad hoc combinations of amdinocillin with ampicillin, ticarcillin, piperacillin, azlocillin, cefazolin, cefamandole, cefoxitin, and moxalactam in ratios similar to those in serum after parenteral administration of normal dosages of these agents were tested in vitro against over 200 strains of bacteria, including Staphylococcus aureus, Streptococcus faecalis, representative genera of the Enterobacteriaceae, and Pseudomonas aeruginosa. Independent of amdinocillin's activity, it was possible to show marked enhancement of the spectrum of all the beta-lactam agents tested, except moxalactam, against members of the Enterobacteriaceae. Little or no enhancement or antagonism of activity was seen against gram-positive bacteria or P. aeruginosa. The enhancing activity of amdinocillin on three beta-lactam antibiotics, ampicillin, cefazolin, and cefoxitin, was also demonstrated in experimental mouse infections with gram-negative bacteria.

Amdinocillin↗

Combinations of antibiotics against Pseudomonas aeruginosa.

Pseudomonas aeruginosa continues to cause serious infections, especially bacteremias, in hospitalized and immunocompromised patients. During the past 10 years, bacteremia due to this organism has increased in frequency in many institutions, and mortality rates in patients with rapidly fatal disease remain as high as 85 percent despite antibiotic therapy. Available data do not allow firm conclusions regarding the in vivo predictive value of in vitro synergy testing for P. aeruginosa, but in vitro demonstration of synergy appears important in selecting therapy for patients with P. aeruginosa infections. Combinations of aminoglycosides (amikacin or tobramycin) with highly active antipseudomonal beta-lactam antibiotics are most likely to be associated with in vitro synergy. Experimental studies in animals models support the use of combination therapy for local and bacteremic infections. Similarly, the retrospective and prospective studies in humans suggest better survival with combinations of antimicrobials, usually including aminoglycosides and beta-lactams, in immunocompromised hosts. At present, the use of newer penicillins, piperacillin, azlocillin, or selected antipseudomonal cephalosporins, in combination with amikacin or tobramycin, appears to be the preferable antimicrobial therapy for serious P. aeruginosa infections.

Aminoglycosides↗

Aminoglycosides plus beta-lactams against gram-negative organisms. Evaluation of in vitro synergy and chemical interactions.

Combination antibiotic therapy has been used mainly to broaden the antibacterial spectrum and prevent the development of resistance. Antibiotic combinations proven to be synergistic in vitro are associated with a significantly better in vivo response, particularly in the compromised host in whom traditional treatment combines an antipseudomonal penicillin plus an aminoglycoside. Several investigators have examined combining new agents, such as the third-generation cephalosporins (cefotaxime, ceftriaxone, ceftizoxime, ceftazidime, cefoperazone, and moxalactam), aztreonam, or the ureidopenicillins, with amikacin. When compared with combinations of an older cephalosporin, carbenicillin or ticarcillin, plus gentamicin or tobramycin, these newer combinations produce higher rates of clinically meaningful synergy and rapid enhancement of in vitro bactericidal activity against the difficult-to-treat Enterobacteriaceae (i.e., Serratia, Citrobacter, Enterobacter, Providencia, and indole-positive Proteus species). This effect, without any evidence of antagonism, has been reported even for strains moderately or completely resistant to the former antibiotics. Unsatisfactory and unpredictable synergistic interactions against both resistant and susceptible strains of Pseudomonas aeruginosa--the most difficult nosocomial pathogen to treat--have been noted with combinations of tobramycin or gentamicin plus cefotaxime, moxalactam, or cefoperazone. Conversely, the use of amikacin plus various beta-lactams against multi-resistant strains is more frequently synergistic. Agents have been observed to exhibit such synergy in the following order of activity, from most to least synergistic: ceftazidime, ceftriaxone, moxalactam, aztreonam, cefotaxime, azlocillin, cefoperazone, cefsulodin, and carbenicillin. The combination of amikacin plus imipenem or ciprofloxacin against strains of P. aeruginosa resistant to the former and moderately resistant to the latter was recently reported to have a low probability of synergy; the combination of two of the newer beta-lactams had mostly an unpredictable or even antagonistic result. In vitro studies have also demonstrated that high concentrations of the antipseudomonal penicillins can inactivate the aminoglycosides. Among the latter compounds, the inactivation order, from most to least inactivated, was as follows: tobramycin, gentamicin, netilmicin, and amikacin. To date, the reports of aminoglycoside inactivation by the newer cephalosporins have been rather contradictory; only moxalactam has been shown produce a significant decrease in activity.

Amikacin↗

Use of ciprofloxacin in cystic fibrosis patients.

In order to evaluate the clinical efficacy and safety of oral ciprofloxacin in the treatment of acute pulmonary exacerbations of cystic fibrosis and trace the possible development of resistance over time, three trials were conducted. In an open-label, uncontrolled trial, 25 courses of ciprofloxacin were administered to 16 patients. Efficacy and safety were assessed based on changes in short-term clinical scores, white blood cell counts, Pseudomonas aeruginosa counts in sputum, pulmonary function tests, and standard serum chemistries and urinalysis that were performed before therapy, weekly during therapy, at the end of therapy, and at a seven-day follow-up visit after therapy. In an open-label, randomized, controlled study, the efficacy and tolerance of oral ciprofloxacin were compared with those of intravenous tobramycin and azlocillin. In another study, the rate of susceptibility of P. aeruginosa isolated from cystic fibrosis patients during more than two years of clinical use was determined. In the uncontrolled trial, ciprofloxacin therapy was associated with clinical improvement in most cases with changes in short-term clinical score and forced expiratory volume in one second being statistically significant (p less than 0.05). Twenty-five patients were entered in the controlled trial with 12 patients in each treatment group being evaluable. The groups were comparable based on admitting demographic and disease characteristics, and no differences in therapeutic response or side effects were noted between the two treatments (p greater than 0.5). Bacterial susceptibility to ciprofloxacin has remained relatively stable over time. Based on these results as well as those from similar evaluations, ciprofloxacin appears to be efficacious in the treatment of acute pulmonary exacerbations in adults with cystic fibrosis, producing responses similar to those observed with standard intravenous antibiotic therapy.

Adult↗

Identification and antibiotic susceptibility of bacterial isolates from burned patients.

We retrieved bacterial blood isolates from 397 adult burned patients admitted over a 7-year period. Sixty-two patients (15.6 per cent) developed true-positive bacterial blood cultures (judged non-contaminants), and of these 30 (48.4 per cent) expired. Pseudomonas aeruginosa (24 isolates), Staphylococcus aureus (19) and Klebsiella pneumoniae (19) were the most frequent isolates. In vitro susceptibilities of 149 isolates were determined to 12 antibiotics (gentamicin, amikacin, ticarcillin, piperacillin, mezlocillin, azlocillin, cefazolin, cefotaxime, ceftazidime, cefoperazone, thienamycin and ticarcillin-clavulinic acid) using agar diffusion assay. Thienamycin proved the most active agent (97 per cent of isolates susceptible). Cefoperazone was the most active cephalosporin (95 per cent susceptible). Twenty-eight organisms demonstrated multiple drug resistance; patients with such organisms had a 71 per cent mortality. Thienamycin was the most active agent against such isolates (27/28 susceptible). Susceptibilities of all 149 isolates to combinations of antibiotics were calculated, assuming no synergism or antagonism; some combinations of third-generation cephalosporins with the newer penicillins may prove to be as effective as combinations including aminoglycosides.

Adolescent↗

Hospital routine analysis of penicillins, third-generation cephalosporins and aztreonam by conventional and high-speed high-performance liquid chromatography.

A high-performance liquid chromatographic procedure for the measurement of fifteen beta-lactam antibiotics in body fluids is described, with special reference to high-speed techniques. The procedure involves a unique sample preparation before analysis for all the following fifteen compounds: benzylpenicillin, ampicillin, cloxacillin, ticarcillin, mezlocillin, azlocillin, piperacillin, cefotaxime and its desacetyl metabolite, cefsulodin, cefoperazone, cefmenoxime, ceftazidime, ceftriaxone and the monobactam aztreonam; thus all biological samples arriving at the laboratory can be treated in batch. Of these fifteen antibiotics, eleven can be chromatographed with the same type of mobile phase, which consists of a mixture of ammonium acetate and acetonitrile in various ratios. Three others need ionpairing chromatography because of their polarity, and ticarcillin requires citric acid. High-speed high-performance liquid chromatography seems to be particularly suitable for the routine analysis of beta-lactam antibiotics because columns equilibrate more rapidly, retention times are much shorter, detection limits are lower and the longer lifetime of columns reduces analysis costs.

Aztreonam↗

Application of high-performance liquid chromatography of some antibiotics in clinical microbiology.

During recent years high-performance liquid chromatography has become an excellent tool for the determination of antibiotics in biological fluids. Compared with biological assays, the major benefits of this method are specificity and rapidity. In particular, the determination of biologically inactive metabolites emphasizes that this technique plays an outstanding role for the analysis of antibiotics. This paper describes how the method can be used in the analysis of several antibiotics and demonstrates the efficacy of this method for clinical microbiology. Methods for the determination in biological fluids of acylaminopenicillins (azlocillin, mezlocillin, piperacillin and aspoxicillin), quinolones (ciprofloxacine, norfloxacine and ofloxacine), a penem (imipenem) and a cephalosporin (cefixime) are summarized. Furthermore, their application to in vitro studies and their trial in clinical studies are described.

Anti-Bacterial Agents↗

Effectiveness of antibiotic removal by the antibiotic-binding blood culture systems.

The effectiveness of antibiotic removal by the BACTEC aerobic resin-containing blood culture medium (16B) and the Antimicrobial Removal Device (ARD) was compared for 12 antibiotics: ampicillin, cephalothin, cefoperazone, cefotaxime, moxalactam, nafcillin, gentamicin, tobramycin, azlocillin, mezlocillin, piperacillin, and ticarcillin. The ability to recover eight commonly encountered species of bacteria from antibiotic-containing serum by these two systems showed that recovery of antibiotic-exposed bacteria was dependent not only upon the amount and rate of the antibiotic removal but also upon the kinetics of bacterial killing by the antibiotic(s). The 16B medium had difficulty recovering organisms exposed to ticarcillin and moxalactam, whereas the ARD had difficulties with moxalactam and sometimes with cefotaxime and cefoperazone. Although neither system was able to recover all species of microorganisms tested, these in vitro results suggest that to use optimally these new culture systems, knowledge of the suspected pathogen(s), the amount and kind of antibiotic(s) administered, and the rate of bacterial killing by the antibiotic(s) is required.

Adsorption↗

Susceptibility of 100 blood isolates of Pseudomonas aeruginosa to 19 antipseudomonal antibiotics: old and new.

The in vitro susceptibility of 19 antipseudomonal antibiotics, old and new, were tested against 100 blood isolates of Pseudomonas aeruginosa. The isolates were taken from consecutive bacteremic patients hospitalized in nine Pittsburgh hospitals from 1983 to 1984. Twelve percent of isolates tested were tobramycin-resistant. All of the tobramycin-resistant isolates were sensitive to apalcillin, azlocillin, aztreonam, cefsulodin, ceftazidime, ciprofloxicin, and imipenem. Two isolates were resistant in vitro to all beta-lactam antibiotics tested, but sensitive to all aminoglycosides. One hundred percent of isolates tested were susceptible to imipenem, whereas, 98% were susceptible to apalcillin and cefsulodin. The lowest MIC90 was 2 mg/L for ciprofloxicin. Tolerance of P. aeruginosa (MIC90/MBC90 ratio greater than 32) was not observed for any antimicrobial agent.

Anti-Bacterial Agents↗

In vitro synergy studies with ciprofloxacin and selected beta-lactam agents and aminoglycosides against multidrug-resistant Pseudomonas aeruginosa.

In order to determine the effect of combining ciprofloxacin with beta-lactam and aminoglycoside agents against multidrug-resistant P. aeruginosa, ciprofloxacin was tested in vitro with the checkerboard broth microdilution technique with various current beta-lactam and aminoglycoside agents. In both inhibitory and bactericidal testing, the combination of ciprofloxacin with a beta-lactam agent (ceftazidime, piperacillin, mezlocillin, azlocillin, or imipenem) was more likely to show a synergistic effect or additive effect and lack of antagonism (96.5% of all inhibitory and cidal tests) than was a combination of ciprofloxacin with an aminoglycoside [(amikacin, tobramycin, or gentamicin), 90% of all inhibitory and cidal tests]. Finding of antagonism in some testing, especially with aminoglycosides, indicates that results are unpredictable. These results may influence selection of agents for selection in clinical situations.

Aminoglycosides↗

Resistance of Xanthomonas maltophilia to antibiotics and the effect of beta-lactamase inhibitors.

We examined the susceptibility of 50 isolates of Xanthomonas maltophilia and the effect of beta-lactamase inhibitors upon the susceptibility. The majority of isolates were resistant to azlocillin, piperacillin, mezlocillin, ticarcillin, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, and ceftazidime. All isolates were resistant to imipenem, CGP 31608, aztreonam, and carumonam. Although disk susceptibility tests showed that the combination of clavulanate with ticarcillin inhibited many isolates, at a ratio of 1:20 few isolates were susceptible to the combination. Addition of clavulanate to aztreonam and to imipenem failed to make organisms susceptible. Sulbactam combined with cefoperazone made some organisms susceptible, but ampicillin-sulbactam was ineffective, whereas tazobactam combined with piperacillin at a ratio of 1:4 made half the isolates have MICs of 32 micrograms/ml or less. The beta-lactamases from the isolates hydrolyzed all of the beta-lactams.

Anti-Bacterial Agents↗

The penicillins.

The penicillin family of antibiotics remains an important part of our antimicrobial armamentarium. In general, these agents have bactericidal activity, excellent distribution throughout the body, low toxicity, and efficacy against infections caused by susceptible bacteria. The initial introduction of aqueous penicillin G for treatment of streptococcal and staphylococcal infections was an important pharmacologic landmark. The emergence of penicillinase-producing Staphylococcus aureus prompted the development of the penicillinase-resistant penicillins (for example, methicillin, oxacillin, and nafcillin), in which an acyl side chain prevented disruption of the beta-lactam [corrected] ring. Subsequently, the aminopenicillins (such as ampicillin and amoxicillin) were developed because of the need for gram-negative antimicrobial activity. Their spectrum included Escherichia coli, Proteus mirabilis, Shigella Salmonella, Listeria, Haemophilus, and Neisseria. The search for a penicillin with additional antimicrobial activity against the Enterobacteriaceae and Pseudomonas aeruginosa led to the development of the carboxypenicillins (carbenicillin, ticarcillin, and temocillin) and the ureidopenicillins (mezlocillin, azlocillin, piperacillin, and apalcillin). Finally, the combination of a beta-lactamase inhibitor (clavulanic acid or sulbactam) and an aminopenicillin or ticarcillin has further extended their antibacterial spectra. The development of an ideal penicillin that is rapidly bactericidal, nonsensitizing, nontoxic, bioavailable, resistant to beta-lactamase, and without inoculum effect and that has a high affinity for penicillin-binding proteins remains the goal.

Humans↗

Carbenicillin and ticarcillin.

Carbenicillin and ticarcillin are penicillins which were initially developed as agents to treat serious Pseudomonas infections in the seriously ill hospitalized patient. These drugs have made a major contribution to improved survival in the neutropenic patients with Pseudomonas infection, the burn patient and to the care of the patient with cystic fibrosis. Areas of use for the compounds have enlarged to include aspiration pneumonitis in hospitalized patients, intra-abdominal and pelvic sepsis, and infections due to Proteus and Enterobacter species. Careful attention to the pharmacology of the agents is necessary to achieve clinical and bacteriologic success and to avoid the toxic side-effects such as bleeding and hypokalemia associated with the use of these agents. A decade of use has shown that the agents have remained effective agents in institutions in which their use has not been abused. It is too early to clearly position azlocillin, mezlocillin, and piperacillin. In the next few years the role of these potent compounds will be established. As noted in this review, these three agents have been used with success to treat all of the aforementioned infections. With these drugs it is also essential that the physician closely correlate in vitro data and the human pharmacology of the drugs if he or she wishes to achieve the most effective response from the agents.

Anaerobiosis↗

New penicillins and their use in pediatrics.

The new penicillins have not been a breakthrough for the pediatrician. Indeed, 20 years ago with the introduction of ampicillin and methicillin and 15 years ago with the introduction of carbenicillin, the pediatrician was given the penicillins that he has used so well to treat trivial and life-threatening infections. The drugs discussed in this article are small steps forward, and whether mezlocillin, azlocillin, or piperacillin will prove more helpful to the pediatrician than have carbenicillin or ticarcillin will be learned in the next few years.

Child↗

Spread of beta-lactam-resistant Pseudomonas aeruginosa in a cystic fibrosis clinic.

BACKGROUND: Pseudomonas aeruginosa colonisation of the airways of patients with cystic fibrosis (CF) is associated with considerable respiratory morbidity. Although segregation of colonised patients from non-colonised patients to prevent cross-infection has been recommended, there is little evidence that such cross-infection is widespread. We observed that a high proportion of children attending our CF clinic were colonised with P aeruginosa that was resistant to ceftazidime and other beta-lactam antibiotics. We used two genomic fingerprinting techniques to see whether this may have arisen from epidemic spread of a single strain. METHODS: The prevalence of P aeruginosa colonisation and the antibiotic susceptibility of the organisms was determined from review of laboratory reports in the case-notes of 120 children with CF. Isolates were cultured from the sputum of 65 children colonised with ceftazidime-resistant P aeruginosa. Polymorphisms in total bacterial DNA from 92 isolates were analysed with two molecular fingerprinting techniques--pulsed-field gel electrophoresis after restriction enzyme digestion and assessment of flagellin gene polymorphisms by amplification of the whole gene and restriction enzyme digestion. RESULTS: 92 (76.7%) of 120 children were colonised with P aeruginosa, and 65 of the 92 harboured isolates that were resistant to ceftazidime. Only three of the 92 children had never been treated with ceftazidime. The results of the two molecular-fingerprinting techniques were concordant and showed that 55 of 65 children harboured the same epidemic strain. This strain was resistant to ceftazidime, azlocillin, and imipenem, and sensitive to tobramycin and ciprofloxacin. INTERPRETATION: This study provides the first molecular evidence of a long-term outbreak of P aeruginosa in a CF centre. We suggest that careful surveillance of the prevalence of antibiotic resistance in CF centres should be instituted with measures to prevent cross-infection. We believe that antipseudomonal monotherapy should be considered with caution.

Adolescent↗

Stability of single and combination antimicrobial agents in various peritoneal dialysates in the presence of insulin and heparin.

The antimicrobial activity of ampicillin, azlocillin, cefotaxime, cephapirin, clindamycin, mezlocillin, nafcillin, piperacillin, tobramycin, and vancomycin was tested in peritoneal dialysate at room temperature for 24 hours and 48 hours. All of the antimicrobial agents were active at 24 hours. The bioactivity of cefotaxime, nafcillin, and vancomycin declined 15% to 20% after 48 hours (P less than 0.001). The addition of heparin or insulin did not affect the activity of any of the study drugs. The combination of cephapirin and tobramycin remained bioactive for 24 hours at room temperature and 35 degrees C. The preparation of drug-dialysate solution every one to two days is feasible for the treatment of peritonitis in patients on continuous ambulatory or continuous cycled peritoneal dialysis.

Anti-Bacterial Agents↗

Natural antibiotic susceptibility of Escherichia coli, Shigella, E. vulneris, and E. hermannii strains.

The natural antibiotic susceptibility of 139 Escherichia coli strains (including 18 enterohemorrhagic E. coli), 73 Shigella strains (S. sonnei (n = 37), S. flexneri (n = 29), S. boydii (n = 6), S. dysenteriae (n = 1)), 23 E. vulneris, and 20 E. hermannii strains toward 71 antibiotics was examined. MICs were determined using a microdilution procedure. All examined taxa were naturally sensitive/intermediate toward tetracyclines, aminoglycosides, some penicillins (amoxycillin/clavulanate, ampicillin/sulbactam, piperacillin [with and without tazobactam], mezlocillin, azlocillin), cephalosporins, carbapenems, monobactams, quinolones, trimethoprim, cotrimoxazole, and chloramphenicol and were naturally resistant/intermediate toward benzylpenicillin, oxacillin, macrolides, lincosamides, glycopeptides, rifampicin, and fusidic acid. No differences in natural antibiotic susceptibility were seen between enterohemorrhagic and other E. coli strains. Likewise, with one exception, no significant differences in natural antibiotic susceptibility were seen either among the Shigella subgroups or between Shigella and E. coli. The natural population of S. flexneri was slightly more susceptible to chloramphenicol than the natural populations of other taxa within the Shigella-E. coli complex. E. vulneris and E. hermannii showed susceptibility patterns to many antibiotics similar to Shigella and E. coli, but there were other antibiotics toward which there were significant differences in natural susceptibility. E. vulneris and E. hermannii were less susceptible to nitrofurantoin and slightly more susceptible to several aminoglycosides than E. coli and Shigella. E. hermannii was the only species that was naturally resistant/intermediate to ticarcillin and amoxycillin (DIN standard). The addition of clavulanic acid to the latter resulted in a decrease of seven twofold dilution steps (E. vulneris: four twofold dilution steps, E. coli/Shigella: two twofold dilution steps) of the MICs of the natural population. With the exception of cefoperazone and cefepime, E. hermannii was more susceptible to cephalosporins than strains of the other species. E. vulneris was the species most susceptible to ticarcillin and the only species that was highly resistant to fosfomycin (MIC > 256 micrograms/mL). The antibiotic susceptibility to fosfomycin was also unique for E. hermannii (MIC 32-128 micrograms/mL), whereas the natural populations of E. coli and Shigella showed lower MIC values. The data of this study represent an assessment of the natural susceptibility of strains of Escherichia spp. and Shigella subgroups to a wide range of antibiotics. These databases can be used for the validation of antibiotic susceptibility test results of Escherichia spp. and shigellae. The conformity of the natural antibiotic susceptibility test results not only among the Shigella subgroups but also between Shigella and E. coli support the status of Shigella as a subgroup of the species E. coli.

Anti-Bacterial Agents↗