CombiANT: Antibiotic interaction testing made easy.

Antibiotic combination therapies are important for the efficient treatment of many types of infections, including those caused by antibiotic-resistant pathogens. Combination treatment strategies are typically used under the assumption that synergies are conserved across species and strains, even though recent results show that the combined treatment effect is determined by specific drug-strain interactions that can vary extensively and unpredictably, both between and within bacterial species. To address this problem, we present a new method in which antibiotic synergy is rapidly quantified on a case-by-case basis, allowing for improved combination therapy. The novel CombiANT methodology consists of a 3D-printed agar plate insert that produces defined diffusion landscapes of 3 antibiotics, permitting synergy quantification between all 3 antibiotic pairs with a single test. Automated image analysis yields fractional inhibitory concentration indices (FICis) with high accuracy and precision. A technical validation with 3 major pathogens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showed equivalent performance to checkerboard methodology, with the advantage of strongly reduced assay complexity and costs for CombiANT. A synergy screening of 10 antibiotic combinations for 12 E. coli urinary tract infection (UTI) clinical isolates illustrates the need for refined combination treatment strategies. For example, combinations of trimethoprim (TMP) + nitrofurantoin (NIT) and TMP + mecillinam (MEC) showed synergy, but only for certain individual isolates, whereas MEC + NIT combinations showed antagonistic interactions across all tested strains. These data suggest that the CombiANT methodology could allow personalized clinical synergy testing and large-scale screening. We anticipate that CombiANT will greatly facilitate clinical and basic research of antibiotic synergy.

A combination of mecillinam and amoxicillin/clavulanate can restore susceptibility of high-level TEM-1-producing Escherichia coli to mecillinam.

Objectives: Mecillinam is recommended in France as a first-line treatment for lower urinary tract infections, due to the large increase in resistance of Escherichia coli to other oral treatments, such as co-trimoxazole or fluoroquinolones, its limited impact on faecal microbiota and its stability in the presence of numerous ß-lactamases. However, we recently identified several mecillinam-resistant E. coli isolates with a high-level expression penicillinase (HEP) phenotype that merit further study. Patients and methods: We studied two isogenic clinical isolates from one patient (one susceptible to mecillinam and one resistant to mecillinam) by WGS to determine the mechanism of mecillinam resistance and compared it with other mecillinam-resistant E. coli . We evaluated the synergistic combination of amoxicillin/clavulanate and mecillinam using a simple test, suitable for daily laboratory practice, to determine the MIC of this combination. Results: We showed that the presence of an SNP in the promoter of the plasmidic TEM-1 ß-lactamase gene is sufficient to confer resistance to mecillinam. This mechanism was present in 67% of HEP-phenotype E. coli tested. Combining mecillinam with amoxicillin/clavulanate abolished resistance, with an MIC compatible with clinical use. This association was not sensitive to the inoculum effect, in contrast to mecillinam alone. Conclusions: An HEP phenotype can confer mecillinam resistance in vitro . This resistance is abolished, regardless of the inoculum, by combining mecillinam with amoxicillin/clavulanate, and can be easily tested in the laboratory. This combination may be used as an oral relay treatment of non-complicated pyelonephritis due to multiresistant E. coli strains.

Mecillinam against genital Chlamydia trachomatis infection: a small-scale proof-of-concept study shows a low cure rate.

OBJECTIVES: Mecillinam is highly active in vitro against Chlamydia spp. We aimed to determine whether mecillinam should be evaluated further as treatment for genital Chlamydia trachomatis infection. PATIENTS AND METHODS: The study was conducted at an open-access clinic for sexually transmitted infections in Oslo, Norway. We planned to include 50 patients. Participants were asymptomatic, heterosexual male patients with a first-void urine sample found to be positive for C. trachomatis by PCR. Treatment consisted of 400 mg of pivmecillinam hydrochloride three times a day for 7 days. A test-of-cure sample, a medication diary and a questionnaire were returned by the participants, and they were used to evaluate treatment outcome, compliance, risk of reinfection and theoretical percentage of time above MIC (t/MIC %). The study was registered in Eudra-CT (no. 2013-002379-179) and clinicaltrals.gov (NCT02083276). RESULTS: The study was discontinued after including 20 patients, due to a high failure rate. Only two of the 17 participants who delivered a test-of-cure sample were cured. Three participants reported condomless sex before the follow-up sample. When the average or most favourable pharmacokinetics (PK)/pharmacodynamics (PD) reported from other studies were applied in a theoretical model, the estimated t/MIC % was above 50% for all of the 15 participants returning a medication diary. Using the least favourable PK/PD, no participant had t/MIC % of >36%. The mean dose interval was 8 h 36 min (standard deviation 3 h 12 min). CONCLUSIONS: A low cure rate combined with uncertainty about intracellular availability and attained t/MIC % makes mecillinam an unattractive candidate for further evaluation as treatment for genital C. trachomatis infection.

Synergistic and bactericidal activities of mecillinam, amoxicillin and clavulanic acid combinations against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in 24-h time-kill experiments.

This study aimed to evaluate the potential synergistic and bactericidal effects of mecillinam in combination with amoxicillin and clavulanic acid against extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Eight clinical E. coli isolates with varying susceptibility to mecillinam [minimum inhibitory concentrations (MICs) of 0.125 mg/L to >256 mg/L] and high-level resistance to amoxicillin (MICs > 256 mg/L) were used. Whole-genome sequencing was performed to determine the presence of ß-lactamase genes and mutations in the cysB gene. The activities of single drugs and the combinations of two or three drugs were tested in 24-h time-kill experiments. Population analysis was performed for two strains before and after experiments. Only one strain had a mutation in the cysB gene resulting in an amino acid substitution. With the two-drug combinations, initial killing was observed both with mecillinam and amoxicillin when combined with clavulanic acid. Synergy was observed with mecillinam and clavulanic acid against one strain and with amoxicillin and clavulanic acid against three strains. However, following significant re-growth, a bactericidal effect was found only with amoxicillin and clavulanic acid against two strains. Pre-existing subpopulations with elevated mecillinam MICs were detected before experiments and were selected with mecillinam alone or in two-drug combinations. In contrast, the three-drug combination showed enhanced activity with synergy against six strains, a bactericidal effect against all eight strains, and suppression of resistance during 24-h antibiotic exposure. This combination may be of clinical interest in the treatment of urinary tract infections caused by ESBL-producing E. coli.

Levels of amdinocillin in human plasma and interstitial fluid following intravenous administration.

Amdinocillin levels in human plasma and interstitial fluid were studied in 12 human volunteers. The results showed that relatively high levels of amdinocillin were detected in the interstitial fluid. The fluid to plasma ratio of the area under the curve was 0.25. This is consistent with high diffusibility as seen in drugs with a low percentage of protein binding.

Randomized trial of cefamandole plus amdinocillin versus cefamandole in serious pediatric infections.

In a randomized, prospective clinical trial cefamandole therapy was compared with cefamandole plus amdinocillin in infants and children with suspected bacterial infections. Fifty-two infections in 50 patients with bone and joint (19 infections), pulmonary (19 infections), soft tissue (eight infections), and urinary tract (6 infections) diseases were treated. Bacterial infection was documented in 31 patients. All isolates were susceptible to cefamandole except one strain of Serratia marcescens, which was susceptible to the combination. In vitro synergy was demonstrated in all coliform bacilli, in three of seven Haemophilus strains, and in six of 16 gram-positive cocci. No correlation between degree of serum bactericidal activity and presence or absence of synergy could be demonstrated. One patient treated with cefamandole died; all other patients responded promptly to therapy without serious adverse drug effects.

Pharmacokinetics of amdinocillin and pivamdinocillin in normal volunteers.

The pharmacokinetic parameters of amdinocillin and pivamdinocillin were studied in 12 normal volunteers. Plasma amdinocillin concentrations were determined by microbiologic assay and urine concentrations by high performance liquid chromatography. Pharmacokinetic parameters were calculated by a two-compartment open model for the intravenous infusion and by a one-compartment model with zero-order absorption for the oral doses. The mean peak serum level after the intravenous infusion of 500 mg was 39 micrograms/ml. At one and a half hours after the oral administration of 250 mg and 500 mg doses, mean peaks were 1.93 and 2.66 micrograms/ml respectively. Half-life was one hour for all doses. Maximal plasma concentration did not increase proportionally with dose. Bioavailability was 45 percent after the 250 mg dose and 38 percent after the 500 mg dose.

Parenteral amdinocillin for treatment of complicated urinary tract infections.

Amdinocillin was administered to 55 male and female patients with complicated urinary tract infections. Forty-eight of these patients (28 men and 20 women) were evaluated to determine the safety and efficacy of amdinocillin. Therapy was instituted intravenously and concluded intramuscularly for a total of 7.8 mean therapy days. Toxicity was negligible and side effects were limited to presumed penicillin-allergic reactions. Escherichia coli was the infecting organism in 60 percent of the patients. In 79 percent of the patients, the infections were cured during therapy and from five to nine days following therapy. It was concluded that amdinocillin is a satisfactory antibiotic for the treatment of complicated urinary tract infections with susceptible organisms.

Combination amdinocillin and cefoxitin therapy of multiply-resistant Serratia marcescens urinary tract infections.

Amdinocillin has previously been shown to be synergistic with other beta-lactam antibiotics against many gram-negative bacteria. We evaluated the safety, efficacy, and microbiologic activity of combination amdinocillin and cefoxitin treatment in 17 patients with complicated urinary tract infections caused by multiply-resistant Serratia marcescens. Patients were treated with amdinocillin, 40 mg/kg per day, and cefoxitin, 100 mg/kg per day, for five to 14 days. In vitro synergistic activity was observed for 17 isolates using broth checkerboard testing and for nine isolates using combination disc diffusion testing. Of the 17 patients treated, 11 were bacteriologically cured, one failed to respond, and five patients had a relapse after initial improvement. Relapses followed short-duration therapy. Amdinocillin with cefoxitin was well tolerated. Combination amdinocillin and cefoxitin therapy was efficacious and safe in treating complicated urinary tract infections caused by multiply-resistant S. marcescens.

Amdinocillin plus cefoxitin versus cefoxitin alone in therapy of mixed soft tissue infections (including diabetic foot infections).

In a randomized comparative trial, 45 patients were treated with amdinocillin plus cefoxitin or cefoxitin alone for bacterial soft tissue infections. Most patients were diabetic and had polymicrobial foot infections. The combination of amdinocillin plus cefoxitin was active in vitro against 71 percent of the isolates obtained before therapy as compared with 65 percent for cefoxitin alone. The combination demonstrated synergy for 29 percent of the isolates tested. A satisfactory clinical response occurred in 90 percent and 71 percent of patients treated with the combination regimen and cefoxitin, respectively, (p greater than 0.1). An increase in serum creatinine thought to be due to interstitial nephritis occurred in one patient treated with the combination regimen. The combination of amdinocillin and cefoxitin was effective in mixed soft tissue infections including diabetic foot infections.

Systemic infections treated with amdinocillin in combination with other beta-lactam antibiotics.

Amdinocillin is a semisynthetic derivative of 6-beta-amidinopenicillanic acid, which has bactericidal activity against a broad spectrum of gram-negative bacteria. We report the results of a multicenter study evaluating the safety and efficacy of amdinocillin in combination with other beta-lactam antibiotics in the treatment of 120 serious gram-negative bacterial infections. Amdinocillin was safe and well tolerated and, in combination with other beta-lactam antibiotics, was effective in the treatment of a broad range of gram-negative bacterial infections. Therapy with amdinocillin and other beta-lactam antibiotics was often associated with a demonstrable synergistic effect. Thus, amdinocillin holds promise as an effective antibiotic with synergistic potential when used in combination with penicillins and cephalosporins.

Efficacy of amdinocillin and lack of nephrotoxicity when combined with a second beta-lactam antibiotic for therapy of serious gram-negative bacillary infections.

Seventy-eight patients with serious gram-negative bacillary infections were assigned at random to receive either amdinocillin or an aminoglycoside. In addition, each patient was also given a broad-spectrum penicillin or cephalosporin antibiotic. The clinical response to treatment was comparable in the two groups. Cures were effected in 35 (92 percent) of 38 patients treated with amdinocillin and a beta-lactam antibiotic, compared with 37 (93 percent) of 40 patients who were treated with an aminoglycoside/beta-lactam combination. For the entire group, only five (7 percent) of the 75 infecting organisms were resistant in vitro to the treatment beta-lactam or amdinocillin combination, and similarly only two (3 percent) organisms were resistant to the treatment aminoglycoside (p = 0.44). Although drug-related toxicity occurred with equal frequency in the two groups, six patients treated with an aminoglycoside experienced nephrotoxicity compared with none of the patients who received amdinocillin (p = 0.034). Thus, amdinocillin plus a broad-spectrum beta-lactam antibiotic may provide suitable empiric therapy for many patients with presumed gram-negative infection and so avoid the risk of aminoglycoside-induced nephrotoxicity.

Amdinocillin: use alone or in combination with cefoxitin or carbenicillin-ticarcillin.

One hundred fifty-five patients with 157 febrile episodes were treated with amdinocillin or amdinocillin and cefoxitin as second-line therapy, or amdinocillin and ticarcillin or carbenicillin as initial therapy in three separate studies. Overall responses were 57 percent, 55 percent, and 54 percent for amdinocillin, amdinocillin-cefoxitin, and amdinocillin-ticarcillin or amdinocillin-carbenicillin, respectively. In all three studies, patients with septicemia responded less often than patients with other infections. Most patients were profoundly neutropenic at the initiation of therapy, and both the initial neutrophil level and neutrophil trend during therapy influenced response. A significant number of superinfections occurred when amdinocillin alone was used. Although amdinocillin, alone or in combination with cefoxitin, appeared effective as second-line therapy in infections with organisms shown sensitive in vitro, the combination of amdinocillin and ticarcillin or carbenicillin was only moderately effective in initial therapy for neutropenic, febrile, cancer patients.

Amdinocillin in combination with beta-lactam antibiotics for treatment of serious gram-negative infections.

Amdinocillin in combination with another beta-lactam antibiotic (ampicillin, cephalothin, cefamandole or cefoxitin) was used to treat 25 patients with pyelonephritis (with or without bacteremia), pneumonia, bacteremia secondary to intravenous devices, and urinary tract infections (with catheter in place) due to gram-negative organisms. The combination resulted in a clinical response in 96 percent of the patients and a bacteriologic response in 100 percent at 72 hours. Few toxic effects were seen. At long-term follow-up, relapse occurred in three of 10 patients with pyelonephritis who were treated with a combination regimen and completed their course of antimicrobial therapy with a beta-lactam antibiotic. Reinfection occurred in one patient who had a urinary tract infection with a catheter in place. In vitro testing showed that the cefamandole-amdinocillin combination most frequently produced synergy against the strains of Escherichia coli isolated. Synergy with the antibiotic combinations was also seen against strains of Klebsiella pneumoniae. It was difficult to correlate the in vitro test results with the in vivo therapeutic effect of these antibiotic combinations.

Single-dose kinetics and dosage of mecillinam in renal failure and haemodialysis.

Mecillinam (amdinocillin) serum concentrations and urinary excretion of the drug and its degradation products were determined in 9 subjects: 1 with normal renal function, 4 with varying degrees of renal failure, and 4 on haemodialysis for end-stage renal failure. The results conform to first-order elimination kinetics. With decreasing glomerular filtration, renal clearance decreases sharply, and in severe renal failure approaches the value of creatinine clearance. However, elimination of the drug also takes place by non-renal clearance, which was found to be 48.8 +/- 9.1 (SD) ml/min. Concentrations of mecillinam and its degradation products were also determined in haemodialysis ultrafiltrate. From these data and other evidence, it is concluded that degradation of the drug is the chief mechanism of non-renal elimination. Based on the relationship between creatinine clearance and plasma clearance of mecillinam, and considering that the drug is a relatively non-toxic bactericidal antibiotic, the following dosage adjustment scheme is proposed: glomerular filtration rate over 30 ml/min: normal dosage; 10-30 ml/min: 50% of normal dosage; under 10 ml/min: 25% of normal dosage. Even when dosage is adjusted, therapeutic concentrations of the drug will appear in urine in most cases. During haemodialysis, which increases clearance of the drug by 100%, the dosage should, in principle, be doubled. Alternatively the treatment schedule may be modified by giving the dose just after each dialysis.

Urinary concentrations and urine ex-vivo effect of mecillinam and sulphamethizole.

Healthy adult volunteers received 1 g of sulphamethizole orally (n = 10) and later 400 mg of pivmecillinam (274 mg of mecillinam) (n = 9). All urine was collected in defined periods over 24 h, and the drug concentrations in urine were determined. For sulphamethizole, the maximum urine concentration for seven subjects was reached in 0-3 h, and for the remaining three in 3-6 h. For mecillinam, eight of the nine subjects attained a maximum urine concentration in 0-3 h, after which the concentration declined rapidly for six subjects in 3-6 h. Strains of Escherichia coli with different MICs for sulphamethizole and mecillinam were exposed to collected urine for 2.5 h and 5 h. The results indicated that a sensitive E. coli population should be suppressed by sulphamethizole in urine for two-thirds of the time (with 1 g twice-daily) and by mecillinam in urine throughout the 24-h period (with 400 mg three times a day). There was a slight but significant correlation between the ex-vivo effect (Delta log10 CFU/mL) and the log10 concentration/MIC ratio after exposure to sulphamethizole for 5 h (r2 = 0.27, p < 0.0001), and a significant correlation between the variables with mecillinam (r2 = 0.66, p < 0.0001).

Preliminary experience with parenteral mecillinam.

Case reports are reviewed of 26 patients, mainly with severe Gram-negative infections, treatment with parenteral mecillinam. Twenty-three patients received other antibiotics in addition to mecillinam. In 19 patients, potentially synergistic antimicrobial therapy was given. Overall, a beneficial clinical effect was recorded in 17 (68%) of the 25 patients assessed. A better response was observed in those patients who had not received other antibiotics before treatment with mecillinam was instituted. It is concluded that parenteral mecillinam may have an important role in the treatment of severe Gram-negative infections.

A comparative trial of pivmecillinam/pivampicillin and amoxycillin/clavulanate in the therapy of urinary tract infection in a general practice population.

Fifty-eight patients seen in general practice presenting with symptoms of acute urinary tract infection were entered consecutively into an open randomized trial of 200 mg pivmecillinam plus 250 mg pivampicillin twice daily or 250 mg amoxycillin plus 125 mg clavulanate 3-times daily for 5 days. The results were analyzed in 41 patients with significant bacteriuria (23 on pivmecillinam/pivampicillin and 18 on amoxycillin/clavulanate). Both antibiotic combinations produced good overall bacteriological cure, but there were a considerable number of persisting symptoms despite the absence of significant bacteriuria. Eight patients in the pivmecillinam/pivampicillin group and 5 in the amoxycillin/clavulanate group had side-effects, principally thrush, vomiting and abdominal pain, and 1 patient from each group ceased treatment for this reason. Sensitivity profiles of urinary isolates (41 trial, 135 non-trial) to both combinations and to ampicillin and mecillinam showed that the majority were fully sensitive to amoxycillin/clavulanate and to a lesser extent to pivmecillinam/pivampicillin; resistance was highest to ampicillin.

Pharmacokinetics of mecillinam after a single intravenous dose in patients with impaired renal function.

Twelve male patients with impaired renal function and six healthy male volunteers were given a single i.v. dose of mecillinam (400 mg). Serum concentrations of mecillinam were determined by a microbiological assay. The pharmacokinetic parameters were obtained using an open, two-compartment model. Results showed that the median elimination t 1/2 (beta) of mecillinam was longer in patients (5 h) than in volunteers (3.4 h) (p less than 0.1). The median AUC0----infinity was greater in patients (72.44 micrograms ml-1.) than in volunteers (32.96 micrograms ml-1 X h) (p less than 0.001). Moreover, the median clearance was decreased in patients (5.5 L h-1) as compared with volunteers (12.17 L h-1) (p less than 0.001). Thus the study showed prolonged half-life and decreased elimination of mecillinam in patients with impaired renal function.

[Absorption, distribution, metabolism and excretion of bacmecillinam. III. Absorption, metabolism and excretion of 14C-bacmecillinam following oral administration to dogs].

The pharmacokinetics (i.e., blood level, biological half-lives and excretion) of bacmecillinam (KW-1100) was investigated. KW-1100 was orally administered to dogs at the dose of 20 mg/kg (as mecillinam). Biological half-lives (radioactivity) of 14C-KW-1100 in plasma were 1.2 hours (T1/2 alpha) and 52 hours (T1/2 beta). The Cmax and Tmax were 8.4 micrograms/ml and 2 hours. The biological half-life (microbiological activity) of KW-1100 in plasma was 0.9 hour. The Cmax and Tmax were 5.6 micrograms/ml and 1 hour. The urinary and fecal excretion of 14C-KW-1100 were approximately 46% and 49% (0 approximately 72 hours), respectively. The major metabolites in the urine (0 approximately 8 hours) were mecillinam, 5,5-dimethyl-2-(1'-formamidomethyl)-thiazolidine-1',4-dicarboxy lat e (M-1) and 6-beta-[(hexahydro-1 H-azepin-1-yl)-methyleneamino]penicilloic acid (M-6), each distribution ratio of which was 57.2, 24.2 and 12.0% of the total radioactivity in the sample, respectively. The major metabolite in the plasma at peak time (2 hours) was mecillinam (56.2%).