Combination antimicrobial susceptibility testing of multidrug-resistant Stenotrophomonas maltophilia from cystic fibrosis patients.

Stenotrophomonas maltophilia is increasingly being isolated from the respiratory tract of individuals with cystic fibrosis, and, because of its multidrug-resistant nature, the selection of suitable treatment regimens can be problematical. Etest methodology was used to facilitate MIC and antimicrobial combination testing on 80 isolates of S. maltophilia cultured from the respiratory tract of Scottish individuals with cystic fibrosis between 2001 and 2010. The overall rate of susceptibility for the 1,410 MIC tests was 23.1%, and resistance was 68.9%. The most active antimicrobials were minocycline, co-trimoxazole, and doxycycline, with 92.4%, 87.3%, and 58.8% of isolates being susceptible, respectively. Of the 517 combinations, 13.2% were synergistic, with the most synergistic being ticarcillin/clavulanate plus aztreonam (91.7% synergistic), ticarcillin/clavulanate plus colistin (40%), and ticarcillin/clavulanate plus levofloxacin (19.4%). Colistin plus tobramycin was the only antagonistic combination (0.2%). By the median susceptible breakpoint index, the most active combinations were minocycline plus co-trimoxazole (median index, 20), minocycline plus piperacillin-tazobactam (median, 20), and co-trimoxazole plus ceftazidime (median, 16.5). The increasing problem of multidrug resistance in organisms recovered from the respiratory tracts of individuals with cystic fibrosis is not going to go away. Current susceptibility testing methods do not address the slow-growing organisms associated with chronic infection, and interpretive standards are based on achievable blood levels of antimicrobials. Addressing these issues specifically for organisms recovered from the respiratory tracts of individuals with cystic fibrosis should lead to better therapeutic outcomes and improved wellbeing of individuals with cystic fibrosis.

Combination testing of multidrug-resistant cystic fibrosis isolates of Pseudomonas aeruginosa: use of a new parameter, the susceptible breakpoint index.

OBJECTIVES: The microbiology laboratory at Aberdeen Royal Infirmary operates an extended susceptibility testing service for multidrug-resistant Gram-negative non-fermenting isolates from the sputum of Scottish cystic fibrosis patients. The service aims to provide clinicians with useful treatment options and developed the use of a novel parameter-the susceptible breakpoint index (SBPI), which allows easy ranking of the antimicrobial combinations in order of their possible in vivo effectiveness. METHODS: Three hundred and fifteen isolates of Pseudomonas aeruginosa were submitted for testing. MICs of 14 antimicrobials were determined using the Etest and the results categorized using CLSI guidelines. Usually, six antimicrobial pairs were tested in combination also using the Etest. The results were assessed using the fractional inhibitory concentration index (FICI) and also by a novel parameter, the SBPI. RESULTS: Some 4173 MICs and 1663 combination pairs were performed. The most active individual antimicrobials were colistin, tobramycin and amikacin, with 84%, 69% and 32% of isolates susceptible, respectively. Twenty-eight of 44 antimicrobial combinations were tested >10 times. Of the combinations, 3.6% were synergistic (FICI < or = 0.5) and 0.1% were antagonistic (FICI > 4.0). Amikacin + ceftazidime (17%), ciprofloxacin + ceftazidime (12.9%) and ciprofloxacin + piperacillin/tazobactam (12%) were the most synergistic combinations. By median SBPI, the most effective combinations in vitro were colistin + ticarcillin/clavulanate, colistin + piperacillin/tazobactam and colistin + meropenem. CONCLUSIONS: The Etest is a useful tool for determining MICs and testing antimicrobial combinations. The SBPI is more discriminatory than the FICI, allowing easy ranking of the combinations, and is likely to have clinical relevance.

Antibiograms of resistant Gram-negative bacteria from Scottish CF patients.

BACKGROUND: Over a 19-month pilot phase, 93 multiply resistant Gram-negative isolates from Scottish cystic fibrosis patients were sent to a referral laboratory for further investigation. METHODS: In common with the referring diagnostic laboratories, disc diffusion testing was carried out. Antibiotic susceptibility testing was also established by MIC methodology. NCCLS methods were used throughout. Twenty antibiotics were tested. RESULTS: Comparing disc diffusion results against MIC results, there were 167 (14%) major errors. By MIC, Pseudomonas aeruginosa (n = 59), Stenotrophomonas maltophilia (n = 16), Burkholderia cepacia (n = 10) and Alcaligenes xylosoxidans (n = 7) were susceptible to 18%, 11%, 4% and 35% of the antibiotics tested, respectively. Colistin and tobramycin were the most active agents against P. aeruginosa with 60% and 49%, respectively, testing susceptible. Minocycline and gentamicin were most active against S. maltophilia with 58% and 18%, respectively, testing susceptible. B. cepacia were most susceptible to co-trimoxazole (10%) and ciprofloxacin (10%). Five and six of the seven A. xylosoxidans isolates were susceptible to piperacillin and imipenem, respectively. CONCLUSIONS: Improved methods for susceptibility testing of such clinical isolates need to be employed in routine diagnostic laboratories. Levels of resistance in referred isolates were very high and similar to those described in the USA.

Reassessment of cefaclor breakpoints for Haemophilus influenzae.

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

The post-exposure response of Enterobacteriaceae to ceftibuten.

The responses of ten isolates of Enterobacteriaceae to ceftibuten exposure were monitored by measuring several parameters. Post-antibiotic effect (PAE), control-related effective regrowth time (CERT) and post-antibiotic sub-MIC effect (PA-SME) were determined by bacterial enumeration carried out either by impedance in combination with viable counting (IMP/VC) or by impedance in combination with bioluminescence (IMP/BIOL). Kill curves were carried out by bioluminescence, viable counting and direct microscopy and post-exposure morphology was established. Ceftibuten primarily provoked filamentation. Over 24 h, kill of up to 3.6 log10 was evident by viable counting and direct microscopy at and above the MIC. Minimal kill, of up to 0.26 log10, was shown by bioluminescence. PAE was found to be method dependent, with statistical differences established by Student's t-test. PAE values of up to 0.48 h and 1.47 h (by IMP/BIOL and IMP/VC respectively) were not concentration dependent above 1 x MIC. CERT values were not method dependent, with values of up to 1.71 h also showing a lack of concentration dependence above 1 x MIC. PA-SME may reflect the situation in vivo more accurately than either PAE or CERT. In PAE and CERT studies the antibiotic is eliminated almost immediately, whereas in vivo there is gradual decrease in antibiotic levels. These persisting levels are reflected more accurately by PA-SME. Compared with PAE and CERT, significantly longer values, of up to 7.27 h, were obtained by PA-SME, although this parameter was also found to be method dependent. The results of the PA-SME studies, which may be the most clinically relevant pharmacodynamic parameter, confirm the appropriateness of the current once- or twice-daily dosing schedules despite the lack of PAE.

The effects of the morphological response of Enterobacteriaceae to cephalosporins on PAE and CERT.

PAE and CERT values of cefaclor, loracarbef and cefuroxime (0.1-100 x MIC) were established for 8 E. coli, 3 K. pneumoniae and 2 P. mirabilis isolates. Cell enumeration was by impedance (IMP) monitoring in combination with either bioluminescence (BIOL) or viable counting (VC). Morphology was determined by interference contrast microscopy. After 2 h exposure to cefaclor, loracarbef and cefuroxime; concentration-dependent differences in counts were seen by BIOL and VC, varying from a mean value of 0.07 x log10 after exposing the P. mirabilis isolates to 0.1 x MIC cefaclor to a mean value of 2.24 x log10 after exposing the E. coli strains to 100 x MIC cefuroxime. Higher concentrations gave rise to fragile morphological forms including spheroplasts and lower concentrations to less fragile forms such as long bacilli. The longest PAE and CERT values were obtained after exposing the E. coli strains to 100 x MIC cefaclor with mean values of 4.07 and 4.87 h, respectively. Corresponding values were PAE and CERT values of 2.17 and 2.60 h for 100 x cefuroxime and 3.45 and 2.91 h for 100 x MIC loracarbef. By the Student's t-test, PAE values determined by IMP/BIOL and IMP/VC were found to be significantly different, whereas CERT values were found not to be significantly different. PAE and CERT are concentration dependent and vary with specific antibiotic/organism combinations.