Tigecycline

Antimicrobial susceptibility of the SAR Bari strain was determined by a BD PHOENIX^™ 100 instrument (Becton Dickinson, Franklyn Lake, NJ). Data were elaborated by the BD Epicenter Expert System according to EUCAST rules (http://www.eucast.org). The PMIC/ID-88 (BD) panel was used to test susceptibility to ampicillin, cefoxitin, ceftaroline, ciprofloxacin, clindamycin, daptomycin, erythro-mycin, fosfomycin, fusidic acid, gentamicin, imipenem, linezolid, moxifloxacin, mupirocin, nitro-furantoin, oxacillin, penicillin, rifampin, teicoplanin, tetracyclin, tigecycline, trimethoprim/ sulfamethoxazole, and vancomycin. The Epsilometer Test (ETest) was used for testing resistance to ciprofloxacin, daptomycin, erythromycin, gentamicin, moxifloxacin, tetracyclin, tigecycline, trimethoprim/sulfamethoxazole, and vancomycin (bioMérieux, Marcy-L’Étolie, France and Liofilchem, Roseto degli Abruzzi, Italy). All tests were repeated on four independent technical replicates. MIC interpretative breakpoints were defined according to EUCAST recommendations. Staphylococcus aureus ATCC 29213 was used as a control strain.

MICs were determined by the Etest method according to the manufacturer’s instructions. Antimicrobial agents included aztreonam, ceftazidime, ciprofloxacin, colistin, gentamicin, imipenem, nalidixic acid, tetracycline, tigecycline, tobramycin, and trimethoprim (bioMe’rieux, Marcy-l’_Etoile, France). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. Interpretation of antimicrobial susceptibility was based on guidelines of the Clinical Laboratory Standards Institute (CLSI) [49 ].

The antibiotic resistance profile of the strain was evaluated using the E‐test method and the following molecules: benzylpenecilin, amoxicillin, cefotaxime, ceftriaxone, imipenem, rifampicin, minocycline, tigecycline, amikacin, teicoplanin, vancomycin, colistin, daptomycin, and metronidazole (Biomerieux, France).

From the soil extract obtained in saline solution (NaCl 0.45%), it was verified that the density of viable microorganisms was >10⁸ ufc mL⁻¹ (optical density [OD] = 0.5 McFarland). The bacterial extraction was sown in Mueller–Hinton agar (Condalab^®, Madrid, Spain), and the minimum inhibitory concentration (MIC) was evaluated by the Kirby–Bauer method, using ε-test antibiotic strips, in triplicate, for the following antibiotics: cefuroxime, cefuroxime axetil, cefoxitin, cefotaxime, ceftazidime, cefepime, ertapenem, imipenem, amikacin, gentamicin, nalidixic acid, ciprofloxacin, tigecycline and trimethoprim/sulfamethoxazole (BioMérieux^®, Marcy l’Etoile, France). Plates were then incubated according to the manufacturer’s instructions. For the quantification of the MIC, the most restrictive halo was used as reference.

Antimicrobial sensitivity testing was performed on Mueller-Hinton agar (MHA) plates by Kirby-Bauer disc diffusion method, according to Clinical Laboratory Standards Institute (CLSI) guidelines [9 ]. The antibiotics used were: ampicillin (10 μg), cephalexin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefpodoxime (10 μg), ceftriaxone (30 μg), cefepime (30 μg), aztreonam (30 μg), cefoxitin (30 μg), piperacillin/tazobactam (100/10 μg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), co-trimoxazole (25 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), gentamicin (10 μg), amikacin (30 μg), tigecycline (15 μg) and colistin (10 μg). All the antibiotic discs and the media were procured from Hi-media, Mumbai, India. E. coli ATCC 25922 was used as quality controls in antibiotic susceptibility testing. The results were interpreted as per CLSI guidelines [9 ] except, tigecycline and colistin. The results for colistin were interpreted by following the criteria proposed by Galani et al. [10 (link)] and for tigecycline by following the breakpoints for Enterobacteriaceae as suggested by Food and Drug Administration (FDA). The minimum inhibitory concentration (MIC) values for imipenem, meropenem, ertapenem, tigecycline and colistin were determined by using Etest strips (bioMerieux, France) as per the manufacturer’s protocol.

The antimicrobial susceptibility testing of K. pneumoniae was examined using Vitex II cards (AST-N292) (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s guidelines. The following antimicrobial agents were tested: amikacin (AK), ampicillin (AMP), amoxicillin/clavulanate (AMC), aztreonam (ATM), ceftriaxone (CRO), cefepime (CFPM), cefuroxime (CXM), ciprofloxacin (CIP), trimethoprim/sulfamethoxazole (SXT), colistin (CST), gentamicin (GN), imipenem (IPM), meropenem (MEM), piperacillin/tazobactam (TZP), and tigecycline (TGC) (bioMérieux, Marcy l’Etoile, France). Clinical and Laboratory Standards Institute (CLSI) recommendations were used to determine the minimum inhibitory concentration (MIC) and breakpoints [13 ]. MDR K. pneumoniae was defined as non-susceptibility to at least one agent in three or more antimicrobial categories [14 (link)]. K pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as quality control strains. The isolates were immediately stored in brain heart infusion broth containing 20% glycerol at −86 °C for further genetic analysis.