Piperacillin tazobactam

Antimicrobial susceptibility testing (AST) of E. coli and K. pneumoniae isolates was performed by the Kirby Bauer disk diffusion method according to the CLSI guideline [19 ]. The antimicrobial disks used for testing were ampicillin (10 µg) [tested only for E. coli], Amoxicillin-clavulanic acid (20/10 µg), piperacillin-tazobactam (100/10 µg), cefazolin (30 µg), cefuroxime (30 µg). cefixime (5 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), imipenem (10 µg), ciprofloxacin (5 µg), trimethoprim sulphamethoxazole (1.25/23.75 µg), nitrofurantoin (300 µg), and amikacin (30 µg). Amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftazidime, ciprofloxacin, and imipenem disks were purchased from the manufacturer Mast (Mast group Ltd, Liverpool, UK) with the remainder from HiMedia (HiMedia, India). The minimum inhibitory concentration (MIC) of ciprofloxacin was determined by E-test (0.002-32 µg/mL) (HiMedia, India), and those isolates with MIC ≥ 32 µg/mL were further tested by agar dilution method following the procedures described by CLSI [20 ]. An isolate was defined to display multidrug resistance (MDR) if non-susceptible to ≥ 1 agent in ≥ 3 antimicrobial categories [21 (link)]. Escherichia coli ATCC 25922 was used for quality control.

Eight K. pneumoniae isolates were received from the Medical Microbiology laboratories of three hospitals in Armenia between January 2019 and August 2019. All isolates were recovered from various clinical specimens (urine, sputum, throat, blood, and stool) of hospitalized patients. The isolates were identified using a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS) as described previously (59 (link)).
All isolates were tested using a disk diffusion method for susceptibility to a panel of 11 antibiotics, including ampicillin (10 mg), piperacillin-tazobactam (30/6 mg), amoxicillin-clavulanic acid (20 and 10 mg, respectively), ceftazidime (10 mg), cefepime (30 mg), norfloxacin (10 mg), levofloxacin (5 mg), amikacin (30 mg), imipenem (10 mg), meropenem (10 mg), and chloramphenicol (30 mg) (Mast Group, Merseyside, United Kingdom) according to the European Committee on Antimicrobial Susceptibility Testing protocol (EUCAST v.6.0, 2017) (60 ). The antibiotics chosen were those most frequently used in clinical settings in Armenia. Isolates resistant to three or more antibiotic classes were considered multidrug resistant.

The antibiotic susceptibility of all the isolates was tested by employing the Kirby-Bauer’s technique as suggested by the CLSI (10 ). The eleven antibiotic disks used include: imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), cefepime (30 μg), cefotaxime (30 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin/tazobactam (100/10 μg), aztreonam (30 μg) (Mast Group Ltd, UK). Isolates with resistance against a minimum of three groups of antibacterial agents were considered as MDR (11 (link)). To detect ESBL phenotype combined disk method using disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK) was applied to all positively screened isolates by modified hodgE test (MHT) (11 (link)). A growth in the area diameter of ≥5 mm around ceftazidime disc with and without clavulanic acid was expected to be a positive result for ESBL production (12 (link), 13 (link)). The MHT was performed for all isolates as recommended by CLSI (10 ). The E test (imipenem 0.002–32μg/mL) (Liofilchem, Roseto degli Abruzzi, Italy) was applied (according to the manufacturer’s instructions) to all positively screened isolates by PCR test for bla_NDM gene, to determine minimum inhibitory concentrations (MICs).

The resistance pattern of isolates against 15 antibiotics including 5 fluoroquinolones was determined by disc diffusion method on Mueller-Hinton agar (Merck, Germany) as described by the Clinical Laboratory Standards Institute (CLSI 2017) guidelines³⁶ . The antibiotic disks used were as follows: amikacin (30 μg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), gatifloxacin (5 μg), norfloxacin (5 μg), gentamycin (10 μg), imipenem (10 μg), levofloxacin (5 μg), ofloxacin (5 μg), piperacillin (100 μg), piperacillin/tazobactam (100 μg/10 μg), and tobramycin (10 μg) (MAST Co., Berkshire, UK). Drug-resistant patterns were defined as follows: MDR isolates (resistant to at least three antibiotics belonging to different chemical classes), XDR strains (resistant to at least one agent in all but two or fewer antimicrobial groups), and PDR strains (resistant to all antimicrobial classes)^{37 (link)}. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control strains.

The profile of this test was investigated based on CLSI [30 (link)] and EUCAST guidelines [31 ] using VITEK 2 compact system (Biomerieux, Craponne, France) in accordance with the manufacturer’s instructions and Kirby–Bauer disk diffusion method [32 (link)] for different antibiotics, including Ampicillin, Piperacillin-Tazobactam, Amoxiclav, Ceftriaxone, Cefotaxime, Ceftazidime, Cefepime, Meropenem, Amikacin, Ciprofloxacin and Levofloxacin (Mast Group, Bootle, England).

The antibiotic susceptibility pattern of the isolates was determined by the disk agar diffusion method on Muller Hinton agar (Merck, Germany) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines²¹. The antibiotics included piperacillin (100 µg), piperacillin-tazobactam (100/10 µg), imipenem (10 µg), meropenem (10 µg), doripenem (10 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), trimethoprim-sulfamethoxazole (1.25-23.75 µg), ceftazidime (30 µg), cefotaxime (30 µg), and cefepime (30 µg) (MAST Co., England). The susceptibility pattern of the isolates against aminoglycosides including kanamycin, amikacin, spectinomycin, netilmicin, gentamicin, streptomycin, and tobramycin was determined using the micro-broth dilution method according to the CLSI guidelines²¹. For interpretation of the minimum inhibitory concentration (MIC) values, we referred to the CLSI guidelines and previous studies^1,21,22. Escherichia coli ATCC 25922 and A. baumannii ATCC 19606 were used as control strains for antibiotic susceptibility testing.