Tetracycline

The antimicrobial susceptibilities of 175 E. faecalis and 67 E. faecium strains were examined by using the disk agar diffusion (DAD) method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines.17 18 Erythromycin (15 µg), Tetracycline (30 µg), Ciprofloxacin (5 µg), Vancomycin (30 µg), Teicoplanin (30 µg), Norfloxacin (10 µg), Nitrofurantoin (300 µg), Quinopristin-Dalfopristin [Synercid (15 µg)] (Mast Co., UK), Chloramphenicol (30 µg), Gentamicin (10 µg), Linezolid (30 µg), and Ampicillin (10 µg) (HiMedia Mumbai Co., India) were used for antimicrobial susceptibility testing (AST).
In addition, minimum inhibitory concentrations (MIC) of the glycopeptide antibiotics i.e. Vancomycin and Teicoplanin (Sigma-Aldrich, Poole, Co., UK) against the E. faecalis and E. faecium isolates were determined using the microdilution broth method.17 18
E. faecalis ATCC 29212 (Vancomycin sensitive), E. faecalis ATCC 51299 (vanB positive), E. faecalis E206 (vanA positive) were used as quality control.

The disk diffusion method using cefoxitin (30 μg) disk in Mueller-Hinton agar (Merck, Germany) according to the CLSI was applied for the screening of methicillin resistance isolates. In addition, PCR assay was used for the detection of mecA gene.12
The Kirby–Bauer disk diffusion method was used to determine the susceptibility of the isolates against penicillin, ceftriaxone, amikacin, gentamicin, tobramycin, kanamycin, tetracycline, linezolid, teicoplanin, ciprofloxacin, rifampicin, quinupristin-dalfopristin, and trimethoprim-sulfamethoxazole (Mast Co., UK) based on the CLSI recommendation (CLSI 2019). The minimum inhibitory concentration (MIC) value for vancomycin, mupirocin, tigecycline, and fusidic acid was determined using the broth microdilution method. Results for fusidic acid and tigecycline were interpreted according to the European Committee for antimicrobial susceptibility testing (EUCAST) breakpoints (http://www.eucast.org). Low-level and high-level mupirocin resistance (LLMUPR, HLMUPR) were defined if MIC values of 8–256 µg/mL and ≥512 µg/mL were obtained. S. aureus strains ATCC 25923, ATCC 43300 and ATCC 29213 were used as reference strains.

Antibiotic susceptibility testing was performed by using Kirby–Bauer disc diffusion method according to Clinical and Laboratory Standards Institute recommendations (CLSI) [8 ]. The antimicrobial disks tested were ampicillin (AMP), cefoxitin (FOX), cefazolin (CFZ), ceftriaxone (CRO), cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), amoxicillin-clavulanate (AMC), aztreonam (ATM), gentamicin (GEN), streptomycin (STR), amikacin (AMK), nalidixic acid (NAL), ofloxacin (OFX), ciprofloxacin (CIP), levofloxacin (LVX), chloramphenicol (CHL), trimethoprim-sulfamethoxazole (TMP/SMX), tetracycline (TET), azithromycin (AZM), imipenem (IPM) and meropenem (MEM) (Mast Co., Bootle, Merseyside, UK). Escherichia coli ATCC 25922 was used as the quality control strain. The MDR was defined as resistance to 3 or more unrelated antibiotics [9 ].

The Kirby–Bauer disc-diffusion method on Mueller–Hinton agar was used to test the susceptibility of the isolates against amikacin, gentamicin, tobramycin, kanamycin, tetracycline, erythromycin, clindamycin, linezolid, teicoplanin, ciprofloxacin, rifampicin, quinupristin-dalfopristin and trimethoprim-sulfamethoxazole (Mast Co., Bootle, UK) based on the CLSI guideline. Susceptibility to vancomycin, mupirocin, tigecycline and fusidic acid was assessed by the broth microdilution method to determine the MIC titre. The European Committee for Antimicrobial Susceptibility Testing (EUCAST) breakpoints was used to determine MIC titres of fusidic acid and tigecycline (EUCAST 2018). The results of other antibiotics were interpreted by using the CLSI 2018 breakpoints. Low-level and high-level mupirocin resistance (LLMUPR, HLMUPR), inducible macrolide-lincosamide-streptogramin group B (iMLS_B) and constitutive (cMLS_B) macrolide-lincosamide-streptogramin group B were identified based on the CLSI guideline. Susceptibility testing was quality controlled using S. aureus ATCC 25923, ATCC 43300 and ATCC 29213 strains. Powders of antibiotics were all obtained from Sigma Chemical Co. (St Louis, MO, USA).

The antimicrobial susceptibility test was done with the disk diffusion method (Bauer et al, 1966) using Mueller–Hinton agar (Difco). Initially, an emulsion of sample in saline solution was prepared by adjustment to the 0.5 McFarland turbidity standards. The susceptibility of the E. coli strains was tested in relation to several antibiotics, including: chloramphenicol (CAF) (30 μg), ciprofloxacin (5 μg), gentamycin (10 μg), trimethoprim (1.25 μg-sulfamethoxazole 23.75 μg), erythromycin (15 μg), streptomycin (10 μg) and tetracycline (30 μg) (Mast Group Ltd., Merseyside, UK). Using sterile tweezers, commercially available antibiotic disks were placed individually on the surface of Mueller-Hinton agar. After 24 h of incubation at 35°C, the strains were scored as “susceptible”, “intermediate”, or “resistant” to each antibiotic based on the measurement of the inhibition zone, as recommended by clinical laboratory standard institute (CLSI).26