A checkerboard assay was performed in a 96-well plate to examine the antibacterial synergy effect of methicillin (Thermo) and the identified compounds as described (Rajagopal et al., 2016 (link)). An overnight culture to an OD600 of 0.1 in TSB was reinoculated at 30°C to reach an OD of 1.0 for the assay. Following a twofold serial dilution of antibiotics and compounds in 150 μL of MHB media, the culture was inoculated to an OD600 of 0.002. After incubation at 30°C for 16 h with shaking, bacterial growth was measured at OD600 (BioTek Synergy HTX).
Methicillin
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Methicillin is a synthetic penicillin antibiotic used in laboratory settings. It functions as a bactericidal agent, inhibiting the synthesis of the bacterial cell wall.
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Lab products found in correlation
Ciprofloxacin, by Thermo Fisher Scientific (5 mentions)
Erythromycin, by Thermo Fisher Scientific (4 mentions)
Clindamycin, by Thermo Fisher Scientific (3 mentions)
Vancomycin, by Thermo Fisher Scientific (3 mentions)
Oxacillin, by Thermo Fisher Scientific (2 mentions)
Azithromycin, by Thermo Fisher Scientific (2 mentions)
Imipenem, by Thermo Fisher Scientific (2 mentions)
Tetracycline, by Thermo Fisher Scientific (2 mentions)
Ampicillin, by Thermo Fisher Scientific (2 mentions)
Trimethoprim sulfamethoxazole, by Thermo Fisher Scientific (2 mentions)
Ceftriaxone, by Thermo Fisher Scientific (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Gentamicin, by AppliChem (1 mentions)
Fusidic acid, by Merck Group (1 mentions)
Oxacillin, by Merck Group (1 mentions)
Mupirocin, by AppliChem (1 mentions)
Tryptic soy broth (tsb), by BD (1 mentions)
Mueller hinton broth (mhb), by BD (1 mentions)
Methicillin, by Merck Group (1 mentions)
11 protocols using methicillin
1
Antibacterial Synergy Assay in 96-well
2
Antibiotic Preparation and Disk Assay
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Fusidic acid (Sigma-Aldrich, St. Louis, MO, USA), mupirocin (AppliChem, GmbH, Darmstadt, Germany), methicillin (Thermo Fisher Scientific, Waltham, MA, USA), oxacillin (Sigma-Aldrich, St. Louis, MO, USA) and gentamicin (AppliChem, GmbH, Darmstadt, Germany) were also purchased as pure compounds. Stock solutions of Fusidic acid, methicillin, oxacillin and gentamicin were prepared in sterile distilled water. For mupirocin, the stock solution was prepared in dimethyl sulfoxide (DMSO, 100%). The percentage of DMSO never exceeded 10% (v/v) of the final volume. The mass of antibiotics on the disc used was selected according to Clinical and Laboratory Standards Institute (CLSI) guidelines (Fusidic acid: 10 µg/disc; mupirocin: 200 µg/disc; methicillin: 5 µg/disc; oxacillin: 1 µg/disc; gentamicin: 10 µg/disc).
3
Cultivation and Antibiotic Susceptibility of MRSA
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The MRSA strain S. aureus USA300 (TCH1516) was used in this study. S. aureus strains were maintained aerobically in tryptic soy broth (TSB, BD) or TSB 1.5% agar at 30°C with shaking at 250 rpm. Methicillin was purchased from Thermo Scientific and used as denoted. Mueller Hinton Broth (MHB, BD) was used for antibiotic susceptibility testing.
4
Evaluating Methicillin Resistance in S. aureus
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A total of 24 isolates consisting of a minimum of two isolates from a cluster of MRSA or MSSA as determined by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) clustering of the MDEs distribution in each isolate Fig. (S1 ), were used for this assay. Single outliers that did not cluster with any other isolate were also included. The methicillin (Sigma Aldrich; South Africa) SCs for the 24 clinical MRSA and MSSA isolates were determined using the micro-dilution method as described in the Clinical and Laboratory Standards Institute (9). Briefly, 5 X 104 CFU/ml of each isolate was incubated in Tryptone soya broth (Oxoid, South Africa) in the presence of 0.5, 1, 2, 4, 8 and 16 µg/ml methicillin for 16 hr at 37 ˚C. The visual SCs were defined as susceptible (no visual growth observed); resistant (micro-well completely covered with bacteria; and intermediate resistance (about 1-4 dot-like growth observed in the micro-well). Two independent tests were performed to assess reproducibility. The MIC was taken as the minimum concentration in which no visual bacterial growth was observed. The growth of the 24 S. aureus isolates was also determined by quantitative analysis of spectrophotometric measurements of the optical density (absorption) at 600nm after the 16 h incubation period. The S. aureus MU50 ATCC 700699 was used as control in the spectrophotometric measurements.
5
Antibiotic Resistance Profiling of S. aureus
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The pattern of antimicrobial resistance was studied using the simple disk diffusion technique. The Mueller-Hinton agar (Merck, Darmstadt, Germany) medium was used for this purpose. Antibiotic resistance of S. aureus strains against 16 commonly used antibiotics in the cases of urinary tract infections was determined using the instruction of clinical and laboratory standards institute (CLSI) guidelines (19 ). Susceptibilities of S. aureus isolates were tested against ampicillin (10 u/disk), gentamycin (10 μg/disk), amikacin (30 u/disk), imipenem (30 u/disk), methicillin (30 μg/disk), tetracycline (30 μg/disk), vancomycin (5 μg/disk), ciprofloxacin (5 μg/disk), norfloxacin (30 μg/disk), cotrimoxazole (30 μg/disk), clindamycin (2 μg/disk), trimethoprim-sulfamethoxazole (25 μg/disk), penicillin G (10 u/disk), oxacillin (1 μg/disk), erythromycin (15 μg/disk) and azithromycin (15 μg/disk) antibiotic agents (Oxoid, Basingstoke, UK). The plates containing the discs were allowed to stand for at least 30 minutes before incubation at 35°C, for 24 hours. The diameter of the zone of inhibition produced by each antibiotic disc was measured and interpreted using the CLSI zone diameter interpretative standards (19 ). S. aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as quality control organisms in antimicrobial susceptibility determination.
6
Antibiotic Resistance Testing of Flavonoids
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The antibiotic discs included amoxicillin (AMO; 25 μg), ampicillin (AMP; 10 μg), ceftriaxone (CET; 30 μg), cefixime (CEF; 5 μg), cephradine (CEPH; 30 μg), erythromycin (ERY; 15 μg), vancomycin (VAN; 30 μg), methicillin (ME; 10 μg), ciprofloxacin (CIP; 5 μg), levolfloxacin (LEV; 5 μg), sulfamethaxozole-trimethoprim (S-T; 25 μg) and imipenem (IMP; 10 μg) were from Oxoid, UK while blank discs were purchased from Himedia, India. Test flavonoids; rutin, morin, and quercetin were purchased from Sigma-Aldrich, UK. Stock solutions of flavonoids morin, rutin, and quercetin were made at concentrations of 50 g/l, 6 g/l, and 10 g/l respectively, using ethanol. Bacterial culturing medias such as nutrient agar (NA; CM0003B), muller hinton agar (MHA; CM0337B), nutrient broth (N.B; CM0001B) and muller hinton broth (MHB; CM0405B) were from Oxoid, UK. mannitol salt agar (MSA; LAB007) was obtained from Lab M Limited, UK.
7
Antimicrobial Susceptibility of MRSA
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The Kirby–Bauer disk diffusion method was used to detect the antimicrobial susceptibility of all MRSA strains. Thirteen types of antimicrobials discs (Oxoid, UK), including methicillin (ME; 5 μg), cefoxitin (FOX; 30 μg), vancomycin (VA; 30 μg), clindamycin (DA; 2 μg), rifamycin SV (RF; 30 μg), trimethoprim-sulfamethoxazole (SXT; 1.25/23.75 μg), imipenem (IPM; 10 μg), erythromycin (E; 15 μg), ciprofloxacin (CIP; 5 μg), ceftriaxone (CRO; 30 μg), amoxicillin-clavulanic acid (AMC; 20/10 μg), gentamicin (CN; 10 μg), and chloramphenicol (C; 30 μg), were used and the susceptibility of the tested strains was determined according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) [50 ]. The MDR was defined as resistance to three or more different classes of the evaluated antimicrobials [51 ]. The minimum inhibitory concentration (MIC) of vancomycin was determined phenotypically using the broth micro dilution method [52 ].
8
Antibiogram of S. aureus Isolates
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Antibiogram of S. aureus isolates was performed by the disk diffusion method. All isolates were tested against 12 antimicrobials from 10 classes. The disks were purchased from Oxoid (Hampshire, England) and include: methicillin (ME, 5 μg), cefotaxime (CTX, 30 μg), vancomycin (VA, 30 μg), azithromycin (AZM, 15 μg), erythromycin (E, 15 μg), gentamicin (CN, 10 μg), tetracycline (TE, 10 μg), ciprofloxacin (CIP, 5 μg), clindamycin (DA, 2 μg), sulfamethoxazole-trimethoprim (SXT, 25 μg), chloramphenicol (C, 30 μg) and linezolid (LZD, 3 μg). The inoculum was optimized to 0.5 McFarland turbidity standard and inoculated on Muller Hinton Agar (Oxoid, Hampshire, England) as recommended by the Clinical and Laboratory Standards Institute (CLSI). The plates were incubated at 37°C for 18 h after placing antimicrobial disks. Zones of inhibition were measured, and the results were interpreted according to the criteria of CLSI (13 ).
9
Isolation of S. aureus from Burn Wounds
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The experiment was carried out in the Microbiology Laboratory of the Department of Microbiology, Stamford University Bangladesh, Dhaka, Bangladesh from April 23, 2012 to January 15, 2013. A total of 40 wound samples from patients with tertiary burns of partial or full thickness (deep reticular dermis) were collected aseptically with a sterile cotton swab by a clinician wearing gloves (US Safety & Supply Co.) and a mask in the burn unit of Dhaka Medical College Hospital, Dhaka, Bangladesh [7 24 ]. The patients were under treatment with antibiotics including trimetho-prim–sulfamethoxazole, methicillin, and ceftriaxone (Oxoid, UK). All patients were men aged between 20 years and 45 years from a lower middle-class community. The samples were inoculated on Mannitol salt agar (MSA) (HiMedia, India) plates immediately after sample collection for isolation of S. aureus and transported to the laboratory as early as possible [19 (link)23 (link)25 ].
10
Identification and Screening of MRSA Isolates
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All swab samples were incubated for 48 h at 37℃ in pre-enrichment media containing tryptic soy broth (Oxoid) with 10 mL of 10% NaCl. Next, the samples were streaked onto mannitol salt agar (Merck, USA) with 6 mg/L of oxacillin (Oxoid) and incubated at 37℃ for 24 h. Three single colonies suspected to be S. aureus derived from each sample were selected and identified by Gram staining with Gram-positive cocci and catalase activity.
The colonies were re-streaked onto tryptic soy agar plates (Oxoid) and incubated at 37℃ for 24 h. A coagulase test was carried out, and the presumptive positive samples were further screened for methicillin resistance by disc diffusion with 1 µg oxacillin (diameter of the inhibition zone for MRSA must be less than 10 mm.) [4 ]. Identification of MRSA isolates was further confirmed by multiplex PCR specific for the mecA gene. All MRSA isolates were kept in brain-heart infusion broth (Oxoid) with 15% glycerol at -70℃ and sent for molecular testing at central laboratory at Chiang Mai University, Faculty of Veterinary Medicine.
The colonies were re-streaked onto tryptic soy agar plates (Oxoid) and incubated at 37℃ for 24 h. A coagulase test was carried out, and the presumptive positive samples were further screened for methicillin resistance by disc diffusion with 1 µg oxacillin (diameter of the inhibition zone for MRSA must be less than 10 mm.) [4 ]. Identification of MRSA isolates was further confirmed by multiplex PCR specific for the mecA gene. All MRSA isolates were kept in brain-heart infusion broth (Oxoid) with 15% glycerol at -70℃ and sent for molecular testing at central laboratory at Chiang Mai University, Faculty of Veterinary Medicine.
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