Patterns of antibiotic resistance of the E. coli isolates were assessed using the simple disc diffusion method. The isolates were cultured onto the Mueller–Hinton agar (HiMedia Laboratories, Mumbai, India; MV1084). Antibiotic discs including kanamycin (1000 μg/disc), tetracycline (30 μg/disc), ampicillin (10 μg/disc), gentamycin (10 μg/disc), imipenem (30 μg/disc), amikacin (30 μg/disc), mezlocillin (30 μg/disc), cefotaxime (30 μg/disc), piperacillin (30 μg/disc), ciprofloxacin (5 μg/disc), cotrimoxazole (30 μg/disc), norfloxacin (30 μg/disc), ceftazidime (30 μg/disc), nitrofurantoin (300 μg/disc), ofloxacin (5 μg/disc), ceftriaxone (30 μg/disc), nalidixic acid (30 μg/disc), tobramycin (30 μg/disc), clindamycin (2 μg/disc) and cephalothin (30 μg/disc) (Oxoid) were placed on the cultured Mueller–Hinton agar and all media were incubated aerobically at 37°C for 24 hours. All examinations and also interpretation of the findings were performed according to the instructions and guidelines of the CLSI [37 ]. Escherichia coli ATCC 8739 was used as a control organism.
Norfloxacin
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Norfloxacin is a synthetic antibacterial agent used in laboratory settings. It functions as a broad-spectrum fluoroquinolone antibiotic, inhibiting the bacterial enzymes DNA gyrase and topoisomerase IV, which are essential for bacterial DNA replication, transcription, repair, and recombination.
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Lab products found in correlation
Ciprofloxacin, by Thermo Fisher Scientific (38 mentions)
Gentamicin, by Thermo Fisher Scientific (31 mentions)
Ampicillin, by Thermo Fisher Scientific (26 mentions)
Trimethoprim sulfamethoxazole, by Thermo Fisher Scientific (23 mentions)
Erythromycin, by Thermo Fisher Scientific (23 mentions)
Tetracycline, by Thermo Fisher Scientific (22 mentions)
Amikacin, by Thermo Fisher Scientific (19 mentions)
Cefoxitin, by Thermo Fisher Scientific (19 mentions)
Cefotaxime, by Thermo Fisher Scientific (17 mentions)
Chloramphenicol, by Thermo Fisher Scientific (16 mentions)
Clindamycin, by Thermo Fisher Scientific (15 mentions)
Ceftriaxone, by Thermo Fisher Scientific (14 mentions)
Amoxicillin clavulanic acid, by Thermo Fisher Scientific (13 mentions)
Meropenem, by Thermo Fisher Scientific (12 mentions)
Nalidixic acid, by Thermo Fisher Scientific (11 mentions)
Imipenem, by Thermo Fisher Scientific (11 mentions)
Gentamycin, by Thermo Fisher Scientific (10 mentions)
Nitrofurantoin, by Thermo Fisher Scientific (10 mentions)
Ceftazidime, by Thermo Fisher Scientific (10 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
62 protocols using norfloxacin
1
Antibiotic Resistance Profiling of E. coli
2
Antibiotic Susceptibility Profiling
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The susceptibility to 12 different antimicrobial agents was tested according to the instructions of NCCLS [22 ] manuals; using disk diffusion technique depending on the diameter of the inhibition zone [23 ]. The following antibiotics were tested; enrofloxacin (5 μg), norfloxacin (10 μg), ciprofloxacin (5 μg), gentamycin (10 μg), amoxicillin (25 μg), neomycin (30 μg), erythromycin (15 μg), streptomycin (10 μg), oxytetracycline (30 μg), trimethoprim-sulfamethoxazole (25 μg), ampicillin (10 μg), and penicillin (10 I.U); (Oxoid, USA).
3
Antibiotic Susceptibility of Feline Pasteurella multocida
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The susceptibility profile was established by a disc diffusion test as recommended by the Clinical and Laboratory Standards Institute (VET01-A4, 2013). The antimicrobial agents tested included ceftiofur, penicillin, amoxicillin, flofenicol, norfloxacin, enrofloxacin, ciprofloxacin, tetracycline, doxycycline, sulfizoxazole, trimethoprim-sulphamethoxazole and erythromycin (Oxoid Ltd., Cambridge, UK). The reference strains Escherichia coliATCC 25922 and Staphylococcus aureusATCC 29213 were used as quality control organisms in all antimicrobial susceptibility tests. There are no CLSI approved breakpoints applicable specifically to feline Pasteurella multocida;therefore, most of the values used here originated from values described in CLSI document VET01-A4 and supplement VET01-S2. The breakpoints used for doxycycline, ciprofloxacin and norfloxacin were adopted from CLSI document M100- S19 (2009).
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The susceptibility profile was established by a disc diffusion test as recommended by the Clinical and Laboratory Standards Institute (VET01-A4, 2013). The antimicrobial agents tested included ceftiofur, penicillin, amoxicillin, flofenicol, norfloxacin, enrofloxacin, ciprofloxacin, tetracycline, doxycycline, sulfizoxazole, trimethoprim-sulphamethoxazole and erythromycin (Oxoid Ltd., Cambridge, UK). The reference strains Escherichia coliATCC 25922 and Staphylococcus aureusATCC 29213 were used as quality control organisms in all antimicrobial susceptibility tests. There are no CLSI approved breakpoints applicable specifically to feline Pasteurella multocida;therefore, most of the values used here originated from values described in CLSI document VET01-A4 and supplement VET01-S2. The breakpoints used for doxycycline, ciprofloxacin and norfloxacin were adopted from CLSI document M100- S19 (2009).
4
Antibiotic Susceptibility of P. aeruginosa
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Ten antibiotics (purchased from Oxoid Ltd., Basingstoke, UK), namely imipenem (10 µg), meropenem (10 µg), levofloxacin (5 µg), ceftazidime (30 µg), gentamycin (10 µg), amikacin (30 µg), aztreonam (30 µg), piperacillin (100 µg), cefepime (30 µg), and norfloxacin (10 µg), were selected for the antibiotic susceptibility test with P. aeruginosa using the disk diffusion susceptibility method [48 (link)]. Overnight bacterial culture in Tryptic Soy Broth (TSB) (Difco, San Diego, CA, USA) was adjusted to 0.5 McFarland turbidity standards. Subsequently, 0.1 mL of bacterial suspension was spread, using sterile swabs, on Mueller–Hinton agar plates (HI Media Lab. Pvt. Ltd. Ref. M173). Duplicate plates were prepared for each isolate. The antibiotic discs were placed on the agar plates (within 15 min of the inoculation) using sterile forceps, to apply the discs at a distance of 2 cm apart from each other, and incubated for 16–18 h at 37 °C. After incubation, inhibition zones were visible and measured with a ruler, with the measurements recorded in mm [49 (link)].
5
Antimicrobial Susceptibility Testing via Kirby-Bauer Disk Diffusion
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Antimicrobial susceptibility testing was done using the Kirby-Bauer disk diffusion method on Mueller Hinton agar (Oxoid Ltd.) prepared with 4mm thickness.13
Panels of eleven antimicrobial disks including ampicillin (10µg), amoxicillin-Clavulanic acid (10µg), cefoxitin (30µg), ciprofloxacin (5µg), gentamicin (10µg), norfloxacin (10µg), cefepime (30µg), ceftriaxone (30µg), nitrofurantoin (F) 300µg, amikacin (10µg), and azithromycin (30µg) (Oxoid Ltd.) were used for susceptibility tests. Then, the bacterial isolates were classified as sensitive (S), intermediate (I), or resistance (R) by comparing against the inhibition zone diameter of interpretative standards as indicated in the CLSI guideline.13
Panels of eleven antimicrobial disks including ampicillin (10µg), amoxicillin-Clavulanic acid (10µg), cefoxitin (30µg), ciprofloxacin (5µg), gentamicin (10µg), norfloxacin (10µg), cefepime (30µg), ceftriaxone (30µg), nitrofurantoin (F) 300µg, amikacin (10µg), and azithromycin (30µg) (Oxoid Ltd.) were used for susceptibility tests. Then, the bacterial isolates were classified as sensitive (S), intermediate (I), or resistance (R) by comparing against the inhibition zone diameter of interpretative standards as indicated in the CLSI guideline.13
6
Antimicrobial Resistance Profiling of MRSA
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The pattern of antimicrobial resistance was studied using the simple disk diffusion technique. The Mueller–Hinton agar (Merck, Germany) medium was used for this purpose. Antibiotic resistance of the MRSA strains against 18 commonly used antibiotics was determined using the instructions provided by the Clinical and Laboratory Standards Institute guidelines (18 ). Susceptibility of MRSA isolates was tested against ampicillin (10 µg/disk), gentamycin (10 µg/disk), lincomycin (2 µg/disk), cephalothin (30 µg/disk), imipenem (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 (10 µg/disk), oxacillin (1 µg/disk), erythromycin (15 µg/disk), azithromycin (15 µg/disk), ceftriaxone (30 µg/disk) and cefixime (5 µg/disk) antibiotic agents (Oxoid, 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 (18 ). Staphylococcus aureus ATCC 25923 and Escherichia coli (E. coli) ATCC 25922 were used as quality control organisms in antimicrobial susceptibility determination.
7
Antibiotic Susceptibility of Aeromonas hydrophila
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The A. hydrophila isolate was also subjected to antibiotic susceptibility tests, according to the guidelines proposed by the Clinical and Laboratory Standards Institute (CLSI) [13 ]. The commercial antibiotics used were amikacin (30 μg), ampicillin (10 μg), amoxicillin (30 μg), levofloxacin (5 μg), norfloxacin (10 μg), cefotaxime (30 μg), gentamicin (10 μg), kanamycin (30 μg), streptomycin (30 μg), erythromycin (15 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), chloramphenicol (30 μg), tetracycline (30 μg), and nalidixic acid (30 μg) (Oxoid, London, UK). First, a fresh culture of A. hydrophila with a turbidity of 0.5 McFarland was swabbed onto the surface of Mueller-Hinton agar (MHA) (HiMedia, Mumbai, India) using sterile cotton buds. The antibiotic disks were fixed on the MHA surface using sterile forceps, and the agar plates were incubated at 35°C for 24 h. The inhibitory zones were interpreted according to the measurements provided by the CLSI guidelines [14 ]. The multiple antibiotic resistance (MAR) index was determined [15 ], and a MAR index value of >0.2 suggested a high-risk exposure to these antibiotics.
8
Antimicrobial Susceptibility Testing Protocol
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Antimicrobial susceptibility was determined using the disc diffusion method, in accordance with the Clinical and Laboratory Standard Institute (CLSI) [27 (link)] for the following antibiotics: Ampicillin (AMP, 10 µg), cefoxitin (FOX, 30 µg), cefotaxime (CTX, 30 µg), ciprofloxacin (CIP, 5 µg), norfloxacin (NOR, 10 µg), tobramycin (TOB, 10 µg), gentamicin (GEN, 10 µg), doxycycline (DOX, 5 µg), amikacin (AMK, 30 µg), azithromycin (AZM, 15 µg), nitrofurantoin (NIT, 300 µg), amikacin, vancomycin (VA, 30 µg), linezolid (LZD, 30 µg) and sulfamethoxazole/trimethoprim (SXT, 1.25/23.75 µg) (Oxoid, Hampshire, England). The results were interpreted using the criteria outlined in CLSI guidelines based on the inhibition zone produced, which correlate with susceptibility levels [27 (link)]. The obtained results were used to identify the percentage of MDR among the tested isolates. As previously documented, multidrug resistance (MDR) is defined as resistance to three or more antimicrobial classes [28 (link)]. The phenotypic identification of the isolates as MRSA was performed against cefoxitin through the disk diffusion method, while the standard strain of S. aureus (ATCC 29312) was included as a control isolate.
9
Antimicrobial Susceptibility Testing Protocol
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For susceptibility tests, the following antimicrobial agents were used: ampicillin, amikacin, norfloxacin, chloramphenicol, gentamicin, sulfamethoxazole/trimetoprim, sulfamethylisoxazole, ofloxacin, cefotaxime, and ceftriaxone sodium (Oxoid Ltd., England). The susceptibility tests were performed according to standard protocols.
10
Antibacterial Peptides and Antibiotics Evaluation
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LL-37 peptide was purchased from Lipopharm.pl (Zblewo, Poland), and the purity of LL-37 was > 98% (as determined by high-performance liquid chromatography [HPLC]). The ceragenins, CSA-13 and CSA-131, were synthesized as described previously [30 (link)]. Conventional antibiotics (sulfamethoxazolum/trimethoprimum, ciprofloxacin, norfloxacin, gentamicin, fosfomycin, meropenem, amoxicillin/clavulanic acid, cefepime, cefotaxime, ampicillin and doxycyclinum) were purchased from OXOID (England) and Polfa Tarchomin (Poland).
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