Amoxicillin clavulanate

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Amoxicillin/clavulanate is a combination antibiotic medication used in laboratory settings. It consists of the active ingredients amoxicillin and clavulanic acid. Amoxicillin is a beta-lactam antibiotic that inhibits bacterial cell wall synthesis, while clavulanic acid is a beta-lactamase inhibitor that enhances the effectiveness of amoxicillin against certain bacteria.

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26 protocols using amoxicillin clavulanate

Screening ESBL-producing E. coli and K. pneumoniae

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Cefuroxime resistant E. coli and K. pneumoniae isolates were screened for ESBL-production by the double disc synergy test using ceftazidime (30 µg), cefotaxime (30 µg) and amoxicillin/clavulanate (30 µg discs) (Oxoid Ltd) as the inhibitory substance. Test was considered positive for ESBL when there was a synergy between any two antibiotics (Fig. 1) with amoxicillin/clavulanate.Figure 1

Image showing double-disk diffusion method (photo by Nahid Karim).

Susceptibility for eleven antibiotics (ampicillin, chloramphenicol, amikacin, ciprofloxacin, trimethoprim-sulfamethoxazole, gentamicin, piperacillin-tazobactam, amoxicillin-clavulanate, ceftazidime, ceftriaxone, Cefuroxime and meropenem) in all ESBL-positive fecal isolates and a subset of arbitrary selected non-ESBL producing fecal E coli was determined using the disc diffusion method (Oxoid Ltd) and breakpoints according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines at the time of the study. Resistant and intermediate isolates were considered as resistance.

Letara N., Ngocho J.S., Karami N., Msuya S.E., Nyombi B., Kassam N.A., Skovbjerg S., Åhren C., Philemon R, & Mmbaga B.T. (2021). Prevalence and patient related factors associated with Extended-Spectrum Beta-Lactamase producing Escherichia coli and Klebsiella pneumoniae carriage and infection among pediatric patients in Tanzania. Scientific Reports, 11, 22759.

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Antibiotic Susceptibility Testing of GNB

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Susceptibility testing of the GNB was examined by the Kirby–Bauer disk diffusion assay on Mueller-Hinton agar medium (Oxoid, England) against 18 antibiotic disks following the Clinical and Laboratory Standard Institute guidelines [17 ]. The following antibiotics were examined: amikacin (30 μg), amoxicillin/clavulanate (20/10 μg), aztreonam (30 μg), cefepime (30 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), cefuroxime (30 μg), ciprofloxacin (5 μg), colistin (10 μg), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), nitrofurantoin (50 μg), piperacillin(100 μg), piperacillin/tazobactam (100/10 μg), tobramycin (10 μg), and trimethoprim/sulfamethoxazole (23.75 μg/1.25 μg) (Oxoid, England). In brief, standardized suspension of each isolate conforming 0.5 McFarland turbidity was inoculated onto two Mueller-Hinton agar plates. Then, nine antibiotic disks were placed onto each plate with recommended distance, followed by overnight incubation at 37°C. The strain of E. coli ATCC 25922 was used as control and was tested each time when susceptibility testing was performed.

Ibrahim M.E., Abbas M., Al-Shahrai A.M, & Elamin B.K. (2019). Phenotypic Characterization and Antibiotic Resistance Patterns of Extended-Spectrum β-Lactamase- and AmpC β-Lactamase-Producing Gram-Negative Bacteria in a Referral Hospital, Saudi Arabia. The Canadian Journal of Infectious Diseases & Medical Microbiology = Journal Canadien des Maladies Infectieuses et de la Microbiologie Médicale, 2019, 6054694.

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Antimicrobial Susceptibility Testing of Enterobacteriaceae

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Antimicrobial susceptibility testing (AST) was carried out using Kirby Bauer’s Disc Diffusion method and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) guidelines (2018).
The following antibiotics for the Enterobacteriaceae were tested: Ceftriaxone (30μg), Sulfamethoxazole-trimethoprim (25μg), Piperacillin-tazobactam (110 μg), Ticarcillin-clavulanate (85μg), Tetracycline (30μg), Amikacin (30μg), Gentamicin (10μg), Ciprofloxacin (5μg), Meropenem (10μg), Azithromycin (15μg), Chloramphenicol (30μg), Nitrofurantoin (300μg), Nalidixic acid (30μg), Ceftazidime (30μg) and Amoxicillin-clavulanate (30μg) (Oxoid, Basingstoke, Hants, UK). Quality control was ensured by using standard strains (Klebsiella pneumoniae, ATCC 700603 and Escherichia coli, ATCC 25922) in determining susceptibility or otherwise of stool isolates. The definition of multidrug resistance (MDR) was based on the resistance to three or more classes of antimicrobial agents [27 (link)].

Dela H., Egyir B., Majekodunmi A.O., Behene E., Yeboah C., Ackah D., Bongo R.N., Bonfoh B., Zinsstag J., Bimi L, & Addo K.K. (2022). Diarrhoeagenic E. coli occurrence and antimicrobial resistance of Extended Spectrum Beta-Lactamases isolated from diarrhoea patients attending health facilities in Accra, Ghana. PLoS ONE, 17(5), e0268991.

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Antimicrobial Susceptibility of Bacterial Isolates

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The antimicrobial susceptibility was determined by the disc diffusion method according to EUCAST, 2017 guidelines (http://www.eucast.org/clinical_breakpoints/). The following antimicrobial agents were tested: amoxicillin-clavulanate, cefotaxime, ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem, tobramycin, gentamicin, amikacin, trimethoprim/sulfamethoxazole (Oxoid, Basingstoke, UK). MICs of nalidixic acid, ciprofloxacin, and levofloxacin were determined by MIC strips (Liofilchem, Roseto degli Abruzzi, Italy).

Markovska R., Stoeva T., Dimitrova D., Boyanova L., Stankova P., Mihova K, & Mitov I. (2019). Quinolone resistance mechanisms among third-generation cephalosporin resistant isolates of Enterobacter spp. in a Bulgarian university hospital. Infection and Drug Resistance, 12, 1445-1455.

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Antibiotic Susceptibility of E. coli and P. aeruginosa

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Pure colonies from each sample with E. coli and P. aeruginosa contamination were randomly selected for antibiotic susceptibility testing. Pure isolates of E. coli, and P. aeruginosa were subjected to antibiotic susceptibility testing using the Kirby–Bauer Disc Diffusion method on Mueller–Hinton agar, as recommended by Clinical Laboratory Standards Institute (CLSI) guidelines [23 ]. Zones of inhibition were measured in millimeters and recorded for each antibiotic.
Antibiotics used for E. coli isolates included amoxicillin–clavulanate (20/10 µg), aztreonam (30 µg), ertapenem (10 µg), gentamicin (10 µg), chloramphenicol (20/10 µg), ciprofloxacin (5 µg), cefuroxime (30 µg), ceftriaxone (30 µg) and trimethoprim–sulphamethoxazole (1.25/23.75 µg) (Oxoid, Hampshire, UK). For P. aeruginosa, piperacillin–tazobactam (100/10µg), aztreonam (30 µg), gentamicin (10 µg) and ciprofloxacin (5 µg) (Oxoid, Hampshire, UK) were used.

Ahmed H., Zolfo M., Williams A., Ashubwe-Jalemba J., Tweya H., Adeapena W., Labi A.K., Adomako L.A., Addico G.N., Banu R.A., Akrong M.O., Quarcoo G., Borbor S, & Osei-Atweneboana M.Y. (2022). Antibiotic-Resistant Bacteria in Drinking Water from the Greater Accra Region, Ghana: A Cross-Sectional Study, December 2021–March 2022. International Journal of Environmental Research and Public Health, 19(19), 12300.

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Antimicrobial Susceptibility Testing of Isolates

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Antimicrobial susceptibility tests were performed using the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2018 ). The antibiotic disks (Oxoid, UK) ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg), piperacillin-tazobactam (100/10 µg), cefoperazone-sulbactam (75/30 µg), cefazolin (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), gentamicin (10 µg), amikacin (30 µg), netilmicin (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), norfloxacin (10 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), and fosfomycin (200 µg) were used. Escherichia coli ATCC 25922 was used as a control in all antibiogram tests. Whether a strain was MDR was determined on the basis of acquired non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012 (link)).

Tewawong N., Kowaboot S., Pimainog Y., Watanagul N., Thongmee T, & Poovorawan Y. (2020). Distribution of phylogenetic groups, adhesin genes, biofilm formation, and antimicrobial resistance of uropathogenic Escherichia coli isolated from hospitalized patients in Thailand. PeerJ, 8, e10453.

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Antibiotic Susceptibility Testing by Disk Diffusion

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Antimicrobial susceptibility test by the disk diffusion method was performed to determine the resistance patterns of the isolates to 17 antibiotics: cefotaxime, ceftriaxone, ceftazidime, aztreonam, ceftazidime/clavulanate, cefotaxime/clavulanate, cephalothin, cefuroxime sodium, cefepime, gentamicin, amikacin, ciprofloxacin, tetracycline, ampicillin, amoxicillin/clavulanate, piperacillin, piperacillin/tazobactam, imipenem, meropenem, ertapenem, and sulfamethoxazole/trimethoprim [Oxoid, England; disc contents according to Clinical and Laboratory Standards Institute (CLSI) guidelines]. All antimicrobial testing was performed on Mueller-Hinton agar by the flooding technique and data interpreted according to the CLSI guidelines.

Tokajian S., Eisen J.A., Jospin G., Farra A, & Coil D.A. (2015). Whole genome sequencing of extended-spectrum β-lactamase producing Klebsiella pneumoniae isolated from a patient in Lebanon. Frontiers in Cellular and Infection Microbiology, 5, 32.

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Antibiotic Susceptibility Testing Protocol

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Blood bacteria enrichment medium, blood agar plate, M-H agar and M-H broth were purchased for Hangzhou Tianhe Microorganism Reagents Company. Tested antimicrobial agents included cefazolin, cefepime, cefotaxime, cefoxitin, ampicillin, piperacillin/tazobactam, amoxicillin clavulanate, meropenem, imipenem, amikacin, tetracycline, ciprofloxacin, trimethoprim–sulfamethoxazole, tigecycline (Oxoid, British). The PCR detection kits and DNA marker were from Beijing Tiangen Company. Ethylenediaminetetraacetic acid (EDTA), Tris, ethidium bromide and agarose were for Shanghai Shenggong Company. MicroScanWalkAway 40 Plus Microbiology System (Siemens, Newark, DE, USA). SP1000 PCR amplification instrument (Bio-Rad Company, USA), DYY-III electrophoresis apparatus (Beijing Liuyi Instrument Factory), Tanon1600 gel imaging system (Shanghai Tianneng Company).

Lin D., Chen J., Yang Y., Cheng J, & Sun C. (2018). Epidemiological Study of Carbapenem-resistant Klebsiella Pneumoniae. Open Medicine, 13, 460-466.

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Antimicrobial Resistance Profiling of Isolates

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The antimicrobial susceptibility was performed using standard disc diffusion method19 . Tested antimicrobials were belongs to β-lactams, FQs, aminoglycosides, tetracycline, folate pathway inhibitors and nitrofurans classes. Amoxicillin-clavulanate (AMC, 20/10µg), piperacillin-tazobactam (TZP, 100/10 µg), ceftriaxone (CRO, 30 µg), cefpodoxime (CPD, 10 µg), cefuroxime (CXM, 30 µg), aztreonam (ATM, 30 µg), imipenem (IMP, 10 µg), meropenem (MEM, 10 µg), gentamicin (CN, 10 µg), doxycycline (DO, 30µg) and ciprofloxacin (CIP, 5µg) were purchased from Oxoid (Hampshire, UK). The other antimicrobial discs including levofloxacin (LEV, 5 µg), ofloxacin (OFX, 5µg), trimethoprim-sulfamethoxazole (SXT, 1.25/23.75 µg) and nitrofurantoin (F, 300 µg) were obtained from Bioanalysis (Ankra, Turkey). Isolates which were resistant to at least three antimicrobial classes were considered as MDR isolates.

Kadry A.A., Al-Kashef N.M, & El-Ganiny A.M. (2020). Distribution of genes encoding adhesins and biofilm formation capacity among Uropathogenic Escherichia coli isolates in relation to the antimicrobial resistance. African Health Sciences, 20(1), 238-247.

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Antimicrobial Susceptibility of Shigella

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Antimicrobial susceptibility was performed on Mueller–Hinton Agar using the Kirby–Bauer method.^{25 (link)} The total Shigella strains were analysed according to the interpretation criteria proposed by the CLSI.²⁶ The antibiotics used were: ampicillin (10 μg), amoxicillin/clavulanate (20/10 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), tetracycline (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), nalidixic acid (30 μg) and nitrofurantoin (300 μg) (Oxoid, England). For quality control, the reference strains Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603 and Pseudomonas aeruginosa ATCC 27853 were used. The strains were considered MDR when they presented resistance to more than two classes of antimicrobials.^{25 (link)} Additionally, the determination of the production of ESBL was carried out using the Jarlier method.^{27 (link)} The presence of ESBL was manifested by the synergistic effect between the inhibitor (amoxicillin/clavulanate) and the cephalosporin antimicrobial discs (cefotaxime and ceftazidime).

Quino W., Bellido G., Flores-León D., Caro-Castro J., Mestanza O., Lucero J, & Gavilan R.G. (2023). Trends in antimicrobial resistance of Shigella species in Peru, 2011–2020. JAC-Antimicrobial Resistance, 5(5), dlad110.

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