Extensively drug resistant Typhoid is emerging as a significant problem in developing countries like Pakistan. Since 2016, Pakistan is going through a major outbreak of this disease (Klemm et al, Rasheed et al). These XDR isolates are resistant to all recommended antibiotic leaving behind options of azithromycin as an oral and carbapenems as parenteral treatment option. Recently few reports of azithromycin resistance in Salmonella Typhi have emerged from India, Nepal, Bangladesh and Pakistan (Shoaib et al, Fida et al, Sajib et al, Iqbal et al, Pham et al, Hussain et al). World health organization has classified Salmonella Typhi as the priority pathogens against which there is dire need to look for the new treatment options. Manuka honey is known for its healing properties against wide range of multi drug resistant bacteria and viruses.
Its antibacterial activity is attributed to the presence of methylglyoxal (MGO), which, in combination with acidic pH, high osmotic pressure, immune modulation, and the presence of trace elements, enhances its antibacterial effects (Carter et al). In-vivo, manuka honey causes extensive bacterial lysis, and it is difficult to believe that bacteria can develop resistance to its multifactorial effects. Manuka honey contains a variety of chemical compounds that may work together to overcome antimicrobial resistance. Several in vitro studies on the antibacterial properties of manuka honey against multidrug-resistant pathogens have been published (Roberts et al, Jenkins et al). In a previous study at our center, BALB/c mice were infected with methicillin-resistant Staphylococcus aureus (MRSA) and treated intravenously with Manuka honey. Surprisingly, all of the mice were saved with no reported deaths (Saleem S, 2013). A study conducted in our laboratory revealed that the MIC of Manuka honey was 7 percent (v/v) for both Salmonella paratyphi A and Salmonella paratyphi B, and 7 percent to 8 percent for S. typhi (Hannan et al., 2009). According to Molan's research, the MIC of Manuka honey against S. typhimurium is 7% (v/v) (Molan PC. 2010).
Although the therapeutic potential of manuka honey has been widely investigated against a variety of bacterial infections, no study has been conducted to date to investigate the antibacterial activity of manuka honey against XDR Salmonella Typhi clinical isolates. As a result, the purpose of this study was to determine the in vitro activity of manuka honey against azithromycin resistant XDR Typhoid clinical isolates.
This was a Cross-sectional study.
This study was conducted at The Department of Microbiology, University of Health Sciences, Lahore.
6 months (January 2021 to June 2021).
A total of 150 positive blood cultures for XDR Salmonella Typhi were collected from blood different tertiary care hospitals of Lahore. Out of 150, 3 were found to be azithromycin resistant.
The isolates were cultured and purified on Tryptic Soya agar. The blood culture bottle detected as positive was sub-cultured onto Blood agar (Oxoid, UK) and MacConkey agar (Oxoid, UK) incubated at 35-37C°.The isolate identification was initially performed by Gram-staining. Biochemical identification was done by using Analytical Profile Index-20 Enterobacterales system (BioMerieux, France) and VITEK2 (bioMérieux) consistent with the manufacturer’s instructions. S.Typhi was confirmed by agglutination with genus and serotype-specific antisera (Salmonella poly antiserum A-I (Difco), Salmonella O antiserum (Difco), and Salmonella Vi antiserum (Difco) (Hussain et al., 2015).
Antimicrobial susceptibility testing was performed by Standard Kirby-Bauer Disk Diffusion method using cation adjusted Mueller-Hinton agar (MHA) (Oxoid UK), according to Clinical Laboratory Standards Institute (CLSI) guidelines 2021 and zones of inhibition were interpreted according to the breakpoints. The reference strains of E. coli ATCC® 25922 and P. aeruginosa ATCC® 2785 were used for consistency (Hussain et al., 2015).
Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentration of all the recommended antibiotics was determined by Vitek 2 (bioMérieux) fully automated system. S.Typhi that demonstrated high MICs against ceftriaxone and cefotaxime (>4ug/mL) was considered as XDR. For Azithromycin MIC was determined by E-Strip (liofilchem, Italy) method initially. A value of ≥ 32 ug/L was taken as sensitive according to CLSI 2021 criteria. The tests were done in duplicate and the MICs were recorded as the higher of the two values for each isolate. For broth micro dilution Azithromycin (AZM) dihydrate used in the study was procured from Sigma Aldrich Chemicals Pvt Ltd. Broth Micro dilution method (BMD) described by Clinical and Laboratory Standard Institute was adopted. The BMD method was carried out on sterile 96 well polystyrene round bottom micro titer plates using two-fold dilutions ranging from 0.03 μg/mL to 32 μg/mL, prepared in Cation Adjusted Mueller Hinton Broth (CAMHB) (BBL Becton, Dickinson & Company) The test was performed in duplicates to ensure repeatability. Control wells were maintained in each row for growth control and media control (Kokare et al., 2021). The quality of every batch was assessed using the standard strains of Escherichia coli ATCC 25922 & Klebsiella pneumoniae ATCC 700603.
Interpretation of results
A definite turbidity or button formation in the growth well was considered as positive. MIC was recorded as the lowest concentration at which the isolate was completely inhibited (absence of visible bacterial growth). The CLSI recommended MIC breakpoints for AZM (susceptible ≤ 16 μg/mL and resistant ≥ 32 μg/mL) were used for results interpretation (CLSI, 2021).
Agar well diffusion assay of Manuka honey
Antibacterial activity of undiluted Manuka honey (+20UMF) was evaluated by agar well diffusion assay against azithromycin resistant isolates adopted from punch plate assay (Boorn et al., 2010). Briefly, 0.5 McFarland bacterial suspensions were inoculated on Mueller Hinton agar plate. A sterile 6 mm cork borer was used to make wells on each plate. Subsequently, 120 μL undiluted Manuka honey was poured in each well and plates were incubated at 37°C overnight. Zone of inhibition (mm) was measured by Vernier caliber. The assay was performed in duplicate (Qamar et al, 2017).
Determination of MIC and minimum bactericidal concentration (MBC) of Manuka honey
Microbroth dilution assay was used to determine the MIC (%v/v) and MBC (%v/v) of the manuka honey as described previously (Qamar et al, 2017). MDR S. typhi was sub-cultured onto sheep blood agar plates and incubated for 24 hours at 37°C. Morphologically identical colonies were picked and suspended in sterile 10 ml tryptic soya broth (TSB) and incubated for approximately 5 hours at 37°C to attain a fully logarithmic phase culture. The culture was adjusted to 0.5 McFarland turbidity calculated at 540 nm using sterile TSB as a blank and a diluent with a 1 cm pathway. The suspension was further diluted 1:100 with double strength TSB to achieve a final concentration of 1 x 105 CFU/mL. Briefly, serial dilutions of manuka honey (5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% and 50%) were prepared in sterile distilled deionized water and 100μL of each dilution was added in 96-wells, flat bottom micro titer plates (Thermo Fisher Scientific, UK). Subsequently, 100μL of bacterial suspension was added into each well. Negative control wells contained 100μL of TSB broth only while positive control wells contained TSB medium inoculated with bacteria suspension. Micro titer plate was incubated at37°C overnight at shaking incubator and visually observed for the presence or absence of growth by comparing each well with negative and positive control wells. All the procedure was performed in triplicate. The MBC is defined as first dilution with no growth on agar plate. A 10μL sample was taken from the no visible growth wells of microtiter plate and was inoculated on the nutrient agar plates (Oxoid, UK) which were incubated aerobically at 37°C for 24 h. Plates were examined for cell viability. Any colonies that developed were scored as bacterial growth and no bacterial growth. All the procedures repeated in duplicate (Qamar et al, 2017).