Antibacterial Activity of Manuka Honey Against Azithromycin Resistant Extensively Drug Resistant Salmonella Typhi Clinical Isolates: In-Vitro Study


 Typhoid fever is a significant health problem in developing countries like Pakistan. Salmonella Typhi the causative agent of typhoid has developed resistant to almost all recommended antibiotics. Emergence of resistance to third generation cephalosporins has further complicated the situation and such strains are called as extensively drug resistant (XDR) Salmonella Typhi. Currently only available options are azithromycin and cabapenems. Recently few reports of azithromycin resistance have emerged from countries like Pakistan, India, Bangladesh and Nepal. As azithromycin is the only oral option available to treat XDR Typhoid, development of resistance may change treatment strategy altogether from out patient management to hospitalization of every patient. This may increase the burden on already weak health care system of countries like Pakistan. So there is dire need to look for the alternative treatment options. Manuka honey is well known for its therapeutic potential against wide range of bacteria including Salmonella Typhimurium. In this study 3 azithromycin resistant isolates were isolated and identified using disc diffusion, E-test and broth micro dilution methods and antibacterial activity, MIC and MBC of manuka honey was performed by agar well diffusion assay and broth micro dilution assay respectively. Manuka honey manifested significant antibacterial activity against all test isolates with zone of inhibition ranging from 7.3mm to 7.5mm, MIC and MBC values were between 10 to 15% v/v Here, we conclude that Manuka honey possess potent antibacterial activity and might be used as an alternative treatment option against azithromycin resistant XDR Typhid. However, further clinical trials are mandatory to validate our initial findings.

using disc diffusion, E-test and broth micro dilution methods and antibacterial activity, MIC and MBC of manuka honey was performed by agar well diffusion assay and broth micro dilution assay respectively.
Manuka honey manifested signi cant antibacterial activity against all test isolates with zone of inhibition ranging from 7.3mm to 7.5mm, MIC and MBC values were between 10 to 15% v/v Here, we conclude that Manuka honey possess potent antibacterial activity and might be used as an alternative treatment option against azithromycin resistant XDR Typhid. However, further clinical trials are mandatory to validate our initial ndings. 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 di cult 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.

Study design
This was a Cross-sectional study.

Setting
This study was conducted at The Department of Microbiology, University of Health Sciences, Lahore.

Sample Collection:
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.

Bacterial identi cation
The isolates were cultured and puri ed 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 identi cation was initially performed by Gram-staining. Biochemical identi cation was done by using Analytical Pro le Index-20 Enterobacterales system (BioMerieux, France) and VITEK2 (bioMérieux) consistent with the manufacturer's instructions. S.Typhi was con rmed by agglutination with genus and serotype-speci c antisera (Salmonella poly antiserum A-I (Difco), Salmonella O antiserum (Difco), and Salmonella Vi antiserum (Difco) (Hussain et al., 2015).

Antimicrobial Susceptibility
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 (lio lchem, 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 de nite 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). Brie y, 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 Page 5/10 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 nal concentration of 1 x 10 5 CFU/mL. Brie y, 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, at bottom micro titer plates (Thermo Fisher Scienti c, 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 de ned as rst 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).

Results:
A total of three azithromycin resistant isolates were isolated from blood culture samples collected from tertiary care hospitals of Lahore. These isolates were identi ed on the basis of morphology, biochemical tests and serology. Further antibiotic susceptibility 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 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 (lio lchem, Italy) method initially. A value of ≥ 32 ug/L was taken as sensitive according to CLSI 2021 criteria. Broth Micro dilution method (BMD) described by Clinical and Laboratory Standard Institute was adopted. Antibacterial activity of Manuka honey was determined by agar well diffusion assay. MIC and MBC were determined by broth micro dilution assay. Manuka honey showed signi cant anti-bacterial activity against all the three isolates. All three isolates had zone of inhibition 7.4±0.4, 7.5±0.0, 7.3±0.4 mm±SD respectively (Table1). MIC (%v/v) and MBC (%v/v) of the manuka honey against 2 isolates was 10% v/v, while third isolate was Inhibited and killed at 15%.  (Pratibha and Manita, 2015). Similarly, a UK study reported the MIC (9.5%) and MBC (12%) of Mnauka honey against P. aeruginosa (Henriques et al). To summarize, manuka honey exhibits promising bactericidal activity against azithromycin-resistant XDR Salmonella Typhi. As a result, using manuka honey to treat such infections may be worth trying. In the future, this research will aid in determining the antibacterial e cacy of our local honeys in vitro and in vivo in comparison to manuka honey against various pathogens, particularly gut pathogens.

Conclusion
We concluded that Manuka honey has strong inhibitory effect against the azithromycin resistant extensively drug resistant Salmonella Typhi. Therefore, it might be used as a potential treatment option against XDR Typhoid cases following the carefully designed clinical trials.

Declarations
Ethical approval The study was ethically approved by the "Ethical Review Board" under reference number UHS/REG-19/ERC/398 from University of health sciences, Lahore, Pakistan.

Consent for publication
Not applicable.
Competing interests