Ethical permission and patient’s consent
Before starting the study, we obtained ethical approval from the Ethical Review Committee at Government College University Faisalabad. As required by the legal guardians of the study population, we obtained consent in the national language, which was also provided in writing when necessary. We assured the participants that their confidential information would be kept secure and used only for research purposes. After that, we transferred the materials to the researcher for further processing. Throughout the study, we processed the samples as anonymous data and analyzed them, ensuring the confidentiality of individuals' personal information. Collection of bacterial isolates from clinical wards
Using aseptic techniques, we collected 100 blood culture isolates of S. Typhi from patients suspected of having septicemia. These isolates were collected from various public and private sector Hospitals in Lahore and Faisalabad, Pakistan. The study participants comprised patients of all age groups from various hospital wards, including the intensive care unit (ICU), emergency, outdoor patient (OPD), and medical and surgery wards. The study included patients ranging from infants to adolescents, without any consideration for gender discrimination. The study included patients who exhibited two or more of the following signs and symptoms: body temperature exceeding 38°C or falling below 35°C, pulse rate surpassing 90 beats per minute, and respiration rate exceeding 20 breaths per minute.
Determination of minimum inhibitory concentration of antibiotics against S. Typhi
All 100 isolates of S. Typhi were analyzed to determine their resistance profile against different antimicrobials using the Vitek® 2 compact system. Antibiogram to determine the susceptibility pattern of identified strains by using ampicillin, co-trimoxazole, ciprofloxacin, chloramphenicol, ceftriaxone, azithromycin, meropenem, imipenem was performed using CLSI guidelines, 2021.
Molecular Characterization of S. Typhi Isolates
DNA extraction of the 100 blood culture isolates of S. Typhi was done using a DNA extraction kit (Seongam, South Korea) as per the manufacturer’s instructions (Bahari et. Al, 2021). This is based on silica membrane spin column-based technology for purifying genomic DNA. This technology comprises of lysis step, Binding Step, wash step and final Elution steps with the use of GT1 buffer, GT2 buffer, W1 Buffer, W2 Buffer, Elution Buffer and Proteinase K enzyme for performing various steps of genomic DNA extraction.
Molecular detection of ARGs in S. Typhi
Molecular detection of antimicrobial-resistant genes in S. Typhi was carried through Monoplex and Multiplex PCR Assays were conducted to clone resistant genes of different antimicrobials using a Thermal cycler machine (Bio-Rad T100™ Thermal cycler, USA) as per manufacturer’s guidelines. The PCR was a 25ųl of the PCR reaction mixture prepared in a PCR reaction tube containing 4ng of DNA, 12 ųl of 2X PCR Green Tag MasterMix (Thermo Fischer Scientific, UK), 1.5 ųM of each forward and reverse primer and 6ųl of nuclease-free water to attain a final volume of 25ųl. All the tubes containing the PCR reactions mixture were run in a thermal cycler machine ( BioRad Laboratories Inc., USA) with the following conditions; Initial denaturation at 95oC for about 120 sec, final denaturation at 95oC for 30 sec, annealing at 68oC for 60 sec and final extension at 68oC for 10 mins. Each step was repeated for 30 times (Khokhar et. al., 2022).
The PCR product from the thermal cycler was put onto 1.1% agarose gel (Thermo Fischer Scientific, UK) to separate resistant gene bands based on charge and size. Ethidium bromide dye was added to visualize bands under UV light as the gel gets stained with this fluorescent dye. DNA bands are visualized on an ultraviolet transilluminator. Amplicon size was compared to the 100bp DNA ladder (Zokeyo, UK). The list of primers used in this study is given in Table 1.
Table 1
Primers sequences and PCR conditions
Target genes | Primer Sequence (5’-3’) | Amplicon size (bp) | Annealing temp (OC) |
pltB | TAAACCATGATAGACTGG | 657 | 55 |
GAAAGTTACGGTTATACC |
catA1 | GAAGATCACTTCGCAGAATAA | 1003 | 45 |
CAGCAATAGACATAAGCG |
dhfR7 | GCAACGTCAGAAAATGGC | 405 | 60 |
AAACTGCTCAAAAAGGAAATTGA |
sul1 | GTATTGCGCCGCTCTTAGAC | 500 | 60 |
AGGGTTTCCGAGAAGGTGAT |
qnrS | TATAATGGTAGTCTAGCCC | 722 | 52 |
GATGTGTGATTTTAAACG |
S. Typhi | GGCAGATATACTTTCGCAGGCA | 227 | 60 |
CCCAGAACCAAATTTGCTTACA |
XDR-S. Typhi | TGAATGGTTCTGGTCTGGCG | 425 | 60 |
CTAAACCACGACGGCTCAGT |
H58 | GGGCTTGATGGCTTCATTAGT | 509 | 60 |
ACAGGTTGTACGCCTTTCCA |
gyr A | TACCGTCATAGTTATCCACA | 313 | 53 |
GTACTTTACGCCATGAACGT |
gyr B | GCGCTGTCCGAACTGTACCT | 345 | 53 |
GATCAGCGTCGCCACTTCC |
parC | ATAGGGTATTATCTGCGGC | 2473 | 55 |
GAATAAACAACGGTTTTACG |
parE | TGCACAGTTGCTGACAATC | 1990 | 55 |
TCGGATTCTCTTATCCGGCCTG |
macA | CTGTAAGCTGTGTCATGATCG | 1308 | 52 |
CTCACATTGCACAGTTCAAGC |
acrB (N-term) | GGTTAAAGTGCAGGAAATTACCG | 1695 | 50 |
CTACGCTATCGGTGTAGTGAT |
acrB (C-term) | GACGATGCTCAAACCCGT | 1815 | 50 |
GCCAACTTTCCTAAGAAAAAGCC |
acrR | CACCGACATATGGCACGAA | 633 | 52 |
CAGCGTCGGACACAATTGATA |
blaCTX−M−15 | CAATGTGCAGCACCAGTAA | 540 | 50 |
CGCAATATCATTGGTGGTG |
bla TEM-1 | CAGCGGTAAGATCCTTGAA | 643 | 55 |
ACTCGCCGTCGTGTAGATAA |
blaIMP | GGAATAGAGTGGCTTAATTCTC | 510 | 52 |
CCAAACCAC TACGTTATC |
blaVIM | GATGGTGTTTGGTCGCATA | 450 | 52 |
CGAATGCGCAGCACCAG |
In vitro activity of Manuka Honey against S. Typhi
The manuka honey (Watson & Son, New Zealand) (250g) with + 24 UMF (unique manuka factor) with MGO was commercially purchased. The honey was serially diluted at the concentrations of 10%, 20%, 30%, 40%, 50%, and 100% as described previously (Ejaz et al. 2023). In short, the bacterial suspension (0.5McFarland) was evenly spread onto Muller Hinton Agar (MHA) plates. Agar wells were created on the plates using a sterile cork borer with a diameter of 6mm, and each dilution was poured into its corresponding well. The plates were then incubated at a temperature of 37°C for 24 hours. The zone of inhibition (mm) of manuka honey against MDR, and XDR S. Typhi strains was calculated using a vernier callipe.
The minimum inhibitory concentration of Manuka Honey against S. Typhi
Fresh 2–3 colonies of S. Typhi bacterial culture were emulsified into 2-3ml of concentrated lysogeny broth solution in 50 falcon tubes. The mixture was then incubated at 37oC to achieve 0.5 McFarland turbidity standard at an optical density of 0.07 and wavelength of 600nm. Further dilutions were made by adding 1:100th double-strength LB to achieve a concentration of 1-105 CFU/ml. Each well of a microtiter plate (Thermo Scientific, UK) was filled with 100ml of each dilution except for the negative control that contained only 100ųl of lysogeny broth (Kim et. al., 2021). 100ųl of + 24 UMF concentrated manuka honey was added to the first well and then further dilutions were added to the next wells, respectively except for the positive control well. Both bacterial suspension and lysogeny broth were added into the positive control wells of the microtitration plate. Following overnight incubation at 37oC in a shaking incubator (MaxQTM Mini 4450, Thermo Fisher Scientific) at 3g, MIC (v/v %) was read comparing the wells with positive and negative controls (Ejaz et al. 2023).