Medically important bacteria isolated from commercial herbal medicines in Kampala city indicate the need to enhance safety frameworks

The high global bacterial infection burden has created need to investigate the neglected potential drivers of pathogenic bacteria, to inform disease prevention. Kampala is facing a proliferation of herbalists, selling herbal medicine (HM), of largely unregulated microbiological quality. We evaluated the bacterial contamination burden in HM sold in Kampala, to support evidence-based redress. The total viable loads (TVL), total coliform counts (TCC), E. coli counts, and prevalence of selected bacterial strains in 140 HM were examined using conventional culture, following the guidelines of World Health Organization (WHO), and Uganda National Drug Authority (NDA). Data were analyzed using D'Agostino-Pearson test, frequencies, proportions, Chi-square, and Mann–Whitney U test with STATA version-15.0. Fifty (35.7%), fifty-nine (42.1%), and twelve (8.6%) HM were unsafe for human use because they exceeded WHO’s permissible limits for TVL, TCC, and E. coli counts respectively. Solids had significantly higher mean TVL than liquids. Violation of NDA’s guidelines was significantly associated with high TVL. Fifty-nine bacteria, viz., Klebsiella pneumoniae (n = 34; 57.6%), Escherichia. coli (12; 20.3%), Staphylococcus aureus (7; 11.9%), Klebsiella oxytoca (3; 5.1%), Bacillus cereus, Pseudomonas aeruginosa, and Enterobacter spp. (1; 1.7% each), were isolated from 45 (32.1%) samples. These bacteria can cause severe clinical diseases, and promote deterioration of HM potency.

Characteristics of the herbal medicines from which bacterial contaminants were isolated. Out of the 140 herbal medicine (HM) samples, 114 were liquids while 26 were solids. In addition, two conventional antibiotic drugs were included as controls. The HM samples were availed on market in different packaging materials namely; bottles (116; 82.6%), polyethene (12; 8.6%), Tins (6; 4.3%), papers (5; 3.6%), and bare (unwrapped) (1; 0.9%). Figure 1 below shows some of the samples that were investigated in this research. Most of the HM (87; 62.1%), were packaged in original material (without evidence of package recycling), the re-used packages were (53; 37.83%), while the bare/unwrapped was (1, 0.07%). One hundred and one (101, 72.1%) samples were visibly clean, while 39 (27.9%) appeared dusty. Though most (77, 55.0%) samples satisfied at least (4 of the10) conditions enshrined in herbal medicine packaging and branding guidelines provided by Uganda National Drug Authority (NDA) 20 , 63 samples (63,45.0%) were in absolute disconformity (did not satisfy any of the 10 conditions), while none of the samples (0, 0%) conformed to all the 10 conditions. The ailments suggested to be treated by these HM belonged to 12 disease categories; gastro-intestinal tract infections being the largest category (constituting 14 diseases), followed by respiratory infections (10 diseases), and urinogenital and reproductive disorders (8 diseases); the smallest categories were the metabolic disorders and bone diseases with one ailment each ( Table 2).
to the WHO standards were found in Rubaga, a Chi-square test revealed that such HM did not significantly differ from those traded in the other divisions of the city (p ≤ 0.05) (Fig. 2).
The mean TVL from all the 140 samples (solids and liquids) was 126.407 × 10 4 CFU/ml or g, and it exceeded the WHO's permissible levels. The mean TVL values were (286.334, 125.919, 77.351, 52.789, and 35.821) × 10 4 CFU/ml or g for Rubaga, Kampala central, Nakawa, Makindye, and Kawempe divisions, respectively. There were no significant variations in the mean TVL of the five divisions, as well as the overall mean, except for Nakawa and Makindye (Mann-Whitney U; Z = 2.467, p = 0.0136) (Fig. 3).
For the liquid HM, the lowest TVL was 0.000 × 10 4 CFU/ml (recorded in 14 samples), while the highest was 1420.000 × 10 4 CFU/ml. For the solid HM, the highest was TVL was 1670.000 × 10 4 CFU/g and the least was 0.180 × 10 4 CFU/g. (Table 3). Overall, the highest TVL was 1670.000 × 10 4 CFU/g (observed in one solid sample), while the lowest was 0.000 × 10 4 CFU/ml (recorded in 14 liquid samples). Consequently, the mean TVL of the solid samples (215.660 × 10 4 CFU/g) was significantly higher than that (106.051 × 10 4 CFU/ml) in liquids (Mann-Whitney U; Z = − 2.136, p = 0.0326). Similarly, the mean TVL was significantly higher in solids than liquids in Makindye (Z = − 2.135, p = 0.0327). On the contrary, the mean TVL was significantly higher in liquids than the solid samples in Rubaga division (Z = − 2.673, p = 0.0075), while there was no significant variation in the mean TVL of solids and liquids in Kawempe, Kampala central, and Nakawa divisions ( Table 3).
The samples that were sold in recycled packaging materials possessed significantly higher mean TVL than those packed in nonrecycled packages except in Kawempe (Z = 0.343, p = 0.7313), and Rubaga divisions (Z = 0.1267, p = 0.1267). Both the Mann-Whitney U test and the t-test revealed no significant differences in the mean TVL values among the types of establishments where they were procured (market stalls, herbal shops, roadside and/or mobile stalls) (p < 0.05). The herbal medicines that scored ≤ 5 out of 10 (≤ 50%) in regard to conformity to NDA's herbal medicine packaging and labeling guidelines possessed significantly higher TVL than those with > 50% (Table 3). Herbal medicines sold at the lower prices (UGX 1000 ≤ 5000) contained greater mean TVL compared to those with high prices (> UGX 5000) but the differences were not significant (Table 3). counts that exceeded the World Health Organization's maximum permissible limit of ≤ 10 3 CFU/ml or g 18,19 .

Discussion
Socio-demographic profiles of participants. The majority of the participants recruited in this study were men. The predominance of men in HM trade was reported earlier in some parts South Africa 22 , Tanzania 23 , and Malawi 24 . However, in some provinces of South Africa such as KwaZulu-Natal, and Mpumalanga 25 , the majority of commercial herbalists were reported to be women, and this aligned with findings of most studies that were conducted in the rural herbalist communities in several parts of Uganda [26][27][28][29] . Though men's predominance as commercial herbalists in urban settings, as opposed to the rural areas, has been well documented, the factors that underpin this gender disproportion remain largely unexplained. Partly, this gender disparity may be attributed to societal norms 28 , and also to the fact that females are commonly responsible for their families' primary health care. Hence treating people becomes a responsibility of mainly the female gender most evidently in the rural settings 28 . www.nature.com/scientificreports/ Our observation, that over 90% of the participants in the current study had attained a certain level of formal education is of great significance, since basic education is believed to be a critical component of the health 30 . The high profits gained from HM sales, as observed in this study, highlight the potential role herbal medicines play towards Uganda's national economy, as well as supporting the attainment of community livelihood needs, primary health care, and cultural heritage. Consequently, medicinal plants have been acknowledged as an important feature of the cultural, economic, medicinal, and ecological components of all cities in the world 31 .
Characteristics of the herbal medicines from which bacterial contaminants were isolated. Both the solid and liquid HM were included in this study and were procured from herbal shops, market stalls, as well as roadside and/or mobile stalls. Likewise, such types of commercial HM establishments were reported in urban settings in other parts of Africa and beyond 6,[32][33][34][35] . The abundant presence of roadside and/or mobile commercial herbal medicines might pose herbal quality challenges since adequate regulation and monitoring of such establishments can be complicated 36 . Also, our observation that some HM were placed in recycled packaging materials (previously discarded as wastes) points to further potential threats to herbal safety since the re-use of these packages has been linked to the introduction of pathogenic microbial contaminants into the drugs elsewhere 33,34,36,37 . Even though the proportion of HM that absolutely violated the Uganda National Drug Authority (NDA)'s herbal packaging and labeling guidelines was generally high, it demonstrated a twofold decrease in the percentage that was reported in Makindye division of Kampala over a decade ago 5 . The NDAguidelines provide for ten conditions that should be fulfilled to attain the desired herbal packaging and labelling standards, viz; (1) tight packaging (absence of leakage), (2) presence of a brand name, (3) active ingredients (contents), (4) indications (diseases/therapeutic claims), and contraindications (warnings and precautions), (5) name of manufacturer, (6) full address of manufacturer with physical location, (7) batch number, (8) date of manufacture and expiry, (9) dosage (for adults and children), and (10) directions for use and storage 38     www.nature.com/scientificreports/ lated innovations not only in the marketing/advertisement but also improvement in packaging and branding, nearly comparable to the standards that are acceptable in pharmacies 39 . Nonetheless, some hinderances to the standard labelling of herbal medicines have been reported in some parts of the world; for example, most herbalists decline to specify the active ingredients/contents present in their herbal products because they wish to keep their traditional medicinal knowledge confidential 5,40 . Unfortunately, this may lead to in adverse drug reactions if members of the community use HM having ingredients to which they are allergic. Much as our inclusion criteria sought a random sample of 140 herbal medicines used for cough, diabetes, Ulcers, and/or diarrhea treatment, the same remedies were found to be used in the management of 62 health complications, including HIV/AIDS, different types of cancers, and reproductive disorders among others. On  www.nature.com/scientificreports/ average therefore, each remedy was suggested to treat over 15 ailments. Though the "one drug for many diseases" approach may not only reduce the cost of treatment of multiple infections 41 , it can also increase the number of individuals (suffering from different ailments) who may consume the same drug, and hence get exposed to its associated risks such as pathogenic microbial contaminants.

Total viable loads (TVL) of bacteria isolated from selected herbal medicines sold in Kampala
City. According to the quality control guidelines set by World Health Organization, herbal medicines with    18,19 . Even though most of these unsuitable samples were found in Rubaga division, the mounts did not significantly differ from those found in the other parts of the city (χ 2 , p < 0.05). The proportion of HM which exceeded the permissible TVL of bacteria in Kampala that were revealed in this study is over threefold higher than that (11.7%), reported by another study in 2009 5 . The difference might partly be explained by the fact that the latter 5 , was less comprehensive since the sampling was limited to liquid HM for cough in Makindye division alone. Other few previous studies that assessed the bacterial contamination of herbal medicines in Kampala did not examine this parameter (the total viable loads of bacteria) 14,44,45 .
Our findings revealed that HM with ≤ 50% level of conformity to Uganda National Drug Authority's packaging and labeling guidelines contained significantly higher TVL values than those with > 50%, and that there were no significant differences in the contamination levels between the highly priced and the low-cost HM. This implies that irrespective of the price, some of the commercial HM in Kampala may disseminate viable bacteria in the community, but conformity to the Uganda National Drug Authority guidelines can play a considerable role in resolving this burden. Most bacteria have the potential not only to impair human health but also spoil and reduce the potency of the herbal medicines they contaminate 46 .

Total Coliform Counts (TCC), and E. coli counts.
In the present study, the proportion of HM (42.1%), which were found to exceed the permissible level of total coliform counts was high give the perceived public health importance of most coliform bacteria 5,46,47 . The term "total coliforms" represents a group of non-sporeforming, gram-negative, facultatively anaerobic, bacillus bacteria that strongly ferment lactose to acid and gas following 42-48 h incubation at 35 ± 2 °C 48 . They mainly belong to two groups. The 1st group includes bacteria that often form part of the natural flora of most vegetation, such as some strains of Enterobacter spp., Serratia spp., and Erwinia spp. among others. The bacteria belonging to this group do not have any historicaly linkages to feces and are commonly prevalent in environmental reservoirs such as water and soil. The presence of these strains in consumable products like HM may not necessarily infer fecal contamination, hence they do not necessarily indicate a substantial public health threat 49 . The 2nd group constitutes thermotolerant (fecal) coliforms, which are commonly found in the intestines of man and other warm-blooded animals 48 . These include some strains of Escherichia spp, Klebsiella spp, and Citrobacter spp. among others. The strains belonging to this group largely constitute the intestinal flora of man and other warm-blooded animals, hence they signify potential contamination by fecal matter and its associated human pathogens, threatening public health 16 . Escherichia coli is believed to be one of commonest flora that inhabit the intestinal tract of vertebrates 50 . Therefore, the presence of this coliform is widely used as a reliable basis to infer the degree of fecal contamination.
In the current study, over eight in very one hundred herbal medicines were found to possess higher loads of E. coli than the permissible level (10 CFU/ml or g), as per the guidelines of WHO 18 . From the public health point of view, over 8% of the samples screened in the current study were therefore unsafe for human use on the basis of this parameter. A chi-square test revealed that the overall number of samples with unacceptable total coliform counts were significantly higher than those harboring undesirable E. coli loads (p = 0.0291). Consequently, it was surprising to note that Rubaga division did not possess any samples with unacceptable levels of E. coli even though over 35% harbored high total coliform counts. These observations demonstrate the potential role other coliform bacteria (most likely nonfecal coliforms that are commonly prevalent on plants, and in environmental resources like soil and water), play in contaminating herbal remedies, hence the need to observe both the good manufacturing practices as well as the good agricultural practices by the herbalists.

Specific bacterial strains isolated from selected herbal medicines sold in Kampala city.
We report a 32.1% proportion of HM that were contaminated with at least one bacterial species of potential medical  The failure to attain a stable level of microbiological safety in the herbal medicines sold in Kampala city, and/ or some other similar settings (Fig. 4), reflects intervention gaps which could be associated with the policy and/ or regulatory environment, hygiene, and sociographic factors of herbalists among others. In the present study, it was outrageous to observe the concurrence of three bacterial strains of potential medical importance in two of the herbal liquids (packed in dusty, re-used mineral water bottles), one for cough and the other for diarrhea treatment. The former was grossly adulterated with P. aeruginosa and the latter harbored S. aureus, while both were positive for E. coli and K. pneumoniae. Such HM might promote the community spread of mixed bacterial infections which can be complicated to treat. It was commendable to note that in this study, many of the primary pathogens such as Salmonella spp. and Shigella spp., some of which have been associated with persistent outbreaks of diarrheal diseases like typhoid fever in Kampala city were absent in all the samples screened 15 . Nonetheless, the strains that have been revealed by the current study to be highly prevalent in HM in Kampala, particularly K. pneumoniae, E. coli and S. aureus have the potential to impair human health in several ways. For example, S. aureus, and several pathotypes of E. coli are involved in severe diarrhea and/or urinogenital infections 16,52,53 . Besides, these pathogens are on the global list of critical multi-drug-resistant bacteria which require urgent redress 54 . Although bacterial contaminants have been reported in some conventional pharmaceutical drugs elsewhere 55 , the modern drugs included as controls in the present study where safe in reference to all the microbiological parameters examined. This implies that the modern drugs sold in pharmacies in Kampala might be of suitable microbiological quality but this can only be confirmed following a comprehensive investigation.

Conclusions
The prevalence of commercial herbal medicines that are bacterially contaminated in Kampala city remains relatively high. Some strains belonging to the bacterial species involved in this contamination, particularly, E. coli, K. pneumoniae, P. aeruginosa and S. aureus have the potential to cause severe clinical diseases, spoilage of HM, and deterrence of therapeutic potency. Also, these strains have been implicated in the community spread of antibiotic resistance. There is need to conduct further research on bacteria isolated from HM in Uganda, especially to examine their antibiotic resistance traits, to fully elucidate the risk incurred. Quality assurance should be enhanced to promote public health and to harness herbal medicine sector development.  Line for the proporƟon reported in the current study Figure 6. % of herbal medicines (HM) reported to be contaminated with potentially pathogenic bacteria in Kampala and its neighborhoods 1,5,6,14,17,44,45,51 .

Study design. A cross sectional survey was conducted on 65 traditional herbalists that sell herbal medicines
in Kampala, between April and September 2021. Pre-tested, semi-structured questionnaires were used to record the socio-demographic profiles of respondents, and the attributes of herbal medicines which were procured from the participants. Such attributes included, prices, quantity and label information among others. In addition, HM packaging properties, categories of traditional HM outlets, cleanliness, and pharmaceutical forms of the HM were examined through field observations which were supplemented using photography, with a highresolution digital camera inbuilt in a Phantom-9 Mobile Phone, model AB7/2019, Techno Mobile Limited 57 . The laboratory experiments were aimed at assessing the Total Viable Loads (TVL), Total Coliform Counts (TCC), as well as the isolation and characterization of selected diarrheal and/or respiratory bacterial pathogens from www.nature.com/scientificreports/ the herbal medicine samples. The targeted bacterial species were those that had been commonly implicated in disease causation in Kampala city and/or Uganda at large, such as; Salmonella spp., E. coli and K. pneumoniae among others 15,16,58 .

Study population.
The study units of interest in this research were the herbal medicines for diarrhea, cough, diabetes, and high blood pressure, sold by herbalists in herbal shops, market stalls, and roadside/mobile stalls in Kampala city. These herbal medicines were selected because of their perceived high frequency of use, and the likely carriage of bacterial contaminants of mostly unidentified profiles 5,14 . Sample size. Due to high number of largely unregulated HM marketed with various therapeutic claims in Kampala, a representative sample (N), was determined using the formula for unknown population size at 95% confidence interval; N = Z 2 1−α [P (1 − P)] ÷ D 259 . Where; N = The desired sample size, Z 1−α = Standard error of the mean = 1.96 at 95% confidence level, P = Estimated prevalence of bacterial contamination in HM above the WHO permissible levels = 90% 14 , and D = Tolerable sampling error = 0.05 at 5% significance level.
Hence, sample size = 1.96 2 [0.9 (1 − 0.9)] ÷ 0.05 2 = 138.3 ≃ 138. Therefore, the minimum sample size required was 138. However, since the current study was conducted in five administrative divisions of Kampala, a sample size of 140 herbal medicines was adopted, and was divided into strata (of 28 samples each), that were obtained from each division. In addition, two conventional pharmaceutical drugs (one powder for diarrhea, and one liquid for cough), from pharmacies were included as controls; hence the total of medicines processed for bacteriological analysis was 142.
Sampling technique. Prior informed consent was obtained from all the herbalists that offered their herbal medicine samples to be used in this study. Ethical approval for the study was obtained from the School of Health Sciences Research and Ethics Committee of Makerere University. The sampling frame exclusively involved herbalists that were engaged not only in trade, but also in harvesting, and/or preparation of the HM. The stratified sampling technique was adopted; viz, each administrative division of Kampala constituted a stratum 28 samples which were procured from herbal shops, market stalls, roadside stalls, and mobile stalls. Then, the random selection of individual herbal medicine samples from each stratum was done following the guidelines for research on HM products, established by the Uganda National Drug Authority (NDA) and the World Health organization 38,60 . Specimen collection and transportation. We randomly selected some of the HM (ready for consumption), that had been proposed to potentially possess largely unknown species of bacterial contaminants in earlier studies, particularly those for diarrhea, cough, diabetes, and high blood pressure 5,14 . Upon procurement, the samples were aseptically wrapped in sterile zipped specimen bags, and transported on a cold chain for bacteriological analysis, in the microbiology laboratory at Makerere University, School of Veterinary Medicine. Since microbial loads may depend on temperature and humidity 47 ; the samples were collected during dryness as well as frequent rains, from April to September 2021.
Media preparation, and bacteriological analysis. Three types of bacteriological analyses were performed, viz: (1) determination of bacterial total viable counts and total coliform counts, and (2)  Preparation of serial dilutions of herbal medicine samples. For the liquid samples (10° dilution), five test tubes (labeled 1-5) were arranged and 9.0 mls of sterile peptone water introduced into each tube. Then 1.0 ml of a sample was pipetted into test tube number 1 followed by thorough mixing, hence constituting the 10 -1 dilution. www.nature.com/scientificreports/ 1.0 ml of the 10 -1 dilution (in test tube 1), was pipetted into test tube 2 using a fresh pipette tip, followed by thorough mixing, hence constituting the 10 -2 dilution, and the procedure was repeated up to test tube number 5, thus constituting the dilutions 10 -3 , 10 -4 and 10 -5 , respectively. For the case of solid samples, 10 g of each sample were introduced into a stomach bag containing 90 mls of peptone water. The mixture was homogenized using a Pulsifier (Serial No: 30660282, Manufactured by Kalyx Biosciences Inc, Canada), and this constituted the 10 0 dilution which was then serially diluted up to the 10 -5 through the same procedure applied above on liquid samples. The CFU of the different dilutions were computed and the mean was evaluated and recorded as the TVC of that sample. The mean TVC for each sample was compared with the permissible limits recommended by the World Health Organization 18,19 , to assess whether the samples were of good quality and safe for human consumption.

Determination of total viable counts (TVC
Determination of Total Coliform count (TCC). 0.1 ml of each dilution was pipetted onto VRBA media plates, spread using a glass spreader, and incubated for 24 h at 37 °C. Colonies that grew were counted using a colony counter (CAT No: SC6+, Serial number: R570003270, made in United Kingdom, www. staurt. equip ment. com) and expressed as Colony Forming Units per gram (CFU/g) for solid samples, or per milliliter (CFU/ml) for liquids. The coliforms were considered as pale pink, moist/mucoid colonies that grew on VRBA media, except for E. coli which presented as hot pink, dry, flat/umbonate colonies. The CFU of the different dilutions were computed and the mean was evaluated and recorded as the TCC of the sample. The mean TCC for each sample was compared with the permissible limits recommended by the World Health Organization 18 , to assess whether the samples were of good quality and safe for human consumption. All samples with a mean TCC > 0.0 CFU/ml, or > 0.0 CFU/g were classified as unsafe for human consumption (WHO, 2007). For the case of Salmonella spp. and Shigella spp., prior to culturing on XLD agar, the samples were first taken through a pre-enrichment stage, followed by an enrichment process. Pre-enrichment was done by incubating the 10 -1 dilution of each sample at 37 °C for 24 h. The enrichment culture procedure was then done by introducing 1.0 ml of the 24-h old pre-enriched culture into 9.0 mls of Rappaport-Vassiliadis Soya Peptone broth, followed by incubated for 24 h at 42 °C. One loopful of the enriched cultures were then streak plated on XLD.
All the inoculated agar plates were incubated at 37°C for 24 h. In the case of E. coli, the pink colonies that grew on VRBA were sub-cultured on sorbitol MacConkey and incubated at 37 °C for 24 h to screen for the potential presence of translucent colonies of Enterohaemorrhagic E. coli (E. coli: 017H7). The observed bacterial species in each sample were compared with the guidelines of the World Health Organization to assess if the samples were safe for human consumption (WHO 10,18 ).
The bacterial colonies that grow were identified based on their cultural characteristics (growth and colony morphotypes on various forms of media), gram staining, and biochemical traits 62 66 . The MR test examines the ability of bacterial strains to catabolize glucose in a buffered media hence yielding ethanol, CO 2 , H 2 , and a mixture of organic acids such as succinic acid, lactic acid, formic acid, and acetic acid. The acids cause a drop in the pH of the culture below 4.2, and this is detected using a suitable indicator 65,66 . 2.0 mls of sterile methyl red-Voges-Proskauer (V-P) medium (glucose-phosphate) base in screw-cap test tubes were inoculated with a loopful of 24-h old bacterial colonies and incubated at 37 °C for 4 days, and then a drop of methyl red reagent (0.25 g of methyl red in 100 mls of ethanol) was added. The results were interpreted basing on the emergent colour changes, i.e., red colour (MR positive), any shade of orange or yellow was taken as a negative reaction. (e) V-P test (Voges-Proskauer test): This test is used to identify fermentative microbes that catabolize glucose via the butanediol pathway. During the glucose fermentation, acetoin (acetylmethylcarbinol) exists as an intermediate in the formation of 2,3-butanediol, which gets oxidized (in the presence of KOH and O 2 ) to a diacetyl. The diacetyl reacts with a guanidine group present in amino acids such as arginine (contributed by peptone in the V-P media), to form a pink-to red-colored product and this colour may be intensified by α-naphthol 63 ). The detection of H 2 S is achieved through its reaction with Iron salts (present in the culture media), which yields black precipitates of either Iron(ii) Sulfide (FeS) or Iron(iii) Sulfides (Fe 2 S 3 ) 63,67 . Using a stabbing wire, a 24-h old culture was stabbed into SIM (Sulfite Indole Motility) media in screw-cap test tubes and incubated at 37 °C for 24 h. The formation of black coloration in the media indicated positive results. This same test was used to assess the motility and indole production. Motile organisms were identified based on their observable growth away from the stabbing line, while indole production was deduced by adding a drop of Kovac's reagent and observing the formation of pink colouration. (g) Triple Sugar Iron test (TSI): This test identifies bacterial strains basing on their differences in the carbohydrate fermentation patterns and hydrogen sulfide production ability 66 . The TSI agar contains sodium thiosulfate, peptone, three fermentative carbohydrates [lactose (1%), sucrose (1%) and glucose (0.1%)], and the phenol red acid base indicator. The accumulation of acids associated with carbohydrate fermentation, lowers the pH hance the phenol red indicator turns from orange-red to yellow, while the oxidative decarboxylation of peptone yields alkaline products hence the indicator turns deep red. The production of H 2 S is indicated by the black colour of the butt 66 . The TSI agar in the screw-cap test tubes was inoculated with 24-h old bacterial colonies, first by stabbing through the center of the media up to the bottom of the butt, followed by streaking on the surface of the slant. The tube was loosely capped and incubated at 37 °C for 24 h. Alkaline/acid (red slant/yellow butt) was interpreted as glucose fermentation only; acid/acid (yellow slant/yellow butt) as metabolization of glucose, lactose and/or sucrose; alkaline/alkaline (red slant/red butt) as absence of any carbohydrate fermentation; blackening of the media as H 2 S production, while bubbles or cracks in the agar were interpreted as gas (CO 2 and H 2 ) production.
Data analysis. Data were entered in MS-Excel and exported to STATA version 15.0 software. The data were tested for normality by using D' Agostino-Pearson test and normal distribution was accepted at (p > 0.05). Data were then analyzed using descriptive statistics (frequencies and percentages), and inferential statistics [Pearson's Chi-square (in case of normality), or Mann-Whitney U test (for nonparametric data)], at 0.05 significancy level.
Ethical approval and consent to participate. The 68 . Informed consent to participate in this study was obtained in writing from the study participants. Respondents' identifiers were recorded in form of assigned codes instead of names to ensure anonymity.