Exploring the potential human pathogenic bacteria in selected ready‐to‐eat leafy greens sold in Dhaka City, Bangladesh: Estimation of bacterial load and incidence

Abstract This study was designed to investigate the presence of potential human pathogenic bacteria, bacterial load, and their incidence in ready‐to‐eat leafy greens viz., coriander, lettuce, and mint leaves sold at diverse marketplaces in Dhaka City. Multiple identification methods including cultural, morphological, biochemical, and molecular analysis were employed in the Plant Pathology Laboratory of Sher‐e‐Bangla Agricultural University to identify the human pathogenic bacteria. In molecular analysis, the DNA samples were put through PCR using bacterial primer 27F: AGAGTTTGATCMTGGCTGAG and universal primer 1942R: CGGTTACCTTGTTACGACTT. Initially, nine different bacterial genera viz. Bacillus, Escherichia, Pseudomonas, Neisseria, Klebsiella, Enterobacter, Shigella, Vibrio, and Staphylococcus were detected, and their incidence was 93%, 67%, 44%, 30%, 26%, 26%, 11%, 7%, and 7% respectively. A total of twelve bacteria have been identified from these genera out of which 7 bacteria viz. Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Staphylococcus aureus, and Shigella spp., were reported as human pathogenic bacteria in several pieces of literature. The highest colony‐forming units per gram were shown in mint (4.27 ± 2.35 × 109) followed by lettuce (2.87 ± 0.76 × 109) and coriander (2.43 ± 1.32 × 109). Considering marketplaces, the highest colony‐forming units per gram were observed in the samples of street markets (5.0 ± 1.72 × 109) and the lowest was in supermarkets (1.87 ± 0.46 × 109) followed by local markets (2.7 ± 0.91 × 109). All the leafy green samples crossed the acceptable level of bacterial load (106 CFU/g). The findings of the study highlight the urgency for improved food safety protocols in their production and distribution in Dhaka city.


| INTRODUC TI ON
Leafy greens have excellent nutritional value in the diet and can be used for medicinal benefits.Different phytochemicals are present in leafy greens including phenolic acids, flavonoids, carotenoids, polyphenols, glucosinolates, isothiocyanate, allylic sulfides, phytosterols, and monoterpenes.Leafy greens mostly contain antioxidants, dietary fibers, minerals, α-linoleic acid, and vitamins.It has different health benefits such as anti-diabetic properties, preventing CVD, anti-hypertensive, anti-carcinogenic, anti-anemic, and improving gut health (Aslam et al., 2020).
Leafy greens consumption may have health benefits, but recent reports of infectious disease outbreaks from the Centers for Disease Control and Prevention (CDC), US Food and Drug Administration (FDA), World Health Organization (WHO), and Center for Science in the Public Interest (CSPI) have raised questions about the safety and quality of vegetables (Nithya & Babu, 2017).Over the past 20 years, more ready-to-eat (RTE) leafy greens have been consumed, and at the same time, more outbreaks of foodborne illness have been linked to the intake of leafy green vegetables (Castro-Ibanez et al., 2017;Olaimat & Holley, 2012).In such outbreaks, poor sanitation practices were invariably mentioned.The use of chemical fertilizers, hormones, pesticides, and their residues in food are additional possible risks that could raise the risk of foodborne illnesses linked to fresh produce (Chen, 2007).RTE leafy greens are a convenient approach to ensuring the consumption of vegetables, but consumers must feel convinced that the food is safe to intake.Due to RTE leafy greens are consumed raw and there is no killing step of pathogens (e.g., heating) at any stage in the chain to prevent transmission so the contaminated pathogenic bacteria are still alive after consumption.Therefore, ensuring the microbiological safety of fresh RTE leafy greens offers a special difficulty, and the consumers might get sick if they intake contaminated leafy greens.(Gil et al., 2015).
Fresh leafy greens are generally known to have significant bacterial populations, some of which may be plant endophytes, plant pathogens, or human pathogens (Leff & Fierer, 2013).Several pathogens, like Salmonella, Shigella, Escherichia coli, Campylobacter, Yersinia, Listeria, Staphylococcus, Bacillus cereus, Vibrio spp., etc., pose a significant risk to public health, particularly with the consumption of raw produce.According to the Centers for Disease Control and Prevention (2022), and World Health Organization (1998), efforts have been made to address the concern of vulnerabilities arising from the cultivation of leafy greens, which continue to be susceptible to contamination by bacteria like E. coli O157, Salmonella, Shigella, Listeria, etc., despite attempts at washing.Ahamad (2020) released a survey report conducted by the Department of Tourism in 2017 found that around 6 million people in Dhaka city consume street food every day which highlighted the concerns regarding hygiene practices surrounding street food preparation and consumption.These street foods, frequently prepared with leafy greens like lettuce in Burgers, coriander in Noodles, and mint in Juices, and served in unsanitary urban environments, stand vulnerable to microbial contamination, as suggested by both the lack of hygiene measures during their preparation and the bustling urban context in which they are sold.
The current study first aims to isolate and identify the range of bacteria present on the surfaces of selected leafy greens, and then, the study aims to specifically detect any human pathogenic bacteria among the isolated bacterial species.Lastly, the research intends to quantitatively assess the colony-forming units and overall bacterial incidence on these leafy greens to shed light on the safety and hygiene of consuming foods prepared by leafy greens.

| Study site and period
The study was carried out in the MS lab of the Department of Plant Pathology at Sher-e-Bangla Agricultural University (23°46′13.68″N, 90°22′39.42″E) Sher-e-Bangla Nagar, Dhaka-1207, during 2021-2022.In this study, samples were collected from 9 different locations in Dhaka metropolitan city (Figure 1).

| Samples selection and collection
Three different leafy greens viz., Coriander, (Coriandrum sativum L.) Mint (Mentha sp.), and Lettuce (Lactuca sativa L.) were selected which are frequently eaten raw (without cooking) that are considered ready-to-eat (RTE) products.A total of 27 working samples (9 for each leafy green) were collected in fresh and disease-free conditions from 9 different supermarkets, local markets, and street markets (Table 1).Each sample was collected 3 times during each visit from the selected marketplaces and as a result, the data were obtained from a total of 81 samples of 27 working samples.During each visit, 100 grams of each sample were taken at random from each market.An insulated box with ice and a sterile zipper bag was used to collect all the samples, which were then thoroughly examined and analyzed within an hour of being collected.For further research, the collected samples were placed in sterile zipper bags and kept in the normal chamber of the refrigerator at 4-6°C.

| Preparation of stock solutions
Within an hour of collection, the samples were taken into the laboratory, and prepared stock solutions were by submerging each 100 g sample with 1000 mL of distilled water, then diluted 5 folds in serial dilution by using a vortex mixture.

| Isolation and identification of bacteria
A 0.05 mL of 1:10 −5 diluted solutions were spread over the Nutrient Agar (NA) petri plates where each step was completed in triplicate and incubated for 24 h at 30°C.After 24 h, these overnight cultures in NA plates were observed where well-distinct colonies with distinctive morphology were preferably selected and isolated on NA slants.Using streak plates, the chosen isolates were purified.

| Biochemical tests
As per standard protocol of the Society of American Bacteriologists (1957), morphological and biochemical tests were done that were Gram-staining, KOH solubility, Oxidase, Catalase, Motility, Simmon's citrate utilization, Casein hydrolysis, Starch hydrolysis, Gelatin liquefaction, and Levan production tests.

| Culture on selective media
Four selective and differentiated media were used to identify bacterial species that were Eosin Methylene Blue (EMB) Agar, Salmonella Shigella (SS) Agar, Cetrimide Agar, and Bacillus cereus Agar.After  culturing the isolates of the bacteria on these media, the growth and color of colonies were observed to identify bacteria.

| Molecular analysis
The molecular detection was done at the Plant Pathology Laboratory using a combination of techniques including DNA extraction, DNA quantification, PCR amplification, gel electrophoresis and documentation, DNA purification, DNA sequencing, and bioinformatics analysis.

| Extraction of genomic DNA from pure cultures
Initially, 1 mL of overnight cultures were centrifuged (Model: Z-216 M, HERMLE, Germany) at 15,000 × g for 2 min, and the supernatant was discarded, leaving behind the cell pellet.Next, the cells were resuspended in 480 μL of 50 mM EDTA, and 120 μL of lytic enzymes (lysozyme) was added, followed by incubation at 37°C for 60 min.
The lysed cells were then centrifuged at 15,000 × g for 2 min, and the supernatant was discarded.For cell lysis, 600 μL of nuclei lysis solution was added, and the mixture was incubated at 80°C for 5 min, cooled to room temperature, and treated with 3 μL RNase solution at 37°C for 60 min.Protein precipitation was achieved by adding 200 μL of protein precipitation solution, vortexing vigorously at high speed for 30 s to mix the protein precipitation solution with the cell lysate.
The vortex mixture was incubated on ice for 5 min and centrifuged at 15,000 × g* for 3 min.The resulting supernatants were transferred to another clean tube containing 600 μL of isopropanol, and DNA precipitation was facilitated by inverting the tube until visible DNA strands formed.After centrifugation at 15,000 × g* for 2 min the pellet cells were removed.Then, 600 μL of room temperature 70% ethanol was added and gently inverted the tubes several times to wash the DNA pellet and centrifuged at 15,000 × g* for 2 min.The DNA pellet was air-dried for 15 min.Lastly, the DNA pellet was rehydrated in 100 μL of rehydration solution and incubated overnight at 4°C.
Finally, the genomic DNA was stored at 20°C until use.

| Electrophoresis and gel documentation
In the gel electrophoresis, Agarose powder (Cat: V3125) was mixed with Tris Borate EDTA (TBE) buffer (Cat: V4251) and heated to 80°C for 5 min to ensure complete dissolution, and then, ethidium bromide (Cat: H5041) was added for binding of the molecule to the DNA (Raji et al., 2008).The gel was poured onto a Horizontal Gel Electrophoresis (Model: Mini, CBS Scientific, USA) gel tray.The DNA samples, along with a 1 kb ladder (Cat: G754B) as a size marker, were loaded into the wells.Electrophoresis was run at 90 volts for 30 min to separate the DNA fragments based on their sizes.After the electrophoresis, the gel was transferred to an Alpha Imager Gel Documentation System (Model: Mini, Protein Simple, USA) for visualization and imaging of the DNA bands.

| DNA purification
DNA bands excised from the gel were dissolved using Membrane Binding Solution.Similarly, PCR amplification products were mixed with this solution.The DNA solutions were then transferred to SV Minicolumns for binding and subsequently washed using Membrane Wash Solution with ethanol.After ethanol evaporation, elution was performed by transferring Minicolumns to clean tubes and adding nuclease-free water, and centrifuged.The purified DNA was obtained and ready for downstream applications.

| DNA sequencing
The purified DNA samples were sent to Bioneer (Seoul, Korea) for partial sequencing.The DNA sample was subjected to Sanger sequencing analysis.After getting the Sanger sequences, Chromas 2.6 software was used to generate the FASTA file containing the partial sequence of the 16S rRNA gene.

| DNA analysis
The sequences were analyzed by using the BLAST (Basic Local Alignment Search Tool) on the NCBI website (https:// www.ncbi. nlm. nih.gov) for matching with existing nucleotide sequences in the NCBI GenBank database and obtained accession numbers.
Phylogenetic analysis was performed using MEGA 11 (Molecular Evolutionary Genetics Analysis) software, where the maximum likelihood algorithm was selected to construct the phylogenetic tree with 1000 bootstraps and nucleotide Tamura-Nei substitution model.

| Estimation of bacterial load
After 24 h of inoculation, colony-forming units (CFU) were first counted and recorded on the same day.At first, CFU was estimated for each mL of stock solution after that this value was multiplied by 10 (100 g samples submerged in 1000 mL stock solutions) to determine the CFU per gram of leafy green samples by following the equation: Here, Total dilution factor = 10 5 (Five-fold serial dilution was performed).Volume of culture per plate = 0.05 mL.

| Estimation of bacterial incidence
Measurement of the incidence of different types of bacteria in leafy greens was determined by the following equation:

| Statistical analysis
The laboratory study was performed following Complete Randomized Design (CRD).The data were analyzed using the computer-based software Statistix-10.Differences in the means were statistically analyzed using ANOVA at a confidence level of 95% (p ≤ .05).

| Bacterial colony-forming units' count
The present study investigates the microbial contamination levels in three selected leafy greens viz.coriander, lettuce, and mint collected from 3 different types of marketplaces.Among the three leafy greens, the highest average (n = 9) bacterial load was observed in the mint leaves (4.27 ± 2.35 × 10 9 CFU/g) similar to lettuce leaves (2.87 ± 0.76 × 10 9 CFU/g), whereas the lowest was in the coriander leaves (2.43 ± 1.32 × 10 9 CFU/g).Despite observable differences in mean counts, the results did not reach statistical significance (p = .0603)at a 95% confidence level, suggesting that the microbial populations in these leafy greens were not significantly different from each other (Figure 2).
In terms of different marketplaces, the street markets samples showed the highest bacterial load (5.0 ± 1.72 × 10 9 CFU/g) with statistically significant differences (p = .0000)compared to the supermarket samples (1.87 ± 0.46 × 10 9 CFU/g) and the local market samples

| Isolation and identification of bacteria
In this study, different cultural, morphological, and biochemical tests were analyzed and identified bacterial genera following Bergey's Manual of Systematic Bacteriology Vol.I (Krieg & Holt, 1984) and Vol.II (Sneath et al., 1986) et al. (2018) indicated that Klebsiella pneumoniae is not only a major hospital-acquired pathogen but also an important foodborne pathogen, capable of causing septicemia, liver abscesses, and diarrhea in humans.Enterobacter aerogenes is multi-drug resistant to antibiotics and can be involved in urinary tract, gastrointestinal, and bloodstream infections, posing a potential risk of adult meningitis (Pradel & Pages, 2002).The study conducted by Kirk et al. (2014) sheds light on the significant impact of Enterotoxin-producing Staphylococcus aureus as a causative agent of toxin-mediated food poisoning.This identification adds to the growing body of knowledge concerning the microbial diversity associated with leafy greens and their potential implications for food safety and public health.

| Study on the cultural characteristics
At first, the pure colony growths of different isolated bacteria were obtained by culturing them on the nutrient agar plate through streaking methods (Figure 4).The cultural characteristics like shape, size, margin, pigment, elevation, and texture of the bacterial pure cultures were examined to identify bacterial genera initially.The observed findings of the bacterial colony characters are presented in (Table 3).

| Study on the morphological characteristics
A comprehensive study of the morphological characteristics of the identified bacteria was performed after Gram staining and subsequent observation under a compound microscope.Among the identified bacteria, Escherichia and Pseudomonas showed small rodshaped; Bacillus, Klebsiella, Shigella, and Enterobacter rod-shaped; Vibrio coma-shaped; Neisseria bean/road-shaped and Staphylococcus spherical-shaped (Figure 5).These findings combined with results from cultural characterization, strengthen the elementary identification of the bacterial genera.

| Study on the biochemical analysis
The biochemical tests conducted on the different isolates of bacteria revealed significant variations in the biochemical characteristics.
Among the 30 isolates of bacteria, 60% were Gram-negative bacteria which means they have a distinctive double membrane structure, and due to this structure, Gram-negative bacteria are more resistant than Gram-positive bacteria and cause significant morbidity and mortality worldwide (Breijyeh et al., 2020).The motility test displayed a positive result in 67% of the isolates, indicating the  4).These diverse results of biochemical analysis highlight the heterogeneity of the bacterial populations present in the leafy greens.

| Study of the bacterial cultures on selective media
By observing the appearance of bacterial colonies on four different selective and semi-selective (differentiated) media, 6 different bacteria were identified and confirmed (Figure 6).E. coli showed a blue colony without a metallic green sheen, Klebsiella pneumoniae showed a pink mucoid colony, and Enterobacter aerogenes showed a pink dull colony on EMB agar.These color variations are indicative of lactose fermentation, with each bacterium showing a unique color response due to the differential properties of the EMB agar.Aryal (2022a) corroborated these findings, explaining that EMB agar is a differential medium that can inhibit the growth of Gram-positive bacteria while aiding in distinguishing lactose fermenters based on colony colors.
The results from this study align with Aryal's description of the EMB agar's capabilities.

Pseudomonas aeruginosa showed a cream colony on Cetrimide
Agar.This selective medium is known for its ability to isolate and identify Pseudomonas species, particularly Pseudomonas aeruginosa, due to its characteristic growth and pigment production.The observed cream colony appearance is consistent with previous research on the distinctive colonial appearance of Pseudomonas aeruginosa on Cetrimide Agar (Brown & Lowbury, 1965).
Bacillus cereus exhibited a blue colony on Bacillus cereus agar.Holbrook and Anderson (1980) supported these findings, reporting that Bacillus cereus agar is a selective medium specifically designed for the identification of Bacillus cereus.The peacock blue color produced by Bacillus cereus on this agar medium is considered a distinguishing feature of the species.
Shigella sp.produced a transparent colony on SS Agar.SS agar is a semi-selective medium that inhibits the growth of most Grampositive bacteria while allowing the growth of Gram-negative enteric pathogens like Shigella and Salmonella.The transparent colony appearance aligns with the reported colorless characteristics of Shigella spp. on SS agar as described by Aryal (2022b).TA B L E 4 Analysis of different biochemical test results of selected isolates for the identification of different bacteria.The results demonstrate a consistent detection of an approximately 1465 ± bp DNA band in the gel doc system, which aligns with Nurfitri et al. (2019) findings.This similarity is observed when using 27F and 1492R primers along with the 1 kb DNA ladder as a marker.

Bacteria
The presence of this band indicates that the used primers and PCR conditions were amplified well in accordance with the primary target of the 16S rRNA gene.

| Analysis of DNA sequences
The sequences were analyzed by using the BLAST on the NCBI website (https:// www.ncbi.nlm.nih.gov) for matching with existing nucleotide sequences in the NCBI GenBank database.The partial sequences of 16S rRNA of isolated bacteria were submitted to NCBI for deposition in the GenBank and obtained accession number (Table 5).(Accession no.OQ740286.1)showed 99.75% identity to the Bacillus velezensis strain ND06 16 s ribosomal RNA gene with accession no.ON386277.1.Lastly, Bacillus altitudinis strain SAUBD-B3 (Accession no.OQ740287.1)showed 99.83% identity to the Bacillus altitudinis strain LY43 16 s ribosomal RNA gene with accession no.

It has been observed that isolated
MZ067888.1.According to Pangastuti (2006), the identity values exceeding 97% demonstrate that the species discovered in the samples is not a novel species and is more identical to those found in the GenBank.

| Analysis of phylogenetic tree
The phylogenetic analysis was performed using genetic sequences of B. subtilis, B. velezensis, and B. altitudinis strains.The sequences were aligned, and a maximum likelihood phylogenetic tree was constructed using MEGA 11 software.The tree was rooted using an outgroup sequence and bootstraps values were calculated to assess the statistical support for the branching patterns (Figure 8).The identification of shared clades indicates a close evolutionary relationship between certain strains, supporting the notion that they belong to the same species.
Comparing these results with the findings of Srisamran

| CON CLUS ION
The study aimed to detect potential human pathogenic bacteria in ready-to-eat leafy greens sourced from diverse marketplaces

ACK N OWLED G M ENTS
The author would like to express his heartfelt gratitude and appreciation to the Ministry of Science and Technology, Government of the People's Republic of Bangladesh, for bestowing the prestigious

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I G U R E 1 MAP of the study area and locations of the samples collected sites in Dhaka metropolitan city.
(2.7 ± 0.91 × 10 9 CFU/g) where the supermarket and local market samples showed no statistically significant differences between them at CFU ∕ mL = No of colonies per plate × Total dilution factor Volume of culture per plate in mL CFU ∕ g = CFU ∕ mL × 10.Incidence of bacteria ( % ) = No. of samples contaminated by bacteria Total no. of samples × 100 F I G U R E 2 Mean colony-forming units of bacteria isolated from per gram of mint, lettuce, and coriander leaves.Columns with a similar uppercase letter showed no significant differences (F-test, p = .0603> .05)among the mean bacterial load (Tukey test, n = 9).U R E 3 Mean CFU of bacteria isolated from per gram of different marketplace samples of leafy greens.Columns with different uppercase letters showed significant differences (F-test, p = .0000< .05)among the mean bacterial load (Tukey test, n = 9).Different Markets Samples a 95% confidence level (Figure 3).These findings suggest that market type significantly influences microbial populations in the samples, possibly due to varying handling practices or environmental conditions.Compared with other research findings, Yafetto et al. (2019) reported a bacterial load of lettuce leaves (1.23 × 10 8 CFU/mL) in Cape Coast, Ghana.Houngla et al. (2019) studied selected leafy greens in Porto-Novo, Republic of Benin, and found the highest total coliform counts in lettuces (3.21 × 10 3 CFU/g).Nipa et al. (2011) observed viable bacterial colonies in coriander leaves ranging from 5.87 × 10 5 to 1.8 × 10 6 CFU/g and in peppermint leaves ranging from 2.2 × 10 5 to 7.7 × 10 5 CFU/g in Chittagong, Bangladesh.The current results indicate a very high frequency of bacteria in all leafy greens samples, highlighting the unhygienic conditions in Dhaka city's markets where it crosses the acceptable level of bacterial load (>10 6 CFU/g) for consumption of the leafy greens in raw condition according to the guidelines provided by Santoset al. (2005).These guidelines can serve as valuable references for ensuring food safety in the production and distribution of leafy green salads.
opportunistic pathogen that can induce diarrheal and emetic symptoms.Additionally, E. coli and Shigella spp.have been responsible for numerous human deaths worldwide (Inderbinen & Stephan, 2016; Tomás-Callejas et al., 2011).Mitov et al. (2010) highlighted that Pseudomonas aeruginosa is an opportunistic pathogen and one of the main bacteria causing nosocomial infections in hospitals, particularly affecting immunocompromised individuals.Furthermore, Zhang

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Cell morphology of the identified bacteria in gram staining test under a compound microscope (100×).presence of motile bacteria.Moreover, the starch hydrolysis and casein hydrolysis tests exhibited positive outcomes in 58% and 67% of the isolates, respectively, indicating the ability of certain bacteria to break down these substrates.The gelatin liquefaction test showed positive results in 50% of the isolates, suggesting the presence of gelatinase-producing bacteria.Interestingly, all isolates exhibited a positive reaction in the catalase test, indicating the presence of catalase-producing bacteria in every case.On the other hand, only 50% of the isolates displayed a positive result in the oxidase test, signifying the presence of oxidase-producing.The levan production test and citrate utilization test revealed positive results in 33% and 67% of the isolates, respectively, suggesting that certain bacteria could produce levan and utilize citrate as a carbon source.The findings of 10 biochemical tests are shown in (Table Gel electrophoresis of PCR productsThe Gel-Doc system of PCR products using primers 27F and 1492R resulted in the amplification of a DNA band with a size of approximately 1465 base pairs (bp).The obtained DNA band was very close in size to the target band, indicating successful PCR amplification.A Bench Top 1 kb DNA ladder was used as a size marker, confirming the size of the amplified DNA fragment.Lanes 1, 2, and 3, (Figure 7) represent the bacterial strains of B. subtilis, B. velezensis, and B. altitudinis, respectively, which exhibited the amplified DNA band.The results suggest that all 3 bacteria isolates contain the targeted DNA region, as indicated by the presence of the amplified band at the expected size.The Gel-Doc system confirms the specificity and successful amplification of the desired DNA fragment in the bacterial samples.
The resulting phylogenetic tree revealed the relationships among the studied strains.Notably, B. subtilis strain SAUBD-B1 and B. subtilis strain Cu31 were found to form a single clade with strong bootstrap support.Similarly, B. velezensis strain SAUBD-B2 and B. velezensis strain ND06; B. altitudinis strain SAUBD-B3 and B. altitudinis strain LY43 were also grouped together in a well-supported clade.
emphasize the variability in bacterial incidences in leafy greens across different studies and regions.Further research is essential to investigate the contributing factors behind these variations and to identify effective strategies to mitigate bacterial contamination in leafy greens.Implementing strict food safety measures and raising awareness among producers, vendors, and consumers about proper hygiene practices can play a crucial role in reducing the risk of bacterial contamination in leafy greens and ensuring food safety.

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Analysis of phylogenetic tree of Bacillus subtilis, Bacillus velezensis, and Bacillus altitudinis strains based on 16S rRNA sequences.The numbers at the branches are bootstrap confidence percentages from 1000 bootstrap trees.The Escherichia coli strain U 5/41 NR_0245701 was used as the outgroup.FI G U R E 9 Bar chart showing the incidence of identified bacterial genera in leafy greens.

Types of marketplaces Location no. Name of the marketplaces Moisture condition of samples
. A total of 30 isolates of bacteria were Pseudomonas aeruginosa in Cetrimide agar, Bacillus cereus in Bacillus Cereus Agar and Shigella spp. in SS Agar.Besides, molecular analysis was done and identified Bacillus subtilis, Bacillus velezensis, and Bacillus altitudinis.From this study, a total of 12 bacteria species were identified from these 9 genera isolated from collected leafy green samples of different markets (Table2).
Yu et al. (2019)20)separately reported the occurrence of E. coli, Bacillus sp., Pseudomonas sp., Shigella sp., Staphylococcus sp., Vibrio sp., Klebsiella sp., Neisseria sp., Enterobacter sp., and other bacteria in fresh fruits and leafy greens.In this study, the absence of certain bacteria like Salmonella spp., Listeria monocytogenes, and Campylobacter jejuni were identified by green samples which were B. cereus, E. coli, P. aeruginosa, K. pneumoniae, E. aerogenes, S. aureus, and Shigella spp.The presence of these pathogenic bacteria raises significant concerns about the potential risks they pose to public health.These seven bacteria were reported as human pathogenic bacteria in several pieces of literature.For example,Yu et al. (2019)reported that Bacillus cereus is a foodborne F I G U R E 4 Growth of pure cultures on NA plates of the identified bacterial genera.TA B L E 3 Cultural characteristics of identified bacteria on NA plates.
Results of bacterial 16S rRNA gene identification with BLAST Program.
Turki et al. (2020)also investigated the prevalence of pathogenic bacteria in leafy greens.Their study reported a 100% incidence of Staphylococcus, Bacillus, Enterobacter, Shigella, and Escherichia coli.Klebsiella and Salmonella also showed a high incidence at 92%, and Pseudomonas were found in 80% and 72% of the samples, respectively.Neisseria had a lower incidence of 12%.TA B L E 5