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Antibiotic-resistant bacteria contaminating leafy vegetables in Saudi Arabia’s eastern region

BMC Microbiology volume 24, Article number: 303 (2024) Cite this article

Abstract

Background

Food-associated antibiotic-resistant bacteria can cause infections that may critically impact human health. The objectives of this study were to determine the microbial contamination level of green leafy vegetables and their antibiotic resistance pattern.

Methods

Sixty-three samples of leafy vegetables were collected from Dammam Central Fruit and Vegetables Market from January to June 2023. The vegetables included lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium officinale). Samples were tested by standard microbiological techniques for identification and antibiotic susceptibility testing.

Result

Eight types of bacteria belonging to six different genera were detected. Enterobacteriaceae family was represented by four genera: Klebsiella, Proteus, Morganella, and Enterobacter. The other two genera were Pseudomonas and Aeromonas. Enterobacter cloacae was the most abundant organism, followed by Pseudomonas putida and Aeromonas sobria. On the other hand, Morganella morganii, Aeromonas hydrophila, and Proteus mirabilis were the least abundant. The three vegetable types had different levels of bacterial contamination. All isolated organisms were sensitive to penicillin, cephalosporin, aminoglycoside, and fluoroquinolone. However, Klebsiella oxytoca, M. morganii, and K. pneumonia showed resistance to ampicillin. A. hydrophila, Morganella morganii, and E. cloacae showed resistance to amoxicillin. M. morganii and E. cloacae were found to be resistant to cefalotin. Moreover, A. hydrophila, M. morganii, and E. cloacae were resistant to cefoxitin. Again, A. hydrophila was found to be resistant to imipenem. Only M. morganii was resistant to Ciprofloxacin. Two isolates, P. mirabilis and M. morganii were resistant to tigecycline. Another two, M. morganii and P. mirabilis were resistant to Nitrofurantoin. Only M. morganii was found to be resistant to trimethoprim.

Conclusion

This study aligns with the broad consensus in the literature about the significance of bacterial contamination in vegetables and the public health implications. The unique focus on antibiotic resistance patterns adds an essential dimension to the existing body of knowledge.

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Background

Vegetables are vital components of a balanced diet and are essential in maintaining good health and well-being. Because of their nutritional advantages, eating raw veggies has received widespread encouragement. The World Health Organization recommends that the minimal amount of fruit and vegetables required by an individual per day is at least 400 g [1]. However, the potential danger of acquiring several infectious diseases increases when fruits and vegetables are prepared and consumed uncleanly. Recent evidence suggests that human infections caused by plant-associated pathogenic bacteria may critically impact human health and safety [2]. Notably, the rise in fresh produce consumption has paralleled the increase in foodborne outbreaks.

Consuming uncooked or unwashed fruits and vegetables significantly increases the chance of disease transmission [3, 4]. The centralized production of fresh produce has resulted in widespread foodborne outbreaks that typically involve many cases [5].

Of the 1,400 microbial species that could potentially contaminate food, 58% are zoonotic [6], including infections that rank among the most significant pathogens to generate food safety concerns [1]. A considerable amount of worldwide morbidity has been brought on by enteric bacterial infections, especially in poor environmental sanitation and personal hygiene [7].

Vegetable contamination may be caused at various stages of production through multiple practices. Irrigation with inadequately treated wastewater is a major cause of contamination of grown vegetables [8]. The produce contamination may also be caused by animal wastes contaminated soil and inadequately composted manure [9].

In recent decades, there have been more instances of food-borne illnesses, which have been attributed mainly to increased vegetable consumption, centralized or global supply chains, better surveillance, and an increase in the number of vulnerable individuals [10].

Most commonly, fresh produce, with sprouting seeds, tomatoes, and leafy greens, has been linked to many high-profile outbreaks of foodborne disease [11]. Though Salmonella and E. coli O157:H7 are the pathogens of primary concern, fresh produce might theoretically be contaminated by a wide variety of pathogenic microorganisms at any stage of the chain. Evidence that certain pathogens are evolved to survive on various kinds of vegetables is being supported by an increasing amount of research. Accordingly, the prevailing bacterial phyla in leafy vegetable and melon and fruit vegetable soils were Proteobacteria, Acidobacteriota, Actinobacteriota, and Chloroflexi [12].

Leafy vegetables have a high potential for contamination at any stage in the production chain, making them a potential reservoir for various pathogens. Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae, Stenotrophomona maltophilia, and Citrobacter braakii have been isolated in a previous study [13]. Examples of other pathogens which have been previously recovered from vegetables included Aeromonas, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella, Shigella, Staphylococcus, and Vibrio cholera [10].

Among the green leafy vegetables, Chinese cabbage had the greatest contamination rate of 54.2%, followed by a rate of 13.5% and 5.2% for lettuce and cabbage, respectively [14].

The epidemiology of microbial contamination, its sources, and the methods by which fruits and vegetables are contaminated are still poorly understood. The importance of this work lies in its identification of potential risks associated with the consumption of raw vegetables as well as urging practical methods to lower the frequency of infections brought on by vegetable contamination.

Vegetable contamination by pathogenic bacteria has become a global health concern due to the emergence and rapid spread of antibiotic resistance in humans, animals, and the environment. Bacteria that colonize terrestrial plants’ leaves frequently appear in clusters that range in size from a few to hundreds of cells [15, 16]. Raw fresh vegetables can be a source of antimicrobial-resistant microorganisms and clinically significant antimicrobial resistance genes. According to previous investigations, vegetables may harbor the extended-spectrum beta-lactamase-producing Enterobacteriaceae, mcr1-positive E. coli, Pseudomonas aeruginosa resistant to carbapenem and colistin, staphylococci and enterococci resistant to linezolid, and enterococci resistant to vancomycin [16, 17]. Therefore, for the sake of quantitative risk assessment, knowledge concerning antibiotic-resistant bacteria of plant origin is vital. The study aimed to determine the level of contamination of green leafy vegetables and detect the resistance pattern of the contaminating pathogens.

Methods

Study area

A total of 63 samples of fresh green leafy vegetables were collected from the local market, Dammam Central Fruit and Vegetables Market during seven visits at three-week intervals from Jan 2023 to June 2023. Dammam City, the capital city of the Eastern Province of Saudi Arabia, is located between latitude 26°20′18″ to 26°32′51″ and longitude 49°49′54″ to 50°09′17″, with a population of 1,305,000, as of 2022. Dammam Central Vegetables Market is the largest one in the area and almost supplies the entire city and its metropolitan with a diverse variety of vegetables from various sources.

Sample collection

The samples included lettuce (Lactuca sativa), parsley (Petroselinum crispum), and watercress (Nasturtium officinale). These were selected because they are the most consumed vegetables in the study area as raw. All samples, about 500 mg of each type, were collected in sterile polythene bags, labeled, and transported in an insulated icebox before being transported to the laboratory where immediately processed at the Medical Microbiology Laboratory of Prince Sultan Military College of Health Sciences. All analyses were performed under sterile conditions to eliminate any potential sources of contamination.

Identification of the isolates and determination of antibiotic susceptibility

Bacterial pathogens were isolated and identified based on morphological, cultural, and different biochemical tests following standard protocol as described before [18]. Each vegetable sample was rinsed with 100 ml of distilled water, and a 1 ml aliquot of the resulting aqueous mixture was combined with 90 ml of Luria Bertani (LB) broth. The samples were then incubated overnight at 37 °C in the LB broth to promote bacterial growth. Following incubation one loop full of the LB broth was streaked onto non-selective and selective agar media and incubated overnight at 37 °C for growth and identification. Media used were Blood Agar (BA), MacConkey Agar (MA), Xylose Lysine Deoxycholate (XLD), and Salmonella-Shigella Agar (SSA).

After culture and preliminary identification through colony morphology and Gram stain, the isolates were tested for species identification, detection of extended-spectrum β-lactamase (ESBL), and antimicrobial susceptibility testing using the automated Vitek®2 Compact system (bioMérieux, Marcy L’etoile, France) according to the manufacturer’s instructions [19]. Gram-positive, GP cards and Gram-negative, GN cards containing different substrates were utilized for species identification. AST-P580 and AST-N291 were used for Gram-positive and Gram-negative antimicrobial susceptibility testing, respectively, using software version 06.01.

The isolates were tested for susceptibility for the following antimicrobial agents: amikacin, cefepime, ceftazidime, ciprofloxacin, gentamycin, imipenem, meropenem, tigecycline, trimethoprim, amoxicillin, ampicillin, cefalotin, cefoxitin, ceftriaxone, nitrofurantoin, and pipracillin. For the microbial identification and susceptibility tests E. coli ATCC 25,922 and K. pneumoniae ATCC 700,603 strains were tested as quality control strains with each cycle. The interpretation of the antimicrobial susceptibility testing results is based on the Clinical and Laboratory Standards Institute (CLSI) methods [20].

Statistical analysis

Descriptive statistical analysis was performed using Microsoft Excel.

Results

Bacterial contamination in different vegetables

Table 1 shows the isolated bacterial species and the percentage contamination in the different types of vegetables: Lettuce, Parsley, and Watercress. Eight spices of bacteria were isolated including Aeromonas sobria, Klebsiella oxytoca, Morganella morganii, Aeromonas hydrophila, Enterobacter cloacae, Pseudomonas putida, Klebsiella pneumonia, and Proteus mirabilis.

Table 1 Frequency and percentage of bacterial contamination in different types of vegetables collected from Dammam central vegetables market during January to June 2023 (n = 63)
Full size table

E. cloacae was the most abundant bacteria isolated from 33% of the total samples followed by 19.0 and 14.3% contamination rate of P. putida and A. sobria, respectively. M. morganii, A. hydrophila, and P. mirabilis were the least abundant, represented by a contamination rate of 4.8%.

Parsley vegetable represented the most contamination rate of 71.4% of the total samples while lettuce and watercress had an equal contamination rate of 57.1%. The analysis of bacterial species present in the vegetables revealed varied contamination levels.

Antimicrobial resistance of bacterial isolates

Table 2 presents the sensitivity data of various bacteria to a range of antibiotics. All isolated organisms were sensitive to penicillin (piperacillin), cephalosporin (ceftazidime and cefepime), aminoglycoside (amikacin and gentamicin), and fluoroquinolone (ciprofloxacin). Ten bacterial isolates were resistant to cefoxitin while seven and six isolates were resistant to amoxicillin and cefalotin, respectively. Five isolates were resistant to each of ampicillin and imipenem. Four isolates were resistant to trimethoprim. Only two bacterial isolates were resistant to each of Piperacillin, ceftazidime, cefepime, amikacin and gentamicin.

Table 2 Antibiotic resistance pattern of isolated bacteria from vegetables of the Dammam central vegetables market during January to June 2023
Full size table

M. morganii, the most antibiotic-resistant bacteria, was resistant to seven antibiotics including aminopenicillins cephalosporin, glycylcycline, and nitrofuran, and diaminopyrimidines. E. cloacae was resistant to three antibiotics. P. putida, and A. sobria were sensitive to all antibiotics. K. oxytoca, M. morganii, and K. pneumonia showed resistance to ampicillin. A. hydrophila, M. morganii, and E. cloacae showed resistance to amoxicillin. M. morganii and E. cloacae were found to be resistant to cefalotin. Moreover, A. hydrophila, M. morganii, and E. cloacae were resistant to cefoxitin. In addition, A. hydrophila was found to be resistant to imipenem while A. sobriae was partially resistant to it. Only M. morganii was resistant to Ciprofloxacin. Two isolates, P. mirabilis and M. morganii were resistant to tigecycline. Another two, M. morganii and P. mirabilis were resistant to Nitrofurantoin. Only M. morganii was found to be resistant to trimethoprim.

Discussion

Fresh produce consumption has increased because of the desire for healthy and being quickly and easily prepared. Thus vegetables are becoming vital components of a balanced diet and have a prominent position in the food pyramid [21].

Eight types of bacteria from six different genera were detected in green leafy vegetables. Four of these genera belong to the family Enterobacteriaceae namely: Klebsiella, Proteus, Morganella, and Enterobacter. Pseudomonas and Aeromonas made up the other two genera.

E. cloacae, the mostly encountered bacteria is gram-negative pathogens, endotoxin-producing, fermentative, catalase-positive, oxidase- and DNAase-negative [5]. E. cloacae are environmental pathogens that usually cause nosocomial pneumonias, postsurgical peritonitis, bloodstream and urinary tract infections outbreaks and have been frequently isolated in human clinical specimens [5, 22]. The contamination rate of E. cloacae in this study was 33.3%. Similarly, other studies have reported Enterobacter spp. in fresh vegetables, more importantly, is the high levels of E. cloacae detected in raw eaten green leafy vegetables, such as parsley, lettuce and chard [23]. While the majority of E. cloacae complex isolates are susceptible to aminoglycosides, cephalosporin, carbapenems, and fluoroquinolone, they are intrinsically resistant to cefalotin, cefoxitin, and amoxicillin antibiotics because they produce constitutive AmpC b-lactamase [5].

Pseudomonas putida was the second most encountered bacteria in this study with a contamination rate of 19.0%. P. putida is a gram-negative, rod-shaped, saprophytic soil environmental bacterium capable of causing nosocomial infections including ventilator associated pneumonia and catheter infections in immunocompromised patients [24].

P. putida is well known for spoiling fresh produce in cold storage, and it has more recently been linked to diseases in medical facilities [25]. Horizontal genetic transfer may be used to acquire and spread antibiotic resistance genes, raising the risk of their persistence in food [26].

Clinical isolates of P. putida demonstrated minimal resistance to most antibiotics in earlier investigations. Resistance rates of 20.2% and 21.7% were reported for levofloxacin and ciprofloxacin, respectively [27]. Another study showed that its resistance rates to trimethoprim/sulfamethoxazole were up to 90%, and quinolones and cefoperazone/sulbactam were > 50%; however, P. putida has a high susceptible rate to amikacin (86.4) [28]. However, P. putida maintained a higher sensitivity to imipenem and amikacin than other antimicrobials [28].

The genus Aeromonas comprises ubiquitous emerging pathogens known to play several roles in the environment. Aeromonas Sobria, the third prevalent bacteria in this study with a contamination rate of 14.3%, is a gram-negative heterotrophic facultative anaerobic bacteria found in warm climates that cause human and animal diseases through contamination of water, seafood, meat, and vegetables [29]. A. Sobria is a causative agent of a variety of illnesses, including gastrointestinal infections and extra-intestinal localizations in open wounds, hepatobiliary system, or eyes [30]. Aeromonads were isolated from 26% of the vegetable samples [31]. Although the clinical isolates of A. Sobria was resistant to antibiotics is a huge challenge for the existence of a plasmid encoding β-lactamase [32]; the environmental strain of the current study was resistant only to the carbapenem, and imipenem.

Enterobacteriaceae Klebsiella oxytoca and Klebsiella pneumoniae were encountered at a contamination rate of 9.5% each. Enterobacter and Klebsiella species were isolated from fresh vegetables at higher contamination rates with 43% extended-spectrum β-lactamase producers, 24% AmpC, and 20% carbapenemase producers [33]. The human commensal and opportunistic K. oxytoca is a gram-negative bacterium reported as a pathogen that causes several illnesses, including bacteremia, antibiotic-associated hemorrhagic colitis, and urinary tract infections [34]. To mediate low-level resistance to quinolones, K. oxytoca possesses several intrinsic antimicrobial resistance genes, such as the beta-lactamase-encoding blaOXA−2 and efflux pump-encoding oqxAB [35].

The current study revealed that K. oxytoca was resistant only to amoxicillin and imipenem. K. oxytoca complex had respective antibiotic resistance rates of 1.8%, 12.5%, 7.1%, 0.8%, and 0.1% to carbapenems, ceftriaxone, ciprofloxacin, colistin, and tigecycline [34]. Over the past seven years, the rates of nonsusceptibility to cephalosporins and carbapenems have grown [34].

K. pneumoniae is a significant Enterobacteriaceae and one of the opportunistic pathogens causing broad spectra of diseases such as urinary tract infections, cystitis, pneumonia, surgical wound infections, endocarditis, and septicemia [36]. The prevalence rate of drug resistance in K. pneumoniae was; amikacin (40.8%), aztreonam (73.3%), ceftazidime (75.7%), ciprofloxacin (59.8%), colistin (2.9%), cefotaxime (79.2%), cefepime (72.6) and imipenem (65.6%). However, the current study has shown that K. pneumoniae was resistant only to aminopenicillins, ampicillin, and amoxicillin.

Morganella morganii, Aeromonas hydrophila, and Proteus mirabilis were the least encountered at a contamination rate of 4.8% each. M. morganii is a gram-negative bacterial pathogen that causes bacteremia, urinary tract infections, intra-abdominal infections, chorioamnionitis, neonatal sepsis, and newborn meningitis [37]. Although M. morganii has a wide distribution, it is considered an uncommon cause of community-acquired infection, and it is most often encountered in postoperative and other nosocomial infections, such as urinary tract infections [38]. P. mirabilis is a gram-negative bacillus with urease activity that frequently causes catheter-related urinary tract infections [39].

The rod-shaped gram-negative bacterium A. hydrophila can be found in food, drinking water, and sewage (35). It is a developing pathogen that can cause skin infections, gastroenteritis, bacteremia, meningitis, hemolytic uremic syndrome, necrotizing fasciitis, and other systemic illnesses. It is regarded as an emerging pathogen that can cause gastroenteritis, skin infections, hemolytic uremic syndrome, peritonitis, bacteremia, meningitis, cholera-like sickness, necrotizing fasciitis, and other more systemic illnesses [40].

When comparing the different types of vegetables and the corresponding resistance patterns, similar levels of resistance, intermediate susceptibility, and susceptibility were noted across the vegetable types.

A diverse range of microorganisms can naturally colonize fresh raw produce. The analysis of bacterial species associated with vegetables revealed varied contamination levels. Gram-negative bacteria including Klebsiella spp., Enterobacter cloacae, Citrobacter freundii, and Aeromonas hydrophila were detected among the leafy vegetable contaminants. Another study in South Africa indicated a contamination rate of 17.4% of the vegetable samples analyzed with presumptive ESBL/AmpC-producing Enterobacteriaceae dominated by Escherichia coli, Enterobacter cloacae, Enterobacter asburiae, and Klebsiella pneumonia .

Similar to a previous finding, none of the examined samples contained Bacillus cereus, Salmonella, and Escherichia coli O157:H7 [33].

Staphylococcus aureus, Escherichia coli, and Shigella, the frequently reported in other studies were not encountered in this study [41, 42].This could be explained by the fact that we collected produce from the central market, which gets it straight from the farms as these bacteria can be transferred from people’s hands to food.

The current study aligns with the broad consensus in the literature about the significance of bacterial contamination in vegetables and the public health implications of such contamination. Raw vegetables are closely related to the soil, so they usually have a high natural contamination and a high risk of contamination from irrigation water [43]. The isolated pathogenic bacteria genera from the irrigation water and fresh vegetable samples include Citrobacter, Enterobacter, Escherichia, Klebsiella, Pseudomonas, and Salmonella [44]. leafy vegetables are often contaminated with a large microbial population [31, 45].

According to a recent study, P. mirabilis and K. pneumonia accounted for the majority of the 26.81% ESBL-producing infection prevalence among clinical samples in Saudi Arabia [46]. Clinical isolates from the study area indicated antimicrobial resistance patterns that included extended-spectrum beta-lactamase (30.13%), carbapenemase-resistant Enterobacter (1.94%), and vancomycin-resistant Enterococci (0.18%) [47].

Conclusion

The unique focus on antibiotic resistance patterns adds an essential dimension to the existing body of knowledge. Moreover, considering the influences of the production and distribution process would make future studies more holistic. Vegetable washing after harvest is still the most effective way to eliminate contaminants from the field. The comparative effectiveness of various sanitizers, such as hypochlorite, chlorine dioxide, and peroxyacetic acid, has been the subject of several articles and reviews [48].

This study aligns with the broad consensus in the literature about the significance of bacterial contamination in vegetables and the public health implications. The unique focus on antibiotic resistance patterns adds an essential dimension to the existing body of knowledge. Moreover, considering the influences of the production and distribution process would make future studies more holistic.

Our study revealed knowledge gaps that need to be filled in order to fully comprehend the extent of the hazards to the public’s health posed by the spread of bacteria resistant to antibiotics through leafy vegetables. The sampling strategy and the protocols for pathogen identification represent the study’s main limitations.

Data availability

Not applicable (this manuscript does not report data generation or analysis).

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Acknowledgements

We thank Ms. Bidayah Micunog for helping in the processing of the specimens at the microbiology laboratory. We thank Dr. Tasneem Elsafi for reading the manuscript.

Funding

This study received no funding.

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Authors and Affiliations

  1. Clinical Laboratory Sciences, Prince Sultan Military College of Health Sciences, P.O. Box 33048, Dammam, 31448, Saudi Arabia

    Salah H. Elsafi, Raneem F. Al Zaid, Shahad A. Alshagifi, Taif A. Farghal, Khlood B. Alshamuse, Aseel S. Albalawi, Faisal Alkhalaf, Amr A. Sumaily, Saleh Almusabi & Siju K. George

  2. Physical Therapy Department, Prince Sultan Military College of Health Sciences, Dhahran, Saudi Arabia

    Eidan M. Al Zahrani

Authors
  1. Salah H. Elsafi
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  2. Eidan M. Al Zahrani
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  3. Raneem F. Al Zaid
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  4. Shahad A. Alshagifi
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  5. Taif A. Farghal
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Contributions

SHE did the Investigation, data curation, and writing - EME Did the conceptualization, methodology, and writing. RA, ShA. TF, KA, AA, and FA performed the sampling collection and methodology, AAS performed the data analysis, review, and editing. SA data analysis, writing - review and editing, and visualization. SG contributed to writing - the original draft, review, and editing.

Corresponding author

Correspondence to Salah H. Elsafi.

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Elsafi, S.H., Al Zahrani, E.M., Al Zaid, R.F. et al. Antibiotic-resistant bacteria contaminating leafy vegetables in Saudi Arabia’s eastern region. BMC Microbiol 24, 303 (2024). https://doi.org/10.1186/s12866-024-03456-5

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Keywords

BMC Microbiology

ISSN: 1471-2180

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