Special Issue Nepal J Biotechnol. 2 0 2 0 O c t ; 8 (2): 69-75 DOI: https://doi.org/10.3126/njb.v8i2.31893 Research article ©NJB, BSN 69 Comparative Assessment of Antibiotic Resistance in Lactic Acid Bacteria Isolated from Healthy Human Adult and Infant Feces Rasika Pawar1 , Vasudeo Zambare2 , Bela Nabar1 1Department of Microbiology, Smt Chandibai Himathmal Mansukhani College, Ulhasnagar, Thane, Maharashtra, India 2Department of Life Sciences, School of Science, Sandip University, Nashik, Maharashtra, India Article history:- Received: 20 Jun 2020; Revised: 23 Sep 2020; Accepted: 30 Sep 2020; Published online: 22 Oct 2020 Abstract Lactic acid bacteria are normal inhabitants of the gastrointestinal tract of humans. Their occurrence in infant and adult feces is abundant. The current study assesses and compares the antibiotic resistance in lactic acid bacteria isolated from healthy human adult and healthy infant fecal samples. A total of 255 lactic acid bacteria isolates (126 from adult feces and 129 from infant feces) were isolated and characterized from 60 fecal samples. Lactobacillus spp., Pediococcus spp. and Enterococcus spp. were included in the study. The study was done using the WHONET software for the analysis of antibiotic susceptibility data of lactic acid bacteria. Most of the Lactobacillus and Pediococcus strains were sensitive to vancomycin. Enterococcus strains showed resistance against vancomycin. Ampicillin, ciprofloxacin and cefuroxime resistance were significantly (p<0.05) higher in Lactobacillus strains isolated from adult fecal samples than those isolated from infant fecal samples. A similar pattern was observed in Enterococcus strains with erythromycin, gentamycin and tobramycin resistance. Pediococcal isolates from adult feces showed significantly higher resistance against tobramycin, ciprofloxacin, gentamycin, cefotaxime and cefuroxime in comparison with infant fecal isolates. Antibiotic resistance was exhibited by lactic acid bacteria against most commonly used antibiotics and it was higher in strains isolated from adult fecal samples than in the strains isolated from infant fecal samples. The increasing trend in antibiotic resistance from infant to adult might be due to food habits and antibiotic intakes. Thus, the widespread antibiotic resistance in different lactic acid bacteriamay pose a food safety concern as well. Keywords: Lactic acid bacteria, Antibiotic resistance, Lactobacillus, Feces, Fecal microbes Corresponding author, email: belanabar23@gmail.com Introduction The lactic acid bacteria (LAB) originate from a taxonomically diverse group of microorganisms, which are non-sporing rods and cocci, usually non- motile that ferment carbohydrates and form lactic acid. Lactic acid bacteria contain the genera namely Lactobacillus, Lactococcus, Pediococcus, Streptococcus, Enterococcus, Oenococcus, Leuconostoc, Carnobacterium, Vagococcus, Tetragenococcus, and Weissella [1]. The microflora of humans and animal gut is complex and it is primarily dominated by lactic acid bacteria. There is high density and rich diversity of microorganisms in the gut, and the microflora complexity increases from the upper gastrointestinal tract to the colon [2]. The human gut contains more than a thousand bacterial species and some of them start to colonize the gut during infancy [3]. Soon after the birth of a newborn infant, the gut flora begins to develop and microbes start to colonize the small intestine and large intestine. Aerobic and facultative anaerobic bacteria (Enterobacteria, Enterococci and Streptococci) are the early colonizers in the human gut. After they colonize, they create anaerobic environment in the gut. This helps anaerobic bacteria (Bifidobacteria, Bacteroides and Clostridia) to start with their colonization majorly in the large intestine [4]. The development of complex, diverse and stable microflora continues from infancy to one year of age. After a year it is similar to adults and it is stable [4]. Many factors are governing the development, diversity, composition and colonization gut microflora of infants, out of which mother’s gut microflora, food and environment are the deciding ones [5]. During birth, an infant is exposed to the mother’s vaginal microflora and also to fecal microflora, and with this exposure colonization of the gut in infants begins [6]. Infant gut microflora is affected by colostrum and later by breast milk. After the introduction of formula and solid foods, Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 Journal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 mailto:belanabar23@gmail.com https://orcid.org/0000-0003-3739-3752 https://orcid.org/0000-0001-9064-8289 https://orcid.org/0000-0002-4370-3510 mailto:belanabar23@gmail.com mailto:belanabar23@gmail.com Nepal J Biotechnol. 2 0 2 0 O c t ; 8 (2) [Special Issue]: 69-75 Pawar et al. ©NJB, BSN 70 complexity and diversity is generated in the gut microflora of infants. Microbes present in the environment and those present directly on the skin of the infant also enter the gut and create a complex niche [7]. Colonization of the gut with diverse microflora creates continuous impacts on the immune system; and in this process, it strengthens the immune system [8]. Over the past few decades, there has been a huge interest developed in LAB physiology and genetics, involving their increasing importance as starter cultures in different industrial fermentation processes and also as probiotics. Since probiotics are directly administered in humans and animals it is very necessary to determine the level of antibiotic resistance. This is a part of the assessment of the safety of the probiotic cultures which are administered as therapeutics. In the past 60 years, approximately 10 million tons of antibiotics have been utilized and released into the environment. As presented in the reports of European Commission there is a huge probability of the spread of antibiotic resistance in the biosphere [9]. Hence,there is a very strong selective pressure in the development of antibiotic resistance in bacterial strains [10]. Lactic acid bacteria dominate the gastrointestinal tract of humans. They are present in large amounts in the gut and are also added or sometimes additionally consumed along with the regular diet. Hence, it is speculated that the presence of antibiotic resistance in lactic acid bacteria used as probiotics can be dangerous. Probiotics are generally administered to maintain microbial balance during gastrointestinal tract infections such as diarrhea. They are administered as therapeutic agents along with antibiotics. If probiotics harbor antibiotic- resistant genes, it could be beneficial in sustaining the antibiotics during the treatment; however, there is a risk of antibiotic-resistant probiotic strains to transfer the resistance genes to the pathogenic bacteria. This could complicate the treatment of a patient with an antibiotic-resistant bacterial infection or disease. Additionally, there is the possibility of the transfer of antibiotic resistance from beneficial lactic acid bacteria, in the food chain. Therapy with any antibiotic, particularly long term and especially oral administration is liable to alter the balance of antibiotic-resistant to sensitive organisms in the intestine [11]. Certain strains of these genera are more commonly used in the food and especially dairy industries or as probiotics [12]. The World Health Organization (WHO) has established a program known as the Antimicrobial Resistance Monitoring (ARM) program for monitoring antimicrobial resistance. WHO has also devised an electronic format WHONET, freely available to download. A special focus of antimicrobial susceptibility test results is available on windows-based database software, developed for the management and analysis of microbiology data [13]. This study aimed to determine the antibiotic susceptibility of lactic acid bacteria (using WHONET software) isolated from adult and infant feces to various groups of antibacterial agents that are mainly isolated from the feces of breastfed infants. Also, the comparative assessment was done to determine the isolates that are more resistant to antibiotics. Materials and Methods Sample collection and ethics statement Thirty healthy adult human volunteers (from Mumbai and Suburbs, India) aged between 25 and 30, who were not suffering from any chronic disease, had not taken antibiotics, proton pump inhibitors, bismuth compounds, Histamine H2-receptor, nonsteroidal anti-inflammatory drugs within the previous 6 months, were selected for the study. Similarly, fecal samples were also collected from thirty healthy infants aged between 3 months to 9 months. Infants who were exclusively breast-fed, healthy and free from acute or chronic disease were selected in the study. The study protocol was approved by an independent ethical committee and performed in compliance with the US Code of Federal Regulations on Good Clinical Practices (21 CFR 10.90, 50, 56 and 812) and the World Medical Association Declaration of Helsinki (1996 amendment) [14]. All adult volunteers and parents of infants signed informed consent before samples were collected. Isolation of lactic acid bacteria from the fecal sample Fecal samples were collected in sterile polypropylene containers and processed immediately as follows. A 0.5 g portion of feces was taken from mid sample, added in 4.5 ml of sterile Nepal J Biotechnol. 2 0 2 0 O c t ; 8 (2) [Special Issue]: 69-75 Pawar et al. ©NJB, BSN 71 saline solution, and completely homogenized. A dilution series (10–1 to 10–7) was made and 100 µl aliquots of each dilution were inoculated on the agar plates by spread plating. Rogosa SL agar (Hi- Media, Mumbai, India) was used to isolate LAB and the plates were incubated micro-aerobically for 3 days at 37°C. Kenner fecal (KF) agar was used for the isolation of Enterococcus and incubated aerobically at 37°C for 24 h [15]. Enumeration and selection of bacterial isolates After incubation, the plates that showed discrete colonies were selected and the colonies were counted. The total count of Lactic acid bacteria in feces was expressed as colony-forming units/g (wet weight). From each fecal sample, 10-20 colonies of LAB were randomly selected. A provisional identification of genera was made based on Gram’s staining, and catalase reaction using 3% (v/v) H2O2 on single colonies. Putative Lactobacilli colonies (Gram-positive, catalase test-negative, rod-shaped) were chosen and further purified using MRS agar. Similarly, putative colonies of Enterococci and Pediococci (Gram-positive, catalase test-negative, cocci, able to grow at 10oC and 45oC, and in 18% NaCl and at pH 4.4) from KF agar plates were purified by re-streaking on the MRS agar. The cultures were stored in MRS broth with 15% glycerol at –20°C [15]. Antibiotic resistance The antibiotic resistance/susceptibility patterns of isolated strains of lactic acid bacteria were studied using the Kirby-Bauer disk diffusion method (according to the CLSI document M2-A9 suggestions) [16]. The antibiotics used in this study were penicillin (10 µg), ampicillin (10 µg), vancomycin (30 µg), cefuroxime (30 µg), cefotaxime (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), tobramycin (10 µg), erythromycin (15 µg) and chloramphenicol (30 µg). The culture densities were adjusted to McFarland 1.5; they were spread on MRS agar plates. Antibiotic discs (Hi-Media, Mumbai, India) were placed on the surface of the agar plates, which were incubated at 37°C for 24 h. The diameters of the clearance zones around the discs were measured and the result (the average of 2 readings) was expressed as susceptible, intermediate, or resistant according to the standard disc diffusion method [16]. The experiment was done in triplicates. Microsoft Excel (2013) was used to obtain data in the appropriate format for BacLink 2019, used to format data to be used in WHONET 2019, which automatically calculates the % resistance using a data analysis tool. Statistical analysis The data was analyzed to check the significant difference between groups using Student’s T-test with a probability level of 0.05 (P < 0.05) using Microsoft Excel (2013). Results Isolation of lactic acid bacteria from the fecal sample A total of 255 LAB isolates were isolated from 30 human adult and 30 human infant fecal samples. Out of the 255 isolates, 126 isolates were from the adult fecal sample, and 129 from the infant fecal sample, the results are presented in Table 1. The isolates were identified phenotypically and characterized. Based on the characters, the LAB isolates were characterized as mesophilic homofermentative cocci, able to grow at 10oC and 45oC as Enterococcus (81 isolates). Homofermentative cocci in tetrads, unable to grow in 18% NaCl, and showing growth at pH 4.4 were characterized as Pediococcus (84 isolates). Lactobacilli (90 isolates) were represented as catalase-negative, slender gram-positive rods. All strains grew at 4oC and 6.5% NaCl concentration. Antibiotic resistance of lactic acid bacteria Data of diameter of zone of clearance in mm of LAB isolated from adult and infant feces was entered in Microsoft Excel and via BacLink software incorporated into WHONET software (Table 2). Table 1. Count of LAB isolates in the adult and infant fecal samples. Sample source Number of samples (n = 60) LAB isolates (n = 255) Lactobacillus spp. (n=90) Pediococcus spp. (n=84) Enterococcus spp. (n=81) Adult feces 30 126 46 41 39 Infant feces 30 129 44 43 42 *LAB= Lactic Acid Bacteria Nepal J Biotechnol. 2 0 2 0 O c t ; 8 (2) [Special Issue]: 69-75 Pawar et al. ©NJB, BSN 72 Figure 1. Antibiotic resistance pattern of Pediococcus spp. (a), Lactobacillus spp. (b) and Enterococcus spp. (c) isolated from adults and infant feces, respectively [AMP- Ampicillin, CHL-Chloramphenicol, CIP-Ciprofloxacin, CTX-Cefotaxime, CXM-Cefuroxime, ERY-Erythromycin, GEN-Gentamicin, PEN-Penicillin G, TOB-Tobramycin, VAN-Vancomycin]. All experiments were performed in triplicates and the error bar represents the standard deviation of independent performs experiments (n=3). Pediococcus spp. isolated from adult feces was comparatively more resistant to antibiotics than those isolated from infant feces. Significantly higher resistances (P < 0.05) were found against ampicillin (7.3%), cefotaxime (22.0%), cefuroxime (36.6%), penicillin (12.2%) gentamycin (26.8%), erythromycin (19.5%), tobramycin (29.3%) and ciprofloxacin (26.8%) from isolates from adult feces than those isolated from infant feces, 7.0%, 9.3%, 0.0%, 11.6%, 9.3%, 0.0%, 2.3% and 20.9% respectively (Figure 1a). All the isolates from both adult and infant samples were sensitive to vancomycin and chloramphenicol. Pediococcus spp. were intrinsically resistant to high levels of glycopeptides and penicillin. Resistance to erythromycin was also reported and was due to a plasmid with an erythromycin resistance methylase B [erm(B)] gene [17]. Discussion To develop probiotics for human or animal consumption, it is necessary to distinguish strains harboring antibiotic resistance genes from other strains because of potential risk for the dissemination of resistance genes. In this study, it was demonstrated that strains isolated from infants were more sensitive than those isolated from adult feces. Lactobacilli and Pediococciare widely used as probiotics and promoters for biological growth. Lactobacilli are reported to be resistant to several antibiotics [18]. In the present study, Lactobacillus spp. isolated from adult feces were more resistant to antibiotics than those isolated from infant feces. Significantly higher resistance was found against cefuroxime (26.1%) and ciprofloxacin (32.6%) from isolates from adult feces than those isolated from infant feces, 4.5%, and 2.3% respectively. Lactobacillus spp. isolated from feces also showed moderate resistance to cefotaxime (13.0%), penicillin (10.9%), chloramphenicol, (10.9%), gentamycin (23.9%), erythromycin (13.0%) and tobramycin (26.1%). Whereas those isolated from infant feces showed comparatively lesser resistance 4.5%, 11.4%, 9.1%, 20.5% and 9.1%, respectively (Figure 1b). Resistance to gentamycin and ciprofloxacin was earlier documented [19, 20]. Concerning cell wall synthesis inhibitors, Lactobacilli are reported to be resistant to oxacillin and cephalosporins (cefoxitin and ceftriaxone) [21]. They were also found to show resistance to aminoglycosides (neomycin, kanamycin, 0 10 20 30 40 50 % R e si st a n t Antibiotics [a] Pediococcus spp. Adult 0 10 20 30 40 50 % R e si st a n t Antibiotics [b] Lactobacillus spp. Adult Infant 0 10 20 30 40 50 % R e si s ta n t Antibiotics [c] Enterococcus spp. Adul t Nepal J Biotechnol. 2 0 2 0 O c t ; 8 (2) [Special Issue]: 69-75 Pawar et al. ©NJB, BSN 73 streptomycin, and gentamicin) [22]. There are many species of Lactobacilli which contain intrinsic resistance to vancomycin, erythromycin and tetracycline. The matter of concern is that since Lactobacilli are added to infant food, they can act as reservoirs of antibiotic resistance genes, which could be transferable [23]. Enterococcus spp. also followed a similar pattern where the antibiotic resistance associated with adult fecal samples was higher than those isolated from infant feces. Adult fecal isolates were 30.8% resistant to erythromycin, 20.5% resistant to tobramycin, and 41% resistant to gentamycin. This was significantly higher (P < 0.05) than infant fecal isolates, which were sensitive to erythromycin, 9.5% resistant to tobramycin, and 4.8% to gentamycin. Higher resistance was also found against vancomycin (30.8%), ciprofloxacin (33.3%), ampicillin (28.2%), cefuroxime (20.5%) and cefotaxime (28.2%); however, it was not statistically significant in comparison to infant fecal isolates which showed 11.9%, 9.5%, 21.4%, 9.8% and 26.2% resistance against above-mentioned antibiotics, respectively. All the isolates (fecal and adult) were susceptible to chloramphenicol. Infant isolates were 11.9% resistant to penicillin; this was higher than adult isolates, which showed 10.3% resistance (Figure 1c). Enterococci showed intrinsic and acquired resistance against many antibiotics [24, 25]. Such intrinsic resistance was reported inlincosamides, nalidixic acid penicillin, polymyxins, quinupristin– dalfopristin, monobactams, and low levels of aminoglycosides. Resistance to high levels of aminoglycosides, high levels of trimethoprim, and high levels of clindamycin, chloramphenicol, tetracyclines, penicillins (due to β- lactamase), fluoroquinolones, macrolides (e.g. erythromycin), glycopeptides and oxazolidinones (linezolid) were acquired [26-27]. Acquired resistance is a major threat in treatment, such a trait was found to be transferred to other Enterococci in the gut [28]. Vancomycin resistance is especially important as vancomycin is the last drug option for treating diseases caused by multidrug resistance Enterococci [29]. Apart from probiotic use, Pediococci are also widely used for the fermentation of meat and vegetables and also in cheese production [30]. According to the EFSA’s FEEDAP Panel [31] (European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed), the bacterial cultures which are used for the production of animal feed should be susceptible to antibiotics used in treating humans bacterial infections. Hence, it is extremely necessary to distinguish antibiotic susceptible and resistant strains. This also emphasizes the importance of safe source or niche of a selection of strains used as probiotics. The results of the study indicate that infant feces could be a better source for isolation of LAB cultures intended to be used as probiotics. Apart from being used traditionally as starter cultures in dairy products, LAB are also used for the production of animal feed. They also belong to normal flora of the human gut and confer health benefits to the host. During the process of food manufacturing and passage of food through gut, there is a possibility of antibiotic resistance, carried by LAB getting transferred to human pathogenic bacteria [32]. Hence, it is imperative to select strains Table 2. Percent antibiotic resistance in target microorganisms isolated from adult and infant fecal samples. Expressed in percentage (%) Mechanism of Action Antibiotic Lactobacillus spp. Pediococcus spp. Enterococcus spp. Adult Infant Adult Infant Adult Infant Cell Wall Inhibitors Ampicillin 10.9 11.4 7.3 7.0 28.2 21.4 Cefotaxime 13.0 4.5 22.0 9.3 28.2 26.2 Cefuroxime 26.1 4.5 36.6 0.0 20.5 9.8 Penicillin 10.9 11.4 12.2 11.6 10.3 11.9 Vancomycin 0.0 0.0 0.0 0.0 30.8 11.9 Protein Synthesis Inhibitor Chloramphenico l 10.9 9.1 0.0 0.0 0.0 0.0 Erythromycin 13.0 9.1 19.5 0.0 30.8 0.0 Gentamycin 23.9 20.5 26.8 9.3 41.0 4.8 Tobramycin 26.1 6.8 29.3 2.3 20.5 9.5 DNA Synthesis Inhibitor Ciprofloxacin 32.6 2.3 26.8 20.9 33.3 9.5 Nepal J Biotechnol. 2 0 2 0 O c t ; 8 (2) [Special Issue]: 69-75 Pawar et al. ©NJB, BSN 74 that have low resistance against antibiotics for human and animal use. From the results of the antibiotic susceptibility in the current study, obtained from a broad range of antibiotics, it was found that the isolated strains of Lactobacillus, Pediococcus and Enterococcus were resistant to various antibiotics. However, antibiotic resistance was lesser in strains obtained from infant fecal samples than adult fecal samples. Conclusion Lactobacillus, Pediococcus and Enterococcus as LAB were isolated from the human fecal samples exhibiting more antibiotic resistance from adult fecal isolate than the infant. The development of antibiotic resistancein LAB can be attributed to the long term exposure of antibiotic as therapeutic agents as well as food habits which pose food safety concerns. Thus, it is essential to see safety measure during antibiotic uptake in day to day life. In addition to this, the low antibiotic-resistant strains from infant could be the choice of strain to avoid the risk of transferof LAB linked antibiotic resistance to human pathogenic bacteria. Authors Contribution RP has made a substantial contribution to data analysis and its interpretation. VZ contributed in designing the experiments. BN contributed to data interpretation and all authors RP, VZ and BN contributed equally to drafting and reviewing of the manuscript followed by final approval from BN. Competing Interests No competing interests were disclosed. Funding The author(s) declared that no grants were involved in supporting this work. Acknowledgements RSP is thankful to Principal, Smt CHM College and VZ is thankful to Sandip University for providing the laboratory facilities and chemicals. 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