A Study on the Assessment of the Antimicrobial Susceptibility Pattern of Microorganisms Present in Yogurt
POONAM JAISWAL, ALOK KUMAR SRIVASTAV Department of Microbiology, Dr. A.P.J. Abdul Kalam University,
Indore, Madhya Pradesh, India.
[For Correspondence: [email protected]]
ABSTRACT
The aim of our study is to assess the antibiotic resistance among commercially available probiotic lactobacilli. Those probiotics which are commercially available are generally considered as safe for humans but for their high consumption there is a need to put sufficient safeguards to protect the consumers from any adverse effects. The safety of these probiotic strains are becoming essential with antibiotic resistance as an upcoming issue and their potentiality to transfer antibiotic resistance genes to pathogenic/commensally bacteria cannot be neglected.
All the Lactobacillus isolates are characterized in vitro for their probiotic characteristics and antimicrobial activities against pathogens. The isolates will be resistant to NaCl (1–6%), bile salts (0.5–3%) and show good growth in the acidic conditions, while maximum growth will be observed at pH around 6.0. All the isolates will be susceptible to clinical antibiotics; also the isolates will exhibit effective aggregation and hydrophobicity studies. Based on the results, selected Lactobacillus isolates will be considered as novel and potential probiotic bacteria. Thus, further extensive research on isolation and characterization of probiotic bacteria from local dairy product and their growth optimization may be necessary for development of probiotic enriched food supplement and human health benefits through prevention and control of bacterial infections.
The starter cultures generally belongs to the lactic acid bacteria group (LAB) and are considered to be safe by the US Food and Drug Administration (FDA). But, it may have role in intrinsic and extrinsic reservoirs for antibiotic resistance gene (AR). This rises the concern regarding the safety of the product as resistance gene can transfer vertically.
Nevertheless, external genetic elements may induce changes that favor the horizontal transfer of resistance gene from pathogens as well as from the human intestinal micro biota, which represents a severe safety issue. Some genus of AR LAB includes Enterococcus, Lactobacillus, Lactococcus, and Streptococcus isolated from fermented probiotic yogurt.
The present study revealed about the prevalence of antibiotic resistance in various species of probiotic strains, which may impose a concern of food safety. Therefore, antibiotic sensitivity must be considered as a crucial part of safety assessment for the evaluations of probiotics.
Keywords: Lactic Acid Bacteria, Antibiotic Resistance Transfer, Probiotics, Cell Free Supernatant, Antibacterial Activity.
INTRODUCTION
The term “probiotics” was introduced in 1953 by Werner kollath. As a contrast to antibiotics, probiotics are defined as factors derived from microbial cells that can stimulate the growth of other microorganism. In 1989, Roy fuller suggested a definition of probiotics which has been used on a large extent as: “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”.
Probiotics are living, health-promoting microorganisms that are incorporated into various kinds of foods. A number of human studies have clearly demonstrated that yogurt containing viable bacteria Streptococcus thermophillusand Lactobacillus delbrueckii spp. Bulgaricuscan
improve digestion of lactose and also can eliminate the symptoms of lactose intolerance. The probiotic bacteria isa component of “thermophilic” starter cultures which can be used in products that are commercially available today. They belong to members of the genera Lactobacillus and Bifidobacterium. They have the ability to withstand the bactericidal properties of bile salts, acidic environment of gastric juices and also can produce lactic acid which can inhibit the growth of other microorganisms, allowing themselves to establish in the intestinal tract.
Probiotics produce a wide range of Antimicrobial metabolites, i.e., organic acid, diacetyl, acetoin, hydrogen peroxide and bacteriocins. These activities contribute to microbiological safety by controlling the growth of other microorganisms and inhibition of pathogenic bacteria.
Lactobacilli represent a significant part of our intestinal micro flora and their role in the general state of human health is being seriously investigated. The genus Lactobacillus is one of the major groups of lactic acid bacteria that have an application in food fermentation and hence are economically significant. The various strains of L. acidophilus or closely related species Lactobacilluscasai, L. paracasei subsp. paracasei and subsp. Tolerans and Lactobacillus rhamnosusare being increasingly used in novel yogurts.
Figure 1: Effect of Probiotics on Pathogens
The taxonomy of Lactobacilli group has undergone significant changes in recent years causing confusion. A number of studies have been conducted for the identification and classification of LAB including conventional biochemical tests, such as carbohydrate fermentation patterns using commercially available kits, physiological tests as well as more complex molecular-biology methods. Recently, Bio-log and API Kit are used on a large scale for identification of LAB. Various cultural media have been designed, developed and evaluated for the differential calculation of probiotic LAB in fermented milk and yogurt.
Generally, Lactobacilli are not associated with any disease and are considered as non- pathogenic members of urogenital and intestinal flora. They are supposed to maintain the gastrointestinal ecosystem in a healthy state through antagonistic interactions with pathogenic bacteria. The regulatory process is carried out by species belonging to Lactobacillus which produce antibacterial compounds, such as organic acids (example lactic acid), bacteriocins and hydrogen peroxide (H2O2). Bacteriocins are low-molecular weight proteins or peptides which are biologically active and can inhibit the growth of a variety of pathogenic bacteria.
Probiotics are live microorganism which when administered in adequate amounts confer health benefit in the host. The main probiotic bacteria are strains belonging to the genera lactobacillus. Lactobacilli are common inhabitants’ of the human intestine micro biota also, some species used in food fermentation processed starters, or as adjunct cultures in the food industry. One of the most important selection criteria for bacterial strains intended use in the food industry concern for their safety. In developed countries such as Europe, an approach has been established by EFSA (European Food Safety Authority) to determine the nature of
Homolactic
Heterolactic
any antibiotic resistance determinant present in a candidate microorganism prior to approval for QPS (Qualified Presumption of Safety) status.
Therefore, the aim of the present investigation is to identify the potential Lactobacilli from genotypic and phenotypic methods in order to optimize their growth using incubation methods such as pour plate and spread plate, under cultivation conditions like anaerobic and aerobic. Focus has been given to determine the in-vitro characteristics of probiotics such as antibiotic susceptibility profile, antimicrobial activity, aggregation studies, NaCl tolerance, pH, bile and cell-surface hydrophobicity capacity of selected probiotic Lactobacillus spp.
established with pathogenic strains and growth control.
MORPHOLOGY OF LACTIC ACID BACTERIA
The term lactic acid bacteria refers to a taxonomically diverse group of gram positives bacteria, facultative anaerobic bacteria, non-spore forming, non-motile and acid-tolerant The shapes may vary from cocci, coccobacili or rods which appear as single cells or forming couples, tetrads, or long chains. LAB species are found in two phyla, the firmicutes and the Actinobacteria for the first genus, aerococcus, alloiococcus, carnobacterium, enterococcus, lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, Vegococcus, and Weissella that are low G+C (31-49%) belong to the Bacilli Class and the Lactobacillus order.
While, the Bifidobacterium genus with a high content G+C (58-61%) belongs to the Actinobacteria phylum.
Figure 2: Best Probiotic Strains Documented Till Date TYPES OF FERMENTATION IN LAB
HOMOLACTIC FERMENTATION
Homolactic LAB catabolize one mole of glucose in the Embden-Mayerhof pathway to yield two moles of pyruvate. Intracellular redox balanced is maintained through the oxidation of NADH concomitants with pyruvate reduction to lactic acid. This process yield two moles of ATP per glucose consumed. Representative Homolactic LAB genera include
Lactobacillus
Lactococcus
Enterococcus and
Streptococcus species
Transport of free glucose and phosphorylation by an ATP dependent hexose kinase.
The phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS), in which PEP is the phosphoryl donor for the uptake of sugar.
Strains That Represent Homolactic Fermentation are as follows:
The transport and phosphorylation of sugars occur by:
Figure 3: The Pathway of Homolactic Acid Fermentation in Lactic Acid Bacteria (LAB) HETEROLACTIC FERMENTATION
Heterofermentative LAB utilizes the phosphoketolase pathway (pentose phosphate pathway) to dissimilate sugars. One mole of glucose-6-phosphate is initially dehydrogenated to 6-
Obligate Heterofermentative
Leuconostoc
Oeonococcus
Weissella
Lactobacilli
phosphogluconate and subsequently decarboxylated to yield of one mole of CO2. The resulting pentose-5-phosphate is cleaved into one mole GAP (glyceraldehydes phosphate) and one mole acetyl phosphate. GAP is again metabolized to lactate as in homofermentation and acetyl phosphate is reduced to ethanol via acetyl-CoA.
Strains That Represent Heterolactic Fermentation are as follows:
Figure 4: The Pathway of Heterolactic Acid Fermentation in Lactic Acid Bacteria Lactobacilli
In addition LAB is capable to produce antimicrobial metabolites such as lactic and acetic acid, ethanol, hydrogen peroxide, diacetyl, antifungal (short-chain fatty acids derived from lipolysis reactions), and antimicrobial peptides known as bacteriocins, and other antibacterial
MODULATE INFLAMMATION INDUCES ANTIBIOTIC THERAPY
TREATMENT OF DIARRHEA IMPROVE IMMUNE FUNCTION
REDUCTION IN BLOOD PRESSURE
proteins like peptidoglycan hydrolases (PGH) capable to cleave the peptidoglycan cell wall of gram-positive and gram-negative bacteria.
POTENTIAL EFFECTS OF PROBIOTICS
Experiments into the potential effects for probiotics, including the molecular biology and genomics of lactobacillus in immune function, cancer, and antibiotic-associated diarrhea, travelers’ diarrhea, pediatric diarrhea, inflammatory bowl diseases, and irritable bowel syndrome.
CHART 1: Potential Effects of Probiotics
TREATMENT OF DIARRHEA
In preliminary research some probiotics have been found to treat various forms of gastroenteritis. They might reduce both the duration of illness and frequency of stools.
Fermented milk products (such as yogurt) also reduce the duration of symptoms.
INDUCES ANTIBIOTIC THERAPY
Antibiotic-associated diarrhea results from an imbalance in the colonic microbiota caused by antibiotic therapy. Microbiota alteration changes carbohydrate metabolism with decreased short-chain fatty acid absorption and an osmotic diarrhea as a result. The consequence of the antibiotic therapy causing diarrhea is due to overgrowth of potentially pathogenic organisms such as Clostridium difficile.
From investigations madein laboratory it has been demonstrated that few strains of LAB (Lactobacillus bulgaricus) have anti-mutagenic effects due to their ability to bind with heterocyclic amines, which are carcinogenic substance formed in cooked meat. Most human trials have found that the strains tested may exert anti-carcinogenic effects by decreasing the activity of an enzyme called β-glucuronidase (which can generate carcinogens in the digestive system). Lower rates of colon cancer among higher consumers of fermented dairy products have been observed in one population study, but confirmation of such an effect does not exist.
Physiological state of the added probiotic in the food
Physiochemical conditions of food processing
Physical conditions of product storage, like temperature
Chemical composition of the product, such as content of nutrients, oxygen or pH
REDUCTION IN BLOOD PRESSURE
Though not a confirmed effect, few studies have revealed that consumption of fermented milk with different strains of LAB may lead to reductions in blood pressure, and possibly cause an effect related to the ACE inhibitor-like peptides produced during fermentation.
IMPROVE IMMUNE FUNCTION
LAB may affect pathogens by means of competitive inhibition and there are evidence that suggests that immune function may be improved by an increase in the number of IgA- producing plasma cells, improving phagocytosis or by increasing the proportion of T lymphocytes and Natural Killer cells. Clinically it has been demonstrated that probiotics can reduce the occurence of respiratory tract infections and dental caries in children.
MODULATE INFLAMMATION
LAB may modulate inflammatory and hypersensitivity responses, an observation thought to be at least part due to the regulation of cytokine function. Clinical studies have suggested that they are able to prevent the reoccurrence of inflammatory bowel disease in adults and improve milk allergies. They are not effective for treating eczema, a persistent skin inflammation.
A LIST OF FACTORS AFFECTING VIABILITY IN FOODS
Both intrinsic and extrinsic factors, can influence the survival of probiotics in food, and have been considered in all stages of manufacturing process of probiotic food:
TEMPERATURE
The temperature at which probiotics organisms grow is an important factor in food applications where fermentation is required, is also a critical factor influencing probiotics survival during manufacture and storage. As it is told above, the lower the temperature the more stable probiotic viability in the food product will be.
pH
Some bacteria like lactobacilli and bifidobacteria can tolerate lower pH levels because produce organic acid and products from carbohydrate metabolism. In fermented milk and yogurts with pH values between 3.7 and 4.3. Lactobacilli are able to grow and survive, while Bifidobacteria tend to be less acid tolerant, with most species surviving poorly in fermented products at pH levels below 4.6.
WATER ACTIVITY
For quiescent probiotic bacteria water activity is a crucial determinant of survival in food products during storage. Higher the moisture levels and water activity, lower is the survival of probiotics.
OXYGEN
Both Bifidobacteria and Lactobacilli are considered strict anaerobes and oxygen can be detrimental to its growth and survival. However, the degree of oxygen sensitivity varies considerably between different species and strains, for example, Lactobacilli, which are mostly microaerophilic, are more tolerant of oxygen than bifidobacteria, to the point where oxygen levels are not an important consideration in maintaining the survival of Lactobacilli. Most probiotic bifidobacteria do not grow well in the presence of oxygen, although, many bifidobacteria have enzymatic mechanisms to limit the oxygen toxicity.
TOXICITY OF INGREDIENTS
Interaction between probiotics and other ingredients could happen and those interactions can be protective, neutral, or detrimental to probiotic stability. Obviously, the inclusion of antimicrobial preservatives can inhibit probiotics survival and elevated levels of ingredients such as salt, organic acid, and nitrates can inhibit probiotics during storage, while starter cultures can sometimes inhibit the growth of probiotics during fermentation through the production of specific bacteriocins.
SIDE-EFFECTS
In situations, such as if the person who is consuming probiotics is critically ill, probiotics could be harmful. In a clinical trial of therapeutics, conducted by the Dutch pancreatitis study group, has revealed that the consumption of a mixture of six probiotics bacteria increases the death rate of patients with predicted severe acute pancreatitis.
It has been reported by some hospitals that Lactobacillus septicemia, which is a potentially fatal disease is caused in people by the consumption of probiotics who have lower immune systems or those who are already very ill. There is no such published evidence that supplements consisting of probiotics can replace the body’s natural flora when these have been killed off. But, indeed bacteria levels in feces have been found to disappear within days when supplementation ceases.
REGULATION OF THE USE OF PROBIOTICS
Probiotics are defined by the food and agriculture organization of the world health organization as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.
1. The term Probiotic can be subdivided into various forms such as Probiotic drugs, Probiotic foods, designer Probiotics (genetically modified Probiotics) and Probiotics for animal use.
2. In the United States, probiotic products are marketed to healthy population in the form foods or dietary supplements.
3. Recently, there is an increase in the incidence and severity of infection from Clostridium difficile (CDI) which have led the clinicians to consider the use of probiotics as “drug” either alone or in combination with traditional antimicrobial agents for the prevention of CDI.
According to the food and drug administration (FDA) definition, a drug is defined as a substance recognized by an official pharmacopoeia that is intended for use in the diagnosis, cure, mitigation, treatment, or prevention of diseases.
e
N
d
h
r
p Identificati
In vitro
Substanti
trials ation of the clinical healt benefit of probiotic agents with hum an otic effect of the probi
mechanism testing to d lineate the
genotypic test henotypic and
the strain by on of the genus and species of
Patterns of antimicrobial drug resistance
Metabolic activities
Side effects noted in humans during clinical trials
Toxin production and hemolytic potential if the probiotic strain is known to possess those properties
Lack of infectivity in animal studies
EVALUATION OF ANTIMICROBIAL PROPERTIES FROM PROBIOTICS
CHART 2: The Proposed Guidelines to Evaluate Antimicrobial Properties From Probiotics
Additionally, safety assessment of the probiotic strain should be at a minimum to determine:
MATERIALS AND METHODS
Lactobacillus is selectively grown on Elliker medium. The recipe for preparation of elliker broth for 1 liter of distilled water is as follows in Table 1.
Table 1: Recipe For Elliker Medium in 1Liter of Distilled Water Serial Number Serial umber
Component
G/Litre
01. Trypton 20
02. Yeast Extract 0.5
03. Sodium Acetate 1.5
04. Dextrose 5.0
05. Ascorbic Acid 0.05
06. Gelatin 2.5
07. So ium Chlo ide 4.0
100 ml Elliker broth was prepared for the isolation of lactobacillus. The broth was kept in test tubes. Common yogurt samples were taken from the local farms of Indore. The sample was kept for one day in a closed tube in order to obtain whey. 1ml of the sample was mixed in the first test tube of the broth, and then serially diluted up to 4th test tube. Test tubes containing broth and sample were then placed in an incubator. The tubes were kept for 48 hours. Then 50 μl of the sample was spread over the elliker medium.
The culture plates that were spread were kept in incubator for 24 hours. Visible growth of lactobacilli strain was being observed. The plates were then further streaked in order to obtain pure colony and also kept in incubator for 48 hours. Pure colony of lactobacillus was obtained which have been confirmed by applying gram staining and biochemical tests such as catalase test, oxidase test.
ANTIBIOTIC SUSCEPTIBILITY TEST
After getting the isolated strain, the antibiotics were applied to the strain.10 ug of each antibiotic (Penicillin, Streptomycin and Norfloxacin) were applied to the isolated strain. The discs were placed in accordance of disc diffusion method. The plates were then kept in incubator for 48 hours to find out the results.The same procedure was repeated for the rest of the two samples taken from separate locations.
Table 2: Antibiotic Suceptibilty Chart of Lactobacillus Serial
Number
Antibiotic Concentration
Resistance Intermediate Sensitive
Sample 1 Norfloxacin 10 ug <12 14-16 >17 Sample 2 Penicillin 10 ug <11 11-21 >22 Sample 3 Streptomycin10ug <12 15-17 >18
Sample Details. (Sample #01: From Rajwada Indore) (Sample #02: FromVijaynagar, Indore) (Sample#03: From Mhow Indore)
RESULTS AND DISCUSSIONS
Table 3: Antibiotic Susceptibility Pattern
Serial No. Antibiotic Resistant Intermediate Sensitive
01. Norfloxacin 0 0 3
Penicillin 2 1 0
02.
03. Streptomycin 1 2 1
All of the three isolates from different samples were tested against the action of the following antibiotics (Norfloxacin, Penicillin, Streptomycin).
Norfloxacin was found to be most effective against the three isolates of lactobacillus isolated from yogurt samples. All of the three isolates were sensitive to the action of Norfloxacin.
Streptomycin had an intermediate activity. Two of the three strains were found to be intermediate sensitive against the action of streptomycin. While one was sensitive.
Penicillin had the lowest activity against all the three isolated strains of lactobacillus. Only one out of three was found to be sensitive against the action of penicillin and remaining of the two were found to be resistant. The resistance of lactobacillus against the action of penicillin is considered to be due to horizontal gene transfer between bacteria and excessive
Figure 1: Zone of Inhibition of Antibiotics Table Table 4: Results of a Single Isolate
FUTURE PROSPECTS
The application of probiotic LAB and research has received great attention for many years resulting in the development of various food products that possess diverse health benefits.
This review has highlighted some of the areas where these beneficial bacterial strains have been applied, thus increasing the prospects of their use now and in the nearest future. Anti- obesity and anti-diabetic effects of LAB have been suggested in some of the studies reviewed. Their use as functional starter cultures in dairy products like yoghurt and ice cream show promising outcomes in the nearest future. Some more concerted in vivo studies may greatly lower current ethical, cultural and religious bottlenecks to probiotic lactic acid bacteria research. The government and various research institutes have investigated the mechanisms behind the effects of probiotic LABs to increase their application. Modern techniques such as microencapsulation are needed for fine-tuning to optimize the survival of large numbers of viable probiotic LAB cells. Bioinformatics of probiotic LAB strains need further studies as well.
CONCLUSION
An intriguing phenomenon noted in many previously cited cases of lactobacillemia was the therapeutic difficulty in eradicating this organism from deep-seated infected foci, especially the endocardium, despite seemingly appropriate treatment regimens and serum antibiotic concentrations exceeding the minimal inhibitory concentration (MICs). Few strains of oral lactobacilli developed resistance considerably when grown in maximal concentrations of
Serial No. Unit Zone of Inhibition Antibiotic Disc
01. 10μg 17mm Streptomycin
10μg 21 mm Norfloxacin
02.
03. 10μg 11 mm Penicillin
penicillin; but others did not. But in contrast, none of the strains which have been tested developed resistance upon prolonged growth in the initial concentration of penicillin to which they were resistant. The high resistance of the lactobacilli has been found to be penicillin was not maintained indefinitely without continued cultivation in the presence of a maximal concentration of antibiotics. The decrease in resistance of the lactobacilli to penicillin during cultivation in the absence of the antibiotic was gradual and such strains did not completely recover their former level of susceptibility to the antibiotic during the period of experimentation. The rate of antibiotic resistant bacteria has increased lately where resistance among bacteria common to the human GI tract is of no exception. Drug resistance presents a health concerning issue, especially if resistance is pointed towards clinical antibiotics and when genetic elements carrying resistance genes are mobile and as such possibly transmitted to health harming microbes. As it is well known, LAB naturally shows both antibiotic resistance and susceptibility and since they have a long history of safe use with no indications of transfer resistance to other.
LABS are of great importance in the food industry for the preparation of fermented foods and also as probiotics to regulate the intestinal microbiota in animals and humans. However, it is important to carry out the appropriate test to identify the presence of antibiotic resistance genes. Whether pathogenic or those present in the gastrointestinal microbiota, which can cause a health problem because of the continuous exposure to the environmental conditions that favor the resistance spread to the public health.
Lactobacillus is generally considered as a harmless bacteria. However association of lactobacillus with certain gastro intestinal diseases and the utilization of it as an adjunct suggest that its antibiotic susceptibility and resistance should be checked. In this experiment lactobacillus was exposed to the action of three antibiotics namely, Penicillin, streptomycin, and Norfloxacin. Norfloxacin has the highest activity streptomycin has the intermediate activity while penicillin has the lowest activity among these three antibiotics.
ACKNOWLEDGEMENTS
I express my deepest gratitude to my Research Supervisor, Dr. Alok Kumar Srivastav, Dr.
A.P.J. Abdul Kalam University, Indore for his never ending guidance and direction through valuable suggestions along with enthusiastic encouragement through-out the period of my work and preparation of this research paper.
Also I am extremely thankful to Dr. Priyanka Das, Dr. A.P.J. Abdul Kalam University, Indore for her guidance and cooperation.
I pay tribute to My Parents for lifting me up till this phase of life. I thank them for their love, trust, patience, support and bearing all kind of stress to make me what I am.
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