Identifying Antibiotic Resistant Bacteria in Curd with Plating & PCR

Introduction

Production of curd by using Bacterial Probiotics

Acidification of milk is one of the methods used in preservation of milk. Acidification, gives the milk a sour taste and it is achieved by bacterial fermentation or by the addition of an acid (for example: lemon juice or vinegar). The acid causes milk to coagulate and solidify fully or partially. Coagulation of milk inhibit the growth of harmful bacteria and improves the dairy product's shelf life. Soured milk that is produced by bacterial fermentation is more specifically called fermented milk or cultured milk. Curd is one of the famous dairy product made by Lactic Acid fermentation of milk. Cow’s milk or buffalo milk is used for the production of cured. Curd is stored in clay pots or plastic containers in the local market. As we all know metal containers are not used to store curd. Because acidic substances in curd can react with metals. It contaminates and make curd inconsumable.

Probiotics present in Curd

Curd is considered as probiotic or functional food as it possesses live lactic acid bacteria. Probiotics are live microorganisms which are harmless and beneficial to health. The Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO) defined a probiotic as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’ (FAO/WHO, 2002). The definition of probiotics changes together with the development of knowledge about them. Before considering a bacterium as a probiotic, it must encounter several conditions, including the ability: To survive in the presence of acids and bile salts, to produce antimicrobial compounds, and to colonize the intestines and resist antibiotics. Some probiotics which have been already isolated from buffalo milk in India are Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus lactis and Bifidobacterium longum; from milk in Islamabad are L. acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. cremoris, L. Lactis ssp. lactis, and Streptococcus thermophilus.

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Lactic Acid Bacteria (LAB)

In the production of curd specific type of probiotic bacteria known as lactic acid bacteria(LAB) are added to milk. LAB are a heterogeneous group of bacteria which can be found naturally in gastrointestinal and urogenital tracts of humans and animals. Lactic acid is produced as a result breakdown of carbohydrates by these bacteria therefore these bacterias are known as LAB. Besides lactic acid, other bi products such as acetate, ethanol, CO2, formate and succinate are also formed by these bacterias. This lactic acid lead to coagulation of milk proteins such as casein by giving a sour taste thereby producing cultured milk products such as curd.

LAB is classified into homofermentative and heterofermentative according to the end products of their glucose metabolism. The homofermentative converts glucose mainly into lactic acid by the Embden-Meyerhof pathway, while the heterofermentative transforms glucose into lactic acid, carbon dioxide, and ethanol or acetic acid by the 6-phosphogluconate pathway.

Lactic acid bacteria are not only used in food production but also in pharmaceutical and special dietary applications. The most commonly used strains of different LAB species in food including the genera of Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus and Bifidobacterium A typical lactic acid bacterium is Gram-positive, non-spore forming, catalase negative, facultatively anaerobe requiring complex media, acid tolerant, fermentative and it lacks cytochromes and produces lactic acid as the major end product.

These bacterias enhances the digestibility of milk. Therefore, fermented dairy products such as curd and yogurt can be used as a substitute for the people with lactose intolerance since a large part of the milk sugar has already been digested by the lactic acid bacteria in the curdling process. As far as the microbial picture of the curd is concerned, according to the Sri Lankan Standards (SLS) regulations recommend that the curd shall be the fermented milk products obtained from coagulation of cow or buffalo milk or a mixture by the involvement of following species of organisms ; Streptococcus lactis, S. diacetylactis, S. cremoris singly or in combination with Leuconostoc spp., Laciobacillaus bulgaricus, Sireptcoccus thermophiles.

Commercial preparation of curd

Traditionally, soured milk was simply fresh milk that was left to ferment and sour by keeping it in a warm place for a day, often near a stove. Modern commercial production methods of fermented dairy products are differing from milk that has become sour naturally. Since milk can get contaminated by other types of bacteria when it left to ferment naturally. Therefore, aseptic conditions and inoculation of specific bacteria from starter culture methods are used at the present day. A starter culture is a microbial preparation with large numbers of bacterial cells of at least one microorganism to be added to a raw material to produce a fermented food by accelerating and directing its process of fermentation. Typical method for the preparation of curd is mentioned below.

Standardized milk (milk product, whose fat and/or solids-not-fat content have been adjusted to a certain pre-determined level. Under the PFA Rules) was heated to 850C for 30 minutes and cooled to 400C. The milk samples were inoculated individually at 1% level with a specific species of LAB from a starter culture sample under the aseptic condition to avoid contamination. All samples were incubated at 370C for 48 hours(this time can be vary according to the starter culture used) for activation of culture. Steps followed in the production of curd is shown in the (Figure 1) flow chart below.

Antibiotic resistant bacteria present in curd

Antibiotic resistance has become a major issue in medical field nowadays. Antibiotics which are used to feed animals (eventually get in to animal manure) and used in starter cultures contribute to additional resistance.

Most starter cultures of lactic acid bacteria have been recognized as safe by the US Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA). An assessment system to analyse the pre-market safety of selected groups of microorganisms also has been proposed by EFSA. Yet, since LAB consist of antibiotic resistant (AR) genes they can act as intrinsic or extrinsic reservoirs for antibiotic resistance (AR) genes. Even though the resistance gene transfer is vertical, the external genetic factors may promote the horizontal transfer of AR genes through the food chain to pathogens (via bacteriophages). Although at present, the WHO recommends that LAB which are used in the food industry should be free of resistance, some genus of AR LAB species such as Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus have been isolated from several fermented meat and milk products.

LAB possess a high probability of developing resistance to antibiotics. Recent studies have questioned the safety of some strains of Lactococcus lactis, Enterococci, and Lactobacillus isolated from fermented foods which showed resistance to tetracycline, erythromycin, and vancomycin.

With regard to specific antibiotics, lactobacilli are typically sensitive to the cell wall-targeting Beta-lactam antibiotics such as penicillin and to protein synthesis inhibitors such as chloramphenicol, macrolides, lincosamide, tetracycline. Beta-lactam antibiotics consist a beta-lactam ring in their chemical structure. This class includes Penicillins, cephalosporins, Carbapenems, Monobactams, Beta-lactamase inhibitors. The beta-lactam antibiotics function is to destroy bacterial by interfering with the structural crosslinking of peptidoglycans in bacterial cell walls. Production of extended-spectrum β-lactamases enzymes (ESBL) by bacteria inhibits the effect of Beta-lactam antibiotics by breaking the antibiotics' structure through hydrolysis.

But they are more resistant to cephalosporins (which is a beta-lactam antibiotic) and vancomycin. And also LAB are generally resistance toward inhibitors of DNA synthesis such as trimethoprim, sulfonamides and fluoroquinolones. Resistance to streptomycin, clindamycin, gentamicin, oxacillin and lincosamide is also reported in some of these species. Most mutual antibiotic resistant genes found in lactobacilli are the tetracycline resistance genes. However, resistance and susceptibility are greatly varying with the different LAB species.

Health benefits of consuming curd

Curd is associated with several health benefits due to its probiotic content. One of the beneficial effects of curd are assembly of the natural antibacterial components such as nisin and acidophilin formed during the fermentation (therapeutic benefits) which has the ability to restoration of normal gut flora following oral therapy. Further it balances the faecal enzymes and intestinal micro flora, treatment of traveler’s diarrhoea, antibiotic therapy, and control of ulcer and reduction of serum cholesterol. The low pH or acidic condition that prevents the growth of putrefactive and other pathogenic organisms and possesses an increased digestibility are some other benefits of the consumption of curd. Several experimental observations have indicated a potential effect of lactic acid bacteria against the development of colon tumours.

Adverse effects of consuming curd

Sometimes antibiotic resistance bacteria can be present in curd. Consumption of curd with such bacteria may lead to antibiotic resistance as mentioned above. Other than that the major adverse effects of consumption of curd are caused by consuming contaminated curd. Since curd is a high nutritious medium many fastidious bacterias are prone to grow on curd. Curd can get contaminated by using contaminated milk, from a contaminated starter culture, in the manufacturing process (loose packing), in storing process and even by consumers. foodborne pathogens can get in to milk by several influences such as size of the farm, number of animals on the farm, hygiene management practices, variation in sample collecting methods (by hand or by machines), geographical location, and season. Milk pasteurization is regarded as an effective method to eliminate foodborne pathogens. Yet pathogens such as Listeria monocytogenes can survive and thrive even after pasteurization.

S. aureus, B. cereus, Klebsiella and coliforms like bacterias have been isolated from different curd samples around the world. According to the type of bacteria which has contaminated the curd the adverse health effects can be varied.

Isolation and identification of Antibiotic resistant bacteria present in curd.

In the process of isolation and identification of Antibiotic resistant bacteria present in curd following steps should be performed respectively.

• Collection of curd samples
• Serial dilution of samples
• Plating on a media
• Antibiotic resistant test
• DNA extraction
• Performing Polymerase Chain Reaction (PCR)
• Electrophoresis
• DNA Sequencing and identification of bacteria (using NCBI- BLAST).

Collection of curd samples

There are several types of curd served in different methods in the local market. After collecting samples and separating small amounts from them to culture bottles or sterile containers serial dilution is performed.

Serial dilution of samples

In serial dilution known amount of sample is diluted with sterile water and they are pipetted to several test tubes and diluted further by increasing sterile water amount to reduce the concentration of microbes. The objective of the serial dilution method is to determine the concentration of an unknown sample by counting the number of colonies cultured from serial dilutions of the sample, and then back track the measured counts to the unknown concentration.

Plating techniques

There are several plating techniques. streak-plating, pour-plating, spread-plating are some examples for plating methods. The streak-plate procedure has been designed to isolate pure cultures of bacteria, or colonies, from mixed populations by simple mechanical separation. Pour plate method often is used to count the number of microorganisms in a mixed sample, which is added to a molten agar medium before it gets solidified. Spread plate technique is usually used to separate microorganisms confined within a small sample volume, which is spread over the surface of an agar plate. Colonies formed after plating is then subjected to antibiotic sensitivity test (ABST).

Brain Heart Infusion Broth (BHI), Man Rogosa Sharpe (MRS) agar and Luria-Bertani (LB) are some different types of medias which are used in plating. MRS is the most common broth used to culture LAB since it is a selective culture medium for Lactobacilli.

Antibiotic sensitivity test (ABST)

There are several types of Antibiotic sensitivity tests done under the principles of diffusion (qualitative methods) and dilution (quantitative methods). Kirby-Bauer disk diffusion method has been used to determine antibiotic susceptibility of some Turkish fermented dairy products. Disc diffusion method was used to screen for the antibiotic susceptibility of Lactic Acid Bacteria isolated from several Foods and Drugs in china.

Minimum inhibitory concentration (MIC) of various antimicrobial susceptibility testing are categorized by various international agencies. These MIC guidelines determine the susceptibility of organisms to particular antibiotic used.

DNA extraction

Then DNA is extracted from the bacteria obtained from antibiotic resistant colonies. According to the method of cell membrane lysis there are several methods of DNA extraction. They chemical, enzymatic and mechanical membrane lysis. In most of the methods enzymes or chemicals are used for the lysis of bacterial cell membranes (Cell wall, cell membrane and nuclear membrane). SDS, CTAB, phenol, Tris and EDTA are several common chemicals used in the solution based DNA extraction method and extraction buffers are used in enzymatic lysis.

Polymerase chain reaction

Then PCR is done to multiply obtained DNA samples or fragments. It can be LAB specific ESBCL (Extended-spectrum beta-lactamase enzymes producing bacteria which are resistant to beta lactam antibiotics) specific or universal PCR.

Electrophoresis and Identification

Then agarose gel electrophoresis is performed. If the electrophoresis results do not show a bright band again (universal) PCR is performed. Finally, the identified bacteria strands are sequenced and their specific part of genome is identified by using NCBI-BLAST.

Conclusion

Curd is very popular among Sri Lankans due to its taste and nutritional value. Curd is a fermented dairy product which can be found in almost all the super markets in Sri Lanka. Lactic Acid Bacteria is the main group of probiotic bacteria which is used to produce curd. Lactic Acid Bacteria contain antibiotic resistant genes in their genome. Antibiotic resistant bacteria contain Antibiotic resistant genes in their genome. Usually these antibiotic resistant genes transfer vertically they can be transferred horizontally by viruses (such as bacteriophage virus) through food chains and infections. Since these genes can promote antibiotic resistance presently, the WHO recommends microorganisms used in the food industry should be free of antibiotic resistant genes. The main aim of this study is to find the antibiotic resistant bacteria present in curd samples of Sri Lanka by using Plating and PCR detection. Other than Lactic acid producing bacterias there can be other bacterias grow due to contamination of curd. This research helps to discover those bacterias also.