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ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 3  |  Page : 263-268

Susceptibility of Cronobacter sakazakii to plant products, antibiotics, and to lactic acid bacteria


Department of Zoology, Environmental Biotechnology Laboratory, Dayalbagh Educational Institute, Dayalbagh Agra, Uttar Pradesh, India

Date of Submission06-Oct-2012
Date of Acceptance23-Nov-2012
Date of Web Publication10-Jul-2013

Correspondence Address:
Alka Prakash
Department of Zoology, Environmental Biotechnology Laboratory, Dayalbagh Educational Institute, Dayalbagh Agra - 282 110, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.114847

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   Abstract 

Introduction: Cronobacter sakazakii is an emerging and opportunistic pathogen, related with outbreaks of necrotizing enterocolitis, bacteremia, and infant meningitis. Powdered infant formula (PIF)-products contaminated by C. sakazakii cause severe infections in infants. Materials and Methods: In the present study, C. sakazakii isolates were tested for their susceptibility to various antibiotics using the Kirby-Bauer disk diffusion method. Alternatively, C. sakazakii infections can be controlled by various plant products and probiotics. Aqueous and alcoholic extracts of Citrus sinensis, Cinnamomum zeylanicum, Piper chaba, Terminalia chebula, Terminalia arjuna, Mangifera indica, Zingiber officinale, Allium sativum, Azadirachta indica, Ocimum sanctum, Syzygium aromaticum, Terminalia bellirica and Emblica officinalis were prepared and their antimicrobial activities were checked against C. sakazakii by the Muller-Hinton Agar well diffusion method. For probiotic control, an in vitro study was conducted to determine the antibacterial activity of lactic acid bacteria against C. sakazakii. Results: Results showed the antimicrobial activity of plant products and lactic acid bacteria against C. sakazakii. It is recommended that these plant products and lactic acid bacteria could be utilized as natural antimicrobials in baby food-formulations to inhibit C. sakazakii infections in neonates. Conclusion: Emergence of multi-drug resistance and side-effects of antibiotics, plant products can serve as effective antimicrobial agents against C. sakazakii. Lactic acid bacteria can also serve in the control of infections caused by C. sakazakii. Specific probiotics may be useful for healthy intestinal colonization and barrier function to fight pathogen adhesion.

Keywords: Antibiotics, Cronobacter sakazakii, Mueller-Hinton Agar, probiotics


How to cite this article:
Sharma G, Prakash A. Susceptibility of Cronobacter sakazakii to plant products, antibiotics, and to lactic acid bacteria. Int J Nutr Pharmacol Neurol Dis 2013;3:263-8

How to cite this URL:
Sharma G, Prakash A. Susceptibility of Cronobacter sakazakii to plant products, antibiotics, and to lactic acid bacteria. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2019 Nov 20];3:263-8. Available from: http://www.ijnpnd.com/text.asp?2013/3/3/263/114847


   Introduction Top


Cronobacter sakazakii is a gram negative, rod-shaped bacterium of the family Enterobacteriaceae. [1] C. sakazakii infections cause meningitis, septicemia, and necrotizing enterocolitis. [2],[3] The organism is ubiquitous in the water and the soil environment [4],[5] and is also prevalent in infant milk formulae, [4] milk cartons, and fermented bread. [6] Moreover, many cases of infection by C. sakazakii have been reported in neonatal intensive care units around the world. [3] Mortality rates vary from 40 to 80% [3],[7] and meningitis survivors suffer severe neurological squeals such as hydrocephalus, quadriplegia, and retarded neural development. Immuno-compromised, premature, low-birth-weight infants, and those aged <28 days are considered to be more at risk than the older infants. [8],[9]

C. sakazakii is found to be resistant to certain antibiotics like vancomycin, penicillin, oxacillin, and lincosamides. [10] In India, bacterial isolates from neonates have been found to be resistant to cephalosporins, fluoroquinolone, and aminoglycosides. [11] With the ubiquitous nature of the organism, high severity of infection in the immunocompromised individual, and resistance to antibiotics, there is a need for a technique that enables fast and reliable detection of C. sakazakii worldwide. Diminishing options of antimicrobial therapy emphasize the herbal treatment of disease. Pluralities of natural antibiotic agents and their preparation are being used in treating the deadly disease in the current medical field. Seed extracts of water-soluble muscadine were found to be effective against C. sakazakii. [12] The volatile oils obtained from the leaves and flowers have been reported to have antimicrobial activity against C. sakazakii. [13] Trans-cinnamaldehyde (TC), an ingredient in Cinnamomum could be used for controlling C. sakazakii. [14] TC also inhibits biofilm synthesis, and thus can be used to control C. sakazakii biofilms on infant formula-feeding equipment. [15] Certain other natural products like Terminalia chebula, [16] Glycyrrhiza glabra[17] have been found to be effective against a wide range of micro-organisms.

The interest in using lactic acid bacilli for gastrointestinal disturbances came as early as 1907 through the work of Metchnikoff. [18] Earlier studies investigated the use of the oral preparation of viable Lactobacillus acidophilus in the treatment of functional gastrointestinal disturbances. [19] The casein-derived caseicin A and B peptides were shown to exhibit antimicrobial activity against C. sakazakii and E. coli. [20] Norberg et al. reported the importance of specific residues (charged and nonpolar aliphatic) residues within the caseicin peptides and their antimicrobial effect on C. sakazakii and other micro-organisms. [21]

The present study was conducted in order to isolate C. sakazakii from milk and milk products and to study its control using antibiotics, traditional plant products, and lactic acid bacteria.


   Materials and Methods Top


Collection of samples

A total of 55 different milk and milk product samples were collected from different areas of Agra which included 15 raw milk samples each of buffalo, cow, goat, and 10 samples of milk products.

Isolation and identification of bacteria

A total of 55 samples obtained from different regions of Agra city were tested for the presence of C. sakazakii. For this, 0.5 g or 0.5 ml of a sample was added to 4.5 ml of Enterobacter Enrichment (EE) broth in the ratio of 1:9 and incubated overnight at 37°C. Cultures from EE broth were streaked on Violet Red Bile Glucose Agar (VRBGA) media and pink-colored colonies were examined microscopically. These typical colonies were further streaked on Tryptic Soy Agar plates and incubated for 24 h at 37°C. Characteristic yellow-colored colonies were picked up and the organisms were identified by standard microbiological procedure (Gram staining, catalase test, indole test, oxidase reaction, triple sugar iron test, motility test, DNAase test, methyl red test, and sugar fermentation tests). C. sakazakii MTCC-2958 used as positive control was obtained from the Institute of Microbial Technology, Chandigarh.

Preparation of genomic DNA

C. sakazakii MTCC-2958 and 5 isolates were cultured in 3 ml of EE broth for 24 h at 37°C. 1.5 ml of the culture was transferred to a micro-centrifuge tube, boiled at 100°C in a heating block for 10 min, and centrifuged at 1500 g for 30 s. Primers Enterobacter Sakazakii Specific Forward and Enterobacter sakazakii Specific Reverse [22] were used to amplify a 469-bp fragment of the ompA gene specific to C. sakazakii. The polymerase chain reaction mix consisted of 1x Gene Amp PCR buffer II (50 mM potassium chloride and 10 mM tris-HCl, pH 8.3). 2.5 mM MgCl 2 , 200 μM (each) dNTP, 1 μM (each) primer, 1U of Taq DNA polymerase, 50 ng of template DNA, and sterile deionized water to make the volume up to 50 μl. The samples were subjected to PCR cycles consisting of denaturation at 94°C for 15 s, 60°C for 15 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. A 5 μl aliquot of the amplified product was characterized on a 1.5% agarose gel. The amplicons were detected by staining with ethidium bromide (0.5 μg/ml).

Antibiotic susceptibility test procedure

Antibiotic susceptibility test was performed by using the Bauer-Kirby disk diffusion technique. [23] A total volume of 100 μl of the culture from 2 × 10 8 colony forming units/ml was plated on each of the Mueller-Hinton Agar plates. These were incubated along with controls for 24 h at 37°C aerobically. The results were interpreted according to the criteria set by the Clinical and Laboratory Standards Institute. [24]

Antimicrobial activity of plant products against C. sakazakii

Collection of plant products

Citrus sinensis leaves (Mousami), Cinnamomum zeylanicum stem bark (Dalchini), Piper chaba stem bark (Choti Peepal), T. chebula fruits (Hime), Terminalia arjuna leaves (Arjuna), Mangifera indica seed kernel (Mango), Zingiber officinale rhizome (Ginger), Allium sativum bulbs (Garlic), Azadirachta indica leaves (Neem leaves), Ocimum sanctum leaves (Tulsi leaves) Syzygium aromaticum buds (Clove), Terminalia bellirica fruits (Beheda), Emblica officinalis fruits (Amla) were collected from a local retail market in Agra, where these plant products are easily available as herbal medicines. The plant products were washed thoroughly with running water and once with sterile distilled water and finally air-dried on sterile blotter under shade.

Extraction of plant material

25 g of each of the powered plant material was soaked in 100 ml of ethyl alcohol (70%) and 25 g of the plant material was soaked in 100 ml of distilled water for 72 h and filtered. The filtrates obtained were concentrated in a double boiler at 45°C for 5 days and finally dissolved in dimethyl sulphoxide to yield 0.2 g/ml of the herbal product extract.

Sterilization of materials

The extract was exposed to Ultra Violet light (UV rays for 24 h). The sterility was checked by streaking the extract on nutrient agar plates and incubated at 37°C for 24 h. It was confirmed that there were no artifacts to contaminate the sensitivity testing.

Determination of Minimum Inhibitory Concentration (MIC) of plant products

MIC of the extracts for the standard strain MTCC-2958 of C. sakazakii and an isolates of C. sakazakii was determined by using the agar well diffusion method. Mueller-Hinton Agar was prepared and 100 μl of the test culture (6 × 10 7 CFU/ml) was inoculated in the media. A total volume of 40 μl of crude extracts of different dilutions (such as 2 × 10 5 -0.02 μg/ml) were added in the well of the media and incubated overnight at 37°C. The inhibition zones were measured and recorded. Sterile dimethyl sulfoxide without herbal extract was used as a negative control. All the experiments were set in triplicates. The lowest concentration of herbal extracts showing the clear zone of inhibition was considered as its MIC.

Probiotic control of C. sakazakii

Equal amount (50 μl) of Lactobacillus species and the test organism at 10 3 CFU/ml was mixed in a sterile test tube. The mixture was stirred gently. A total volume of 100 μl was transferred to a VRBGA petri plate and incubated at 37°C for 24 h. This procedure was repeated at intervals of 5 min up to 60 min. Standard plate count was done after incubation. The experiment was conducted in a duplicate set.


   Results Top


Out of the total of 55 different milk and milk product samples, 300 isolates were obtained, of which 5 were biochemically confirmed as C. sakazakii. These 5 isolates (lab isolates number Jal 1, Jal 2, Jal 3, Jal 4, and Jal 5) included 2 isolates from raw buffalo milk, 1 from raw cow milk, and two from milk products. All 5 isolates were Gram negative, catalase positive, motile, DNAase positive, indole production negative, methyl red negative, oxidase negative, and were negative for hydrogen sulfide production in triple sugar iron media. Sugar fermentation tests showed that these isolates have the ability to ferment sucrose, glucose, raffinose but not adonitol and arabinose. The primer pair ESSF and ESSR was successfully used to amplify a 469-bp DNA fragment unique to C. sakazakii to confirm these 5 isolates.

Antimicrobial control of C. sakazakii

[Table 1] gives the inhibition zone sizes for 5 isolates and the mean for each antibiotic. C. sakazakii isolates were found to be 100% susceptible to ciprofloxacin, gentamycin, spectinomycin and ceftriaxone, whereas they were resistant to rifampicin and vancomycin.
Table 1: Antimicrobial susceptibility pattern of C. sakazakii isolates

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Plant products on C. sakazakii

The antimicrobial activity of the ethanolic and aqueous extracts of various plant products was assayed in vitro by the agar diffusion method against MTCC-2958 and a randomly selected isolate Jal-2. The results also revealed a decrease in the inhibition zone on diluting the concentration of the extract. [Table 2] and [Table 3] give the minimum inhibitory concentration of plant products against standard MTCC-2958 and an isolate Jal-2 respectively. Alcoholic extract of T. chebula was found to be most effective against C. sakazakii as its MIC value is 0.2 μg/ml. MIC value for aqueous extract of A. indica, S. aromaticum, T. bellirica is 2 μg/ml. There exists no significant difference between the responses of standard MTCC-2958 and that of the isolate Jal-2 but there exists a significant difference between susceptibility to different herbal products (P < 0.05).
Table 2: MIC of natural products against standard C. sakazakii MTCC 2958

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Table 3: MIC of natural products against C. sakazakii isolate Jal-2

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Plant products on C. sakazakii

The antimicrobial activity of the ethanolic and aqueous extracts of various plant products was assayed in vitro by the agar diffusion method against MTCC-2958 and a randomly selected isolate Jal-2. The results also revealed a decrease in the inhibition zone on diluting the concentration of the extract. [Table 2] and [Table 3] give the minimum inhibitory concentration of plant products against standard MTCC-2958 and an isolate Jal-2 respectively. Alcoholic extract of T. chebula was found to be most effective against C. sakazakii as its MIC value is 0.2 μg/ml. MIC value for aqueous extract of A. indica, S. aromaticum, T. bellirica is 2 μg/ml. There exists no significant difference between the responses of standard MTCC-2958 and that of the isolate Jal-2 but there exists a significant difference between susceptibility to different herbal products (P < 0.05).

Probiotic control of C. sakazakii

The antimicrobial activity of Lactobacillus fermentum against C. sakazakii incubated for various time durations is shown in [Figure 1]. At 0 contact time, there were 38 × 10 3 CFU/ml with significant reduction in the colony count to 6 × 10 3 CFU/ml in the first 5 min. [Figure 2], shows the effect of Pediococcus acidilactici on the growth of C. sakazakii. There were 35 × 10 3 CFU/ml present at 0 contact time with a reduction in the colony count to 12 × 10 3 CFU/ml at 5 min contact time. [Figure 3] shows the effect of L. casei on the growth of C. sakazakii. There were 36 × 10 3 CFU/ml present at 0 contact time with a reduction in the colony count to 16 × 10 3 CFU/ml at 5 min contact time. In [Figure 4], the effect of L. lactis on the growth of C. sakazakii is shown. There were 32 × 10 3 CFU/ml present at 0 contact time with colony count reduced to 20 × 10 3 CFU/ml at 5 min contact time. Five minutes was the effective contact time to cause more than 50% reduction in colony counts of C. sakazakii by L. fermentum, L. casei and Pediococcus acidilactici used in the experiment except for L. lactis.
Figure 1: Antimicrobial activity of Lactobacillus fermentum against C. sakazakii

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Figure 2: Antimicrobial activity of Pediococcus acidilactici against C. sakazakii

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Figure 3: Antimicrobial activity of L. casei against C. sakazakii

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Figure 4: Antimicrobial activity of L. lactis against C. sakazakii

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   Discussion Top


In the present study, buffalo raw milk and milk products were more susceptible to C. sakazakii than cow and goat raw milk. These isolates were found to be 100% susceptible to ciprofloxacin, gentamycin, spectinomycin and ceftriaxone, whereas they were resistant to rifampicin and vancomycin. Our findings are in accordance with the results of Stock and Wiedman. [10] Alcoholic extract of T. chebula was found to have more effective antimicrobial activity against C. sakazakii. The contact time effective to cause more than 50% reduction in colony counts of C. sakazakii by L. fermentum, L. casei, and Pediococcus acidilactici was five minutes.

The emergence of multi-drug resistance in human and animal pathogenic bacteria and undesirable side-effects of certain antibiotics has triggered immense interest in the search for new antimicrobial drugs of plant origin. Traditional systems of medicine have been used for centuries for treating human diseases. [25],[26],[27] The present study suggests a potential application of plant crude extracts as antimicrobial agents. These herbal products contain flavonoids, phenols, glycosides, saponins, oleic acids, linolenic acid, and gallic acids etc., which affect the microbial susceptibility. Previous studies show the beneficial effect of plant products and lactic acid bacteria against C. sakazakii. Red muscadine (Vitis rotundifolia Michx.) juices with natural organic, phenolic acids, and polyphenol compounds were tested against C. sakazakii. [12] These results suggest that red muscadine juice could be utilized as a natural antimicrobial in baby food formulations to inhibit C. sakazakii. Apple cider extract exhibited antimicrobial activity against C. sakazakii, Escherichia coli, and Bacillus cereus. [28] Volatile extracts of Cardaria draba had been reported to have antibacterial activity against C. sakazakii and other human pathogenic microorganisms. [29] TC (an ingredient of Cinnamomum) effect on the expression of genes for C. sakazakii biofilm formation was studied by reverse transcription quantitative PCR. [15] These plant products can be easily incorporated by individuals in the daily diet. Their side-effects are also negligible compared to antibiotics.

Besides the antimicrobial activity of plant products, various clinical trials have been conducted using viable lactic acid bacilli against bacteria. Antimicrobial peptide generated by L. acidophilus DPC6026 against pathogenic strains E. sakazakii and E. coli has been studied by Hayes et al. [20] This peptide has a potential role in milk-based formula, which has been linked to C. sakazakii infections in neonates. The administration of probiotics in infant milk formula can promote the intestinal health and immune development when breast feeding is not possible. [30] Thus, the use of safe probiotic bacteria gives new options to infant formula development safety and better infant health promotion. This approach may also allow the development of new probiotic combinations to counteract the risks associated with other pathogens.

The preceding studies have shown the therapeutic effect of plant products and lactic acid bacteria against C. sakazakii. The result of our study indicates that plant products and lactic acid bacteria can be used for the control of C. sakazakii infections and could be utilized as a natural antimicrobial in baby food formulations to inhibit C. sakazakii infections in neonates.

Special emphasis should be given for proper milk pasteurization and prevention of subsequent contamination as C. sakazakii can lead to serious neonatal infections, associated with a high mortality rate. The infections can be treated with antibiotics. However, due to the emergence of multi-drug resistance and side effects of antibiotics, plant products can serve as effective antimicrobial agents against C. sakazakii. Lactic acid bacteria can also serve in the control of infections due to C. sakazakii. Specific probiotics may be useful for healthy intestinal colonization and barrier function to fight pathogen adhesion.


   Conclusion Top


The present study focused on the susceptibility of C. sakazakii to antibiotics, plant products, and probiotics. Plant products and probiotics can serve in effective control of C. sakazakii infection, especially against drug-resistant strains. Therefore, consumption of a diet supplemented with the studied plant products and probiotics could be used to provide protection against C. sakazakii infection. Identification of specific molecules of lactic acid bacteria and plant products responsible for antimicrobial activity against C. sakazakii infection needs further investigations.

 
   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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[Pubmed] | [DOI]



 

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