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Detection of Viable but ^ncu^ra^e Microbial Cells in Chicken Mince
Asiyat M. Abdullaeva
Moscow State University of Food Production 11 Volokolamskoe highway, Moscow, 125080, Russian Federation
E-mail: abdullaevaam@mgupp.ru
Larisa P. Blinkova
I.I. Mechnikov Research Institute of Vaccines and Sera 5а, Maliy Kazenniy pereulok, Moscow, 105064, Russan Federation
E-mail: b.larus@mail.ru
Boris V. Usha
Moscow State University of Food Production 11 Volokolamskoe highway, Moscow, 125080, Russian Federation
E-mail: vet-san-dekanat@yandex.ru
Rumiya K. Valitova
Moscow State University of Food Production 11 Volokolamskoe highway, Moscow, 125080, Russian Federation I.I. Mechnikov Research Institute of Vaccines and Sera 5а, Maliy Kazenniy pereulok, Moscow, 105064, Russan Federation
E-mail: rumiya.valitova@gmail.com
Yuriy D. Pakhomov
I.I. Mechnikov Research Institute of Vaccines and Sera 5а, Maliy Kazenniy pereulok, Moscow, 105064, Russan Federation
E-mail: yury-pakhomov@yandex.ru
Daria B. Mitrofanova
Moscow State University of Food Production 11 Volokolamskoe highway, Moscow, 125080, Russian Federation I.I. Mechnikov Research Institute of Vaccines and Sera 5а, Maliy Kazenniy pereulok, Moscow, 105064, Russan Federation
E-mail: dar.gushina@yandex.ru
There is a potential for existence of hazardous viable but nonculturable (VBNC) cells of pathogenic microorganisms in foodstuffs that can be formed under the influence of various factors. Their detection and determination of conditions for formation of VBNC cells of various bacteria are relevant for preventing contamination of meats. This was the aim of the present study. The search was conducted for VBNC cells in chicken mince in real time and during experimental infection of it by Staphylococcus aureus 209P. In order to detect VBNC cells in chicken mince, total number of microbes, number of bacterial colonies (CFU), and the portion of living (dead) cells were determined in 1g of the product using a commercial set of fluorescent dyes. A second study was carried out after 5 h of incubation of tested samples at room temperature. In samples of minced meat on the 4th day after production, more than 99 % of all detected living cells were VBNC. After 5-hour incubation of the sample, the number of CFU/g increased by 22.5 times, but the portion of VBNC cells remained higher than 99 % of viable bacteria. During artificial infection of the same batch of mince with S. aureus in broth culture at the stage of logarithmic growth, the amount of VBNC cells for 0 hours was 97.33 %. After 5 hours their number increased to 99.99 %. Probably, in the introduced culture
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How to Cite
This article is published under the Creative Commons Attribution 4.0 International License.
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Abdullaeva, A. M., Blinkova, L. P., Usha, B. V., Valitova, R. K., Pakhomov, Y. D., & Mitrofanova, D. B. (2019). Detection of Viable but Nonculturable Microbial Cells in Chicken Mince. Health, Food & Biotechnology, 1(4). https://doi.org/10.36107/ hfb.2019.i4.s281
of Staphylococcus at the stage of active reproduction, formation of VBNC bacteria did not occur, which initially reduced their number in the sample. After 5-h incubation, transition of bacteria to VBNC state was accelerated, possibly due to unfavorable conditions for the cell population (changes in trophic substrate, temperature, pH, etc.). Experimental data confirm presence of VBNC bacteria in chicken products that don't grow on traditional nutrient media and showing a false negative result in traditional microbiological expertise. Because of the biohazard of such dormant cells, it is advisable to provide regulated testing of foodstuffs for presence of VBNC cells.
Keywords: viable but nonculturable cells, chicken mince, detection, biohazard
Introduction
The environment, in which many microorganisms exist, can stimulate their growth, or on the contrary, limit it or contribute to the cessation of cell reproduction. The latter process is most often associated with emerging stress due to non-optimal values of temperature (Chaisowwong et al., 2012) and pH (Cunningham et al, 2009), osmotic pressure (Asakura et al., 2002), oxygen concentration and other gases (Oh et al, 2015), UV and radiation (Zhang et al, 2015), pulsed light and pulsed energy fields (Kramer, B., & Muranyi, P., 2014), lyophilization (Blinkova et al, 2014), antibiotics (Hu, & Coates 2012), chemical preservatives, disinfectants, metal ions, nutrient deficiencies (Zhao et al, 2017), etc.
The consequence of the stressful effects of these factors on non-sporeforming microorganisms is their transition to a viable but nonculturable (VBNC) state (Abdullaeva, Blinkova & Pakhomov, 2019; Ayrapetyan & Oliver, 2016; Li, Mendis, Trigui, Oliver & Faucher 2014; Pinto, Santos & Chambel, 2015; Ramamurthy, Ghosh, Pazhani & Shinoda, 2014). This is considered the "cell strategy for their survival" (Nystrom, 2003; Oliver, 2010). When favorable conditions appear, cells can return to their normal physiological state, restoring metabolism and the ability to grow and multiply (Rao, Shashidhar & Bandekar, 2014).
Because of the formation of VBNC cells, dangerous contaminant bacteria that remain undetected in foods, when conventional methods of microbiological control are used, can multiply to dangerous levels and cause diseases (Abdullaeva, Blinkova & Pakhomov, 2019; Ayrapetyan & Oliver, 2016; Fakruddin, Mannan & Andrews, 2013).
Non-sporeforming microorganisms contaminating food products, water, that are found in of VBNC state, include Salmonella spp., Shigella spp., E. coli, Staphylococcus spp., Streptococcus spp., Listeria monocytogenes, Yersinia spp., Aeromonas hydrophila,
Vibrio spp., Campylobacter jejuni, Pseudomonas aeruginosa et al. By 2015, researchers reported 68 species of microorganisms that can be found in the state of VBNC state (Zhao, Zhong, Wei, Lin, & Ding, 2017; Bates, & Oliver, 2004; Ding, Suo, Xiang, Zhao, Chen, & Liu, 2016; Rao, Shashidhar, & Bandekar, 2014).
Infection of food products with VBNC cells is possible not only during the manufacture of food products on infected equipment, but also during storage, transportation and with contaminated raw materials (Abdullaeva, Blinkova & Pakhomov, 2019; Zhao, Zhong, Wei, Lin, & Ding, 2017).
Among the food sources of VBNC microorganisms, are meats of various animals and birds, fish and seafood, eggs, vegetables, fruits, juices, pickles and even kitchen utensils and dust is known (Bates & Oliver, 2004; Dinu & Bach, 2013; Nicolo et al., 2010; Zhao, Zhong, Wei, Lin, & Ding, 2017; Ziprin, Droleskey, Hume & Harvey, 2003).
The existence of VBNC cells in Campylobacter jejuni under conditions of increased oxygenation (Oh, McMullen & Jeon, 2015), low-temperature exposure in L. monocytogenes (Chaisowwong et al., 2012), after UV irradiation in P. aeruginosa and enteritis causing, E. coli was reported (Ding et al., 2017; Dinu & Bach, 2013; Zhang, Ye, Lin, Lv & Yu, 2015). In the spore-forming B. cereus VBNC cells appear after treatment with a pulsating electric field in the presence of potassium sorbate at low pH (Cunningham, O'Byrne & Oliver, 2009; Rowan, 2004), in Lactobacillus lindneri, L. harbinensis in beer causing its spoilage (Liu et al., 2017). There are known cases of wine spoilage in barrels after their treatment with sodium metabisulfite, which are caused by the VBNC yeasts Brettanomyces bruxellensis, Saccharomyces cerevisiae, Candida albicans var. stellatoidea (Capozzi et al., 2016; Divol & Lonvaud-Funel, 2004; Serpaggi, Remize & Recorbet, 2012). Unusual outbreaks of food poisoning have also been described. Thus, a recorded foodborne bacterial infection connected to the consumption
of salted salmon roe is associated with the VBNC cells of the enteropathogenic strain of E. coli O157. H7 (Makino et al., 2000). In another case (Asakura et al., 2002), infection was reported with evidence for Salmonella enterica serovar Oranienburg, isolated from dried salted squid after reversing the from VBNC cells to a vegetative form. However, there was no direct detection of the original VBNC bacteria in seafood.
The presence of VBNC microorganisms in the fresh bodies of water and seas (Masmoudi, Denis & Maalej, 2010; Rowan, 2004), in tap water treated with chlorinating disinfectants, can pose a threat to human health. This was reported, for example, by Al-Oadiri, Lu, Al-Alami & Rasco, (2011) who detected enterohemorrhagic E. coli O157: H7 and C. jejuni in tap water using molecular detection methods.
Under the influence of temperature pasteurization, microbes in the VBNC state were present in milk (Gunasekera, S0rensen, Attfield, S0rensen & Veal, 2002), and live nonculturable cells of Cronobacter sakazakii were found in dry dairy products (Barron & Forsythe, 2007). When potassium sorbate was used for antibacterial processing of food products at pH 4.0 after 24 hours, L. monocytogenes VBNC cells formed under the influence of this substance were detected (Cunningham, O'Byrne & Oliver, 2009). By simulating the processing conditions of production equipment at a meat processing plant with help of disinfectants (Peneau, Chassaing & Carpentier, 2007), VBNC Pseudomonas fluorescens was obtained. The question of the occurrence of VBNC cells after various methods of decontaminating exposure remains insufficiently studied (Schottroff, Fröhling, Zunabovic-Pichler, Krottenthaler, Shlüter & Jäger, 2019).
More than 250 diseases associated with food contaminated with microorganisms are known (Nicolo & Guglielmino, 2012). As follows from the literature sources cited in our article, the main direction of research was to establish stress factors for the transition of microorganisms to a nonculturable state. Studies on the conditions and features of the existence of VBNC bacteria in food products, as well as improving the microbiological expertise for the presence of VBNC bacteria-contaminants, were fragmentary. The use of food products imported with long-distance transportation from geographically remote countries with low hygienic standards may increase the likelihood of microbial contamination of products with transition of bacteria into VBNC forms.
The issue of accurately conducted microbiological expertise on the safety of both domestic and imported
food products and raw materials has a high level of relevance, especially in the conditions of a pandemic spread of infection. In this situation, food poisoning can be an aggravating factor. Therefore, the detection and the population characteristics of dangerous VBNC cell of contaminant pathogens, such as S. aureus (Zhao, Wei, Zhong, & Jin, 2016) in one of the types of meat products - widely consumed chicken mince, was the purpose of this research.
Methodology
Materials
The object of the study was minced chicken from one of the producers in the Moscow region, 4 days after the date of manufacture that was purchased in a retail network. For microbiological analysis of products - determination of the number of anaerobic and facultative anaerobic microorganisms (KMAFAnM) according to State Standard 10444.15-54; we used 1.5 % nutrient agar and nutrient broth produced by the State Scientific Center for Applied Microbiology in Obolensk and Petrifilm plates manufactured by 3M USA, in in accordance with P54354-2011 "Meat and meat products." Staining of minced meat samples for the purpose of detection of bacterial contaminant was performed using a commercial kit of DNA- tropic dyes Live/Dead (Baclight™, USA).
Equipment
The following equipment was used during the experiments: Goryaev chamber, Russia, for microscopy of minced meat suspensions; when detecting microorganisms in mince meat suspensions, a Mikmed-5 light microscope from LOMO, Russia (magnification x 400) was used; Opton fluorescence microscope, Germany (magnification x 320); centrifuge "Eppendorf", Germany; high-precision scales by A & D, Japan, refrigerator Atlant, Belarus; Vortex Biosan, Latvia; Petri dishes, test tubes and plastic sterile pipettes of various volumes of the company "Costar", USA, automatic "Eppendorf" pipettes, Germany.
Methods of research
When conducting experiments on the microbiological examination of minced chicken, the main provisions of SanPiN 2.3.21078-01 "Hygienic requirements for safety and nutritional value of food products" and TR TS 034/2013 "On the safety of meat and meat products" were used.
In the experiments, the methods of microbiological studies were used: growing broth cultures, incubation of a minced meat sample of with broth or broth culture S. aureus 209P at a temperature of (21 ± 1) °C for 0 h and 5 h; 10-fold serial dilutions of chicken mince samples in saline; subsequent plating on nutrient agar and Petrifilm; counting colony forming units (CFU/ml) on nutrient media; determination of the total number of bacteria in suspensions in the Goryaev chamber in a light microscope; staining of bacterial cells in minced meat samples with Live/ Dead dye (Baclight™); counting differentially stained living (green) and dead (red) cells in a fluorescent microscope; calculation of the percentage of VBNC cells in the studied samples.
Data analysis
For a statistical evaluation of the reliability of differences in experimental data, we used the calculation of the arithmetic mean of 6 determinations (M), its error (m), and the variation limit (I95) at p <0.05.
Samples were taken from mixtures at the following points: 0 h, 5 h. In each of the mixtures (minced meat mixture with meat-peptone broth; minced meat mixture with S. aureus 209P broth culture), the total number of bacteria was determined using a Goryaev chamber, and inoculation was made from dilutions of these mixtures in saline on 1.5 % meat-peptone agar and on the Petrifilm to determine CFU/ml To detect the level of viable bacteria in 1 ml of tested minced meat suspensions the number of living and dead cells was determined by staining with Live/Dead which was counted using a luminescent microscope. Based on the total data, the amount of VBNC cells was calculated.
Then, a statistical analysis of the experimental results was carried out with an assessment of the reliability of the average concentrations of microorganisms and the calculation of the variation limit using the Student t-test.
Results and Discussion
Procedures of microbiological study
For study, 5 g samples were taken from the package of minced meat, which were suspended in 5 ml of sterile nutrient broth, then the mixture was stirred on a vortex and a series of 10-fold dilutions was made in 0.9 % saline solution, with subsequent inoculation of a nutrient medium. Artificially infected with staphylococci chicken mince samples were prepared by adding 5 g of minced chicken to test tubes with 5 ml of fresh broth culture Staphylococcus aureus 209P. Further procedures were performed as described previously.
The data obtained in the experiments were supposed to demonstrate whether the products we tested contained bacterial VBNC cells. The results are presented in table 1.
According to the results of the study of minced chicken, which was stored for 4 days in a retail network in refrigerator, after opening the package (0 h), the amount of CFU/g of product on meat-peptone agar was (4.0 ± 0.44) x 103 in 1 ml of suspension. This value corresponded to the CFU/g parameter (103 - 104) that is regulated by the documentation i.e. <105 for KMAFAnM in food. This value characterizes a fresh high quality product.
Table 1
Results of microbiological examination of minced chicken from a retail network
Sample incubtion time (h)
Total number of bacteria x108/ml
(W
Parameters (n=6) (M ± m)
CFU/ml xio3
(W
Portions of cells (%) (I)
viable
dead
Mean portion of VBNC (%)
10,76 ± 1,1 (8,10 ^ 13,42)
9,2 ± 1,09 (6,57 ^ 11,83)
4 ± 0,44 (2,94 ^ 5,06)
90 ± 9,9 (66,1 ^ 113,9)
86 ± 5,0 (76,3 ^ 95,7)
99 ± 0,41 (98,01 ^ 99,99)
14 ± 1,5 (10,37 ^ 17,6)
1 ± 0,24 (0,76 ^ 1,24)
99,99 99,99
Reliability of the differences (p95)
>0,05
<0,05
<0,05
<0,05
Inoculating Petrifilm showed values close to the obtained CFU/ml values on Petri dishes.
Microscopic examination of a sample from a mixture of tested minced chicken in a sterile meat-peptone broth (sample 0 hour) was performed to determine the total number of microorganisms (TNM). It revealed (10.76 ± 1.1) x 108 cells/ml. After 5 h, the TNM remained statistically not different from the initial level (9.2 ± 1.09) x 108 cells/ml (p> 0.05). Comparison of 2 values (CFU/g/ml and TNM/ml) for the initial sample of minced chicken at 0 h, and also after 5 h showed that the value difference for each period was 5 orders of magnitude.
In order to determine the presence of VBNC cells in minced meat, we studied the number of live and dead bacterial cells in the same samples of suspended minced meat after staining with the Live/Dead DNA tropic dye. As can be seen from the table. 1, the average number of viable cells in the field of view was (86 ± 4.0) %, and for dead bacteria - (14 ± 0.15) %. Consequently, there were 6.1 times fewer dead than living cells capable of forming colonies. Based on the calculations, the amount of VBNC cells we found in the studied minced meat permitted for consumption was 99.99 % of the population. After 5 hours of incubation of the mixtures of minced meat with nutrient broth at room temperature, the determination of TNM/ml, CFU/ml and VBNC was again carried out.
As a result, the data show that after a 5-hour incubation, the total number of microbes in the minced meat remained at a statistically unambiguous level compared to the same parameter at 0 h (p> 0.05). At the same time, the value of CFU/ml increased by 22.5 times (p <0.05). The average number of stained living bacteria capable of forming colonies, (86 ± 4.0) %
and (99 ± 0.41) %, was statistically significant (p <0.05), with significantly different limits of variation (I95) for average values: (76.3 -f 95.7) and (98.01 -f 99.99), respectively. Besides, the number of bacterial VBNC cells remained at the same level - 99.99 %.
Similar results were obtained by plating chicken samples on nutrient agar and on Petrifilm after 0 hours and 5 hours of incubation at room temperature (Figure 1 and Figure 2).
These quantitative values allow us to assume that during the 5 hours of incubation at a temperature more favorable for microorganisms (21 ± 1) ° C, the physiologically active part of the resident population of microbes from minced chicken stored in the refrigerator apparently had several cycles of reproduction (generation), reducing the number of dead cells. In this case, there was no transition of cells to the nonculturable state or reversion of VBNC microbes into the vegetative stage.
In connection with the obtained results, it was interesting to study the behavior of bacterial cells by their ability to enter the state of VBNC in the conditions of fresh contamination of minced meat with the most common food pathogens.
For this purpose, minced meat was infected with a S. aureus 209P culture.
The results of the microbiological examination of chicken mince experimentally infected using broth culture of S. aureus 209P are presented in table 2.
The studied parameters of the microbial population at 0 h indicate that the level of TNM in the Staphylococcus infected meat, was (11.36 ± 1.3) x 108 bacterial cells per
Figure 1. Inoculation of mince sample at 0 h on Petrifilm, dilution 10-2
Figure 2. Inoculation of mince sample at 5 h on Petrifilm, dilution 10-2
Table 2
Results of microbiological examination of minced chicken experimentally contaminated with S. aureus 209P
Sample incubtion time (h)
Total number of bacteria *108/ml 0,5)
Parameters (n=6) (M ± m)
CFU/ml xl03
d,5)
Portions of cells (%) (I95)
viable
dead
Mean portion of VBNC (%)
5
Reliability of the differences (p95)
11,36 ± 1,3 (8,21 f 14,5)
9,56 ± 1,1 (6,90 f 12,22)
>0,05
300,0 ± 32,0 (222,6 f 377,4)
8,0 ± 0,87 (5,89 f 10,10)
<0,05
99 ± 0,41 (98,01 f 99,99)
99 ± 0,40 (98,03 f 99,97)
<0,05
1 ± 0,1 (0,76 f 1,24)
1 ± 0,1 (0,76 f 1,24)
>0,05
97,33 99,99
ml. From this KMAFAnM population, (300 ± 32) x 103 CFU/ml was detected, i.e. only 0.0265 % of cells were cultured on a traditional medium. Testing this sample with cells of microorganisms stained with Live/ Dead dye kit in a luminescent microscope revealed (99 ± 0.41) % of live bacteria and (1 ± 0.1) % oa dead cells, (p <0.05). The portion of the VBNC microbes in this population was 97.33 %.
The determination of a complex of parameters in Staphylococcus infected chicken mince after 5 hours of incubation at room temperature showed a 37.5fold decrease in CFU/ml. Moreover, the total number of cells in suspension, as well as the number of live and dead bacteria, were statistically unambiguous (p> 0.05). Consequently, increasing the percentage of VBNC bacteria from 97.33 % to 99.99 % (an increase by 2.6 %) and the indicated decrease in the number of CFU/ml after 5 hours of incubation are associated with the loss of the ability of this part of the population to grow and form colonies on a normal nutrient medium and transition to a nonculturable state. Similar trends
in colony formation were found on Petrifilm (Figure 3 and Figure 4).
As it is known, this loss of culturability in bacteria, as a rule, is caused by stress in the environment. Apparently, for S. aureus 209P cells grown in the broth at 37 °C to the logarithmic phase and artificially contaminated minced chicken, changes in the nutrient substrate and temperature regime from 37 °C to (21 ± 1) °C could become stressful factors. The result of this stressful effect was the transition of cells to the VBNC state. Similar data on VBNC bacteria grown in nutrient broth were obtained by Peneau, Cassaing & Carpentier (2007) when studying the effectiveness of antibacterial treatments in the food processing premises.
Thus, a comparison of the data we obtained with the results of other researchers showed that the original part of our experiments is the detection of VBNC contaminant bacteria in ready to eat products from chicken meat in the amount of up to 99.9 % of the total number of living and dead cells. In addition,
Figure 3. Inoculation of Petrifilm with a mince sample contaminated with S. aureus 209P, 0 h, dilution 10-5
Figure4. Inoculation of Petrifilm with a mince sample contaminated with S. aureus 209P, 5 h, dilution 10-5
0
new information is also the relatively rapid (during 5 h) transition of vegetative cells of S. aureus to a nonculturable form after artificial contamination of minced chicken meat.
Conclusion
Thus, the experiments performed on minced chicken (intact, ready-to-eat, and experimentally infected with S. aureus 209P) allowed us to prove the presence of VBNC bacteria there (up to 99.9 %). The introduction of S. aureus cells into the minced meat in the logarithmic growth phase led to a quantitative increase in bacterial VBNC cells after 5 hours of incubation of the samples under non-optimal conditions. Therefore, we have shown the possibility of the presence of nonculturable bacteria in fresh poultry products.
Since S. aureus is a dangerous pathogen, the realization of the potential of the viable nonculturable microbe cells that we discovered can be reverted to an active physiological state and can lead to severe toxicoinfection after eating minced chicken, in which Staphylococcus was present in a dormant state.
The biohazard of such food products associated with the detection of VBNC bacterial cells indicates the feasibility of introducing mandatory expertise for the presence of dormant forms of microorganisms-contaminants and subsequent antibacterial treatment of products with a high level of infection with viable nonculturable microbes.
References
Abdullaeva, A. M., Blinkova, L. P., & Pakhomov, Yu. D. (2019). Significance of viable but nonculturable bacteria for safety of food products. Problemy Veterinarnoy, Sanitarii, Gigieny i Ekologii [Problems of Veterinary, Sanitation, Hygiene and Ecology] 2(30), 183-189. http://dx.doi.org/10.25725/vet.san. hyg.ecol.201902012 Al-Oadiri, H. M., Lu, X., Al-Alami, N. I., & Rasco, B. A. (2011). Survival of Escherichia coli O157: H7 and Campylobacter jejuni in bottled purified drinking water under different storage conditions. Journal of Food Protection, 74(2), 254-260. https://doi. org/10.4315/0362-028X.JFP-10-368 Asakura, H., Makino, S.-I., Takagi, T., Kuri, A., Kurazono, T., Watarai, M., & Shirahata, T. (2002). Passage in mice causes a change in the ability of Salmonella enterica serovar Oranienburg to survive NaCl osmotic stress: resuscitation
from the viable but non-culturable state. FEMS Microbiology Letters, 212(1), 87-93. https://doi. org/10.1111/j.1574-6968.2002.tb11249.x Aurass, P., Prager, R., & Flieger, A. (2011). EHEC/ EAEC O104: H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief. Environmental Microbiology, 13(12), 3139-3148. https://doi. org/10.1111/j.1462-2920.2011.02604.x Ayrapetyan, M., & Oliver, J. D. (2016). The viable but non-culturable state and its relevance in food safety. Current Opinion in Food Science, 8, 127-133. https://doi.org/10.1016Zj.cofs.2016.04.010 Barron, J. C., & Forsythe, S. J. (2007). Dry stress and survival time of Enterobacter sakazakii and other Enterobacteriaceae in dehydrated powdered infant formula. Journal of Food Protection, 70(9), 2111-2117.https://doi.org/10.4315/0362-028X-70.9.2111 Bates, T. C., & Oliver, J. D. (2004). The viable but nonculturable state of Kanagawa positive and negative strains of Vibrio parahaemolyticus. Journal of Microbiology, 42(2), 74-79. Blinkova, L., Martirosyan, D., Pakhomov, Yu., Dmitrieva, O., Vaughan, R., & Altshuler M. (2014). Nonculturable forms of bacteria in lyophilized probiotic preparations. Functional Foods in Health and Disease, 4(2), 66-76. Capozzi, V., Di Toro, M. R., Grieco, F., Michelotti, V., Salma, M., Lamontanara, A., Russo, P., Orru, L., Alexandre, H., & Spano, G. (2016). Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: new insights on molecular basis of VBNC behaviour using a transcriptomic approach. Food Microbiology, 59, 196-204. https:// doi.org/10.1016/j.fm.2016.06.007 Chaisowwong, W., Kusumoto, A., Hashimoto, M., Harada, T., Maklon, K., & Kawamoto, K. (2012). Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat. Journal of Veterinary Medical Science, 74(1), 43-50. https://doi.org/10.1292/ jvms.11-0305
Cunningham, E., O'Byrne, C., & Oliver, J. D. (2009). Effect of weak acids on Listeria monocytogenes survival: evidence for a viable but nonculturable state in response to low pH. Food Control, 20(12), 1141-1144. https://doi.org/10.10Wj. foodcont.2009.03.005 Ding, T., Suo, Y., Xiang, O., Zhao, X., Chen, S., Ye, X., & Liu, D. (2017). Significance of viable but nonculturable Escherichia coli: induction, detection, and control. Journal of Microbiology and Biotechnology, 27(3), 417-428 https://doi. org/10.4014/jmb.1609.09063.
Dinu, L. D., & Bach, S. (2013). Detection of viable but non-culturable Escherichia coli O157: H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays. Food Control, 31(2), 268-273. Divol B. & Lonvaud-Funel A. (2004). Evidence for viable but nonculturable yeasts in botrytis -affected wine. Journal of Applied Microbiology, 99(1), 85-93.
Fakruddin, M., Mannan, K. S., & Andrews, S. (2013). Viable but nonculturable bacteria: food safety and public health perspective. ISRN Microbiology, 2013:703813. https://doi.org/10.1155/2013/703813 Gunasekera, T. S., S0rensen, A., Attfield, P. V., S0rensen, S. J., & Veal, D. A. (2002). Inducible gene expression by nonculturable bacteria in milk after pasteurization. Applied and Environmental Microbiology, 68(4), 1988-1993. https://doi. org/10.1128/AEM.68.4.1988-1993.2002 Hu, Y., & Coates, A. (2012). "Nonmultiplying bacteria are profoundly tolerant to antibiotics," in Antibiotic Resistance, eds R. M. Anthony and A. Coates (Berlin: Springer Press), 99-119. https://doi.org/ 10.1007/978-3-642-28951-4_7 Kramer, B., & Muranyi, P. (2014). Effect of pulsed light on structural and physiological properties of Listeria innocua and Escherichia coli. Journal of Applied Microbiology, 116(3), 596-611. https://doi. org/10.1111/jam.12394 Li, L., Mendis, N., Trigui, H., Oliver, J. D., & Faucher, S. P. (2014). The importance of the viable but non-culturable state in human bacterial pathogens. Frontiers in Microbiology, 5, 258. https://doi. org/10.3389/fmicb.2014.00258 Liu, J., Li, L., Li, B., Peters, B. M., Deng, Y., Xu, Z. & Shirtliff, M. E. (2017). First study on the formation and resuscitation of viable but nonculturable state and beer spoilage capability of Lactobacillus lindneri. Microbial Pathogenesis, 107, 219-224. https://doi.org/ 10.1016/j.micpath.2017.03.043 Makino, S.-I., Kii, T., Asakura, H., Shirahata, T., Ikeda, T., Takeshi, K., & Itoh, K. (2000). Does Enterohemorrhagic Escherichia coli O157: H7 enter the viable but nonculturable state in salted salmon roe? Applied and Environmental Microbiology, 66(12), 5536-5539. https://doi.org/10.1128/ AEM.66.12.5536-5539.2000 Masmoudi, S., Denis, M., & Maalej, S. (2010). Inactivation of the gene katA or sodA affects the transient entry into the viable but non-culturable response of Staphylococcus aureus in natural seawater at low temperature. Marine Pollution Bulletin, 60(12), 2209-2214. https://doi. org/10.1016/j.marpolbul.2010.08.017 Nicolo, M. S., Gioffre, A., Carnazza, S., Platania, G., Silvestro, I. D., & Guglielmino, S. P. (2010). Viable
but nonculturable state of foodborne pathogens in grapefruit juice: a study of laboratory. Foodborne Pathogens and Disease, 8(1), 11-17. https://doi. org/10.1089/fpd.2009.0491 Nicolo, M. S., & Guglielmino, S. P. P. (2012). "Viable but nonculturable bacteria in food," in Public Health-Methodology, Environmental and Systems Issues, ed. J. Maddock (Rjeka: InTech), 189-216. https://doi.org/10.5772/38118 Nystrom, T. (2003). Nonculturable bacteria: programmed survival forms or cells at death's door? Bioessays, 25(3), 204-211. https://doi. org/10.1002/bies.10233 Oh, E., McMullen, L., & Jeon, B. (2015). Impact of oxidative stress defense on bacterial survival and morphological change in Campylobacter jejuni under aerobic conditions. Frontiers in Microbiology, 6, 295. https://doi.org/10.3389/fmicb.2015.00295 Oliver, J. D. (2010). Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiology Reviews, 34(4), 415-425. https://doi. org/10.1111/j.1574-6976.2009.00200.x Peneau, S., Chassaing, D., & Carpentier, B. (2007). First evidence of division and accumulation of viable but nonculturable Pseudomonas fluorescens cells on surfaces subjected to conditions encountered at meat processing premises. Applied and Environmental Microbiology, 73(9), 2839-2846. https://doi.org/10.1128/AEM.02267-06 Pinto D., Santos M. A., & Chambel L. (2015). Thirty years of viable but nonculturable state research unsolved molecular mechanisms. Critical Reviews in Microbiology, 41(1), 61-76. Ramamurthy, T., Ghosh, A., Pazhani, G. P., & Shinoda, S. (2014). Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria. Frontiers in Public Health, 2, 103. https://doi.org/10.3389/ fpubh.2014.00103 Rao, N. V., Shashidhar, R., & Bandekar, J. R. (2014). Induction, resuscitation and quantitative realtime polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water. World Journal of Microbiology and Biotechnology, 30, 2205-2212. https://doi. org/10.1007/s11274-014-1640-1 Rowan, N. J. (2004). Viable but non-culturable forms of food and waterborne bacteria: quo vadis? Trends in Food Science & Technology, 15(9) 462-467. https:// doi.org/10.1016/j.tifs.2004.02.009 Rowan, N. J., Valdramidis, V. P., & Gomez-Lopez, V. M. (2015). A review of quantitative methods to describe efficacy of pulsed light generated inactivation data that embraces the occurrence of viable but non culturable state microorganisms. Trends in Food Science & Technology, 44(1), 79-92. https://doi.org/10.1016Zj.tifs.2015.03.006
Schottroff, F., Fröhling, A., Zunabovic-Pichler, M., Krottenthaler, A., Shlüter, O., & Jäger, H. (2018). Sublethal injury and viable but nonculturable (VBNC) state in microorganisms during preservation of food and biological materials by non-thermal processes. Frontiers in Microbiology, 9, article 2773, p. 1-19. https://doi.org/10.3389/ fmicb.2018.02773.
Serpaggi, V., Remize, F., & Recorbet, G. (2012). Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces. Food Microbiology, 30(2), 440-447.
Zhang, S., Ye, C., Lin, H., Lv, L., & Yu, X. (2015). UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa. Environmental Science & Technology, 49(3), 1721-1728. https://
doi.org/ 10.1021/es505211e Zhao, X., Zhong, J., Wei, C. Lin C.-W., & Ding T. (2017). Current perspectives on viable but non-culturable state in foodborne pathogens. Frontiers in Microbiology, 8, 1-32. https://doi.org/10.3389/ fmicb.2017.00580 Zhao, X., Wei, C., Zhong, J., & Jin, S. (2016). Research advance in rapid detection of foodborne Staphylococcus aureus. Biotechnology & Biotechnological Equipment, 30(5), 1-7. https://doi. org/ 10.1080/13102818.2016.1209433 Ziprin, R. L., Droleskey, R. E., Hume, M. E., & Harvey, R. B. (2003). Failure of viable nonculturable Campylobacter jejuni to colonize the cecum of newly hatched leghorn chicks. Avian Diseases, 47(3), 753-758. https://doi.org/10.1637/7015
