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Comparison of preservation enrichment media for long storage duration of Campylobacter jejuni§
Korean J. Microbiol. 2023;59(3):192-196
Published online September 30, 2023
© 2023 The Microbiological Society of Korea.

O'la AL-Fawares1*, Rozan O. Al-Khresieh1, Walid Aburayyan1, Nesrin Seder2, Hala I Al-Daghistani3, Nasser El-Banna1, and Emran M. Abu-Taleb4

1Department of Medical Laboratory Analysis, Faculty of Science, Al-Balqa Applied University, Al-salt 19117, Jordan
2Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmacy, Applied Science Private University, Amman 11937, Jordan
3Department of Medical Laboratory Sciences, Faculty of Medical Allied Sciences, Al-Ahliyya Amman University, Amman 19328, Jordan
4Department of Medical Laboratory Sciences, AL-Salt Government Hospital, Jordan Ministry of Health, Al-Salt 19117, Jordan
Correspondence to: *E-mail: ola.alfawares@bau.edu.jo; Tel.: +9627997622457
§Supplemental material for this article may be found at http://www.kjom.org/main.html
Received July 31, 2023; Revised August 22, 2023; Accepted August 23, 2023.
Abstract
Research on Campylobacter jejuni has been greatly impaired due to poor culturing and storage techniques. This study aims to evaluate different preservation methods for C. jejuni for long-term periods of preservation. Three different preservation methods (i, ii, and iii) were tested for their potential for long-term storage of C. jejuni which could be recommended as a simple and reliable method. In the first preservation method (i), 15% glycerol was added to 85% of the bacterial suspension of 4 McFarland standards (McF) and yeast extract. In the second method (ii) the bacterial suspension was supplemented with defibrinated lacked horse blood along with skim milk. Skim milk and 50% glycerol were added to the bacterial suspension in method (iii). Results indicated that method (ii) maintained the highest preservation rate of C. jejuni and the bacteria was capable to grow even after 12 months of storage at -80°C. Method (i) was less efficient than method (ii) where isolates eventually lost their ability to grow after 8 months. Method (iii) showed the least successful preservation rate as many isolates started to die after six months of preservation. This study emphasizes the importance of using defibrinated lacked horse blood along with skim milk during the preservation of bacterial isolates of C. jejuni.
Keywords : Campylobacter jejuni, defibrinated lacked horse blood, growth recovery, preservation methods, skim milk, ultra-cold mechanical freezers
Body

Campylobacteraceae, a commensal bacteria in both humans and animals, is a diverse group that includes three distinct genera: Campylobacter, Arcobacter, and Helicobacter (Facciolà et al., 2017). Campylobacter is small gram-negative bacteria ranging from 0.2 to 0.9 µm in width and 0.5 to 5 μm in length. They are characterized as spiral and non-spore-forming fastidious bacteria that are motile in a corkscrew through a single polar flagellum at one or both of the two ends of the bacterial cell (Samie et al., 2009; Bronowski et al., 2014). Campylobacter is classified as thermophilic bacteria due to their ability to grow between 37°C and 42°C, with 41.5°C being the optimal temperature. The optimal pH for Campylobacter survival is about 6.5–7.5. Campylobacteracea is microaerophilic, growing in a low oxygen tension atmosphere (5% oxygen, 10% carbon dioxide, 85% nitrogen) (Davis and DiRita, 2008; Silva et al., 2011; Al-Khreshieh et al., 2023).

For the isolation of Campylobacter species, different selective agars can be used such as Butzler, Preston, and charcoal cefoperazone deoxycholate (CCDA) agars. Yet, Campylobacter detection methods are not commonly used in routine laboratory practices, because it is fastidious and difficult to be cultivated (Zanetti et al., 1996). Campylobacter jejuni is not very tolerant to several non-optimal conditions thus little is known about the mechanisms that enable cell’s survival under different environmental and stress settings, in contrast to other enteric bacteria such as Salmonella, Pseudomonas, and E. coli (Park, 2002; Kim et al., 2021; Seder et al., 2021). For precise and reliable detection of Campylobacter species in biological samples, several techniques have been developed, including filtering, latex agglutination, and fluorescence in situ hybridization (FISH). The most efficient confirmation procedures rely on the polymerase chain reaction (PCR) and other molecular approaches, including Pulse-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequencing (MLS) (Silva et al., 2011; Al-Khresieh et al., 2022; Rousou et al., 2023).

One of the major challenges of microbial studies is the unavailability of a reliable preservative media for the long-term storage of microbes to protect the integrity and structure of the microorganism cells without resulting in partial lysis or cellular leakage (Ranjan et al., 2020; AL-Fawares et al., 2023). The best method for storing bacteria depends on the compatibility of the bacteria and the cell viability. In general, as the storage temperature decreases, the time that bacteria can remain viable increases (O’Connell et al., 2016). Cryoprotectants, on the other hand, are necessary to lessen cell damage brought on by freezing once the temperature has fallen below the freezing point. It depends on the bacterial strain for how long a culture will keep alive in a particular storage environment. Several scientists reported disturbances in the cultivation and preservation of Campylobacter bacterial stock for further investigation (Kaakoush et al., 2015).

In the present study, we describe three different methods of preserving Campylobacter species in research labs at two conventional freezing temperatures of -20°C and -80°C and conducted a reliable comparison for these conditions at different storage periods of 1, 3, 6, 9, and 12 months.

Materials and Methods

Materials

A modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was used for the isolation of C. jejuni, Columbia agar base for purification. Nutrient broth no. 2 was used along with the different preservatives including yeast extract, skim milk, defibrinated lacked horse blood, and molecular glycerol for preservation. CampyGenTM microaerophilic sachets and mCCDA campylobacter growth supplements such as sodium pyruvate, sodium metabisulphite, and ferrous sulfate were used. All materials described in this section were products of Oxoid. A total of 20 samples, including raw chicken meat, unpasteurized milk, vegetables, and human samples, were collected from different sources in Jordan. Only the necks and wings of the hens, which were sourced from both legal and unlicensed slaughterhouses, were examined. Samples were enriched in a under microaerobic conditions before being cultivated on cefoperazone deoxycholate agar with modified charcoal. Staining, biochemical testing, and polymerase chain reaction molecular identification were used to identify bacteria. All samples other than the clinical ones were processed according to the FDAs Bacteriological Analytical Manual (Al-Khresieh et al., 2022).

Bacterial strains and culture conditions

The strains studied were 20 fresh isolates of C. Jejuni by which, 17 were obtained from chicken samples, and the remaining 3 isolates were obtained from patients suffering from gastroenteritis. All 20 cultures were stored using three different preservation methods, at both -20°C and -80°C using a deep freezer (Laboquest. Inc). Each strain was monitored for viability at 1, 3, 6, 9, and 12-month intervals by sub-culturing on Columbia Blood Agar (CBA) and incubated at 42°C for 48 h under microaerophilic conditions (5% O2, 10% CO2, 85% N2) using Campygen Oxoid (Gorman and Adley, 2004).

Preservation methods

Three different preservation methods were investigated. In method (i): The preservation medium was prepared by autoclaving nutrient broth no. 2, 15% glycerol, and 0.1 g of yeast extract. A quantity of 850 µl of fresh C. jejuni culture (McFarland 3-4) was aseptically transferred into two vials of 150 µl sterile glycerol. Two vials were stored at -20°C and -80°C.

In method (ii): The second preservation medium; 15% glycerol/85% nutrient broth no.2 was added with 10% defibrinated lacked horse blood and 0.1 mg of skim milk, along with culture. A quantity of 850 µl of fresh C. jejuni culture (McFarland 3-4) was aseptically transferred into two vials of 150 µl sterile glycerol. The mixtures were emulsified by vortex and the two vials were then stored at -20°C and -80°C.

The third preservation method (iii) contained 50% glycerol/50% nutrient broth no. 2 culture and 0.1 mg of skim milk. 500 µl of culture broth was emulsified with 500 µl of sterile glycerol. As with the previous methods, the culture reached (McFarland 3-4) and the two vials were stored at -20°C and -80°C.

Results were obtained by examining the cultural growth of the tested isolates after sub-culturing them on Colombia Blood Agar. Based on the following scale, an estimation of the growth of the tested strains was reported: +++, confluent growth when colonies < 50.000 CFU/ml; ++, semi-confluent growth when colonies < 30.000 CFU/ml; +, weak growth <5.000 CFU/ml and no growth 0 CFU/ml. Results of C. jejuni viability over 12 months following storage at -20°C and -80°C.

Statistical analysis

All bacterial samples were repeated three times independently. Bacterial recovery data were analyzed by two-way ANOVA followed by Turkey’s post hoc analysis using the SPSS program, version 20 (SPSS, IBM). Values with P < 0.05 are regarded as statistically significant.

Results

Mean recovery of C. jejuni was expressed as CFU/ml for 20 sample at different storage temperatures of -20°C and -80°C in period of 1, 3, 6, 9, and 12-month were detected in Fig. 1. In the first studied preservation method (i), the growth of the isolates started to abrogate after six months of preservation especially those that were preserved at -20°C. After 12 months of preservation, all isolates lost their growth ability when sub-cultured. In the second preservation method (ii), shows that 40% of the testing isolates stored at -80°C maintained confluent growth when subcultures on Colombia blood agar over the first three months. After six months, 5% maintained confluent growth, and 60% maintained semi-confluent growth. Nine months later, despite that none of the isolates exhibited their confluent growth, 65% maintained semi-confluent growth. At the end of the testing period (12 months) only 15% of the testing isolates, were semi-confluent, 75% were weak growth, and 10%, lost their complete growth ability (Supplementary data Table S1). Method (iii) was the least efficient when compared to the other two preservative methods since no isolates grew after 6 months of preservation.

Fig. 1. The recovery of C. jejuni samples using different preservation methods ‎during several periods.
Preservation methods: (i) 15% glycerol/85% nutrient broth no. 2 culture; (ii) Cryobank microbial ‎preservation system using defibrinated lacked horse blood; (iii); 50% glycerol/50% nutrient broth no. 2 culture. *P<0.05, **P<0.01, and ***P<0.001, One-way ANOVA test was conducted between the three methods in reference to method ii.

When considering storage temperatures employed in the current study, and regardless of the preservation conditions, 65% of the total number of isolates, stored at -20°C lost their viability after 12 months of preservation. In addition, when considering the best preservation method (ii) in our results, isolates that were stored at -20°C, it was noticed that about 55%, 25%, 15%, 5%, and 0% of the isolates were capable to grow in semi-confluent growth over a period of time 1, 3, 6, 9, and 12 months, respectively (Supplementary data Table S1).

Discussion

Preservation and storage of microbial strains are essential steps for both research-based and clinical applications. Campylobacter jejuni could lose its viability usually within 3 days after bacterial isolation. Indeed, C. jejuni may enter its viable non-culturable state, generating a non-motile coccoid form that will no longer be able to grow on culture media (Lv et al., 2020). In our investigation, we systematically compared the viability of C. jejuni using a variety of preservation methods to find a simple and reliable methods. Following the addition of a known concentration of preservatives, the isolates were stored immediately by freezing at -20°C and -80°C and evaluated for a period of 1, 3, 6, 9, and 12 months. Rogol et al. (1990) have developed a medium that contains 5% human blood. In this context, results show blood as an excellent supplement for Campylobacter spp. growth, storage, and recovery since iron and detoxifying enzymes that are found in blood demonstrated to reduce the toxicity of media (Gorman and Adley, 2004). Moreover, to maintain the in vitro viability of C. jejuni, different preservatives were tested to be added and aid in the maintenance of the cell during various storage conditions.

Our recent work has demonstrated that blood plays a crucial role in preserving the viability of C. jejuni when stored at low temperatures for long periods of up to 12 months. Remarkably, using 10% defibrinated lacked horse blood and 0.1 mg of skim milk, along with culture at -80°C resulted in stable storage conditions and a high recovery percentage. Our findings are consistent with previous research conducted by Gorman and Adley (2004). The authors justify that blood containing many detoxifying enzymes such as catalase, peroxidase, and superoxide dismutase that will in turn reduce the toxicity of the media during long preservation periods. Worth to mention, the National Committee for Clinical Laboratory Standards recommends using Mueller-Hinton broth mixed with lysed horse blood for measuring the quantitative antimicrobial susceptibility of Streptococcus pneumoniae (D’Amato et al., 1987).

Unsurprisingly, C. jejuni recovery from any of the tested methods and stored at -20ºC showed poor growth compared with -80ºC storage under the same conditions. Storage in ultra-cold mechanical freezers (-70°C to -90°C) is highly effective for the majority of microorganisms to ensure the strains’ survival. In this context, a previous study revealed that 100% of Neisseria gonorrhea isolates remained viable after eight years of storage at -80°C, which indicates that preserving at -80°C is a good temperature (Guo et al., 2020). Notable, storage at -196°C is considered ideal because also that the genotype and, by extension, the bacterial phenotype, do not alter as the chances of DNA mutations are almost zero at that temperature (Sanderson and Zeigler, 1991; Prakash et al., 2013; Aburayyan, 2023). However, even though ferrous sulfate, sodium metabi-sulfite and sodium pyruvate (FBP) medium was annotated by (Gorman and Adley, 2004) as a simple and reliable method for keeping C. jejuni after 12 months at -20°C, storage at this temperature is inadequate for many bacteria and is not recommended for long-term storage. Therefore, there is a high demand for the optimization of cryoprotectants and other conditions employing a variety of microorganisms needs to receive further consideration.

Conclusion

The long-term sustainable use of microbial strains is directly impacted by the storage conditions. To keep cultures viable in a stable genetic form, suitable maintenance methods are required. For each type of presentative strain, a unique preservation strategy should be developed, taking into consideration the variety of strains and the various effects of preservation. This strategy should include optimizing preservatives as well. This research demonstrates that adding blood, as a supplement to C. jejuni preservation at -80°C is an easy, affordable preservation method that will enable a quick and reliable recovery.

Acknowledgments

The research reported in this publication was funded by the Deanship of Scientific Research and Innovation at Al-Balqa Applied University in Jordan under Award Number 432/2020/2021.

Conflict of Interest

The authors declare that there is no conflict of interest.

Data Availability

The data that support this study will be shared upon reasonable request to the corresponding author.

Author Contributions

ROA and EMA conducted the experiments, WAR, NS and NE performed and edited the analysis, HIA supplied the strains, and OA wrote, edit, and supervised the overall project.

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