Molecular diagnostics of <i>Salmonella</i> and <i>Campylobacter</i> in human/animal fecal samples remain feasible after long-term sample storage without specific requirements

CB Harder; S Persson; J Christensen; A Ljubic; EM Nielsen; J Hoorfar; CB Harder; S Persson; J Christensen; A Ljubic; EM Nielsen; J Hoorfar

doi:10.3934/microbiol.2021024

AIMS Microbiology

2021, Volume 7, Issue 4: 399-414. doi: 10.3934/microbiol.2021024

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Research article

Molecular diagnostics of Salmonella and Campylobacter in human/animal fecal samples remain feasible after long-term sample storage without specific requirements

1.
Statens Serum institut, Dept. Bacteriology, Parasitology and Fungi, Artillerivej 5, 2300 Copenhagen, Denmark
2.
Molecular Ecology, Microbial Ecology and Evolutionary Genetics, Lund University, Sölvegatan 37, 223 62 Lund
3.
Danish Veterinary and Food Administration, Microbiological department, Søndervang 4, 4100 Ringsted
4.
AGC Biologics, Process Transfer, Vandtårnsvej 83, 2860 Søborg, Denmark
5.
Technical University of Denmark, National Food Institute, 2800 Kgs. Lyngby, Denmark

Received: 17 March 2021 Accepted: 05 September 2021 Published: 14 October 2021

Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives.

This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 10⁶ CFU/mL of both Salmonella and Campylobacter strains stored at −20 °C, 5 °C and 20 °C (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.
- Detection threshold,
- Campylobacter,
- Salmonella,
- gastritis,
- DNA stability,
- diagnostics,
- preservation methods,
- pathogens
Citation: CB Harder, S Persson, J Christensen, A Ljubic, EM Nielsen, J Hoorfar. Molecular diagnostics of Salmonella and Campylobacter in human/animal fecal samples remain feasible after long-term sample storage without specific requirements[J]. AIMS Microbiology, 2021, 7(4): 399-414. doi: 10.3934/microbiol.2021024

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Abstract

Rapid advances in the development of sequencing technologies, numbers of commercial providers and diminishing costs have made DNA-based identification and diagnostics increasingly accessible to doctors and laboratories, eliminating the need for local investments in expensive technology and training or hiring of skilled technicians. However, reliable and comparable molecular analyses of bacteria in stool samples are dependent on storage and workflow conditions that do not introduce post-sampling bias, the most important factor being the need to keep the DNA at a stable detectable level. For that reason, there may remain other prohibitively costly requirements for cooling or freezing equipment or special chemical additives.

This study investigates the diagnostic detectability of Salmonella and Campylobacter DNA in human, pig and chicken stool samples, stored at different temperatures and with different preservation methods. Stool samples were spiked with 10⁶ CFU/mL of both Salmonella and Campylobacter strains stored at −20 °C, 5 °C and 20 °C (Room temperature, RT) and treated with either RNAlater, EDTA or Silica/ethanol. DNA was extracted at 9 different time points within 30 days and quantified by Qubit (total DNA) and qPCR (Salmonella and Campylobacter DNA). We found no statistically significant differences among the different preservation methods, and DNA from both species was easily detected at all time points and at all temperatures, both with and without preservation. This suggests that infections by these bacteria can be diagnosed and possibly also analysed in further detail simply by taking a stool sample in any suitable sealed container that can be transported to laboratory analysis without special storage or preservation requirements. We briefly discuss how this finding can benefit infection control in both developed and developing countries.

Acknowledgments

This research was supported by InnovationsFonden (formerly called The Danish National Advanced Technology Foundation) to the project ‘Metagenome Kit’ (Grant number: 85-2013-1). CBH was supported by the Carlsberg Foundation (grants no. CF18-0809/CF19-0197) at the time of writing. This publication reflects the views only of the author/(s) and neither InnovationsFonden nor the Carlsberg Foundation can be held responsible for any use which may be made of the information contained therein. We would like to thank Christian Vråby Pedersen for excellent technical assistance.

Conflict of interest

The authors declare no conflict of interest.

References

[1]	WHO WHO estimates of the global burden of foodborne diseases: foodborne disease burden epidemiology reference group 2007–2015 (2015) .
[2]	Chlebicz A, Śliżewska K (2018) Campylobacteriosis, Salmonellosis, Yersiniosis, and Listeriosis as zoonotic foodborne diseases: A Review. Int J EnvironRes Public Health 15: 863. doi: 10.3390/ijerph15050863
[3]	Fletcher SM, McLaws ML, Ellis JT (2013) Prevalence of gastrointestinal pathogens in developed and developing countries: systematic review and meta-analysis. J Public Health Res 2: 42-53. doi: 10.4081/jphr.2013.e9
[4]	Chokshi A, Sifri Z, Cennimo D, et al. (2019) Global contributors to antibiotic resistance. J Global Infect Dis 11: 36. doi: 10.4103/jgid.jgid_110_18
[5]	Bonardi S (2017) Salmonella in the pork production chain and its impact on human health in the European Union. Epidemiol Infect 145: 1513-1526. doi: 10.1017/S095026881700036X
[6]	Awad WA, Molnár A, Aschenbach JR, et al. (2014) Campylobacter infection in chickens modulates the intestinal epithelial barrier function. Innate Immunity 21: 151-160. doi: 10.1177/1753425914521648
[7]	Landers TF, Cohen B, Wittum TE, et al. (2012) A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep 127: 4-22. doi: 10.1177/003335491212700103
[8]	Weerakoon K, Gordon C, McManus D (2018) DNA diagnostics for schistosomiasis control. Tropical Med Infect Dis 3: 81. doi: 10.3390/tropicalmed3030081
[9]	Schultze A, Akmatov MK, Andrzejak M, et al. (2014) Comparison of stool collection on site versus at home in a population-based study. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 57: 1264-1269. doi: 10.1007/s00103-014-2051-z
[10]	Platts-Mills JA, Liu J, Gratz J, et al. (2014) Detection of Campylobacter in stool and determination of significance by culture, enzyme immunoassay, and PCR in developing countries. J Clin Microbiol 52: 1074-1080. doi: 10.1128/JCM.02935-13
[11]	Büscher P, Deborggraeve S (2015) How can molecular diagnostics contribute to the elimination of human African trypanosomiasis? Expert Rev Mol Diagn 15: 607-615. doi: 10.1586/14737159.2015.1027195
[12]	Love BC, Rostagno MH (2008) Comparison of five culture methods for Salmonella isolation from swine fecal samples of known infection status. J Vet Diagn Invest 20: 620-624. doi: 10.1177/104063870802000514
[13]	Buss JE, Thacker E, Santiago M (2020) Culture methods to determine the limit of detection and survival in transport media of Campylobacter Jejuni in human fecal specimens. J Vis Exp 157.
[14]	Herikstad H, Motarjemi Y, Tauxe RV (2002) Salmonella surveillance: a global survey of public health serotyping. Epidemiol Infect 129: 1-8. doi: 10.1017/S0950268802006842
[15]	Bale J, Meunier D, Weill FX, et al. (2016) Characterization of new Salmonella serovars by whole-genome sequencing and traditional typing techniques. J Med Microbiol 65: 1074-1078. doi: 10.1099/jmm.0.000325
[16]	Bereswill S, Jerome JP, Bell JA, et al. (2011) Standing genetic variation in contingency loci drives the rapid adaptation of Campylobacter jejuni to a novel host. PLoS One 6: e16399. doi: 10.1371/annotation/5247af81-4595-44b7-9c3f-2e45ad85abfa
[17]	Achtman M, Wain J, Weill FX, et al. (2012) Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathogens 8: e1002776. doi: 10.1371/journal.ppat.1002776
[18]	Turnbaugh PJ, Ley RE, Hamady M, et al. (2007) The human microbiome project. Nature 449: 804-810. doi: 10.1038/nature06244
[19]	Qin J, Li R, Raes J, et al. (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464: 59-65. doi: 10.1038/nature08821
[20]	Leser TD, Amenuvor JZ, Jensen TK, et al. (2002) Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol 68: 673-690. doi: 10.1128/AEM.68.2.673-690.2002
[21]	Gill SR, Pop M, DeBoy RT, et al. (2006) Metagenomic analysis of the human distal gut microbiome. Science 312: 1355-1359. doi: 10.1126/science.1124234
[22]	Faith JJ, Guruge JL, Charbonneau M, et al. (2013) The long-term stability of the human gut microbiota. Science 341: 1237439. doi: 10.1126/science.1237439
[23]	Jenkins SV, Vang KB, Gies A, et al. (2018) Sample storage conditions induce post-collection biases in microbiome profiles. BMC Microbiol 18. doi: 10.1186/s12866-018-1359-5
[24]	Choo JM, Leong LEX, Rogers GB (2015) Sample storage conditions significantly influence faecal microbiome profiles. Sci Rep 5.
[25]	Anderson EL, Li W, Klitgord N, et al. (2016) A robust ambient temperature collection and stabilization strategy: Enabling worldwide functional studies of the human microbiome. Sci Rep 6.
[26]	Wu GD, Lewis JD, Hoffmann C, et al. (2010) Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags. BMC Microbiol 10: 206. doi: 10.1186/1471-2180-10-206
[27]	Hale VL, Tan CL, Knight R, et al. (2015) Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8weeks. J Microbiol Methods 113: 16-26. doi: 10.1016/j.mimet.2015.03.021
[28]	Claassen S, du Toit E, Kaba M, et al. (2013) A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. J Microbiol Methods 94: 103-110. doi: 10.1016/j.mimet.2013.05.008
[29]	Kongmuang U, Luk JM, Lindberg AA (1994) Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR. J Clinical Microbiol 32: 3072-3074. doi: 10.1128/jcm.32.12.3072-3074.1994
[30]	McOrist AL, Jackson M, Bird R (2002) A comparison of five methods for extraction of bacterial DNA from human faecal samples. J Microbiol Methods 50: 131-139. doi: 10.1016/S0167-7012(02)00018-0
[31]	Persson S, de Boer RF, Kooistra-Smid AMD, et al. (2011) Five commercial DNA extraction systems tested and compared on a stool sample collection. Diagn Microbiol Infect Dis 69: 240-244. doi: 10.1016/j.diagmicrobio.2010.09.023
[32]	Rapp D (2010) DNA extraction from bovine faeces: current status and future trends. J Appl Microbiol 108: 1485-1493. doi: 10.1111/j.1365-2672.2009.04606.x
[33]	Wesolowska-Andersen A, Bahl MI, Carvalho V, et al. (2014) Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis. Microbiome 2: 1-11. doi: 10.1186/2049-2618-2-19
[34]	Bahl MI, Bergström A, Licht TR (2012) Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS Microbiol Lett 329: 193-197. doi: 10.1111/j.1574-6968.2012.02523.x
[35]	White BA, Clooney AG, Fouhy F, et al. (2016) Comparing apples and oranges?: next generation sequencing and its impact on microbiome analysis. Plos One 11: e0148028. doi: 10.1371/journal.pone.0148028
[36]	Nsubuga AM, Robbins MM, Roeder AD, et al. (2004) Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method. Mol Ecol 13: 2089-2094. doi: 10.1111/j.1365-294X.2004.02207.x
[37]	Vlčková K, Mrázek J, Kopečný J, et al. (2012) Evaluation of different storage methods to characterize the fecal bacterial communities of captive western lowland gorillas (Gorilla gorilla gorilla). J Microbiol Methods 91: 45-51. doi: 10.1016/j.mimet.2012.07.015
[38]	Wasser SK, Houston CS, Koehler GM, et al. (1997) Techniques for application of faecal DNA methods to field studies of Ursids. Mol Ecol 6: 1091-1097. doi: 10.1046/j.1365-294X.1997.00281.x
[39]	Nechvatal JM, Ram JL, Basson MD, et al. (2008) Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces. J Microbiol Methods 72: 124-132. doi: 10.1016/j.mimet.2007.11.007
[40]	Bennett JE, Dolin R, Blaser MJ (2014) Mandell, douglas, and bennett's principles and practice of infectious diseases: 2-volume set (Vol. 2). Elsevier Health Sciences .
[41]	Anderson NW, Buchan BW, Ledeboer NA (2014) Comparison of the BD MAX enteric bacterial panel to routine culture methods for detection of Campylobacter, Enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella isolates in preserved stool specimens. J Clini Microbiol 52: 1222-1224. doi: 10.1128/JCM.03099-13
[42]	Buss JE, Cresse M, Doyle S, et al. (2019) Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods. European J Clini Microbiol Infect Dis 38: 1087-1093. doi: 10.1007/s10096-019-03499-x
[43]	Lund M, Nordentoft S, Pedersen K, et al. (2004) Detection of Campylobacter spp. in chicken fecal samples by real-time PCR. J Clin Microbiol 42: 5125-5132. doi: 10.1128/JCM.42.11.5125-5132.2004
[44]	Josefsen MH, Krause M, Hansen F, et al. (2007) Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat. Appl Environ Microbiol 73: 3040-3048. doi: 10.1128/AEM.02823-06
[45]	Josefsen MH, Löfström C, Hansen TB, et al. (2010) Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Appl Environ Microbiol 76: 5097-5104. doi: 10.1128/AEM.00411-10
[46]	R Core Team: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria (2017) .Available from: http://wwwR-projectorg/.
[47]	de Mendiburu F Package ‘agricolae’. R Package, Version, 1–2 (2019) .
[48]	Songane M (2017) Challenges for nationwide vaccine delivery in African countries. Int J Health Econ Manage 18: 197-219. doi: 10.1007/s10754-017-9229-5
[49]	Yahia EM (2009) Cold chain development and challenges in the developing world. VI International Postharvest Symposium 877: 127-132.
[50]	Oura CAL, Edwards L, Batten CA (2013) Virological diagnosis of African swine fever—comparative study of available tests. Virus Res 173: 150-158. doi: 10.1016/j.virusres.2012.10.022
[51]	Dorsaz S, Charretier Y, Girard M, et al. (2020) Changes in microbiota profiles after prolonged frozen storage of stool suspensions. Front Cell Infect Microbiol 10: 77. doi: 10.3389/fcimb.2020.00077
[52]	Milde A, Haas-Rochholz H, Kaatsch HJ (1999) Improved DNA typing of human urine by adding EDTA. Int J Legal Med 112: 209-210. doi: 10.1007/s004140050237
[53]	Fremin BJ, Bhatt AS (2020) A combined RNA-Seq and comparative genomics approach identifies 1,085 candidate structured RNAs expressed in human microbiomes. BioArxiv preprint.
[54]	The Human Microbiome Project Consortium (2012) A framework for human microbiome research. Nature 486: 215-221.
[55]	Frølund M, Wikström A, Lidbrink P, et al. (2018) The bacterial microbiota in first-void urine from men with and without idiopathic urethritis. Plos One 13: e0201380. doi: 10.1371/journal.pone.0201380
[56]	Jung CE, Chopyk J, Shin JH, et al. (2019) Benchmarking urine storage and collection conditions for evaluating the female urinary microbiome. Sci Rep 9: 13409. doi: 10.1038/s41598-019-49823-5
[57]	Prevention CfDCa Guidelines for Specimen Collection: Instructions for Collecting Stool Specimens (2021) .Available from: https://www.cdc.gov/foodsafety/outbreaks/investigating-outbreaks/specimen-collection.html.
[58]	Andersen SC, Kiil K, Harder CB, et al. (2017) Towards diagnostic metagenomics of Campylobacter in fecal samples. BMC Microbiol 17: 133. doi: 10.1186/s12866-017-1041-3
[59]	Abou Tayoun AN, Burchard PR, Malik I, et al. (2014) Democratizing Molecular Diagnostics for the Developing World. Am J Clin Pathol 141: 17-24. doi: 10.1309/AJCPA1L4KPXBJNPG
[60]	Indian Council of Medical Research Available from: https://www.icmr.gov.in/.
[61]	Parashar UD, Nelson EAS, Kang G (2013) Diagnosis, management, and prevention of rotavirus gastroenteritis in children. BMJ 347: f7204. doi: 10.1136/bmj.f7204

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