Characterization of microflora in Latin-style cheeses by next-generation sequencing technology

BMC Microbiology

Julie A Kase³  Eric W Brown³  Marc W Allard³  James R White¹  Andrea R Ottesen³  Tina S Lusk² 

[1] Institute for Genome Sciences, University of Maryland School of Medicine, 801 W. Baltimore St., Baltimore, MD 21201, MD, USA;Oak Ridge Institute for Science and Education, Oak Ridge, TN 38730, Tennessee;Division of Microbiology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740, MD, USA

关键词: Exiguobacterium;    Bacteria;    Microflora;    Next Generation Sequencing;    Latin-style cheese;   

Others  :  1145204
DOI  :  10.1186/1471-2180-12-254

 received in 2012-05-29, accepted in 2012-10-24,  发布年份 2012

【 摘 要 】

Background

Cheese contamination can occur at numerous stages in the manufacturing process including the use of improperly pasteurized or raw milk. Of concern is the potential contamination by Listeria monocytogenes and other pathogenic bacteria that find the high moisture levels and moderate pH of popular Latin-style cheeses like queso fresco a hospitable environment. In the investigation of a foodborne outbreak, samples typically undergo enrichment in broth for 24 hours followed by selective agar plating to isolate bacterial colonies for confirmatory testing. The broth enrichment step may also enable background microflora to proliferate, which can confound subsequent analysis if not inhibited by effective broth or agar additives. We used 16S rRNA gene sequencing to provide a preliminary survey of bacterial species associated with three brands of Latin-style cheeses after 24-hour broth enrichment.

Results

Brand A showed a greater diversity than the other two cheese brands (Brands B and C) at nearly every taxonomic level except phylum. Brand B showed the least diversity and was dominated by a single bacterial taxon, Exiguobacterium, not previously reported in cheese. This genus was also found in Brand C, although Lactococcus was prominent, an expected finding since this bacteria belongs to the group of lactic acid bacteria (LAB) commonly found in fermented foods.

Conclusions

The contrasting diversity observed in Latin-style cheese was surprising, demonstrating that despite similarity of cheese type, raw materials and cheese making conditions appear to play a critical role in the microflora composition of the final product. The high bacterial diversity associated with Brand A suggests it may have been prepared with raw materials of high bacterial diversity or influenced by the ecology of the processing environment. Additionally, the presence of Exiguobacterium in high proportions (96%) in Brand B and, to a lesser extent, Brand C (46%), may have been influenced by the enrichment process. This study is the first to define Latin-style cheese microflora using Next-Generation Sequencing. These valuable preliminary data will direct selective tailoring of agar formulations to improve culture-based detection of pathogens in Latin-style cheese.

【 授权许可】

   
2012 Lusk et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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【 参考文献 】

• [1]USDA: Dairy Products 2010 Summary. http://usda01.library.cornell.edu/usda/nass/DairProdSu//2010s/2011/DairProdSu-04-27-2011.pdf webcite
• [2]Gould B: Understanding Dairy Markets - Per Capita Hispanic Cheese Consumption. http://future.aae.wisc.edu/data/annual_values/by_area/2196?tab=sales webcite
• [3]Dairy Management Inc: Understanding the Dairy Opportunity Among Hispanic Consumers. http://www.dairyfoods.com/ext/resources/Digital_Brochures/DF-Hispanic-White-Paper-FINAL.pdf webcite
• [4]Ortman JM, Guarneri CE: United States Population Projections: 2000 to 2050. http://www.census.gov/population/www/projections/analytical-document09.pdf webcite
• [5]Clark S, Costello M, Drake M, Bodyfelt F: Chapter 16 Latin American Cheeses. In Sensory Evaluation of Dairy Products. 2nd edition. Edited by Clark S, Costello M, Drake M, Bodyfelt F. Springer - Verlag, New York; 2009:493-494.
• [6]Genigeorgis C, Carniciu M, Dutulescu D, Farver TB: Growth and survival of Listeria monocytogenes in market cheeses stored at 4 to 30 degrees C. J Food Prot 2012, 54:662-668.
• [7]Centers for Disease Control and Prevention: Food Outbreak Online Database. http://wwwn.cdc.gov/foodborneoutbreaks webcite
• [8]Centers for Disease Control and Prevention: Outbreak of Multidrug-Resistant Salmonella enterica serotype Newport Infections Associated with Consumption of Unpasteurized Mexican-Style Aged Cheese. MMWR 2008, 57(16):432-435.
• [9]Jackson K, Biggerstaff M, Tobin-D’Angelo M, Sweat D, Klos R, Nosari J, Garrison O, Boothe E, Saathoff-Huber L, Hainstock L: Multistate outbreak of Listeria monocytogenes associated with Mexican-style cheese made from pasteurized milk among pregnant, Hispanic women. J Food Prot 2011, 74:949-953.
• [10]Linnan MJ, Mascola L, Lou XD, Goulet V, May S, Salminen C, Hird DW, Yonekura ML, Hayes P, Weaver R: Epidemic listeriosis associated with Mexican-style cheese. N Engl J Med 1988, 319:823-828.
• [11]MacDonald P, Whitwam R, Boggs J, MacCormack J, Anderson K, Reardon J, Saah J, Graves L, Hunter S, Sobel J: Outbreak of listeriosis among Mexican immigrants as a result of consumption of illicitly produced Mexican-style cheese. Clin Infect Dis 2005, 40:677-682.
• [12]Thompson TL, Marth EH: Changes in Parmesan cheese during ripening: Microflora - coliforms, enterococci, anaerobes, propionibacteria and staphylococci. Milchwissenschaft Milk Science International 1986, 41:201-204.
• [13]Ordonez JA, Barneto R, Ramos M: Studies on Manchego cheese ripened in olive oil. Milchwissenschaft Milk Science International 1978, 33:609-612.
• [14]Terzic-Vidojevic A, Vukasinovic M, Veljovic K, Ostojic M, Topisirovic L: Characterization of microflora in homemade semi-hard white Zlatar cheese. Int J Food Microbiol 2007, 114:36-42.
• [15]Litopoulou-Tzanetaki E: Changes in Numbers and Kinds of Lactic Acid Bacteria During Ripening of Kefalotyri Cheese. J Food Sci 1990, 55:111-113.
• [16]Centeno J, Menendez S, Rodriguez-Otero J: Main microbial flora present as natural starters in Cebreiro raw cow’s-milk cheese (Northwest Spain). Int J Food Microbiol 1996, 33:307-313.
• [17]Berthier F, Beuvier E, Dasen A, Grappin R: Origin and diversity of mesophilic lactobacilli in Comte cheese, as revealed by PCR with repetitive and species-specific primers. Int Dairy J 2001, 11:293-305.
• [18]Trovatelli LD, Schiesser A: Identification and significance of enterococci in hard cheese made from raw cow and sheep milk. Milchwissenschaft Milk Science International 1987, 42:717-719.
• [19]Beresford TP, Fitzsimons NA, Brennan NL, Cogan TM: Recent advances in cheese microbiology. Int Dairy J 2001, 11:259-274.
• [20]Quigley L, O’Sullivan O, Beresford TP, Ross RP, Fitzgerald GF, Cotter PD: Molecular approaches to analyzing the microbial composition of raw milk and raw milk cheese. Int J Food Microbiol 2011, 150:81-94.
• [21]O’Sullivan DJ: Methods for analysis of the intestinal microflora. Curr Issues Intest Microbiol 2000, 1:39-50.
• [22]Redford AJ, Bowers RM, Knight R, Linhart Y, Fierer N: The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves. Environ Microbiol 2010, 12:2885-2893.
• [23]Telias A, White JR, Pahl DM, Ottesen AR, Walsh CS: Bacterial community diversity and variation in spray water sources and the tomato fruit surface. BMC Microbiol 2011, 11:81. BioMed Central Full Text
• [24]Lewis T, Loman NJ, Bingle L, Jumaa P, Weinstock GM, Mortiboy D, Pallen MJ: High-throughput whole-genome sequencing to dissect the epidemiology of Acinetobacter baumannii isolates from a hospital outbreak. J Hosp Infect 2010, 75:37-41.
• [25]Quigley LF, O’Sullivan OF, Beresford TP, Ross RP, Fitzgerald G, Fitzgerald GF, Cotter P, Cotter PD: High-throughput sequencing for detection of subpopulations of bacteria not previously associated with artisanal cheeses. Appl Environ Microbiol 2012, 78:5717-5723.
• [26]Alegria A, Szczesny P, Mayo BF, Bardowski JF, Kowalczyk M, Kinde HF, Mikolon AF, Rodriguez-Lainz AF, Adams CF, Walker RL FAU, Cernek-Hoskins S, et al.: Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and -independent approaches. Appl Environ Microbiol 2012, 78:1890-1898.
• [27]Masoud WF, Vogensen FK, Lillevang S, Abu Al-Soud W, Sorensen SJ, Jakobsen M: The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR. Int J Food Microbiol 2012, 153:192-202.
• [28]White JR, Nagarajan N, Pop M: Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS Comput Biol 2009, 5:e1000352.
• [29]Renye J Jr, Somkuti G, Vallejo Cordoba B, Van Hekken D, Gonzalez-Cordova A: Characterization of the microflora isolated from queso fresco made from raw and pasteurized milk. Journal of Food Safety 2008, 28:59-75.
• [30]Saubusse MF, Millet LF, Delbes CF, Callon CF, Montel MC: Application of Single Strand Conformation Polymorphism –PCR method for distinguishing cheese bacterial communities that inhibit Listeria monocytogenes. Int J Food Microbiol 2007, 116:126-135.
• [31]Callon CF, Duthoit FF, Delbes CF, Ferrand MF, Le Frileux YF, De Cremoux RF, Montel MC: Stability of microbial communities in goat milk during a lactation year: molecular approaches. Syst Appl Microbiol 2007, 30:547-560.
• [32]Liu F, Wang D, Du L, Zhu Y, Xu W: Diversity of the Predominant Spoilage Bacteria in Water-Boiled Salted Duck during Storage. J Food Sci 2010, 75:M317-M321.
• [33]Collins MD, Lund BM, Farrow JA, Schleifer KH: Chemotaxonomic Study of an Alkalophilic Bacterium, Exiguobacterium aurantiacum gen. nov., sp. nov. J Gen Microbiol 1983, 129:2037-2042.
• [34]Fruhling A, Schumann P, Hippe H, Straubler B, Stackebrandt E: Exiguobacterium undae sp. nov. and Exiguobacterium antarcticum sp. nov. Int J Syst Evol Microbiol 2002, 52:1171-1176.
• [35]Chocolatewala N, Chaturvedi P, Desale R: The role of bacteria in oral cancer. Indian J Med Paediatr Oncol 2010, 31:126-131.
• [36]Rodrigues DF, Tiedje JM: Multi-locus real-time PCR for quantitation of bacteria in the environment reveals Exiguobacterium to be prevalent in permafrost. FEMS Microbiol Ecol 2007, 59:489-499.
• [37]Vishnivetskaya TA, Petrova MA, Urbance J, Ponder M, Moyer CL, Gilichinsky DA, Tiedje JM: Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods. Astrobiology 2006, 6:400-414.
• [38]Siddikee MA, Chauhan PS, Anandham R, Han GH, Sa T: Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil. J Microbiol Biotechnol 2010, 20:1577-1584.
• [39]Borsodi A, Kiss R, Cech G, Vajna B, Toth E, Marialigeti K: Diversity and activity of cultivable aerobic planktonic bacteria of a saline Lake located in Sovata, Romania. Folia Microbiol 2010, 55:461-466.
• [40]Okeke B: Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1. Journal of Industrial Microbiology & Biotechnology 2008, 35:1571-1579.
• [41]Jameson JE: A discussion of the dynamics of salmonella enrichment. Journal of Hygiene, Cambridge 1962, 60:193-207.
• [42]Mellefont LA, McMeekin TA, Ross T: Effect of relative inoculum concentration on Listeria monocytogenes growth in co-culture. Int J Food Microbiol 2008, 121:157-168.
• [43]Tran T, Stephenson P, Hitchins A: The effect of aerobic mesophilic microflora levels on the isolation of inoculated Listeria monocytogenese strain LM82 from selected foods. Journal of Food Safety 1990, 10:267-275.
• [44]Al-Zeyara SA, Jarvis B, Mackey BM: The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths. Int J Food Microbiol 2011, 145:98-105.
• [45]Nocker A, Richter-Heitmann T, Montijn R, Schuren F, Kort R: Discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by 454 pyrosequencing. Int Microbiol 2010, 13(2):59-65.
• [46]Pan Y, Bodrossy L, Frenzel P, Hestnes AG, Krause S, Luke C, Meima-Franke M, Siljanen H, Svenning MM, Bodelier PL: Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses. Appl Environ Microbiol 2010, 76:7451-7458.
• [47]Angiuoli SV, Matalka M, Gussman A, Galens K, Vangala M, Riley DR, Arze C, White JR, White O, Fricke WF: CloVR: a virtual machine for automated and portable sequence analysis from the desktop using cloud computing. BMC Bioinforma 2011, 12:356. BioMed Central Full Text
• [48]Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI: QIIME allows analysis of high-throughput community sequencing data. Nat Methods 2010, 7:335-336.
• [49]Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ: Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 2009, 75:7537-7541.
• [50]Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R: UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011, 27:2194-2200.
• [51]Wang Q, Garrity GM, Tiedje JM, Cole JR: Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 2007, 73:5261-5267.
• [52]White JR, Arze C, Team TC, Matalka M, White O: CloVR-16S: Phylogenetic microbial community composition analysis based on 16S ribosomal RNA amplicon sequencing - standard operating procedure, version 1.1. http://precedings.nature.com/documents/5888/version/2 webcite