Role of whole genome se­quenc­ing in food safety

Food safety is be­com­ing a public health pri­or­ity and gov­ern­ments are mak­ing ef­forts to de­velop poli­cies and reg­u­la­tory frame­works, es­tab­lish and im­ple­ment ef­fec­tive food safety sys­tems to en­sure that food pro­duc­ers and sup­pli­ers along the en­tire food chai

Progressive Grocer (India) - - Contents - By Dr. K. V. Satya­narayana

Ways to es­tab­lish and im­ple­ment food safety sys­tems for food sup­pli­ers to op­er­ate re­spon­si­bly and sup­ply safe food to con­sumers.

Food safety is be­com­ing a public health pri­or­ity and gov­ern­ments are mak­ing ef­forts to de­velop poli­cies and reg­u­la­tory frame­works, es­tab­lish and im­ple­ment ef­fec­tive food safety sys­tems to en­sure that food pro­duc­ers and sup­pli­ers along the en­tire food chain op­er­ate re­spon­si­bly and sup­ply safe food to con­sumers.

The epi­demi­o­log­i­cal in­ves­ti­ga­tion of a food-borne out­break, which in­cludes iden­ti­fi­ca­tion of pathogen, source at­tri­bu­tion, re­moval of con­tam­i­nated food items from the sup­ply chain and de­vel­op­ment of other in­ter­ven­tion strate­gies, de­pends on the abil­ity to sub­type the eti­o­log­i­cal agent at a high enough res­o­lu­tion to dif­fer­en­ti­ate re­lated from non-re­lated cases. The tra­di­tional phe­no­typic sub­typ­ing meth­ods used in­clude serotyp­ing, phage typ­ing and bio­typ­ing. Since 1990, the field of sub­typ­ing has been rev­o­lu­tion­ized with the ad­vent of molec­u­lar and Dna-based sub­typ­ing meth­ods, which al­low more sen­si­tive dis­crim­i­na­tion than the tra­di­tional meth­ods. Com­monly used molec­u­lar sub­typ­ing/geno­typ­ing meth­ods in­clude band­ing pat­tern-based Pulse Field Gel Elec­trophore­sis (PFGE), and DNA se­quenc­ing­based Multi Lo­cus Se­quence Typ­ing (MLST), and Mul­ti­ple Lo­cus Vari­able Number Tan­dem Re­peat Anal­y­sis (MLVA). The es­tab­lish­ment of Pulsenet in USA in 1996 and its ex­pan­sion as “Pulsenet In­ter­na­tional” was in­stru­men­tal in ad­vanc­ing the adop­tion of molec­u­lar sub­typ­ing for bac­te­rial food-borne dis­ease sur­veil­lance. Pulsenet se­lected PFGE as the pri­mary geno­typ­ing method­ol­ogy, which sub­stan­tially ad­vanced food-borne dis­ease sur­veil­lance and out­break in­ves­ti­ga­tion.

Whole genome se­quenc­ing (WGS)

Whole genome se­quenc­ing (WGS) is a lab­o­ra­tory pro­ce­dure that de­ter­mines the com­plete DNA se­quence in the genome of an or­gan­ism in one process. Re­cent ad­vances in se­quenc­ing tech­nolo­gies and bioin­for­mat­ics tools have made WGS a vi­able and ad­vanced so­lu­tion for epi­demi­o­logic in­ves­ti­ga­tion and sur­veil­lance of food-borne bac­te­rial pathogens. Due to its ad­van­tages over PFGE, WGS is now be­com­ing the pre­ferred method for or­gan­ism iden­ti­fi­ca­tion and com­par­i­son among iso­lates.

Ad­van­tages/ben­e­fits of WGS

• WGS pro­vides sig­nif­i­cantly im­proved sub­type dis­crim­i­na­tion as well as in­ter­pre­ta­tion of evo­lu­tion­ary re­lat­ed­ness of iso­lates • WGS can pro­vide lo­ca­tion speci­ficity for dis­ease out­break in­ves­ti­ga­tions, help in un­der­stand­ing how pathogens spread within and be­tween ge­o­graphic ar­eas

• WGS is be­com­ing in­ex­pen­sive and is eas­ier to use since it has iden­ti­cal sam­ple prepa­ra­tion for all pathogens • The data gen­er­ated by WGS yields ad­di­tional in­for­ma­tion on vir­u­lence fac­tors, an­tibi­otic re­sis­tance, mo­bile ge­netic el­e­ments and ge­o­graphic lo­ca­tion

Ap­pli­ca­tions of WGS in food safety (a) Reg­u­la­tors and public health agen­cies

Whole Genome Se­quenc­ing (WGS) has al­ready been used in a few coun­tries to sub­type com­mon food­borne pathogens wherein the high-res­o­lu­tion WGS sub­typ­ing data has en­hanced the out­break de­tec­tion and fa­cil­i­tated epi­demi­o­log­i­cal in­ves­ti­ga­tions. The high speci­ficity and sen­si­tiv­ity of WGS pro­vides greater con­fi­dence in reg­u­la­tory de­ci­sions made by au­thor­i­ties on food safety, public health etc.

(i) USA

US FDA is uti­liz­ing WGS since 2008 and is co­or­di­nat­ing ef­forts by fed­eral, state, and in­ter­na­tional public health agen­cies to se­quence pathogens col­lected from food-borne out­breaks, con­tam­i­nated food prod­ucts, and en­vi­ron­men­tal sources and make their ge­nomic se­quences pub­licly avail­able in a data­base called Genome­trakr. Genome­trakr, es­tab­lished by the FDA in late 2012, is the first dis­trib­uted net­work of labs to uti­lize WGS for pathogen iden­ti­fi­ca­tion and can be used to help pin­point the con­tam­i­na­tion sources of cur­rent and fu­ture out­breaks. As on Septem­ber 2016, this net­work has se­quenced more than 71,000 iso­lates, and closed more than 175 genomes.

Since 2012, FDA is us­ing WGS of food-borne pathogens for reg­u­la­tory pur­poses in var­i­ous ways (http://www.fda.gov): • Dif­fer­en­ti­at­ing sources of con­tam­i­na­tion, even

within the same out­break • De­ter­min­ing which in­gre­di­ent in a mul­ti­in­gre­di­ent food har­bored the pathogen as­so­ci­ated with an ill­ness out­break • Nar­row­ing the search for the source of a con­tam­i­nated in­gre­di­ent, even when the source is far off place • As a clue to the pos­si­ble source of ill­nesses - even be­fore a food has been as­so­ci­ated with ill­nesses by tra­di­tional epi­demi­o­log­i­cal meth­ods WGS and po­ten­tial role in FSMA com­pli­ance: Un­der US FDA’S new Food Safety Mod­ern­iza­tion Act (FSMA), food in­dus­try is ex­pected to have en­vi­ron­men­tal mon­i­tor­ing pro­grams (EMPS), es­pe­cially when ready-to-eat food is ex­posed to en­vi­ron­ment prior to pack­ag­ing, to ver­ify the ef­fec­tive­ness of their pro­cess­ing and san­i­ta­tion con­trols in their pro­cess­ing plants. With sev­eral re­cent cases, wherein FDA has linked en­vi­ron­men­tal pos­i­tives in fa­cil­i­ties from pre­vi­ous years with ill­nesses oc­cur­ring to­day, food in­dus­try is ex­pected to em­brace WGS tech­nol­ogy to check if they have res­i­dent strain of a pathogen in their ready-toeat prod­uct fa­cil­i­ties. Re­cently, Cal­i­for­nia based Clear Labs Inc. re­leased WGS test based on their pro­pri­etary next gen­er­a­tion se­quenc­ing (NGS) plat­form. The test will al­low food com­pa­nies in iden­ti­fy­ing pathogen strains in sam­ples, de­ter­mine how dif­fer­ent pathogen strains are evo­lu­tion­ar­ily re­lated, what re­gions they come from, and from which food groups they orig­i­nate.

Whole Genome Se­quenc­ing (WGS) has al­ready been used in a few coun­tries to sub­type com­mon food-borne pathogens wherein the high­res­o­lu­tion WGS sub­typ­ing data has en­hanced the out­break de­tec­tion and fa­cil­i­tated epi­demi­o­log­i­cal in­ves­ti­ga­tions.

(ii) Canada

In Canada, WGS is be­ing rou­tinely ap­plied in par­al­lel to other meth­ods like PFGE dur­ing clus­ter in­ves­ti­ga­tion or out­break re­sponse; to mon­i­tor trends in emerg­ing pathogens, anti-mi­cro­bial re­sis­tance and to iden­tify novel vir­u­lence fac­tors. Cana­dian Food In­spec­tion Agency is in the process of in­cor­po­rat­ing in­for­ma­tion from WGS into health risk as­sess­ments and epi­demi­o­log­i­cal sur­veil­lance and the ex­pan­sion of the Pulsenet Canada lab­o­ra­tory net­work refers to the tran­si­tion to in­clude WGS as a pri­mary sub­typ­ing method.

(iii) Europe

Public Health Eng­land (PHE) has been us­ing WGS rou­tinely as part of spe­cial­ist mi­cro­bi­ol­ogy and epi­demi­ol­ogy ser­vices and has ben­e­fit­ted from the ap­pli­ca­tion of WGS through im­prove­ments in sur­veil­lance and out­break in­ves­ti­ga­tion. In June 2014, a Wgs-based in­ves­ti­ga­tion by PHE iden­ti­fied the root cause of a Sal­monella out­break through eggs and pre­vented fur­ther out­breaks. By 2014, over 28,000 bac­te­ria and virus genomes were se­quenced in­clud­ing over 3,500 sal­monella genomes (www.gov. uk). PHE is also us­ing whole genome se­quenc­ing (WGS) to de­tect an­tibi­otic re­sis­tance genes. In Den­mark, food and en­vi­ron­ment mon­i­tor­ing for Lis­te­ria mono­cy­to­genes to­gether with health sur­veil­lance us­ing WGS was im­ple­mented in 2013.

(b) In­dus­try

In­dus­try play­ers like Mars, Nes­tle, Con­a­gra & Dupont are im­ple­ment­ing WGS in their own food safety pro­grams. In­dus­try can use WGS to mon­i­tor in­gre­di­ent sup­plies, to de­ter­mine the ef­fec­tive­ness of preven­tive and san­i­tary con­trols and de­ter­mine the per­sis­tence of pathogens in the en­vi­ron­ment. WGS can pro­vide a much faster re­sponse time for out­breaks and re­calls in the food in­dus­try.

In­ter­na­tional ef­forts on WGS for food safety

When com­pared to de­vel­oped coun­tries, the ap­pli­ca­tion of WGS for food safety man­age­ment in de­vel­op­ing coun­tries has been very lim­ited. In the de­vel­oped coun­tries, a well-es­tab­lished re­port­ing sys­tem used by public health agen­cies is re­sult­ing in the proper doc­u­men­ta­tion of in­ci­dence of food-borne ill­ness in pop­u­la­tions. This re­port­ing sys­tem is ab­sent or al­most neg­li­gi­ble in the de­vel­op­ing coun­tries and there is a need to es­tab­lish this re­port­ing/ sur­veil­lance sys­tem in the de­vel­op­ing coun­tries be­fore WGS can be ef­fec­tively used in food safety man­age­ment. The Food and Agri­cul­tural Or­ga­ni­za­tion (FAO) is lead­ing global ef­forts to build ca­pac­ity to de­tect, con­trol, and pre­vent food-borne dis­eases. As part of th­ese ef­forts, FAO con­vened a tech­ni­cal meet­ing on the im­pact of WGS on food safety man­age­ment within a “One Health Frame­work” in May 2016, which was at­tended by par­tic­i­pants from 50 coun­tries. “One Health Frame­work” ad­vo­cates the idea of fully con­nect­ing the clin­i­cal, food and farm en­vi­ron­ment as an in­te­grated whole rather than be­ing dis­con­nected parts.

Global out­reach for WGS in food safety will re­quire con­certed ef­forts and co­or­di­na­tion among var­i­ous or­ga­ni­za­tions like WHO, FAO, In­ter­na­tional Stan­dards Or­ga­ni­za­tion, (ISO) etc. There is a great need for train­ing in WGS tech­nol­ogy, its val­i­da­tion, har­mo­niza­tion, ef­fec­tive data-shar­ing and in­te­gra­tion across the global com­mu­nity.

In­dus­try play­ers like Mars, Nes­tle, Con­a­gra & Dupont are im­ple­ment­ing WGS in their own food safety pro­grams. In­dus­try can use WGS to mon­i­tor in­gre­di­ent sup­plies, to de­ter­mine the ef­fec­tive­ness of preven­tive and san­i­tary con­trols and de­ter­mine the per­sis­tence of pathogens in the en­vi­ron­ment.

How can In­dia use WGS for food safety

In­dian food in­dus­try has been adopt­ing var­i­ous Food Safety Man­age­ment Sys­tems (mostly pri­vate stan­dards like FSSC 22000, BRC, SQF, etc) largely to meet the cus­tomer/ ex­port coun­try re­quire­ments. US FDA is al­ready us­ing WGS in nar­row­ing its search for the source of a con­tam­i­nated in­gre­di­ent. As part of the 2012 Sal­monella out­break in­ves­ti­ga­tion, FDA found that the Sal­monella Bareilly DNA for the sam­ples tied to the 2012 out­break was very sim­i­lar to the Sal­monella Bareilly DNA iso­lated from shrimp that came from a pro­cess­ing plant in south­west In­dia sev­eral years ear­lier. With FSMA in force, US FDA is ex­pected to in­crease its use of WGS to link en­vi­ron­men­tal pos­i­tives in ready-to-eat prod­uct fa­cil­i­ties from pre­vi­ous years with ill­nesses oc­cur­ring to­day. In the next few years, there is pos­si­bil­ity of WGS be­com­ing manda­tory for ex­ports, specif­i­cally to few coun­tries hav­ing WGS sup­ported food sur­veil­lance sys­tems in place. This could af­fect the econ­omy of sev­eral coun­tries

in­clud­ing In­dia, which do not have this tech­nol­ogy for food safety.

There is an im­per­a­tive need for as­sess­ing In­dia’s readi­ness in de­ploy­ing WGS for food safety. For im­ple­ment­ing WGS, In­dia al­ready has good in­fra­struc­ture and pre-req­ui­sites in terms of se­quenc­ing ca­pac­ity, high ca­pac­ity stor­age & data anal­y­sis, good in­ter­net con­nec­tiv­ity and bio-in­for­mat­ics sup­port. There are sev­eral re­search in­sti­tutes in In­dia with WGS fa­cil­i­ties and re­search pro­grams. There are also a few pri­vate ser­vice providers for WGS, sup­port­ing re­search in agri­cul­ture and health care ar­eas.

The ma­jor chal­lenge for In­dia in adopt­ing WGS for food safety would be the data­base con­struc­tion and the val­i­da­tion since food-borne dis­ease sur­veil­lance in In­dia is lack­ing. Food-borne dis­ease sur­veil­lance is es­sen­tial for mon­i­tor­ing of food-borne dis­eases and their trends, eval­u­at­ing strate­gies for the con­trol and pre­ven­tion of food-borne dis­eases, de­tec­tion, etc. In­dia lacks sys­tem­atic stud­ies in un­der­stand­ing the types of foods in­volved and the agent caus­ing the dis­ease. Most of the food-borne dis­ease out­breaks in In­dia go un­re­ported or are not in­ves­ti­gated and some of them are only no­ticed af­ter a ma­jor health is­sue or eco­nomic loss has oc­curred.

The Food Safety and Stan­dards (FSS) Act 2006, stip­u­lates the du­ties and func­tions of Food Safety and Stan­dards Author­ity of In­dia (FSSAI), which in­clude reg­u­lat­ing and mon­i­tor­ing the man­u­fac­ture, pro­cess­ing, dis­tri­bu­tion, sale and im­port of food so as to en­sure safe and whole­some food. As per the Act, the Food Author­ity shall also search, col­lect, col­late, an­a­lyze and sum­ma­rize rel­e­vant sci­en­tific and tech­ni­cal data, par­tic­u­larly re­lat­ing to (i) food con­sump­tion and the ex­po­sure of in­di­vid­u­als to risks re­lated to the con­sump­tion of food; (ii) in­ci­dence and preva­lence of bi­o­log­i­cal risk; (iii) con­tam­i­nants in food. The Food Author­ity shall also pro­mote, co-or­di­nate and is­sue guide­lines for the de­vel­op­ment of risk as­sess­ment method­olo­gies and mon­i­tor and con­duct and for­ward mes­sages on the health and nu­tri­tional risks of food to the gov­ern­ment.

Epi­demi­o­log­i­cal, en­vi­ron­men­tal, and clin­i­cal com­po­nents should in­te­grate closely for car­ry­ing out food-borne dis­ease in­ves­ti­ga­tion. Rou­tine col­lec­tion and anal­y­sis of clin­i­cal, food, and en­vi­ron­men­tal sam­ples is a pre-req­ui­site for im­ple­ment­ing WGS. Hope­fully, FSSAI will co­or­di­nate with the agen­cies con­cerned and take ini­tia­tives in build­ing ba­sic epi­demi­ol­ogy, sur­veil­lance, food mon­i­tor­ing and test­ing in­fra­struc­ture for im­ple­ment­ing ef­fec­tive food safety sys­tems in the coun­try.

Con­clu­sion

Whole Genome Se­quenc­ing is a pow­er­ful tool that can be used for a wide range of public health and food safety ap­pli­ca­tions. WGS is now be­ing used to mon­i­tor emerg­ing food pathogens and un­der­stand how pathogens spread within and be­tween ge­o­graphic ar­eas. It can help the food in­dus­try to mon­i­tor in­gre­di­ent sup­plies, de­ter­mine the ef­fec­tive­ness of preven­tive and san­i­tary con­trols and de­ter­mine the per­sis­tence of pathogens in the en­vi­ron­ment. The high speci­ficity and sen­si­tiv­ity of WGS pro­vides greater con­fi­dence in reg­u­la­tory de­ci­sions made by au­thor­i­ties on food safety, public health as well as de­ci­sions made by food in­dus­try. How­ever, ba­sic food-borne dis­ease sur­veil­lance sys­tems need to be es­tab­lished in de­vel­op­ing coun­tries in­clud­ing In­dia, be­fore WGS can be used for food safety.

There is an im­per­a­tive need for as­sess­ing In­dia’s readi­ness in de­ploy­ing WGS for food safety. For im­ple­ment­ing WGS, In­dia al­ready has good in­fra­struc­ture and pre­req­ui­sites in terms of se­quenc­ing ca­pac­ity, high ca­pac­ity stor­age & data anal­y­sis, good in­ter­net con­nec­tiv­ity and bio-in­for­mat­ics sup­port.

The writer is a Se­nior Man­ager at Sathguru Man­age­ment Con­sul­tants and leads the Food Pro­cess­ing and Re­tail prac­tice. He can be reached at satya­narayanak@sathguru.com.

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