Part 2: What are the best control strategies?Dr. Bassam A. Annous, Eastern Regional Research Center, USDA–ARS–NEA, and Dr. Ruth Eden, BioExpert. Biofilms are usually formed in a wet environment and in the presence of nutrients. Once biofilms are formed, the cleaning of the food and food contact surfaces becomes more difficult to remove the extracellular polymeric substances (EPS). Therefore, prevention of biofilm formation, using regularly scheduled cleaning and disinfecting protocols is an important first step in preventing cells from attaching and forming biofilms on surfaces. High-temperature washing can reduce the need for the physical force required to remove biofilms. Chemical cleaners suspend and dissolve food residues by decreasing surface tension, emulsifying fats, and denaturing proteins. The mechanism by which cleaning agents remove EPS associated with biofilms has not been determined.
Fruit and Vegetable SurfacesConventional methods of washing fresh produce with hypochlorite or other sanitizing agents cannot assure microbial safety because they can only achieve 1-2 log reduction. This is because of many enteric pathogens, such as Escherichia coli O157:H7, cause illness at very low infectious doses. For apples, using water, detergents or sanitizing agents, produced a maximum of 3-log (99.9%) reduction in the levels of E. coli. The same treatment using brush washer surprisingly gave less than a 1-log (90%) reduction in E. coli.
A commercial-scale surface pasteurization treatment developed at ERRC (Annous, Burke, and E. Sites), resulted in 4-log (99.99%) reduction in the population of Salmonella Poona on the surface of artificially contaminated cantaloupe. The process involved the immersion of melons in water at 168.8°Fahrenheit for three minutes and then rapidly cooling them. This pasteurization process not only enhances the safety of the fruit but increases the product shelf life by reducing the native microflora that may cause spoilage.
EquipmentBacterial cells could attach and form biofilms on food processing equipment. The complexity of processing equipment makes it difficult to remove and/or inactivate the bacterial cells in biofilms on food processing surfaces. Since it is extremely difficult to remove these biofilms, food processors should prevent biofilm formation in the first place. This could be done by developing and maintaining a thorough sanitation regiment to help prevent a biofilm layer from attaching to equipment surfaces. Product residues due to spills or debris in the facility support bacterial proliferation and subsequent biofilm formation. Consequently, regular removal of food residues is a key to preventing biofilm formation. Oko, 2013 suggested three important steps to removing biofilm in a food processing facility: (i) Cleaning with appropriate sanitizing agents at the required concentrations (ii) Allowing enough exposure time at the appropriate temperature (iii) Applying mechanical action This combination is claimed to penetrate and/or remove the biofilm, and thereby to kill the embedded bacterial cells. An effective cleaning procedure would break up or dissolve EPS allowing sanitizers to gain access to viable bacterial cells. Alkaline cleaners, especially those with chelators like EDTA, are more effective at removing biofilms than acidic cleaners. Bacteria become far more susceptible to sanitizers once the biofilm matrix has been destroyed, certain enzymes have been proven effective in disrupting EPS matrixes, thus allowing for the removal of biofilms. Recently, novel methods that can serve as alternatives to the current methodology for the disinfection of microbial contamination, such as essential oils and bacteriophages have been successfully tested. Poly ethylene glycol (PEG) has been shown to inhibit protein adsorption and bacterial attachment to surfaces. Cold plasma was used to deposit PEG-like structures on the surfaces of stainless steel 304 and 316L using 12-crown-4 ether and tri (ethylene glycol) dimethyl ether (triglyme), and ethylene glycol divinyl ether as starting materials. The plasma modified surfaces significantly reduced biofilm formation by about 80%. When 1% beef hot dog was added to the base medium, biofilm formation on stainless steel 304 was reduced further. Plasma modification of the surfaces did not interfere with the efficacy of cleaning by the chlorinated alkaline detergent. Newer physical methods of biofilm removal include super-high magnetic fields, ultrasound treatment, high pulsed electrical fields, combined use of high pulsed electrical fields in conjunction with organic acids, and low electrical fields alone or in combination with biocides, such as silver, carbon, platinum, and antibiotics.
SummaryMost bacterial cells in nature exist in biofilms instead of planktonic single cells. Organic and inorganic material (nutrients) attach to surfaces of food and/or equipment and thus creates a conditioning layer whereby microorganisms attach to. Microbial cells then start secreting EPS that further help in the attachment of the biofilm to the food and food processing surfaces. The ESP formation acts as a barrier from sanitizing compounds, making the biofilm stronger. Bacterial activity within the biofilm community can be coordinated through cell-to-cell signaling (Quorum sensing). Biofilm formation is associated with many foodborne outbreaks. As a result, biofilm has become a problem in food industries as it renders its inhabitants resistant to antimicrobial agents and cleaning agents. The growth of biofilms in food processing environments leads to an increased opportunity for microbial contamination of the processed product. Pathogenic microorganisms in biofilms are the major source of food contaminations. Biofilms were created by various bacteria on fruit and vegetable surfaces, on various meats, and especially on all types of processing equipment. Once created the biofilm is very difficult to remove, and offer significant protection to the bacteria residing in it. Breaking up EPS is important in eliminating the biofilm protection and making the bacterial cells more susceptible to the cleaning sanitizers. Several novel methods are available to better clean surfaces containing bacteria in biofilms.
The RecallsSince our last report on July 23, there have been several additional recalls associated with the outbreak of Salmonella in papayas. On July 26 after many people got sick, Grande Produce issued a recall for its imported papayas from the Carica de Campeche farm in Mexico. This farm seems to be the primary source of the outbreak. On August 4, a second papaya recall was issued by Agroson’s LLC, for more than 2,000 boxes of Cavi-brand Maradol Papaya imported from the same Mexican farm. A few days later, a third papaya recall was issued by Freshtex Produce. These papayas sold under the Valery brand were distributed in Illinois. Carica de Campeche farm produced papayas under the following brands: Caribeña, Cavi, and Valery. The FDA first recalled the Maradol papayas from the Carica de Campeche farm in Mexico.
Carica de Campeche farm under the brand Caribeña: This first recall came after extensive testing and trace back. Papayas from the Carica de Campeche farm tested positive for Salmonella Kiambu, Salmonella Thompson, Salmonella Agona, Salmonella Senftenberg, and Salmonella Gaminara. The Caribeña brand was distributed by Grande Produce between July 10 and 19, 2017.
Cavi brand papayas distributed by Agroson’s: Agroson’s LLC, recalled certain Maradol Papaya Cavi Brand, grown and packed by Carica de Campeche. The papayas were distributed on July 16-19 and were available to consumers until July 31. No illness has been reported due to the Maradol Papaya Cavi Brand, and the recall was voluntary in cooperation with the FDA.
Valery brand distributed by Freshtex: The FDA announced that Freshtex Produce of Alamo, TX was voluntarily recalling “Valery” brand Maradol Papayas grown and packed by Carica de Campeche. The papayas were distributed to the State of Illinois from July 10 to July 13, 2017. No illness has been reported. The FDA increased its testing to see if papayas from other farms from Mexico could be contaminated. More brands and distributors are expected to be linked to the investigation.
The OutbreakThe CDC found that as of August 9 a total of 141 people were infected with the outbreak strains of Salmonella Kiambu (51) or Salmonella Thompson (90) in 19 states. Among 103 people with available information, 45 (44%) have been hospitalized. The data from the testing laboratory and from epidemiological investigation indicated that the most likely source of the outbreak was Maradol papayas from Carica de Campeche farm in Mexico.
Why did it Happen?Papayas from Mexico are being screened at the border for Salmonella since the outbreak in 2011, by a third party laboratory. Only papayas that have tested negative for Salmonella are allowed into the USA. The question needs to be asked: how was it possible that the contaminated papayas were able to get into the USA? As shown in our recent post, research indicated that bacteria can attach and colonize on the surfaces of plants, ultimately forming biofilms. Bassam A. Annous 2005 shows Salmonella produces fimbriae and cellulose, commencing biofilm formation, which helps Salmonella attach and colonize on melon and cantaloupe surfaces. Once attached the cells survive better on the surface. As mentioned in our previous post, the outbreaks can be due to the ability of Salmonella to attach or internalize into fruits. Survival and multiplication of Salmonella on fresh fruits is considerably increasing once the protective epidermal barrier has been broken. FSMA is designed to help minimize the risk of illness from foodborne pathogens in fresh fruits, and include requirements for water quality, employee hygiene, and equipment and tool sanitation. However, to date, all these new measures did not prevent the current papaya outbreak.
The RecallThe Department of Agriculture’s Food Safety and Inspection Service (FSIS-USDA ) announced that Good Food Concepts, LLC., a Colorado Springs, Colorado recalled 1,290 pounds of raw beef products, processed Aug. 3 and Aug. 4, 2017, due to potential contamination with E. coli O26. The contamination was discovered on Aug 5, 2017, by the company who had notified the FSIS inspection. A lot of carcasses received from Callicrate Ranch on July 31, 2017, were found to be contaminated with STEC (Shiga toxin producing E. coli) E. coli O26. Good Food Concepts company and FSIS are concerned that some beef may have been frozen and in consumers’ freezers.
Pathogenic E. coliThe most prevalent strain of E. coli causing recalls and illnesses is E. coli O157:H7. However, there are seven serotypes of E. coli that cause most of the food recalls and diseases, called the Top 7. They include: in addition to O157:H7 also O26, O45, O103, O111, O121, and O145.
E. coli O26In the recent past, E. coli O26 resulted in several recalls and illnesses. E. coli O26 is one of the six most common strains of non-O157 E. coli found to cause foodborne illnesses. It is difficult to identify in the laboratory the O26, in particular by the new rapid methods, because most laboratories are looking specifically for E. coli O157:H7. However, it is a pathogenic strain that caused disease. E. coli STEC O26 shows that the extensive genetic diversity and pathogenic clonal subgroups can emerge soon.
Disease SymptomsSymptoms usually begin two to eight days after exposure. People infected with O26 often develop bloody diarrhea, dehydration, and abdominal cramps. Most people recover within a week. However, some patients can develop hemolytic uremic syndrome or HUS. Children and older people, with suppressed immune systems, are more susceptible to HUS.
Recall due to E. coli O26 by Costco in CanadaThe Canadian Food Inspection Agency (CFIA) announced a few days ago that Costco Wholesale Canada Ltd. recalled Gold Coast brand Broccettes – Broccoli Florettes due to possible E. coli O26 contamination. The products had been sold in British Columbia, but no illnesses were reported in association with these commodities.
Previous Recalls Due to E. coliE. coli O26 and O21were found in flour from General Mills. The FDA investigation identified General Mills flour as the source of this outbreak which led to a voluntary recall in May 2016. On September 29, 2016, the CDC reported (https://www.cdc.gov/ecoli/2016/o121-06-16/index.html) that 63 people were infected with the outbreak strains of E. coli O121 and O26 in 24 states. Illnesses started on dates ranging from December 21, 2015, to September 5, 2016. Seventeen ill people were hospitalized, and one person developed the hemolytic-uremic syndrome.
Multistate Outbreak of E. coli O26 Infections Linked to Chipotle Mexican Grill RestaurantsThe FDA and the Centers for Disease Control and Prevention (CDC) along with state and local officials investigated two separate outbreaks of E. coli O26 infections. Both were linked to Chipotle Mexican Grill restaurants in several states The CDC reports indicated that as of January 27, 2016, a total of 55 people had been infected with the outbreak strain of E. coli O26. Eleven states were involved: California (3), Delaware (1), Illinois (1), Kentucky (1), Maryland (1), Minnesota (2), New York (1), Ohio (3), Oregon (13), Pennsylvania (2), and Washington (27). There have been 21 reported hospitalizations. The majority of these cases were reported from Oregon and Washington during October 2015. Chipotle Mexican Grill closed 43 restaurants in Washington and Oregon in early November 2015 in response to the initial outbreak.
National Meat and Provisions Recalls Beef and Veal Products Due To Possible E. Coli O26 ContaminationThe FSIS-USDA announced the recall of approximately 2,349 pounds of beef and veal products that may have been contaminated with E. coli O26, on Oct. 14, 2016. The products were shipped to a distributor, as well as hotels, restaurants, and other institutions in Louisiana.
Part 1: What are Biofilms?In nature, most bacteria do not exist as suspended (planktonic-free floating) cells. Bacteria live in a group (mass of bacterial cells) attached to each other and to surfaces, in a biofilm form. A biofilm is as a complex community of microorganisms, embedded in self-created extracellular polymeric substances (EPS). Therefore, the biofilm is a microbial population adherent to each other and to surfaces or interfaces enclosed in the matrix. In this complex biofilm network of EPS, the bacterial cells perform less as individual cells and more as a collective living system, frequently creating channels to deliver nutrients and water to the cells located inside the biofilm. Bacteria create biofilm as a protection mechanism, for better survival in the environment. Cells in a biofilm are more resistant to cleaning and disinfection processes in the food industry. The bacteria in the biofilm attaches so firmly to the equipment’s surface that it becomes resistant to conventional sanitation procedures used by the food industry. Various techniques such as molecular methods, chemical methods, and physical methods have been used to better understand the complex mechanism of biofilm formation, and to get an insight into how to create a process that will eliminate the biofilm formation and/or inactivate cells within the biofilm. Moisture and nutrients from food (organic and inorganic material) are commonly found on production lines. These two elements bond together to create a conditioning layer. This layer allows for the initial attachment of the bacterial cells to the surface of the layer and the secretion of EPS. The production of ESP enhances the biofilm attachment to the food contact surfaces and protects the cells within the biofilm from the external stresses such as sanitizing agents.
How is Biofilm Formed?The formation of biofilm can be described as a stepwise process, as shown in Figure 1, consisting of:
- Initial reversible attachment of the planktonic (free-floating) bacteria cell to the surface
- Irreversible attachment by the production of EPS
- Bacteria multiplication and development of biofilm structure
- Development of a microcolony covered by mature biofilm, stabilizing the microcolony from environmental stress
- Dispersion of cells from the biofilm into the surrounding, and the return of cells to their planktonic form.