Research article Topical Sections

A machine learning algorithm for identifying and tracking bacteria in three dimensions using Digital Holographic Microscopy

  • Digital Holographic Microscopy (DHM) is an emerging technique for three-dimensional imaging of microorganisms due to its high throughput and large depth of field relative to traditional microscopy techniques. While it has shown substantial success for use with eukaryotes, it has proven challenging for bacterial imaging because of low contrast and sources of noise intrinsic to the method (e.g. laser speckle). This paper describes a custom written MATLAB routine using machine-learning algorithms to obtain three-dimensional trajectories of live, lab-grown bacteria as they move within an essentially unrestrained environment with more than 90% precision. A fully annotated version of the software used in this work is available for public use.

    Citation: Manuel Bedrossian, Marwan El-Kholy, Daniel Neamati, Jay Nadeau. A machine learning algorithm for identifying and tracking bacteria in three dimensions using Digital Holographic Microscopy[J]. AIMS Biophysics, 2018, 5(1): 36-49. doi: 10.3934/biophy.2018.1.36

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  • Digital Holographic Microscopy (DHM) is an emerging technique for three-dimensional imaging of microorganisms due to its high throughput and large depth of field relative to traditional microscopy techniques. While it has shown substantial success for use with eukaryotes, it has proven challenging for bacterial imaging because of low contrast and sources of noise intrinsic to the method (e.g. laser speckle). This paper describes a custom written MATLAB routine using machine-learning algorithms to obtain three-dimensional trajectories of live, lab-grown bacteria as they move within an essentially unrestrained environment with more than 90% precision. A fully annotated version of the software used in this work is available for public use.


    1. Introduction

    Microbes can either be desirable or undesirable in foods and may have positive or negative impacts on impact human’s health. Undesirable microbes that are pathogenic enter the food chain either through contamination from soil, water, air or contact with unhygienic equipments including humans. The most common types of bacteria that have caused outbreaks resulting in food poisoning are Campylobacter spp, Salmonella spp, Escherichia coli, Staphylococcus aureus, Toxoplasma, Clostridium perfrigens and Listeria monocytogenes. L. monocytogenes is a facultative, Gram-positive, intracellular and ubiquitous bacterium which is widespread in the soil environment, decaying vegetation, water and may be part of the fecal flora of many mammals, including healthy human adults [1]. Out of the currently known seventeen species of Listeria in the natural environment, the two pathogenic species are L. monocytogenes and L. ivanovii, the remaining non-pathogenic species are L. welshimeri, L. innocua, L. seeligeri, L. grayi, L. marthii, L.aquatic, L. booriae, L. cornellensis, L. fleischmannii, L. floridensis, L. grandensis, L. newyorkensis, L. riparia, L. rocourtiae and L. weihenstephanensis [2,3].

    Listeriosis results when a person is infected and develops symptoms of Listeria infection, the illness is associated with high mortality [4]. Before the 1980s, human listeriosis was an obscure disease although there were large outbreaks of considerable morbidity and mortality [5]. Listeriosis was first recognized as a foodborne pathogen in the early 1980s and since then the disease has risen among human and animals in Europe and North America. It is the second most common cause of death from food borne disease in the US &France andthe fourth in England & Wales [6]. Most of the people at high risk of listeriosis are pregnant women, newborn babies, elderly people (over 60 years). The bacterium is known to cross the intestinal barrier, disseminate via the bloodstream and can reach the liver, spleen, central nervous system and foetus [7]. Since the early 1980s when the food pathogen status of Listeria was established, there have been rigorous efforts by the food industry and regulatory agencies to control the growth of this ubiquitous organism in food production, processing, distribution and consumption chain [8].

    L.monocytogenes, unlike most other foodborne pathogens, can grow at refrigeration temperatures and in foods with fairly low moisture content and high salt concentration. This ability to persist and multiply in the food environment makes L.monocytogenesespecially difficult to control. They have been reported in smoked seafoods, hot dogs, soft cheeses from unpasteurised milk, and deli-meats. With prompt investigations into Listeria contaminations, adequate reports of listeriosis cases and their interventions will help policy makers in ensuring the safety of consumers from Listeria outbreaks worldwide. For instance, an investigation into a large multi-state outbreak of listeriosis reported in the US traced to turkey deli-meats in 2002 helped policy makers to make changes that helped to prevent future L. monocytogenes contamination in ready-to-eat meat (RTE) and poultry products [9]. By ensuring that the food contact surfaces where RTE foods are processed or served in retail or home environment is clean and sanitized will further help to prevent Listeria cross-contamination.

    This article reviews the outbreaks L. monocytogenes in foods with special emphasis on deli meats and cheese worldwide. The first section is a brief introduction on its characteristics as a foodborne pathogen. Section 2 highlights some of the outbreak of Listeria in several foods in many countries worldwide in the last twenty years. In addition, this section reports on the prevalence of these outbreaks in deli-meats and cheese. Section 3 describes their identification, sampling methods and characterization in foods. Section 4 describes efforts towards the prevention of Listeria and other containment strategies.


    2. Global Outlook of Listeria Outbreaks (1995 to 2016)

    Listeria are frequentlypresent in raw foods of both plant and animal origin around the world and they are also present in cooked foods due to post-processing contamination. They have been isolated in foods such as raw and pasteurized fluid milk, cheeses (particularly soft-ripened varieties), ice cream, raw vegetables, fermented raw meat and cooked sausages, raw and cooked poultry, raw meats, and raw and smoked seafood [10]. RTE foods such as cold cuts or deli meats, cheeses and other dairy products are ideal sources for contamination since they are not cooked before consumption. In food handling operations, L. monocytogenes are able to grow at refrigeration temperature between 4 °C to 10 °C, freezing temperatures have little detrimental effect on this ubiquitous microbe. In addition, they can still survive when the desired temperature is not reached during pasteurization especially in large packages. L. monocytogenes have also been isolated from food processing environments, particularly those that are cool and wet.

    L. monocytogenes was estimated to infect 23,150 people globally and killed 5,463 (23.6%) of the infected people in 2010 [11]. However, most of the cases involving Listeria are not reported in developing countries due to ineffective control or surveillance strategies. This makes it problematic in having a comprehensive global outlook on the outbreaks of Listeria and listeriosis in humans. Snapshots of some cases involving Listeria contamination worldwide from 1995 to 2016 are shown in Table 1. These cases were either from the raw food samples, the processing plants of such foods or the processed foods in different countries. The method of detection varied from country to country. In the year 2016, a legal tussle is being waged against a major US food company in the contamination of salad products by L. monocytogenes.

    Table 1.Select reports of Listeria outbreaks globally from 1995 to 2016
    Country Year Food source No. of cases References
    France1995Cheese36[12]Goulet et al.,1995
    Israel1995-1999N.A. aN.A.[13]Siegman-Igra et al.,2002
    Italy1997Corn salad2[14]Aureli et al.,1997
    Sweden1997Rainbow trout9[15] Ericsson et al.,1997
    Finland1998-1999Butter25[16]Lyytikäinen et al.,2000
    USA1998-1999Frankfurter108[17]Kathariou et al.,2006
    France2000Salmon fishN.A.[18] Dauphin et al.,2001
    Japan2001Cheese86[19]Makino et al.,2005
    France2002Raw sausage11[20]de Valk et al.,2005
    Canada2002Soft cheese130[21]McIntyre et al.,2002
    USA2004Deli meats108[22]Mead et al.,2006
    Switzerland2005Cheese10[23]Bille et al.,2005
    Germany38899Jellied pork189[24]Pichler et al.,2009
    Austria2008Milk and cheese12[25]Koch et al.,2010
    Canada2008Deli meats57[26]Currie et al.,2015
    Netherlands2008-2013N.A.406[27]Friesema et al.,2015
    Austria2009-2010Acid curd cheese25[28]Pichler et al.,2011
    Australia2010Chicken wraps36[29]Popovic et al.,2010
    USA2010Celery10[30]Gaul et al.,2013
    Belgium2011Cheese12[31]Yde et al.,2012
    USA2011Cantaloupe147[32]McCollum et al,2013
    South Africa2012RTE foods51[33]Nyenje et al.,2012
    South Korea2014Ham and sausage25[34]Park,M. et al.,2014
    Macedonia2014Home made sausage7[35]MCID,2014
    UK2014N.A.169[36]Whitworth,2015
    USA2014-2015Caramel apples35[37]CDC,2015
    USA2016Lettuce salad19[38]CDC,2016
    anot available
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    These reported cases were from clinical listeriosis tests on patients or through a national survey.

    L. monocytogenes are known to have13 distinct O-antigenic patterns, which comprises the serovars; 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, 4ab, and 7 [39]. However, the pathogenicity of L. monocytogenes are divided into three lineages [40]; Lineage I (Highly pathogenic, with epidemic clones and responsible for most outbreaks e.g. 1/2b, 3b, 4b, 4d, 4e), Lineage II (medium pathogenic, sporadic cases e.g. 1/2a, 1/2c, 3c, 3a) and Lineage III (low and rarely cause human diseases e.g. 4a, 4c). These strains of L. monocytogenes show differences in their virulence and epidemic potential, it was shown that a limited subset of clones that mostly belong to serotypes 1/2a, 1/2b and 4bwere responsible for a large part of clinical cases worldwide, they also account for 98% of listeriosis outbreaks [39,41].

    Although the disease burden of listeriosis on population level is low, on individual level the impact is high, largely due to severe illness and a high case fatality [27]. Despite this low incidence of listeriosis that represents less than 0.1% of all food-borne illnesses they cause infections with very high mortalities 20 to 30% deaths [42,43].

    The approximate infective dose of L. monocytogenes was estimated to be 10-100 million colony forming units (CFU) in healthy hosts, and only 0.1-10 million CFU in people at high risk of infection. On the mild end of the spectrum, listeriosis usually consists of the sudden onset of fever, chills, severe headache, vomiting, and other influenza-type symptoms [44]. Along these same lines, the US Centre for Disease Control notes that infected individuals may develop fever, muscle aches, and sometimes gastrointestinal symptoms such as nausea or diarrhea [45].

    Most cases of listeriosis are sporadic and have been reported in high-income countries, where incidence is quite low but fatality rate is high. Most healthy adults and children who consume Listeria contaminated food experience only mild to moderate symptoms. The infection is usually self-limited, since in healthy hosts, exposure to Listeria stimulates the production of tumor necrosis factor and other cytokines, which activate monocytes and macrophages to eradicate the organism. Few people with normal immune function go on to have more severe, life-threatening forms of listeriosis, which is often characterized by septic shock, meningitis and encephalitis [1].With ageing population and many immune-compromised individuals in high income countries, listeriosis has become a major issue. Since the US Center for Disease Control and Prevention(CDC) began tracking outbreaks in the 1970s, L monocytogenesis the third most costly foodborne pathogen in the USA per case in 2010, after Clostridium botulinum and Vibrio vulnificus [46]. The global burden of human listeriosis was estimated for 2010 to result in 172,823 disability-adjusted life years(DALYs) [11]. DALYs are useful in comparing diseases and health conditions thereby it will help policy makers to allocate resources in the prevention of listeriosis [47]. It was estimated that the annual benefit from safety measures of L. monocytogenes in the USA far exceeds its cost. The estimated benefit and cost was US2.3billionto22billion,andUS 0.01 billion to 2.4 billion respectively [48]. Foodborne outbreaks due to severe pathogens, such as L. monocytogenes and those that result in product recalls, are typically the most costly from the individual and societal perspectives. In Canada, the costs that were associated with the cases of Listeria outbreaks (including medical costs, nonmedical costs, and productivity losses) and those incurred by the implicated plant and federal agencies responding to the outbreak were estimated to be nearly $ 242 million Canadian dollars [49].

    Some RTE products may not undergo thermal or other processing sufficient to inactivate L. monocytogenes. In those products receiving a listericidal treatment, the presence of the pathogen is generally associated with recontamination from environmental sources prior to final packaging. Other RTE foods may be contaminated at the point of sale, for example, due to slicing of processed meats. Within the home, opened packages may be contaminated from L. monocytogenes present within the refrigerator or in other refrigerated foods, from the kitchen environment or from family members. In a risk assessment by the U.S. Food and Drug Administration (FDA) and the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS), deli meats were identified as having the highest predicted relative risk for causing listeriosis cases and deaths among 23 categories of RTE foods that are potentially contaminated with L. monocytogenes, on both a per serving and per annum basis. The risk assessment estimated that about 90% of human listeriosis cases in the United States are caused by consumption of contaminated RTE deli meats [50].


    2.1 Listeria in Deli meats

    Deli shortened from delicatessen is a delicacy or specialty food that is popular in many developed countries around the world.

    The three major categories of deli meats are 1) freshly cooked meat with salt and spices as in turkey and roast beef 2) moderately processed, ground meat that has been emulsified into a soft cake-like batter where flavors, additives, binders are added, it is then cased in a cylindrical package and cooked for several hours as in turkey, bologna and chicken deli 3) very processed, as the second category but aged for several days and fermented as in hot dogs, liver sausage [51].

    The prevalence of L. monocytogenes outbreaks in deli meats and other meat products in 16 countries are shown in Table 2.

    Table 2.Prevalence of L. monocytogenes outbreaks in deli meats and meat products
    Country Product Prevalence (%) Reference
    Canada frankfurters & wieners 5 [52] Bohaychuk et al., 2006
    China ready-to-eat meat 1.4 [53] Yang et al., 2016
    Spain pate 5.4 [54] Dominguez et al., 2001
    Switzerland beef 6.3 [55] Fantelli & Stephan, 2001
    Belgium cooked meat products 1.1 [56] Uyttendaele, 2009
    Sweden meat products 1.2 [57] Lambetz, et al., 2012
    Northern Spain store packaged deli-meat 8.5 [58] Garrido et al., 2009
    Estonia raw meat products 18.7 [59] Kramarenko et al., 2013
    UK RTE salad with meat 6.0 [60] Little et al., 2009
    Spain RTE cooked ham 12.5 [61] Cabedo et al., 2008
    Spain RTE dried sausage 16.9 [61] Cabedo et al., 2008
    Italy vacuum packed salami 20.5 [62] Di Pinto et al., 2010
    China RTE meat products 7.4 [63] Yu & Jiang, 2014
    Greece RTE meat products 1.9 [64] Materagas et al., 2010
    USA deli meats 0.4 [65] Pradhan, et al., 2011
    Trinidad & Tobago RTE deli meats 8.1 [66] Syne et al., 2011
    Korea RTE kimbab 6.7 [67] Cho et al., 2008
    Mexico RTE turkey frankfurters 11.7 [68] Casteneda et al., 2013
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    The incidents of nationwide or multi-state Listeria outbreaks linked to deli-meats are commonly reported in Canada and USA. In 2004, there was a nationwide outbreak in 24 states of the US with 108 reported cases that led to 14 deaths, 4 miscarriages in pregnant women [22]. In nine US states, there were 54 cases with 8 deaths reported in 2002 [9]. In 2000, 30 cases were also reported in eleven US states, with 4 deaths and 3 miscarriages [69]. In 2008, 57 cases of listeriosis and 24 deaths in Canada were linked to contaminated delicatessen meat from one meat processing plant [26].

    In 2003, there was a recall of ready-to-eat (RTE) meat products by a large processing plant in Trinidad as a result of contamination by L. monocytogenes. Of the 32 processed RTE meat products tested from this plant, 11 (34.4%) were positive for Listeria spp., E. coli, Salmonella spp. and Campylobacter spp. either in combination or singly [70].

    The USDA reported that approximately 83% of Listeria cases and deaths in the US are associated with deli meats sliced and packaged at retail outlets [71]. Mechanical slicers were reported to be an important source of Listeria cross-contamination in deli meats at retail outlets. It was recommended that these outlets should hold their products at a temperature of 5 °C or below, maintain person hygiene and wear personal gloves while serving customers, use growth inhibitors and maintain a log on the methods and frequency of cleaning surfaces [50]. The Food and Drug Administration (FDA) Food Code specified a minimum frequency of every four hours for slicers to be fully disassembled, cleaned, and sanitized by retailers to reduce Listeria illnesses and outbreaks [72]. Further details on prevention and intervention strategies are discussed in section 4.


    2.2 Listeria in Cheese

    Foods can be contaminated after processing by the introduction of unpasteurized materials. This can occur during the preparation of some cheeses especially when unpasteurized raw milk is used. Listeria can also be spread by contact with contaminated hands, equipments and counter tops. The centralized production of prepared RTE food products increases the risk of higher levels of contamination, since it requires that foods be stored for long periods at refrigerated temperature which favors the growth of Listeria. During the preparation, transportation and storage of foods, the organism can multiply to reach a threshold needed to cause infection. The ability to identify the critical control points in the manufacture of cheeses will help to prevent Listerial contamination and how it behaves under varying post-processing packaging conditions [73].

    It is important to adhere to strict hygienic operations, rigorous safety procedures as the equipments or additives may be a source of Listeria contamination. Artisan cheesemakers believe that raw milk makes tastier cheese, but when the raw milk is not pasteurized, the risk of Listeria contamination is greater [74].The prevalence of Listeria outbreaks in cheese from different countries are shown in Table 3.

    Table 3.Prevalence of Listeria monocytogenes outbreaks in cheese
    Country Product Prevalence (%) Reference
    Ethiopia cottage cheese 1.0 [75] Gebretsadik et al., 2011
    Portugal soft cheese 46 [76] Pintado et al., 2005
    Portugal hard & semi-hard cheese 24 [77] Guerra et al., 2001
    Spain soft cheese 1.0 [78] Vitas et al., 2004
    Turkey soft cheese 5.0 [79] Akpolat et al., 2004
    Turkey Tulum cheese 4.8 [80] Colak et al., 2007
    Brazil soft & semi-soft cheese 6.7 [81] Abrahao et al., 2008
    Iran white cheese 9.2 [82] Arslan & özdemir, 2008
    Mexico Quesco Fresco cheese 3.4 [83] Moreno et al., 2007
    Portugal soft cheese (from raw ewe’s milk) 46 [84] Pintado et al., 2005
    Turkey white cheese 2.4 [85] Aygun & Pehlivanlar, 2006
    Ethiopia cottage cheese 1.7 [86] Derra et al., 2013
    USA fresh soft cheese 0.17 [87] Gombas et al., 2003
    Sweden soft & semi-soft cheese 0.4 [58] Lambetz et al., 2012
    Spain RTE fresh salty cheese 1.3 [62] Cabedo et al., 2008
    Italy cream cheese 1.9 [63] Di Pinto et al., 2010
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    Sporadic cases of neonatal listeriosis have been reported in Japan. Since L. monocytogenes has been often isolated from foods in Japan, food-borne outbreaks potentially could have occurred. In 2001, L. monocytogenes serotype 1/2b was isolated from a washed-type cheese during routine Listeria monitoring of 123 domestic cheeses [18]. Similarly in Switzerland 10 cases of Listeria outbreaks in a locally made and distributed soft cheese (known as 'tomme') were reported in 2005. Out of the 10 cases, 8 were in older immune compromised patients who became ill with bacteraemia (three deaths), and two cases were in pregnant women who had septic abortion [24]. All the cases were due to a serotype 1/2a isolate with one of two pulsovars found by PFGE.


    3. Identification, Sampling and Characterization of L. monocytogenes

    The employed detection methods will play vital roles in a better understanding of the control programs and the interventions to be applied. In the identification of Listeria spp. based on Gram-staining, catalase, oxidase and motility tests, were used in the past as they can be distinguished by haemolysis test, CAMP test and sugar fermentation [88]. L. monocytogenesis is known to show a narrow β-haemolysison blood media and ferments D-rhamnose but not D-xylose. The CAMP test is positive with β-haemolytic Staphylococcus aureus, showing enhanced haemolysis at the intersection of the cultures of L. monocytogenes and S. aureus.L. monocytogenes can be distinguished from other Listeria spp. with commercial test kits such as API Listeria [89]. There are current modified rapid methods of enrichment, these include biosensory and bacteriophage based detection methods. These molecular tests are rapid and reproducible enabling multi-parameter testing with automation possibilities [90].

    Several methods are available for typing L. monocytogenes strains in the surveillance of foodborne disease outbreaks including serotyping, genoserotyping (PCR serogrouping), polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP), and pulsed-field-gel-electrophoresis(PFGE). PFGE has a high discriminatory power in outbreak surveillance, but it provides limited information about phylogenetic relationships and overall clonal structure [91].

    Even though the cosmopolitan distribution of L. monocytogenes is worldwide, the geographic distribution of its clones remains largely unknown. Sequence-based typing methods such as multi locus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST) have been used to classify isolates into high level groups (epidemic clones and clonal complexes), allowing researchers to group geographically and temporally unrelated isolates into a common ancestor strain [92]. The European Food Safety Association Panel on Biological Hazards recommended the application of molecular typing methods [93].

    Their recommendations include PFGE, which was established as the gold standard approach for epidemiological studies in foodborne outbreaks. PFGE has the advantage of being widely used for small-scale epidemiology, listeriosis surveillance and outbreak investigations but it is considered to be time-consuming and requires stringent standardization for inter-laboratory data comparison. PFGE also provides limited information on the phylogenetic relationships among strains, which limits the understanding of the evolution of important phenotypic traits such as virulence [94]. EFSA also recommended MLST as a useful method for microbial ecology which is based on core genome sequences that can be used to investigate broad population structure of several bacterial species. In the case of L. monocytogenes, several studies have used MLST including a recent analysis of more than 1000 isolates, across wide temporal, geographical and source diversity [95]. However, MLST has a drawback since is neither rapid nor cheap and has limited discriminatory power within L.monocytogenes.

    Other molecular typing methods that have been suggested include multi-locus variable-number tandem repeat analysis (MLVA), multi-virulence-locus sequence typing (MVLST), single nucleotide polymorphism (SNP) and clustered regularly interspaced short palindromic repeats (CRISPR) [96].

    Although the genetic markers that are associated with enhanced virulence of L.monocytogenes are yet to be identified, however previous studies have identified multiple distinct mutations that leads to a premature stop codon (PMSC) in the key L. monocytogenes virulence gene, internalin A (in1A) [97,98]. This virulence factor (interlin InlA; encoded by inlA) facilitates the uptake of L. monocytogenes by non-professional phagocytic cells that express the human isoform of E-cadherin, and this was noted to play a critical role in crossing the intestinal barrier at the initial stages of an infection [99].

    In recent years, high-throughput next-generation sequencing (HT-NGS) has become increasingly popular, replacing classical microbiology and first-generation bio-molecular technologies in several fields, as HT-NGS are known to deliver sequence data thousands folds more cheaply than Sanger sequencing [100]. By combining the laboratory and epidemiological data from PFGE and whole genome sequencing (WGS) have markedly improved the detection of listeriosis and have increased the number of solved outbreaks. WGS is known to compliment the time consuming PFGE and leads to a faster detection of Listeria as shown in the ‘PULSE NET and LISTERIA’ initiative [101].


    4. Prevention and Containment Strategies

    It is important that hygienic practices are regularly maintained by those in contact with foods, the food contact surfaces need to be clean in order to prevent cross-contamination from the processing facilities. The growth of Listeria in cold and damp area of food factories can be prevented by controlling the flow of water in these processing facilities. An interagency report on the risk assessment of L. monocytogenes in deli meats and cheeses identified measures that will reduce their outgrowth to include: cleaning and sanitizing the slicer which is a key route of contamination, use of growth inhibitors to mitigate their growth, strict temperature control during refrigeration and wearing of gloves [102].

    Listeria are known to survive for extended periods in acidic conditions and can form biofilms to grow in the absence of oxygen. In recent years, biofilms have been shown to be important for harborage of various food-borne pathogens thereby increasing the risk for food safety violations [103]. Contamination of food products are fostered by biofilms on equipments and their formation is favored by the accumulation of food residues, spoilage organisms and food-borne pathogens. Many food manufacturing processes may provide niches of low sanitation level, where biofilms can prosper, including stainless steel (e.g. ultrafiltration membrane, valves, air separator), rubber surfaces (e.g. belt, packing machine, air separator, liner, short milking tube) and floors [104].

    L. monocytogenes may increase or decrease in biofilms, depending on the competing microbes that are present. This organism was shown to increase in mixed biofilms with Pseudomonasfragi [105], Pseudomonas fluorescens [106] and Flavobacterium spp. [107]. In addition, large amounts of exopolysaccharides (EPS) may improve the conditions for adherence by entrapment [105]. The number of L. monocytogenes cells has been observed to decrease in a mixed biofilm with Staphylococcus sciuri [108] or with Staphylococcus xylosus and Pseudomonasfragi [109]. In a recent study on L. monocytogenes from retail deli environments, it was shown that L. monocytogenes vary in their ability to adhere and form biofilms [110]. The results also indicated that persistent strains had enhanced adhesion on day 1 of a 5-day adhesion-biofilm formation assay and there was no significant difference in sanitizer tolerance between persistent and transient strains. The major findings from this study suggested that foods contaminated with persistent L. monocytogenes strains from the retail environment are likely to have wild-type virulence potential which may persist due to increased adhesion and biofilm formation capacity rather than sanitizer tolerance, thus posing a significant public health risk since L.monocytogenes are able to form biofilms and tolerate sanitizers for their survival. Sanitizers extend the lag time of Listeria growth and are less effective against the biofilms formed thereby increasing the risk of outbreaks.

    Since research has proven that the control of L.monocytogenes is directly associated with the removal of biofilm and the persistence of L. monocytogenes in areas that are used for food storage, processing, production and transportation. Therefore, it is important to mitigate biofilms in food production environments [111]. The interplay of these mechanisms along with their virulence potential and persistence in deli environments is an important area that needs to be studied further. During routine hygiene monitoring in food processing environments, drain water samples are often taken without inclusion of drain biofilm matrix. As part of an efficient monitoring program, it was hypothesized that by including both drain water and drain biofilm samples in Listeria monitoring programs, this might yield more reliable insights into the presence of Listeria and the composition of microbial communities originating from floor drains [112].

    There has been a lot of work carried out to prevent or reduce Listeria contamination in USA foods. The Listeria rule as described in [113] established three alternative ways to address post -lethality contamination of L. monocytogenes in RTE meat and poultry products. Under Alternative 1, the establishment applies a treatment to the product after its exposure to the processing environment (post-lethality treatment) and uses a growth inhibitor (antimicrobial agent or process) to prevent the growth of L. monocytogenes in the product up to its declared shelf life. Under Alternative 2, the establishment can use either a post-lethality treatment or an antimicrobial agent/process to control L. monocytogenes. Alternative 3 requires the establishment to have a sanitation program controlling L. monocytogenes contamination in the processing environment and on the product. The three USDA alternatives decreased the incidence of Listeria to less than 0.1% in intact packages. The prevention and containment intervention strategies can be classified as:


    4.1 Physical treatments

    They include thermal and non-thermal treatments such as irradiation UV light, pulsed light, and the use of high pressures. Thermal treatment above 70 °C is the most common traditional intervention strategy against L. monocytogenes. However, it is well known that the microorganism may develop an adaptive response during exposure to sublethal food processing interventions and the pathogenic potential is preserved [114]. The use of UV light as an alternative treatment to traditional thermal treatment to disinfect surfaces of equipments in meat plants is gaining acceptance. Studies have demonstrated that UV light has the potential to reduce bacterial contamination in food surfaces and as post-lethality treatment to control L. monocytogenes in meat and poultry processing facilities [115].

    The efficacy of Pulsed Light as an alternative to thermal treatment in reducing L. monocytogenes on the surface of 2 RTE dry cured meat products was investigated. The results showed a maximum log reduction of 1.5-1.8 cfu/cm2 at 11.9 J/cm2 pulsed light [116] irradiation employs the use of ionizing radiation from 60Cobalt, 137Caesium, or machine generated electron beams, it can either be used alone or in combination with other treatments. The applied dosage is important as too high dosage can have negative impacts on the sensory quality of the product [117].

    It was possible to control L. monocytogenes on frankfurters and cooked pork chops with irradiation and modified atmospherepackaging (MAP) containing a high concentration of CO2 [118]. The potential use of pectin-nisin films alone or in combination with ionizing radiation for preventing listeriosis due to post-processing contamination of ready-to-eat meat products was very promising [119].

    In an investigation on the application of Intense Light Pulses (ILP), ILP was shown to reduce L. monocytogenes and E. Coli 0157:H7 on the surface of stainless steel knife in meat contacts. The results further suggested ILP can be effective in meat processing lines as an intervention strategy to prevent cross-contamination between the equipment and the final product [120]

    High hydrostatic pressure (HHP) has been applied to decrease the count of L. monocytogenes in foods. Recently, it was reported that treatment at 600 MPa for 3 min resulted in an immediate 3.9-4.3 Log CFU/g reduction of L. monocytogenes count in RTE sliced ham [121]

    HHP was shown to be effective in slowing the growth of microorganisms that will shorten the shelf life of Queso Fresco cheese. The effect of HHP on the survival of a 5-strain rifampicin-resistant cocktail of L.monocytogenes in Queso Fresco cheese was evaluated as a post-packaging intervention. The result from this evaluation showed that HHP at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log10 cfu/g when held at either 20 or 40 °C for 5, 10, 15 or 20 min [122].


    4.2 Chemical treatments

    Under the USDA/FSIS rule, alternative 3 in the control of L. monocytogenes rigid sanitation control measures are to be incorporated into the Hazard Analysis Critical Control Point (HACCP) plan, Sanitation Standard Operating Procedures (SSOP), or prerequisite program. The potential for the contamination of Refrigerated or Frozen-Ready-To-Eat (RF-RTE) food and food-contact surfaces with L. monocytogenes, can be minimized through the establishment and use of a sanitation program that includes a written SSOP and a sanitation maintenance schedule for areas where RF-RTE foods or food-contact surfaces are processed or exposed [123].

    Sanitizers that contain quaternary ammonium compounds (QACs), peroxyacetic acid, iodine, or chlorine have been used to control L. monocytogenes in various situations. The Food and Drug Administration (FDA) recommends the use of QACs for many applications, because QACs have been found to be effective against L. monocytogenes and leave a residual germicidal effect on surfaces. FDA also noted that peroxyacetic acid sanitizers have been shown to be effective against biofilms containing L. monocytogenes [124].

    In order to discourage the excessive use of sanitizers such as chlorine in limiting the growth of L. monocytogenes in deli meats and cheese, electrolyzed oxidizing water (EOW) is a suitable alternative as an effective sanitizing agent that finds application in post-processing interventions. However, it was observed that treatments with the acidic EOA or basic EOB were not enough to meet regulatory requirements for control of L. monocytogenes on RTE meats. As such, there is a need for additional interventions to control the pathogen on RTE meats [125]. Lauric arginate is labeled as GRAS and it may be used successfully as part of an integrated cleaning and sanitation program [126].

    Sanitizer on food contact surface by definition is required to bring about a 5 log reduction after a 30s contact time. Lauric arginate tested at 100 and 200 ppm concentrations showed a maximum of 2.5 log CFU/coupon at 100 ppm concentration with exposure time of 15 min at low bacterial concentration levels of 3 log CFU/coupon; therefore, it does not meet the requirements of being either a cleaner or sanitizer when used alone [126]


    4.3 Natural interventions

    These include the use of probiotics, probiotic metabolites and organic acids, phytochemicals. Plant based secondary metabolites with generally recognized as safe (GRAS) bioactive substances can be used to control the growth of L. monocytogenes. The antimicrobial activity of plant extracts was attributed to the presence of different compounds especially the low molecular weight phenols, terpenes and ketones that also show antimicrobial activities in their pure form [127]. The essential oils (EOs) from different plants are known to have different minimum inhibitory concentration against L. monocytogenes in vitro. EOs with the strongest anti-bacterial properties against food pathogens contain a high percentage of phenolic compounds such as carvacrol, thymol and eugenol [128]

    The antimicrobial effects of natural occurring peptides such as nisin were shown to have inhibitory effects on L. monocytogenes. However, some Gram-positive bacteria that are repeatedly exposed to increasing nisin concentrations can acquire nisin resistance. The emergence of nisin tolerance has been reported in several species of bacteria, including L.monocytogenes [129,130]. As a result of this resistance, the use of natural antimicrobials from a broad variety of natural sources is been explored. Soy peptide in combination with nisin was shown to restrict the motility and aggregation of L. monocytogenes [131]. Recently, it was shown that there is a synergistic interaction of nisin and p-Anisaldehyde (AS) which is extracted from anise seed oil, derived from Pimpinella anisum L. seeds can be used to control a diverse array of L.monocytogenes strains [132].

    Organic acids such as acetic, lactic, malic, and citric are natural constituents of many foods and they have been used in food preservation since ancient time. Cured meats, such as sausage, ham and frankfurters are more susceptible to the listericidal effects of organic acids probably as a result of the presence of salt, nitrite and other preservatives [133]. By dipping frankfurters with a solution containing 2.0% of acetic acid, 1.0% of lactic acid, 0.1% propionic acid and 0.1% of benzoic acid followed by a steam prevented the growth of L. monocytogenes for 14 weeks at 7 °C [134]

    The applications of plant-based strategies for L. monocytogenes control in foods are likely to grow, due to the consumer demand for natural products (green label) and the industry interest for additive decrease (clean label) [114].


    4.4 Active packaging

    A combination of these intervention methods, incorporated preservatives into edible films for active packaging are of interest to researchers. In addition, commercial anti-listeral phage preparations which can be employed in the presence or absence of chemical preservatives are also

    The use of chitosanedible films in active packaging is useful in reducing L. monocytgenes in foods. However, as chitosan are not able to migrate from the film to the food matrix and it is important to add other preservatives such as bacteriocins, EOs and organic acids since chitosan film allow the gradual release of these preservatives into foods [133].

    Trinetta and co-workers demonstrated the effectiveness of sakacin A-containing pullulan films to control L.monocytogenes growth and the applicability of active pullulan films as a means of delivering a bacteriocin directly to a food surface. Pullulan films require less antimicrobials, they demonstrate longer antimicrobial activity and allow for controlled migration of the molecule from film to the food matrix, when compared with the direct addition of sakacin A to RTE meat products [135].


    5. Conclusion

    The prevention of the outbreaks of listeriosis associated with deli meats and cheese during their processing, storage and distribution is vital. Listeriosis as a food-borne disease is a big economic burden to the food industry and the public health services. Modern methods of identification that are rapid, accurate and reliable will help in its control. In order to improve the chances of listeriosis to be included as a disease in the international priorities of the World Health Organization, the quantification of the burden of Listeria and listeriosis especially in developing countries need to improve.


    Conflict of Interest

    The author declares no conflict of interests in this paper.


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