Not the Usual Suspects: 5 Gaps in Your Environmental Monitoring Program

Even the best-planned cleaning and disinfection monitoring programs have their blind spots. Stefan Widmann takes a closer look at five of the most likely – and most dangerous – gaps in your environmental monitoring program and explains what they are, why you should care about them, and what you can do about it.

#1 Viable but non-culturable (VBNC) microorganisms

For a long time, microbiologists assumed that any bacteria that failed to grow on normal culture media were dead. Subsequent research revealed that there is a third state beyond culturable and dead: viable but non-culturable (VBNC).

In general, bacteria in the VBNC state do not multiply but are still alive, as shown by their metabolic activity. Most relevant to us is the fact that they can become culturable after resuscitation and thus proliferate in food. Moreover, some pathogenic bacteria will not grow in the absence of a host and need only to survive in food until ingestion to cause illness.

There are many reasons why bacteria can go into the VBNC state; starvation, incubation outside the temperature range optimal for growth, elevated osmotic concentrations, levels of oxygen concentration, or exposure to white light are just some causes. The specific traits of the bacteria strain in question determine what exactly causes bacteria to enter this state. 

Why should you care?
Some bacteria able to enter the VBNC state are of concern for food manufacturing. While we do not yet know all bacteria species that can become VBNC, we know some that do; they count indicator organisms (such as Klebsiella aerogenes and Klebsiella pneumoniae), adulterants (such as Lactobacillus plantarumand Lactococcus lactis) and pathogens (such as Salmonella Typhimurium, Campylobacter coli or Listeria monocytogenes)among their numbers.

Having identified them, we must now ask whether these bacteria could return to a fully culturable and potentially pathogenic state. Microbiologists were, for a long time, in the dark on this question, as it is difficult fully to separate VBNC bacteria from culturable ones. Researchers have solved this problem, in part, by using a statistical approach: they dilute high numbers of VBNC bacteria to the point that it is nearly impossible for any culturable bacteria to remain. The bacteria are then counted after a defined period of time. If high degrees of growth are observed, the only possible conclusion is that bacteria have left the VBNC state and have become culturable.

A further corollary is that if they can return to a culturable state, they can also become pathogenic again. There are examples of exactly this phenomenon leading to outbreaks. For example, VBNC E. coli O157 were suspected in an outbreak in Japan in 1997, as the total numbers of  E. coli were insignificant and shigatoxigenic strains such as O157 could cause illness in very low numbers.

#2 Anaerobic and microaerophilic bacteria

Anaerobic bacteria or, more generally, anaerobic microorganisms, can be divided into three groups: obligate, aero-tolerant and facultative. As their names indicate, they each have special requirements regarding the air, or more precisely, the oxygen, surrounding them. Obligate anaerobes such as Clostridioides difficile are harmed by oxygen and will die shortly after exposure. Aero-tolerant bacteria such as Clostridium botulinum cannot make use of oxygen and will neither die nor grow in its presence. Facultative anaerobes can use oxygen but do not need it for growth, as is the case with E. coli. There is also the group of microaerophilic bacteria such as Campylobacter that need some oxygen to grow, albeit in much smaller amounts (1-2%) than in normal air but can be inhibited in aerobic conditions. 

Why should you care?
Several pathogenic bacteria have these special growth requirements. Currently, thermotolerant Campylobacter species are cause for worry among public health professionals. On average, every other chicken is infected with Campylobacter, making poultry meat one of the most common causes of food poisoning. In the EU, illnesses caused by Campylobacter species occur twice as often as those caused by Salmonella. Of the anaerobic group, a Clostridia species such as C. botulinum is responsible for the foodborne illness known as botulism, often transmitted through canned (i.e., oxygen-poor) food, in which C. botulinum can thrive and produce the compound botulinum, which is toxic to humans. Another Clostridia species, C. perfringens, is the most common source of food poisoning in the US and Canada and causes symptoms such as abdominal cramping and diarrhea. The risk of C. perfringens infection correlates especially strongly with food kept or stored in warm conditions for longer periods of time, which favors their growing to infectious numbers (104 cfu/g).

#3 The Great Plate Count Anomaly

Some estimates indicate that only 1% of bacteria can be cultivated with the knowledge and techniques currently at our disposal. The “great plate count anomaly” is the term we use to describe the observation that microscopic cell counts are significantly higher than corresponding counts of “colony forming units” on agar plates. A couple of examples can illustrate this phenomenon best: while 50% of the microorganisms of the oral flora can be cultured with agar plates, most of the gastrointestinal flora cannot be cultured at all. The reasons for this are numerous, but the organism community surrounding the species in question, including other bacteria as well as plants and animals, may play an important role. 

Aerobic plate count methods rely on very general media, which do not support the growth of most bacteria groups. Technically, this is not really part of the great plate count anomaly, as some bacteria are able to grow on special agar plates under special conditions (such as anaerobic or microaerophilic conditions).

Why should you care?
The great plate count anomaly does not pose significant problems in day-to-day testing runs, as aerobic plate counts for indicator microorganisms are specific to a given production environment and, as such, are always relative to an established baseline determined for that production environment.
However, plate methods are very time-consuming, requiring an incubation period of up to three days, depending on the protocol in effect. There are direct methods that do not require a cultivation step to count bacteria; microscopes provide a comprehensive view of bacteria but are also very time-consuming. While direct methods such as flow cytometry are common in water treatment facilities, they are not common in the food industry.

#4 Psychrotrophic bacteria

Psychrotrophic bacteria can grow at temperatures as low as 0 °C, with optimal and maximal growth temperatures above 15 °C. This makes such microbes especially problematic for foodstuffs and beverages such as raw meat and milk stored at low temperatures for longer periods of time. The psychrotrophic groups of bacteria most commonly found in food are the Gram-negative genera Pseudomonas, Aeromonas, Achromobacter, Serratia, Alcaligenes, Chromobacterium and Flavobacterium as well as Gram-positive genera such as Bacillus, Clostridium, Corynebacterium, Streptococcus, Lactobacillus and Microbacteria. Listeria monocytogenes and some strains of Clostridium botulinum are also known to be able to proliferate at refrigeration temperatures.

Why should you care?
Psychrotrophic bacteria are adulterants and can significantly diminish the quality and the shelf life of food. Chilled production facilities and storage tanks offer a favorable environment for the multiplication of these bacteria species. In chilled milk, for example, Pseudomonas fluorescens can produce both proteases and lipases. Hence, species belonging to the Pseudomonas genus are regarded as typically responsible for technological difficulties, as the proteases and lipases they produce can cause milk fat and proteins to degrade, giving milk a greyish color and bitter taste. Pseudomonas species are the microorganisms most often responsible for spoilage in aerobically stored chilled meat. It is well known that Pseudomonas species are very robust and able to withstand stressful environmental conditions that would inhibit the growth of other spoilage microorganisms. In vacuum-packed, refrigerated raw meat, the microflora is dominated in most cases by psychrotrophic lactic acid bacteria. Moreover, growth of pathogens during refrigerated storage could lead to serious illness.

#5 Biofilms

Microorganisms are able to colonize surfaces by forming a polymeric matrix in which multiple microbial species may be present; this is known as a biofilm. Evidence shows that the ability to form and survive in biofilms is not restricted to specific groups of microorganisms. In fact, the vast majority of bacteria are able to form biofilms. Biofilms may therefore be composed either of monocultures or of several different microorganism species. Some researchers have suggested that the complex structure of mixed biofilms renders them more stable and more resistant to cleaning chemicals. The initial population that binds to the surface can change the properties of that surface, allowing for those that come later to adhere via cell-to-cell association; in some cases, the attachment of a second species may increase the stability of the biofilm population. For example, studies show that L. monocytogenes is more likely to adhere to steel in the presence of Pseudomonas.

Why should you care?
Biofilms that form on food processing equipment and other food-contact surfaces act as a persistent source of contamination, threatening the overall quality and safety of food products and possibly resulting in foodborne diseases as well as economic losses. 
Spoilage microorganisms are known to be responsible for almost a third of losses in food supply chains, making biofilm prevention and control a priority in the food industry. Microorganisms that form or thrive in biofilms are more resistant to disinfection, making them problematic in a wide range of food industries. Other effects of biofilms such as the corrosion of metal surfaces are a further critical concern in the food industries. In either case, the presence of biofilms in a food factory puts human health at risk. The degree of risk is dependent on the bacterial species forming this three-dimensional, living structure.

How do you close these gaps? The potential of flow cytometry
Food producers generally do not have many options at their disposal. Those that offer a modicum of precision, such as vital staining in combination with microscopes, can quantify VBNC bacteria but are time-consuming and require special equipment. All groups of anaerobic and microaerophilic bacteria – with the notable exception of facultative anaerobes – can grow on classic agar plates, but only under carefully controlled oxygen levels.

Yet agar plates are no panacea. Agar plates are able to count only approximately 1% of known species of bacteria and take days to deliver results – up to 10 days in the case of psychrotrophic bacteria. ATP methods, while fast, do not quantify bacteria and are of only limited use in detecting bacteria from biofilms; the kinetic data from freely suspended planktonic cells should not be used as a reference as the release of ATP is much lower for biofilms. Moreover, ATP traces coming from food residue or fungi can easily overshadow the ATP released by bacteria, as eukaryotic cells contain 10 million times more ATP than prokaryotic cells. Accordingly, ATP devices used to detect biofilms tend to have a much higher limit of detection, meaning that they are not as sensitive as they would be when detecting free-floating bacteria. 

Each of these five cases has shown just how difficult it can be to detect bacteria and residues on food production surfaces; the shortcomings of the most common detection methods, such as plating and ATP testing, are as stubborn as they are well-documented.

What can food producers do to close the gaps that cultural methods and ATP testing leave behind? In the next article, my colleague Cristian Ilea discusses the potential of impedance flow cytometry and the CytoQuant^® flow cytometer, a new solution that immediately quantifies bacteria and residue particles on surfaces.