Dive into the world of microbes and temperatures with us! Understanding how bacteria grow is crucial, especially since many foodborne pathogens are mesophiles that thrive at moderate temperatures. There's a myth, however, that psychrophiles, those that love the cold, only grow in fridge-like conditions. We'll clarify this by exploring the growth characteristics of these temperature-sensitive microbes, including the versatile Listeria, and the adaptations that allow some to prosper in the cold. Get ready to deepen your knowledge on microbiological principles vital for food safety.
Growth Temperature of Food Poisoning Bacteria – Most Bacteria Causing Infectious Food Poisoning are Mesophilic
When considering the relationship between microbial growth and temperature in food microbiology, it's beneficial to start with the understanding that most bacteria causing infectious food poisoning cannot proliferate at refrigerator temperatures.
Why is this the case? The bacteria that cause infectious food poisoning in humans are originally from similar creatures such as cattle, pigs, sheep, and birds, living in the intestines of these warm-blooded animals. Therefore, their natural habitat is such that they cannot multiply in the low temperatures typical of the natural world, especially below 10°C during winter.
In Nature, Psychrophiles Dominate
In food microbiology, microbes that cannot grow at refrigerator temperatures are called mesophiles. As students learn about food microbiology, it's easy to mistakenly believe that mesophiles are abundant. However, they are exceptionally rare in the natural environment. The majority of microbes living in nature can indeed proliferate at refrigerator temperatures because, especially in temperate regions, temperatures often drop below 10°C in winter. When crows die or fish perish in ponds during such conditions, they decompose and are broken down naturally. Thus, without bacteria that can actively multiply at these low temperatures, the elemental cycling of the Earth’s ecosystem would not be sustainable.
Meat and vegetables naturally harbor environmental microbes. Therefore, storing them in the refrigerator will lead to spoilage, which is only natural considering their original environmental temperatures. Contrarily, the bacteria that cause infectious food poisoning in our bodies, as mentioned earlier, cannot grow under refrigerator conditions.
Misunderstandings Arising from the Minimum Growth Temperatures of Salmonella and E. coli
The understanding discussed above is fundamental when considering the relationship between microbial growth and temperature in food microbiology. However, some readers of this article might still believe that even the bacteria causing infectious food poisoning can proliferate at refrigerator temperatures. Indeed, internet sources and textbooks often list the minimum growth temperatures for bacteria causing infectious food poisoning as below 10°C.
For example, reliable data from the U.S. FDA lists the minimum growth temperatures for enterohemorrhagic E. coli at 6.5°C, and for Salmonella at 5.2°C. At first glance, such figures might lead beginners in food microbiology to believe that food poisoning bacteria can actively multiply inside a refrigerator. This understanding, however, is incorrect.
Let's look at some research paper data. The following figure shows the growth behavior of enterohemorrhagic E. coli and psychrophilic bacteria as general spoilage bacteria in beef:
- At 4°C, the psychrophilic spoilage bacteria have multiplied significantly after 72 hours. However, there is no noticeable growth of enterohemorrhagic E. coli.
- Raising the temperature to 7.2°C, the growth of psychrophilic bacteria becomes even more vigorous (not shown in the figure), but enterohemorrhagic E. coli still shows no growth after at least 72 hours.
- At the upper limit of refrigerator temperature management, which is 10°C, enterohemorrhagic E. coli shows a minimal growth of about 0.5 log after 72 hours.
What I want to emphasize from this figure is to avoid the misconception based on just the numerical data of minimum growth temperatures, such as thinking "enterohemorrhagic E. coli can actively multiply in a refrigerator because its minimum growth temperature is 5.6°C." The growth speed in a refrigerator of so-called psychrophilic spoilage bacteria and enterohemorrhagic E. coli is markedly different. It is crucial to grasp this concept firmly as a clear mental image.
The graph above was drawn by this blog operator from the following paper, with the necessary data extracted.
Journal of Food Protection, Vol. 69, No. 8, 2006, Pages 1978–1
The Mechanism Enabling Psychrophiles to Grow at Low Temperatures
Let’s delve a bit into why psychrophiles can proliferate at low temperatures, from a biological perspective. Although somewhat foundational for food microbiology, understanding this can aid in grasping various phenomena within the field.
First, what does it mean for a temperature to be "high" or "low"? A high temperature indicates that water molecules are very active, while a low temperature means they are less active. Considering this, let’s think about how temperature affects microbial activity. The life activities of microbes are governed by the components that make up their cells, such as lipids, proteins, and carbohydrates.
Focusing on fatty acids controlled by temperature, there are unsaturated fatty acids, which include double bonds, and saturated fatty acids, which do not. Unsaturated fatty acids bend at the double bond sites, causing a kink in their structure. If you try to align them, their 3D structure—full of kinks—makes them prone to scattering, like restless children during a school assembly. In contrast, saturated fatty acids line up neatly.
As mentioned earlier, higher temperatures mean more vigorous water molecule activity. When water molecules become active, even neatly aligned saturated fatty acids are shaken, becoming slightly unstable. Imagine the noise from outside a schoolyard during assembly, unsettling the students. Fatty acids react similarly: saturated ones, normally well-behaved, remain aligned despite some "noise" (increased molecular activity from higher temperatures). However, the already unstable unsaturated fatty acids become even more agitated with just a bit of added "noise" or molecular activity, making their 3D structure highly unstable.
In other words, at high temperatures, organisms composed mainly of unsaturated fatty acids become very unstable. Conversely, cells composed of unsaturated fatty acids are relatively stable at low temperatures, while those made up of saturated fatty acids maintain a stable arrangement even at higher temperatures but may become too rigid and solidify at low temperatures.
This means unsaturated fatty acids have lower melting points, while saturated fatty acids have higher ones. For instance, the white fat on beef seen in supermarkets isn’t white while the cow is alive; at a body temperature of 37°C, the saturated fatty acids in the cow’s fat are melted and clear. Once the beef is cooled below 10°C post-mortem, these fats solidify.
Conversely, fish fat, made of unsaturated fatty acids, remains liquid even in the chilled section of a supermarket. If a fish's body were composed of saturated instead of unsaturated fatty acids, consider the inconvenience it would face. Imagine a fish swimming near the warm surface of the ocean at 25°C during summer, then diving into the deep sea where temperatures are often below 10°C. If its fatty acids were saturated, they would solidify, potentially preventing the fish from resurfacing.
Lastly, consider the relationship between proteins, which are also composed of amino acids linked by peptide bonds, and temperature. The bonds themselves are not affected by temperature, but the hydrogen bonds pinning the chains into their secondary structure are. Increased water molecule activity makes these hydrogen bonds more likely to break, causing the protein's structure to fluctuate. A moderate fluctuation enhances enzymatic activity as explained earlier with heating, but too much fluctuation can permanently denature the protein. As temperatures decrease, these fluctuations settle down nicely, yet if the temperature is too low and there is no molecular movement, the flexibility needed for enzymatic activity is lost.
Thus, the structural flexibility of the enzymes' proteins in microbial cells, which varies among different microbes, determines their optimal and minimal growth temperatures. Psychrophiles can grow at low temperatures because the amino acid sequences in their protein structures are flexible enough to maintain activity even in such conditions.
Microbes Capable of Growing Across a Wide Range of Temperatures Are Rare
The lengthy explanations using analogies so far aim to equip you with the understanding that factors like the proportions of saturated and unsaturated fatty acids in microbial cells, as well as the amino acid composition of enzyme proteins (which influences their structure), are crucial when considering the relationship between microbial growth and temperature. To grow at low temperatures, the fundamental composition of these cellular elements must change. However, altering the composition of microbial cell components requires specific genes that encode for such designs. For example, Listeria monocytogenes can proliferate at both mammalian body temperatures and within the cooler confines of a refrigerator. In contrast, most bacteria that cause infectious food poisoning thrive near mammalian body temperatures but cannot multiply at refrigerator temperatures.
Some readers might wonder why not all microbes possess the capability to proliferate across such a broad temperature spectrum like Listeria, as it seems advantageous. If Listeria can grow at low temperatures, it must possess genes that facilitate such adaptation by altering its fatty acid and protein compositions.
This means Listeria must possess more genes compared to bacteria that can only grow within a mesophilic range. While having more genes could seem beneficial as it allows adaptation to a wide range of environments, microbial life is not that simple. Microbes usually do not inhabit a variety of environments; rather, they are often adapted to a specific niche. While having a broad range of capabilities might aid survival outside their preferred environments, in most cases, such as E. coli residing in mammalian intestines, genes enabling growth at low temperatures would be superfluous. Carrying unnecessary genes means that these bacteria would take longer to divide within the intestines.
Thus, microbes generally carry the minimum number of genes necessary for the most efficient division within their specific domain. Just as in professional sports, where a baseball player focuses on pitching or batting, not both, or a football player specializes as a goalkeeper or a forward but not both, professional sports rarely accommodate "all-rounders" because the competition is too intense. Similarly, the world of bacteria does not indulge microbes with unnecessary genes. Understanding this helps clarify why microbes like Listeria, capable of proliferating across a wide temperature range, are indeed rare.
Methods for Testing Psychrophilic Bacteria
How do we measure psychrophilic bacteria? Of course, general viable count methods can be used to measure bacteria that can grow at low temperatures, such as Listeria.
In Japan, the standard method for testing general viable counts in food hygiene inspections is set at 35°C for 48 hours. Globally, the methods are mainly divided between the American AOAC method and the European ISO method.
- American (AOAC method): 35°C for 48 hours (equivalent to Japan's official method)
- European (ISO method): 30°C for 72 hours
Japan's official method aligns with the American approach. Each method has its pros and cons. Generally, lowering the incubation temperature and extending the incubation time allows for counting more bacteria. It has been reported that the EU (ISO method) at 30°C for 72 hours results in a count that is 0.18 log higher than that of the American (AOAC method) at 35°C for 48 hours, which suggests an advantage for the ISO method. However, when it comes to indicators like general viable counts, speed is crucial. From this perspective, the AOAC method has the upper hand.
https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/jam.13227
The issue then arises: what cultivation temperature should be used to measure bacteria that can only grow at low temperatures? The standard methods for general viable counts cannot measure psychrophilic bacteria, especially those that typically adhere to seafood and vegetables in temperate or cold regions.
So, what is the most suitable temperature for testing psychrophilic bacteria? By definition, psychrophiles are not classified by taxonomic or characteristic features but are broadly defined as bacteria that can proliferate at low temperatures during food preservation. The definition of psychrophiles is subject to various interpretations online, causing confusion. Some define psychrophiles as "obligate psychrophiles that can grow only at low temperatures, proliferating best between 10°C and 20°C, or even lower," while others might define them as having an optimal growth temperature below 20°C.
Indeed, many bacteria encountered in food handling cannot grow at 30°C. For instance, when measuring bacteria on fish, a task often performed by the author, the general viable count method at 35°C for 48 hours significantly underestimates the actual count. Bacterial counts could be underestimated by two orders of magnitude if not cultured below 25°C. In other words, attempting to analyze the flora from agar plates used under general viable count conditions (35°C, 48 hours) will miss 99.9% of the bacteria, rendering actual flora analysis unfeasible. Bacteria such as Alteromonas, Psychrobacter, and Shewanella are frequently detected in fish, many strains of which cannot grow above 30°C (unpublished by the authors).
Thus, bacteria that "cannot grow at 30°C" do indeed exist in the routine of food microbiology testing. However, considering that bacteria that can only grow at temperatures as low as 15°C are commonly present in food microbiology testing environments is misleading and causes confusion. The history of discovering psychrophiles dates back to the 1960s when the search for low-temperature bacteria was a global trend in the early stages of research. However, most of these bacteria turned out to grow at 25°C or 20°C, leading to widespread disappointment among researchers. Amidst this confusion and debate, researchers like Dr. Richard Morita went to extreme environments like Antarctica, where they finally discovered true psychrophiles, defined, for example, by a maximum growth temperature of 20°C.
https://pubmed.ncbi.nlm.nih.gov/1095004/
In summary, while bacteria that cannot proliferate at 30°C are indeed frequently encountered in routine food testing, encountering those that cannot grow below 25°C is not as common. Bearing this in mind, even for products like fresh fish where psychrophiles are expected, adopting a practical cultivation at 25°C could allow for timely and effective measurement of low-temperature-growing bacteria. This article aims to clarify the common misconception that psychrophilic bacteria can only grow at refrigerator temperatures. It is important to remember that 30°C is a critical temperature, at which many low-temperature bacteria cannot proliferate. Understanding this point is key.
he Relationship Between Microbial Growth Rate and Temperature
When discussing psychrophiles, I often receive questions from young quality control professionals at private companies, expressing concerns about low-temperature-growing foodborne pathogens. The underlying concern usually stems from an overestimation of the characteristics of such bacteria, with a belief that "low-temperature-growing foodborne pathogens can proliferate at considerable speeds at low temperatures, posing significant risks." Indeed, low-temperature-growing pathogens like Listeria, Bacillus cereus, and Clostridium botulinum type II do require careful management under low-temperature conditions compared to mesophilic foodborne pathogens.
However, it's crucial to recognize that while psychrophilic foodborne pathogens can indeed grow at low temperatures, their growth rate is fundamentally constrained by the kinetics of enzyme reactions. As temperatures decrease, their proliferation rates also decline. It's a common misconception that these low-temperature bacteria can rapidly multiply at cold temperatures. In fact, while food poisoning caused by bacteria like Bacillus cereus can occur in food that has been stored at low temperatures for an extended period, more frequently, food poisoning cases are due to improper temperature management rather than the inherent capabilities of the bacteria.
It's essential for those involved in food safety to understand that the capability of psychrophilic pathogens to grow at lower temperatures doesn't equate to rapid proliferation under such conditions. Thus, effective temperature management remains critical in preventing foodborne illnesses, rather than overestimating the growth capabilities of these bacteria at low temperatures. This clarification helps in addressing the concerns and ensuring that food safety practices are based on accurate understanding of microbial behavior in different temperature environments.
Composite View from Plotted Actual Growth Rates of Multiple Bacteria Including E. coli and Staphylococcus aureus (Modified from McMeekin, 1996 Diagram)
Int J Food Microbiol Nov;33(1):65-83.(1996)
