Sources, transmission, and tracking of sporeforming bacterial contaminants in dairy systems

Graphical Abstract

Summary Sporeforming bacteria are important causative agents of dairy product spoilage, conformance deviations, and rarely, foodborne safety issues. The 4 primary sources for spores in processed dairy products include raw milk, ingredients, biofilms present in the processing equipment, and environmental niches in the processing facility. The transmission of bacterial spores from dairy farm sources, especially manure, bedding, and feed, where they are often present in high levels, into raw milk is modulated by milking hygiene factors. The detection, enumeration, and tracking of sporeforming bacteria through the dairy system present unique challenges and often require specialized training and molecular methods to differentiate between closely related organisms.

Highlights

• •Bacterial spores are present throughout the dairy continuum.
• •Spores are resistant to a variety of stressors, including heat treatment and drying.
• •Sporeforming bacteria may cause quality, conformance, or even food-safety issues.
• •Detecting and tracking spores often requires specialized training and methods.

Abstract:

Bacterial endospores, or simply spores, are formed by a diverse group of members within the phylum Bacillota and include notable genera such as Bacillus, Paenibacillus, and Clostridium. Spores are distributed ubiquitously in natural environments, with soil being an important primary reservoir for these microbes. As such, spores are present throughout the dairy farm environment, and transmission into raw milk occurs through several pathways that coalesce at the point of milk harvest. Despite the very low spore concentrations typically found in bulk tank raw milk, the impact of spores on dairy product quality, safety, and product conformance is widely documented. Processed dairy products affected by the presence of sporeforming bacteria include milk, cheese, dairy powders, ice cream mix, and more. Although raw milk is a major source of spores leading to quality, safety, and conformance issues in dairy products, the impact of other sources should not be discounted and may include ingredients (e.g., cocoa powder), contamination originating from biofilms in processing equipment, and even cross-contamination from the processing environment itself. Addressing spore contamination in the dairy system is complicated by this widespread distribution and by the diversity of these organisms, and successful source tracking often requires discriminatory molecular subtyping tools. Here, we review the key sources of sporeforming bacteria in the dairy system, the factors leading to the transmission of this diverse group of microbes into processed dairy products, and methods employed to enumerate and track spore contaminants.

Sporeforming bacteria present a series of challenges to the dairy industry, the most considerable of which is their resistance in spore form to several stressors encountered throughout the dairy continuum that are typically lethal to vegetative cells, including exposure to heat (e.g., pasteurization), chemicals (e.g., sanitizers), and desiccation (Setlow, 2016). This resistance paired with the phenotypic diversity of sporeforming bacteria allows certain subsets of these microbes to grow under dairy product-relevant conditions (e.g., low temperatures, low oxygen, and so on) following processing hurdles commonly used in the dairy industry. During subsequent vegetative growth in processed dairy products, sporeforming bacteria may lead to several undesirable outcomes, including (1) product spoilage either through the production of degradative enzymes (De Jonghe et al., 2010) or through fermentation pathways (e.g., lactate fermentation; Brändle et al., 2016), (2) deviations in product conformance to regulatory standards or customer specifications potentially resulting in rejected product lots (Reich et al., 2017), and (3) rarely, food safety incidents (e.g., toxin production leading to foodborne illness; Spanu, 2016; Table 1).

Table 1.

Potential sources of bacterial spore contamination throughout the dairy system leading to quality, conformance, and safety concerns in processed dairy products

Item	Example of product affected	Key organism of concern	Potential spore source
			Raw milk	Ingredients	Biofilms in processing equipment	Processing environment
Dairy product spoilage	HTST fluid milk	Psychrotolerant aerobic sporeformers (e.g., Paenibacillus odorifer)	Likely	Likely	Possible	Possible
	UHT fluid milk	Highly heat-resistant thermotolerant mesophilic or thermophilic sporeformers (e.g., Geobacillus stearothermophilus)	Possible (rare)	Likely	Likely	Possible
	Ice cream mix	Psychrotolerant aerobic sporeformers	Likely	Likely	Possible	Possible
	Cheese	Anaerobic butyric acid bacteria (e.g., Clostridium tyrobutyricum)	Likely	Possible	Unlikely	Unlikely
Conformance deviations	Dry dairy powders	Thermophilic aerobic sporeformers (e.g., Anoxybacillus spp.)	Unlikely	Possible	Likely	Possible
Foodborne illness	Ready-to-eat dairy products	Aerobic sporeforming pathogens (e.g., Bacillus cereus sensu stricto)	Likely	Possible	Possible	Possible
		Anaerobic sporeforming pathogens (e.g., Clostridium botulinum)	Likely	Possible	Possible	Unlikely

Sporeforming bacteria that cause dairy product spoilage fall predominantly into 3 groups, (1) psychrotolerant sporeformers (e.g., Paenibacillus sp.) capable of growing at refrigeration temperatures in HTST milk and other refrigerated fluid dairy products (e.g., soft serve ice cream mix), (2) butyric acid–producing anaerobic sporeformers (e.g., Clostridium tyrobutyricum) that grow and produce late-blowing defect in certain styles of hard and semi-hard cheeses, and (3) highly heat-resistant sporeformers (e.g., Geobacillus stearothermophilus) that survive UHT processing and go on to cause spoilage in shelf-stable milk. Dairy product spoilage resulting from the growth of sporeforming bacteria has been recently thoroughly reviewed (Murphy et al., 2016; Martin et al., 2021); therefore, this topic will not be further discussed here.

The impact of spores on dairy product conformance primarily affects dry dairy products (e.g., whey and milk powder) as these products typically have customer-driven specifications for mesophilic or thermophilic spores (Burgess et al., 2010). Spores, and in particular thermophilic spores (for example, those produced by Anoxybacillus sp. and Geobacillus sp.), are important hygiene indicators in dry dairy products as they may persist in biofilms in processing equipment that is not adequately cleaned and sanitized (Seale et al., 2015). Although not all dry dairy products have stringent spore specifications, certain end product manufacturers (e.g., powdered infant formula) may have extremely low specifications for thermophilic spores (e.g., <500 spores/g; Watterson et al., 2014) leading to a high degree of importance placed on controlling thermophilic spores in these products.

Briefly, while foodborne illnesses resulting from the consumption of dairy products contaminated with sporeforming bacteria are reportedly very rare and will not be discussed in depth here, there are several sporeforming pathogens of concern in the dairy system. In particular, Bacillus cereus s.s. and Clostridium botulinum are the primary sporeforming pathogens that have been implicated in foodborne disease outbreaks originating from dairy products (Spanu, 2016). Although less common, other sporeforming pathogens have been reported in dairy products including Clostridium perfringens. Several Bacillus sp. have been shown to produce functional toxins (Gopal et al., 2015; Miller et al., 2016), although there are no reported dairy-related foodborne disease outbreaks from these strains.

The primary reservoir for spores in the natural environment is considered to be soil (Heyndrickx, 2011); however, the major contamination pathways from the environment to bulk tank raw milk on modern conventional dairy farms are directly and indirectly primarily through manure (Borreani et al., 2019), bedding (Murphy et al., 2019), and feed (Driehuis et al., 2016). Spore concentrations in these materials are often quite high, with one study reporting mean mesophilic spore counts (MSC) in manure, bedding, and feed of 5.87, 4.62, and 3.50 log cfu/g, respectively (Martin et al., 2019). Despite these high spore concentrations in key sources, spores are generally present in raw milk at much lower levels. For example, recent studies indicate that the mean psychrotolerant spore count (PSC), MSC, and thermophilic spore count are −0.72 (Buehler et al., 2018), 0.26 and 0.26 log cfu/mL (Murphy et al., 2019), respectively, whereas the butyric acid bacteria most probable number (MPN) for enumeration of anaerobic gas–producing spores is 1.79 log MPN/L (Shi et al., 2023). The level of spore contamination in the bulk tank raw milk is ultimately driven by the practices implemented at the point of milk harvest, including teat and udder hygiene (Zucali et al., 2015), milking routine (Bava et al., 2017), and consistently applied procedures by milking parlor employees (Evanowski et al., 2020).

Beyond the contribution of raw milk itself, another important source of spores is ingredients used in the manufacture of processed dairy products (Stoeckel et al., 2016). For example, cocoa powder mixes (typically composed of cocoa powder, sucrose [or other sugar], starches, gums, flavorings, and so on) have been shown to contribute high levels of spores to chocolate milk and other chocolate-flavored dairy products (e.g., chocolate ice cream mix; Douglas et al., 2000; Cadirci et al., 2018). Cocoa beans are an agricultural product that can become contaminated with spores while being harvested from their natural environment. The most likely vectors for contamination are soil, insects, tools used during their harvest, the fermentation process, and poor sanitary conditions (Lima et al., 2012; Pereira and Sant'Ana, 2018). Bacillus species have been found during the fermentation process of cocoa beans as it usually takes place where the fruit is harvested and the bean from which the cocoa powder is made is exposed to the naturally present microorganisms (Lima et al., 2011; Pereira and Sant'Ana, 2018; Eijlander et al., 2019). According to Lima et al. (2012), cocoa nibs have a higher concentration of mesophilic and thermophilic spores, varying between 4.3 and 5.6 log cfu/g before they are exposed to predrying processes, which reduces them significantly. Once the cocoa nibs enter the manufacturing process to be converted into cocoa powder, there are additional chances for spore contamination to occur. Due to the biofilm-generating ability of common sporeforming bacteria found in cocoa nibs, machinery and other instruments are susceptible surfaces for them to adhere to and contaminate the cocoa powder in the process (Lima et al., 2012; Pereira and Sant'Ana, 2018; Ubong, 2020). Studies indicate that the mesophilic strains most commonly found in cocoa powder include Bacillus cereus and Bacillus subtilis groups, Bacillus coagulans, Bacillus licheniformis, Paenibacillus, and others, whereas thermophilic strains including Geobacillus pallidus and Geobacillus stearothermophilus are also commonly identified (Pereira and Sant'Ana, 2018), all of which represent quality, and potentially safety, concerns for HTST and UHT chocolate milk products. Indeed, HTST chocolate milk is known to exhibit faster outgrowth of sporeforming bacteria during shelf-life than unflavored milk (Douglas et al., 2000), in part due to the contribution of spores through the cocoa powder.

Milk and whey powders, while themselves processed dairy products, also represent important ingredients used in the manufacture of a variety of other processed dairy products from milk to processed cheeses (Oliveira et al., 2016). While raw milk is still an important source of spores in milk and whey powders, thermophilic spores may also originate from biofilms in processing equipment (e.g., separators, heat exchangers, and evaporators) that are not adequately cleaned and sanitized (Flint et al., 2020) as the conditions inside dairy powder processing equipment is ideally suited to select for thermophiles (Seale et al., 2015). The use of dried dairy ingredients in reconstituted and heat-treated beverages (e.g., sports beverages, nutritional beverages, and so on) is of particular importance because obligate thermophiles represent a major proportion of the spore contaminants in many dairy powders (Yuan et al., 2012; Miller et al., 2015) and spores of these organisms exhibit much higher heat resistance than other groups of spores (Stoeckel et al., 2016), potentially leading to spore survival during UHT processing in end product applications. Additionally, nondairy ingredients including spices, dried herbs, vanillin, stabilizers, egg products, and so on have all been shown to be important potential sources of spores in processed dairy products (Lücking et al., 2013). Dairy processors using these and other ingredients with the potential to be contaminated with spores and are manufacturing a processed dairy product that may be susceptible to spoilage by a subgroup of sporeforming bacteria (e.g., psychrotolerant sporeforming bacteria) should include targeted spore assessments in their routine testing plan. The enumeration of spores in dairy products and ingredients will be covered in more detail later in this review.

As noted, spore transmission resulting from biofilm formation within dairy processing equipment represents a major pathway for contamination of dairy products. This transmission pathway has been largely studied in dry dairy product processing (Dettling et al., 2020; Flint et al., 2020); however, there is evidence that biofilms containing sporeforming bacteria may result in contamination of a variety of processed dairy products including extended shelf-life milk (Doll et al., 2017). Sporeformers have been shown in numerous studies to produce sanitation-resistant biofilms, with spores exhibiting higher adhesion tendency than vegetative cells (Jindal and Anand, 2018), which is likely due to the hydrophobic nature of spores (Bourdichon et al., 2021). The impact of spores originating from the processing environment is often readily observed in dry dairy products as the groups of spores originating from biofilms in the equipment are largely obligate thermophilic sporeformers (e.g., Anoxybacillus spp.), which are rarely identified from raw milk sources (Miller et al., 2015; Dettling et al., 2020). Detection and tracking the transmission of spores from biofilms in other dairy products, for example, HTST milk, present unique challenges because the predominant sporeforming bacteria that cause quality deterioration in this product (i.e., psychrotolerant sporeformers) are commonly found in raw milk supplies and finished products. In these types of products, successful differentiation between spores originating from raw milk and those originating from processing equipment requires the use of discriminatory subtyping tools, such as those discussed later in this review.

Finally, although the processing environment (e.g., floors, drains, ceilings, and so on) has not widely been considered a substantial source of spores in processed dairy products, there is evidence that this is an area for further examination. For example, Lücking et al. (2013) isolated 93 psychrotolerant, mesophilic, thermotolerant mesophilic, and thermophilic sporeforming bacteria from dairy processing environments and identified the environment as an important contamination source for processed dairy products (Lücking et al., 2013). Highlighting the potential importance of the processing environment as a source of spores in dairy manufacturing, it is well known that the characteristics of the environment in which spores are produced affect the ability of those spores to survive under different conditions. Namely, there is a positive correlation between the temperature of the environment in which the spores were formed and the thermal resistance of the resulting spores (Carlin, 2011). This could be particularly concerning in the case of milk and whey powders as these products are manufactured in dry processing environments. The introduction of highly heat-resistant spores into these products from environmental sources could pose a high risk of spore survival in end product applications even with high heat processing (e.g., UHT).

There are several considerations for the practical implementation of spore testing to improve quality and conformance throughout the dairy system. First, as discussed previously, spores are often present at very low levels, especially in raw milk, which presents challenges for enumeration when establishing a baseline of target spores in a raw milk supply. Unlike for other bacterial enumeration in raw milk (e.g., total bacteria count), due to their low levels, enumeration of spores cannot be conducted by microscopic techniques (e.g., direct microscopic count) or by rapid automated methods (e.g., flow cytometry), instead requiring labor-intensive traditional microbiological methods. Due to the labor-intensive nature of spore enumeration, coupled with the need for equipment not found widely in contemporary in-house laboratories (e.g., water baths), spore testing is often conducted at third-party laboratories. Second, spore enumeration methods vary widely globally and from customer to customer (for products with customer-driven specifications), and these variations can lead to considerable differences in the resulting spore count. For example, Murphy et al. (2021) evaluated 10 commercially processed dairy powders using 48 unique spore test method combinations consisting of commonly used heat shock treatments, plating methods, medium types, and incubation temperatures and found significant variation in the resulting spore count, which was primarily driven by the original heat shock treatment (Murphy et al., 2021). While several groups have recommended one or more standard spore test methods for use by the dairy industry (Eijlander et al., 2019; Murphy et al., 2021), and in dry dairy products, the use of numerous methodologies to enumerate spores in dry dairy products is still commonplace as specific parameters are often customer driven. It should also be noted that it is important for dairy industry stakeholders that use third-party laboratories for spore testing to clearly indicate what method (i.e., the heat shock temperature and time, medium, plating method, and incubation temperature) should be used to evaluate their products, or minimally, request the exact protocol from the third-party laboratory as the method used has a significant impact on the result and interpretation.

A final consideration for spore enumeration is the spreading and swarming phenotype, or the ability of the cells to migrate across a solid surface, exhibited by many sporeforming bacteria. Surface migration plays a key role in bacterial fitness and biofilm establishment, as it allows cells to locate and occupy environmental niches that may not be accessible to other microorganisms (Liu et al., 2020). The spreading and swarming phenotype exhibited by many sporeforming bacteria found in dairy products also represents a major challenge for enumeration as colonies spread rapidly across the surface of agar plates, obscuring visualization of other colonies (Murphy et al., 2021). Further, some sporeforming bacteria (e.g., Paenibacillus glucanolyticus) produce what are known as “microcolonies” (De Vos et al., 2011; Figure 1), which are motile cell aggregates that form across the surface of an agar plate. These microcolonies are difficult to distinguish from individual colony-forming units to the untrained analyst and may result in considerable overestimation of the spore concentration. Murphy et al. (2021) reported that spreading and swarming phenotypes were likely partially responsible for the interlaboratory variability in spore count outcomes in dry dairy powders and highlighted the need for specialized training for analysts performing spore count methods.

Figure 1.

Screenshot of a timelapse video showing Paenibacillus glucanolyticus microcolony development whereby a single colony-forming unit spreads rapidly across the surface of an agar plate. Motility phenotypes such as microcolonies, spreading, and swarming can often make the enumeration of spores in dairy products challenging. The original video can be found on the Milk Quality Improvement Program YouTube channel (https://youtu.be/PpJhk3iOib8).

Last, as previously mentioned here, there is a substantial need for discriminatory subtyping methods to apply to sporeforming bacteria throughout the dairy system, as effectively tracking this diverse group of organisms from environmental locations, either in the primary production system or in the processing facility, through the finished product represents a major challenge. A variety of approaches have been used for tracking sporeforming bacteria in dairy systems including single gene sequencing methods (e.g., rpoB gene sequencing; Huck et al., 2008), random amplified polymorphic DNA PCR (Dettling et al., 2020), pulsed field gel electrophoresis (Dréan et al., 2015), and others. However, despite the advantages of using these subtyping tools over traditional microbiological methods, including improved differentiation between related sporeforming bacteria, there are still limitations with these methods, especially for some taxonomic groups. For example, the Bacillus cereus group is a complex of closely related species (Carroll et al., 2022) that represents a major challenge to the dairy industry both because some members of this group are foodborne pathogens and others are causative agents of dairy product spoilage, but also because this group is difficult to resolve using commonly applied subtyping methods (Carroll et al., 2017). These limitations can be overcome by whole-genome sequencing, which is increasingly cheaper, faster, and more accessible to dairy industry stakeholders. For example, bacterial genome sequencing services such as Plasmidsaurus (https://www.plasmidsaurus.com/) offer whole bacterial genome sequencing services with genomic DNA extraction with a turnaround time of less than 1 wk for $105 per bacterial isolate at the time of this writing. Services such as these will transform the use of whole-genome sequencing for source tracking in the dairy industry, although bioinformatics and interpretation of genomic data will continue to be a need to effectively use these data.

As the dairy industry continues to evolve to the changing needs of domestic and international consumers, adopts new technologies, and strives for continuous improvement in quality, safety, and conformance, addressing spores throughout the dairy system will be more important than ever. Successful implementation of a spore control approach will require adherence to best practices at the farm, in the processing facility, and through ingredient suppliers to reduce the transmission of spores from primary sources into the dairy continuum. This will require training, education, and outreach at all levels of the dairy industry and, importantly, will also require collaboration across all sectors.