Abstract

Due to its nutritional value and physicochemical characteristics, milk is susceptible to detrimental and pathogenic microorganisms. Therefore, the dairy industry needs to control the factors that pose risks to the safety and quality of the product. Thus, a microbiological analysis of the air was carried out in the milking area of two dairy farms in Puerto Rico. Triplicate air samples were collected from May to September 2021. The results showed that farm B had higher bacteria concentrations in most months. Sixty-three genera of bacteria were identified among all the samples collected from both farms using MALDI-TOF MS. Additionally, several important sanitary bacteria were detected in the samples, albeit at a low frequency. In conclusion, the results of this study demonstrate that the concentration of microorganisms in the air of milking areas can be influenced by different factors, such as the location of the farm, as well as temperature and relative humidity. Furthermore, MALDI-TOF MS proved to be a helpful and fast technique for identifying the isolated bacteria in the samples.

1. Introduction

Cows’ milk is widely used product, because of its nutritional value. However, its physicochemical characteristics make it susceptible to detrimental and pathogenic microorganisms that may affect its quality ¹. In dairy farms, the microorganisms can vary by the facility’s location, environmental conditions, management practices, storage, and the effectiveness of the cleaning processes ². Therefore, the dairy industry aims to control those factors that would put the safety and quality of the product at risk by implementing regulations and guidance for cleaning and disinfection, animal health, employee hygiene, and the farm's natural environment ³.

Identification of microorganisms in bioaerosols of farms have been proposed as a tool to identify possible detrimental and pathogenic microorganisms before the product reaches its destination. Currently, several microbiological methods are used to analyze the presence of microorganisms in the process environments that occur on livestock farms. Most of those methods sought to isolate, purify, classify, and identify the microorganisms found during environmental sampling ^{4, 5, 6}. However, many of these processes take long to obtain results, especially when identifying a microorganism. For this reason, instrumentation such as Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-TOF MS) is being proposed as an accurate and fast technique to identify microorganisms ⁷. MALDI-TOF-MS uses protein analysis to identify bacteria, yeasts, viruses, and fungi, surpassing conventional identification methods that depend on a pure and fresh culture.

MALDI-TOF has been used successfully in several studies to identify microorganisms in livestock farms ⁸, including milk processing farms ⁹. However, some/all these studies identified potentially pathogenic microorganisms such as Aerococcus viridans, Corynebacterium striatum, Staphylococcus epidermidis, Staphylococcus saprophyticus, and Enterococcus avium ⁸. Also, other bacteria that pose a risk of spoilage, such as Staphylococcus and Bacillus spp. ⁹.

Milk is a widely used human food product susceptible to deterioration caused by microbial contamination. Microorganisms in the environment surrounding livestock industries may affect the animals and the raw material (milk) obtained ¹⁰. Therefore, the dairy industry needs to know the microbiological relationship between the environment and the process to ensure milk and animal health quality and safety. However, identifying this relationship is challenging in livestock farming because microbial identification is a slow and complex process ¹¹. Therefore, this study aimed to identify the bacteria suspended in aerosols from the milking area of two dairy farms in Lajas (farm A) and Hatillo (farm B) Puerto Rico by using MALDI-TOF MS.

2. Methodology

Microbiological sampling was conducted in the milking area of two dairy farms to isolate and identify the bacteria in bioaerosols using the MALDI-TOF MS technique. Samples were obtained monthly from May to September 2021 in three different areas of the milking process (entrance, middle, and exit).

The microbiological sampling was carried out according to Łukaszuk et al. ¹² with some modifications. The bacteria suspended in the air were collected by using the SAS-Super 100/180 (International pbi Spa, Milan, Italy) air sampling system previously disinfected with 70% isopropyl alcohol. The equipment was programmed with a speed of 100L/min, and RODAC (replicate organism detection and counting) plate with Tryptic Soy Agar (TSA) / Lecithin was used for the isolation. The collected samples were covered with paraffin, placed in sterile bags, and transported at refrigeration temperatures of 4 - 6°C to the laboratory to be processed as soon as possible in a biological safety cabinet (Nuaire NU-440-400 Class II).

Before working with the samples, the laboratory and the equipment were disinfected to avoid sample contamination. Next, the RODAC plates were incubated at 30 - 35°C for 48 - 72 hours. After the incubation period, the colonies were counted, and the concentration of microorganisms was calculated using the following formula:

(1)

Where Colonies, Pr is the adjusted value of probable colony counts, 28.3 is used to express the CFU/m³ value in CFU/ft^3, and sample volume was 100 L.

The colonies obtained from the microbiological sampling were subcultured on the TSA culture medium and incubated at 30 - 35°C for 24 - 48 hours. The most isolated colonies were selected, transferred with a sterile needle, and streaked on the TSA plate. In addition, Gram staining was performed with the pure cultures.

Isolated and purified bacteria were identified with Bruker MALDI-TOF Biotyper (positive ion mode) by the direct method (colony placed with matrix solution of α-Cyano-4-hydroxycinnamic acid) and bacterial DNA extraction ¹³.

In the direct method, fresh (24 - 48 hours) and pure cultures of the microorganisms isolated and subcultured in TSA medium were used. Using a sterile needle, a small part of the colony was placed in the stainless-steel sample plate used by the MALDI-TOF-MS ¹⁴. The colony was allowed to dry at room temperature in the previously disinfected biosafety cabinet for one minute. After drying, 1 µl of formic acid and 1 µl of the matrix were added. In addition, a BTS standard was used. This standard contains an extract of Escherichia coli DH5 alpha and worked as a positive control to warrant that the MALDI TOF worked properly and identified the characteristic profile of peptides and proteins ¹⁵. The peptide mass fingerprinting (PMFs) was then passed to the Accugenix® portal database to identify the microorganisms. DNA extraction was performed for isolates that were not properly identified within the database.

In a 1.5 mL microtube, 300 µl of molecular-grade water and the isolated bacteria were added and mixed in a vortex for one minute. Next, 300 - 900 µl of ethyl alcohol was added and centrifuged (Benchmark MC-12 microcentrifuge) for 2 minutes at 13,000 rpm. The resulting supernatant was discarded, and the centrifugation step was repeated. Subsequently, the supernatant was discarded, and 50 µl of 70% formic acid was added and mixed on the shaker to dissolve the precipitate. After this step, 20-50 µl of acetonitrile was added, mixed, and centrifuged. Finally, 1 µl of the supernatant was placed in the cover plate of the MALDI-TOF-MS and allowed to dry at room temperature in the biosafety cabinet, after which 1 µl of the matrix was added. When the plate dried, it was placed in the MALDI-TOF MS for analysis following the same indications as in the direct method ¹⁵.

A paired t-test at 0.05 significant level was used to determine if the concentration of airborne microorganisms was statistically different in each dairy farm during the sampling months.

3. Results

The results obtained in farms A and B during the sampling months are presented.

Farm B consistently exhibited higher levels of bacteria throughout the sampling months. The t-test analysis conducted on the microorganism concentrations between both farms from June to September showed a significant difference, which is clearly displayed in Table 1.

A total of 200 isolated organisms were identified using MALDI-TOF MS, of which 17 families and 63 genera of bacteria were identified. The families found most frequently were Bacillaceae, Staphylococcaceae, Moraxellaceae, and Planococcaceae (Figure 1). Gram-positive microorganisms predominated with 58% occurrence, while 42% of the organisms isolated were Gram-negative.

Table 2 shows the bacterial families and species occurrences during the sampling months in both farms. Thus, the Bacillaceae was the predominant family, with 15 species in all analyzed samples. The most frequent microorganisms of this family in the five sampling months were Bacillus cereus and Bacillus altitudeinis/pumilus/safensis, followed by Exiguobacterium acetylicum and E. indicum isolated during the months of July and August in both farms. Also, in farm A was detected Bacillus subtilis and Exiguobacterium aurantiacum from May to July.

The Staphylococcaceae was the second most frequently isolated group of microorganisms with 10 species. Of this family, the genus found the most in the five samplings was Staphylococcus chromogenes in both farms, followed by Staphylococcus sciuri, which was detected in May, July, and August in farm A. Something similar happened with Staphylococcus gallinarum, which was detected in farm B in May, June, and August.

The third most frequent group was the Moraxellaceae family, with seven species isolated and identified by the MALDI-TOF technique. All the bacteria identified were from the Acinetobacter genera. In this group, A. indicus was the most frequent bacteria, detected in May and September in both farms. The occurrence of the other species was random during the five sampling months in both farms.

Five species of bacteria were detected during the sampling months in farms A and B in the Enterobacteriaceae and Micrococcaceae families. For the Planococcaceae family, three species of Kurthia were identified, being Kurthia massiliensis the most frequent in both farms. Other families such as Alcaligenaceae, Caulobacteraceae, Comamonadaceae, Corynebacteriaceae, Erwiniaceae, Flavobacteriaceae, Microibacteriaceae, Paenibacillaceae and Xanthomonadaceae.

In this study, most of the sanitary-important bacteria detected in the sampling had a low occurrence (Figure 2), except for Staphylococcus chromogenes and Bacillus cereus. The other microorganisms detected in low frequency were Enterobacter cloacae, Enterococcus casseliflavus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Serratia marcescens.

4. Discussion

In this research, the number of bacteria suspended in the process area air may be influenced by some factors related to the conditions of this area. For example, temperature, relative humidity, and ventilation directly affect the quantity and distribution of bacteria in these process environments ¹⁶. Also, in the case of milk-producing farms, the conditions of the milking place, the place available for the animals, and its design influence the concentration of microorganisms suspended in the air ¹⁷. Additionally, other factors related to practices on the farm contribute to the concentration of air-suspended microorganisms, such as the frequency with which the facilities and equipment are cleaned before and after each milking process ¹¹. In this investigation, when comparing the number of cattle owned by both farms studied, farm B has a higher number than farm A. Also, farm A has a more rigorous cleaning system to perform the milking process. These practices may be responsible for farm B obtaining a higher number of bacteria suspended in the air in the milking area.

The most frequent bacterial family in this study was the Bacillaceae. This family is commonly found in milking farm environments, contaminating dairy products and affecting their quality and shelf life with the presence of endospores produced by some genera belonging to this family ¹⁸. Controlling this type of spore-producing microorganism in places of large-scale milk production is an important challenge for producers ¹⁹.

In this study, the most frequent Bacillaceae isolated was B. cereus. This result is comparable with the study by ²⁰, where the Bacillus genus was most abundant during sampling on a chicken farm. In another study presented by ²¹, the two bacteria with the highest frequency in the microbiological monitoring of seven dairy farms were B. cereus and Clostridium perfringens, with the microorganism B. cereus appearing first in 93% of the samples taken.

B. cereus is commonly found in the environment (soil, water, air). It is a bacterium of interest in food microbiology due to its ability to produce enterotoxins that affect people's health ²². Also, B. cereus is resistant to food cooking and pasteurization processes ²³. Additionally, it can cause infections such as bacteremia, meningitis, and brain abscesses ²⁴. The other bacteria with high frequency in the Bacillaceae family were Bacillus altitudeinis/pumilus/safensis. These microorganisms contaminate food and pharmaceutical facilities, causing product quality problems ²⁵. In addition, Bacillus pumilus has been implicated in food poisoning, human infections, and bacteremia in humans ²⁶.

Exiguobacterium was another genus frequently isolated in farm sampling and belonging to the Bacillaceae family. This genus is versatile and present in various environments, temperature ranges, pH, heavy metals, and salinity ²⁷. Exiguobacterium can be an essential resource for natural alternatives to the environment and agricultural practices (for its ability to promote plant growth and improve productivity) ²⁸. However, this genus of bacteria has been isolated as a contaminant from colostrum samples on dairy farms in the Czech Republic ²⁹. The researchers characterized this microorganism as one of the environmental contaminants present in 75% of the samples analyzed.

During this research, the Staphylococcaceae family was the second most frequent group isolated in both farms. The members of this family are characterized by being bacteria with a coccus morphology, Gram-positive, catalase-positive, and non-motile. They are antibiotic-resistant organisms and do not produce spores. These organisms survive various non-physiological environmental conditions and can grow in oxygen-rich (aerobic) environments or oxygen-depleted (anaerobic) conditions. Some genera of Staphylococcus spp. have been isolated from bovine udders, raw milk, storage tanks, and cheese samples ³⁰. This bacterium is highly related to the development of mastitis in susceptible cattle ³¹.

Some species of the Staphylococcaceae family (i.e., S. aureus, S. chromogenes, S. sciuri, S. xylosus) inhabit the skin, oral cavity, respiratory system, and intestine also are opportunistic pathogens. These bacteria can cause suppurative lesions and septicemia in humans and animals ³². Multiple studies have used MALDI-TOF to identify Staphylococcus spp. in raw milk and cheese ^{30, 33, 34}. These studies demonstrate that the Staphylococcus identification technique by MALDI is reliable for identifying this family.

In this research, the bacterial species found most frequently in the five samplings was Staphylococcus chromogenes, with a frequency of 80% for farm A and 40% for farm B. This bacterium is a coagulase-negative, Gram-positive coccus, which can be isolated from dairy cattle with mastitis. S. chromogenes was isolated in the study by ²⁰ from air samplings over several months in different areas of a poultry plant. Furthermore, S. chromogenes was isolated in bovine milk samples from 76 farms in Austria and identified using MALDI-TOF MS ³⁵.

The family Moraxellaceae comprises a heterogeneous group of bacteria widely distributed in different natural habitats and have diverse ecological and clinical significance ³⁶. Additionally, this family has members that can colonize the mucosa and skin of humans and animals, causing infections. Among the bacteria genera in this family are Moraxella spp., Acinetobacter spp., and Psychrobacter spp. ³⁷.

Studies abundantly detected microorganisms belonging to the Moraxellaceae family in milk from farms in Henan and Heilongjiang Province ¹⁰. The authors suggest that these microorganisms in milk may be due to transfer from feces into milk. These results relate to those in this investigation, where several species of Acinetobacter were isolated from the milking area air. Species of this genus, such as A. baumannii, isolated from raw milk, have demonstrated the ability to resist multiple antibiotics ³⁸. This adaptation risks the health of dairy farm animals due to the bacteria's ability to survive, potentially infecting animals and spreading.

The Enterobacteriaceae family are Gram-negative bacteria associated with warm-blooded animals' gastrointestinal tract. Several bacteria in this family are pathogenic microorganisms related to food spoilage and safety. Therefore, they are considered a high-risk factor in raw milk. This family includes genera such as Escherichia, which inhabits the feces of animals; Enterobacter, Citrobacter, Serratia, and Klebsiella, present in soils, water, and grains ¹⁴. Mastitis in cattle is associated with Gram-negative bacteria, especially those of the Enterobacteriaceae family. These bacteria mainly affect cows after milking due to contact of the udder with contaminated water and farm areas.

However, phenotypic methods often misidentify Enterobacteriaceae at the genus and species level. Therefore, there is an interest in using more precise techniques for identifying enterobacteria, such as MALDI-TOF MS. This interest was demonstrated in the study by ¹⁴, where 183 members of Enterobacteriaceae were identified with MALDI-TOF MS in milk, cow feces, water samples, and from the process area in a dairy farm in Rio de Janeiro, Brazil. The authors' statements are related to the findings of our investigation, in which we isolated several genera of the Enterobacteriaceae family (including Cronobacter sp., Enterobacter sp., Enterococcus sp., and Klebsiella spp.) from air samples collected in milking areas.

The Micrococcaceae family comprises a diverse group of microorganisms exhibiting various morphological and chemical-taxonomic properties that distinguish between genera and species ³⁹. These bacteria are found in the skin and mucous membranes of humans and animals and can also be in the environment. Some species in this family can cause opportunistic infections in hosts. A study by ⁴⁰ detected Micrococcaceae in environments associated with a dairy farm, including the bedding area and air during summer and winter. Our study's findings align with Nguyen et al.'s results, as we isolated Arthrobacter spp., Glutamicibacter sp., and Rothia spp. from the air in the milking area.

This bacterial family comprises Gram-negative rod-shaped bacteria, saprophytic microorganisms in soil and marine waters, and some pathogens of animals, humans, and plants ⁴¹. In addition, members of this family, such as Pseudomonas spp., are classified as plant growth promoters through certain growth factors, nutrients, and disease prevention ⁴². Studies have shown the presence of Pseudomonas spp. in milk samples and at various sampling points in the analyzed milk dairies ⁴³. Its presence in milk was related to inadequate handling of milking processes, hygiene, and product storage. These results are associated with this study since one of the bacteria isolated from this family was Pseudomonas aeruginosa during May in both farms. Furthermore, this P. aeruginosa bacterium has been related, isolated, and identified with the MALDI-TOF technique in an outbreak of mastitis in a herd of dairy cows where it was determined that the outbreak occurred with a single strain of the microorganism using a cleaning solution that contained the bacteria ⁴⁴.

The Planococcaceae family is taxonomically heterogeneous. According to ⁴², more than one hundred species of bacteria are grouped into fourteen genera. These are primarily bacilli and Gram variables; some can produce motile and aerobic spores. Also, the Planococcaceae family may be prevalent in the microbiota of cheese ripening. It was a family of organisms found in the sampling of dairy farms ⁴⁶. A study isolated and molecularly characterized microorganisms from the Planococcaceae family, which was possible in a sampling carried out in dairy farm storage tanks ⁴⁷. In this study, Kurthia massiliensis was the family member most prevalent during the samplings. One author described K. massiliensis as a Gram-positive, aerobic, encapsulated, motile rod shape; no evidence of pathogenicity exists for this microorganism ⁴⁸.

In both farms, a group of sanitary-importance microorganisms was found with variable occurrence. Although the capacity of these microorganisms to cause problems is moderate, they are agents that can cause diseases in humans and animals or represent a risk to the environment ⁴⁹. The sanitary-important microorganisms identified were Enterobacter cloacae, a nosocomial pathogenic bacterium responsible for bacteremia and respiratory, urinary, and intra-abdominal tract infections. Several strains of this microorganism have already been detected as resistant to antibiotics, which has caused a significant increase in infections ⁵⁰. In addition, this microorganism in dairy products can indicate unsanitary production or improper handling, which constitutes a safety problem for the consumer ⁵¹.

Enterococcus casseliflavus is a bacterium commonly found in human and animal intestinal tracts. It is one of the species of enterococci widely found in dairy cows ⁵². This organism has been found in bulk tanks and milking equipment even after they have been disinfected. A study found the bacteria E. casseliflavus in a few of the 2,000 samples of cows suspected of mastitis in Poland ⁵³. In addition, cases of human infections have been reported where E. casseliflavus has shown antibiotic resistance ⁵⁴.

Klebsiella pneumoniae is found on mucosal surfaces in mammals and the environment (water and soil). In humans, it is located in the gastrointestinal tract and nasopharynx. However, in immunocompromised people, it can cause pneumonia ⁵⁵. K. pneumoniae, an opportunistic pathogen, infects the mucosal epithelium and spreads to deep tissues and the bloodstream. It causes severe infections such as meningitis, ophthalmitis, bacteremia, and liver abscesses. K. pneumoniae is clinically challenging as it is resistant to certain antibiotics ⁵⁶. This bacterium, also associated with mastitis, was accurately identified by MALDI-TOF in isolates from the Mastitis Pathogen Culture Collection of the Canadian Bovine Mastitis and Milk Quality Research Network ⁵⁷.

Acinetobacter baumannii is an opportunistic pathogenic bacterium causing nosocomial infections, such as pneumonia, skin infections, and meningitis. A. baumannii is one of the pathogens most resistant to antibiotics ⁵⁸. In addition, it causes mastitis, pneumonia, and sepsis in animals for consumption ⁵⁹. Therefore, a study recommends analyzing bulk tank milk for foodborne pathogens and Acinetobacter spp. due to their potential risk to milk quality and public health ⁶⁰.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium, ubiquitous and persistent in the environment ⁶¹ Found in water, soil, and various physical media. P. aeruginosa is the main microorganism and the fifth worldwide cause of nosocomial infections. It is the second and fourth cause of urinary and surgical infections, respectively ⁶¹. In addition, it is one of the pathogenic agents that cause mastitis ⁶². One study described an outbreak of mastitis in 20 herds of dairy cows caused by P. aeruginosa related to poor cleaning practices. The investigation showed that the microorganism's transmission occurred with a contaminated cleaning solution used during the milking process ⁴⁴.

Serratia marcescens is an opportunistic pathogenic bacterium belonging to the Enterobacteriaceae family and causing various lung and urinary tract infections, meningitis, and sepsis. One study described their results from two herds of dairy cows, where positive samples of S. marcescens were obtained in milk with mastitis ⁶³. It was concluded that this microorganism and its transmission are due to the mishandling of equipment and chemical compounds used during cleaning.

Bacillus cereus and Staphylococcus chromogenes were the sanitary-important microorganisms with a frequency of over 60% in the samplings on both farms. B. cereus is a bacterium commonly found in the environment and capable of producing toxins related to foodborne illnesses. Consuming these toxins can cause symptoms such as vomiting and diarrhea. In addition, these bacteria can contaminate animal feed ²³. As a spore-former, B. cereus resists pasteurization and adverse environmental conditions. This bacterium has been linked (although not as frequently) to mastitis in cows used in the dairy industry ⁶⁴. S. chromogenes is a bacterium classified as a pathogen for humans, resistant to antibiotics, and associated with nosocomial infections ⁶⁵. It is a pathogenic microorganism that can coagulate plasma and has been isolated from dairy cow mastitis ⁶⁶. Several of these microorganisms have already been isolated in previous research. For example, in the study by ⁶⁷, conventional dairy farms in Canada were microbiologically sampled, reporting pathogens such as Bacillus spp., Streptococcus spp., Staphylococcus aureus, and Escherichia coli most related to mastitis disease in cows.

5. Conclusions

The results of this study show that the concentration of microorganisms in the air of milking areas can be influenced by different factors, such as the location of the farm, as well as temperature and relative humidity. The study also demonstrates the effectiveness of using MALDI-TOF MS to identify microorganisms quickly and accurately in dairy farms. This study successfully isolated and identified numerous bacteria that are commonly associated with animal production environments. Some of the identified bacteria are known to be part of the normal environmental flora, while others have been linked to livestock diseases. Additionally, MALDI-TOF MS accurately identified bacteria classified as opportunistic pathogens, capable of affecting the health of both animals and humans. These findings contribute to the understanding of the significance of comprehending and identifying the microbial flora present in dairy farms, thereby facilitating the development of new protocols to prevent and reduce microbial contamination.

Acknowledgments

The authors are grateful to Pace Analytical Laboratory, Inc. for allowing the use of the laboratories to conduct this research.

References