Tracking heat-resistant, cold-thriving fluid milk spoilage bacteria from farm to packaged product

Control of psychrotolerant endospore-forming spoilage bacteria, particularly Bacillus and Paenibacillus spp., is economically important to the dairy industry. These microbes form endospores that can survive high-temperature, short-time pasteurization; hence, their presence in raw milk represents a major potential cause of milk spoilage. A previously developed culture-dependent selection strategy and an rpoB sequence-based subtyping method were applied to bacterial isolates obtained from environmental samples collected on a New York State dairy farm. A total of 54 different rpoB allelic types putatively identified as Bacillus (75% of isolates), Paenibacillus (24%), and Sporosarcina spp. (1%) were identified among 93 isolates. Assembly of a broader data set, including 93 dairy farm isolates, 57 raw milk tank truck isolates, 138 dairy plant storage silo isolates, and 336 pasteurized milk isolates, identified a total of 154 rpoB allelic types, representing an extensive diversity of Bacillus and Paenibacillus spp. Our molecular subtype data clearly showed that certain endospore-forming bacterial subtypes are present in the dairy farm environment as well as in the processing plant. The potential for entry of these ubiquitous heat-resistant spoilage organisms into milk production and processing systems, from the dairy farm to the processing plant, represents a considerable challenge that will require a comprehensive farm-to-table approach to fluid milk quality.     

Development of Molecular Typing Methods for Bacillus spp. and Paenibacillus spp. Isolated from Fluid Milk Products

Bacillus spp. and related sporeformers are important food spoilage organisms. While use of molecular subtyping methods has provided important information on the ecology and transmission of foodborne pathogens, the lack of rapid, reliable, and affordable subtyping methods for Bacillus spp. has limited our ability to understand and control their transmission throughout the food chain. We used a previously described collection of Bacillus spp. and Paenibacillus spp. isolated from dairy products to develop a DNA sequencing‐based subtyping approach for these spoilage microorganisms. After optimization of polymerase chain reaction (PCR) parameters, primers targeting the rpoB housekeeping gene allowed for successful amplification in all isolates. rpoB sequencing allowed differentiation of 29 subtypes (that is, sequence types) among the 57 isolates characterized. Phylogenetic analyses of rpoB sequences revealed distinct monophyletic lineages that correlated with bacterial genera (Bacillus and Paenibacillus) as well as with species or species‐like assemblages within each genus. rpoB sequencing provided improved subtype discrimination over 16S rDNA sequencing; therefore, rpoB sequencing allows for both sensitive subtype discrimination as well as for species and genus identification. Analysis of subtypes isolated over time in dairy products revealed the presence of both persistent and transient bacterial subtypes, indicating that application of these methods can improve our understanding of the ecology of these spoilage organisms and can help in identification of bacterial niches that may contribute to the persistence of these spoilage organisms in food systems.     

Prevalence,antimicrobial resistance,and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100–499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30–99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.     

Short communication: Bacterial ecology of high-temperature, short-time pasteurized milk processed in the United States

To determine the microbial ecology of pasteurized milk within the United States, 2% fat pasteurized fluid milk samples were obtained from 18 dairy plants from 5 geographical areas representing the Northeast, Southeast, South, Midwest, and West. Of the 589 bacterial isolates identified using DNA sequence-based subtyping methods, 346 belonged to genera characterized as gram-positive endospore-forming bacteria (i.e., Bacillus and Paenibacillus). Of the 346 gram-positive endospore-forming bacteria isolated in the present study, 240 were classified into 45 allelic types identical to those previously identified from samples obtained in New York State, indicating the widespread presence of these microbes in fluid milk production and processing systems in the United States. More than 84% of the gram-positive spore-forming isolates characterized at d 1, 7, and 10 were of the genus Bacillus, whereas more than 92% of isolates characterized at d 17 of shelf life were of the genus Paenibacillus, indicating that the predominant gram-positive spoilage genera shifts from Bacillus spp. to Paenibacillus spp. during refrigerated storage.     

A century of gray: A genomic locus found in 2 distinct Pseudomonas spp. is associated with historical and contemporary color defects in dairy products worldwide

Some gram-negative bacteria, including Pseudomonas spp., can grow at refrigeration temperatures and cause flavor, odor, and texture defects in fluid milk. Historical and modern cases exist of gray and blue color defects in fluid milk due to Pseudomonas, and several recent reports have detailed fresh cheese spoilage associated with blue-pigment-forming Pseudomonas. Our goal was to investigate the genomes of pigmented Pseudomonas isolates responsible for historical and modern pigmented spoilage of dairy products in the United States to determine the genetic basis of pigment-forming phenotypes. We performed whole genome sequencing of 9 Pseudomonas isolates: 3 from recent incidents of gray-pigmented fluid milk (Pseudomonas fluorescens group), 1 from blue-pigmented cheese (P. fluorescens group), 2 from a historical blue milk spoilage incident (Pseudomonas putida group), and 3 with no evidence for blue or gray pigment formation (2 from P. fluorescens group and 1 from Pseudomonas chlororaphis group). All 6 isolates collected from products with a gray or blue pigment defect were confirmed to produce pigment using potato dextrose agar or pasteurized milk. A subset of 2 isolates was selected for inoculation into milk and onto the surface of a model cheese for subsequent color measurement. These isolates produced different colors on potato dextrose agar, but produced nearly identical color defects in milk and on model cheese. For the same subset of 2 isolates, the gray color defect in milk was produced only in containers with ample headspace and not in full containers, suggesting that oxygen is vital for pigment formation. This work also demonstrated that a Pseudomonas isolate from cheese can produce a pigment defect in milk, and vice versa. Comparative genomics identified an accessory locus encoding tryptophan biosynthesis genes that was present in all isolates that produced gray or blue pigment under laboratory conditions and was only previously reported in 2 P. fluorescens isolates responsible for blue mozzarella in Italy. Because this locus was found in genetically distant isolates belonging to different Pseudomonas species groups, it may have been acquired via horizontal gene transfer. These data suggest that several past and present gray- or blue-pigmented dairy spoilage events share a common genetic etiology that transcends species-level identification and merits further investigation to determine mechanistic details and modes of prevention.     

Short communication: Outbreak of Nocardia neocaledoniensis mastitis in an Italian dairy herd

Nocardia spp. are an uncommon cause of mastitis, and outbreaks have typically been reported in dairy farms with poor hygienic and management conditions. The outbreak described herein involved a dairy farm with 43 lactating cows that, after a long period with low bulk milk somatic cell counts (<180,000 cells/mL), experienced an increasing incidence of clinical mastitis with bulk milk somatic cell counts greater than 300,000 cells/mL. Fifteen mastitic quarters milk samples from 9 dairy cows were found to be infected by a member of the genus Nocardia, as identified on the basis of selected phenotypic and chemotaxonomic characteristics. The isolates were confirmed as Nocardia neocaledoniensis by 16S rDNA gene sequencing. Average quarter milk somatic cell count for infected udders was 863,057 cells/mL, significantly greater than the average value in noninfected quarters (189,710 cells/mL).     

Tracking spore-forming bacterial contaminants in fluid milk-processing systems

The presence of psychrotolerant Bacillus species and related spore formers (e.g., Paenibacillus spp.) in milk has emerged as a key biological obstacle in extending the shelf life of high-temperature, short-time pasteurized fluid milk beyond 14 d. A recently developed rpoB DNA sequence-based subtyping method was applied to characterize spoilage bacteria present in raw milk supplies for 2 processing plants, and to assess transmission of these organisms into pasteurized products. Thirty-nine raw milk samples and 11 pasteurized product samples were collected to represent the processing continuum from incoming truck loads of raw milk to packaged products. Milk samples were held at 6°C for up to 16 d and plated for bacterial enumeration at various times throughout storage. Among the 88 bacterial isolates characterized, a total of 31 rpoB allelic types representing Bacillus and Paenibacillus spp. were identified, including 5 allelic types found in both raw milk and finished product samples. The presence of the same bacterial subtypes in raw and commercially pasteurized milk samples suggests that the raw milk supply represents an important source of these spoilage bacteria. Extension of the shelf life of high-temperature, short-time pasteurized fluid milk products will require elimination of these organisms from milk-processing systems.     

Molecular identification of mesophilic and psychrotrophic bacteria from raw cow's milk

The aim of this study was to use molecular techniques to assess the microbiota of eight raw cow's milk samples at biotype and species level. Sixty-six isolates from raw milk samples were screened by Randomly amplified polymorphic DNA–PCR (RAPD–PCR) biotyping and representative strains of RAPD–PCR profiles were identified by 16S rRNA gene sequencing. Pseudomonas spp. were the most commonly occurring contaminants along with Enterobacteriaceae such as Hafnia alvei, Serratia marcescens and Citrobacter freundii. Moreover, Gram-positive isolates belonging to the genera Staphylococcus and Lactococcus were also found. Experiments of growth at different temperatures showed that more than 50% of the Gram-negative isolates could grow at chill temperatures and that 65% of the Pseudomonas spp. strains grew at 7 °C within 5 days. Only 13 Gram-negative isolates displayed proteolytic activity on milk agar, suggesting that not all the biotypes of milk contaminating species are able to perform this spoilage-associated activity. Among the Gram negative, the proteolytic strains were mainly Peudomonas spp. that displayed the activity at both 7 °C and 20 °C. A reliable molecular identification of raw milk microbiota is important for the study of the microbiological quality of raw milks and for the assessment of the ecology at species level in order to develop improved systems, preventing contamination and having the best conditions for the storage of milk.     

Short communication: Pseudomonas azotoformans causes gray discoloration in HTST fluid milk

Pseudomonas species are well recognized as dairy product spoilage organisms, particularly due to their ability to grow at refrigeration temperatures. Although Pseudomonas-related spoilage usually manifests itself in flavor, odor, and texture defects, which are typically due to production of bacterial enzymes, Pseudomonas is also reported to cause color defects. Because of consumer complaints, a commercial dairy company shipped 4 samples of high temperature, short time (HTST)-pasteurized milk with distinctly gray colors to our laboratory. Bacterial isolates from all 4 samples were identified as Pseudomonas azotoformans. All isolates shared the same partial 16S rDNA sequence and showed black pigmentation on Dichloran Rose Bengal Chloramphenicol agar. Inoculation of one pigment-producing P. azotoformans isolate into HTST-pasteurized fluid milk led to development of gray milk after 14 d of storage at 6°C, but only in containers that had half of the total volume filled with milk (～500 mL of milk in ～1,000-mL bottles). We conclusively demonstrate that Pseudomonas can cause a color defect in fluid milk that manifests in gray discoloration, adding to the palette of color defects known to be caused by Pseudomonas. This information is of considerable interest to the dairy industry, because dairy processors and others may not typically associate black or gray colors in fluid milk with the presence of microbial contaminants but rather with product tampering (e.g., addition of ink) or other inadvertent chemical contamination.     

Pseudomonas spp. and Serratia liquefaciens as Predominant Spoilers in Cold Raw Milk

The storage of fresh raw milk at low temperature does not prevent proliferation of psychrotrophic bacteria that can produce heat‐resistant proteolytic enzymes contributing to the reduced shelf life of dairy products. This study aimed to identify the dominant psychrotrophic proteolytic enzyme‐producing population of raw milk from Brazil. Raw milk samples collected in 3 different cooling tanks in Brazil were stored at optimal (45 h at 4 °C followed by 3 h at 7 °C) and suboptimal (45 h at 7 °C followed by 3 h at 10 °C) conditions to simulate farm storage and transportation allowed by Brazilian laws. The highly proteolytic enzyme‐producing strains isolated from stored cold raw milk were characterized by repetitive sequence‐based Polymerase Chain Reaction (PCR) analysis. This clustering resulted in 8 different clusters and 4 solitary fingerprints. The most proteolytic isolates from each rep‐cluster were selected for identification using miniaturized kit, 16S rDNA and rpoB gene sequencing. Serratia liquefaciens (73.9%) and Pseudomonas spp. (26.1%) were identified as the dominant psychrotrophic microorganisms with high spoilage potential. The knowledge of milk spoilage microbiota will contribute to improved quality of milk and dairy products.     

Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.     

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.     

Molecular subtyping and characterization of psychrotolerant endospore-forming bacteria in two New York State fluid milk processing systems

Psychrotolerant endospore-forming bacteria Bacillus and Paenibacillus spp. are important spoilage organisms in fluid milk. A recently developed rpoB subtyping method was applied to characterize the diversity and phylogenetic relationships among Bacillus and related sporeformers associated with milk processing systems. Milk samples representing the processing continuum from raw milk to pasteurized products were collected from two fluid milk processing plants, held at 6 degrees C up to the code date that had been established by each processing plant (i.e., either 18 or 21 days), and plated for bacterial enumeration throughout storage. Bacterial colonies selected to represent the visible diversity in colony morphology on enumeration plates were examined further. Among 385 bacterial isolates characterized, 35% were Bacillus spp., and 65% were Paenibacillus spp. A total of 92 rpoB allelic types were identified among these isolates, indicating considerable diversity among endospore-forming spoilage organisms present in fluid milk systems. Of the 92 allelic types identified, 19 were isolated from samples collected from both processing plants. The same rpoB allelic types were frequently identified in paired raw milk and packaged product samples, indicating that Bacillus and Paenibacillus spp. can enter dairy processing systems through raw milk. Certain subtypes were found exclusively in pasteurized samples, including those that were temporally independent, suggesting the possibility of in-plant sources for these spoilage organisms, including through the persistence of selected subtypes in processing plants. Development of effective control strategies for the diverse array of psychrotolerant endospore-forming organisms that currently limit the shelf lives of high-temperature short-time fluid milk products will require comprehensive, integrated efforts along the entire milk processing continuum.     

Siderophore production and utilization by milk spoilage Pseudomonas species

Many bacteria respond to potentially growth-limiting availability of iron by producing low-molecular-weight iron chelators (siderophores). The aim of this work was to examine the siderophores synthesized and utilized by Pseudomonas spp. implicated in milk spoilage. Twenty isolates of Pseudomonas spp. previously shown to have significant milk spoilage potential were tested for the ability to produce siderophores. Of these, 14 produced pyoverdin and 2 of these also produced pyochelin; 1 produced only pyochelin; 1 produced only salicylate; 2 produced non-pyoverdin, hydroxamate-containing siderophore; and 2 produced chrome azurol sulfonate reactive material that was neither pyoverdin nor pyochelin. There was considerable diversity among the pyoverdins produced. All isolates were shown to utilize iron complexed with exogenous pyoverdin, but usage of particular exogenous pyoverdins differed among isolates. Interference with the iron-uptake systems of the Pseudomonas spp. may be a means by which food spoilage can be slowed, and the pyoverdin system would appear to be a potential target. However, given the diversity of pyoverdins produced and utilized, and the presence of other siderophores, successful interference with bacterial iron acquisition in this context may be challenging.     

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities—A case study

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.     

An indirect ELISA system for the detection of heat-stable Pseudomonas endopeptidases (AprX) in milk

Heat-stable endopeptidases in raw milk, especially the alkaline metallopeptidase AprX secreted by Pseudomonas spp., are a well-known challenge for the dairy industry. They can withstand UHT treatment and may cause quality defects over the shelf life of milk products. Therefore, we established an indirect ELISA for the detection of Pseudomonas AprX in milk. We developed a 2-step sample treatment for milk contaminated with AprX to avoid the interference of milk proteins with the detection system. First, casein micelles were destabilized by the detraction of Ca²⁺ using trisodium citrate; then, AprX was concentrated 10-fold using hydrophobic interaction chromatography. The recovery of AprX in spiked milk samples after the 2-step treatment was 43 ± 0.1%. Specific antibodies for purified AprX from Pseudomonas lactis were produced to establish the ELISA. Western blot experiments showed that the binding affinity of these antibodies depended on the sequence homology of the AprX from P. lactis and several other Pseudomonas spp. The indirect ELISA, which was completed in 6 to 7 h, had a limit of detection of 21.0 ng mL^?1 and a limit of quantification of 25.7 ng mL^?1. Milk proteins or milk endogenous peptidases were not detected by the antibodies. The ELISA had high precision, with a CV between 0.2 and 0.8% measured on the same day (intraday) and 5.6 and 6.8% measured on 5 separate days (interday). Milk samples were spiked with different AprX activity levels [7.5–150 nkat Na-caseinate/o-phthalaldehyde (OPA) mL^?1] and evaluated by ELISA. The recovery of the ELISA was 92.3 ± 1.6 to 105 ± 4.7%. The lowest AprX activity quantifiable in the spiked milk samples was 500 pkat Na-caseinate/OPA mL^?1. The proof of concept to detect heat-stable Pseudomonas AprX in milk by ELISA was established.     

Microbial biodiversity of raw milk collected from Yangzhou and the heterogeneous biofilm-forming ability of Pseudomonas

This study aimed to reveal microbial populations in Chinese raw milk using both 16S rRNA sequencing and high-throughput sequence (HTS) methods. Psychrotrophic isolates were clustered into 23 genera and 57 species, and the abundant genera in raw milk were Pseudomonas, Acinetobacter and Lactococcus. Genetic diversity and biofilm-forming capacity of Pseudomonas isolates were analysed using random amplified polymorphic DNA (RAPD) and crystal violet staining assay, respectively. Heterogeneous biofilm-forming ability of Pseudomonas isolates with the same RAPD patterns was observed. HTS analysis revealed a 33-fold higher diversity at the genus level, and a different bacterial population was predominated by Flavobacterium, Pseudomonas and Acinetobacter. The results provide insight into the microbial contamination of raw milk.     

Quantification of the proteolytic and lipolytic activity of microorganisms isolated from raw milk

Heat-resistant enzymes of psychrotolerant microorganisms are associated with the spoilage of UHT-milk and milk products. In this study, we investigated the extracellular peptidase, esterase and lipase activity after submerged cultivation at 6 °C of 231 recently isolated microorganisms in milk medium. In contrast to the widely used agar diffusion tests for secretion of hydrolases, here a more realistic liquid screening approach was used. The advantages of the latter are the possibility of quantifying the enzyme activity in volumetric units and the opportunity to simulate the growth conditions of the microorganisms to the storage conditions of raw milk. The majority of enzymatically active isolates belonged to Gram-negative bacteria, especially the genus Pseudomonas. Surprisingly, among them, twelve novel Pseudomonas species were discovered. In this study, we demonstrated that numerous raw milk isolates, including bacteria and yeasts, produce extracellular enzymes that may cause spoilage problems for the dairy industry.     

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.     

Diversity of gram-positive catalase-negative cocci in sheep bulk tank milk by comparative 16S rDNA sequence analysis

The species diversity of 192 isolates of a collection of Gram-positive catalase-negative cocci (GPCNC) from sheep bulk tank milk was investigated using 16S rDNA sequencing. A molecular approach enabled the identification of 23 species of GPCNC of sanitary and technological importance within the Enterococcus, Streptococcus, Lactococcus, Aerococcus and Trichococcus genera. This is relevant since ovine dairy products might be a source of commensal and potentially pathogenic or drug resistant GPCNC. Control strategies are needed in the food chain to optimise food safety and product development.