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

Abstract

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 graypigmented 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.

Draft Genome Sequences and Genomic Analysis for Pigment Production in Bacteria Isolated from Blue Discolored Soymilk and Tofu

“…carnis strains UCD_MED3 and UCD_MED7 both contain at least two homologs of each of the trpABCDF genes. We also detected multiple homologs of these genes in confirmed blue pigment-producing Pseudomonas strains, as well as two Pseudomonas carnis strains that have been reported to produce black and grey diffusible pigments on multiple types of media, including refrigerated pig flesh [24,27,28]. These results corroborate previous findings and suggest that our analysis is comparable to others.…”

“…A homolog was considered present if there was a hit with an E-value better than 1×E -10 . Ten isolates confirmed to produce blue pigment contain at least two homologs of each gene 24 , 27 . Two isolates contain two homologs of each trp gene but have not been reported to produce blue pigment 28 .…”

“…Five isolates that have not been reported to produce a blue pigment contain only one homolog of each gene 28 , 29 . In addition, five isolates for which blue pigment production was tested but not observed contain only one gene 24 , 27 (Table 5 ).…”

“…They are in a sister group to Pseudomonas lactis and Pseudomonas paracarnis. Additionally, P. lactis and P. fluorescens are among those species previously reported as present in blue-colored dairy products such as milks, cheeses and queso fresco [27,33]. Pseudomonas carnis UCD_MED3 and Pseudomonas carnis UCD_MED7 lie within a highly blue pigmented clade, however some other species designations in this clade remain debatable, and strains that produce a blue pigment are found outside of this main clade.…”

Identification, subtyping, and tracking of dairy spoilage-associated Pseudomonas by sequencing the ileS gene

“…Pseudomonas are ubiquitous in natural environments, nutritionally versatile, often psychrotolerant, and capable of producing undesirable odors, flavors, and pigments that render affected food spoiled and possibly inedible (Hayes et al, 2002;Remenant et al, 2015). For these reasons, Pseudomonas has long been identified as a predominant spoiler of many fresh foods, including uncultured dairy products such as fluid milk and mozzarella cheese (del Olmo et al, 2018;Reichler et al, 2019). Although Pseudomonas spp.…”