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.     

When cheese gets the blues: Pseudomonas fluorescens as the causative agent of cheese spoilage

A bacterial contamination of fresh, low-acid cheese that resulted in production of a blue fluorescent pigment on the surface of the cheese was determined to be caused by Pseudomonas fluorescens biovar IV, a gram-negative bacteria that produces a blue, nondiffusible pigment as well as the soluble pigment pyoverdin, which fluoresces under UV light. Ten isolates collected from contaminated cheese and environmental samples were initially identified as P. fluorescens using 16S rDNA sequencing, but only 8 of the isolates produced blue pigment and fluoresced under UV light when re-inoculated onto fresh, low-acid cheese. The Biolog Metabolic Fingerprint system (Biolog Inc., Hayward, CA) and the Analytical Profile Index (BioMerieux Vitek Inc., Hazelwood, MO) for nonenteric gram-negative species as well as EcoRI ribotyping did not differentiate between the isolates that produced blue color and those that did not. Pulsed field gel electrophoresis with the enzyme XbaI was able to distinguish between the isolates that produced pigment and those that did not and allowed for identification of a specific environmental site (i.e., an overhead cheese vat agitator system) as the likely source of product contamination.     

Identification of the Pseudomonas fluorescens group as being responsible for blue pigment on fresh cheese

New cases of blue cheese discoloration has led to recent research to identify the causal agent and factors that favor blue pigment appearing. Nonetheless, very few reports have described the source of contamination and the measurements to eradicate the microbiological source on cheese farms by determining the relation between blue discoloration on fresh cheese and the Pseudomonas fluorescens group. Thus, 60 samples from a cheese farm (cheese, equipment surfaces, tap water, and raw and pasteurized milk) were analyzed by phenotypical, MALDI-TOF, 16S rRNA sequencing and pulsed-field gel electrophoresis tests to determine the causal agent. The results obtained by pulsed-field gel electrophoresis with restriction enzymes XbaI and SpeI confirmed tap water as the initial contaminated source. The above-mentioned result was essential to avoid Pseudomonas contamination due to the most residual microorganisms being inactivated through a new disinfection program.     

Spoilage potentials and antimicrobial resistance of Pseudomonas spp. isolated from cheeses

Pseudomonas spp. are aerobic, gram-negative bacteria that are recognized as major food spoilage microorganisms. A total of 32 (22.9%) Pseudomonas spp. from 140 homemade white cheese samples collected from the open-air public bazaar were isolated and characterized. The aim of the present study was to investigate the biochemical characteristics, the production of extracellular enzymes, slime and β-lactamase, and antimicrobial susceptibility of Pseudomonas spp. isolated from cheeses. The identified isolates including Pseudomonas pseudoalcaligenes, Pseudomonas alcaligenes, Pseudomonas aeruginosa, Pseudomonas fluorescens biovar V, and P. pseudoalcaligenes ssp. citrulli were found to produce extracellular enzymes, respectively: protease and lecithinase production (100%), and lipase activity (85.7, 42.9, 100, and 100%, and nonlipolytic, respectively). The isolates did not produce slime and had no detectable β-lactamase activity. The antimicrobial susceptibility of the isolates was tested using the disk diffusion method. Pseudomonas spp. had the highest resistance to penicillin G (100%), then sulphamethoxazole/trimethoprim (28.1%). However, all Pseudomonas spp. isolates were 100% susceptible to ceftazidime, ciprofloxacin, amikacin, gentamicin, and imipenem. Multidrug-resistance patterns were not observed among these isolates. In this study, Pseudomonas spp., exhibiting spoilage features, were isolated mainly from cheeses. Isolation of this organism from processed milk highlights the need to improve the hygienic practices. All of the stages in the milk processing chain during manufacturing have to be under control to achieve the quality and safety of dairy products.     

Evolution under different storage conditions of anomalous blue coloration of Mozzarella cheese intentionally contaminated with a pigment-producing strain of Pseudomonas fluorescens

Several widespread occurrences of anomalous blue coloration of Mozzarella cheese have been recorded in the United States and some European countries. Official laboratory analysis and health authorities have linked the occurrences to contamination of the processing water with strains of Pseudomonas fluorescens, although several experts questioned how to unequivocally link the blue color to the presence of the microorganism. To establish a method to determine whether a given Pseudomonas spp. strain is responsible for the defect and study the evolution of the coloration under different storage conditions, we developed an in vitro system for the evaluation of blue coloration of Mozzarella cheese intentionally contaminated with strains of P. fluorescens. The purpose of the system was to determine whether P.fluorescens strains, isolated from Mozzarella cheese with anomalous blue coloration, were able to reproduce the blue coloration under controlled experimental conditions. Thirty-six trials of experimental inoculation of Mozzarella cheese in different preservation liquids were conducted using various suspensions of P.fluorescens (P. fluorescens ATCC 13525, P.fluorescens CFBP 3150, and P. fluorescens 349 field strain isolated from blue-colored Mozzarella cheese) at different concentrations and incubated at different temperatures. Growth curves of all tested P.fluorescens strains demonstrated that after 3 d of incubation the concentration was generally >10⁶ cfu/g of Mozzarella cheese incubated in either tryptic soy broth (control) or conditioning brine. Prolonged incubation for 5 d at either 20°C or 8°C led to concentrations up to 10⁹ cfu/g of Mozzarella cheese incubated in tryptic soy broth and up to 10⁸ cfu/g of Mozzarella cheese incubated in preservation liquid. All Mozzarella cheeses inoculated with the field strain of P. fluorescens, except those opened 1 h after packaging and stored at 8°C, showed the characteristic anomalous blue coloration, which appeared from 1 to 72 h after opening the packaging, and was proportional to colony count, duration of storage, and storage temperature. With the proposed system, which enabled a larger number of samples to be analyzed under controlled experimental conditions and a large amount of data to be generated in a short time, we described precisely how and under which conditions the presence of P. fluorescens in Mozzarella cheese is responsible for the anomalous blue coloration. The system will help producers intercept contaminated batches and help consumers avoid the conditions under which the defect can appear.     

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

Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors, flavors, and pigments. They are frequently found in dairy plant environments and enter dairy products predominantly as postpasteurization contaminants. Current subtyping and characterization methods for dairy-associated Pseudomonas are often labor-intensive and expensive or provide limited and possibly unreliable classification information (e.g., to the species level). Our goal was to identify a single-copy gene that could be analyzed in dairy spoilage-associated Pseudomonas for preliminary species-level identification, subtyping, and phenotype prediction. We tested 7 genes previously targeted in a Pseudomonas fluorescens multilocus sequence typing scheme for their individual suitability in this application using a set of 113 Pseudomonas spp. isolates representing the diversity of typical pasteurized milk contamination. For each of the 7 candidate genes, we determined the success rate of PCR and sequencing for these 113 isolates as well as the level of discrimination for species identification and subtyping that the sequence data provided. Using these metrics, we selected a single gene, isoleucyl tRNA synthetase (ileS), which had the most suitable traits for simple and affordable single-gene Pseudomonas characterization. This was based on the number of isolates successfully sequenced for ileS (113/113), the number of unique allelic types assigned (83, compared with 50 for 16S rDNA), nucleotide and sequence diversity measures (e.g., number of unique SNP and Simpson index), and tests for genetic recombination. The discriminatory ability of ileS sequencing was confirmed by separation of 99 additional dairy Pseudomonas spp. isolates, which were indistinguishable by 16S rDNA sequencing, into 28 different ileS allelic types. Further, we used whole-genome sequencing data to demonstrate the similarities in ileS-based phylogenetic clustering to whole-genome-based clustering for 27 closely related dairy-associated Pseudomonas spp. isolates and for 178 Pseudomonas type strains. We also found that dairy-associated Pseudomonas within an ileS cluster typically shared the same proteolytic and lipolytic activities. Use of ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry identify, characterize, and track Pseudomonas in their facilities and 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.     

Interactions in biofilms of Lactococcus lactis ssp. cremoris and Pseudomonas fluorescens cultured in cold UHT milk

Three Lactococcus lactis ssp. cremoris isolates from refrigerated bulk raw milk were cultured separately and in association with a known psychrotrophic dairy Pseudomonas fluorescens strain, in skim UHT milk for 72 h at 7°C, to determine mutual influences in both the planktonic and biofilm phases. Two levels of inoculum of each culture partner were combined. Protocooperation and commensalism cases were found, all of them in the biofilm phase. Type and intensity of the interactions depended on Lactococcus strain and on the cell density of each partner. Maximum enhancement of attachment was observed to be approximately 100-fold for P. fluorescens and 20,000-fold for one of the L. lactis strains. Confocal scanning laser microscopy images show compact masses of Pseudomonas trapping lactococci cells in cooperative biofilms.     

Growth of Pseudomonas weihenstephanensis,Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life

One of the reasons for spoilage of UHT milk during shelf-life is the presence of residual proteolytic activity produced from Pseudomonas spp. during storage of raw milk. The aim of this study was to describe the product defects occurring in indirectly heated UHT milk during shelf-life, and to establish a correlation between proteolytic activity and onset of product spoilage. UHT milk was produced from raw milk incubated with different Pseudomonas strains, and examined over four months during storage at 20 °C. Inactivation kinetics of the peptidases were determined. In UHT milk, product defects occurred in the order: bitterness – particles – creaming – sediment – gelation in all the samples containing peptidases (apparent enzyme activity ≥ 0.03 pkat mL⁻¹). A linear correlation was found between proteolytic activity and onset of product defects, apart from onset of gelation.     

The effect of legume protease inhibitors on native milk and bacterial proteases

Protease inhibitors from legume seed extracts (soybean, cowpea and marama beans) and purified soybean protease inhibitor were evaluated with regards to their abilities to inhibit proteases produced by important milk contaminating bacteria, i.e. Bacillus spp. and Pseudomonas spp., and native milk protease, plasmin. Although heat treatment is the most common mean of inactivating enzymes, some heat-stable enzymes can survive the ultra-high temperature (UHT) processing of milk and cause sensory and consistency defects during storage at room temperature. The legume protease inhibitors reduced the activity of plasmin and proteases produced by Bacillus spp. by up to 94% and 97%, respectively, while it showed low inhibitory activity towards Pseudomonas fluorescens proteases (19%) in a buffer system. The protease inhibitors reduced the activity of plasmin (41%) and Bacillus proteases (50%) in UHT milk, however to a lesser extent as compared to inhibition in the buffer system; while it had little or no effect on proteases form Pseudomonas spp. Legume protease inhibitors show great potential in preventing or reducing proteolytic activity of Bacillus proteases and plasmin and may be exploited in various applications where these proteases cause sensory or consistency defects in the product.     

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.     

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.     

Kinetic models for pulsed electric field and thermal inactivation of Escherichia coli and Pseudomonas fluorescens in whole milk

Inactivation of Escherichia coli ATCC 11775 and Pseudomonas fluorescens ATCC 948 in UHT whole (4% fat) milk during thermal processing at 56–62 °C and pulsed electric field (PEF) processing at 30 or 35 kV cm⁻¹ at approximately 30, 40 or 50 °C was investigated. E. coli ATCC 11775 was more heat-resistant than P. fluorescens ATCC 948, but more susceptible to PEF processing. All inactivation kinetics showed strong deviations from log-linearity. Thus, a simplified logistic (log-decay) regression model was used to accurately predict thermal and PEF inactivation of E. coli ATCC 11775 and P. fluorescens ATCC 948 under various treatment conditions. This is a useful tool for identifying processing conditions to inactivate pathogenic and spoilage microorganisms in whole milk at sub-pasteurisation temperatures.     

The microflora of fresh and spoiled sardines (Sardina pilchardus) caught in Adriatic (Mediterranean) Sea and stored in ice

A total of 1500 strains were isolated from the skin and gills of fresh, ice-stored (4 days) and spoiled (8 days) Adriatic sardines, and were identified at different taxonomic levels. Fresh sardine microflora found included mostly non-fermenting Gram-negative bacteria, (Pseudomonas, Flavobacterium, Psychrobacter, Acineobacter, Shewanella), and a minor proportion of Enterobacteriaceae and Gram-positive bacteria. The highest increase in microflora frequency, after 8 days in ice, was observed for thePseudomonas fragigroup (8–30·8%) andShewanella putrefaciens(1·8–11%). A significant presence was also noted for fluorescentPseudomonas(15·6–18·4%) andPsychrobacter immobilis(16·4–23·4%). The isolation frequency of the other groups decreased during storage. The most important proteolytic species werePseudomonas fluorescensandShewanella putrefaciensand the most lipolytic bacteria werePsychrobacter immobilisand again,P. fluorescensandS. putrefaciens. A total of 288 isolates, representative of the main groups, were tested for potential spoiling activity (H₂S and off-odour production, TMAO reduction).Shewanella putrefacienswas the strongest spoiler, followed byPseudomonas fluorescens. A weaker activity was observed for strains ofPseudomonas fragi, enterobacteriaceae and non-saccharolytic pseudomonads. The contribution of weak spoiler bacteria can be undermined by the activity of the strongest spoilers, but in some cases the considerable incidence of the former group suggests their effective role.Morganella morganiiwas the only histamine-producing species among 57 screened strains representative of different taxa.     

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.     

Tyrosine metabolism in pigment-forming Yarrowia lipolytica strains isolated from English and European speciality mould-ripened cheese exhibiting a brown discolouration defect

Yarrowia lipolytica s4fd was isolated from a UK-manufactured speciality mould-ripened cheese as the causative micro-organism of a brown surface discolouration spoilage defect. Comparative studies utilizing Y. lipolytica isolates indicated that inter-strain variations in pigment accumulation were attributable to differences in tyrosine uptake and metabolism to homogentisic acid. The range and activity profiles of proteolytic enzymes involved in tyrosine release and turnover in isolates that differed in pigment formation were, however, similar. Homogentisic acid formation was affected by physiological parameters, and approaches that may be developed by the cheesemaker to prevent spoilage losses associated with the development of the brown discolouration defect are discussed.     

Production of biogenic amines during the ripening of Pecorino Abruzzese cheese

The production of biogenic amines (BA) during the manufacturing and ripening of sheep milk Pecorino Abruzzese cheeses prepared from raw milk without starter culture (A) and from pasteurized milk with added starter (B) were compared. At the end of ripening (60 days), the total BA contents of cheeses of batches A and B were 697 and 1086 mg kg⁻¹, respectively; the dominant BA were different. Single isolates of enterococci, pseudomonads and Enterobacteriaceae were screened for their potential to produce BA. Qualitative tests indicated a large spread of BA-forming cultures among the members of the Enterobacteriaceae and lactic acid bacteria (LAB). Differences among the levels of BA produced in UHT milk by representative isolates of coliforms, Pseudomonas and LAB were observed in relation to the microbial group or the isolate. The results emphasize the need to improve the general hygienic conditions of Pecorino Abruzzese cheese manufacture and control the indigenous bacterial population.     

Isolation and characterisation of a heat-resistant peptidase from Pseudomonas panacis withstanding general UHT processes

A secreted peptidase from Pseudomonas panacis was identified and purified. Genome sequencing of the producer strain allowed identification of the peptidase as AprA based on a comparison to peptide sequences of mass spectra obtained from the purified enzyme. The amino acid sequence of the 49.4 kDa peptidase was 98% similar to the metallopeptidase AprX from a Pseudomonas fluorescens strain. The peptidase showed maximum activity at pH 8 and 40 °C and withstood general ultra-high temperature (UHT) processing (138 °C for 18 s) in skim milk, with 88.0 ± 7.7% of the initial enzyme activity remaining after heating. The peptidase showed considerable enzyme activity under storage conditions of UHT milk. The potential for spoilage of milk might during storage was verified by adding very low enzyme activities to UHT-treated milk. The addition of 1 pkat mL⁻¹ peptidase activity resulted in a destabilisation of the milk during four weeks storage.     

Invited review: Controlling dairy product spoilage to reduce food loss and waste

Food loss and waste is a major concern in the United States and globally, with dairy foods representing one of the top categories of food lost and wasted. Estimates indicate that in the United States, approximately a quarter of dairy products are lost at the production level or wasted at the retail or consumer level annually. Premature microbial spoilage of dairy products, including fluid milk, cheese, and cultured products, is a primary contributor to dairy food waste. Microbial contamination may occur at various points throughout the production and processing continuum and includes organisms such as gram-negative bacteria (e.g., Pseudomonas), gram-positive bacteria (e.g., Paenibacillus), and a wide range of fungal organisms. These organisms grow at refrigerated storage temperatures, often rapidly, and create various degradative enzymes that result in off-odors, flavors, and body defects (e.g., coagulation), rendering them inedible. Reducing premature dairy food spoilage will in turn reduce waste throughout the dairy continuum. Strategies to reduce premature spoilage include reducing raw material contamination on-farm, physically removing microbial contaminants, employing biocontrol agents to reduce outgrowth of microbial contaminants, tracking and eliminating microbial contaminants using advanced molecular microbiological techniques, and others. This review will address the primary microbial causes of premature dairy product spoilage and methods of controlling this spoilage to reduce loss and waste in dairy products.     

Microbial composition of defect smear – A problem evolving during foil-prepacked storage of red-smear cheeses

To elucidate the problem of smear defect in foil-prepacked fully-ripened red-smear cheese portions, the microbial composition of 47 smear samples from 2 cheese varieties was analyzed. Cheese variety M was characterized by a diversity of 27 different bacterial species of which 11 represented typical smear microorganisms belonging to Arthrobacter, Brachybacterium, Brevibacterium, Corynebacterium, Microbacterium and Staphylococcus. For variety T 29 different species were identified with 12 isolates representing typical smear microorganisms. Further isolates represented members of Bacillus, Citrobacter, Enterococcus, Facklamia, Morganella, Proteus and Vagococcus. Culture-dependent analysis revealed a stable microbial composition that was confirmed by culture-independent techniques. However, several bacteria with potential to contribute to smear defects by off-flavor or exopolysaccharide production were identified. Our results indicate that the cause of the defect might not be assigned to a single microbial group but rather to the contribution of various microorganisms and their metabolic activity within the foil.