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Pseudomonas fluorescens: a potential food spoiler and challenges and advances in its detection

Abstract

Purpose

This review focuses on the spoilage strategies used by the Pseudomonas fluorescens, and in addition, it also discusses various diagnostic approaches used for its identification in food items. Some challenges faced and advances in the detection of P. fluorescens and also discussed in this review.

Methods

An extensive literature search was performed with published work and data was analyzed in detail to meet the requirements of the objectives.

Results

P. fluorescens are unicellular rods, with long straight or curved axis, but not helical, motility by one or more polar flagella, Gram-negative, non-spores former, stalks, or sheaths. P. fluorescens is represented by seven biotypes denoted by the letters A, B, C, D, E, F, and G. The microbe shows wide choice of growth temperature and causes contamination and spoilage in ordinary and refrigerated food items by its enzymes and pigment production. The biofilm formation by P. fluorescens poses another serious threat to the food industries.

Conclusion

Molecular identification of P. fluorescens is generally done by 16S rRNA, intergenic spacer (ITS1) utilizing traditional polymerase chain reactions (PCR). Nowadays, qPCR and multiplex PCR are largely utilized in identification of P. fluorescens based on AprX gene (extracellular caseinolytic metalloprotease) in the milk and meat spoilage strains. The available methods still show some disadvantages with accuracy and specificity of detection. Rapid detection of P. fluorescens in food samples is the need of hour to improve the detection efficiency.

Introduction

In nineteenth Century, Dr. Migula, Professor at Karlsruhe Institute of Germany, first proposed the name Pseudomonas and it read as cells with polar organs of motility, with development of spores in a few categories (for instance: Pseudomonas violacea) (Palleroni 2010). In 1926, Den Dooren de Jong stressed on microbes of soil and featured the extreme adaptability of Pseudomonas (Palleroni 2010). It was represented by unicellular rods, with the long axis curved or straight, motility by one or more polar flagella, Gram-negative, non-spore forming, sheaths, or stalks (Stanier et al. 1966). The respiration is the only process involved in energy-yielding metabolism and all species utilize oxygen as a terminal oxidant, whereas some species use denitrification as an anaerobic respiratory system. All Pseudomonas spp. are chemoorganotrophs, while few are facultative chemolithotrophs which use H2 as energy source. Pseudomonads has three subgeneric group: fluorescent group having species Pseudomonas aeruginosa, P. fluorescens, P. putida; acidovorans group, with P. acidovorans, P. testosterone; and, alcaligenes group representing P. alcaligenes, P. pseudoalcaligenes sp.nov., P. mutivorans sp.nov, P. stutzeri, and P. maltophilia (Stanier et al. 1966). Pseudomonas fluorescens is represented by seven biotypes denoted by the letters A, B, C, D, E, F, and G (Stanier et al. 1966). Consequently, DNA/RNA hybridization confirmed the presence of five diverse rRNA groups (rRNA groups I–V) (Palleroni et al. 1972; Palleroni 1993; Kersters et al. 1996). Pseudomonas rRNA group I contained P. aeruginosa, all the fluorescent species (P. fluorescens, P. putida, P. syringae), and some non-fluorescent species (P. stutzeri, P. alcaligenes, P. pseudoalcaligenes, and P. mendocina) (Palleroni et al. 1973). Various types of spoilage caused by P. fluorescens and its spoilage agents are shown in Fig. 1. The various types of spoilage caused the Pseudomonas fluorescens, and various diagnostic approaches used for its identification are discussed in the following sections:

Fig. 1
figure 1

Spoilage of different food items by Pseudomnas fluorescens

Pseudomonas fluorescens as a food spoiler

Pseudomonas fluorescens is a regular contaminant of ready to-eat foods. These microscopic organisms show wide choice of growth temperature, and contamination is a key issue in ordinary and refrigerated food items as shown in Table 1. A report from Italy also confirmed the presence of P. fluorescens in packed ready to-eat vegetables (Caldera and Franzetti 2014). Raw vegetables quality, processing, packaging system, and storage temperature are essential factors that influence the microbial composition in the final product. Pinkeye disease in potato tubers was caused by the pectic enzyme of P. fluorescens (Folsom and Friedman 1959; Huether and McIntyre 1969). Biotypes I and II of P. fluorescens were isolated from celery, cabbage, and chicory stored at 1° and 4 °C, and on the other hand, biotypes III and V were isolated from incompletely processed lettuce gathered from the processing plants (Brocklehurst and Lund 1981; Sellwood et al. 1981; Magnuson et al. 1990). Head spoil disease in broccoli had additionally confirmed the relationship of surfactant-positive strains of P. fluorescens biovar II, IV, which reduces the water surface tension and enables surfactant-deficient strains to colonize over water-soaked areas even in the absence of physical injury (Hildebrand 1989). Biosurfactants producing P. fluorescens are also responsible for the spoilage of aerobically stored chicken meat (Mellor et al. 2011). Different volatile compounds, for example, trimethylamine, methyl mercaptan, and dimethyl disulfide, were produced by P. fluorescens in sterile fish muscles under in vitro condition at 0 °C after 32 days of incubation (Miller et al. 1973). P. fluorescens also produced 2-butenal, methyl thiol n-butyrate, 3-octanol in spoiled chicken breast stored at 2 to 6 °C for 14 days (Pittard et al. 1982). Similarly, alcohols, aldehydes, esters, hydrocarbons, toluene, ketones, few sulfur-containing compounds were identified, when P. fluorescens biotype I was inoculated under in vitro conditions in beef stored at 6 °C at pH 5.5–5.7 (Edwards et al. 1987). P. fluorescens isolated from sea bream stored aerobically in Greece market at 0°, 10°, and 20 °C and modified-atmosphere packaging (MAP) conditions (40% CO2–30% N2–30% O2) (Tryfinopoulou et al. 2002). An investigation in Belgium has confirmed the growth of P. fluorescens in tofu packed in modified conditions and showed that some strain can survive under 100% CO2 (Stoops et al. 2012). The postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk with P. fluorescens continues to be an issue with processor defects like lower flavor scores, coagulation, and fruity fermented milk (Reichler et al. 2018).

Table 1 Specific spoilage causes by Pseudomonas fluorescens in various food items

P. fluorescens having protease, lecithinase, and lipase activity were isolated from the raw and pasteurized milk in four dairy processing plants in New York (USA), from chicken in Santiago (Chile) and cheese from El-Menofia (Egypt) (Dogan and Boor 2003; Hammad 2015; Morales et al. 2016). P. fluorescens acts as most common contaminants in Italian bulk milk tank and shows lipolytic, proteolytic, and lecithinase activity (Decimo et al. 2014). A research report from Brazil additionally confirmed the deterioration of goat milk by P. fluorescens that was strongly associated with its proteolytic activity at different temperatures (Scatamburlo et al. 2015). A study in Iraq revealed the presence of P. fluorescens in raw cow and buffalo’s milk due to protease activity (Al-Rodhan and Nasear 2016). P. fluorescens biotypes I, II, and III were likewise identified due to proteolytic and lypolytic activities in the milk, minced beef, chicken, and fish sold at the different sale points in Izmir, Turkey (Keskin and Ekmekçi 2007). P. fluorescens isolated from the pork meat in Budapest, Hungary, showed quite intense proteolytic activity as compared to lipase (Márta 2012).

Mystery of blue cheese

P. fluorescens can induce troublesome changes in food items by producing pigment molecule (Andreani et al. 2014). A notable model is blue mozzarella cheese events that happened in Italy, when shoppers observed blue stains on mozzarella cheese in the wake of opening the bundles (RASFF 2010). About 70,000 mozzarella cheese chunks were tested and P. fluorescens group was found associated with this specific spoilage event (Andreani et al. 2015). Substance responsible for blue color in contaminated mozzarella cheese was indigoidine compound produced by P. fluorescens (Caputo et al. 2015). Later on, some reports confirmed that the blue shade created by P. fluorescens strains was not because of indigo or indigoidine; however, it was a substantial particle, and an indigo-derivative (Andreani et al. 2015). Besides, in the ongoing finding, additionally it was observed that blue shade is most probably an indigoid molecule (Fasolato et al. 2018). A recent study reported that the genes involved in blue pigment production possibly play role in tryptophan biosynthetic pathway and also provide antioxidant protection (Andreani et al. 2019).

Enzymes of P. fluorescens

Lipases, i.e., triacylglycerol hydrolases, act on food fat molecules and cause the release of unsaturated glycerol and fatty acids (Andreani 2016). Free short-chain unsaturated fats give unpleasant flavors, stated to be rancid, whereas medium-chain unsaturated fats are associated with bitter, foamy, or unclean flavors (Samaržija et al. 2012). Various report stated that lipolytic activity is more noteworthy at refrigeration temperatures (Woods et al. 2001; Rajmohan et al. 2002). In cheese, lipases get absorbed within fat globules and remain in the cheese, inciting decay impacts amid ripening of hard and semi-hard cheese (Samaržija et al. 2012). Presence of different lipases in spoiled food improves the heat stability of lipase (Teh et al. 2014). Few strains of P. fluorescens cause rancidity in cheddar cheese due to lipase that retained 20–25% of their lipolytic activity at 100 °C for 10 min (Law et al. 1976). Extracellular lipase enzymes from P. fluorescens responsible for the off flavoring of the milk are treated at ultra-high-temperature (UHT) (Andersson et al. 1981). In meat, lipases break down glycerides forming free fatty acids and produce off-flavor, frequently referred to as rancidity (Huis 1996). Novel lipase from P. fluorescens C9 strain, in which lipA gene was reported, strongly suggested the presence of second lipase in this strain (Dieckelmann et al. 1998). On the other hand, in P. fluorescens B52, genes encoding for thermostable lipase (lipA) and protease (aprX) situated within same operon, differed by associated genes with discharge of the protease (extracellular) and gene expression of both hydrolases, are interlinked (Woods et al. 2001; McCarthy et al. 2004). In another report, P. fluorescens grown in refrigerated raw milk with lipase enzyme had high activity at 25 °C and a broad pH optimum extending from 7.0 to 10 (Martins et al. 2015).

Gelation of UHT milk by proteinases of Pseudomonas fluorescens strain isolated from raw milk depends on its amount before heat treatment (Law et al. 1977). Shelf-life of UHT milk is much lower than processed raw milk stored at 6 °C (Griffiths et al. 1988). The proteinase produced in the milk leads to extensive breakdown of k-casein to para-k-casein, an event similarly to the action of rennet. Additionally, modification of milk casein is encouraged because of the activity of milk proteinase (endogenous), particularly in plasmin (Datta and Deeth 2001).

Production of proteinases destabilizes the casein and influences the cheese yield (Mitchell and Marshall 1989). In this process specifically, plasminogen and plasmin are liberated from casein micelles, altering the cheese yield and influencing the texture and flavor of the final product (Samaržija et al. 2012). A recent study based on the phenotypic examination of 87 P. fluorescens species revealed that each strain (94%) could stimulate proteolysis on nutrient agar plates with 2% UHT milk at 22 °C, while around 72% could initiate this at refrigeration temperatures, showing high predominance of proteolytic strains inside the P. fluorescens group (Andreani et al. 2014). The most common family of thermostable proteases within the genus Pseudomonas is serralysin protease family, a much conserved protein group from the AprX with an alkaline zinc metalloprotease family with molecular masses in the range of 39.2 and 45.3 kDa (Dufour et al. 2008; Marchand et al. 2009; Teh et al. 2014). Extracellular protease from the P. fluorescens CY091 with sub-atomic weight of 50 kDa retained 20% of its activity even after heating at boiling temperature for 10 min, revealing its high resistance to heat inactivation (Liao and McCallus 1998). Another zinc-metalloacid protease produced by Pseudomonas fluorescens RO98 was isolated from raw milk with molecular weight of 52 kDa and demonstrated its activity between 15 and 55 °C and pH 4.5–9.0 (Koka and Weimer 2000). In a few Pseudomonas spp. strains, AprX has been identified as the only protease associated with food decay (Woods et al. 2001). Proteases are chiefly synthesized at the end of the exponential stage, when thickness of cell is high, featuring the contribution of quorum sensing mechanism in spoilage activity (Liu et al. 2007; Pinto et al. 2010; Bai and Rai 2011).

Pectic lyase is additionally produced by soft rot causing strains of P. fluorescens (Liao 1989). Yield of pectin lyase produced by P. fluorescens W51 was progressively increased when glycerol was utilized as a sole source of carbon, whereas thermal stability of pectate lyase produced by P. fluorescens CY091 expanded when CaCl2 or positively charged molecules, for example, polylysine was used at 48 °C in the culture medium (Schlemmer et al. 1987; Liao et al. 1997). Another critical class of extracellular enzymes present in spoiled food is constituted by lecithinases and different phospholipases that disturbs the fat globules of milk and makes fat substances accessible for further lipase action (Samaržija et al. 2012). The most common enzyme of this family is phospholipase C (lecithinase) which is produced by most of the pseudomonads (Fox et al. 1976).

Biofilm formation by P. fluorescens

P. fluorescens has been regarded as the predominant and most harmful microbiota during the cold storage of raw milk (Machado et al. 2015; Von-Neubeck et al. 2015; Vithanage et al. 2016). During storage of the dairy product, not only the P. fluorescens heat-resistant enzymes will remain active after heat sterilization, causing milk bitterness, sediment, and gelation, but they also form biofilm on the surface of equipments and tools in dairy production line (Shpigel et al. 2015; Stoeckel et al. 2016). Once the biofilm is formed by the Pseudomonas bacteria, it is hard to remove it by the hygienic treatments during the processing of raw milk (Ksontini et al. 2013). The biofilm formation will not only cause the pipelines corrosion, but also provides appropriate substratum for the growth of other bacteria including pathogens, which may threaten the health of consumers (Costerton 1999; Aswathanarayan and Vittal 2014). The availability of nutrient in the environment will also affect biofilm growth and P. fluorescens can form biofilms under any nutrient concentration that allows growth (Teh et al. 2014). Biofilm formation by P. fluorescens strains was temperature dependent, and lower incubation temperature (± 10 °C) favored the formation of biofilm after 48 h (Rossi et al. 2016).

Methods used for diagnosis of P. fluorescens

Isolation of P. fluorescens on all universal (supplement agar) medium and in specific media is a normal practice in food industries. Toward the end of nineteenth century, ELISA-based methodologies have given effectively recognizable proof of microscopic organisms from different foods, particularly the meat items. Various ELISA-based techniques were developed for the identification of P. fluorescens using meat surface inhibition ELISA with affectability level as low as 3 × 105 microscopic organisms for each milliliter (Eriksson et al. 1995). Polyclonal antibodies against F protein of P. fluorescens cell envelope were produced and were dependent on indirect ELISA approach for the detection of refrigerated microscopic organisms at an affectability of 104–105 cfu cm−2 (González et al. 1996). Another indirect ELISA kit was developed against the live cells of P. fluorescens in the refrigerated meat with sensitivity level of 104 cfu cm−2 (Gutierrez et al. 1997).

Molecular identification of the P. fluorescens and its biotype dependent on 16S rRNA and intergenic spacer (ITS1) utilizing traditional polymerase chain reactions (PCR) were extensively developed (Scarpellini et al. 2004; Franzetti and Scarpellini 2007; Márta 2012; Ardura et al. 2013; Caldera and Franzetti 2014; Hammad 2015; Al-Rodhan and Nasear 2016; Morales et al. 2016). P. fluorescens enzyme-specific gene identification approaches are also regularly used in its identification as shown in Table 2 (Martins et al. 2005; Decimo et al. 2014; Hammad 2015; Al-Rodhan and Nasear 2016). PCR-denaturing gradient gel electrophoresis (DGGE) was additionally used to examine the V3 and V6-V8 areas of 16S rRNA quality; however, this strategy was not a valid proof to distinguish Pseudomonas in the biological meat community (Jiang et al. 2011). Nowadays, qPCR and multiplex PCR techniques are commonly utilized to identify the P. fluorescens on the basis of AprX gene coding for extracellular caseinolytic metalloprotease in the meat and milk spoilage strains (Dufour et al. 2008; Chiang et al. 2012). Enhanced multiplex PCR was also developed to identify the food microorganisms producing biogenic amines as presence of P. fluorescens odc gene is normally recognized to produce ornithine decarboxylase (De las Rivas et al. 2005, 2006).

Table 2 Common molecular approaches used in Pseudomonas fluorescens detection

Challenges and advances in diagnosis of P. fluorescens

Immunological approaches using ELISA for the detection of P. fluorescens in meat products were well designed but could not get commercialized as most of the experiments were conducted under in vitro conditions. On the other hands, the test showed the variations in their sensitivity level, and this was one of the challenges in diagnosis of the potential food spoiler (Eriksson et al. 1995; González et al. 1996; Gutierrez et al. 1997).

Molecular identification methods based on 16S rRNA gene sequencing provide low resolution and cannot discriminate Pseudomonas at the species level (Ait Tayeb et al. 2005). Although methods based on rpoB gene sequencing are widely used in identification of P. fluorescens strains (Ait Tayeb et al. 2005; Machado et al. 2015), markers targeting protein-coding sequences have also been used to improve the resolution of molecular detection methods for example gene aprX; coding the alkaline protease was used for the identification of P. fluorescens in dairy products, but presence of same gene in other species of Pseudomonas makes it difficult to distinguish between them and poses another challenge in its accurate identification (Martins et al. 2005; Decimo et al. 2014).

To overcome the challenges faced in accurate identification of P. fluorescens from spoiled food items, some advanced methods have been used as shown in Table 3. The taxonomic resolution of 16S rRNA gene-based study is generally limited to the genus level, and the common use of operational taxonomic units (OTUs) based on 97% sequence similarity cut-off often results in phylogenetically mixed units (Koeppel and Wu 2013). These approaches in some cases fail to resolve ecologically meaningful differences between closely related organisms in complex environments (Eren et al. 2014, 2015). An alternative approach used to overcome this problem is using oligotyping, which decomposes a given taxon, or 97% OTU, into high-resolution units (‘oligotypes’) by only using the most information-rich nucleotide positions identified by Shannon entropy calculations (Eren et al. 2013; Schmidt et al. 2014). This approach was successfully used in meat and dairy processing environment for the isolation of P. fluorescens oligotypes (Stellato et al. 2017). Another study reported the use of multiplex PCR for the detection of P. fluorescens showing the ability of biofilm formation with a detection limit of target strain to 102 cfu/ml (Xu et al. 2017). AdnA protein, a transcriptional activator related to biofilms formation in P. fluorescens, is very important for its spreading and survival in soil (Marshall et al. 2001).

Table 3 Some advances in detection of Pseudomonas fluorescens causing food spoilage

In one of the studies, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the P. fluorescens in raw milk (cow), as most of the frequently reported heat-resistant lipase-producing bacterial species with detection limit of 4.8 × 102 cfu/reaction of the template DNA and 7.4 × 101 cfu/reaction of P. fluorescens led to contamination of pasteurized cow milk (Xin et al. 2017). LAMP assay cannot distinguish between DNA from viable cells and to that from dead cells (Chen et al. 2011; Wan et al. 2012). This serves as an advantage to accurately assess the potential contamination of heat-resistant lipase produces in milk as these microorganisms remain active in contaminated dairy products even after P. fluorescens has been destroyed. Therefore, LAMP detection method is more accurate than culture-dependent method.

Raman spectroscopy, an alternative cultivation-free verification method with 15-min analysis time, was developed to detect the P. fluorescens in water samples. According to this test, test colonies were screened under UV light at 365 nm, and fluorescent and nonfluorescent colonies were specifically marked (Yilmaz et al. 2015). In another study, TaqMan assay was developed that showed results better than 16S rRNA for the identification and enumeration of closely related species and strains with a sensitivity 10 cfu/ml. The assay was also successful in determining the concentration of the test preparation within 2 h (Saha et al. 2012).

Conclusion

It is observed that the rates of P. fluorescens are steadily growing and making them perfect spoiler of food items. There is dire need for improvements in accurate detection of this organism. There is still scope for improvement in the presently available method of P. fluorescens detection. Some biosensors have already been developed for detecting food-related diseases, e.g., ultrasensitive transglutaminase-based nano-sensor used for early diagnosis of celiac diseases in human (Gupta et al. 2017). Further, thought is required for more capability in Pseudomonas recognition techniques with new developments in accuracy and specificity to meet the future demand. New approaches using biosensors with high specificity and sensitivity can be developed for robust identification of P. fluorescens present in food and plant samples.

References

  • Ait Tayeb L, Ageron E, Grimont F, Grimont PA (2005) Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates. Res Microbiol 156(5–6):763–773

    Article  CAS  PubMed  Google Scholar 

  • Al-Rodhan AM, Nasear HA (2016) PCR- based detection of Pseudomonas fluorescens in cow and buffaloes’ raw milk. Bas J Vet Res 15(1):194–208

    Google Scholar 

  • Andersson RE, Danielsson G, Hedlund CB, Svensson SG (1981) Effect of a heat-resistant microbial lipase on flavor of ultra-high-temperature sterilized milk. J Dairy Sci 64(3):375–379

    Article  CAS  Google Scholar 

  • Andreani N (2016) Into the blue: spoilage phenotypes of Pseudomonas fluorescens in food matrices (Doctoral dissertation, University of Padova) https://paduaresearch.cab.unipd.it/9109/1/Andreani_NadiaAndrea_tesi.pdf.pdf. Accessed 10 Dec 2018

  • Andreani NA, Martino ME, Fasolato L, Carraro L, Montemurro F, Mioni R, Bordin P, Cardazzo B (2014) Reprint of ‘tracking the blue: a MLST approach to characterise the Pseudomonas fluorescens group’. Food Microbiol 39:116–126

    Article  CAS  PubMed  Google Scholar 

  • Andreani NA, Carraro L, Martino ME, Fondi M, Fasolato L, Miotto G, Magro M, Vianello F, Cardazzo B (2015) A genomic and transcriptomic approach to investigate the blue pigment phenotype in Pseudomonas fluorescens. Int J Food Microbiol 213:88–98

    Article  CAS  PubMed  Google Scholar 

  • Andreani NA, Carraro L, Zhang L, Vos M, Cardazzo B (2019) Transposon mutagenesis in Pseudomonas fluorescens reveals genes involved in blue pigment production and antioxidant protection. Food Microbiol 82(9):497–503

    Article  CAS  PubMed  Google Scholar 

  • Ardura A, Linde AR, Garcia-Vazquez E (2013) Genetic detection of Pseudomonas spp. in commercial Amazonian fish. Int J Environ Res Public Health 10(9):3954–3966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Aswathanarayan JB, Vittal RR (2014) Attachment and biofilm formation of Pseudomonas fluorescens PSD4 isolated from a dairy processing line. Food Sci Biotechnol 23(6):1903–1910

    Article  CAS  Google Scholar 

  • Bai AJ, Rai VR (2011) Bacterial quorum sensing and food industry. Compr Rev Food Sci Food Saf 10(3):183–193

    Article  CAS  Google Scholar 

  • Bedeltavana A, Haghkhah M, Nazer A (2010) Phenotypic characterization and PCR-ribotyping of Pseudomonas fluorescens isolates, in tracking contamination routes in the production line of pasteurized milk. Iran J Vet Res 11(3):222–232

    Google Scholar 

  • Brocklehurst TF, Lund BM (1981) Properties of pseudomonads causing spoilage of vegetables stored at low temperature. J Appl Bacteriol 50(2):259–266

    Article  Google Scholar 

  • Caldera L, Franzetti L (2014) Effect of storage temperature on the microbial composition of ready-to-use vegetables. Curr Microbiol 68(2):133–139

    Article  CAS  PubMed  Google Scholar 

  • Caputo L, Quintieri L, Bianchi DM, Decastelli L, Monaci L, Visconti A, Baruzzi F (2015) Pepsin-digested bovine lactoferrin prevents mozzarella cheese blue discoloration caused by Pseudomonas fluorescens. Food Microbiol 46:15–24

    Article  CAS  PubMed  Google Scholar 

  • Chan WKM, Joo S-T, Faustman C, Sun Q, Vieth R (1998) Effect of Pseudomonas fluorescens on beef discoloration and oxymyoglobin in vitro. J Food Prot 61(10):1341–1346

    Article  CAS  PubMed  Google Scholar 

  • Chen S, Wang F, Beaulieu JC, Stein RE, Ge B (2011) Rapid detection of viable Salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification. Appl Environ Microbiol 77(12):4008–4016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chiang YC, Tsen HY, Chen HY, Chang YH, Lin CK, Chen CY, Pai WY (2012) Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157: H7, Vibrio parahaemolyticus, Salmonella spp and Pseudomonas fluorescens in milk and meat samples. J Microbiol Methods 88(1):110–116

    Article  CAS  PubMed  Google Scholar 

  • Costerton JW (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322

    Article  CAS  Google Scholar 

  • Datta N, Deeth HC (2001) Age gelation of UHT milk-a review. Food Bioprod Process 79(4):197–210

    Article  Google Scholar 

  • De las Rivas B, Marcobal Á, Muñoz R (2005) Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines. FEMS Microbiol Lett 244(2):367–372

    Article  CAS  PubMed  Google Scholar 

  • De las Rivas B, Marcobal A, Carrascosa AV, Munoz R (2006) PCR detection of foodborne bacteria producing the biogenic amines histamine, tyramine, putrescine, and cadaverine. J Food Prot 69(10):2509–2514

    Article  CAS  PubMed  Google Scholar 

  • Decimo M, Morandi S, Silvetti T, Brasca M (2014) Characterization of gram-negative psychrotrophic bacteria isolated from Italian bulk tank milk. J Food Sci 79(10):M2081–M2090

    Article  CAS  PubMed  Google Scholar 

  • Dieckelmann M, Johnson LA, Beacham IR (1998) The diversity of lipases from psychrotrophic strains of Pseudomonas: a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens. J Appl Microbiol 85(3):527–536

    Article  CAS  PubMed  Google Scholar 

  • Dogan B, Boor KJ (2003) Genetic diversity and spoilage potentials among Pseudomonas spp. isolated from fluid milk products and dairy processing plants. Appl Environ Microbiol 69(1):130–138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dufour D, Nicodème M, Perrin C, Driou A, Brusseaux E, Humbert G, Gaillard JL, Dary A (2008) Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them. Int J Food Microbiol 125(2):188–196

    Article  CAS  PubMed  Google Scholar 

  • Edwards RA, Dainty RH, Hibbard CM (1987) Volatile compounds produced by meat pseudomonads and related reference strains during growth on beef stored in air at chill temperatures. J Appl Bacteriol 62(5):403–412

    Article  CAS  PubMed  Google Scholar 

  • Eren AM, Maignien L, Sul WJ, Murphy LG, Grim SL, Morrison HG, Sogin ML (2013) Oligotyping: differentiating between closely related microbial taxa using 16S rRNA gene data. Methods Ecol Evol 4(12):1111–1119

    Article  PubMed Central  Google Scholar 

  • Eren AM, Borisy GG, Huse SM, Mark Welch JL (2014) Oligotyping analysis of the human oral microbiome. Proc Natl Acad Sci U S A 111(28):E2875–E2884

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eren AM, Morrison HG, Lescault PJ, Reveillaud J, Vineis JH, Sogin ML (2015) Minimum entropy decomposition: unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences. ISME J 9(4):968–979

    Article  CAS  PubMed  Google Scholar 

  • Eriksson PV, Di-Paola GN, Pasetti MF, Manghi MA (1995) Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces. Appl Environ Microbiol 61(1):397–398

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fasolato L, Andreani NA, De-Nardi R, Nalotto G, Serva L, Cardazzo B, Balzan S, Carraro L, Fontana F, Novelli E (2018) Spectrophotometric techniques for the characterization of strains involved in the blue pigmentation of food: preliminary results. Ital J Food Saf 7(1):6928

    PubMed  PubMed Central  Google Scholar 

  • Folsom D, Friedman BA (1959) Pseudomonas fluorescens in relation to certain diseases of potato tubers in Maine. Am Potato J 36(3):90–97

    Article  Google Scholar 

  • Fox CW, Chrisope GL, Marshall RT (1976) Incidence and identification of phospholipase C-producing bacteria in fresh and spoiled homogenized milk. J Dairy Sci 59(11):1857–1864

    Article  CAS  PubMed  Google Scholar 

  • Franzetti L, Scarpellini M (2007) Characterisation of Pseudomonas spp. isolated from foods. Ann Microbio 57(1):39–47

    Article  CAS  Google Scholar 

  • Freeman LR, Silverman GJ, Angelini P, Merrit C Jr, Esselen WB (1976) Volatilesproduced by microorganismsisolated from refrigeratedchicken at spoilage. Appl Environ Microbiol 32(2):222–231

    CAS  PubMed  PubMed Central  Google Scholar 

  • González I, Martín R, García T, Morales P, Sanz B, Hernandez PE (1996) Polyclonal antibodies against protein F from the cell envelope of Pseudomonas fluorescens for the detection of psychrotrophic bacteria in refrigerated meat using an indirect ELISA. Meat Sci 42(3):305–313

    Article  PubMed  Google Scholar 

  • Griffiths MW, Phillips JD, West IG, Muir DD (1988) The effect of extended low-temperature storage of raw milk on the quality of pasteurized and UHT milk. Food Microbiol 5(2):75–87

    Article  Google Scholar 

  • Gupta S, Kaushal A, Kumar A, Kumar D (2017) Ultrasensitive transglutaminase based nanosensor for early detection of celiac disease in human. Int J Biol Macromol 5(1):905–911

    Article  CAS  Google Scholar 

  • Gutierrez R, Gonzalez I, Garcia T, Carrera E, Sanz B, Hernandez PE, Marti R (1997) Monoclonal antibodies and an indirect ELISA for detection of psychrotrophic bacteria in refrigerated milk. J Food Prot 60(1):23–27

    Article  CAS  PubMed  Google Scholar 

  • Hammad AM (2015) Spoilage potential of Pseudomonas spp. isolated form domiati cheese. Assiut Vet Med J 61:18–23

    Google Scholar 

  • Hildebrand PD (1989) Surfactant-like characteristics and identity of bacteria associated with broccoli head rot in Atlantic Canada. Can J Plant Pathol 11(3):205–214

    Article  Google Scholar 

  • Huether JP, McIntyre GA (1969) Pectic enzyme production by two strains of Pseudomonas fluorescens associated with the pinkeye disease of potato tubers. Am Potato J 46(11):414–423

    Article  Google Scholar 

  • Huis JHJ (1996) Microbial and biochemical spoilage of foods: an overview. Int J Food Microbiol 33(1):1–18

    Article  Google Scholar 

  • Jiang Y, Gao F, Xu XL, Ye KP, Zhou GH (2011) Changes in the composition of the bacterial flora on tray-packaged pork during chilled storage analyzed by PCR-DGGE and real-time PCR. J Food Sci 7(1):M27–M29

    Article  CAS  Google Scholar 

  • Kersters K, Ludwig W, Vancanneyt M, De-Vos PD, Gillis M, Schleifer KH (1996) Recent changes in the classification of the pseudomonads: an overview. Syst Appl Microbiol 19(4):465–477

    Article  Google Scholar 

  • Keskin D, Ekmekçi S (2007) Investigation of the incidence of Pseudomonas sp. in foods. Hacet J Biol Chem 35(3):181–186

    Google Scholar 

  • Koeppel AF, Wu M (2013) Surprisingly extensive mixed phylogenetic and ecological signals among bacterial operational taxonomic units. Nucleic Acids Res 41(10):5175–5188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koka R, Weimer BC (2000) Isolation and characterization of protease from Pseudomonas fluorescens RO98. J Appl Microbiol 89(2):280–288

    Article  CAS  PubMed  Google Scholar 

  • Ksontini H, Kachouri F, Hamdi M (2013) Dairy biofilm: impact of microbial community on raw milk quality. J Food Qual 36(4):282–290

    Article  Google Scholar 

  • Law BA, Sharpe ME, Chapman HR (1976) The effect of lipolytic gram-negative psychrotrophs in stored milk on the development of rancidity in Cheddar cheese. J Dairy Res 43(3):459–468

    Article  CAS  Google Scholar 

  • Law BA, Andrews AT, Sharpe ME (1977) Gelation of ultra- high-temperature- sterilized milk by proteases from a strain of Pseudomonas fluorescens isolated from raw milk. J Dairy Res 44(1):145–148

    Article  CAS  Google Scholar 

  • Liao CH (1989) Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables. Appl Environ Microbiol 55(7):1677–1683

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liao CH, McCallus DE (1998) Biochemical and genetic characterization of an extracellular protease from Pseudomonas fluorescens CY091. Appl Environ Microbiol 64(3):914–921

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liao CH, Sullivan J, Grady J, Wong LJ (1997) Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables. J Appl Microbiol 83(1):10–16

    Article  CAS  Google Scholar 

  • Liu M, Wang H, Griffiths MW (2007) Regulation of alkaline metalloprotease promoter by N-acyl homoserine lactone quorum sensing in Pseudomonas fluorescens. J Appl Microbiol 103(6):2174–2184

    Article  CAS  PubMed  Google Scholar 

  • Machado SG, da Silva FL, Bazzolli DM, Heyndrickx M, Costa PM, Vanetti MC (2015) Pseudomonas spp. and Serratia liquefaciens as predominant spoilers in cold raw milk. J Food Sci 80(8):M1842–M1849

    Article  CAS  PubMed  Google Scholar 

  • Magnuson JA, King AD, Török T (1990) Microflora of partially processed lettuce. Appl Environ Microbiol 56(12):3851–3854

    CAS  PubMed  PubMed Central  Google Scholar 

  • Marchand S, Vandriesche G, Coorevits A, Coudijzer K, Jonghe VD, Dewettinck K, Vos PD, Devreese B, Heyndricks M, Block JD (2009) Heterogeneity of heat-resistant proteases from milk Pseudomonas species. Int J Food Microbiol 133(1–2):68–77

    Article  CAS  PubMed  Google Scholar 

  • Marshall B, Robleto EA, Wetzler R, Kulle P, Casaz P, Levy SB (2001) The adnA transcriptional factor affects Pf0-1 motility and biofilms-formation. Appl Environ Microbiol 77(12):4318–4329

    Google Scholar 

  • Márta D (2012) Molecular monitoring of meat spoiling Pseudomonas species analysis of Staphylococcal enterotoxin expression and formation(Doctoral dissertation, Budapesti CorvinusEgyetem). https://pdfs.semanticscholar.org/c655/56eac5d83c36a50f2895876c31bd440c4b85.pdf. Accessed 14 Nov 2018

  • Martin NH, Murphy SC, Ralyea RD, Wiedmann M, Boor KJ (2011) When cheese gets the blues: Pseudomonas fluorescens as the causative agent of cheese spoilage. J Dairy Sci 94(6):3176–3183

    Article  CAS  PubMed  Google Scholar 

  • Martins ML, Araju EFD, Mantovani HC, Moraes CA, Vanetti MCD (2005) Detection of apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated milk. Int J Food Microbiol 102(2):203–211

    Article  CAS  PubMed  Google Scholar 

  • Martins ML, Pinto UM, Riedel K, Vanetti MC (2015) Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk. Braz J Microbiol 46(1):207–217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McCarthy CN, Woods RG, Beacham IR (2004) Regulation of the aprX–lipA operon of Pseudomonas fluorescens B52: differential regulation of the proximal and distal genes, encoding protease and lipase, by ompR–envZ. FEMS Microbiol Lett 241(2):243–248

    Article  CAS  PubMed  Google Scholar 

  • Mellor GE, Bentley JA, Dykes GA (2011) Evidence for a role of biosurfactants produced by Pseudomonas fluorescens in the spoilage of fresh aerobically stored chicken meat. Food Microbiol 28(5):1101–1104

    Article  CAS  PubMed  Google Scholar 

  • Miller A, Scanlan RA, Lee JS, Libbey LM (1973) Volatile compounds produced in sterile fish muscle (Sebastes melanops) by Pseudomonas putrefaciens, Pseudomonas fluorescens, and an Achromobacter species. Appl Microbiol 26(1):18–21

    CAS  PubMed  PubMed Central  Google Scholar 

  • Mitchell SL, Marshall RT (1989) Properties of heat-stable proteases of Pseudomonas fluorescens: characterization and hydrolysis of milk proteins. J Dairy Sci 72(4):864–874

    Article  CAS  Google Scholar 

  • Mlipano CL, Alistair G, Michael L (2018) Detection of proteolysis in milk by Pseudomonas fluorescens using urea PAGE method. J Food Stud 7(1):14–25

    Article  Google Scholar 

  • Morales PA, Aguirre JS, Troncoso MR, Figueroa GO (2016) Phenotypic and genotypic characterization of Pseudomonas spp. present in spoiled poultry fillets sold in retail settings. LWT Food Sci Technol 73:609–614

    Article  CAS  Google Scholar 

  • Palleroni NJ (1993) Pseudomonas classification. Antonie Van Leeuwenhoek 64(3–4):231–251

    PubMed  Google Scholar 

  • Palleroni NJ (2010) The Pseudomonas story. Environ Microbiol 12(6):1377–1383

    Article  CAS  PubMed  Google Scholar 

  • Palleroni NJ, Ballard RW, Ralston E, Doudoroff M (1972) Deoxyribonucleic acid homologies among some Pseudomonas species. J Bacteriol 110(1):1–11

    CAS  PubMed  PubMed Central  Google Scholar 

  • Palleroni NJ, Kunisawa R, Contopoulou R, Doudoroff M (1973) Nucleic acid homologies in the genus Pseudomonas. Int J Syst Bacteriol 23(4):333–339

    Article  CAS  Google Scholar 

  • Parlapani FF, Kormas KAR, Boziaris IS (2015) Microbiological changes, shelf life and identification of initial and spoilage microbiota of sea bream fillets stored under various conditions using 16S rRNA gene analysis. J Sci Food Agric 95(12):2386–2394

    Article  CAS  PubMed  Google Scholar 

  • Pinto UM, Costa ED, Mantovani HC, Vanetti MCD (2010) The proteolytic activity of Pseudomonas fluorescens 07A isolated from milk is not regulated by quorum sensing signals. Braz J Microbiol 41(1):91–96

    Article  PubMed  PubMed Central  Google Scholar 

  • Pittard BT, Freeman LR, Later DW, Lee ML (1982) Identification of volatile organic compounds produced by fluorescent pseudomonads on chicken breast muscle. Appl Environ Microbiol 43(6):1504–1506

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rajmohan S, Dodd CER, Waites WM (2002) Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage. J Appl Microbiol 93(2):205–213

    Article  CAS  PubMed  Google Scholar 

  • RASFF (2010) Available at https://ec.europa.eu/food/sites/food/files/safety/docs/rasff_annual_report_2010_en.pdf. Accessed 10 Oct 2018

  • Reichler SJ, Trmčič A, Martin NH, Boor KJ, Wiedmann M (2018) Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life. J Dairy Sci 101(9):7780–7800

    Article  CAS  PubMed  Google Scholar 

  • Rossi C, Chaves-Lopez C, Serio A, Goffredo E, Goga BTC, Paparella A (2016) Influence of incubation conditions on biofilm formation by Pseudomonas fluorescens isolated from dairy products and diary manufacturing plants. Ital J Food Saf 5(3):154–157

    Google Scholar 

  • Saha R, Bestervelt LL, Donofrio RS (2012) Development and validation of a real-time TaqMan assay for the detection and enumeration of Pseudomonas fluorescens ATCC 13525 used as a challenge organism in testing of food equipments. J Food Sci 77(2):M150–M155

    Article  CAS  PubMed  Google Scholar 

  • Samaržija D, Zamberlin Š, Pogačić T (2012) Psychrotrophic bacteria and milk and dairy products quality. Mljekarstvo 62(2):77–95

    Google Scholar 

  • Scarpellini M, Franzetti L, Antonietta G (2004) Development of PCR assay to identify Pseudomonas fluorescens and its biotype. FEMS Microbiol Lett 236(2):257–260

    Article  CAS  PubMed  Google Scholar 

  • Scatamburlo TM, Yamazi AK, Cavicchioli VQ, Pieri FA, Nero LA (2015) Spoilage potential of Pseudomonas species isolated from goat milk. J Dairy Sci 98(2):759–764

    Article  CAS  PubMed  Google Scholar 

  • Schlemmer AF, Ware CF, Keen NT (1987) Purification and characterization of a pectin lyase produced by Pseudomonas fluorescens W51. J Bacteriol 169(10):4493–4498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schmidt VT, Reveillaud J, Zettler E, Mincer TJ, Murphy L, Amaral-Zettler LA (2014) Oligotyping reveals community level habitat selection within the genus Vibrio. Front Microbiol 5:563

    PubMed  PubMed Central  Google Scholar 

  • Sellwood JE, Ewart JM, Buckler E (1981) Vascular blackening of chicory caused by a pectolytic isolate of Pseudomonas fluorescens. Plant Pathol 30(3):179–180

    Article  Google Scholar 

  • Shpigel NY, Pasternak Z, Factor G, Gottlieb Y (2015) Diversity of bacterial biofilm communities on sprinklers from dairy farm cooling systems in Israel. PLoS One 10(9):e0139111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads a taxonomic study. Microbiol 43(2):159–271

    CAS  Google Scholar 

  • Stellato G, Utter DR, Voorhis A, De Angelis M, Eren AM, Ercolini D (2017) A few Pseudomonas oligotypes dominate in the meat and dairy processing environment. Front Microbiol 8:264

    Article  PubMed  PubMed Central  Google Scholar 

  • Stoeckel M, Lidolt M, Achberger V, Glück C, Krewinkel M, Stressler T, Von-Neubeck M, Wenning M, Scherer S, Fischer L, Hinrichs J (2016) 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. Int Dairy J 59:20–28

    Article  CAS  Google Scholar 

  • Stoops J, Maes P, Claes J, Van Campenhout L (2012) Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu. Lett Appl Microbiol 54(3):195–202

    Article  CAS  PubMed  Google Scholar 

  • Teh KH, Flint S, Palmer J, Andrewes P, Bremer P, Lindsay D (2014) Biofilm-an unrecognised source of spoilage enzymes in dairy products? Int Dairy J 34(1):32–40

    Article  CAS  Google Scholar 

  • Tryfinopoulou P, Tsakalidou E, Nychas GJ (2002) Characterization of Pseudomonas spp. associated with spoilage of gilt-head sea bream stored under various conditions. Appl Environ Microbiol 68(1):65–72

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vithanage NR, Dissanayake M, Bolge G, Palombo EA, Yeager TR, Datta N (2016) Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential. Int Dairy J 57:80–90

    Article  CAS  Google Scholar 

  • Von-Neubeck M, Baur C, Krewinkel M, Stoeckel M, Kranz B, Stressler T, Fischer L, Hinrichs J, Scherer S, Wenning M (2015) Biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential. Int J Food Microbiol 211:57–65

    Article  CAS  PubMed  Google Scholar 

  • Wan C, Yang Y, Xu H, Aguilar ZP, Liu C, Lai W, Xiong Y, Xu F, Wei H (2012) Development of a propidium monoazide treatment combined with loop-mediated isothermal amplification (PMA-LAMP) assay for rapid detection of viable Listeria monocytogenes. Int J Food Microbiol 47(11):2460–2467

    CAS  Google Scholar 

  • Woods RG, Burger M, Beven CA, Beacham IR (2001) The aprX–lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production. Microbiol 147(2):345–354

    Article  CAS  Google Scholar 

  • Xin L, Zhang L, Meng Z, Lin K, Zhang S, Han X, Yi HX, Cui Y (2017) Development of a novel loop-mediated isothermal amplification assay for the detection of lipolytic Pseudomonas fluorescens in raw cow milk from North China. J Dairy Sci 100(10):7802–7811

    Article  CAS  PubMed  Google Scholar 

  • Xu Y, Chen W, You C, Liu Z (2017) Development of a multiplex PCR assay for detection of Pseudomonas fluorescens with biofilm formation ability. J Food Sci 80(10):2337–2342

    Article  CAS  Google Scholar 

  • Yilmaz AG, Temiz HT, Soykut EA, Halkman K, Boyaci IH (2015) Rapid identification of Pseudomonas aeruginosa and Pseudomonas fluorescnes using Raman spectroscopy. J Food Saf 35(4):501–508

    Article  Google Scholar 

  • Zhang S, Lv J (2014) Purification and properties of heat-stable extracellular protease from Pseudomonads fluorescens BJ-10. J Food Sci Technol 51(6):1185–1190

    Article  CAS  PubMed  Google Scholar 

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Kumar, H., Franzetti, L., Kaushal, A. et al. Pseudomonas fluorescens: a potential food spoiler and challenges and advances in its detection. Ann Microbiol 69, 873–883 (2019). https://doi.org/10.1007/s13213-019-01501-7

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Keywords

  • Pseudomonas fluorescens
  • Spoilage
  • Enzymes
  • Diagnosis
  • Advances