A study of the incidence of different fluorescent Pseudomonas species and biovars in the microflora of fresh and spoiled meat and fish, raw milk, cheese, soil and water

M. GENNARI AND F. DRAGOTTO. 1992. Of 182 various foodstuffs and environmental samples examined, 86% had microflora containing fluorescent Pseudomonas in differing proportions. A computer-aided technique was used to identify most of the 445 fluorescent strains. Pseudomonas fluorescens biovar V-1 was most frequently isolated (24%); it either predominated or was present in all types of samples. Other strains, belonging to the other subgroups of biovar V (V-2, V-4, V-5, V-6 and V-7), together represented 14.3%. We also identified Ps. fluorescens biovars I-1 and I-2 (13.9%), II-1 and II-3 (3.6%), III-1 and III-2 (8.7%), IV-2 (0.7%); Ps. putida A and B (11%); Ps. lundensis (10.3%); group B3 (2%) and Ps. aeruginosa (0.7%). Unidentified strains accounted for 10.6% of the flora, many resembling Ps. fluorescens biovar V. Although the presence of Ps. fluorescens V-1 was often marked, other taxa predominated or were present in large quantities in some particular samples, such as Ps. fluorescens I-1 in raw milk and cheese, Ps. lundensis in spoiled meat and Ps. fluorescens III-1 in spoiled fish. Pseudomonas putida A and B were evident in environmental rather than in food samples.     

Species of Pseudomonas obtained at 7°C and 30°C during aerobic storage of lamb carcasses

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30°C were Ps.fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7°C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% S _SM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group ( Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (>74% S _SM) and Ps. lundensis (>80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Species of Pseudomonas obtained at 7 degrees C and 30 degrees C during aerobic storage of lamb carcasses.

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30 degrees C were Ps. fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7 degrees C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% SSM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group (Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (> 74% SSM) and Ps. lundensis (> 80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Classification of the spoilage flora of fish, with special reference to Shewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes, were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (SSM) and Jaccard (SJ) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% SJ similarity level which correspond to the SSM level of 86%. SJ-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10(8) cfu/g. Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.     

A numerical taxonomic study of the Pseudomonas flora isolated from poultry meat

Pseudomonas strains were isolated from both fresh and cold-stored broiler skin. Phenotypically-based numerical taxonomic techniques were used to characterize the isolates and 36 reference strains. For this purpose, Biolog GN Microplates, API 20NE and a number of other biochemical tests were used. Jaccard clustering revealed the predominance of four major Pseudomonas groups: Ps. fragi, Ps. lundensis, strains belonging to Ps. fluorescens biovars and an unidentified group of strains displaying a high degree of similarity to Ps. fluorescens biovars. Within Ps. fluorescens, biovar A was best represented. The marked proteolytic character of members of Ps. fluorescens biovars A, B and C, as well as of members of the unidentified cluster, supports their possible role in the origin of organoleptic defects. In the Ps. lundensis cluster, a distinct group of Ps. lundensis-like species was found. Further genotypic studies should be carried out to clarify the taxonomic status of the Ps. lundensis-like strains and that of the unidentified group resembling Ps. fluorescens biovars A and B.     

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage

A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.     

Classification of the spoilage flora offish, with special reference to Shewanella putrefaciens

One hundred and fifty-nine Gram-negative strains isolated from refrigerated fish, taken from the Baltic Sea or Swedish inland waters, together with 32 reference strains of Shewanella, Pseudomonas, Aeromonas and Alcaligenes , were phenotypically classified using 124 unit characters. Data were processed by the Simple Matching (S_SM) and Jaccard (S_J) coefficients, and unweighted pair group algorithm with arithmetic averages. Fourteen clusters were defined at the 75% S_J similarity level which correspond to the S_SM level of 86%. S_J-based clusters containing field strains were designated Pseudomonas fragi (cluster 1; 31% of the field strains), Ps. lundensis (cluster 2; 2% of the field strains), Ps. fluorescens biovar III (cluster 4; 4% of the field strains), Ps. putida biovar A (cluster 5; 3% of the field strains), Ps. fluorescens/putida (clusters 3 and 6; 6% of the field strains), Psychrobacter (clusters 8 and 9; 3% of the field strains), Shewanella putrefaciens (clusters 10, 11, 12 and 13; 44% of the field strains) and Aer. sobria (cluster 14; 6% of the field strains, all isolated from fresh water fish). Each field strain represented the spoilage flora of refrigerated fish at a total aerobic count of about 10⁸ cfu/g.
Phenotypic characteristics of major clusters are given. The four S. putrefaciens clusters may be separated by key characteristics. Shewanella putrefaciens ATCC 8071^T and reference strains from sources other than fish, did not group in any of the clusters. The mol % guanine + cytosine content was on average 47.6 for cluster 10, and 45.3 for cluster 13.     

Classification of the spoilage flora of raw and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus

Eighty-one bacterial strains isolated from refrigerated raw milk, 124 from pasteurized milk and cream stored at 5°C and 7°C, and 19 type and reference strains of Pseudomonas spp. and Bacillus spp. were characterized by numerical phenotypic analysis. Data were processed with simple matching ( S _SM) and Jaccard ( S _J) coefficients, and UPGMA clustering. Fourteen clusters of Gram-negative bacteria were formed at S _J= 79% ( S _SM= 90%). Raw milk was exclusively spoilt by Gram-negative bacteria, the majority of which were Pseudomonas fluorescens biovar I, Ps. fragi, Ps. lundensis and Ps. fluorescens biovar III. Minor groups in raw milk included Enterobacteriaceae spp. and Acinetobacter spp. Pasteurized milk was spoilt by essentially the same Gram-negative organisms in 65% (5°C) and 50% (7°C) of the cases. The phenotypic characteristics of Gram-negative bacteria are given. Bacillus polymyxa (both temperatures) and B. cereus (only at 7°C) were responsible for 77% of samples spoiled by the Gram-positive organisms. Minor milk spoilage groups included other Bacillus spp. and lactic acid bacteria. All Bacillus spp. grew fermentatively in milk, and most strains denitrified. It is suggested that: (i) industrial recontamination tests of pasteurized milk are directed against Pseudomonas; (ii) milk is stored at 5°C or lower to avoid growth of B. cereus ; and (iii) the significance of gas-producing and nitrate/nitrite-reducing Bacillus strains is recognized in cheese production.     

A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source tests

A computer-assisted probabilistic identification technique employing 18 carbon source utilization tests has been developed and applied to 787 Pseudomonas strains isolated from beef, pork and lamb stored under aerobic conditions. Seven hundred and twelve (89.7%) were identified using these tests alone and a further six (0.8%) with extra tests. Taxa detected were Ps. fragi cluster 2, 390 strains (49.6% of all isolates); Ps. fragi cluster 1, 191 strains (24.9%); meat cluster 3, 87 strains (11.1%); Ps. fluorescens biotype I, 31 strains (3.9%); Ps. fluorescens biotype III, 7 strains (0.9%); and Ps. putida , 1 strain (0.1%). The relative incidence of members of the various taxa was similar on beef, pork and lamb, and was unaffected by storage temperature in the range 0°–10°C. Each taxon was also detected at similar rates before and after spoilage. Meat origin (abattoir) affected the frequency of detection of meat cluster 3 and Ps. fluorescens biotype I strains but did not affect the incidence of detection of either cluster of Ps. fragi.     

A study of the relative incidence of different Pseudomonas groups on meat using a computer-assisted identification technique employing only carbon source tests

A computer-assisted probabilistic identification technique employing 18 carbon source utilization tests has been developed and applied to 787 Pseudomonas strains isolated from beef, pork and lamb stored under aerobic conditions. Seven hundred and twelve (89.7%) were identified using these tests alone and a further six (0.8%) with extra tests. Taxa detected were Ps. fragi cluster 2, 390 strains (49.6% of all isolates); Ps. fragi cluster 1, 191 strains (24.9%); meat cluster 3, 87 strains (11.1%); Ps. fluorescens biotype I, 31 strains (3.9%); Ps. fluorescens biotype III, 7 strains (0.9%); and Ps. putida, 1 strain (0.1%). The relative incidence of members of the various taxa was similar on beef, pork and lamb, and was unaffected by storage temperature in the range 0 degrees-10 degrees C. Each taxon was also detected at similar rates before and after spoilage. Meat origin (abattoir) affected the frequency of detection of meat cluster 3 and Ps. fluorescens biotype I strains but did not affect the incidence of detection of either cluster of Ps. fragi.     

Isolation of fluorescent pseudomonads from the rhizosphere of banana plants antagonistic towards root necrosing fungi

Total aerobic bacteria and fluorescent pseudomonads were counted in bulk and rhizospheric soils of banana plants of 14 plantations in Martinique (French West Indies). Fluorescent Pseudomonas isolates were then identified and investigated for in vitro antagonism towards Cylindrocladium sp., a fungal pathogen of banana roots. Total aerobic bacteria and fluorescent pseudomonads were significantly more abundant in rhizospheric soils than in bulk soils. Among 58 fluorescent Pseudomonas isolates, 41 were identified as Pseudomonas fluorescens biovar V and 17 as Ps. putida biovar A. Six strains exhibited an antagonism towards Cylindrocladium isolates. Among them, Ps. putida strain 93.1 totally blocked fungal growth. No relationship was established between the antifungal effect and enzyme or hydrogen cyanide production by bacteria, suggesting that siderophores and other compounds were involved in fungal inhibition. Antagonistic fluorescent pseudomonads represent a potential for the biological control of banana root infections by Cylindrocladium sp.     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4°C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.     

Cross-reactivity of antibodies raised to Pseudomonas fluorescens protease with extracellular proteins produced by meat-spoiling pseudomonads

The cross-reactivity patterns of antibodies to Pseudomonas fluorescens protease with the extracellular proteins produced by a number of meat-spoiling pseudomonads were studied. Immunoblotting studies showed that purified IgG to Ps. fluorescens protease cross-reacted with extracellular proteins in the cell culture supernatant fluids of Pseudomonas spp., including Ps. fragi and Ps. lundensis. In the case of Ps. lundensis and Pseudomonas spp. 11390, the cross-reactive moieties were of similar molecular weight to the Ps. fluorescens protease (46 kDa). However, in Ps. fragi the cross-reactive moiety was a lower molecular weight protein (8 kDa). This may represent a fragment of the active enzyme. These results indicate the presence of common antigenic determinants among the proteases of meat spoiling pseudomonads.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

Characterization of Pseudomonas spp. associated with spoilage of gilt-head sea bream stored under various conditions.

The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparus aurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S. aurata stored under different temperatures (at 0, 10, and 20 degrees C) and packaging conditions (air and a modified atmosphere of 40% CO(2)-30% N(2)-30% O(2)). Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently. Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4 degrees C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.     

Effect of rhizosphere fluorescent Pseudomonas strains on plant-parasitic nematodes Radopholus similis and Meloidogyne spp.

Three Pseudomonas fluorescens strains and the type strain Pseudomonas putida CFBP2066 inhibited invasion of the plant-parasitic nematodes Radopholus similis and Meloidogyne spp. in banana, maize and tomato roots. Results were, however, not always significantly different from controls. One Ps. fluorescens strain kept R. similis numbers significantly lower in banana roots after the initial invasion stage. All strains also showed an in vitro repellent effect towards the nematodes, with Meloidogyne spp. being more affected than R. similis. As Ps. putida CFBP2066 was negative for the enzymatic activities tested as well as HCN productivity, it was concluded that either other chemical bacterial compounds affected nematode infectivity or strains elicited induced systemic resistance in plants.     

Factors contributing to the survival of poultry associated Pseudomonas spp. exposed to a quaternary ammonium compound

Resistance to benzalkonium chloride (BC) among Pseudomonas spp. isolated from poultry carcasses was determined and strategies for elimination of resistant strains evaluated. This investigation showed that resistance was quite common, about 30% of the isolates being able to grow in 200 μg ml⁻¹ BC. Pseudomonas fluorescens strains were generally less susceptible than strains of Ps. lundensis and Ps. fragi. An overnight incubation in medium containing 200 μg ml⁻¹ BC was sufficient to reduce the susceptibility of two Pseudomonas strains to the lethal effect of BC significantly. Adding EDTA enhanced the lethal effect of BC, but the effect was reduced after growing cells in medium containing BC and EDTA. Growth in medium with a quaternary ammonium compound (QAC) rendered the cells more susceptible to chlorine, phenolics and alkylaminoacetate. These results indicate that alternating use of QACs with these compounds can be used to avoid build-up of resistant strains. In addition, increased temperatures improved the lethal effect of BC and should be considered when planning disinfection routines.     

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.     

Metabolic activities of pseudomonads in batch cultures in extract of minced lamb

E.H. DROSINOS AND R.G. BOARD. 1994. Pseudomonas fragi, Ps. lundensis and Ps. fluorescens were studied in axenic batch cultures growing in a lamb juice (pH 6.0) aerobically or in an atmosphere ( Ps. fragi only) enriched with carbon dioxide at 4C. With all but a glucose dehydrogenase-deficient strain of Ps. fluorescens there was a sequential catabolism of glucose and lactate. Diauxic growth was observed in a nutrient-deficient meat juice supplemented with glucose and lactate. A transient peak in the concentration of gluconate and pyruvate was associated with the catabolism of glucose and lactate respectively. With Ps. fluorescens deficient in glucose dehydrogenase there was simultaneous catabolism of glucose and lactate. The stereoisomers of lactate were catabolized simultaneously in a laboratory medium. Glucose-6-phosphate was oxidized to 6-phosphogluconate by Ps. fragi and Ps. lundensis under aerobic conditions only. Creatine and creatinine were catabolized by Ps. fragi under aerobic conditions only. There was a slight decrease in the concentration of total amino acids (ninhydrin-reactive material) during the exponential phase of growth. The results suggest that the dominance of Ps. fragi in the climax populations in meat is due to catabolism of amino acid related substrates, creatine and creatinine.