The Bacterial Flora of some Queensland Fish and its Ability to cause Spoilage

The bacterial flora were determined qualitatively and quantitatively on samples taken at various stages of handling several species of fish of commercial importance in Queensland. There was an overall increase in the number of bacteria during handling and processing; both the composition and quantity of the bacterial flora of individual samples taken at each stage of handling varied widely. Members of the genus Micrococcus formed the major proportion of the flora of freshly caught fish. Pseudomonas and Moraxella spp. were predominant amongst the bacterial flora able to grow at 2° and constituted the bulk of the population in samples with high bacterial counts. This psychrophilic population was markedly reduced at the filleting stage. A medium prepared by the action of trypsin on a fish muscle homogenate was used to test bacterial isolates for their ability to produce odours. Forty-three per cent of the pseudomonad isolates produced sulphydryl odours at 5°. Only small proportions of the other groups produced detectable odours. Members of the genus Pseudomonas were considered the most important fish spoilage bacteria under the conditions found in Queensland.     

Characterization of some fish and shrimp spoiling bacteria

The classification of some important groups of bacteria involved in fish and shrimp spoilage was studied. Trimethylamine is produced byPseudomonas putrefaciens, a “non-defined” group resemblingPs. putrefaciens, Photobacterium spp. and someMoraxella-like bacteria. Hypoxanthine is produced by the same groups of bacteria except the last named and also by the “typical shrimp spoilers” (presumptiveAlteromonas). Strong off-odours are produced on fresh fish byPs. putrefaciens, dextroseoxidativePseudomonas spp. (Groups I and 11 according to Shewan, Hobbs and Hodgkiss, 1960), the above mentioned “non-defined” group and by only some of the “typical shrimp spoilers”, whereasMoraxella-like bacteria andPhotobacterium spp. failed to produce strong odours. Strong off-odours are produced on boiled shrimp by the “typical shrimp spoilers” (presumptiveAltermonas),Ps. putrefaciens, the dextrose-oxidativePseudomonas spp. and the “non-defined” group;Moraxella-like bacteria produced less offensive odours or none, nor didPhotobacterium.     

Production of Off Odours by Isolates from Poultry Skin with Particular Reference to Volatile Sulphides

Poultry skin free from psychrotrophic micro-organisms was used to assess the abilities of isolates from poultry skin to produce off odours. Pseudomonas group I and II strains were the major off odour producers and the predominant volatile produced was methanethiol. Results obtained using skin as a spoilage substrate were similar to those obtained with leg muscle but a higher proportion of strains produced volatile sulphides from methionine and cystine supplemented media. Addition of glucose to the methionine medium inhibited methanethiol production.     

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.     

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.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

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.     

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.     

Extracellular Nuclease Activity of Fish Spoilage Bacteria, Fish Pathogens, and Related Species

The production of extracellular deoxyribonuclease and ribonuclease by 23 marine and 3 dairy strains of Pseudomonas putrefaciens, 15 strains of fish-pathogenic fluorescent pseudomonads, 38 strains of fluorescent pseudomonads isolated from haddock, and 34 related organisms was determined by an agar plate method. All strains of P. putrefaciens produced both deoxyribonuclease and ribonuclease. Of the other 87 organisms examined, 26.5% produced ribonuclease and 14.5% produced deoxyribonuclease. All organisms which produced deoxyribonuclease also produced ribonuclease. Deoxyribonuclease production by P. putrefaciens is suggested as a useful criterion of identity for members of this intense fish spoilage species.     

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.     

Phenotypic heterogeneity of Pseudomonas corrugata strains from southern Italy

Fifty-five strains of Pseudomonas corrugata isolated in southern Italy were characterized phenotypically and compared with 23 strains of different origins. At least two main cultural types with rough or smooth colonies were observed. Strains with rough colonies produced a diffusible pigment in culture. On the basis of their nutritional profiles, Ps. corrugata strains formed a distinct phenon most closely related to fluorescent Pseudomonas spp. isolated from tomato pith necrosis-diseased plants. Three major groups of strains were differentiated within the Ps. corrugata phenon on the basis of utilization of 2-ketogluconate, meso-tartrate, hystamine, DL- glycerate and induction of a hypersensitive reaction on tobacco. Some Ps. corrugata strains belonging to group 1 and 3 which did not produce pigment in culture produced IAA in a colorimetric test. Variability in the serological reaction of the Italian strain was observed. None of the three antisera utilized reacted with all strains. Some strains isolated from diseased plants from the same greenhouse showed different nutritional profiles and reacted with different antisera. Fifteen lipopolysaccharide (LPS) patterns were observed. Strains were divided into two groups on the basis of their protein profiles. The heterogeneity which had already been observed in a world-wide study on Ps. corrugata was confirmed in strains from this restricted area.     

Certain aspects of resistance of plum trees to bacterial canker Part I. Some biochemical characteristics of Pseudomonas morsprunorum (Wormald) and related phytopathogenic bacteria

Several strains of Pseudomonas mors-prunorum (Wormald) and Ps. prunicola (Wormald) isolated from pathological lesions of plum and cherry were studied together with the causal organism of bacterial canker of stone-fruits in California (Ps. syringae from apricot) and other phytopathogenic bacteria obtained from pear and syringa. Comparison was also made with pseudomonas forms pathogenic to pea, bean, lettuce, and tobacco, and with the common saprophytes Ps. fluorescens and Ps. pyocyaneus. With the exception of two yellow organisms (B. pruni and the Pear 8 strain—the latter, however, very occasionally showing fluorescence), all belong to the green-fluorescent group of Pseudomonas (Dowson's Group II). On the basis of their dissimilation of C and N compounds a very close relationship has been established between these fruit-tree and syringa pathogens of the green-fluorescent group. Ps. mors-prunorum is not highly specialized in its nutrient requirements but can satisfy its fundamental C and N requirements from a very large variety of simple substances. The only consistent biochemical differentiation shown by Ps. mors-prunorum (including some of the syringa strains) in comparison with Ps. prunicola (including Ps. syringae from apricot and most of the pear strains) is its more rapid production of add from sucrose. Both the mors-prunorum and prunicola varieties produce a levan from sucrose, which causes a raised gummy growth on solid sucrose-containing media. This applies also to Ps. pisi, Ps. tabaci, and Ps. phaseolicola , but is not the case with the weakly pathogenic forms— Ps. marginalis, cerasi (= trifoliorum , from bean), and the saprophytes— Ps. fluorescens and Ps. pyocyaneus.
On the basis of biochemical characteristics, considered apart from host pathogenicity, there is no justification for erecting to specific rank these various levan-forming. green-fluorescent, phytopathogenic pseudomonads.     

Antibacterial activity among Pseudomonas strains of meat origin

Almost 750 Pseudomonas strains from meat, fish, milk or plant were screened for inhibitory potential towards 10 selected Pseudomonas fragi isolated from meat. Only strains of fish origin and a reference Ps. putida strain produced an inhibitory substance which was either a siderophore or a bacteriocin-like substance. The two systems were efficient in meat extract medium suggesting potential use for controlling meat quality.     

Detection of tabtoxin-producing strains of Pseudomonas syringae by PCR

AIMS: The present study describes a system based on PCR to distinguish tabtoxin-producing strains of Pseudomonas syringae from other Ps. syringae plant pathogens that produce chlorosis-inducing phytotoxins. METHODS AND RESULTS: Thirty-two strains of Ps. syringae and related species were examined. Two sets of PCR primers were developed to amplify genes (tblA and tabA) required for tabtoxin production. Only a PCR product of 829 bp or 1020 bp was produced in PCR reactions with the tblA or tabA primer sets, respectively, and cells from tabtoxin-producing pathovars of Pseudomonas syringae. All known non-tabtoxin producing bacterial species failed to produce an amplification product with either primer set. CONCLUSIONS: PCR of genes required for tabtoxin production is a simple, rapid and reliable method for identifying tabtoxin-producing strains of Ps. syringae. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol can effectively distinguish tabtoxin-producing strains of Ps. syringae from other Ps. syringae pathovars and Ps. syringae pv. tabaci strains from other tabtoxin-producing Ps. syringae pathovars.     

Exopolysaccharides of the plant pathogens Pseudomonas corrugata and Ps. flavescens and the saprophyte Ps. chlororaphis

The rRNA-DNA homology group I pseudomonads Pseudomonas asplenii, Ps. corrugata, Ps. flavescens (plant pathogens), Ps. alcaligenes, Ps. pseudoalcaligenes subsp. pseudoalcaligenes (opportunistic human pathogens), Ps. aureofaciens and Ps. chlororaphis (saprophytes) were examined for their ability to produce exopolysaccharides (EPSs) when cultured on various solid and liquid complex media with glucose, glycerol or gluconate as primary sources of carbon. All three strains (388, 717 and ATCC 29736) of Ps. corrugata produced alginate, a polyuronan. An EPS composed of glucose, fucose, mannose and an unidentified uronic acid substituted with lactic acid was produced by one (B62) of two strains of Ps. flavescens. Of four strains of Ps. chlororaphis tested, only strain NRRL B-2075 produced EPS. The extracellular material purified by anion-exchange chromatography appeared to be a mixture of alginate plus an acidic hexosamine-containing polymer(s). Production of EPS by the other pseudomonads was not supported by any of the media tested.     

Identification of Shewanella baltica as the most important H2S-producing species during iced storage of Danish marine fish

Shewanella putrefaciens has been considered the main spoilage bacteria of low-temperature stored marine seafood. However, psychrotropic Shewanella have been reclassified during recent years, and the purpose of the present study was to determine whether any of the new Shewanella species are important in fish spoilage. More than 500 H2S-producing strains were isolated from iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time. All strains were identified as Shewanella species by phenotypic tests. Different Shewanella species were present on newly caught fish. During the warm summer months the mesophilic human pathogenic S. algae dominated the H2S-producing bacterial population. After iced storage, a shift in the Shewanella species was found, and most of the H2S-producing strains were identified as S. baltica. The 16S rRNA gene sequence analysis confirmed the identification of these two major groups. Several isolates could only be identified to the genus Shewanella level and were separated into two subgroups with low (44%) and high (47%) G+C mol%. The low G+C% group was isolated during winter months, whereas the high G+C% group was isolated on fish caught during summer and only during the first few days of iced storage. Phenotypically, these strains were different from the type strains of S. putrefaciens, S. oneidensis, S. colwelliana, and S. affinis, but the high G+C% group clustered close to S. colwelliana by 16S rRNA gene sequence comparison. The low G+C% group may constitute a new species. S. baltica, and the low G+C% group of Shewanella spp. strains grew well in cod juice at 0 degrees C, but three high G+C Shewanella spp. were unable to grow at 0 degrees C. In conclusion, the spoilage reactions of iced Danish marine fish remain unchanged (i.e., trimethylamine-N-oxide reduction and H2S production); however, the main H2S-producing organism was identified as S. baltica.     

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.     

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.

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.     

Bacterial Spoilage of Thawed Frozen Peas

The main groups of bacteria developing in peas were isolated on differential media. Leuconostoc mesenteroides and Streptococcus lactis predominated and smaller numbers of catalase positive 'coryneform'bacteria were also regularly present. Strep. cremoris , streptococci (group D), catalase positive cocci and Gram negative rods were less regularly isolated.
Bacterial spoilage of untreated peas was usually accompanied by the development of a yellow colour and a reduction in pH. In pure culture in peas leuconostocs and group N streptococci produced an acid pH while the catalase positive organisms produced alkali, sometimes with ammoniacal odours. In mixed cultures in peas the products of a leuconostoc and a 'coryneform'tended to neutralize each other. A leuconostoc and a 'coryneform'did not interfere with each others'growth when grown from similar inocula. The behaviour of streptococci is still being investigated. The type and rate of spoilage probably depends on the balance between ammonia production and acid production in the system.