A Numerical Taxonomic Study of Pseudomonas-like Bacteria Isolated from Fish in Southeastern Queensland and their Association with Spoilage

Pseudomonas -like bacteria isolated from fresh and spoiling fish in southeastern Queensland were subjected to a wide range of physiological and nutritional tests. The results of these tests, together with those of 20 named strains, were analysed numerically, resulting in the formation of 11 groups. Most of the isolates clustered into group 1 and group 2 which also contained the bulk of the strains able to produce spoilage odours when grown in a tryptic digest of fish muscle at 2°C. Almost all of the group 1 organisms produced sulphydryl type odours, had only 50 mol % G + C and were identified as strains of Alteromonas putrefaciens which were deficient in the ability to produce H₂S detectable in Peptone Iron Agar. Certain of the group 2 strains produced fruity and sulphydryl type odours, but these organisms were not distinguishable from other strains in this group not producing odours. Group 2 strains were highly related to Pseudomonas fragi and were intermediate in properties between Ps. fluorescens and Ps. putida. The remaining nine minor groups contained few organisms able to produce spoilage odours.     

Identification of Pseudomonas spp. Isolated from Milk Produced in South Eastern Queensland

S ummary . Bacteria were isolated from raw and pasteurized milks produced throughout S. E. Queensland. Milk samples were plated initially on penicillin agar or on milk agar and incubated at 30° and 7°, respectively. On the basis of primary characterization, 167 of the 330 isolates obtained were identified as Pseudomonas spp. The pseudomonads were further characterized in accordance with the taxonomic studies of the genus by Stanier, Palleroni & Doudoroff (1966). Species designations were ascribed to the Pseudomonas isolates on the basis of distinctive species characteristics in conjunction with similarity coefficients between each isolate and the ideal species phenotype, as follows: Ps. fluorescens (121), Ps. aeruginosa (16), Ps. putida (12), Ps. maltophilia (9), Ps. pseudoalcaligenes (5), Ps. cepacia (3) and Ps. alcaligenes (1). A dendrogram obtained by cluster analysis of the Pseudomonas isolates is included.     

Psychrotrophic bacterial flora of raw ewes'milk, with particular reference to Gram negative rods

The microbial flora of 141 samples of raw ewes'milk was determined, before and after storage for 72 h at 4° and 7°C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated ( ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Aerobic Oral Bacteria in Healthy Captive Snakes

Cotton swab samples were taken from the ventral surface of the mouth and from the proximal esophagus from 23 captive nonpoisonous snakes. The samples were cultured and investigated for aerobic bacteria. Both the mouth and the esophagus) samples of 6 snakes were negative. When the bacterial isolates of the mouth and the esophagus of the whole snake population were compared, it was found that the flora isolated from both locations were similar. However, when the samples of individual snakes were compared it was found that the same isolates were seldom found in both the mouth and the esophagus. The most common bacteria found were Pseudomonas sp., Alcaligenes-like organisms, Gram-positive rods and Gram-positive cocci belonging to the family Micrococcaceae. Important pathogens were seldom isolated. Salmonella virchow could be found from 2 snakes. The presence of bacteriologically negative samples, great variations in the composition of the flora between individual snakes, and the occurrence of typical environmental bacteria in the oral cavity all suggest that snakes lack a specific autochtonous flora: and the bacteria isolated from the oral cavity may be occasional environmental bacteria. The source of pathogens may be the environment, too.     

Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish

The incidence of histamine- or tyramine-producing lactic acid bacteria was examined in several products of vacuum-packed sugar-salted fish (salmon, halibut, mackerel). No histamine-producing isolates were observed, whereas the majority of tyramine-producing isolates were identified as Carnobacterium spp. These organisms were shown to be important members of the microbial flora during storage of vacuum-packed sugar-salted salmon at 5°C. The amount of tyramine produced was reduced by lowering the temperature from 9°C to 4°C for all of five strains of carnobacteria or lactobacilli. The majority of tyramine was produced during the exponential growth phase for Carnobacterium piscicola N 5 and Lactobacillus viridescens N 69. The ability of these bacteria to produce tyramine may be used as an index of microbial quality/acceptability of stored vacuum-packed sugar-salted fish.     

THE SANITATION OF FISH BOXES. I. THE QUANTITATIVE AND QUALITATIVE BACTERIOLOGY OF COMMERCIAL WOODEN FISH BOXES

SUMMARY: The cleaning efficiency of hosing wooden market fish boxes with cold water, the usual commercial practice, was very low, even when high pressure jets were used. After washing, the internal surfaces could still carry up to 20 × 10⁶ bacteria/cm². Even prolonged steaming failed to sterilize the surfaces. Coryneform organisms accounted for over 50% of the total bacterial flora, although these have usually been reported as present in only low proportions on both fresh and spoiling fish. The major subsidiary groups were species of Achromobacter and Pseudomonas , constituting about 18% and 14% of the population respectively. A few micrococci and flavobacteria were also present.
The conditions of counting, i.e. whether roll tubes or Petri dishes were used, whether incubation was at 0°, 20° or 37°, and whether the media were based on sea or tap water, could markedly influence both the magnitude of the count and the proportions of the different types of bacteria which could be isolated.     

Species Composition and Barotolerance of Gut Microflora of Deep-Sea Benthic Macrofauna Collected at Various Depths in the Atlantic Ocean

The bacterial flora of marine animals collected at depths of 570 to 2,446 m was examined for population size and generic composition, and the barotolerant characteristics of selected bacterial isolates were determined. Total numbers of culturable, aerobic, heterotrophic bacteria were found to be low in animals collected at the greatest ocean depths sampled in this study. Vibrio spp. were predominant in 10 of 15 samples examined, and Photobacterium spp. and yeasts were the major components of the remainder. Pseudomonas, Achromobacter, and Flavobacterium spp. comprised minor components of the gut flora of deep-sea fish. Forty-six pure cultures isolated from samples of seven animals were tested for growth or viability after incubation for 1 week under pressures ranging from 100 to 750 atm. Strains of bacteria isolated from samples of fish intestine were more barotolerant than those from the stomach (P<0.01). When incubated at a pressure of 600 atm, viability of bacterial cultures originally isolated from fish caught at a depth of 570 m was significantly decreased in comparison with viability of cultures from animals caught at depths of 1,393 and 2,446 m (P<0.01). From results of this study, it is concluded that the gut microflora of animals that dwell in the deeper regions of the ocean are adapted to an increased hydrostatic pressure environment, that is, the gut microflora is less inhibited by elevated hydrostatic pressure with increasing depth from which the host animal was collected.     

Evolution of bacterial flora from a subterranean saline well by graduated salinity changes in enrichment media

The bacterial flora of samples taken from a subterranean saline well was enriched (1) by periodic salinity increase and (2) by periodic salinity decrease, both at 25° and 35°C. During the enrichment process, bacterial flora, including halotolerant, marine, moderately and extremely halophilic bacteria, were enumerated. Results were similar at both temperatures. Marine bacteria and moderately halophilic microorganisms were the most favoured groups, predominating between 3 and 30% (w/v) salt content; extremely halophilic bacteria also appeared when salinity reached 30% (w/v) and halotolerant bacteria were poorly represented. The taxonomic distribution of 125 selected strains, chosen at random from counting media, was essentially similar to those from other hypersaline environments.     

Cyanophycin-degrading bacteria in digestive tracts of mammals, birds and fish and consequences for possible applications of cyanophycin and its dipeptides in nutrition and therapy

Aims:  To determine the susceptibility of cyanophycin granule polypeptide (CGP) to degradation by several mammalian, avian and fish gut flora.
Methods and Results:  Samples of gut flora were investigated for the occurrence of bacteria capable of CGP degradation. With all samples, a complete anaerobic degradation of CGP was achieved over incubation periods of only 12–48 h at 37°C. CGP-degrading bacteria were detected in all samples, and they occurred in particular high titres in caecum flora from rabbit and sheep and in the digestive tract of carp fish. A total of 62 axenic cultures were isolated. All degraded CGP aerobically, 46 of them degraded CGP also anaerobically over incubation periods ranging from 24 h to 7 days. HPLC analysis revealed that all isolates degraded CGP to its constituting dipeptides. Eight strains were identified by 16S rRNA gene sequencing and were affiliated to the genera Bacillus , Brevibacillus , Pseudomonas , Streptomyces and Micromonospora .
Conclusions:  These data demonstrate for the first time the occurrence of a natural niche for CGP in the digestive tracts of animals.
Significance and Impact of the Study:  The biodegradability of CGP by gut flora provides a first confirmation for the potential applications of CGP and its dipeptides in nutrition and therapy as highly bio-available sources for arginine, lysine, aspartate and possibly also other amino acids.     

Temperature-dependent enumeration and characterization of anaerobic, thermophilic xylan-degrading bacteria present in two Icelandic hot springs

Abstract Anaerobic thermophilic xylan-degrading bacteria present in unenriched and enriched 70°C samples from two Icelandic hot springs were enumerated at 68, 78, 90 and 99°C by the use of the Most-Probable-Number method. Xylan was used as substrate. From the samples taken at 70°C and incubated at the temperatures previously described no growth was observed above 78°C. A total of ten strains were isolated and characterized from the positive MPN enrichment cultures from the MPN experiments. A higher number of different strains could be isolated in the enriched samples compared with the unenriched, control samples from the same hot spring. Introduction of xylan, i.e., in situ enrichment, into one of the hot springs changed the bacterial population, as none of the bacteria isolated from the unenriched samples were isolated from the enriched samples. All the isolated bacteria were asporogenous, non-motile and gram-negative rods. One long thin rod had morphological similarities to members of the genus Dictyoglomus and was found in both hot spring samples.     

The Aerobic Flora of Stored Meat with Particular Reference to the Use of Selective Media

The changes in the aerobic flora of meats stored at 15, 9 and 4° have been compared by means of selective media and by screening representative isolates. By both methods the predominant spoilage flora at the three temperatures belonged to the genus Pseudomonas . Using the medium of Masurvosky, Goldblith & Voss (1963) preliminary investigations have been made into the numbers of pseudomonads in meats.     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Indigenous PAH-degrading bacteria from oil-polluted sediments in Caleta Cordova,Patagonia Argentina

Indigenous bacteria with the capability to degrade polycyclic aromatic hydrocarbons (PAH) were isolated from polluted sediment samples recovered from Caleta Cordova by using selective enrichment cultures supplemented with phenanthrene. Bacterial communities were evaluated by denaturing gradient gel electrophoresis (DGGE) in order to detect changes along enrichment culture and relationships with the representative strains subsequently isolated. Members of these communities included marine bacteria such as Lutibacter, Polaribacter, Arcobacter and Olleya, whose degradation pathway of PAH has not been studied yet. However, isolated bacteria obtained from this enrichment comprised the genus Pseudomonas, Marinobacter, Salinibacterium and Brevibacterium. The ability of isolates to grow and degrade naphthalene, phenanthrene and pyrene was demonstrated by detection of the residual substrate by HPLC. Archetypical naphthalene and catechol dioxygenase genes were found in two isolates belonging to genus Pseudomonas (Pseudomonas monteilii P26 and Pseudomonas xanthomarina N12), suggesting biodegradation potential in these sediments. The successful bacterial isolation with the ability to degrade PAH in pure culture suggest the possibility to study and further consider strategies like growth stimulation in situ, in order to increase the intrinsic bioremediation opportunities in the polluted Caleta Cordova harbor.     

Bacterial Diversity within Feedlot Manure

In this study, we identified the predominant culturable anaerobic bacteria and enumerated the total culturable anaerobic bacterial population present in samples of feedlot manure from Southern Queensland. Sixteen bacterial isolates were cultured from feedlot pad material with species of Lactobacillus, Clostridium and Bacillus predominating. From a library of 123 clones, produced by the amplification, cloning and partial DNA sequencing of the 16S rRNA gene, only 3% were closely related to previously described species and 21% to known genera. Of the total clone library, 96% were apparently Gram-positive and fell within families whose members were generally anaerobes. The majority (71%) of the clone library was related to either members of the family Clostridiaceae or lactic acid-producing bacteria (Lactobacillus or Lactosphaera). It was concluded that Gram-positive clostridial and lactic acid-producing bacteria predominate in feedlot pad manure. The overwhelming majority of species are novel and have not been obtained in culture. It would appear that the most likely source of the sickly-sweet nuisance odours (particularly from butyric acid) that emanate from feedlots is the by-product of anaerobic fermentation by clostridia. Gut-inhabiting and Gram-negative bacteria do not appear to survive for lengthy periods of time under the environmental conditions present in feedlot manure.     

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.     

Characterization of lactic acid bacteria isolated from seafood

Lactic acid bacteria were isolated from various samples of seafood: fresh pollock, brine shrimp, gravad fish, vacuum-packed seafood (surimi, smoked tuna, salted cod), and fish stored under 100% CO₂ at 5°C (smoked tuna, fresh and salted cod, salmon). Eighty-six independent isolates were obtained and were grouped according to cell morphology, presence or absence of diaminopimelic acid in the cell wall, and lactate configuration. Fifty-four isolates were identified as belonging to the genus Lactococcus and most of them exhibited DNA homologies with L. lactis subsp. lactis. Four strains were identified as Lactobacillus plantarum , eight strains as genus Leuconostoc and 16 belonged to the genus Carnobacterium. One facultative heterofermentative Lactobacillus and three other isolates were not identified. Of the strains 47% showed similar patterns of carbohydrate fermentations especially among strains belonging to the genera Lactococcus and Carnobacterium. Most of the strains (64%) grew at 5°C, in salted media and in fish extract medium without added sugar. Carnobacterium piscicola and Carn. divergens were the only reference strains able to grow in the same conditions as well as psychrotroph strains isolated from seafood. A numerical analysis could not be used because of the divergent properties of isolates of the same genus and strong similarities between different genera.     

Bacterial production of histamine in some tropical fish

Quantitative and qualitative distribution of histamine-forming bacteria associated with the fish Rastrelliger kanagurta, Sardinella longiceps, Sillago sihama and Liza subviridis, were investigated. These bacteria constituted a significant portion of the total bacterial population of fish and the values obtained in the present study were higher than those previously reported. The order of quantitative abundance of histamine-forming bacteria in the fish examined was: S. longiceps greater than R. kanagurta greater than S. sihama greater than L. subviridis. The bacterial genera isolated were Vibrio sp., Bacillus sp., Pseudomonas sp., Aeromonas sp. and Micrococcus sp., and among them Vibrio was dominant. Growth of the isolates (Vibrio sp., V. fischeri and Bacillus sp.) at different temperatures, pH and sodium chloride concentrations indicated them to be mesophilic, euryhaline and tolerant to acidic and alkaline pH. Bacillus sp. produced more histamine in R. kanagurta, while V. fisheri produced more histamine in S. longiceps.     

Differential attraction of malaria mosquitoes to volatile blends produced by human skin bacteria

The malaria mosquito Anopheles gambiae sensu stricto is mainly guided by human odour components to find its blood host. Skin bacteria play an important role in the production of human body odour and when grown in vitro, skin bacteria produce volatiles that are attractive to A. gambiae. The role of single skin bacterial species in the production of volatiles that mediate the host-seeking behaviour of mosquitoes has remained largely unknown and is the subject of the present study. Headspace samples were taken to identify volatiles that mediate this behaviour. These volatiles could be used as mosquito attractants or repellents. Five commonly occurring species of skin bacteria were tested in an olfactometer for the production of volatiles that attract A. gambiae. Odour blends produced by some bacterial species were more attractive than blends produced by other species. In contrast to odours from the other bacterial species tested, odours produced by Pseudomonas aeruginosa were not attractive to A. gambiae. Headspace analysis of bacterial volatiles in combination with behavioural assays led to the identification of six compounds that elicited a behavioural effect in A. gambiae. Our results provide, to our knowledge, the first evidence for a role of selected bacterial species, common on the human skin, in determining the attractiveness of humans to malaria mosquitoes. This information will be used in the further development of a blend of semiochemicals for the manipulation of mosquito behaviour.     

Abundance of actinobacteria and production of geosmin and 2-methylisoborneol in Danish streams and fish ponds

Occurrence of the odours geosmin and 2-methylisoborneol (MIB) in freshwater environments indicates that odour-producing organisms are commonly occurring. In the present study, we assumed actinomycetes to be a major source of the odours. Seasonal concentrations of odours and abundance of Actinobacteria, which includes actinomycetes and other G+ and high GC bacteria, were determined in one oligotrophic and two eutrophic freshwater streams, as well as in aquacultures connected to these streams, in Denmark. Concentrations of geosmin and MIB ranged from 2 to 9 ng l(-1) and were lowest in the winter. Passage of stream water in the aquacultures increased the amount of geosmin and MIB by up to 55% and 110%, respectively. Densities of actinobacteria were determined by fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) technique and were found to make up from 4 to 38 x 10(7) cells l(-1), corresponding to 3-9% of the total bacterial populations. The lowest densities of actinobacteria occurred in the winter. Filamentous bacteria targeted by the FISH probe made up about 2.7-38% (average was 22%) of the actinobacteria and were expected to be actinomycetes. Combined microautoradiography and CARD-FISH demonstrated that 10-38% (incorporation of 3H-thymidine) and 41-65% (incorporation of 3H-leucine) of the actinobacteria were metabolically active. The proportion of active actinobacteria increased up to 2-fold during passage of stream water in the aquacultures, and up to 98% of the cells became active. Sequencing of 16S rRNA genes in 8 bacterial isolates with typical actinomycete morphology from the streams and ponds demonstrated that most of them belonged to the genus Streptomyces. The isolated actinomycetes produced geosmin at rates from 0.1 to 35 aggeosmin bacterium(-1)h(-1). MIB was produced at similar rates in 5 isolates, whereas no MIB was produced by three of the isolates. Addition of the odours to stream water demonstrated that indigenous stream bacteria were capable of reducing the odours, and that enrichment with LB medium stimulated the degradation. Our study shows that bacterial communities in freshwater include geosmin- and MIB-producing actinobacteria. However, the mechanisms controlling production as well as degradation of the odours in natural waters appear complex and require further research.     

Bacterial flora of newly caught Antarctic fish Notothenia neglecta

Summary The heterotrophic bacterial flora of Antarctic fish Notothenia neglecta was studied in Potter Cove (King George Island, South Shetland Islands). Quantitative and qualitative analysis of aerobic bacteria from sea water, skin, stomach and intestine were carried out. In addition, anaerobic flora of stomach and intestine was studied and compared. Pseudomonas was dominant in sea water samples but, neither skin nor digestive tract samples showed to be rich in this genus. Stomach flora showed variable results between samples. The gut flora was composed almost exclusively of Vibrio. Despite extreme environmental conditions this Antarctic fish show an intestinal indigenous microflora very similar to warmer waters fish. Several factors, that we discussed, could be determining the Vibrio dominance in the gut of marine teleosts.