Experimental study on the growth of Salmonella and coli-aerogenes bacteria in pure and mixed cultures,in a fermentor,in two different enrichment media: Sodium selenite and tetrathionate broth

Summary Different serotypes of Salmonella and coli-aerogenes bacteria were grown in a fermentor at +43°C. The Culture media used were composed of two different nutrient broths, one supplemented with sodium selenite, the other with potassium tetrathionate. The growth of both bacteria and the following types of mixed bacteria was studied: Escherichia coli-Salmonella brancaster and Klebsiella pneumoniae-Salmonella brancaster. During the first 10 h of incubation, sodium tetrathionate broth inhibited the multiplication of coli-aerogenes bacteria. Similar results were obtained when these bacteria were grown with S. brancaster and incubated for 18 h, regardless of the original concentration of each of the bacteria. In the presence of sodium selenite, after 18 h of incubation growth was inhibited in E. coli, but not in the other coli-aerogenes bacteria.     

Bacteriological Examination of Shell Fish

Aquatic edible molluscs (oysters, wedge-shell, butter-fish, sting-winkle, date-shell and sea-urchins) caught from sea water near Alexandria were found to be grossly polluted. Most of the samples examined showed exceptionally high counts of coli-aerogenes bacteria and enterococci; many salmonellae were also isolated. Discrimination of polluted shell fish may be accomplished by testing the sea water or more conveniently by determining the most probable number index of shell fish liquor. The coli-aerogenes count is probably the best available standard; there is however a positive linear relationship between the coli-aerogenes and the enterococcal counts. This conclusion, drawn from scatter diagrams, was also confirmed by the calculation of the correlation coefficient r =+ 0·9, when the logarithms of the geometric means were used. A seasonal variation in the coli-aerogenes and the enterococcal counts of shell fish liquor was noted which could only be attributed to the temperature differences in summer and winter.     

PSEUDOMONAS AERUGINOSA IN DRINKING WATER

SUMMARY: The literature concerning antagonism between Pseudomonas aeruginosa and Escherichia coli , the natural habitat of the former and its potential pathogenicity, has been briefly surveyed. Experimental work showed that the two organisms can coexist in pure water, but in mixed cultures in peptone water Ps. aeruginosa can inhibit E. coli , though not in the early stages of growth and only within a critical range of peptone concentration. Ps. aeruginosa might therefore reduce the coli-aerogenes content in grossly polluted water but would not normally interfere with the determination of the actual coli-aerogenes content at the time of sampling. Water supplies containing many Ps. aeruginosa but few or no coli-aerogenes bacteria were found to be relatively common in Northern Israel and in view of the faecal origin of the former, and its potential pathogenicity, it is concluded that this organism should be taken into account, as well as coli-aerogenes bacteria, in assessing the suitability of water for drinking purposes.     

SOME BIOCHEMICAL CHARACTERISTICS OF FRESHLY ISOLATED STRAINS OF THE GENUS SERRATIA

SUMMARY: Many of 110 strains of Serratia , isolated from soil, water, milk and dairy equipment, were biochemically closely related to the coli-aerogenes bacteria. Acid and gas was formed from glucose in 14 days at 30° by 53% and from lactose and MacConkey's broth by about 40%. All except one strain gave——++ IMViC reactions.
An inverse relationship was observed between depth of pigmentation and carbohydrate fermentation. Complete loss of pigment in mutant strains was not uncommon, and was associated with loss of proteolytic properties and increase of saccharolytic activity.
The majority of the strains had psychrophilic characteristics: 75% grew at 3–5°. Most strains showed moderate growth at 37°, but only 7 formed red pigment at that temperature.
All strains resembled Serratia marcescens in morphology, containing minute coccoid rods smaller than those of coli-aerogenes bacteria.     

THE DETERMINATION OF THE COLI-AEROGENES CONTENT OF MILK AND DAIRY EQUIPMENT BY PLATING ON VIOLET RED BILE AGAR INCUBATED AT 30°

SUMMARY: Violet red bile agar (VRB) incubated at 30° for 20–24 hr was as good an indicator of coli-aerogenes bacteria in milk as MacConkey's broth. A high proportion (82%) of the large, deep red colonies considered to be formed by coli-aerogenes bacteria were confirmed as such. A British brand of dehydrated VRB agar was as suitable as an American brand of this medium for determining the coli-aerogenes content of milk. All the strains of typical coli-aerogenes bacteria tested formed red colonies. In a small proportion of cases the diameter was less than 0·5 mm. The only other milk bacteria which formed colonies resembling those of coli-aerogenes organisms were some acid forming strains of Gram-negative rods. Coli-aerogenes bacteria, determined on VRB agar at 30°, generally constituted only a small proportion of the microflora of fresh raw milk and of farm dairy equipment.     

FURTHER OBSERVATIONS ON THE BACTERIAL CONTENT OF WATER IN WATERCRESS BEDS

SUMMARY: Observations on two watercress beds in Kent have confirmed previous findings (Jones & Baker, 1955) that even in comparatively well-protected beds the coli-aerogenes content of the water may be high in summer. They have also shown that in spite of circumstances which would lead one to expect pollution, as in the case of one bed, the coli-aerogenes content can be low in winter. The season appears to influence the content in the water of coli-aerogenes bacteria, pectate liquefying bacteria and those capable of growing at 37°, but in the case of those growing at 22° other factors can play a more important part, as higher counts were not uncommon in winter. Seasonal effects were less marked with inlet samples; they were less influenced by atmospheric temperature, and little extraneous contamination occurred. Numbers increased as the sampling point moved from inlet to outlet.     

THE BACTERIOLOGY OF FARM WATER SUPPLIES: A STUDY OF THE COLONY COUNT IN 72 HOURS AT 22°

SUMMARY: The colony count at 22° of farm water supplies from springs and wells was mainly composed of biochemically inactive, non-pigmented, Gram-negative rods. Water from a stream polluted with farmyard sewage showed a similar dominance of Gram-negative rods, but orange or yellow pigmented colonies were more abundant. There were few 37° positive coli-aerogenes bacteria in either the farm water supplies or the sewage polluted stream, and Bact. coli type I was rare.
A high proportion of the bacteria from farm water supplies fermented milk in 3 days at 22°; a third developed acid, 15% proteolysis and 6.4% ropiness.
Contamination of pure spring water with surface soil from a heavily grazed pasture resulted in a hundredfold increase in colony count with aerobic sporing rods replacing Gram-negative rods as the dominant organisms, but coli-aerogenes bacteria were absent.     

CAUSES OF GREENING OF UNEVISCERATED POULTRY CARCASES DURING STORAGE

SUMMARY: The greening of uneviscerated poultry carcases stored at 15° is due to the production of hydrogen sulphide following multiplication of bacteria in the gut. The main groups present in the gut after death are faecal streptococci, coli-aerogenes organisms, lactobacilli and clostridia, but their relative importance in causing greening has not yet been established. The hydrogen sulphide diffuses from the gut into the muscle tissue and there reacts with the haem pigments of blood and muscle to form derivatives which microspectroscopically are indistinguishable from sulphaemoglobin.
The delay of greening at lower storage temperatures is consistent with this view; and it is suggested that mechanically plucked birds green more rapidly over the ribs than hand plucked ones because the shaking distributes bacteria from the caecum along the gut.     

THE VALUE OF THE ASSOCIATION ESCHERICHIEAE-GROUP D STREPTOCOCCI IN THE DIAGNOSIS OF CONTAMINATION IN FOODS

SUMMARY: The tests for faecal contamination in foods, based on 'indicator species', should be reconsidered for the following reasons. First, in Europe, Escherichia coli is regarded as a specific index of such contamination; but, apparently through the use of antibiotics, the proportion of human faeces containing Klebsiella has risen from 5.2% in 1947 to 48.4% in 1956–7. Moreover, E. coli and other coli-aerogenes organisms may be absent from the gut of certain animals, notably the pig. Second, while the usual methods of counting coli-aerogenes bacteria do not permit the isolation of strains which ferment lactose slowly or not at all (i.e. paracolons), these occur in 35.6% of samples of human faeces. Further, the recommended methods of detecting such strains are not wholly satisfactory. Third, the origin of the coli-aerogenes bacteria is uncertain.
It is thus necessary to consider other organisms which normally inhabit the intestinal tract of humans and other animals. As to Clostridium perfringens , its source is doubtful, for it may come from faeces or from soil, and many foods contain only soil strains. The group D streptococci, on the other hand, are excellent indicators of faecal contamination. They are constant or frequent in the intestines of man and animals, and often more numerous there than coli-aerogenes bacteria. Their specificity as an index of faecal contamination is high and their investigation is easy with the selective media now available. Associated with the coli-aerogenes bacteria, they justify the diagnosis of faecal contamination in a foodstuff.     

COLI-AEROGENES BACTERIA OF PASTEURIZED MILKS, WITH SPECIAL REFERENCE TO PATHOGENIC SEROTYPES OF ESCHERICHIA COLI

SUMMARY: A total of 375 samples of retail pasteurized milk in bottles was examined from January to the end of August 1955, for coli-aerogenes bacteria and especially for pathogenic serotypes of Escherichia coli. These serotypes were not found, though 101 of the samples contained bacteria of the coli-aerogenes group. During the summer months, June-August, the percentage of contaminated samples was about 40% and 10% of the samples contained E. coli I or II. The epidemiological significance of these findings is discussed.     

THE MULTIPLICATION OF COLI-AEROGENES BACTERIA IN MILK STORED AT 3–5°

SUMMARY: When 108 samples of individual farm milk supplies were held at 3—5° for 72 hr, 35·2% showed increases in coli-aerogenes organisms of over one-hundredfold and 10·2% increases of more than one-thousandfold. The coli-aerogenes microflora after refrigeration was dominated by Klebsiella cloacae and K. aerogenes I. While some strains of all the coli-aerogenes types isolated showed growth, though sometimes scanty, on yeast-dextrose agar in 5 days at 3—5°, the majority of the strains of K. cloacae showed luxuriant growth under these conditions and can be considered as typical facultative psychrophiles of milk.     

Coli-aerogenes Bacteria on Plants

S ummary . Specimens of plant foliage and flowers collected in the region of Attica from various situations and geographical regions were examined over a period of 13 months for the presence of coli-aerogenes bacteria able to grow at 37°. Twenty-five (12.2%) of 204 specimens carried coli-aerogenes bacteria of which Escherichia coli type I was isolated from 12 (5.8%). Coli-aerogenes bacteria were seldom isolated from plants grown in uninhabited areas, and they occured more frequently during summer and autumn.
Vegetables and fruits sold in the central market of Athens are frequently contaminated with coli-aerogenes bacteria. Of 76 specimens of vegetables examined 66 (86.8%) were positive for coli-aerogenes bacteria, and 17 (22.3%) for E. coli type I. The figures from 41 specimens of fruits were 26 (65.8%) and 4 (9.7%), respectively.     

Vibrio ponticus sp. nov., a neighbour of V fluvialis-V. furnissii clade, isolated from gilthead sea bream, mussels and seawater

A new Vibrio species, Vibrio ponticus, is proposed to accommodate four marine bacteria isolated from sea water, mussels and diseased sea bream (Sparus aurata), at the Mediterranean coast of Spain. Strains are Gram negative, slightly halophilic bacteria that require Na+ ion for growth, oxidase and catalase positive, negative for arginine dihydrolase and ornithine decarboxylase but positive for lysine decarboxylase and indole, and utilize beta-hydroxybutyrate as a sole carbon source. Phylogenetic analysis locate these marine bacteria in the vicinity of the V. fluvialis-V. furnissii clade, sharing with these two species 16S rDNA sequence similarities slightly above 97% (97.1 and 97.3%, respectively). DNA-DNA hybridisation values confirm that the four strains form a genospecies and represent a new species in the genus Vibrio. We propose strain 369T (CECT 5869T, DSM 16217T) as the type strain.     

THE EFFECT OF VARIOUS WATER BACTERIA ON THE GROWTH OF ESCHERICHIA COLI I IN MACCONKEY'S BROTH AT 37° AND 42°

SUMMARY: The growth of a streptomycin resistant strain of Escherichia coli I in MacConkey's broth at 37° or 42° was not inhibited by water organisms of the genera Micrococcus, Proteus, Achromobacter and Pseudomonas , but the presence of large numbers of other members of the coli-aerogenes group resulted in retardation or inhibition of its growth.     

THE OCCURRENCE OF COLI-AEROGENES ORGANISMS ON PLANTS

SUMMARY: Coli-aerogenes bacteria were seldom found on the foliage of a wide variety of garden plants, trees and shrubs, and field plants, including pasture, except where there was a possibility of contamination by insects, animals or dust. They were present in only a few instances on the flowering heads of hay plants (wind pollinated) but on almost all the flowers of garden plants (insect pollinated). Although cereal crops are wind pollinated, coli-aerogenes organisms were found on about one-third of the grains examined. The question of their origin on grain is discussed.
In a series of paired samples of soil and of the foliage of the covering vegetation, coli-aerogenes organisms were present in most of the soil samples, but were absent from the foliage except where animal contamination was possible, as in the case of grazed pasture.
With foliage samples unlikely to have been contaminated by animals, most of the coli-aerogenes organisms isolated were A. cloacae , but with grazed pasture E. coli predominated, indicating contamination by grazing animals.
The coli-aerogenes floras of garden flowers and cereal grains corresponded closely, consisting in each case largely of A. cloacae .     

THE DETERMINATION OF COLI-AEROGENES ORGANISMS IN RAW MILK BY SURFACE SMEARS ON DRIED EOSIN METHYLENE BLUE AGAR PLATES

SUMMARY: The determination of the coli-aerogenes content of raw milk by smearing 0·1 or 0·2 ml of suitable dilutions on dried Levine's eosin methylene blue agar plates and incubating at 37° or 30°, was found too unselective as a routine technique. Only about two-thirds of the colonies showing the characteristic appearance of coli-aerogenes bacteria were confirmed as such by the formation of acid and gas in MacConkey's broth. Appreciable proportions of the numerous rose coloured colonies and of the very small dark colonies with metallic sheen, which are not considered to be coliaerogenes bacteria, formed acid and gas in MacConkey's broth.     

COLD-TOLERANT FERMENTATIVE GRAM-NEGATIVE ORGANISMS FROM MEAT AND OTHER SOURCES

SUMMARY: From various chilled meats, twenty-eight strains of coli-aerogenes bacteria and one Aeromonas were isolated which grew well at +1±5° and some at −1±5°. The optimum growth temperature for most of these strains was nearer 37° than 30°. Nine strains (including the Aeromonas ) fermented lactose rapidly, the remainder slowly or not at all. All the strains which fermented lactose rapidly with the production of gas gave positive presumptive coli-aerogenes tests in MacConkey's broth at 30°, but only five were positive at 37°; none was positive at 44°. Because such organisms can attain populations of millions/cm², they could confuse the interpretation of presumptive coli-aerogenes tests made on chilled meat.     

THE RAPID IDENTIFICATION OF ESCHERICHIA COLI I BY THE PRODUCTION AT 44° OF BOTH INDOLE AND GAS FROM LACTOSE

SUMMARY: The modification of the 44° test proposed by Mackenzie, Taylor & Gilbert (1948) is useful for the rapid identification of Escherichia coli I in water and foods. False positive tests caused by other coli-aerogenes bacteria, or by their association with other organisms, can be considered rare. Only a few E. coli I fail to produce indole or ferment lactose at 44°, and further confirmation for routine purposes is necessary only when the results at 44° are discordant, e.g. indole positive-lactose negative or indole negative-lactose positive.     

Bacteria in bivalve shellfish with special reference to the oyster

The bacterial flora of the Pacific oyster Crassostrea gigas, the sea mussel Perna viridis and the arkshell clam Scapharca cornea differed considerably from that of seawater in both numbers and generic composition. The numbers of heterotrophic bacteria in the bivalve shellfish, including the anaerobes and spore-forming bacteria, were greater than that in the surrounding water. Pseudomonas spp. were the dominant organisms, comprising over one third of the 321 strains characterized after isolation from the bivalves and seawater. Other bacteria isolated from the shellfish included Vibrio, Acinetobacter, and Aeromonas spp., whereas the seawater flora consisted mainly of coliform organisms, coryneform bacteria and Flavobacterium/Cytophaga spp. Bacteria associated with the deposit-feeding clams were higher in density and more distinct in generic composition as compared with those in the suspension-feeding oysters and mussels. Over 90% of the coliform and heterotrophic bacteria in oysters were found in organs associated with the digestive tract. Coliforms were mainly found in the stomach while heterotrophs were present in both stomach and the lower intestine. The results suggest that the stomach flora of oysters are mainly derived from the external environment and, through a process of selection and multiplication, that it may be gradually replaced by a more indigenous population which dominates the lower digestive tract.     

OBSERVATIONS ON THE ISOLATION OF SALMONELLAE FROM SELENITE BROTH

SUMMARY: Studies of growth curves of enterobacteria in selenite broth containing different carbohydrates showed that whereas mannitol and lactose brought about a steep fall in coli-aerogenes bacteria, mannitol improved the growth of Salm. typhi-murium . With mixed cultures of Cit. freundii I and Salm. typhi-murium the presence of lactose, utilizable by the former, adversely affected the viable count of the latter.
Comparative studies with routine faeces specimens showed that selenite broth was an efficient selective medium with MacConkey's agar; but much better results were obtained when it was combined with deoxycholate-citrate agar. Gentian violet introduced into selenite broth improved its selectivity for most Salmonella types when MacConkey's agar was used for final isolation.