Isolation and characterization of coliforms from ... - Wiley Online Library

Journal of Applied Microbiology 1997,82,597-609

Isolation and characterization of coliforms from glacial ice and water in Canada's High Arctic S.J. Dancer, P. Shears' and D.J. Platt University Department of Bacteriology, Glasgow Royal Infirmary, Glasgow, and 'Department of Medical Microbiology, Royal Liverpool University Hospital, Liverpool, UK 5799/06/96: received 10 June 1996,revised 27 September 1996 and accepted 2 October 1996 S.J. DANCER, P. S H E A R S AND D.J. PLATT. 1997.Ellesmere Island is the northern most member of the Canadian Arctic Archipelago with over one-third of the land mass covered by ice. A joint services expedition to the island's Blue Mountains offered a unique opportunity for microbiological studies of resident bacteria in an environment uninhabited by man. Over 100 samples of water and ice were collected from stream, lake and glacier and the filtrate cultured under canvas. Bacterial growth was harvested onto swabs for transport back to the UK and 50 coliforms chosen at random for identification and antibiotic susceptibility testing. Most of the glacial strains were capsulated, pigmented and some over 2000 years old. Genera such as Serratia, Enterobarter, Klehsiella and Yersinia were found; speciation was inconclusive and some organisms remain unidentified. Ampicillin resistance was evident in 80% of water isolates as opposed to 30% of the glacial organisms, but the isolates were generally exquisitely susceptible to antibiotics. T h e facility for ampicillin resistance did not appear to be transferable. Plasmid DNA was found in 33% of the glacial organisms and over 50% of the water isolates. Similar profiles were identified within and apparently between species and required plasmid restriction analysis to help establish identity. Plasmid-free Serratia spp. were subjected to genomic fingerprinting. Indistinguishable patterns were found within sets of isolates both widely spaced by distance and collection date and it was postulated that coliforms able to survive an Arctic environment had spread extensively throughout the expedition area. In conclusion, this study contributes towards knowledge of naturally occurring antibiotic resistance, confirms the presence of plasmids and genotypic data provided evidence that potentially ancient organisms from glaciers can be cultured from water samples significantly distant.

INTRODUCTION

A joint services scientific expedition to Ellesmere Island in Canada's High Arctic offered a unique opportunity for microbiological studies of resident bacteria. T h e expedition site chosen was a valley beside the Blue Mountain range on North West Ellesmere, bounded by Hare Fiord to the west and Borup Fiord to the east (latitude 8O058'N-80"35'N; longitude 087"00'W483"00'W). The area was uninhabited and unex-

3 Dancer, l k i z e r s z t y Depurtment uf Burteizotog)r, Glusgom Royul Infirmury, Castle Strert, Glusgow G4 OSF, UK

Correspnndence to. DY S

0 1997 The Society for Applied Bacteriology

plored and represented an environment unpressurized by the presence of man. Consequently it was decided to collect samples of water and glacial ice in an attempt to isolate and characterize resident coliform bacteria. Ellesmere Island exhibits the meteorological conditions associated with the High Arctic regions. Summertime temperatures range from 0°C to 10°C in the Blue Mountain Valley with 21 h of sunlight and little precipitation. Winter conditions are severe with constant darkness, blizzards and temperatures falling to -50°C. Spring, Summer and autumn follow each other in rapid succession between the months of

598 S . J. D A N C E R E T A L .

June-August, and an abundance of plant and animal life proliferates after the spring melt in mid-June. Once the thaw has begun, the Arctic tundra becomes riddled with fast flowing stream and river as the shallow soil and underlying permafrost reject further melt water. T h e water channels turn seaward down the valley and are constantly fuelled by melting glaciers from the more permanent ice-caps in the north. Puddle, pond and lake further contribute to a plethora of water sources. Expedition members were housed in tents and the largest of these held radio-communications equipment and the microbiology laboratory. The aim of the project was threefold. Firstly, it was hoped to isolate coliforms from varied water sources in order to identify and characterize organisms capable of surviving the Arctic climate. Previous studies have established that certain bacteria have the ability to survive freeze-thaw stress and actively metabolize at the low temperatures characteristic of the High Arctic (Nelson and Visser 1978; Nelson and Parkinson 1978; Hardfield et al. 1992). It has also been reported that coliform bacteria are able to survive continuous freezing conditions notably, viable coliforms have been regenerated from frozen pony faeces from Scott’s and Shackleton’s Antarctic expedition 50 years prior to collection ( h e a t h 1962; Boyd and Boyd 1963). As Ellesmere Island provides a suitable environment for many species of animal and bird, it was expected that the coliform bacteria would be isolated from water sources in the region visited. The second aim was to determine the antibiotic susceptibilities of any organism isolated. Finally, the presence of permanent ice-cap and glaciers on North West Ellesmere Island raised the possibility of revival of bacteria captured by glacial formation several thousands of years ago (Park 1991; Kennedy et d.1994). Liberating such bacteria from a state of suspended animation would allow further comparison with modern day organisms. The antibiotic susceptibilities of these organisms and subsequent genetic studies if appropriate, would provide useful data upon the presence of resistance characteristics before the advent of the ‘antibiotic era’ this century (Smith 1967; Mare 1968; Gardner et al. 1969; Hughes and Datta 1983). ~

MATERIALS AND METHODS Sample numbers and origin

One hundred and twenty-five l00ml samples of water and glacial ice were collected into sterile containers. T h e samples originated from an obliquely defined area z 50 km x 20 km stretching from Blue Mountain coastal regions adjoining the Blue Mountain Valley up to and including glacial extensions from the Van Royen ridges (Fig. 1). The water sources included puddle (six samples), pond (16 samples), stream (45 samples), river (eight samples), lake (22 samples) and glacial

melt water (six samples). There were 22 samples of glacial ice. These were collected from two glaciers extending from the Blue Mountain icefield (14 samples), one glacier from the Blackwelder Mountain icefield (two samples) and one glacier from the Van Royen icefield (six samples) adjacent to and connected with the Krieger Mountain ice-cap. COLLECTION METHODS Water sources

A 500 ml sterile aluminium collection cup (Oxfam Del Agua Water Testing Kit, devised by D r Barry Lloyd, Robens Institute, University of Surrey, Guildford, U K ) was lowered to a depth of 30cm in pond and lake sampling and withdrawn using an attached metal cable. The cup was submerged in puddle sampling if possible, and held against the current flow in stream and river. Glacier melt water was collected from streams located on the glacial surface and from waterfalls at or near the glacier snout. One hundred ml aliquots were decanted into sterile screw-top containers for transport back to the field microbiology laboratory. Sometimes this journey would take up to 4 d to complete. Glacial ice

(i) Loratzon qfsamplzng. T h e collection site for glacial ice samples was the snout, or nose, of the glacier. Since one of the aims of this project was to culture potentially ancient organisms, it was essential to ensure that the ice sampled was as old as possible. For this reason ice was extracted from near the base of the glacier margin, close to the glacier snout (Paterson 1981). T h e lower part of each glacier is made up of a well-defined layer of dirty sediment-rich ice, which is a mixture of ice and rock debris incorporated into the bottom of the glacier from the ground surface over which it flows. It was important to avoid this material when sampling since its relative age and origin are unknown. Samples were taken from clean pure glacier ice directly above this zone (Fig. 2 ) . (ii) Method ojsamplzng. Flame-sterilized ice axes were used to remove superficial snow and ice to a depth of at least 60 cm in order to avoid contamination by surface melt water. It has been established that there is little, if any, penetration by extraneous bacteria from melt water into glacial ice since ice possesses a very high density and also exhibits a core temperature capable of freezing any mobile moisture instantly (Cameron and Morelli 1974; Abyzov et nl. 1982). Underlying hard ice was then chipped into sterile containers and allowed to melt naturally. Ice axes and scalpels used for sample extraction were sterilized with flame or alcohol throughout the procedure as appropriate. As with water sample collection, the return journey to Base camp took up to 4 d before microbiological processing.

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 597-609

CHARACTERIZATION OF ARCTIC COLIFORMS 599

Fig. 1 Map of expedition area, Ellesmere Island, denoting sampling sites and extremes of sampling. Each square represents an area 10 km x 10 km. Map shown by kind permission of the Canada Map Office, Department of Energy, Mines and Resources, Ottawa

Fig. 2 Lateral margin of a glacier extending from the Blackwelder Mountain Icefield showing welldefined layer of dirty sediment-rich ice in the basal ice zone 0 1997 The Society for Applied Bacteriology,Journal of Applied Microbiology 82, 597-609

600 S. J. D A N C E R E T A L

Culture

Plasrnid profile

One hundred ml aliquots of water or melted ice were filtered through sterile membranes as detailed by the Del Agua Water Testing Kit User's Manual. The filtration assembly was sterilized between samples. Following filtration, membranes were placed upon sterile absorbent pads soaked in 0.5% nutrient agar and incubated at 10-15°C for 4-7 d. Bacterial growth was harvested onto charcoal swabs in transport medium for transportation back to the UK. Nutrient agar in preweighed sachets was dissolved in filtered water and placed in culture bottles before boiling for 20 min. All collection containers and Petri dishes were sterilized similarly by boiling; sterilization of the filtration assembly was performed by decanting 1 ml of methanol into the apparatus, igniting it and allowing it to burn in the absence of excess oxygen. This produces formaldehyde vapour which disperses throughout the filtration assembly to sterilize all internal surfaces. Forceps and scalpels were flame-sterilized as required.

Agarose gel electrophoresis of plasmid DNA was performed according to the method described by Birnboim and Doly (1979). Molecular weight standards for comparison of plasmid size were 98, 42, 24 and 4.6 MDa plasmids from E. coli 39R86 1.

Regeneration of cultures. Charcoal swabs were plated onto nutrient, blood and MacConkey agar in a U K hospital microbiology laboratory. Plates were incubated at 4",20", 30" and 37°C for 24-48 h and four colonial types removed from each plate for further study. Identification.Coliform bacteria were provisionally identified by Gram stain and oxidase reaction and bacterial capsules visualized using Indian ink. Slime production was assessed by incubating the organism in tryptic soy broth for 48 h at room temperature - then adding a few drops of safranin to the medium before decanting and examining the inside of the container for a thin film of slime (Ishak rt ul. 1985). A representative collection of 5 1 isolates (29 from water sources, 22 from glacial ice) were selected for full identification. This was performed using API 20 test kits for Enterobacteriaceae and other Gram-negative rods (BioMerieux). T h e strips were incubated at 30°C for 24-48 h as profiles were not obtained at 37°C; some isolates required 48-72 h at room temperature in order to produce reactions. Antibiotic susceptibility testing. T h e following antibiotic discs were tested against the chosen isolates: ampicillin (10 pg, 25 pg), cefazolin (30 pg), cephamandole (30 pg), ceftazidime (10 pg), tetracycline (10 pg), trimethoprim (2.5 p g ) , streptomycin (10 pg), gentamicin (10 p g ) , nalidixic acid (30 pg), ciprofloxacin (1 pg), chloramphenicol (10 p g ) and carbenicillin (100 pg). The Stokes method for antibiotic susceptibility testing was performed using control strains Escherirhza rolz N C T C 10418 and Sevutia finticola N C T C 12147.

Conjugation

Those strains resistant to ampicillin (Amp) and susceptible to nalidixic acid (NA) were shaken in 5 ml of nutrient broth for 4 h at 30°C. Esfherichiu coli K12 (2-5ml) (resistant to NA, susceptible to Amp) and 2 ml of warmed nutrient broth were added to 0-5ml of donor cultures and incubated at 30°C overnight. Conjugation mixtures were plated onto Diagnostic Sensitivity Test (DST) agar containing 3 0 p g ml-' NA and 10 pg ml-' Amp; these were incubated for 24 h at 37°C and mixtures left to stand overnight at 4°C and 20°C. T h e following day, mixtures were plated afresh onto similar antibiotic-containing media for 24 h at 37°C. All plates were examined for recipient E. coli and visible colonies subjected to antibiotic susceptibility testing with Amp (10 p g ) and NA (30 pg) discs. Testing was repeated for those colonies resistant to Amp and morphologically identical to E. coli K12. Potential transconjugants were profiled for plasmids in parallel with original Arctic donor and K12 strains. Plasmid fingerprinting

Restriction fingerprinting of isolates that harboured plasmids was carried out according to the method described by Platt et al. (1986). Briefly, plasmid DNA was extracted and purified from Arctic isolates by an alkaline lysis, phenol extraction and ethanol precipitation method. Restriction enzymes were obtained from Gibco-BRL (Paisley, UK) and used according to the manufacturer's instructions. Each plasmid was cleaved by at least two restriction enzymes chosen to provide an optimum number of fragments to ensure specificity and minimize coincidental matching. Each gel was calibrated with a PstI digest of bacteriophage A DNA and controlled with a II digest of the enzyme under study. Restriction enzyme fragmentation patterns (REFP) were analysed by computer as described below. Genomic DNA fingerprinting

Whole-cell DNA was extracted as described by Platt et al. (1996). Briefly, cultures were grown overnight in lOml of Brain Heart Infusion broth (BHI) (Oxoid), centrifuged at 4000 rev min-' for 5 min and resuspended in 3 x 1 ml volumes of TE50 buffer (Tris 10 mmol 1-', EDTA 50 mmol l-',

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 597-609

CHARACTERIZATION O F ARCTIC COLIFORMS 601

pH 8.0). After centrifugation (30 s, 11 800g) pellets were resuspended in 200 pl of TE50 containing 100 pl of lysozyme (40mg ml-I), vortexed and incubated on ice for 5 min. A 15p1 volume of 20% sodium dodecyl sulphate (SDS) was added followed by 50 p1 of Proteinase K (10 mg ml-I). T h e solution was mixed gently by inversion and then sheared by single passage through a 25G needle and incubated at 37°C for 2 h. Five hundred pl of phenol-chloroform were added, mixed and the emulsion centrifuged for 10 min. T h e upper aqueous layer was removed and an equivalent volume of isopropanol added to precipitate DNA (60 min at room temperature). The precipitate was centrifuged for 10 min and then resuspended in 100 p1 of TElO buffer (10 mmol l-' Tris, 1Ommol 1-' EDTA, p H 7.8). Triplicate tubes were pooled and 1OOpl of ammonium acetate (7.5mol 1-I) were added, followed by 600 pl of 95% ice-cold ethanol. Tubes were left overnight at -20°C. Precipitated DNA was centrifuged for 10 min, resuspended in 30Opl of TE10, digested with 20p1 of RNAase (10mg ml-') at 37°C for 1 h, after which the phenol-chloroform and isopropanol precipitation steps were repeated. The DNA pellet was resuspended in 300 pl ofTElO before treatment with ammonium acetate and 95% ethanol as previously. After centrifugation the purified DNA was resuspended in 60pl of T E (10mmol 1-' Tris, lmmol 1-' EDTA) and stored a t 4°C. Electrophoresis was typically for 24 h at 20 mA in 0.8% agarose gels using standard Tris-borate buffer (89 mmol 1-' Tris, 89 mmol 1-l boric acid, 1 mmol 1-' EDTA) further diluted 2:l in distilled water. Criteria used in the selection of restriction enzymes

That two unrelated reference strains of the species investigated gave REFPs with a chosen enzyme such that fragments within the 5-30kb size range were well resolved in a 0.6-0.8% agarose gel. That a minimum of six fragments were present (normally 12-30 fragments is considered optimal). That a lower fragment size limit could be defined by reference to a specific fragment in a PstI digest of bacteriophage 2. That the reference strains were distinguishable on the basis of the REFPs generated. Computer-aided analysis of restriction fragments

Restriction fragment mobility in ethidium bromide-stained agarose gels was recorded on Polaroid type 665 film and input to a computer using a digitizer and commercially available software (Platt and Sullivan 1992). Each gel was calibrated using restriction fragments from both PstI and K'pnI digests of bacteriophage 2. T h e molecular weight of these fragments was fitted to a robust modified hyperbola (Platt rt ul. 1996) from which fragment sizes in adjacent tracks were estimated

by interpolation. Numerical values were stored for subsequent calculation of similarity coefficients and graphically output; the latter was on a logarithmic scale. RESULTS Bacterial recovery

Each water sample tested produced semiconfluent, if not confluent, growth ( 1000 cfu per 100 ml) on a 50 mm diameter membrane filter at first incubation. Lower numbers were obtained from glacial specimens (100-500 cfu per 100 ml) with no sterile samples observed. Regeneration following return to the UK produced virtually 100% recovery using blood and nutrient agar at temperatures of 4",20" and 30°C; colony counts were significantly reduced on MacConkey agar and growth severely inhibited by incubation at 37°C on all media types.

Morphology Over 400 colonial types were investigated from the original swabs plated. Many of the cfus were mucoid and highly pigmented; both lactose and non-lactose fermenting colonies were present. Some isolates exhibited P-haemolysis. Gram stain identified both Gram-negative and -positive cocci and bacilli, although it was noted that some organisms exhibited variable staining (Table 1). There was more difficulty establishing Gram reaction and shape among the glacial isolates; further subculture onto nutrient agar often aided identification. A greater proportion of organisms from water sources were Gram-negative bacilli (8090) than from glacial ice (5890), mainly because of the difference in numbers of Gram-positive cocci 14"/0 from water and 37% from glacial ice. Approximately half the Gram-negative bacilli from both sources were oxidase-negative. Gram-positive rods were found from all sources in small numbers but spore stains were consistently negative. Twelve per cent of organisms from water were Gram-negative cocci; none were identified from glacial ice. Many of the Gram-positive cocci from glacial ice were large coccoid forms in tetrads and resembled Micrococcus spp.; simple Gram staining suggested that most of the Gramnegative bacilli from all sources possessed capsules. -

Identification

Twenty-nine randomly selected water isolates and all 22 glacial isolates were confirmed oxidase-negative Gram-negative bacilli. They were identified as Serratia, Enteroburter, N e b siellu, Yersiniu and ..lcinetobacter spp. but final speciation remained speculative because the temperature requirements of the organisms did not allow use of API identification strips

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82,597-609

602 S. J. DANCER E T A L .

Table 1 Overall numbers and morphological types of bacteria recovered from water and glacial ice after transportation and revival in the UK

Water sources

Glacial ice

Morphology

Pond, puddle, stream, river, lake, glacial melt water

All samples from snout of g1acier

Gram-negative bacilli (oxidase negative) Gram-negative bacilli (oxidase positive) Gram-positive cocci Gram-positive bacilli Gram-negative cocci Gram-variable cocco-bacilli

140 (4390) 120 (37%) 45 (14%) 10 (3'10) 12 (3%) 0 (O"/o)

22 (290ko) 22 (29%) 28 (3746) 2 (2.So/u) 0 (0%) 2 (2.5%)

Total

327 ( 100?/0)

76 (10000)

according to manufacturer's recommendations. Provisional profiles, however, indicate that most isolates were Serratia spp. (5 lob), followed by Enterobarter spp. (27%), Klebsiellu spp. (lWo), Yersinia (6"/0) and one Arinetobacter isolate (2%). Members of each genus (except .Icinetobacter) were found from both water and glacial ice. Presumptive species and API profiles with percentage confidence are listed in Table 2. Klebsiella and Enterobucter isolates both possessed capsules and Klebsielh isolates also secreted extracellular capsular material slime into the surrounding liquid medium. Serratia isolates did not show visible capsules in Indian ink preparations but most secreted slime into the culture medium. Yersinia and A4cinetobucter isolates did not appear to produce either. Antibiotic susceptibilities

Table 2 lists the antibiotic resistance characteristics of the organisms studied. Five of 22 glacial isolates were fully sensitive to all the antibiotics tested in contrast to only one of 29 water isolates. T h e most common antibiotic resistance demonstrated was to cefazolin (84%0 isolates) often coupled with resistance to cephamandole (71%) and ampicillin (65%). Forty-three per cent of isolates resisted carbenicillin. One glacial Serratia isolate exhibited additional intermediate resistance to trimethoprim and nalidixic acid and the sole .Icinetobactev organism was resistant to trimethoprim, nalidixic acid, tetracycline and ceftazidime, as well as ampicillin, carbenicillin, cefazolin and cephamandole. These latter two isolates were found buried in ice from one of the Blue Mountain glaciers. None of the organisms were resistant to streptomycin, gentamicin, chloramphenicol or ciprofloxacin. Overall, more isolates from water sources showed resistance than from glacial ice. T h e most common combination of resistance - ampicillin, cefazolin, cephamandole and car-

benicillin was present among six of 22 glacial isolates (27%) as opposed to 15 of 29 water isolates ( 5 2 O l O ) . ~

Plasmid profiles

Plasmid profiles in kilobases (kb) are listed in Table 2. Nine of 22 glacial isolates (41%) harboured 2 4 plasmids; among 29 water isolates 1-3 plasmids were detected in 16 organisms (55%). T h e most common sized plasmids among both water and glacial organisms were 122 kb (seven glacial and seven water isolates) and two smaller 6.1 and 3.0 kb plasmids (six glacial and two water isolates). Two plasmids, l40kb and 29 kb, were found solely among water organisms - the larger alone (five isolates) and the smaller usually co-resident with the 122 kb plasmid (four isolates). In total, 26 (51%) isolates appeared to be plasmid free. Some isolates produced identical profiles, e.g. a 122:6.1:3.0 kb profile was found from five glacial and two water organisms. These were identified as Serratia spp. (Ser. marcescens x 3 , Ser. liquefaciens x 4) resistant to cephalosporins only. Four out of five water isolates containing one 140 kb plasmid resisted ampicillin, 1st and 2nd generation cephalosporins and carbenicillin - the fifth failed to resist carbenicillin whilst remaining susceptible to the former antibiotic group. Of three Yersinza spp. identified, one glacial isolate contained four plasmids, one water isolate contained one small plasmid and the third, also from water, had no visible plasmids. Each produced different antibiograms. Conjugation

Four attempts at conjugation failed to produce any transconjugant K12 E. cola carrying donor ampicillin resistance. It was concluded that the ampicillin resistance was probably chromosomally determined.

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 597-609

CHARACTERIZATION O F ARCTIC COLIFORMS 603

Plasmid fingerprinting

The restriction patterns from 25 plasmid-containing isolates are summarized in Table 3 and illustrated digitally (Fig. 3 ) . Twelve Serrutia spp. scattered between water and glacial ice origins produced six distinct patterns. Three plasmidcontaining Klebsiella isolates were each different from each other. Seven Enterobarter isolates divided into three with identical patterns and four with individual REFPs. Sermtia isolates therefore demonstrated less diversity in plasmid restriction analyses than the other organisms, but each genus, Serratia, Enterobacter and Klebsiella, appeared to contain plasmids giving different patterns of restriction despite producing similar plasmid profiles originally. REFP analysis of genomic DNA was carried out on eight plasmid-free Serrutia isolates (Fig. 4) from both glacial ice and water. The REFP of glacial isolates 77 A and 91 A were identical, though each was isolated from independent glaciers more that 40 km apart. Three isolates, 1l4C (glacial origin), 35B (stream) and 122D (river), generated REFPs either identical (1 14C/35B) or that differed in a single restriction fragment. These two groups of isolates were not closely related (Dice coefficient of similarity < 55%). Isolates 31 A (glacier), 89 A (glacier) and 79 A (stream) each produced unique REFP patterns unrelated to each others and to the two groups described above (Dice coefficients ranged from 34 to 60%0).

DISCUSSION

The extent to which coliform bacteria would be recoverable from an Arctic environment was not known as they do not appear to survive in Antarctic soil (Boyd and Boyd 1963). Viable coliforms have been reported, however, in frozen pony faeces from the Scott expedition in Antarctica over 50 years previously (Sneath 1962). The initial florid growth from water samples in this study prompted the attempt to isolate bacteria buried deeply within glacial ice. After successful revival preliminary characterization sought phenotypic characters that might relate to survival mechanisms at low temperatures (less than -50°C for most of the year). It is possible that capsules and slime which were present amongst the isolates in such abundance, may have contributed. Klebsiellu pneumoniae and Enteroburter uerogenes produce an increased amount of capsular material when cultivated at temperatures below that required for maximum growth (Moat and Foster 1988). There is currently no explanation for this phenomenon and a molecular mechanism against freezing has not been demonstrated. The protective effect of additives such as glycerol are thought to act by reducing the electrolyte concentration in the residual unfrozen extracellular solute at low temperatures (Lovelock 1953; Mazur 1970). Low molecular weight glycerol, however, bears little resemblance to high molecular weight exopolysaccharides usually associated with slime or

capsular material - indeed, the ability of most polysaccharides to bind free water would actually raise the concentration of electrolytes in residual unbound water and so precipitate cellular damage. It is possible that the protective effect of macromolecules is related to their ability in permitting a reversible influx and efflux of solute during freezing and thawing, thus enabling cells to avoid the effects of excessive osmotic gradients (Lovelock 1953; Mazur 1970; Meryman 1971). Marked pigment production was noted among isolates of Serratiu and Enterobarter spp. Carotenoid pigments protect against harmful photodynamic effects and it has been well documented that not only u.v., but visible light can also kill coliforms (Krinsky 1968). Arctic organisms would have been constantly exposed to both during spring, summer and autumn periods (Moat and Foster 1988; Hardfield et a/. 1992). Organisms buried deeply within glacial ice may not have been so susceptible because light would have been refracted and their near anaerobic environment would also provide protection from light damage (Hollaender et al. 1951). Pigment production, however, did not appear to differ quantitatively between the two groups of bacteria studied from water and from ice. The abundant growth of organisms (100-2 1000 cfu per 100 ml) on initial culture was unexpected considering the environmental conditions. One previous report detailed an average yield of 4.8 cfu per 100ml at 15°C from snow pits dug on the Agassiz ice cap, Ellesmere lsland (Hardfield et al. 1992). Differing proportions of Gram-negative and -positive organisms were recovered using Plate Count and Sea Water agar; the authors commented upon the poor yield and suggested that environmental conditions at the study site permitted only minimal and scattered microbial activity (Hardfield et ul. 1992). The reason for the higher yield reported in this study is not immediately apparent. The origin of the coliform bacteria is thought to be bird and animal sources, of which there was ample evidence. Dead carcasses of indeterminate age were observed upon glacial surfaces, and live wolves, musk-oxen, lemmings and stoat were encountered commonly. No doubt all or most of these animals and numerous bird species have been present for many years and contributed to environmental coliforms ancient and modern in water and glacial ice. T h e Agassiz study also described difficulty in affiliating some organisms to their basic morphological groups by simple Gram stain and was confirmed in this study (Hardfield et al. 1992). Soil bacteria surviving nutrient-poor conditions display two distinct vegetative forms according to their present environment. If nutritionally poor, the coccoid form is manifest, whereas in a rich medium the organism grows as bacilli (Ensign and Wolf 1964; Moat and Foster 1988). Both forms, however, appear to be equally resistant to starvation despite differing biochemical patterns (Boylen and Ensign

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 597-609

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Origin

Snout, Woody Willow Glacier Snout, Woody Willow Glacier Snout, Woody Willow Glacier Snout, Woody Willow Glacier Ice climb ascent, Shirley Glacier Ice climb ascent, Shirley Glacier Snout, Shirley Glacier Snout, Shirley Glacier Snout, Shirley Glacier Snout, Shirley Glacier L. Snout, Christopher Glacier R. Snout, Christopher Glacier Mid Snout, Christopher Glacier L. side Christopher Glacier L. side Christopher Glacier L. side Christopher Glacier L. side Christopher Glacier L. side Christopher Glacier R. side Christopher Glacier Blue Mountain Glacier Blue Mountain Glacier Blue Mountain Glacier Puddle near Base Camp Lake Inlet puddle Base Camp Lake Shirley Glacier Water Hole

Strain designation

1-31A 2-3 1C 3-31D 631E 5-73A 6-73D 7-77A 8-78A 9-78B 10-78D 11-89A 12-91A 13-94C' 1.C95C' 15-95D 16-96A 17-96B 18-96E 19-97D 20-114C 21-116A 22-116C 23-20A 2.C33A 25-72B 97.70 91.50 95.90 88-60 93.00 95.80

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Confidence

Serratia jhntirola Enterobarter ugglomeriins Ser. fnntirnla Klebsiella pneumoniae Ent. ugglomerans Ent. agglomrrans Serratia species Ser. fontirola Ser. marcescens Ser. liquejuriens Ser. finticola Ser. rubidaea Serratia species Ent. agglumeruns 1-ersinia enterorolztira Ser. marresc'ens Ser. liquefariens Ser. liquefariens .4rinetobarter species Ser. finticola Ent. amnigenus Ser. marcescens Ser. rubidaea Ent. aerogenes A% pneumoniar

ID

Ap, Cmd Fully sensitive PAP, Cmd PAP, Cmd Fully sensitive Fully sensitive Cz Cmd Ap Cmd Cz Cmd Cz Cmd Ap Cmd Cz Cmd cz Fully sensitive AP Cz Cmd Cz Cmd pAp Cmd T m Nal pAp Cmd T m T c Nal Ap Cmd Fully sensitive CZ Ap Ap Ap Cmd

Resistant to:

Table2 The origin, identification, API profile, antibiotic susceptibility and plasmid profile of 51 Arctic coliforms from water and glacial ice

0 0 0 0 0 0 0 0 122: 6.1: 3.0 122: 6.1: 3.0 0 0 0 0 65: 43: 12: 5.0 122: 6.1: 3.0 122: 10: 6.1: 3.0 122: 6.1: 3.0 12: 10.8 0 122: 5.8 122: 6.1: 3.0 122: 86: 29 158: 29 0

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Puddle near Wolf Pond Pond near Base Camp Lake Large pond near Base Camp Lake Wolf Pond Inlet stream, Base Camp Lake Outlet stream, Base Camp Lake Craggs Stream Sea Stream Stream behind Record Hill Stream below Record Hill Stream behind Record Hill Escarpment Stream Ravine Stream Valley of Death Stream Valley of Death Stream Mount Blah Stream 1st Inlet Stream, Blue Mt River 2nd Inlet Stream, Blue Mt River Surface Melt Water, Woody Willow Glacier Surface Melt Water, Woody Willow Glacier Blue Mountain River Blue Mountain Rirer Base Camp Lake Base Camp Lake Ice-dammed Lake Cache Lake

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88.20 88.80 88.20 99.20 88.20 88.20 70.30

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81 97.60 58.80

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94.70 77.30 93.20 50.60 50.60 85.20 76.10

-

99.30 58.80 85.20 94.60 95.80

1.: frederiksenii Ser. liyuejizriens Entrrobacter species Ent. intermedium Ent. aggbmerans Ent. intermedium Ser. jbntirola Ent. intermedium Ent. intermrdium 1.: entrmcnlitirn Entrrnbarter species

Ser. fonticolu Ser. liyuejizciens Kl. pnrumoniae Ser. jbntirola Ser. finticola Enterobacter species Kl. ozarnue Kl. ozuenue Ser. jhntirola Srr. $curia Ser. jcariu Kl. pneumoniue Ser. liyuejariens Serrutia species Kl. pneumonzar Ap Cmd Fully sensitive Ap Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd Cz Cmd Cz Cmd Ap Cmd PAP cz Ap Cmd Ap Cmd Cz Cmd Ap Cmd Ap Cmd Ap Cmd Ap Cmd PAP AP

0 65: 43: 6.1 140 122 0 122: 29 0 140 ND 0 0 140 122: 6.1: 3.0 0 0 5.8 122: 6.1: 3.0 122: 29 0 0 0 0 140 1 40 0 122: 29

Ap, Ampicillin including carbenicillin and 1st generation cephalosporins; pAp, low level resistance; variable with respect to 1st generation cephalosporins; Cz, 1st generation cephalosporins only; Tm, trimethoprim; Nal, nalidixic acid; Tc, tetracycline; Cmd, cephamandolc (2nd generation); ND, not determined.

26-85B 27-17A 28-22A 29-88A 30-35B 31-3SA 3244A 3347c 34-54A 35-79A 36-S1B 37-100A 38- 101E 39-105B' 40-106C 41-llOB 42-117B 43-120B 44-30A 45-30B 46-24A 47-1 22D 484B 49-12A 50-27C 51-107B

0 VI

Q)

cn

c

n

0

; n

0 0

1

0

n

>

n

0

2

0

1

D

N

z

rn

0 --I

n >

D

1

0

606 S . J . DANCER E T A L .

Table 3 Restriction enzyme fragmentation patterns (REFPs) of 25 plasmid-containing isolates from water and glacial ice in Canada's High Arctic

Origin and Plasmid

designation

Genus

profile

Plasmid REFP

G1acier 78B 78D 96A 96B 96E 116A 116C

Serratia Serratia Serratia Serratia Serratia Enterobacter Serratia

122: 6.1: 3.0 122: 6.1: 3.0 122: 6.1: 3.0 122: 10: 6.1: 3.0 122: 6.1: 3.0 122: 8.3 122: 6.1: 3.0

Slll s1 s1 s11 s1 El1 s1

Puddle 20A 33A

Serratia Enterobacter

122: 86: 29 158: 29

S1V Ell1

Pond 17A 22A 88A

Serratia Klebsiellu Serratia

65: 43: 6.1 140 122

SVl Kl SV

Stream 38A 47C 54A lOOA lOlE 117B 1208

Enterobacter Klebsiella Serratia Klebsiella Serratia Serratia Enternbacter

122: 29 140 ND 140 122: 6.1: 3.0 122: 6.1: 3.0 122: 29

El K11 S1V K111

Lake 4B 12A 107B

Entero barter Enterobarter Enterobacter

140 140 122: 29

E1V EV El

strain

s1

Slll El

ND, Not determined

1970). In this study individual cfus on isolation exhibited both coccoid and bacillary forms and Gram variability. These organisms were predominantly from glacial ice and reverted to more stable forms on subculture. Some psychrotrophs exhibit filaments at their upper temperature limit of growth and the psychophile Arthrobacter glacialis forms clumps of coccoid cells at 18°C and motile rods at or below 13°C (Gounot 1991). T h e organisms in this study were psychrotrophic and did not display these features. Glacial samples are subject to two main criticisms for the work reported: firstly, possible contamination by superficial melt-water during sampling, and secondly, the estimated age of extracted bacteria in relation to the potential age of the glacier sampled. Although every effort was taken to avoid

contamination during sampling, the collector was occasionally suspended by ropes over the glacial snout and at the mercy of prevailing wind and weather. Despite the adoption of a sterile protocol, semiblizzard conditions may have introduced contaminated water into the collection jar. This may explain some of the similarities between the surface water and glacial organisms studied, rather than recently melted glacial material entering water sources sampled. With regard to age estimates of glacial bacteria, no information is available on the absolute age of the glaciers sampled. Isotopic dating cannot be applied easily to a sample composed of almost pure water and the expedition glaciologist dated the glaciers visited by counting the number of annual accumulation layers. Equipment was not available to drill out core ice from the centre of the glaciers; this method was used by a group examining the Devon Island ice caps in Arctic Canada and confirmed that basal ice from that glacier was probably 6000 years old (Paterson et al. 1977). As the Devon Island ice cap is bigger than the glaciers in this study, this would be a maximum age estimate. The smallest glacier sampled was found to have more than 350 exposed layers, i.e. 350 years old, but this was regarded as an absolute minimum because many individual layers are not visible at a range of several metres. T h e final estimate for the Shirley glacier was 2000 years old - provided ice was taken from the glacial snout, just above the well-defined layer of dirty sediment-rich ice in the basal ice zone (Paterson 1981; Bentley 1996). Figure 5 shows a cross-section of idealized flow pattern in a glacier. Net accumulation of mass occurs in the upper (accumulation) zone of the glacier whilst net loss of mass occurs in the lower (ablation) zone. As snow accumulates it is gradually buried by subsequent years and is progressively transformed to glacial ice. It is the downward velocity resulting from this burial combined with the downhill velocity as the glacier ice deforms under its own weight that leads to the direction of flow of the glacier. Net upward velocities in the ablation zone are the result of the loss of mass from the glacier surface by melt and evaporation. T h e oldest ice occurs near the base of the glacier and only becomes exposed at the glacier margin when it reaches the snout (Paterson 1981). For this reason ice was extracted from the snout of each glacier visited. If the 2000 year age estimate is valid, it is possible that some of the study isolates are of comparable age. Most of the genera identified were Serratia, Klebsiella, Enterobarter and Yersinia; these are all known to grow at lower temperatures (Bergey 1984). Enterobacter spp. grow better at 20-30°C with increased pigmentation at those temperatures. Yersinia can grow at 4°C and Serratia at 10°C with pigment (prodigiosin) production maximal at 20-35°C (range 12-36°C) from Ser. marcescens and Ser. rubidaea. Serratia liquefaciens, Ser.Jicaria and Ser. fonticola can grow at 6 5 ° C . Serratia fonticola was the most common isolate in this study and produced a pink pigment very similar to that

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 597-609

CHARACTERIZATION O F ARCTIC COLIFORMS 607

Fragment size (kb) 2

1

3

5

10

I

I

20 I

I I II I I 1 ' 1 I I I I i i I I I I I I Ill I Ill I I I I I I I I 1 1 I Ill I 111 I I I I I I I I II I Ill I Ill I I I I I I I I I1 I Ill I Ill I I I I I I I I I I I I I I I IIIIII I I I I 1 II I l l IIII 1 I I I II I 11 Ill 1 I I 1 1 1 1 1 II I l l I I

9D8B 16196A 171968

221116c 10/78D 381101E 23120A 421117B

I

I

I

2

1

I

Avall Avall Avall Avall Avall Avall Avall Avall

I

3

I

I

I

10

5

20

Fig. 3 44zuIIREFPs of plasmids from Serratia spp. Water and glacial isolates. Host strain designation as in Table 2

Fragment size (kb) I

I

Lambda 201114c 301358

471722D 7D7A l2I9lA

35D9A

11189A 1131A

I II 11 I I 11 I I 1 1 I I I 1111 II I I I I I II I I I i I I IIII I l l I I I I II I 1 I I I I I 1 I II l l l l I I I 111 I1 I I 1 II I 111 1 I I I I I

I

I ii

I I

3

I

fstl Sau3A Sau3A

1

Sau3A Sau3A Sau3A Sau3A

I

Sau3A Sau3A Sau3A

I

I

2

10

5

3

2

I

5

I

10

Fig. 4 Sau3A REFPs of genomic DNA from plasmid-free Serrutia spp. Strain designation as in Table 2

generated by Ser. marcescens and Ser. rubidaea (Gavani et al. 1979). It is possible that the isolates identified as Kl. pneumoniae could in fact be the environmental Kl. terrigena and/or Kl. planticolu, as these are very like Kl. pneumoniue but grow at 10°C (Bergey 1984). Neither species are included in the standard API 20E schedule. There were discrepancies between quoted antibiotic susceptibility for the genera identified and what was actually found. Most Enterobarter species are resistant to ampicillin and cephalosporins but susceptible to carbenicillin; the isolates in this study varied from fully susceptible (all glacial strains) to differing combinations of resistance including carbenicillin resistance. Serratia marcescens is usually considered

to be intrinsically resistant to ampicillin and cephalosporins (1st generation), but three glacial isolates exhibited ampicillin susceptibility and one isolate (presumed Ser. liguefaciens) was fully susceptible. EPrsinia enteroroliticii usually produces constitutive and inducible p-lactamases which confer resistance to ampicillin, cephalosporins and carbenicillin; two isolates studied resisted ampicillin only. In general, the isolates were remarkably susceptible to antibiotics, which would be expected as regards age and environment. Some, almost certainly, predate the advent of the so-called 'antibiotic era' and therefore illustrate that capabilities for antibiotic resistance already existed naturally. Other studies from collections either before the antibiotic era or from antibiotic virgin territories confirm this observation (Smith 1967; Mare 1968;

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 597-609

608 S. J. D A N C E R E T A L

Accumulation zone

(a)

(b)

*/

I

-,-

Basal ice zone

--

Fig. 5 Cross-section of (a) idealized flow pattern in a glacier, and (b) detail of margin at snout showing relationship of sampling sites to dirty basal ice zone (Paterson 1981)

Gardner et al. 1969; David and Anandan 1970; Hughes and Datta 1983). It is of interest that two organisms showed some resistance to antibiotics such as trimethoprim, nalidixic acid, tetracycline and possibly ceftazidime. Most of the organisms demonstrated production of plactamases. Failure to transfer this capacity by standard broth mating techniques suggested that the enzymes are probably chromosomal in origin, but it would be necessary to try mobilizing a conjugative R-plasmid into and out of the isolates in order to confirm true chromosomal locations (Spratt et al. 1973). Constitutive expression of fi-lactamases from the coliforms is not surprising because virtually all Gram-negative bacteria produce these enzymes and the types produced are often specific for species and sometimes for subspecies (Matthew and Harris 1976). The amount of P-lactamase produced is frequently low but can be readily induced; most of the chromosomally determined enzymes preferentially hydrolyse cephalospor ins. The organisms in this study demonstrated more resistance to 1st or 1st and 2nd generation cephalosporins than to ampicillin. The capacity for hydrolysing carbenicillin may be determined by a further enzyme or could be included within a broad substrate-specific p-lactamase able to hydrolyse ampicillin, cephalosporins and carbenicillin together (Sawai et al. 1982). There were 20 (39%) organisms resistant to this group of antibiotics. Further work will be necessary to determine the nature of the P-lactamases produced by the organisms in this study. Approximately half the isolates contained detectable plasmids according to the method used. T h e function of these is unknown but would be of interest to pursue as they may code for survival mechanisms hitherto unexplored. Almost

identical profiles were recognized within and between crossspecies and required restriction analysis to confirm whether the plasmid(s) were indeed the same. No identical REFPs were found between different genera but fingerprinting gave an indication as to whether final speciation was consistent. This is because it is more likely that identical restriction patterns would exist within the same species, and often within the same serotypes (Brown et al. 1986). T h e results therefore suggest some uncertainty concerning speciation as one restriction pattern produced by five Serrutzu isolates and a second from a further two were organisms designated either Ser. liquefaciens or Ser. murcescens, both within each pattern group. In all cases the percentage confidence API identification was higher for the Ser. nzurcescens isolates (95.998.9%) than for Ser. liquefaczens (58.8-84.6%) and it is possible that all the organisms may ultimately belong to the same species. Alternatively the lower plasmid diversity among Serratia isolates may suggest that these plasmids are particularly mobile - having spread between species. As identical patterns were found in isolates from both glacial ice and water sources sometimes over 40 km apart, it seems most likely that those water isolates with identical plasmids have been derived from recently melted glacial ice. The continuous cycle of freezethaw conditions in the Arctic environment would be expected to release frozen viable organisms within at least a limited area as the thaw period, allowing water-borne mobility, only lasts for about 2 months per year. There were no related plasmids between three Klebsiella isolates, but three Enterobucter spp. demonstrated identical profiles. T h e latter isolates were from two streams 4 km apart and a lake 20 km from the closest stream and were collected over 1 month apart. Whereas plasmid data provided an initial evaluation framework, the small group of plasmid-free Serratza isolates investigated by genomic restriction analysis clearly supports this in that identical patterns were found among one glacial and two water isolates although there were five different patterns illustrated from just eight isolates tested. It is hoped to extend genomic analyses to the remainder of the collections to clarify further relationships between all the isolates studied and this should contribute to a better understanding of the mechanisms operating in this environment. ~

ACKNOWLEDGEMENTS The authors wish to acknowledge the following people, without whose help this study would not have been completed: SAC Murray Spark, RAF Leuchars; F1. Lt. Dr Roger Smith (Leader, JSE Blue Mountains); J.T. Paul White, RAF Sealand, Liverpool; Mr E. Potts, Liverpool School of Tropical Medicine; Professor C.A. Hart, Department of Microbiology, University of Liverpool; D r M. Bentley, Department of Geography, University of Edinburgh; D r D. Baird and

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 597-609

CHARACTERIZATION OF ARCTIC COLIFORMS 609

MLSOs at Monklands and Hairmyres Hospitals; Mrs S. Byrne, Lomond Healthcare NHS Trust, Vale of Leven District General Hospital, Alexandria, Dumbartonshire.

REFERENCES Abyzov, S.S.,Lipenkov, V.Y., Bobin, N.E. and Kudryashov, B.B. (1982) Microflora of central Antarctic glacier and methods for sterile ice-core sampling for microbiological analyses. Biology Bulletin ofthe A4cademyof Sciences of the USSR 9, 304-349. Bentley, M. (1996) University of Edinburgh; personal communication. Berge-ys Manual o f Systematic Bacteriology (1984) Vol. 1, ed. Krieg, N.R. and Holt, J.G. Baltimore/London: Williams 8i Wilkins. Birnboim, H.V. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. NucleicaJcids Research 7, 1513-1523. Boyd, W.L. and Boyd, J.W. (1963) Viability of coliform bacteria in Antarctic soil. Journal of Bacteriology 85, 1121-1 123. Boylen, C.W. and Ensign, J.C. (1970) Intracellular substrates for endogenous metabolism during long-term starvation of rod and spherical cells of i29, 92-101. Gounot, A.M. (1991) Bacterial life at low temperature: physiological aspects and biotechnological implications. Journal o f .@plied Bacteriology 71, 386-397. Hardfield, M., Jones, H.G., Letarte, R. and Simard, P. (1992) Seasonal fluctuation patterns of microflora on the Agassiz ice cap, Ellesmere Island, Canadian Arctic. Musk-ox 39, 119-123. Hollaender, A,, Stapleton, G.E. and Martin, F.L. (1951) X-ray sensitivity of E. culi as modified by oxygen tension. Nature 167, 103- 104. Hughes, V.M. and Datta, N. (1983) Conjugative plasmids in bacteria of the ‘pre-antibiotic’ era. Nature 302, 725-726. Ishak, M.A., Groschel, D.H.M., Mandell, G.L. and Wenzel, R.P. (1985) Association of slime with pathogenicity of coagulase-nega-

tive staphylococci causing nosocomial septicaemia. Journal of Clinical Microbiology 22, 1025- 1029. Kennedy, M.J., Reader, S.L. and Swierczynski, L. M. (1994) Preservation records of micro-organisms: evidence of the tenacity of life. .l/licrobiology 140, 2513-2529. Krinsky, N.I. (1968) The protective functions of carotenoid pigments. In Photnph,ysiology 3 ed. Giese, A.C. pp. 123-195. New York: Academic Press. Lovelock, J.E. (1953) The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochimicu et Bioph.ysica -4cta 11, 28-36. Mare, I.J. (1968) Incidence of R factors among Gram-negative bacteria in drug-free human and animal communities. Nature 220, 1046-1047. Matthew, M. and Harris, A.M. (1976) Identification of 8-lactamascs by analytical isoelectric focusing: correlation with bacterial taxonomy. Journal of General 221icrobiology94, 55-67. Mazur, P. (1970) Cryobiology - the freezing of biological systems. Science 168, 939-949. Meryman, H.T. (1971) Cryoprotective agents. Cryobiology 8, 173183. Moat, A.G. and Foster, J.W. (1988) .Micwhirzl Phystology, 2nd edn. New York-Chichester-Brisbane-TorontwSingapore: WileyInterscience Publication, John Wiley and Sons. Nelson, L.M. and Parkinson, D. (1978) Growth characteristics of three bacterial isolates from an arctic soil. Canadian Journal qf’ ~l/licrohiolog~! 24, 909-9 14. Nelson, L.M. and Visser, S. (1978) Effect of spring thaw on microorganism in an Arctic meadow site. .4rctic and--llpine Research 10, 679-688. Park, P. (1991) Ice age bacteria return from the dead. Nea, Scientist 130(1771), 22. Paterson, W.B. (1981) The Ph.ysics of Glaciers, 2nd edn. Oxford and New Tork: Pergamon. Paterson, W.B., Koerner, R.M., Fisher, D., Johnsen, S.J., Clausen, H.B., Dansgaard, W. et al. (1977) An oxygen-isotope climatic record from the Devon Island ice-cap, Arctic Canada. Nature 266, 508-511. Platt, D.J. and Sullivan, L. (1992) Molecular epidemiology: Molmatch and beyond. International Labmate 16, 11-13, Platt, D.J., Chesham, J.S., Brown, D.J., Kraft, C.A. and Taggart, J. (1986) Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application. Journal of Hygiene 97, 205-210. Platt, D.J., Browning, L.M. and Candlish, D. (1996) Molecular analysis of Salmonella enterica serotype Dublin: building bridges between population genetic and molecular epidemiological studies. Electrophoresis 17, 667-671. Sawai, T., Kanno, M. and Tsukamoto, K. (1982) Characterisation of eight 8-lactamases of Gram-negative bacteria. Journal of Bacteriology 152, 567-571. Smith, D.H. (1967) R factor infection of Escherichia coli lyophilized in 1946.Journal oJ’Bacteriology 94,2071-2072. Sneath, P.H.A. (1962) Longevity of microorganisms. Nature 4842, 643-646. Spratt, B.G., Rowbury, R.J. and Meynell, G.G. (1973) The plasmid of Salmonella typhimuriunz LT2. ,2loleeular and General Genetics 121,317-353.

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 597-609