THE SLOW GROWING GRAM NEGATIVE PIGMENTED WATER BACTERIA

THE SLOW GROWING GRAM NEGATIVE PIGMENTED WATER BACTERIA L.G. Herman, BSA, Ph.D. National Institutes of Health Environmental Safety Branch Bethesda, Maryland 20205 Summar Bacteria, producing yellow to orange pigmented colonies on the surface of natural and artificial media, can be found in virtually every segment of the environment. They can be isolated from the soil, from fresh and salt water, from rain and snow, as well as from man, animals, birds, fish and plants. The majority of pigmented organisms are slow growing and can best be seen on the surface of agar plates and membrane filter pads, but usually only after extended incubation of 5-15 days at room temperatures, Some species appear to be chlorine tolerant and can be readily isolated from domestic water supply outlets such as taps, spigots and drinking fountain heads. They also grow readily in static water holding units such as tanks, water baths and special equipment, commonly noted in laboratories and hospitals. Although most of the yellow pigmented bacterial colonies can be classified as harmless microorganisms, occurring in most natural water supplies; infections can and do occur among infants, debilitated patients, surgical patients and patients on immunosupressive drug therapy, chiefly through careless handling of water supplies contaminated with these organisms, The use and abuse of our natural fresh water supplies is an ever increasing worldwide problem. Not only are the lakes and streams polluted by chemicals, fertilizers, and insecticides from surface waters, but the sewage disposal practices encourage the survival and growth of many undesirable species of microbes. Bayliss /2/ in 1930 foresaw the coming events when he stated, "It is easy to remove microorganisms and avoid other particles by filtration, but it is not easy to reduce the organic content of many waters to the point where it will not support microbial growth." The truth of this statement was verified many times over a period of twenty years of microbiological analyses of water samples in a hospital and research facility. 170 L. G. Herman The samples came from many sources such as dynamic and free flowing and static or stored water supplies especially in dead end or little used pipe lines, water baths, reservoirs, tank and storage units. Most samples at one time or another, contained viable mold spores, yeast cells, acid fast bacilli, aerobic and anaerobic spore formers, micrococci, streptococci and diptheroids in addition to many species of pigmented and nonpigmented gram negative rods /14/—some of which still remain to be classified as this symposium is attempting to demonstrate. Our attention was first drawn to this problem when four open heart surgery patients developed a bacteremia during their convalescent period /4/. Environmental studies demonstrated a similar organism in a water bath for tempering blood in the surgical area, and in the watér cooling system of the heart lung machine used on the four patients, as was isolated from blood cultures of these patients. Subsequent environmental studies showed that various species of pigmented gram negative bacteria could be readily isolated from the water from virtually every area, in varying numbers, of the research complex. Since water is the "almost universal" solvent, it is able to extract both beneficial and harmful components from the soil or surfaces over or through which it flows and percolates that give it flavor, odor, color, hardness, acidity, alkalinity, and the nutrients that support and encourage microbial growth before, during and after routine use and handling. Chambers and Clarke /7/ describe it clearly, "the extent to which water can serve as a bacterial growth medium is one of the least recognized facets of microbiology." The typical water bacteria, i.e., the pigmented gram-negative, non-fermenting, slow growing species require somewhat different conditions for optimum development. Pigment is most readily produced on the surface of agar plates incubated at room temperature for 5-15 days. The slow growth is not limited to artificial media, since samples of water held at room temperature show a slow but steady increase in numbers, while the levels of coliforms or other non-pigmented species usually decrease during similar holding periods. Pigment and Biochemical Characteristics While it is easy to recognize pigmented colonies on the surfaces of agar plates or membrane filter pads, Gilardi /11/ rightly comments that, "using pigment production as a taxonomic criterion is in itself of dubious merit because of the effects of variation in temperature and media on pigment production." Ciegler /8/ noted that over half of the marine bacteria isolated to date, contain unsaponifiable fat soluble yellow to red pigments. Hayes /12/ separated pigmented cultures isolated from fish into Flavobacteria and Cytophaga species. Starr and Stephens /32/ noted that many phytopathogenic bacterial colonies isolated from plants were also yellow in color. Bean and Everton /3/ isolated 172 cultures of orange-yellow pigmented The Slow Growing Gram-Negative Pigmented Water Bacteria 171 bacteria from chlorinated cooling waters and classified most of them as Flavobacteria. On the other hand, Koyama et. al. /19/ noted non-pigmented Flavobacteria and concluded that, "pigmentation of these bacteria is so variable that the description is not rigid and they must therefore be considered to be a heterogeneous species," Although Hayes /12/ and Shewan /28/ feel that the Flavobacteria should be classified as Cytophaga , Weeks /33/ states that "Flavobacterium is more a historic concept than a taxonomic reality." However, Gilardi /11/ defines the minimal features required for the identification of Flavobacterium as: (1) Gram negative asporogenous bacilli, (2) polar flagellated, if motile, (3) oxidize carbohydrates to acid without gas, (4) usually indole positive, (5) catalase positive, and (6) indophenol oxidase positive and (7) gelatin positive. Isolation Methods Of the many problems associated with the slow growing, pigmented, gram negative water bacteria, that of original isolation is probably the most frustrating. Although various species of Flavobacteria and Cytophaga, Table I, can grow over a wide range, 4°c - 44°c, of temperatures, most species grow best at room temperature, 18°C - 32°C, but require extended incubation of 5-15 days to produce visible colonies and measurable pigmentation /14/, hence the plates should be kept in plastic bags to prevent undue loss of moisture from the agar base. Since most species are pigmented and aerobic, swabbed or surface inoculated plates are preferable to poured plates, in that the pigments can be more readily noted and colony growth is more Table I Slow Growing Pigmented Water Bacteria in Flowing and Static Water Supplies % Maximum Source No. Sampled& Positive Colony Count/ml Drinking Fountains 1965 117/150 78 3 x 10° Drinking Fountains 1975 164/217 76 4x 10° Drinking Fountains 1978 334/440 75 Oe 10° Sink Faucets 48/69 70 3 x 10° Dead End Pipe Lines 10/10 100 5 10* Water Baths 5/7 Ta ik 10° X-ray and Photo Wash Tanks 10/20 50 Ding 10° Humidifying’ Unite 36/55 65 eee Dental Chair Spray Units 7/7 100 Dx. 10° Distilled Water Lines 5/10 50 Stx 10° *Dilutions spread on surface of T.S.A. plates and incubated at room temperature 5-15 days. 172 L. G. Herman easily transferred from the agar surfaces. Enrichment methods in broth are not recommended because the slow growing cells are often "lost" in the heavy and more rapid growth of other species of bacteria, However, it has been noted that, in many instances, when lactose broth tubes from M.P.N. determinations of drinking water are held for 5-15 days at room temperature, pigmented growth is readily visible in many gas free tubes and this, when streaked on agar plates, usually also produces pigmented colonies, Since pigmented bacterial colonies can be isolated from water from virtually every corner of the globe, a variety of media /27/ are advisable to provide the proper conditions for optimum growth, pigment production and identification of the various species. Basic nutrient meat or soybean extract agar can be enhanced by the addition of yeast extract, plant proteins and carbohydrates, fish meal, milk casein, as well as soil extract and sea salts, with and without the addition of sheep blood. To prevent overgrowth by gram positive cocci and spore formers, the addition of antibiotics to the media is helpful, since most Flavobacteria are antibiotic insensitive. Where the microbial contamination is minimal, i.e. less than 1 viable organism in 10, 50 or 100 ml, as in the case of distilled, tap, deionized or reverse osmosis treated water, the membrane filter technique should be used with 50-500 ml_ samples so that 10-20 visible colonies can develop per pad on extended incubation of 5-15 days at room temperatures, Table II Water Samples Membrane Filtered Through 47 mm x 0.45p Pads” Source Samples Tested Sample Size Colonies Per Pad Tap water through clean faucet 15 250 ml <10 (5) 10-200 (10) Tap water through rubber hose 20 250 ml <200 (6) ınıcb (14) Distilled water from still (direct) 30 500 ml No Growth Distilled water at end of system; 20 250 ml <200 (5) clean faucet TNTC (15) Distilled water through rubber hose 20 50 ml <200 (5) TNTC (15) Deionized water, central unit 5 250 ml TNTC (5) “pads laid on T.S.A. plates, incubation at room temperature 5-15 days. bonrc -too numerous to count. The Slow Growing Gram-Negative Pigmented Water Bacteria 173 Since some species of Flavobacteria are photochromogenic, incubation should be continued in subdued light rather than in direct sunlight or total darkness and where the temperatures are not likely to vary greatly /19/. Dynamic Water Supplies Drinking Fountains It is amazing that the "drinking fountain head" still remains the most ignored part of the entire water delivery system to the individual in the average community. Table I illustrates the variations and degree of contamination and levels of pigmented water bacteria in one hospital. In 1965 a total of 78% of the drinking fountains were contaminated, yet thirteen years later there was little (75%) signi— ficant difference in the number of contaminated units. Another factor often overlooked when water from drinking fountains is compared with water from sink faucets, is the absence of chlorine in the fountain water. Many public buildings such as schools, hospitals, offices, etc., have a separate water delivery system for drinking fountains which is cooled or chilled, especially in the summertime, and constantly circulated so that the chlorine content usually falls below the effective level of 0.6 ppm, thereby permitting microbial growth and multiplication in the system and especially at the air-water interface on the fountain head, Close examination of many fountains leaves much to be desired in the way of aesthetics. Many have easily visible accumulations of deposited scales, others are heavily rusted, occasional heads have a deposit of slimy growth, often yellowish to brown in color /13/ /14/. Nevertheless, the drinking fountain head, unless routinely brushed and polished, is often little better than the "common cup" that is seldom cleaned or sanitized. Distilled, Deionized and Reverse Osmosis Units The continuing demand for water of high purity in pharmacy, biochemical, microbiological and tissue culture research has been developed to a point where distilled water is now available without demonstrable pyrogens and free from viable bacteria /16/. However, the usual central distilled water supply tank with extended pipe lines and rubber hoses, will always be contaminated with a wide variety of pigmented and non-pigmented organisms /6, 9, 14, 17/. Deionizing units of various sizes are also able to provide water with very low levels of anions and cations, but microbial contamination remains a problem, Stamm et.al., /31/ found Flavobacterium in every deionized and softened water unit and outlet in their laboratories and noted growth in the resin columns of up to 10° cells per ml. DeRoos et. al., /9/ noted that when both deionized water and distilled water was available in the laboratory, the distilled water had a lower bacterial and pyrogen content than the deionized supply. 174 L. G. Herman A water supply, low in sodium and calcium salts and virtually free from Pyrogens and viable bacteria for use in hemodialysis, seems to be possible with a reverse osmosis water production system /15/. However, it was noted that critical maintenance and strict sanitation is necessary to prevent recurring contamination with both pyrogens and viable organisms /25/. Static Water Supplies Water Baths and Wash Tanks The water in this equipment, static, open, seldom changed, and held at varying temperatures from ambient room to 45°C provides an ideal medium for microflora of all species (Table III) but even here, Flavobacterium seem to be the predominant organism - up to 35% of the recognizable types. Table III Predominant Microbial Species Isolated From 100 Laboratory Water Baths, (0°c-45°c) No. Contaminated Flavobacterium sp. 21% Flavobacterium with mixed flora 14 Klebsiella sp. 20 Other Gram negative species 20 Pseudomonas aeruginosa 4 Gram positive spore formers Yeast species 3 Sterile - No growth LZ aswabs on T.S. agar plates held 5-15 days at room temperature. Contaminated water baths are especial hazards in surgical suites where blood packs are tempered /4/ or in dressing rooms where saline transfusions are warmed, and especially in microbiological laboratories when aerosols created by splashing, etc., tend to release contaminants into media, table tops, equipment, etc. The most effective and least hazardous or corrosive inhibitor noted to date is the addition of 0.1% - 0.6% of glutaraldehyde to the water bath. The water should then remain inhibitory for extended periods of time depending on the addition of organic matter through spills, etc, Regular cleaning and the use of distilled water is also recommended, The Slow Growing Gram-Negative Pigmented Water Bacteria 175 Nebulizers and Humidifiers A critical study of micro organisms /29/ in heated nebulizers showed almost complete control of growth of all types of gram negative rods, but chiefly Pseudomonas, Moraxella, Acinetobacter, and Flavobacterium species when the water temperature was held at 46°C or above while those units held at 41°C or below, showed 38% - 52% contamination when held for 5 days or more before cleaning. It was also noted that tap water, when used in humidifying units, was the source of infection for 5 of 8 infants exposed to these contaminated mists /21/. Dental Chair Units Studies of the water spray from a dental hand piece /1, 20/ (Table I), showed a high level of contamination by pigmented gram negative organisms in every unit checked, which could be a hazard to both patient and dentist, especially since some counts exceeded 5,000,000 organisms per ml. This contamination problem is further complicated by the fact that dental reservoirs are usually heated to 35-37°C to provide a warm spray. The possibility that the pigmented colonies often noted in sputum specimens /26/ may in some cases originate from dental sprays, should not be over looked, Hemodialysis Systems Favero et, al./10/ noted that water produced by deionization, distillation, reverse osmosis or softening, supported many species of gram negative bacteria especially when stored or used in the dialysate of artificial (hemodialysis) kidney machines. In many instances, the proliferating organisms were often the cause of pyrogenic and septic reactions in the treated patient. They emphasized the hazards of "naturally occurring" bacteria—in which organisms present in distilled water and diluted in the same "membrane filtered" water grew more rapidly, showed higher disinfectant resistance and longer survival times than did those organisms that were passed through conventional laboratory media and then reintroduced into the same waters, Among the nine gram negative organisms usually isolated from dialysis fluids, Flavobacterium, Pseudomonas and Acinetobacter are the most common and troublesome contaminants. Infection Hazards with Pigmented Water Bacteria Premature and Newborn Infants With the advent of antibiotics, more attention is being paid to the gram negative bacteria capable of surviving in the presence of many antibiotics at higher than average levels required to inhibit the gram positive species. The emergence of a slightly pigmented gram negative non-motile, oxidase positive bacillus that produced indole but had little reaction on sugars and was sensitive only to erythromycin and 176 . L. G. Herman carbenomycin, was the apparent cause of spinal meningitis in 19 newborn infants of which fifteen died /5/. King /18/ studied these organisms further and in 1959 separated them into three serological groups, and because of its light yellow pigment and association with meningitis, tentatively classified it as a new species— Flavobacterium meningosepticum. Following this publication, sporadic cases have been noted in the international literature annually /14, 22, 34/. Mature Patients Infected Following Surgery Olsen /22/ isolated Er meningosepticum from ten cases of post operative infection following thoracic surgery in Denmark, all patients survived. To determine the source of these infections, he was able to isolate F. meningosepticum from brook water, garden soil and from the blood, urine, sputum and spinal fluid of various patients—all similar to the 1959 King isolates. The hazards of indwelling arterial catheters are well described by Stamm et. al., /30/ in which they noted 14 patients with positive blood cultures due to a Flavobacterium. The organisms were isolated from the catheters, stop cocks and ice supply used to cool the syringes. Their recommendations are simple—sterile techniques, handwashing and fresh catheters every 24 hours. Discussion Since the slow growing pigmented water bacteria are usually found in circulating and static water supplies even in the presence of detectable levels of chlorine /13/, it may mean that many are chlorine tolerant and survive to grow and multiply when "naturally occurthe chlorine has been dissipated. Although these organisms may be ring" in water supplies, /10, 23/ they grow so slowly and better on the surface of agar plates, that they can easily be missed because most water analyses tubes and poured plates are discarded after 48 hours incubation. While various species of Flavobacterium have been isolated from patients following use of products contaminated by them, viz /11, 23, 24, 30/ hence, one should not overlook the hospital water supplies as a possible source of those infections, even though they are difficult to identify and trace /1, 4, 10/. Although the average laboratory may not have the personnel or experience available to identify each and every waterborne organism, as has been shown many times during this symposium, they can easily demonstrate the presence of other than coliform species by: (1) swabbing the surface of various types of agar plate media directly, or by (2) using larger amounts, e.g., 50-500 ml of treated water for membrane filtration, both with extended incubation. The ability to recognize microbial growth in any kind of potable, distilled or deionized water supplies when used for domestic, medical or research uses, thus becomes a responsibility of the microbiologist associated with the consuming unit. The Slow Growing Gram-Negative Pigmented Water Bacteria 177 Therefore, depending on the final use of any given water supply, the presence of slow growing, pigmented water bacteria can be (1) harmless indicators of an unsterile condition, or (2) they can be a hazard to the system, the user and the end product. The degree to which this occurs, depends on the pressures created by our changing society on the total environment and eventually on the microflora within it. Then as some species are stimulated or altered by the effect of acids, antibiotics, antibodies, antiseptics, chemicals, disinfectants, gases, petroleum products, etc., their adaptation becomes more easily recognizable as "survivors" in a complex population, which is certainly true of the yellow pigmented gram negative bacteria of the Flavobacterium - Cytophaga group of microorganisms. Bibliography /1/ Abel, L.C., Miller, R.L., Micik, R.E., and Ryge G.: J. Dent. 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