Bacterial growth properties in refrigerated all-in-one TPN mixtures

CLINICAL

NUTRITION

(1987) 6: 25-29

Bacterial Growth Properties in Refrigerated all-in-one TPN Mixtures R. Jeppsson,

M. Johansson*,

KabiVitrum AB,* Microbiological (Reprint requests to M.J.)

J. Tengborn

and Pharmacy Departments,

S-l 12 87 Stockholm,

Sweden

The administration of a single mixture of the components used in total parenteral ABSTRACT nutrition has been considered to minimise the risks for infection in comparison with the conventional system with separate infusions. These all-in-one (AIO)-mixtures are prepared in hospital pharmacies under stringent aseptic conditions but the risk for adventitious contamination cannot be eliminated. The aim of the present study was to evaluate the microbiological growth in AIO-mixtures deliberately contaminated with mesophilic and psycrophilic bacteria. The AIO-mixture was inoculated with the different micro-organisms and the growth was studied at cold temperature during a period of 4 days. The results of the study implicate that with prompt refrigeration after mixing, the AIO-mixture can be stored for at least 4 days without the risk for growth of common contaminants. The investigated AIO-mixture was shown to have intrinsic growth suppressing properties towards a variety of Gram negative psycrophilic organisms during refrigeration. The nature of this properties seems to be a combination of low temperature, low pH and presence of trace elements.

Escherischia co& when incubated at + 22’ C. No growth was obtained at + 4°C. Similar results were found in TPN mixture in 3L bags composed of glucose and amino acid solution, when held at +4”C [6]. Jarvis and colleagues tested growth of Klebsiella pneumoniae and Enterobacter cloacue at + 4°C and at + 25°C and found no proliferation at the low temperature [7]. The aim of the present study was to evaluate the microbiological risks with contaminated all-in-one (AIO) preparations, where intravenous fat emulsion is included. Following the manufacturer’s instructions, the TPN solution shall be refrigerated after mixing, and may be held at +2-8°C for at least 4 days. The main risk for microbial proliferation during refrigeration would be from psycrophilic organisms, mainly nonfermentative Gram negative rods, e.g., Pseudomonas juorescens; therefore the growth pattern of such organisms was specially investigated.

INTRODUCTION Nutrition

support

one (AIO)

preparations

to patients by using premixed all-inparenterally has gained increasing popularity. This technique was first described by Sollasol [l] in France but is now widely used throughout Europe and the USA. Admixtures are prepared in hospital pharmacies under stringent aseptic conditions. However, the risk for adventitious contamination cannot be eliminated. The growth pattern for microorganisms in contaminated intravenous (IV) solutions has been investigated by many authors. Crystalline amino acid solutions with or without the addition of carbohydrates have been found to permit growth of Candida albicans, Pseudomonas aeruginosa and Enterobacter aerogenes. Also dextrose solutions 5-15”,, (w/v) and 0.9O, (w/v) saline permitted growth of several species [2]. The IV solutions examined have been held at room temperature during the tests and the object has been to demonstrate risks during normal or prolonged administration. These investigations and the work of Maki [3] have resulted in guidelines concerning time limits for administration of TPN solutions [4]. The possible microbial risks using admixtures have been studied by Duffett-Smith [5] at room temperature and during refrigeration. The tested admixtures, composed of amino acids, dextrose, vitamins and inorganic salts, permitted growth of Klebsiella pneumoniae and

MATERIALS

AND METHODS

Compounding

One standardised AIO-formula was used as a basis for the tests (Table 1). The admixtures were prepared aseptically in a laminar air flow cabinet. Extra potassium and the trace elements (Addamel’“) were added to the amino acid 25

26

BACTERIAL GROWTH PROPERTIES IN REFRIGERATED ALL-IN-ONE

Table 1 Contents of the AIO-mixture microbiological challenge test Intralipid” 20°,‘l VitalipidR Adult”) Soluvit Ra: Vamin ” 9 Glucose’) Addamel”‘a) Potassium chloride 2 mmol/mlb) Glucose 10°,bJ AddiphosHal Contents per litre: Fat (g) Glucose (g) Nitrogen (g) Na K Ca Mg PC’ Cl Zn Mn Fe CU

(mmol) (mrnol) (mmol) (mmol) (mmol) (mmol) (pmol) (pmol) (pmol) (pmol)

used in the

TPN MIXTURES

Test Solutions 500 ml lOm1 lamp 1000 ml lOm1 lOm1 1000 ml lOm1 39.4 78.7 3.7 25.6 21.7 3.0 1.2 10.8 26.8 7.9 15.7 19.7 2.0

a) KabiVitrum AB, Stockholm, Sweden b) ACO, Stockholm, Sweden c) Including the amounts from Intralipidz’ (15 mmol/l

Intralipid’)

solution. The phosphate (Addiphose) was added to the dextrose solution. Dextrose and amino acid solutions were connected with a 3-litre EVA (ethylene vinyl acetate) plastic bag (the IV bag, KabiVitrum AB) and the solutions were compounded. Finally the lipid emulsion including fat and water soluble vitamins was introduced into the bag. Pressurised sterile filtered (0.22 pm Millex, Millipore) nitrogen was used to speed up the mixing. For adjustment of pH to 7.5 in some of the AI0 mixtures, 50ml sodium hydroxide 1M was added to the Vamin” 9 Glucose prior to mixing. Intralipida 20%, pH 4.7, was obtained by adding 0.3 ml of hydrochloric acid 1M 500ml of the emulsion. Intralipid@ 20% with trace elements was prepared by adding 2 ml Addamel to 500 ml of the emulsion.

Organisms Candida albicans ATCC 10231, Escherischia coli ATCC 8739 and Pseudomonas fluorescens ATCC 13525 were obtained from American Type Culture Collection. Bacillus circulans and Staphylococcus epidermidis were isolated from environmental samples, as were the psycrophilic isolates Pseudomonas maltophilia (2 varieties), Flavobacterium and CDC group VA 1.

Microbiological growth studies were performed in the following media: a) 1. AI6 mixture (pH 5.4, osmolality 923 mOsm/kg) 2. Intralipid@’ 20% (pH 8.1, osmolality 370 mOsm/

e)

kg) 3. Varnir@ 9 Glucose (pH 5.2, osmolality 1350 mOsm/kg) 4. Soybean casein digest medium (SCDM) AI0 mixture (pH 7.5, osmolality 918 mOsm/kg) AI0 mixture minus trace elements (pH 5.4, osmolality 910 mOsm/kg) AI0 mixture minus trace elements and phosphate (pH 5.4, osmolality 901 mOsm/kg) Intralipid”’ 20% (pH 8.1, osmolality 370 mOsm/

0

kg) Intralipidm 20% (pH 4.7, osmolality 363 mOsm/

b) c) d)

g) h)

kg) Intralipids 20% plus trace elements (pH 7.2, osmolality 386 mOsm/kg) Intralipidm 20% plus trace elements (pH4.3, osmolality 386 mOsm/kg)

Preparation

of Seeded Solutions

Mesophilic organisms The microorganisms used in the initial study were harvested from surface growth on TSA (Tryptone Soya Agar, Oxoid) incubated at +33”C or from SDA (Sabouraud Dextrose Agar, Oxoid) incubated at +25X. The cells were centrifugated 5 min at 4000 rpm and washed twice in phosphate buffered saline, pH 7 (PBS). Using aseptic technique, 50 ml of sterile test solution was transferred to sterile 38 x 200 mm test tubes. After a viable count of each suspension of microorganisms, serial dilutions were made to give an inoculum of 5 x lo4 and 5 x lo3 CFU/ml. The resulting starting number of cells would be lo3 and lo2 CFU/ml in 50ml test solution. After inoculation the seeded solutions were incubated at + 8°C (Termaks cooling incubator). Viable counts were performed after 0,24,48, 72 and 96 h of incubation with pour plate technique using TSA or SDA for Candida. The plates were incubated at + 33°C for TSA and at +25”C for SDA.

Psycrophilic Organisms Viable count plates of psycrophilic organisms were incubated at + 33”C, after testing that they were not inhibited by this temperature. A surface spread technique was used, where a 1 ml sample was spread on a 14 cm agar plate surface and allowed to dry for 30 min. The plate was then incubated. In all studies with psycrophilic organisms, the cells

CLINICAL

27

NUTRITION

were taken from a 48 h culture in Soybean Casein Digest Medium (SCDM). The cells were diluted in PBS and immediately added to 50 ml of test solution in 38 x 200 mm test tubes. In each test series 2-4 dilution steps per strain were used for each solution type. Incubation and viable count frequency were made as above.

RESULTS Growth of Mesophilic

Contaminants

In Figure 1 the growth, or lack of growth, is demonstrated for all tested strains. The results from repeat tests with numbers of cells in the interval 50-500 CFU/ml confirm the lack of growth and are not explicitly presented. For mesophilic organisms the low temperature inhibits growth, and the cooling down period is too short for growth to be initiated.

Growth of Psycrophilic

Contaminants

fluorescens ATCC 13525 normally grows to considerable numbers during refrigeration temperature, also in nutrient-poor solution [8]. The growth of this organism was evaluated in SCDM, as a nutrient rich reference, in AIO-mixture, and in two IVsolutions, which are parts of the AIO-mixture (Fig. 2). Intralipid” does not inhibit the growth, it was only slightly less than in SCDM. VaminL Glucose permitted growth of two logs in 96 h, after a 24 h lag phase. No growth appears in the AIO-mixture. In order to further investigate the growth inhibiting properties in the AIO-mixture, a series of five different strains of psycrophilic organisms were studied. The rePseudomonas

I

1

1

1

I

I

1

2

3

4

TIME

(DAYS)

Fig. 2

Growth of P fluorescens in different solutions when stored at 8’C for 4 days. The different solutions were: v SCDM, 0 Intralipid” 20”,,, 0 Vamin” 9 Glucose and n AIO-mixture

3

$2 tl 3

1 0

Fig. 1 Growth of mesophilic microorganisms in AIO-mixture when stored at 8°C for 4 days. Batteries studied were: n B circulans, (3 E coli, 0 S epidermidis and v C albicans

i

i

3

TIME (days)

TIME (days)

Fig. 3 Growth

of psycrophilic microorganisms in AIO-mixture when stored at 8°C for 4 days. Batteries studied were: v P fluorescens, n I? maltophilia 4233 a I? maltophilia 4235, 0 Flavobacterium, l CDC Group VA 1

4

28

BACTERIAL GROWTH PROPERTIES IN REFRIGERATED ALL-IN-ONE

TI’N MIXTURES

sults for both solutions were similar, i.e., no proliferation was observed (Fig. 3). All SCDM reference solutions showed macroscopic growth after 72 h for each strain and are not included in the diagram. A further attempt to trace the mechanisms behind the inhibition was made using AIO-mixture, with variations in the factors pH and trace elements, which were assumed to be responsible for the inhibitory effect. Growth was compared in AIO-mixture, in AIOmixture adjusted to pH7.5, in AIO-mixture without trace elements and phosphate buffer and in SCDM (Fig. 4). The results indicate that these factors take part in the inhibition, possibly in combination. Next, a similar series of variations was made, using Intralipid” instead of a AI0 mixture. The combination of Intralipid” and trace elements inhibits growth more than Intralipid” alone at low pH. At pH 8 the growth of this organisms is unaffected by addition of trace elements. (Fig. 5). In the SCDM reference macroscopic growth was observed after 72 h, and is not included in the figure. A second series, not shown, inoculated to 10 cells/ml, showed similar results.

0

1

3 TIME

:DAY

4

S)

Fig. 5 Growth of P fluorescens in different modifications of Intralipid”’ when stored at 8°C for 4 days. The modifications were: V Intralipid”, 20°: pH 8, 0 IntralipidR 20°i, with trace elements, pH 7.2 n IntralipidX 20% pH 4.7 and 0 IntralipicP 20”; with trace elements, pH 4.3

DISCUSSION

0

1

2 TlME

3

4

(DAYS)

Fig. 4 Growth of P fluorescens in different solutions when stored at 8°C for 4 days. The different solutions were: V SCDM, R AIO-mixture, 0 AIO-mixture pH 7.5, ill AIO-mixture without trace elements and phosphate and 0 AIO-mixtures without trace elements

The risks for growth of microorganisms in TPN mixtures and in single IV solutions when held at room temperature have been well documented. Intravenous fat emulsion alone (or in TPN regimens) can both support growth and prevent the detection of it due to the inherent turbidity of emulsions. The results of the published growth studies at room temperature, especially point out the risk for multiplication of Gram negative contaminants. A maximum time limit of 12 h between start and complete finish of an intravenous fat infusion, which is recommended in the CDC Guidelines for Prevention of Intravascular Infections [4], is well motivated and is supported by the published studies [9, lo]. Other intravenous fluids have a corresponding recommended time limitation of 24 h. Regarding the AI0 systems, where amino acids, fat, glucose and various additives are premixed in a 3L bag, the time limitations of 12 or 24 h become impractical.

CLINICAL

To overcome this, the bag should be refrigerated immediately after mixing. The cooling down time can vary depending on the individual refrigeration conditions but normally the time to reach SC is less than 5h[ll]. The results from our initial study show that the chosen mesophilic contaminants stay at their starting concentration. This is most likely an effect of the temperature drop, and the following low temperature during storage. The findings correspond to published results, e.g., Jarvis [7] who found that the rapid growth at 25 C in lipid emulsion by Enterobacter cloacae and by two serotypes of Klebsiella pneumoniae was completely suppressed when incubated at f5’C. We found that this state of constant level is maintained for at least 96 h. Psycrophilic contaminants would be a potential risk during prolonged refrigeration storage. Their presence in an IV-fluid related infection was reported by McKee and colleagues [ 121 who isolated Pseudomonas maltophilia from a blood sample, as well as from the IV bottle. The fluid, fat emulsion, had previously been dispensed from a 500ml bottle to a 100ml bottle in the hospital pharmacy. When seeding AI0 solution with psycrophilic Pseudomonas fhtorescens it was found that growth was inhibited during the whole of the test period, 96 h. In the bacterial culture medium, SCDM, rapid growth took place, and also in two IV-solutions which were a part of the AIO-mixture. Of these two, the fat emulsion, Intralipid” 20”,, showed rapid growth and Vamin” Glucose moderate growth. The reason for this inhibition which was verified by repeat tests, and also by testing with four other psycrophilic strains, has not been fully explained. Complementary tests were made where different factors in the AIO-mixture were changed and it was found that the comparatively low pH is in part responsible. If the pH was raised from pH 5.4, the normal level, pH7.5, moderate growth took place. This was also the case if the trace element additive, Addamel”, and/or the phosphate buffer was excluded. This did not increase the pH, so the presence of the trace elements seemingly inhibits growth of Pseudomonas fluorescens. When simulating these changes in Intralipid” 20”,, we did not see any complete growth suppression. The low pH partly suppressed growth, whereas pH8 allowed rapid growth to occur. A complementary suppressing effect of trace elements was found at low pH, none at all at pH 7.2. It is known that Pseudomonas sp is inhibited by a low pH [8, 13, 161. Also the trace elements like Zn”, CtP*, MrP+, Fez+ and Fe3 seem to play a role when growth of Pseudomenus jhorescens is suppressed, in AI0 and to some extent in the Intralipid” mixture. In the tested AI0 mixture the concentration of Zn’+ is 7.9pM, Cu”+ 2.0/1M, MrP- 15.7,uM and Fe2+/FeS+ is 19.7pM.

NUTRITION

29

Picket and Dean [ 141 reported inhibition of Pseudomonas fluorescens at a zinc concentration starting at 15 PM and with full inhibition at 500pM. An additive effect between CU*+ and Zn*+ was demonstrated by Albright and colleagues [ 151. Other metal combinations (Cd + Zn) have given synergistic inhibitory effect on Pseudomonas sp [14]. Cd is absent in the AIO-mixture, but other combinations like Fe2+/Fe3* or Cu’+ may show a similar effect. Coliform bacteria can be significantly injured by as little as 2 PM Cu [16]. A counter acting component in the trace element solution (EDTA) may reduce the bacteriostatic effect by forming chelates with the metallic ions [ 161. The results of the study implicate that with prompt refrigeration after mixing, the AIO-mixture can be stored for at least 96 h without risk for growth of common contaminants. The investigated AIO-mixture was shown to have intrinsic growth suppressing properties, towards a variety of Gram negative psycrophilic organisms during refrigeration. The nature of this factor seems to be low temperature, low pH and presence of trace elements. REFERENCES

ill Solassol C L, Joyeux H, Etco L et al 1974 New

techniques for long term intravenous feeding. Annals of Surgery 179: 519-522. PI Holmes C J, AllwoodM C 1979 The growth of microorganisms in parenteral nutrition solutions containing amino acids and sugars. International Journal of Pharmaceutics 2: 325-335 [31 Maki D G 1976 Growth properties of micro-organisms in infusion fluid and methods of detection. In: Philips J, Meers P D, D’Arcy P F (ed) Microbiological Hazards of Infusion Therapy. MTP Press Ltd 13-47 [41 Simmons B P, Hooton T M. Wone E S et al 1982 Guidelines for prevention intravascularinfections. Centers for Disease Control. Guidelines for the prevention and control of nosocomial infections. NITA 5: 40-46 T, Allwood M C 1979 Further studies on [51 Duffet-Smith the growth of micro-organisms in total parenteral nutrition solutions. Journal of Clinical Pharmacy 4: 219-225 [61 Hill D, Tredhee R 1984 The effects of freezing and thawing on micro-organisms in parenteral nutrition solutions. British Journal of Parenteral Therapy 136-140 A K 1984 Bacterial growth and [71 Jarvis W R, Highsmith endotoxin production in lipid emulsion. Journal of Clinical Microbiology 17-20 181 1974 In: Buchanan R E, Gibbons N E (eds). Bergey’s Manual of Determinative Bacterilogy, Eighth Edition. The Williams and Wilkins Company/Baltimore 217-243 D, Mayhall C G, Hall G 0, Pesko L J, [Ql Kaemmerer Thomas R B 1983 Microbial growth patterns in intravenous fat emulsions. American Journal of Hospital Pharmacy 40: 1650-1653 JlOl Cracker K S, Noga R, Filibeck D J et al 1984 Microbial growth comparison of five commercial parenteral lipid

of

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BACTERIAL GROWTH PROPERTIES IN REFRIGERATED ALL-IN-ONE

emulsions. Journal of Parenteral and Enteral Nutrition 8: 391-395 [I l] Lundmark T 1985 Personal communication. [12] McKee K T, Melly M A, Greene H L, Schaffner W 1979 Gram-Negative Bacillary Sepsis Associated with use of Lipid Emulsion in Parenteral Nutrition. American Journal of Diseases in Children 133: 649-650 [ 131 Dainty R H, Shaw B G, Harding C D, Michanie S 1979 The spoilage of Vacuum-packed Beef by Cold Tolerant Bacteria. In: Russel A D, Fuller R (eds) Cold Tolerant Microbes in Spoilage and the Environment. Academic Press, London 83-100

TPN MIXTURES

[14] Pickett A W, Dean A C R 1979 Cadmium and Zinc sensitivity and tolerance in Bacillus subtilis subsp niger and in Pseudomonas sp Microbios 24: 51-64 [15] Albright L J, Wilsson E M 1974 Sub-lethal effects of several metallic salts-organic compounds combinations upon the heterotrophic microflora of a natural water. Water Research 8: 101-105 [16] Domek M J, Le Chevallier M W, Cameron S C, McFeters GA 1984 Evidence for the Role of Copper in the Injury Process of Coliform Bacteria in Drinking Water. Applied and Environmental Microbiology 48: 289-293

Submission date: 25 January 1986. Accepted after revision: 20 June 1986