Blood cultures evaluation by gas chromatography of volatile fatty acids

Transcription

1 Med Sci Monit, ; 6(): 65-6 Blood cultures evaluation by gas chromatography of volatile fatty acids Jaroslav Julák, Eva Stránská, Eva Procházková-Francisci, Vlasta Rosová st Medical Faculty, Charles University, Praha, Czech Republic Faculty Teaching Hospital at Královské Vinohrady, Praha, Czech Republic General Faculty Hospital, Praha, Czech Republic key words: BacT/Alert, bacteremia, blood culture, gas chromatography, sepsis, volatile fatty acids SUMMARY The volatile fatty acid profiles were determined by a simple gas chromatographic method in 75 microbiologically positive and negative blood cultures. Aerobic bacteria yielded profiles with low content of acids, some of which were chromatographically negative. Anaerobic bacteria produced more acids and more distinctive profiles. The method makes it possible to confirm the microbiological findings and to select the blood cultures containing anaerobic bacteria within minutes. This study was supported by grant /97 from the Grant Agency of Charles University, and by a gift from the Jistota Foundation, Komerãní Banka Praha. We are also indebted to Dr. Hrouda from Organon Teknika Co. for valuable advice and material support. INTRODUCTION Blood cultures are applied to diagnose human sepsis, bacteremia, or fungemia, i. e. the invasion of microorganisms into the blood stream. To accomplish the screening procedure, the examined blood is added to the cultivation medium and incubated. The selection of the positive cultures is performed by a number of automated systems based on the colorimetric, fluorimetric, electrochemical or other detection of growth [,], rather than the former visual examination of culture bottles. The blood culture systems were also used for bacteriological examination of other body fluids []. If microbial growth is detected, the micro-organisms are isolated and their identity established by determination of their phenotypic features. A number of analytical methods may also be used for detection and identification of bacteria [4]. Among them, gas chromatographic determination of volatile (VFA) and non-volatile (NVFA) carboxylic acids profiles, i.e. the pattern of the fermentative metabolism end products in spent media, is of great importance in identification of pure cultures of anaerobic bacteria [5], in lesser extent of facultative anaerobic ones [6,7]. These profiles are in defined conditions more or less characteristic of bacterial species. Unfortunately, they are also more or less dependent on the composition of the used cultivation medium, and the straight analysis of clinical body fluids which are a poorly defined 'medium' is thus somewhat limited. Nevertheless, the analyses of blood cultures [8 ] and other fluids [] were previously reported. In this study, we attempted to verify the simple and rapid chromatographic determination of VFA in blood cultures, which may improve the detection of anaerobic infections, life-threatening but often not detected by routine microbiological examination [4,5]. The complementary determination of NVFA including lactic acid, necessary for exact identifica- Received: Accepted:..8 Correspondence address: Jaroslav Julák MD PhD, Department of Medical Microbiology, st Medical Faculty, Charles University, Studniãkova 7, 8 Praha, Czech Republic 65

2 tion of bacteria, was omitted in this study for benefit of simplicity and speed of the test. Preliminary results of this research are also given [6]. MATERIAL AND METHODS Cultivation media The home-made media were prepared from Brain Heart Infusion Broth (Oxoid, Basingstoke, UK) supplemented with saccharose or glucose, and poured into ml bottles. The positive growth was observed visually as a light opalescence. We made use of the automated BacT/Alert (Organon Teknika, Durham, NC, USA) with colorimetric detection of bacterial growth as well. The BacT/Alert bottles are filled with digest of casein and soybean meal supplemented with sodium polyanetholsulfonate and complex of amino acids and carbohydrates. The BacT/Alert FAN bottles contain supplemented Brain Heart Infusion Broth, charcoal is added to suppress the inhibition of growth by antibiotics in the blood samples. Both types of BacT/Alert bottles are delivered in aerobic and anaerobic versions: the first ones containing pyridoxine and oxygen, the second ones menadione, hemin, and a mixture of nitrogen and carbon dioxide as the atmosphere over the filling. Blood cultures In preliminary experiments, bottles were inoculated with the suspensions of pure bacterial cultures, ml amounts of sterile human blood were added, and samples were aseptically taken after 6, 4, 48 hours and 5 days of cultivation at 7 C. Randomly selected positive and negative cultures taken from patients with suspected bacteremia were analyzed as the clinical samples. Their cultivation at 7 C was terminated with first signs of growth in the home-made cultures and after a positive signal in BacT/Alert system, respectively, and a routine microbiological examination took place in the microbiological departments of General Faculty Hospital and Faculty Teaching Hospital at Královské Vinohrady (Praha, Czech Republic). The microbiological findings of cultures with unusual VFA profile were confirmed by further thorough examination by microscopy, aerobic and anaerobic cultivation. Extraction ml samples of blood culture were drawn aseptically into screw-capped test tubes, acidified with H PO 4, and ml of 5mM -methylhexanoic acid solution (Fluka, Buchs, Switzerland) was added as an internal standard (I. S.). VFA were extracted into 5 ml of tert-butyl methyl ether (Sigma-Aldrich, Steinheim, Germany), tubes vortexed and phases separated by brief centrifugation (5 min at g). ml of the organic phase was injected into gas chromatograph. Gas chromatography A Chrom 6 gas chromatograph (Laboratorní pfiístroje, Praha, Czech Republic) with a FID and a NUKOL column ( m x mm I.D.,.5 mm film thickness; Supelco, Bellefonte, PA, USA) was used. The injector temperature was 5 C, the oven and the detector were maintained at 6 C. The split injection :5, nitrogen as carrier gas (flow-rate.6 ml min ) and make-up gas (flow-rate 5 ml min ) were used. The flow-rates of hydrogen and air were and ml min, respectively. VFA were identified by comparing their t' R with those of Volatile Acid Standard Mix (Supelco, Bellefonte, PA, USA). The analyses were terminated after elution of I.S. at t' R = 9. min. Evaluation Peak heights were measured and amounts of VFAs expressed as the ratio to the height of I.S. peak. The background of VFA was determined for each blood culture system by analyzing pure media and negative cultures, and the maximum values for particular acids were subtracted from those found in positive cultures. For the BacT/Alert FAN bottles, the background must have been established for each batch. Particular acids are further abbreviated as follows: acetic A, propionic P, isobutyric IB, butyric B, isovaleric IV, valeric V, isocaproic IC, caproic C. RESULTS The analyses of pure media and microbiologically negative cultures showed small contents of VFA in home-made and BacT/Alert bottles (up to. I.S.). In BacT/Alert FAN bottles, the VFA content was higher and varied considerably among batches (mostly up to.5, but in some batches up to times of I.S.). The analyses of artificially infected blood cultures showed that: a) VFA appeared in some strains after 6 hours, in all chromatographically positive strains after 4 hours of cultivation; b) their content raised 66

3 Julák J et al Blood cultures evaluation by gas chromatography Table. Volatile fatty acid profiles of blood cultures with reference bacterial strains. Content of acids Bacterial species A P IB B IV V IC C Bacillus coagulans Bacillus lentus Bacillus megaterium Bacillus subtilis Bacteroides fragilis Clostridium bifermentans Clostridium tetani Escherichia coli Enterobacter aerogenes Enterococcus faecalis Haemophilus influenzae Klebsiella pneumoniae Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Staphylococcus hominis Staphylococcus warneri Streptococcus anginosus Streptococcus bovis Streptococcus pneumoniae Streptococcus mutans Streptococcus sanguis Veillonella parvula () () () () () () () () () The content of acids is expressed as follows: : peak height / I.S. under ; : peak height / I.S. to ; : peak height / I.S. to ; ( ): depends on cultivation mode. in most cases no longer than 48 hours; c) their quantitative content slightly differed in repeated cultivation of the same strain, not affecting the overall profile; d) no differences were observed between aerobic and anaerobic bottles. The results, given in Table, are expressed semiquantitatively due to the cultivation way and time dependence and the quantitative differences among strains in content of particular acids. Except Ps. aeruginosa, all listed species produce A; in lesser extent, additional P occurred at Gram-negative rods; additional IV at Gram-positive bacilli and at most strains of staphylococci; both P and IV occurred at Enterobacter. Anaerobic Bacteroides and Clostridium displayed rich profiles of higher VFA, depending on the way of cultivation; e.g. for Cl. tetani, IC was found as 5 fold of I.S. in Bact/Alert bottles, but was absent in home-made media. All strains of Acinetobacter calcoaceticus and A. lwoffii and out of strains of Ps. aeruginosa were chromatographically negative. From the total of 75 analyzed clinical blood cultures, 88 were microbiologically positive and 87 negative. The chromatographically positive cultures containing a single bacterial species are summarized in Table. Another 7 cultures contained or more species with superposition of VFA profiles. No production of VFA displayed 9 strains of Candida sp. and most strains of Acinetobacter sp, Corynebacterium jeikeium and Pseudomonas aeruginosa, where occasional incidence of minute amounts of A (under.9 I.S.) was observed. The 8 samples detected as positive by the BacT/Alert system were found to be microbiologically and chromatographically negative. The Stenotrophomonas profile should be verified by further explorations. The VFA profiles of 4 cultures were considered as not corresponding to those given above; examples are given in Table. Their further microbiological examination yielded additional, mostly minority and anaerobic bacteria; unfortunately, we had lim- 67

4 Table. Volatile fatty acid profiles of clinical blood cultures containing a single bacterial species. Bacterial species N o of strains Content of acids A P IB B IV V IC C Acinetobacter baumannii Acinetobacter calcoaceticus Citrobacter freundii Corynebacterium jeikeium Corynebact. pseudodiptheriticum Corynebacterium sp. Corynebacterium xerosis Escherichia coli Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis Enterococcus faecium Klebsiella oxytoca Klebsiella ozenae Klebsiella pneumoniae Peptostreptococcus sp. Proteus mirabilis Pseudomonas aeruginosa Salmonella enteritidis Serratia marcescens Staphylococcus hominis Stenotrophomonas maltophilia Streptococcus agalactiae Streptococcus faecalis Streptococcus millerii Streptococcus mitis Streptococcus viridans () () () () () () () () The content of acids is expressed as follows: : peak height / I.S. under ; : peak height / I.S. to ; : peak height / I.S. to ; : peak height / I.S. over ; ( ): occasional occurrence ited possibility to establish their identity more precisely. The original microbiological finding was not confirmed in cases. DISCUSSION The determination of VFA profile of blood culture can divide aerobic bacteria into the following groups: a) bacteria displaying none or scarce production of A; b) heterogeneous group of bacteria producing always A; c) Gram-negative rods producing A and P; d) staphylococci and bacilli producing A an usually IV as well; e) enterococci with A, P, and IV. As a rule, A was a dominant component. More detailed differentiation of aerobes is not possible, nevertheless, these findings may be an useful adjunct to their identification. Anaerobic bacteria produce larger amounts of the above mentioned acids and at least one of the higher ones. Hence, the presence of IB, B, V, IC or C indicates the presence of anaerobes; the determination of NVFA and/or more detailed study of the medium composition influence on the VFA profiles should be necessary for their more detailed characterization. The dominant P seems to be indicative of propionibacteria. This makes it possible to discover the anaerobes in such samples where they were not found, either due to their minute amounts, or their slow growth preceded by the faster growth of aerobes and the premature termi- 68

5 Julák J et al Blood cultures evaluation by gas chromatography Table. Selected blood cultures with unusual volatile fatty acid profiles. Bacterial species found by routine examination Corrected ratio peak height / I.S. A P IB B IV V IC C Acinetobacter baumannii Candida sp. Corynebacterium jeikeium Corynebact. pseudodiptheriticum Enterococcus faecalis Klebsiella pneumoniae Streptococcus mitis Streptococcus mitis The content of acids is expressed as the ratio peak height / I.S., corrected to the background of negative cultures. The unexpected values are printed in bold and underlined. G, G-: Gram-positive, Gram-negative. Other bacteria found by additional examination anaer. G-coccobacilli (Bacteroides?), anaer. G cocci, solitary anaer. G cocci by microscopy Other anaer. G cocci anaer. G- rods, Peptostreptococcus sp. fusiform G- rods, Bacteroides sp. anaer. G rods anaer. G cocci (Peptostreptococcus?) polymorph anaer. G rods and cocci and microscopy anaer. G cocci (Peptostreptococcus?) anaer. G rods, G cocci chains of anaer. G cocci no Staph., anaer. G-rods by microscopy Klebsiella oxytoca anaer. G rods unidentified anaerobe anaer. G-cocci, G rods unidentified anaerobe nation of primary cultivation, or by routine aerobic subsequent cultivation only. In this study, the gas chromatography corrected the prior microbiological findings in. % of all analyzed samples. This is in good agreement with previous results [], where the high VFA content and subsequently anaerobes were found in.6 % of analyzed peritoneal effluent samples examined by a blood culture system. CONCLUSIONS Bacteria producing volatile fatty acids can be detected in blood cultures by gas chromatography. Among aerobic ones, Gram-negative rods, staphylococci, bacilli and enterococci may be distinguished. The abundance of lower acids and/or detection of IB, B, V, IC or C indicate the presence of anaerobes. Propionibacteria can be distinguished by dominant P. Gas chromatography detected anaerobes omitted by routine microbiological examination in % of examined blood cultures. REFERENCES:. Weinstein MP: Current blood culture methods and systems: Clinical concepts, technology, and interpretation of results. Clin Infect Dis, 996; : Reimer LG, Wilson ML, Weinstein MP: Update on detection of bacteremia and fungemia. Clin Microbiol Rev, 997; : Bourbeau P, Riley J, Heiter BJ et al: Use of the BacT/Alert blood culture system for culture of sterile body fluids other than blood. J Clin Microbiol, 998; 6: Julák J: Identification of bacteria by instrumental chemical analysis (In Czech). Praha: Karolinum, 998: Holdeman LV, Cato EP, Moore WEC et al: Anaerobe Laboratory Manual. Blacksburg: Virginia Polytechnic Institute and State University, Kiechle FL, Starnes RV, Colville JM: Production of lactate by aerobic bacteria. Clin Chem, 98; 8:

6 7. Salverson A, Bergan T: Enterobacteria differentiated by gas-liquid chromatography of metabolites. Zbl Bakt Hyg, 98; A5: 4-8. Wüst J: Presumptive diagnosis of anaerobic bacteremia by gas-liquid chromatography of blood cultures. J Clin Microbiol, 977; 6: Sondag JE, Ali M, Murray PR: Rapid presumptive identification of anaerobes in blood cultures by gas-liquid chromatography. J Clin Microbiol, 98; : Edson RS, Rosenblatt JE, Washington JA, Stewart JB: Gas-liquid chromatography of positive blood cultures for rapid presumptive diagnosis of anaerobic bacteremia. J Clin Microbiol, 98; 5: Larsson L, Mærdh P-A, Odham G, Carlsson M-L: Diagnosis of bacteremia by automated head-space capillary gas chromatography. J Clin Pathol, 98; 5: Goddard WW: Head-space gas chromatography as a potential means of detecting and identifying bacteria in blood cultures. Med Lab Sci, 984; 4: 9. Catchpole CR, Macrae F, Brown JD et al: Use of prototype automated blood culture system and gas-liquid chromatography for the analysis of continuous ambulatory peritoneal dialysis associated infection. J Clin Pathol, 997; 5: Rosenblatt JE: Can we afford to do anaerobic cultures and identification? A positive point of view. Clin Infect Dis, 997; 5(Suppl. ): S7-S 5. Salonen JH, Eerola E, Meurman O: Clinical significance and outcome of anaerobic bacteremia. Clin Infect Dis, 998; 6: Julák J, Rosová V, Procházková-Francisci E, Stránská E: Evaluation of blood cultures by volatile fatty acids determination (In Czech). Klin mikrobiol inf lék, in press 6