Software of the Month Club 1997 April

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║ -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= ║ ║ Welcome to ║ ║ Bacterial Identification Program (B.I.P.) ║ ║ first version (2.0) for DOS ║ ║ ──────────────────── ║ ║ 1996, Murat AYDIN ║ ║ ──────────────────── ║ ║ =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= ║ BIP helps to correct and faster identification of an unknown microorganism. Usually, a microbiologist is able to use this program. Mostly, each menu of BIP contains a self-explanatory words or messages. However this file (README.TXT) can give more descriptive informations and some details about the BIP. This program does not entail windows, mouse, special card, color monitor, external device or any other. It was planned that this program must serve to scientific purposes. What changes in this version? Water contaminants were added, also minor changes were made. Installation of BIP : 1. Create a directory preferable in a harddisk, although, BIP can run in a floppy diskette too, but slower than that HDD. 2. Decompress the original file into a directory with PKUNZIP. Those files must be present : BIP.EXE README.TXT <- this file BAK.BAK INDEX IDENT EDITOR.COM SINIR OKUMA.COM SORT.EXE <- YOU will put this file 3. Copy the current version of SORT.EXE file into this directory 4. Run the BIP.EXE Introduction: To identify a microorganism with genetically, is a safety method. It bases on stationary molecular architecture of bacterial cell instead of transient adaptational motives. However, It takes much more time, effort, instrument and money than that phenotypic identification procedures. Usually, there is no time for both physician and patient, particularly while any hard infection threatening in life. To make FASTER identification of a pathogenic microorganism is necessary for early control of infection. For make a bacterial identification with BIP; a pure and young culture of Suscepted Bacterial Sample (SBS) is prepared. Some physiologic and biochemical tests (see Appendix-A) are performed on the SBS. For make TRUE identification, the tests must be a number of as much as possible . Although, tests can be distinguished as simple and fast resulted ones according to the individual laboratory conditions. Then, the test results are given to the BIP with type on keyboard. BIP will prepare a list of bacteria which are possible. At this point, user can decide which bacterial specimen is pathogenic, or he/she can request to make a further identification from the BIP. In result, BIP gives a report from screen and/or printer about identity of the SBS. Noticeable that, this program can be used for educational purposes too, However, I had not directly aimed to this, when I wrote this program. (for instance, to find some bacteria which are catalase positive and oxidase negative rods, or to find which bacteria are lactose negative but urease positive?). BIP contains those: 1. ENCYCLOPEDIA: It consists of some encyclopedic informations ════════════════ about a selected microorganism. Why necessary an encyclopedic data for any bacterial specimen ?: Color, niff, bulk and shape of bacterial colonies are useful clues of its identity. These observations and other subjective inspections could not be easy translated to a computer language. On the other hand, some times, these informations are highly descriptive for make a bacterial identification. For this reason, this menu must be ready while the program running. There are 2 sub-menus: 1. Read : All bacteria will be alphabetically listed in ───────── a window. User selects one bacterial specimen and presses <enter>. All encyclopedic data (if was input before by user!) will be shown on the screen, user can print these with use of <P> key. These encyclopedic data can be called through two sections of the BIP: i) the 'encyclopedia' menu, ii) the identification menu (see `further identification'). ■ What must be present in encyclopedic file of bacteria: There is never an obligation!. Contents of these files are COMPLETELY individual. Some microbiologists prefer the bellowing examples: ----------------- There is some greenish on Endo agar, but not on blood agar It is smelling as lucky flower Colonies are reflecting the sun-light and they are slimy Colonies are giving fluorescence under U.V. but in first 1/2 hour Don't believe the catalase test, because last four were catalase variable Colonies are looking like Actinobacillus suis, but some times Staph. If this found, give a message to Dr......., he is interesting with this For confirmation of this specimen, make latex fixation, but careful that some species can give false positive cross-reaction with of P. penneri If this found, Carbenicillin can be more potent! etc. ------------------ Each of encyclopedic files of bacteria may not be present in the original .ZIP file, because every user may desire to use an individual text file(s). More important that, any different language can be used in the individual text file (e.g., french, finnish, german, spanish..). The name of encyclopedic file is specific to that bacterial specimen. This is a number between 1 to 434 (it is possible that, this range will enlarge in future versions of BIP). When user needs to manually write an encyclopedic data file instead of through the BIP, he/she must learn the number of relevant bacteria from UTILITY menu (see below). User can write a file with any ASCII editor program under DOS and then, the file must be saved into the current directory as to be format in <number>.BAK. Extension of the file must be .BAK word. (e.g., 1.BAK, 2. BAK, 3.BAK,..... 430.BAK, 431.BAK). 2. Edit: This is used when edit an encyclopedic file of ──────── a selected bacterial specimen. All bacteria will be alphabetically listed in a window when this option was activated. User selects one and presses <enter>. So that, the ASCII editor program which is present in the original .ZIP file will be run by BIP. The current file name will be automatically adjusted to number of current bacterial specimen by BIP. During the editor program is running, user may press <F1> for learn the specific comments of the editor program (Editor V1.3B, Venetek). Any ASCII editor program can be replaced in the directory instead of this editor program (but with rename of OKUMA.<com> or OKUMA.<exe>). 2. UTILITY: There are 2 sub-menus: ═══════════ 1. Add new one: Name of a new bacterial specimen which will ─────────────── be added to BIP, is asked to the user. The name must be input by the user, then, whether or not it is already exist will be checked by BIP. If it is already exist, BIP will alert the user and re-run, otherwise the name will be recorded into the BIP. End of this operation, user must immediately input phenotypic profile of that bacterial specimen. (see next sub-menu). There is not an upper limit for input new bacteria to BIP. Total number of bacteria present are 440 in current Ver 2.0. However, they can be enlarged if any user input new bacterial specimen(s). The program is not static. It is dynamic and very flexible. 2. Change old one: In the original file of this version, ────────────────── there are 62 of standard physiologic and biochemical test responses of 440 clinical significant bacteria. Unfortunately, this test pattern, test names, means and their sequences are unchangeable. Test results of each bacterial specimens which are currently recorded into BIP, were taken from the safety sources (see references). However, user may need to add (or change) a test RESULT which was NOT EXIST. Because, there are not satisfactory informations in literatures about some bacteria and also some particular tests. If user needs to change or re-input any test result for one of bacteria, he/she marks the bacterial specimen from 'change old one' sub-menu, a specific number(*) for that specimen appears in bottom line of screen and another window seems for input (or change) new test results. (* : use this number for manually edit to the encyclopedic file of that bacterial specimen). User input the test results specific for that bacterial specimen through this window. Some line(s) for unknown test result(s) of the new bacterial specimen may be left as blank by user. These lines may be or may not be input in future. For 'change old one' option, those keys are acceptable: <+> character as a positive test result (100%), <-> character as a negative test result (0%), <*> character as an undecided test result (50%), <space> for unknown test results or <ent> for input a numeric data. As you see, user is free input a numerical data. For instance, DNase activity of Fusobacterium naviforme is 44. This is mean, 44% of species of this bacterial specimen have a DNase activity. User presses <ent>, while the bar is place on the «DNase» test line, a new window will be open for input a numeric data, user types 44 and presses <ent>, this value will be accepted to be result of DNase test for F. naviforme. Also, this number will be showed to the user at upper-right corner of the window while the highlighted bar is on the DNase test line. However, in the `identification menu', it will never be permitted to input any numeric data. (See below) 3. IDENTIFICATION: For make a bacterial identification, this menu ══════════════════ must be activated. Two windows will be opened. At right, identification window (IW), it consists of test pattern, a high-lighted bar stops on the first test (gram stain). At left, there is also a window, possible bacteria window (PBW), but it is empty yet. User types the test results in the IW. It is not prerequisite to perform and input all of tests. User may leave some lines as blank if the test was not treated on the SBS despite to fact that test pattern of BIP contains 62 of applicable laboratory trials. In this section, only those keys are welcome; <+> key as a positive test result (100%), <-> key as a negative test result (0%), <*> key as an undecided test result (50%) or <space> key for unperformed tests. However, user CAN NOT TYPE ANY NUMERIC DATA in this section. This is because, A TEST RESULT CAN BE ONLY POSITIVE or NEGATIVE !, rarely, a test result can be undecidable. Hence, any gradual value can not be prompted to a test. Then, user presses <F2> key. This serves to describe a cut-off value. A number from 1 to 99 is acceptable, To describe a threshold (cut-off): ***********************. User can describe a cut-off value between 1 and 99 for preparing the list of suscepted bacteria. When the cut-off value was described as to be 80 (defaultly), BIP will assort the possible bacteria which their similarity percentage is equal or greater than 80%. When a higher value was chosen as a cut-off by the user, for instance 90, BIP will give lesser number of bacteria but more similar to the SBS. So that, current identification will continue in a selectable magnitude of perspective by user. This specificity is not present in many of similar softwares and computer supported microbiologic devices. After a threshold was described, user presses <enter>. BIP will prepare a list of possible bacteria in the PBW. A bacterial specimen can be distinguished from the PBW as a pathogenic microorganism by microbiologist. User gives a report to clinician. In this step, user may request to make a further identification if he/she could not decide yet. Further identification: ********************** Caution: For this section is shareware!. For further identification, user selects a bacterial specimen in the PBW with the high-lighted bar, then, user invites F1 function. BIP calculates their taxometric distances between selected bacterial specimen and the others. (At this point, user can reach the encyclopedic menu of a selected bacterial specimen if he/she uses the <ent> key instead of <F1> key). It is advisable that, before a further identification request, the lowest possible bacterial specimen(s) (if any) must be removed from the list of possible bacteria (in the PBW). (see to remove a bacterial specimen). BIP will design a report from screen or/and printer. This report includes those: i) Which test(s) is more specific in order to make an exact separation of the SBS, all tests were checked, one by one, and tests which will be particular identic, were advised to the user. ii) What is identification power of each of advised tests, iii) In final, gives a list for all tests (not only advised tests) were assorted according to their identification parameters from biggest to less. User must choice one or more of tests which was advised by BIP. And, he/she must perform them in his/her own laboratory to the SBS. (These specificities are not present too in many of similar softwares and computer supported microbiologic devices.) So that, indispensebality and spontaneously, the user will establish a true laboratory strategy for true identification. Further more, lavishness time, chemical substances and also money can be prevented by this way. During the calculation of relationships between bacteria, BIP uses hundreds or thousands mathematical operations. For example: (where ; n = number of tests (1..62); deney, result of the test (0..100); OTU, Operational Taxonomic Unit (0..100); r(), test result of suscepted bacterial specimen; rr(), test result of converse one; t, total similarity of the both; d, taxonomic distance of two bacteria (0..1).) if r(n) and rr(n) then deney = deney + 1: t = t + 100 - ABS (r(n) - rr(n)) OTU = Int (t * deney^-1) end if d^2 = 1 - (OTU x 10^2) ('d' parameter is calculated by the BIP for assortment of bacteria, but not shown on the screen, it is unnecessary for user) To save the data: ****************** End of an identification, user presses <esc> key and gives a protocol number. This number will be asked by BIP when necessary. 'Only' numeric data is acceptable as a file name. DON'T INPUT any patient-name!. The current information of the SBS will be saved in a file (as to be a very specific format). The file name will be recorded as to be <number>.HST. If user wants to save a data in an already exist file, he/she must type <@:> before the protocol number (e.g., @:2381). To load a data: ****************** For loading an old data in order to make some retrospective analyses, user comes to 'identification' menu, and uses <F1> key. BIP asks a protocol number for load it, the number will be input. These informations should be used for statistical purposes. However, there is not a statistical menu in this version of BIP. To remove a bacterial specimen from suscepted list: This operation can apply during in the `identification menu'. This is never completely delete a bacterial specimen from the BIP, but it deletes only its name from the list of possible bacteria. For make this, the high-lighted bar is transferred to the PBW with use of <tab> key. The bar is placed on lowest possibility bacterial specimen (if any) with use of cursor-movement keys. And it can be removed from the list by use of <del> key. Why necessary to delete a bacterial specimen from the list?: Some times, bacterial specimens may have `phenon'. For instance, the both, Streptococcus faecalis and Mistuokella multiacidus responses the tests of fermentations of glucose, sucrose, lactose and mannose as to be positive. However these are highly different bacteria. S. faecalis is facultative, Gram-positive, short cocci chain. The second is strictly anaerobic, Gram-negative and it has a rod shape. A microbiologist is able to easy understand which strain is wrong. Although, BIP includes the both bacteria into the same list. This is normal, if user did not input the results of those 3 tests; 'gram stain', 'anaerobic' and 'coccus'. Already, BIP will exactly advise to perform of above three tests for further identification under these conditions. For this reason, user easy decides that one or more bacterial specimen(s) which are listed, must be deleted from the list of possible bacterial specimens. 4. EXIT TO DOS: User leaves to BIP. AN EXAMPLE SESSION FOR BACTERIAL IDENTIFICATION: -------------------------------------------------- Suppose that, a material was taken from throat of a boy who is 8 ages old. White membranes are present on tonsillar tissue tend to spread over oro-pharynx. A fever in 40-42 °C is fluctuating since 2 days. Non-sporing, non-motile, gram positive rods were dominant in direct stain of fresh material. The material was inoculated to BHI and blood agar, incubated aerobically. Usually, after 18 hours, bacterial colonies appear. One type of colony was apparently dominant on the blood agar plate. These are colonies of pathogen microorganism. They were hemolytic and gram positive rods. One loopful material was taken from that colony, inoculated in BHI broth in order to perform tests. While incubation period, early informs can be given to the BIP. Early clues which we have about this specimen were given to the BIP. (hemolysis, +; coccus, -; spore, -; motility,-; anaerobic, ±; gram stain, +). One-hundred-eight of possible bacteria were listed in the PBW by the BIP. These were removed from the list possible bacteria (PBW): Three of Eubacterium, five of Bifidobacterium, eight of Actinomyces, two of Propinobacterium, seven of Peptostreptococcus, fourteen of Streptococcus, fifty-two of Lactobacillus, two of Haemophylus and one of Actinobacillus. Because their cell shape, clinic symptoms were missing. Whereby, seven of Corynebacterium and one of Nocardia were remained in the list. In fact, Nocardia should be removed, but some species of Nocardia may seem like a diphtheroid. Corynebacterium diphtheria is a quite forbidding specimen in this bacterial population. The bar was placed on the C. diphtheria, and F1 key was pressed. BIP advised that; it is necessary to perform the fermentation of rhamnose, raffinose, maltose, arabinose, trehalose, sucrose and urease activity, and also NO3->NO2 tests. Each of above tests were performed on young and pure culture of the bacteria. The results were rhamnose, -; raffinose, -; urease, +; NO3->NO2, -; maltose, -; arabinose, -; trehalose, - and sucrose, -. These results were given to the BIP. In first bench, Corynebacterium ulcerans was placed with similarity of 86 OTU, then, Corynebacterium renale (80 OTU), Corynebacterium cystitidis (80 OTU) and some anaerobic bacterial specimens. Corynebacterium ulcerans was reported to clinic together with its antibiotic susceptibility test result. -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Appendix-A: ----------- Standard physiologic and biochemical tests used in BIP and their standardizations: _________________ * Gram stain (koopler's modification for anaerobes), * catalase production (3% for facultatives, 15% for anaerobes), * oxidase, coagulase (with rabbit plasma), * sporulation (with spore stain), * capsule forming (with india ink), * flagellar motility (neither twitching nor other one, only flagellar), * hemolysis (on sheep blood agar), * indole production from tryptophan, * methyl red, * Voges-Proskauer (acetyl-methyl-carbinol production), * citrate utilization (as a carbon source), * acid and gas production from glucose, sucrose, lactose, mannose, * fermentation of mannitol, * hydrogen sulphide production, * urease activity, * anaerobic? (strictly anaerobic, neither microaerophilic nor capnophylic), * dextrose fermentation, * NO3─>NO2 (nitrate reduction), * gelatinase activity, * growth in KCN, (15 ml of KCN 5% at 1 liter), * growth bile, (tolerance of 40% bile), * lipase activity, * glycerol fermentation, * trehalose fermentation, * abide production from maltose, * gas production from maltose, * fermentations of arabinose,raffinose, cellobiose, melibiose, rhamnose, xylose, dulcitol, adonitol, sorbitol, erythritol, salicin, myo-inositol, * coccus, (is the cellular shape a coccus?), * tyrosine pellucidation or melanin production, * α-methyl glycoside, * ornithine decarboxylation, * lysin decarboxylation, * aesculin hydrolysis, (not fermentation), * ß-galactosidase production (ONPG), * phenyl alanine de-amination, * arginine hydrolysis, * DNase activity, (on DNase agar), * fermentation of mucate and malonate, * growth at 42°C, 22°C and 5°C, respectively. The standardization of above tests are according to bellowing references: =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= References: =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Ballows A, Hausler WJ, Herrman KL, Isenbirg HD, Shadomy HJ(ed.). Manual of Clinic Microbiology, 4.th ed., American Society for Microbiology, Washington D.C. 1991. Baltimore, Williams & Wilkins, 1980 Steel KJ. The oxidase reaction as a taxonomic tool. J Gen Microbiol 25 : 297-306, 1961. Barritt MM. The intensification of the Voges-Proskauer reaction by the addition of α-naphthol. J. Pathol Bacteriol 42 : 441-454, 1936 Barry AL et al: Improved 18-hour methyl red test. Appl Microbiol 20 : 866-870, 1970. Barry AL, Feeney KL. Two quick methods for Voges-Proskauer test. Appl Microbiol 15 : 1138-1141, 1967. BBL Manual of Products and Laboratory Procedures, 5th ed,pp 115-138. Cockeysville,MD, BioQuest, 1968 Blazevic DJ, Ederer GM. Principles of Biochemical Tests in Diagnostic Microbiology, pp29-36 New York, John Wiley&Sons, 1975. Carlquist PR. A biochemical test for separating paracolon groups. J Bacteriol 71 : 339-341, 1956. Christensen WB. Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other and from Salmonella and Shigella types. J Bacteriol 52 : 461-466, 1946. Clark WM, Lubs HA. The differentiation of bacteria of the colon aerogenes family by the use of indicators. J Infect Dis 17 160, 1915. Elmer WK, Stephen DA, William MJ, Paul CS, Washington CW (eds). Color atlas and textbook of diagnostic microbiology. 4.th ed, Lippincott JB Company, Philadelphia, 1992. Falkow S. Activity of lysine decarboxylase as an aid in the identification of Salmonellae and Shigellae. Am J Clin Pathol 29 : 598-600 , 1958. Finegold SM, Martin WJ, Scoot EG. Bailey and Scoott's Diagnostic Microbiology, 5th ed, p490. St.Louis, CV Mosby, 1978. Gale EF. The bacterial amino acid decarboxylases. In Nord FF(ed), Advances in Enzymology and Related Subjects of Biochemistry Vol.6, New York, Interscience Publishers, 1946. Gordon J, McLeod JW. The practial application of the direct oxidase reaction in bacteriology. J Pathol Bacteriol 31 : 185-190, 1928. Hendriksen SD. A comparison of the phenylpyruvic acid reaction and urease test in the differentiation of Proteus from other enteric organisms. J Bacteriol 60 : 225-231, 1950. Hendriksen SD, Closs K: The production of phenylpyruvic acid by bacteria. Acta Pathol Microbiol Scand 15 : 101-113, 1938. Isenberg HD, Sundheim LH: Indole reactions in bacteria. J Bacteriol 75 : 682-690, 1958. Koser SA. Utilization of the salts of organic acids by the colon-aerogenes groups. J Bacteriol 8 : 493-520, 1923. Lennette EH, Balows A, Hausler WJ Jr, Shadomy EJ(eds). Manual of Clinical Microbiology, 4th ed. Washington, DC, American Society for Microbiology, 1985. MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria, 2nd ed, pp 78-93. Baltimore, Williams&Wilkins, 1980. Miller JM, Wright JW. Spot indole test: Evaluation of four reagents. J Clin Microbiol 15 : 589-592, 1982. Moeller V. Simplified tests for some amino acid decarboxylases and for the arginine dihydrolase system. Acta Pathol Microbiol Scand 36 : 158-172, 1955. Noel RK, John GH. Bergey's Manual of Systematic Bacteriology. Edited by Barbara Tansill, Vol.1,2,3 1984. Prosser JI. Molecular marker systems for detection of genetically engineered micro-organisms in the environment. Microbiology 140 : 5-17, 1994. Shaw C, Clarke PH. Biochemical classification of Proteus and Providencia cultures. J Gen Microbiol 13 : 155-161, 1955. Stuart CA, Van Stratum E, Rustigian R. Further studies on urease production by Proteus and related organisms. J Bacteriol 49 : 437-444, 1945. Vracko R, Sherris JC. Indole-spot test in bacteriology. Am J Clin Pathol 39 : 429-432, 1963. Voges O Proskauer. Beitrag zur Ernährungsphysiologie und zur Differential-diagnose der Bakterien der hämorrhagischen Septicamia. Z. Hyg 28 : 20-32, 1898. Wallace GI, Neave SL. The nitrite test as applied to bacterial cultures. J Bacteriol 14 : 377-384, 1927. Weaver DK, Lee EKH , Leahy MS. Comparison of reagent impregnated paper strips and conventional methods for identification of Enterobacteriaceae. Am J Clin Pathol 49 : 494-499, 1968. - O - =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= This program can be distributed by free. I hope, BIP will expedite laboratory jobs of microbiologists. All comments and suggestions are welcome. Please send your comments and suggestions to the address below or e-mail them to: =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Murat AYDIN, Ph. D. Dent. Department of Microbiology, Faculty of Medicine, Çukurova Üniversity Adana - TÜRKÿYE E-mail: muratay@pamuk.cc.cu.edu.tr =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=