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GB1601689A - Method of diagnosing bacteremia and apparatus therefor - Google Patents

Method of diagnosing bacteremia and apparatus therefor Download PDF

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Publication number
GB1601689A
GB1601689A GB2986477A GB2986477A GB1601689A GB 1601689 A GB1601689 A GB 1601689A GB 2986477 A GB2986477 A GB 2986477A GB 2986477 A GB2986477 A GB 2986477A GB 1601689 A GB1601689 A GB 1601689A
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changes
curve
significant
inoculum
antibiotic
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MARIEL C
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MARIEL C
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Priority to GB2986477A priority Critical patent/GB1601689A/en
Priority to FR7819530A priority patent/FR2397457A1/en
Publication of GB1601689A publication Critical patent/GB1601689A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18Testing for antimicrobial activity of a material

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Description

(54) METHOD OF DIAGNOSING BACTEREMIA AND APPARATUS THEREFOR (71) I, CLAUDE MARIEL of 3, rue du Bel Air, 92190 Meudon, France, a citizen of France, do hereby declare the invention, for which I pray that a patent may be granted to me and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a method allowing a fast detection of bacteremia through pH change measurements in a culture medium inoculated with the blood to be tested, and permitting through the same pH change measurements an appraisal of the bacteriostatic activity of several antibiotics on the detected pathogen.
At the same time the invention relates to an apparatus for carrying out this method through pH measurements.
The already known methods of bacteremia diagnosis include, in addition to standard bacteriological blood cultures which are time consuming, methods based upon centrifugation techniques (Buffy coat method) with a definite risk of contamination, or using membrane filtration techniques, which happen to be toxic for gram negative bacteria: FARMER S.G. Appl.
Microb. 1972, 23 , 500-504, and which share the same risk of contamination.
A radiometric method called "Bactec" system (JOHNSTON Lab.) based upon the release of radioactive CO2 produced bv bacterial respiration in media containing C14 labelled glucose, amongst other criticism, suffers from the problem of radioactive substance disposal.
A recent method based on impedance change measurements (KAGAN L. et col.
J. Clin. Microbiol. 1977 5, 51-55) suffers from the need of a preliminary lysisfiltration with its contamination hazards.
The method according to the invention is based on the fact that any bacterial growth induces pH changes of the culture medium; therefore: A) Any significant pH change in the medium containing blood for cultivation can be considered as indicating bacteremia; B) Any inhibition of pH change by any antibiotic in an inoculated medium can be considered as indicating antibacterial activity of this antiobiotic on the inoculated bacteria.
The invention provides an apparatus which permits a fast diagnosis of bacteremia and a fast determination of sensitivity of so detected bacteria to several antibiotics, this apparatus being a combination of: - several bottles into which a culture medium and blood may be aseptically introduced, each bottle being fitted with a sterilizable electrode; - each electrode being connected to each way of an electronic switching system; - a pH meter connected to the switching system, and to a multichannel potentiometric recorder and a computer; - the computer receiving a program, being connected to its keyboard and commanding an alarm system.
The invention also provides a method of diagnosing a bacteremia in a patient using the apparatus as defined above, in which a sample of the patient's blood is aseptically added into each bottle fitted with an electrode where it is incubated with a culture medium; the pH values in each bottle are registered at regular intervals, and each pH deviation which is considered significant by the computer is indicated by the alarm system.
The invention further provides a method of determining the sensitivity of bacteria detected with the method as defined above to an antibiotic using the apparatus as defined above, in which an aliquot volume from the content of any bottle where a significant pH change has been detected, is inoculated into each tube containing a conventional broth, and different known con centrations of the antibiotic; the pH values in each tube are registered at regular intervals and significant pH changes are recorded for each tube no significant pH change with time corresponding to an efficient concentration of the antibiotics.
The apparatus according to the invention, is an original combination of several features known per se, which permits an automatic registration, at given intervals, of the pH of culture media, in order to detect the medium where a pH modification indicates bacterial growth.
Figure 1 represents a general scheme of the equipment according to the invention: A series of n bottles (1) (three or more bottles) is provided. Each bottle contains a culture medium and a sterilizable electrode (2) and has been steam sterilized.
Each bottle is connected through the electrode to an electronic switching system (3) with n ways (4). This switching system is connected to a pH meter (5), a multichannel potentiometric recorder (6) and a minicomputer (7). The minicomputer receives a program in (8), is connected to its keyboard (9), and commands an alarm system (10).
The apparatus according to the invention can be used for two purposes: A) to detect bacteremia, and B) with minimal modifications, to determine bacterial sensitivity to several antibiotics.
A. Method of fast detection of bacteremia: Into each bottle containing an electrode and a culture medium (for example brainheart-P.A.S. medium from Institut Merieux) a sample of blood (from the patient in whom a bacteremia is suspected) is aseptically added, and the bottles are aerobically inoculated at 370C with agitation. The electrodes are connected to the electronic switching system and the apparatus is set in operation. The pH values in each bottle are registered at given intervals of time (every 10 minutes for instance) and, if judged necessary, can be graphically registered, due to the potentiometric recorder, for a retrospective control.
In fact, the most important point is that the digital outputs of the pH meter are stored in the programmed minicomputer in the proper memory localizations. At the end of every measurement cycle, the minicomputer, after statistical analysis in order to avoid spurious indications, decides whether or not any pH deviation should be considered significant.
If the deviation is considered significant, the alarm system is actuated, permitting a fast diagnosis of bacteremia and the second step B) of the procedure is started.
Every bottle where no pH change is observed within 24h is disconnected and subcultured in the conventional way.
Anaerobic cultures are handled in the conventional way.
B. Method of fast detection of sensitivity to antibiotics: The same apparatus can be used for a fast detection of susceptibility to several antibiotics: n' bottles are replaced by n' tubes containing an electrode in addition to a broth (for example, trypticasesoja broth) and disks delivering known amounts of different antibiotics. One tube is left free of any antibiotic as a reference.
These tubes with different concentrations of one antibiotic are inoculated with an aliquot volume from the content of any bottle where a significant pH change has been detected. Bacteriostatic activity can be detected by the differences in pH curves between the tube without any antibiotic and the others containing various concentrations of the antibiotic. These differences are processed by the same minicomputer. The tubes containing efficient concentrations of antibiotics show no significant pH change versus time.
Experimental studies on the apparatus and the method according to the invention have led to the finding that: 1) pH is stable in non-inoculated bottles: The pH of non-inoculated agitated bottles maintained at 37"C is stable over 24 h, which pH changes never exceeding 0.02 units, the pH meter being sensitive to 0.01 unit changes. A 0.04 pH change is therefore considered as indicative of bacterial growth.
2) The correlation between pH changes and bacterial growth is constant: a) fermentative bacteria: Enterobacteriaceae including numerous strains of Escherichia coli, Klebsiella sp. Proteus sp. Serratia-Enterobacter sp., as well as Gram positive cocci including numerous Staphylococcus aureus and epidermidis, and Streptococci from several Lancefield groups uniformly produce pH decreases following an exponential curve.
b) Among the non-fermentative bacteria, the only genus and species of clinical significance is Pseudomonas aeruginosa. This species induces a pH increase probably due to de-amination and ammonia production.
3) Progressive dilutions of the inoculum induce progressive delay in pH changes.
4) Cultivation in presence of blood but without polyanethol sulfonate (P.A.S.) induces a 2-hour delay in pH changes, this being due to early bacterial inhibition (verified by serial bacterial counts).
P.A.S. completely reverses this inhibition and consequently is added to the conventional culture medium like the one from Institut Merieux.
5) Antibiotics which at sufficient concentration, can stop bacterial growth also stop pH changes.
Reference is now made to the accompanying drawings in which: Figure I is the general scheme of the apparatus and Figure 2, Figure 3 and Figure 4 are pH changes/time graphs.
Figure 2 presents, related to incubation time, pH changes induced by different inocula of E. coil 54, 127 (strain maintained in "Institut Gustave Roussy") into a culture medium (brain-heart-P.A.S. medium + blood): The curve A represents pH changes following an inoculum of 2.106/ml; The curve B represents pH changes following an inoculum of 2. 105/ml The curve C represents pH changes following an inoculum of 2.104/ml; The curve D represents pH changes following an inoculum of 2.103/ml; The curve E represents pH changes following an inoculum of 2.102/my The curve T represents pH changes of non-inoculated reference medium.
The curves A, B, C, D, E show that pH values exponentially decrease after a certain delay and each tenfold dilution of the inoculum increases this delay by 80.5 + 6.18 minutes (i1D.S.); the curve T presenting no pH changes for non-inoculated medium.
From this figure it can be extrapolated that two viable bacteria in 100 ml of broth will induce a significant pH change within 13 hours.
Figure 3 presents the pH changes induced by three clinical strains (curves 1, 2 and 3) of Pseudomonas aeruginosa (non-fermentative genus) inoculated at a 10-2 dilution into a culture medium (brain-heart-P.A.S. + blood). With the three strains, a pH increase (beyond the zone of non-significance shown in strokes) is observed.
Figure 4 presents the pH changes induced by different concentrations of gentamicin on E. coli 54, 127 (10-2 dilution) in trypticasesoja broth. T is the curve obtained with the broth with no antibiotic. A, B, C, D, E are the curves obtained with broths containing respectively 0,5y; ly, 2y; 4y et 8y of gentamicin for 1 ml. The curves C, D, E show no significant pH change versus time.
This graph shows that it is possible to state, within 3 hours, that a gentamicin concentration of 2y/ml or more inhibits bacterial metabolism, and then to predict a probable in vivo antibacterial activity of this antibiotic if it is administered at a proper dose.
The equipment and the method according to the invention permit a diagnosis of bacteremia and the choice of an efficient antibiotic generally within 10 to 12 hours, avoiding excessive manipulations and contamination hazards.
WHAT I CLAIM IS: 1. An apparatus which permits a fast diagnosis of bacteremia and a fast determination of sensitivity of so detected bacteria to several antibiotics, this apparatus being a combination of: - several bottles into which a culture medium and blood may be aseptically introduced, each bottle being fitted with a sterilizable electrode; - each electrode being connected to each way of an electronic switching system; - a pH meter connected to the switching system, and to a multichuter; - the computer receiving a program, being connected to its keyboard and commanding an alarm system.
2. A method of diagnosing a bacteremia in a patient using the apparatus according to claim 1, in which a sample of the patient's blood is aseptically added into each bottle fitted with an electrode where it is incubated with a culture medium; the pH values in each bottle are registered at regular intervals, and each pH deviation which is considered significant by the computer is indicated by the alarm system.
3. A method of determining the sensitivity of bacteria detected with the method of claim 2 to an antibiotic using the apparatus according to claim 1, in which an aliquot volume from the content of any bottle where a significant pH change has been detected, is inoculated into each tube containing a conventional broth, and different known concentrations of the antibiotic; the pH values in each tube are registered at regular intervals and significant pH changes are recorded for each tube, no significant pH change with time corresponding to an efficient concentration of the antibiotics.
4. An apparatus according to claim 1 substantially as herein described with reference to the accompanying drawings.
5. A method according to claim 2, substantially as herein described with reference to the accompanying drawings.
6. A method according to claim 3, substantially as herein described with reference to the accompanying drawings.
**WARNING** end of DESC field may overlap start of CLMS **.

Claims (6)

**WARNING** start of CLMS field may overlap end of DESC **. pH changes. Reference is now made to the accompanying drawings in which: Figure I is the general scheme of the apparatus and Figure 2, Figure 3 and Figure 4 are pH changes/time graphs. Figure 2 presents, related to incubation time, pH changes induced by different inocula of E. coil 54, 127 (strain maintained in "Institut Gustave Roussy") into a culture medium (brain-heart-P.A.S. medium + blood): The curve A represents pH changes following an inoculum of 2.106/ml; The curve B represents pH changes following an inoculum of 2. 105/ml The curve C represents pH changes following an inoculum of 2.104/ml; The curve D represents pH changes following an inoculum of 2.103/ml; The curve E represents pH changes following an inoculum of 2.102/my The curve T represents pH changes of non-inoculated reference medium. The curves A, B, C, D, E show that pH values exponentially decrease after a certain delay and each tenfold dilution of the inoculum increases this delay by 80.5 + 6.18 minutes (i1D.S.); the curve T presenting no pH changes for non-inoculated medium. From this figure it can be extrapolated that two viable bacteria in 100 ml of broth will induce a significant pH change within 13 hours. Figure 3 presents the pH changes induced by three clinical strains (curves 1, 2 and 3) of Pseudomonas aeruginosa (non-fermentative genus) inoculated at a 10-2 dilution into a culture medium (brain-heart-P.A.S. + blood). With the three strains, a pH increase (beyond the zone of non-significance shown in strokes) is observed. Figure 4 presents the pH changes induced by different concentrations of gentamicin on E. coli 54, 127 (10-2 dilution) in trypticasesoja broth. T is the curve obtained with the broth with no antibiotic. A, B, C, D, E are the curves obtained with broths containing respectively 0,5y; ly, 2y; 4y et 8y of gentamicin for 1 ml. The curves C, D, E show no significant pH change versus time. This graph shows that it is possible to state, within 3 hours, that a gentamicin concentration of 2y/ml or more inhibits bacterial metabolism, and then to predict a probable in vivo antibacterial activity of this antibiotic if it is administered at a proper dose. The equipment and the method according to the invention permit a diagnosis of bacteremia and the choice of an efficient antibiotic generally within 10 to 12 hours, avoiding excessive manipulations and contamination hazards. WHAT I CLAIM IS:
1. An apparatus which permits a fast diagnosis of bacteremia and a fast determination of sensitivity of so detected bacteria to several antibiotics, this apparatus being a combination of: - several bottles into which a culture medium and blood may be aseptically introduced, each bottle being fitted with a sterilizable electrode; - each electrode being connected to each way of an electronic switching system; - a pH meter connected to the switching system, and to a multichuter; - the computer receiving a program, being connected to its keyboard and commanding an alarm system.
2. A method of diagnosing a bacteremia in a patient using the apparatus according to claim 1, in which a sample of the patient's blood is aseptically added into each bottle fitted with an electrode where it is incubated with a culture medium; the pH values in each bottle are registered at regular intervals, and each pH deviation which is considered significant by the computer is indicated by the alarm system.
3. A method of determining the sensitivity of bacteria detected with the method of claim 2 to an antibiotic using the apparatus according to claim 1, in which an aliquot volume from the content of any bottle where a significant pH change has been detected, is inoculated into each tube containing a conventional broth, and different known concentrations of the antibiotic; the pH values in each tube are registered at regular intervals and significant pH changes are recorded for each tube, no significant pH change with time corresponding to an efficient concentration of the antibiotics.
4. An apparatus according to claim 1 substantially as herein described with reference to the accompanying drawings.
5. A method according to claim 2, substantially as herein described with reference to the accompanying drawings.
6. A method according to claim 3, substantially as herein described with reference to the accompanying drawings.
GB2986477A 1977-07-15 1977-07-15 Method of diagnosing bacteremia and apparatus therefor Expired GB1601689A (en)

Priority Applications (2)

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GB2986477A GB1601689A (en) 1977-07-15 1977-07-15 Method of diagnosing bacteremia and apparatus therefor
FR7819530A FR2397457A1 (en) 1977-07-15 1978-06-29 METHOD AND APPARATUS FOR DIAGNOSING BACTERIAEMIA AND BACTERIAL SENSITIVITY TO ANTIBIOTICS

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GB2986477A GB1601689A (en) 1977-07-15 1977-07-15 Method of diagnosing bacteremia and apparatus therefor

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992019764A1 (en) * 1991-05-08 1992-11-12 Baxter Diagnostics Inc. Method and apparatus to detect bacterial contamination of transfusable blood
US5173434A (en) * 1990-11-05 1992-12-22 Baxter Diagnostics Inc. Measurement of color reactions by monitoring a change of fluorescence
US5372784A (en) * 1988-08-31 1994-12-13 Baxter Diagnostics Inc. Measurement of bacterial CO2 production in an isolated fluorophore by monitoring an absorbance regulated change of fluorescence
EP1281958A3 (en) * 2001-07-30 2005-06-29 Endress + Hauser Conducta Gesellschaft für Mess- und Regeltechnik mbH + Co.KG. Arrangement to attach a sensor

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD149843A3 (en) * 1979-06-28 1981-08-05 Manfred Kunze METHOD AND DEVICE FOR DETECTING BACTERIAEMIA

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1221251A (en) * 1958-03-06 1960-06-01 Sheppard Electronics Ltd Improvements to electrical testing devices
US3479255A (en) * 1964-01-27 1969-11-18 Beckman Instruments Inc Electrochemical transducer
US3743581A (en) * 1970-10-21 1973-07-03 Bactomatic Inc Microbiological detection apparatus

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5372784A (en) * 1988-08-31 1994-12-13 Baxter Diagnostics Inc. Measurement of bacterial CO2 production in an isolated fluorophore by monitoring an absorbance regulated change of fluorescence
US5173434A (en) * 1990-11-05 1992-12-22 Baxter Diagnostics Inc. Measurement of color reactions by monitoring a change of fluorescence
WO1992019764A1 (en) * 1991-05-08 1992-11-12 Baxter Diagnostics Inc. Method and apparatus to detect bacterial contamination of transfusable blood
EP1281958A3 (en) * 2001-07-30 2005-06-29 Endress + Hauser Conducta Gesellschaft für Mess- und Regeltechnik mbH + Co.KG. Arrangement to attach a sensor

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Publication number Publication date
FR2397457B1 (en) 1981-09-11
FR2397457A1 (en) 1979-02-09

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Effective date: 19920525