Method for microbial species detection, quantification and antibiotic susceptibility identification

Abstract

A method of using microfluidic chips to significantly accelerate the time to identify and quantify microbes in a biological sample and test them for antibiotic resistance, particularly for urinary tract infections. A first microfluidic chip uses antibody or similar probes to identify and quantify any microbes present. The same or a similar chip uses antibody or similar probes to identify microbes with DNA or RNA known to indicate antibiotic resistance. Another microfluidic chip tests for antibiotic susceptibility of any microbes by growing them in very small wells in the presence of antibiotics, reducing the time required for such testing by as much as 95%. Another microfluidic chip runs traditional urinalysis or similar tests.

What is claimed is: 
     
         1 . A method for analyzing a biological sample, comprising:
 a. providing a microfluidic microbe detection chip (MDC), the MDC comprising:
 i. a plurality of microfluidic channels; 
 ii. at least one inlet connected to each of said microfluidic channels for receiving the biological sample and delivering it to said plurality of microfluidic channels; 
   b. providing a plurality of probe reservoirs, each said reservoir containing a supply of probes which recognize microbial surface molecules, microbial intracellular proteins or microbial DNA or RNA and bind thereto, and wherein each said reservoir is connected to at least one of said microfluidic channels on the MDC to deliver said probes in said reservoir to said at least one of said microfluidic channels;   c. providing a biological sample to said at least one inlet of the MDC, which in turn provides the sample to said plurality of microfluidic channels;   d. causing the probes to engage said biological sample in said plurality of microfluidic channels, whereby the probes can react and bind to said microbial surface molecules, microbial intracellular proteins or microbial DNA or RNA recognized by the probes, if such are present in said biological sample;   e. measuring the presence of any of said probes which are bound to said microbial surface molecules, microbial intracellular proteins or microbial DNA or RNA in said biological sample in said plurality of microfluidic channels; and   f. reporting the results of said measurement.   
     
     
         2 . The method of  claim 1 , furthering comprising quantifying the amount of any of said probes which are bound to said microbial surface molecules, microbial intracellular proteins or microbial DNA or RNA in said biological sample in said plurality of microfluidic channels and reporting the results of such quantification. 
     
     
         3 . The method of  claim 1 , further comprising using a lysing solution to lyse any microbes in said biological sample prior to step 1.d. 
     
     
         4 . The method of  claim 1 , further comprising flushing said plurality of microfluidic channels of biological sample and unbound probes between steps 1.d and 1.e. 
     
     
         5 . The method of  claim 1 , further comprising selecting the biological sample from the group consisting of urine, blood, sputum, saliva, mucous and swabs from solid tissue. 
     
     
         6 . The method of  claim 1 , wherein each said probe comprises an attacher-reporter complex. 
     
     
         7 . The method of  claim 6 , further comprising selecting an attacher portion of each attacher-reporter complex from the group consisting of natural or synthetic DNA, RNA, antibodies, aptamers or other amino acid structures, using natural and/or non-naturally occurring amino acids and which recognize microbial surface molecules, microbial intracellular proteins or microbial DNA or RNA. 
     
     
         8 . The method of  claim 7 , further comprising selecting each said attacher portion to attach to a portion of the microbial surface molecules, microbial intracellular proteins or microbial DNA or RNA which identifies a specific microbe, the microbe being selected from the group consisting of the 4-10 microbes most likely to cause an infection in the type of biological sample being tested. 
     
     
         9 . The method of  claim 8 , further comprising selecting said microbes targeted by said probes to match the 4-10 most likely to cause a urinary tract infection in a specific geography. 
     
     
         10 . The method of  claim 7 , wherein each said probe targets a microbe selected from the group consisting of  Acetobacter aurantius, Acinetobacter baumannii, Actinomyces israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Anaplasma phagocytophilum, Azorhizobium caulinodans, Aztobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferia, Brucella, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtherias, Corynebacteriym fusiforme, Coxiella burnetti, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enteroccous faecium, Enterococcus galllinarum, Enterococcus maloratus, Eschericichia coli, Francisella tularenisis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Hamephilus vaginalis, Helicobacter pylori, Klebsilla pneumoniae, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis, Peptostreptococcus, Porphyromonas gingivalis, Prevotella melaninogenica, Pseudomonas aeruginosa, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomas, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophillia, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faecium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mutans, Streptococcus oxalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Viridans streptococci, Wolbachia, Yersinia enterocolitica, Yersinia pestis , and  Yersinia pseudotuberculosis.    
     
     
         11 . The method of  claim 7 , wherein said attacher portion attaches to a portion of the DNA or RNA of said microbe which identifies resistance to a specific antibiotic or antimicrobial. 
     
     
         12 . The method of  claim 11 , further comprising selecting said antibiotic and antimicrobial resistance identifying DNA or RNA from the group consisting of amikacin, aminoglycosides, amoxycillin, amoxycillin-clavulanate, aztreonam, β-lactams, carbapenems, carbenicillin, ceffriaxone, cefixime, cefoperazone, cefotaxime, cefpodoxime, cefprozil, ceftazidime, cefuroxime, coamoxiclav, cephalexin, cephalosporins, chloramphenicols, ciprofloxacin, clindamycin, colistin, cotrimoxazole, doxycycline, erythromycin, flucloxacillin, fluoroquinolones, folic acid inhibitors, foloxacin, fusidic acids, gentamicin, glycopeptides, kanamycin, lipopeptides, lyncosamides, macrolides, meropenem, metronidazoles, monobactams, moxifloxacin, mupirocin, nalidixic acid, neomycin, nitrofurantoins, norfloxacin, ofloxacin, oxazolidinones, penicillin, piperacillin-tazobactam, pivmecillinam, polymyxin b, quinolones, rifampicin, streptogramins, sulfamethoxazole, sulfonamides, tetracyclines, trimethoprim, vancomycin. 
     
     
         13 . The method of  claim 6 , further comprising selecting said reporter portion from the group consisting of fluorescent structure, chemiluminescent structure, radioactive nuclides, magnetic nanoparticles, giant magnetoresistance-based magnetic nanoparticles, coated magnetic nanoparticles and surface plasmon structures. 
     
     
         14 . The method of  claim 1 , further comprising providing 4-50 varieties of said probes. 
     
     
         15 . The method of  claim 1 , further comprising performing an antibiotic susceptibility test by:
 a. providing an antibiotic susceptibility chip (ASC), the ASC comprising:
 i. a plurality of wells, each said well comprising a volume to receive said biological sample and at least some of said wells pre-coated with at least one antibiotic and a reporter to report the presence of microbes in said well; and 
 ii. at least one ASC inlet connected to each of said wells for receiving said biological sample and delivering it to said plurality of wells; 
   b. providing at least one ASC sensor adjacent to said plurality of wells to measure said reporter;   c. providing said biological sample to said ASC inlet, which in turn provides said sample to the plurality of wells;   d. measuring the presence of any microbes in each well as indicated by the reporter in such well; and   e. reporting the results of said measurement.   
     
     
         16 . The method of  claim 15 , further comprising between steps 15.c and 15.d, incubating said ASC for a sufficient time to enable replication of any microbes in each well to a level which is detectable by said at least one ASC sensor. 
     
     
         17 . The method of  claim 15 , furthering comprising quantifying the amount microbes in each well as indicated by the reporter in such well and reporting the results of such quantification. 
     
     
         18 . The method of  claim 15 , wherein said reporter comprises resazurin and wherein said at least one ASC sensor comprises a fluorescent sensor for the detection of resazurin combined with a living microbe. 
     
     
         19 . The method of  claim 15 , wherein 5-25 of said wells are pre-coated with an antibiotic. 
     
     
         20 . The method of  claim 19 , further comprising selecting the pre-coated antibiotics from the group consisting of amikacin, aminoglycosides, amoxycillin, amoxycillin-clavulanate, aztreonam, β-lactams, carbapenems, carbenicillin, ceffriaxone, cefixime, cefoperazone, cefotaxime, cefpodoxime, cefprozil, ceftazidime, cefuroxime, coamoxiclav, cephalexin, cephalosporins, chloramphenicols, ciprofloxacin, clindamycin, colistin, cotrimoxazole, doxycycline, erythromycin, flucloxacillin, fluoroquinolones, folic acid inhibitors, foloxacin, fusidic acids, gentamicin, glycopeptides, kanamycin, lipopeptides, lyncosamides, macrolides, meropenem, metronidazoles, monobactams, moxifloxacin, mupirocin, nalidixic acid, neomycin, nitrofurantoins, norfloxacin, ofloxacin, oxazolidinones, penicillin, piperacillin-tazobactam, pivmecillinam, polymyxin b, quinolones, rifampicin, streptogramins, sulfamethoxazole, sulfonamides, tetracyclines, trimethoprim, vancomycin.