when confronted with a patient battling an infectious disease , elucidation of the offending pathogen requires great effort . time is critical : broad - spectrum antibiotics are initiated after cultures are collected , as identification of the offending microbe requires 2448 hours . to increase the sensitivity of culture , an enrichment step may be added . the workup is not completed once a pathogen is identified : antimicrobial susceptibility must next be ascertained following additional subculture in the presence of select antibiotics for another 2448 hours . several techniques have been developed to aid in reducing the time required to identify a pathogen . these techniques include the use of chromogenic agar / broth and nucleic acid amplification technology ( naat ) [ 3 , 4 ] . while both of these techniques have been employed in screening for antenatal gbs colonization , the centers for disease control and prevention still recommend that culture be performed . this is an absolute requirement for patients allergic to penicillin , as naat is not capable of providing antimicrobial susceptibility profiles . additionally , the resources and highly specialized training required to perform naat are not universally available . recently , the infectious diseases society of america released a paper detailing the current approach towards identifying clinical pathogens . described is a process characterized as having inadequate sensitivity and significant time delays , thereby contributing to the development of greater and greater antimicrobial resistance . these concerns are all too - well illustrated in our approach towards treatment for gbs prophylaxis , in which increasing resistance to penicillin has been observed , and resistance to clindamycin is on the rise [ 8 , 9 ] . additionally , there is concern that this practice will contribute to e. coli resistance observed in premature neonates . already , we have seen the implications of antimicrobial resistance with enterococcus , and hospital acquired infections with this pathogen are estimated to add $ 27,000.00 per infection [ 11 , 12 ] . n. gonorrhoeae resistance has been increasing steadily , and multidrug resistance has recently been confirmed . these three disparate organisms demonstrate a range in the microbial response to our directed approach to both prophylaxis and treatment . through a modification of a recently reported test , we have developed a method for the simultaneous identification of a pathogen and determination of its antibiotic susceptibility . we have modified this test so that gbs , e. faecalis , and n. gonorrhoeae may be detected at dilute concentrations after 6-hour incubation . finally , we show that following an overnight incubation , test organisms may be detected at concentrations rivaling those published for pcr . as this test allows one to simultaneously identify a pathogen and determine its antimicrobial susceptibility , this novel technique provides a change in the clinician 's approach to managing infectious diseases . group b streptococcus clinical isolates 12386 and 01.12.76 were shown by disk diffusion to be susceptible or resistant to clindamycin , respectively . enterococcus faecalis atcc 29212 was confirmed to be susceptible to vancomycin by disk diffusion , and atcc strain 51299 was confirmed to be resistant . e. coli , s. aureus , candida albicans , and beta streptococcus groups a , c , f , and g were all clinical isolates . rabbit polyclonal anti - gbs antibody ( 1521 ) , hrp - conjugated rabbit polyclonal anti - gbs antibody ( 1524 ) , rabbit polyclonal anti - enterococcus antibody ( 3711 ) , and hrp - conjugated rabbit polyclonal anti - enterococcus antibody ( 3714 ) were all obtained from virostat ( portland , maine ) . anti - n . gonorrhoeae antibody ( pa1 - 73144 ) were purchased from thermofisher ( waltham , ma ) . 96-well immulon microtiter plates ( thermofisher , waltham , ma ) were coated with specified antibody . for the gbs assay , anti - gbs antibody was first diluted 1  :  30 in bicarbonate buffer ( sigma , st . louis mo ) . for the n. gonorrhoeae or enterococcus assays , antibodies were diluted 1  :  200 or 1  :  100 , respectively . 100  l of the antibody dilutions was placed in respective wells , and the plates were incubated at 4c overnight . wells were then washed three times with phosphate - buffered saline ( pbs , sigma aldrich , st . louis , mo ) supplemented with 0.05% tween-20 ( fisher scientific , pittsburg , pa ) . the plates were blocked for 30 minutes at room temperature with 200  l of startingblock ( thermoscientific , rockford , il ) followed by washing three times with pbs - tw at room temperature . bacterial isolates were individually prepared at a concentration of approximately 10  bacteria / ml based on 0.5 mcfarland and confirmed by od600  nm . aliquots were plated on appropriate agar ( columbia colistin nalidixic acid blood agar , for gbs and enterococcus , and chocolate agar , for n. gonorrhoeae , fisher scientific , pittsburg , pa ) , and colony forming units ( cfus ) were determined following a 2448-hour culture . inoculates were prepared further in tenfold dilutions down to 10  bacteria / ml . for the time zero test , isolates were diluted in pbs and incubated at room temperature for 20 minutes . for growth experiments , fastidious broth ( remel , lenexa , ks ) was substituted for pbs , and inoculates were diluted out in test tubes and incubated prior to being transferred to wells . varying concentrations of inoculates were added to tubes containing an amount of competing organisms held steady at 10  bacteria / ml in fastidious broth . after a prespecified incubation time - point ( 6 hours for antimicrobial susceptibility testing with gbs and enterococcus , 9 hours for inducible resistance of gbs to clindamycin , or overnight for determining the limit of detection of all three organisms or determination of antimicrobial susceptibility with n. gonorrhoeae ) organisms were transferred to microtiter wells and plates were then incubated for 20 minutes at room temperature . for the time zero assay , this transfer step was omitted , as inoculates were prepared directly in the wells . for growth experiments , 10  l of the incubated bacterial suspensions was added to 90  l of pbs in each well . following 20-minute incubation , horseradish peroxidase ( hrp ) conjugated antibody was next added in 100  l aliquots and plates were incubated for 10 minutes at room temperature . hrp - conjugated antibodies for all tests were diluted 1  :  100 in pbs . after washing three times with pbs - tw , tmb peroxidase substrate ( kpl , gaithersburg , md ) plates were read in an elx 808 biotek ( winooski , vermont ) plate reader at 450  nm . serial dilutions of each bacterium were prepared starting with a 0.5 mcfarland and diluted down to 10  bacteria / ml in pbs . when processed on the pathogen - specific elisas , group b streptococcus , e. faecalis , and n. gonorrhoeae each individually showed detection in the range of 1010  bacteria / ml after 20-minute incubation ( figures 1(a)1(c ) ) . gbs was detected down to 10  bacteria / ml , e. faecalis down to 10  bacteria / ml , and n. gonorrhoeae down to 10  bacteria / ml . in order to determine the specificity of the test , suspensions were tested in which the bacterium to be identified was diluted out , but the level of a competing bacterium was held constant at 10  bacteria / ml in pbs . testing of several commonly found vaginal cocolonizers mixed with the bacterium of interest revealed little to no interference with the assays ( supplemental figures  1(a)1(l ) in supplementary material available online at http://dx.doi.org/10.1155/2016/5293034 ) . the ability to detect the test organism at greater dilutions increased when the incubation times were lengthened . gbs was detected down to 10  bacteria / ml after 2-hour incubation , 10  bacteria / ml after 6 hours , and 10  bacteria / ml after incubating overnight ( figure 2(a ) ) . a series of dilutions of e. faecalis starting at 0.5 mcfarland was prepared similarly to that of gbs , and after incubating overnight ( 18  hrs ) in fastidious broth , the tubes surrounding the turbidity point were assayed . turbidity was observed down to 10  bacteria / ml . this tube was assayed directly , as well as was a series of dilutions of this sample . this same procedure was performed on the subsequent nonturbid dilutions , so that 10 and 10  bacteria / ml tubes were both assayed directly and then when diluted out further . e. faecalis was detected at 10  bacteria / ml , and when this sample was diluted out further , bacteria were capable of being detected following both 10-fold and 100-fold dilution ( figure 2(b ) ) . in the nonturbid overnight culture tubes , no e. faecalis was detected ( 10 and 10  bacteria / ml ) . n. gonorrhoeae was diluted out serially in fastidious broth , first starting with a 0.5 mcfarland . after overnight incubation , the assay was run . n. gonorrhoeae was detected strongly at high concentrations , as well as at very low concentrations , 10 and 10  bacteria / ml ( figure 2(c ) ) . the test was not capable of detecting bacteria less than 10  bacteria / ml following 24-hour incubation . isolates were individually prepared in the presence or absence of selected antibiotics . after culturing for 6 hours , the assay was performed and susceptibility profiles were obtained . a strain of gbs sensitive to penicillin was cultured in the presence of increasing concentrations of the following antibiotics : penicillin , cefazolin , erythromycin , and clindamycin . at all concentrations of penicillin tested , growth of this strain of gbs was inhibited and no gbs was detected at 10  bacteria / ml ( figure 3(a ) ) . incubation of gbs with increasing concentrations of cefazolin showed a dose effect , in which growth was observed at a more dilute concentration of antibiotic , 0.005  g / ml , but not when increasing amounts were used ( supplemental figure  2(a ) ) . similar results were observed when the same strain was tested in the presence of either clindamycin or erythromycin ( supplemental figures  2(b ) and 3(c ) ) . next , a gbs strain resistant to clindamycin but sensitive to erythromycin was prepared , as above . supplemental figure 2(d ) shows that , at 0.25  g erythromycin , growth of the gbs strain was inhibited at 10  bacteria / ml . conversely , at all concentrations of clindamycin tested , gbs was detected at levels similar to that of the no antibiotic control for this strain of gbs ( supplemental figure  2(e ) , strain 165 ) . when vancomycin sensitive e. faecalis was diluted out in the presence of vancomycin , no growth was detected at 10  bacteria / ml ( figure 3(b ) ) . when a vancomycin resistant strain was substituted under test conditions , however , this organism was detected at 10  bacteria / ml after 6-hour incubation ( supplemental figure  2(f ) ) . strains of n. gonorrhoeae were prepared and diluted out as above , in the presence of either penicillin or ceftriaxone . following 24-hour incubation , this strain was detected at 10  bacteria / ml when tested in the absence of penicillin , but no bacteria were detected at this inoculum in wells which received penicillin ( figure 3(c ) ) . when a penicillin - resistant strain of n. gonorrhoeae was tested , bacteria were strongly detected at 10  bacteria / ml following 24-hour incubation in wells receiving less than 2  g / ml , with slight detection at 2  g / ml ( supplemental figure  2(g ) ) . a strain of n. gonorrhoeae sensitive to ceftriaxone was next tested and showed similar results : bacteria cultured in the absence of ceftriaxone were detected at 10  bacteria / ml , but not when cultured in the presence of either 0.0625  g / ml or 0.125  g / ml ceftriaxone at this inoculum ( supplemental figure  2(h ) ) . both antibiotic concentrations used were less than the recommended level of 0.25  g / ml in determining resistance to ceftriaxone . gbs strains previously determined to show either noninducible ( isolate a ) or inducible ( isolate b ) resistance to clindamycin in the presence of erythromycin were tested following 9-hour incubation . in the noninducible resistance strain ( isolate a ) , gbs was detected only in wells which did not receive clindamycin ( table 1 ) . when a strain capable of showing inducible resistance was tested , gbs was detected down to a 10  bacteria / ml dilution , with very strong detection at 10  bacteria / ml , moderate detection at 10  bacteria / ml , and light detection at 10  bacteria / ml . the development of microbial resistance to antibiotics has plagued contemporary clinicians . following the introduction of each new antibiotic , resistant isolates have been detected , with resistance of staphylococcus to methicillin noted 2  yrs after the first use of this antibiotic in 1960 , resistance of enterococcus to vancomycin was noted 16  yrs after its introduction in 1972 , and resistance of staphylococcus to linezolid was noted just 1  yr after its first use in 1996 . the development of microbial resistance to an antibiotic has become the norm , and anticipated resistance has been confirmed again as recently as 2011 with the multidrug resistant n. gonorrhoeae strain reported in japan . that antimicrobial resistance has developed and continues to do so is not surprising . what is concerning is that the approach to managing patients with suspected infection / colonization has remained static , and in fact this approach continues to be reestablished , with the recent example of intrapartum gbs prophylaxis as recommended by the cdc in 2010 . it is agreed that intrapartum gbs prophylaxis has led to a reduction in early - onset gbs sepsis : however , there is concern that this practice may promote resistant strains of e. coli . as enterococci are to a large degree resistant to cephalosporins , use of this class of antibiotic for surgical prophylaxis allows for overgrowth of enterococcus at sites previously colonized by cephalosporin - sensitive organisms and may contribute to the increased number of hospital associated surgical site infections . perhaps nowhere is this battle between pathogen and microbicide more apparent than with n. gonorrhoeae . from the first use of sulfonamides in the 1940s , this bacterium has countered the development of resistant strains . after showing that this pathogen is capable of developing resistance to any antibiotic directed against it , including sulfonamides , penicillin , tetracycline , spectinomycin , quinolones , macrolides , and now cephalosporins , it is apparent that novel approaches are needed beyond the two - drug counterattack recommended just last year . we recently developed an immunoassay in which membranes coated with antibody against gbs are exposed to bacterial suspensions , and after incubating for a series of time - points , bound gbs is detected . this assay was tested on patients ' vaginal - rectal specimens and was found to have a high degree of sensitivity and specificity , with the added benefit of being capable of detecting nonbeta hemolytic streptococcal strains of gbs . in order to decrease the interobserver variation seen with dot - blot assays , we converted the test to an elisa format . in this study , we have substituted the anti - gbs antibodies for those directed against either n. gonorrhoeae or enterococcus . we show that these polyclonal antibodies provide a great deal of sensitivity in a nonselective broth , with little interference from other commonly isolated cocolonizers figure 1 and supplemental figure  1 . following a six - hour incubation period , the limit of detection increased to 10  bacteria per ml for gbs ( figure 2 ) . furthermore , we show that antibiotic susceptibility may be determined ( supplemental figure  2 ) . from this , one may envision a panel in which a series of wells are set up in which multiple clinically relevant antibiotics are tested , providing the clinician with a targeted approach to treatment in a greatly reduced timeframe ( figure 3 ) . a central facet of this assay involves cellular viability : the concentration of bacteria present in the well is related specifically to the incubation times utilized . by incubating overnight , the sensitivity of the test was increased significantly as live bacteria continued to grow and divide , with gbs being detected at 10  bacteria per ml , enterococcus detected at 10  bacteria per ml , and n. gonorrhoeae detected at 10  bacteria per ml ( figure 2 ) . this allows for a sensitivity greater than published results for several pcr - based methods of detection following an enrichment step , with the unique advantage being that this test is capable of detecting viable cells [ 2225 ] . this assay provides the unique approach of targeting the growth of a specific clinically relevant pathogen through the use of a capture antibody . an immediate binding assay demonstrates whether a pathogen is present or not . by then culturing the organism in the presence or absence of selected antibiotics , one may show that cells are viable and susceptible to specific antimicrobials , thereby allowing the clinician to then make an informed decision more rapidly .
background . 
 elucidation of a pathogen 's antimicrobial susceptibility requires subculture after the organism is first isolated . 
 this takes several days , requiring patients to be treated with broad - spectrum antibiotics . 
 this approach contributes to the development of bacterial resistance . 
 methods . 
 microtiter wells were coated with a polyclonal antibody targeting the pathogen of interest . 
 bacterial suspensions were added in the presence / absence of selected antibiotics . 
 after washing , captured bacteria were detected . 
 findings . 
 group b streptococcus ( gbs ) , enterococcus faecalis , and neisseria gonorrhoeae were each detected at 105  bacteria / ml following a 20-minute incubation period . 
 susceptibility to select antibiotics was discernable following a 6-hour incubation period ( gbs and enterococcus ) . 
 sensitivity was increased to 102  bacteria / ml for gbs , 101  bacteria / ml for e. faecalis , and 101  bacteria / ml for n. gonorrhoeae following 1824-hour culture . conclusion . 
 this novel assay allows for the highly sensitive and specific identification of a pathogen and simultaneous determination of its antimicrobial susceptibility in a reduced time .