Rapid differentiation of fermentative from nonfermentative gram-negative bacilli in positive blood cultures by an impedance method

Rapid differentiation of fermentative gram-negative bacilli (fermenters) from nonfermentative gram-negative bacilli (nonfermenters) in positive blood cultures may help physicians to narrow the choice of appropriate antibiotics for empiric treatment. An impedance method for direct differentiation of fermenters from nonfermenters was investigated. The bacterial suspensions (or positive culture broths containing gram-negative bacteria) were inoculated into the module wells of a Bactometer (bioMérieux, Inc., Hazelwood, Mo.) containing 1 ml of Muller-Hinton broth. The inoculated modules were incubated at 35 degrees C, and the change in impedance in each well was continuously monitored. The amount of time required to cause a series of significant deviations from baseline impedance values was defined as the detection time (DT). The percent change of impedance was defined as the change of impedance at the time interval from DT to DT plus 1 h. After testing 857 strains of pure cultures (586 strains of fermenters and 271 strains of nonfermenters), a breakpoint (2.98%) of impedance change was obtained by discriminant analysis. Strains displaying impedance changes of greater than 2.98% were classified as fermenters; the others were classified as nonfermenters. By using this breakpoint, 98.6% (340 of 345) of positive blood cultures containing fermenters and 98% (98 of 100) of positive blood cultures containing nonfermenters were correctly classified. The impedance method was simple, and the results were normally available within 2 to 4 h after direct inoculation of positive blood culture broths.     

Rapid identification of gram-negative bacilli from blood cultures

Blood-culture broths showing macroscopic or radiometric evidence of growth of gram-negative bacilli were examined by a rapid automated bacterial identification system. A differential centrifugation technique was developed to prepare suitable inocula. The identification results obtained were confirmed by the API 20E method, with single colonies of the strains isolated 24 h later. Of 90 organisms tested, seven did not give the same identification by the two systems. With the rapid automated technique a presumptive identification of gram-negative bacilli can be made 24 h earlier than by more conventional methods.     

Evaluation of the VITEK 2 system for rapid direct identification and susceptibility testing of gram-negative bacilli from positive blood cultures

This study explores the possibility of combining the BacT/Alert Microbial Detection System with the VITEK 2 system to achieve rapid bacterial identification and susceptibility testing. Direct inoculation of bacterial suspension to the VITEK 2 ID-GNB card and AST-NO09 card was made by differential centrifugation of blood cultures of organisms with gram-negative enteric bacillus-like morphology. A total of 118 strains were investigated; of these, 97 (82.2%) strains were correctly identified to the species level and 21 (17.8%) strains were not identified; by comparing the results with those of the reference method of API identification systems using a pure culture, it was found that no strain had been misidentified. Among the 21 strains with no identification, 13 (61.9%) strains were nonfermenters. The direct-identification reporting time of VITEK 2 was 3.3 h. Direct testing of susceptibility to 11 antibiotics, i.e., amikacin, cefepime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, netilmicin, piperacillin, piperacillin-tazobactam, and tobramycin, was also performed by using the broth microdilution (MB) method according to the NCCLS guidelines as a reference. After comparing the MICs of the VITEK 2 system with those obtained by the MB method within +/-twofold dilution, it was determined that the 1,067 organism-antibiotic combinations had an overall correct rate of 97.6% (1,041 combinations). The rates of susceptibility to the 11 antibiotics ranged from 88.7 to 100%, respectively. Only two (0.2%) and four (0.4%) combinations of the susceptibility tests gave very major errors (i.e., reported as sensitive by the VITEK 2 system but shown to be resistant by the MB method) and major errors (i.e., reported as resistant by the VITEK 2 system but shown to be sensitive by the MB method), respectively. The reporting time for the direct testing of susceptibility against the 11 antibiotics for 97 blood culture isolates by the VITEK 2 system ranged from 3.3 to 17.5 h. Compared with conventional methods that require 1 or 2 days, this method can make same-day reporting possible and thus permit better patient management.     

Evaluation of the Merlin MICRONAUT system for rapid direct susceptibility testing of gram-positive cocci and gram-negative bacilli from positive blood cultures

Bloodstream infections are life-threatening conditions which require the timely initiation of appropriate antimicrobial therapy. We evaluated the automated Merlin MICRONAUT system for rapid direct microtiter broth antimicrobial susceptibility testing (AST) of gram-positive cocci and gram-negative bacilli from BACTEC 9240 bottles with positive blood cultures in comparison to the standard method for the Merlin MICRONAUT system. This prospective study was conducted under routine working conditions during a 9-month period. Altogether, 504 isolates from 409 patients and 11,819 organism-antibiotic combinations were evaluated for comparison of direct and standard AST methods. For gram-negative bacilli, direct and standard AST of 110 isolates was evaluated and MIC agreement was found for 98.1% of 2,637 organism-antibiotic combinations. Category (susceptible, intermediate susceptible, resistant [SIR]) agreement was found for 99.0%, with results for 0.04% of combinations showing very major errors, those for 0.2% showing major errors, and those for 0.8% showing minor errors. For gram-positive cocci, 373 isolates were evaluated and MIC agreement was found for 95.6% of 8,951 organism-antibiotic combinations. SIR agreement was found for 98.8%, with results for 0.3% of combinations showing very major errors, those for 0.4% showing major errors, and those for 0.5% showing minor errors. Although the number of tested isolates was limited (n = 33), direct AST of streptococci was performed for the first time, yielding promising results with SIR agreement for 98.6% of 363 organism-antibiotic combinations. In conclusion, direct AST of gram-negative bacilli and gram-positive cocci from positive blood cultures with the MICRONAUT system is a reliable technique that allows for the omission of repeat testing of subcultured isolates. Thereby, it reduces the time to results of blood culture testing and may have a positive impact on patient care.     

Evaluation of the Hyplex BloodScreen Multiplex PCR-Enzyme-linked immunosorbent assay system for direct identification of gram-positive cocci and gram-negative bacilli from positive blood cultures

We evaluated the Hyplex BloodScreen PCR-enzyme-linked immunosorbent assay (ELISA) system (BAG, Lich, Germany), a new diagnostic test for the direct identification of gram-negative bacilli and gram-positive cocci from positive blood cultures, with 482 positive BACTEC 9240 blood culture bottles. The test involves amplification of the bacterial DNA by multiplex PCR and subsequent hybridization of the PCR product to specific oligonucleotide probes in an ELISA-based format. The available probes allow the separate detection of Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella spp., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis/Enterococcus faecium, Streptococcus pyogenes, and Streptococcus pneumoniae and the staphylococcal mecA gene. The Hyplex BloodScreen test showed an overall sensitivity of 100% for the identification of gram-negative bacilli and 96.6 to 100% for the identification of gram-positive cocci (S. aureus, 100%; S. epidermidis, 97.2%; Enterococcus faecalis/Enterococcus faecium, 96.6%; and Streptococcus pneumoniae, 100%). The specificities of the test modules ranged from 92.5 to 100% for gram-negative bacilli and 97.7 to 100% for gram-positive cocci (Escherichia coli, 92.5%; Pseudomonas aeruginosa, 98.5%; Klebsiella spp., 100%; Enterobacter aerogenes, 100%; S. aureus, 100%, S. epidermidis, 97.7%; Enterococcus faecalis/Enterococcus faecium, 99.6%; Streptococcus pyogenes, 100%; and Streptococcus pneumoniae, 99.3%). The result of the mecA gene detection module correlated with the result of the phenotypic oxacillin resistance testing in all 38 isolates of Staphylococcus aureus investigated. In conclusion, the Hyplex BloodScreen PCR-ELISA system is well suited for the direct and specific identification of the most common pathogenic bacteria and the direct detection of the mecA gene of Staphylococcus aureus in positive blood cultures.     

Rapid automated identification of gram-negative bacilli from blood cultures with the AutoMicrobic system.

Automated identification of gram-negative bacilli directly from blood culture bottles by using the AutoMicrobic System (AMS) was evaluated with a modified procedure for the AMS Enterobacteriaceae-plus nonfermenter identification card. A total of 150 strains were tested (44 clinical and 106 seeded) and compared with a conventional identification procedure. These strains included 107 Enterobacteriaceae and 43 oxidase-positive or glucose-nonfermenting, or both, organisms. AMS identifications on one of these strains were not interpretable owing to equal probability AMS identification values. Of the remaining 149 strains, 138 (92.6%) were correctly identified within 8 to 13 h of the first reading. Of 69 identifications analyzed after 6 h of incubation, 91% were correct. This procedure was found to be rapid, convenient, and nonlabor intensive and is recommended for presumptive identification of gram-negative bacilli in blood cultures.     

Evaluation of a new selective chromogenic agar medium for detection of extended-spectrum beta-lactamase-producing Enterobacteriaceae

A novel chromogenic agar medium (ESBL-Bx; bioMérieux, Marcy l'Etoile, France) was compared to MacConkey agar supplemented with 2 mg ceftazidime/liter (MCKC) for the selective isolation and presumptive identification of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae directly from clinical samples. Of a total of 644 clinical specimens (including 551 fecal samples), 496 yielded no growth and 148 yielded growth on one or both media. Overall, 44 ESBL-producing Enterobacteriaceae strains (Escherichia coli [n=17], Enterobacter aerogenes [n=17], Klebsiella spp. [n=5], and Citrobacter freundii [n=5]) were isolated from 37 specimens by a combination of both methods after 18 to 24 h of incubation. The sensitivities were 97.7 and 84.1% for ESBL-Bx and MCKC, respectively, with 43 ESBL-positive strains isolated as colored colonies from 36 specimens on ESBL-Bx versus 37 ESBL-positive organisms isolated from 32 specimens on MCKC. The specificities by specimens were 89 and 91% for ESBL-Bx and MCKC, respectively. On either one of the two media, natural AmpC-hyperproducing Enterobacter spp. (n=25) and Citrobacter spp. (n=14) were the most common false positives as well as non-ESBL-producing Klebsiella oxytoca (n=18) on ESBL-Bx and Morganella morganii (n=10) on MCKC. We conclude that ESBL-Bx is a sensitive and specific medium for the isolation of ESBL-producing Enterobacteriaceae from clinical samples. The main advantages of ESBL-Bx over MCKC reside in its chromogenic character and its sensitivity and selectivity, which enabled the recovery and presumptive identification of most ESBL-producing Enterobacteriaceae within 24 h and reduced by 27% the need for unnecessary identification and confirmation of ESBL testing when disregarding all colorless colonies growing on this medium.     

Rapid detection of positive blood cultures

A study of 16,108 blood cultures investigated the value of performing early smears in addition to the inspection of early subcultures after 12 h of incubation. The early smear and subculture detected 44.6% of 1,236 positive cultures; of those detected by the early subculture, 57.3% grew after 12 h of incubation.     

Evaluation of detection of positive blood cultures by pH changes.

We observed that the pH of positive blood culture broths was significantly lower than that of negative broths. However, significantly pH changes were generally not observed for subculture-positive, macroscopically negative blood culture broths. Therefore, although the pH of most positive blood culture broths was reduced significantly, this change was generally not useful for the early detection of positive cultures.     

Rapid identification and antimicrobial susceptibility testing of gram-negative bacilli from blood cultures by the AutoMicrobic system.

A procedure was developed which allows direct identification and antimicrobial susceptibility testing of fermentative and nonfermentative gram-negative bacilli from positive blood cultures. A 10-ml sample was removed from turbid blood culture bottles, and the bacteria were washed and concentrated by centrifugation. The bacterial pellet was used to inoculate an Enterobacteriaceae Plus Identification Card and a Gram-Negative General Susceptibility Card of the AutoMicrobic system. Results with these cards were compared with results obtained with standard technique for 196 blood cultures seeded with recent clinical isolates. Identification of most cultures was available in 8 h, whereas the antimicrobial susceptibility results were available in an average of 4.7 h for all organisms. Direct identification was correct for 95% of the cultures, whereas the antimicrobial susceptibility data had an average agreement of 87% with 3.8% very major and 1.4% major errors. In using this procedure it was possible to provide accurate preliminary identification and results of antimicrobial susceptibility tests for gram-negative bacilli on the same day that a blood culture was determined to be positive.     

Evaluation of four anti-microbic susceptibility testing systems for gram-negative bacilli

A total of 200 clinical isolates were assayed by five anti-microbic susceptibility testing systems. Two frozen minimal inhibitory concentration (MIC) systems (MicroScan and Pasco), an automated MIC system (AMS, Vitek Systems), and the standard disk diffusion were compared with a reference broth dilution method. Organisms tested included 100 resistant clinical stock strains and 100 fresh random clinical isolates. Overall, there were 1,600 anti-microbic-organism combinations analyzed. The Pasco and MicroScan systems had no major discrepancies, the AMS system had seven, and the disk diffusion two. The number of very major discrepancies were as follows: AMS, 11; disk diffusion, 9; MicroScan, 5; Pasco, 2. Of the total 36 major or very major discrepancies in the study, 33% (12 of 36) were with an aminoglycoside and 44% (16 of 36) occurred with a second-generation cephalosporin, of which 10 of 16 were with cefamandole. Overall, there was a greater than 98.8% essential agreement with all systems compared with the reference method.     

Evaluation of PASCO MIC-ID system for identifying gram-negative bacilli.

A production model of the semi-automated PASCO MIC-ID system (PASCO Laboratories, Wheat Ridge, Colo.) was evaluated with 122 groups or species of gram-negative bacilli, which included typical (499 cultures) and atypical (37 cultures) strains of fermenters and nonfermenters. The PASCO system identified 90.9% of 536 cultures accurately; these included 90.8% of 152 nonfermenters, 93.8% of 308 enteric fermenters, and 78.9% of 76 oxidase-positive fermenters. These results were obtained with the aid of serologic tests and a few additional biochemical tests, when recommended by the PASCO system. Of the 14 misidentified nonfermenters, 3 were Pseudomonas paucimobilis, 3 were Weeksella virosa (Centers for Disease Control group IIf), 2 were Xanthomonas (Pseudomonas) maltophilia, and 6 were randomly distributed among the other groups and species tested. The 19 enteric fermenters that were misidentified were randomly distributed among the groups and species tested. Of the 16 misidentified oxidase-positive fermenters, 4 were Pasteurella ureae, and 12 were randomly distributed among the other groups and species. The system identified the most commonly encountered organisms at a rate of 95% or better. The PASCO system is easy to inoculate and read. A slightly improved data base should remedy most of the identification problems.     

Evaluation of the Minitek system for direct identification of anaerobic rods from positive blood cultures

The direct inoculation of the Minitek anaerobe identification system (BBL Microbiology Systems, Cockeysville, Md.) from positive blood cultures was compared with subculture and Minitek results obtained using the manufacturer's recommended procedures. A total of 40 clinical anaerobic blood cultures were processed for rapid identification utilizing bacterial pellets obtained by centrifugation. Of these cultures, 30 yielded pure isolates of anaerobic rods that were used for comparison. In 87% of the pure cultures, identification from the direct inoculum method was identical to the routine procedure using Minitek biochemicals. When the additional test for lecithinase and lipase production was included, the identification agreement was 97%. Direct identification of anaerobic rods from blood cultures utilizing the Minitek system is reliable, easy to perform, and can provide a complete identification in 24 h from the time a blood culture showed growth.     

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.     

Evaluation of three rapid methods for the direct identification of Staphylococcus aureus from positive blood cultures

Staphylococci represent the most commonly encountered blood culture isolates. Differentiating Staphylococcus aureus from coagulase-negative staphylococci (CoNS) is important in guiding empirical therapy, especially since the majority of CoNS are contaminants. This study evaluated three rapid methods for the direct identification of S. aureus from blood cultures. A total of 157 patient blood cultures with gram stains showing gram-positive cocci in clusters were included. The following assays were evaluated: API RAPIDEC staph (API) (bioMerieux, Durham, N.C.), the tube coagulase test (TCT) read at 4 h, and peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) (AdvanDx, Woburn, Mass.). All assays yielded results of S. aureus or non-S. aureus. The direct rapid results were compared to results obtained with isolated colonies using the AccuProbe Staphylococcus aureus Culture Identification Test (Gen-Probe, San Diego, Calif.). API, TCT, and PNA FISH exhibited sensitivities of 96, 84, and 99% and specificities of 99, 100, and 100%, respectively. Direct identification testing by any of these three assays yielded acceptable performance and timely results. This ability to accurately detect S. aureus in blood cultures gives the physician information with which to initiate or tailor antimicrobial therapy. Coupled with direct susceptibility testing of positive blood culture broths, the patient and institution may experience improved outcomes.     

Resistance of gram-negative bacilli and staphylococci from blood cultures to aminoglycoside antibiotics. Comparison of 3 in vitro investigations from Austria 1982-1988

A total of 790 blood culture isolates was collected during 3 study periods in 1982/83, 1984/85 and 1987/88. Staphylococci were the most frequent bacteria in the first two periods (56.5% and 63%, respectively). During the last period, E. coli was the most frequent of all species (27%). Differences in the distribution of bacteria between the laboratories were considerable. In one laboratory in Vienna, coagulase-negative staphylococci dominated in all 3 study periods (32%, 33% and 47%). Susceptibility against gentamicin, tobramycin and netilmicin (MIC less than or equal to 4 mg/l), as well as against amikacin (MIC less than or equal to 8 mg/l) were determined by a microdilution method. The resistance rates against gentamicin (G) were 25%, 21% and 25%, against tobramycin (T) 27%, 19% and 25%, against netilmicin (N) 6%, 4% and 19%, and against amikacin (A), 5%, 2% and 19%. Most resistant strains were staphylococci (G 26%-41%, T 26%-46%, N 3%-31%, A 3%-36%), whereas gram-negative bacilli were more susceptible (G 12%-14%, T 7%-11%, N 7%-9%, A 1%-7.5%). The increase of resistance against netilmicin and amikacin in staphylococci was most striking. Nearly all those strain could be attributed to one laboratory in Vienna. Most of them were coagulase-negative staphylococci. In the first study period, the most frequent pattern was resistance against gentamicin and tobramycin, whereas in the last period resistance to all 4 aminoglycosides dominated. The study demonstrates considerable local differences in antibiotic resistance. Monitoring of antibiotic resistance in collaborative studies using standardized methods should be a valuable tool in planning strategies for controlling an epidemic spread of resistance.     

Infection with an extended-spectrum beta-lactamase-producing strain of Serratia marcescens following tongue reconstruction

We report a case of postsurgical wound infection of polymicrobial etiology caused by Serratia marcescens and Pseudomonas aeruginosa following the use of a radial forearm free flap for oncological tongue reconstruction. S. marcescens was a producer of SHV-12 extended-spectrum beta-lactamase (ESBL). This is the first report from India of this ESBL. S. marcescens and P. aeruginosa were resistant to the empirical perioperative antibiotics administered. Delay in the recognition of the type of infection and in the institution of appropriate therapy resulted in total loss of the free flap.     

Usefulness of the MicroSeq 500 16S rDNA bacterial identification system for identification of anaerobic Gram positive bacilli isolated from blood cultures

Using full 16S ribosomal RNA (rRNA) gene sequencing as the gold standard, 20 non-duplicating anaerobic Gram positive bacilli isolated from blood cultures were analysed by the MicroSeq 500 16S rDNA bacterial identification system. The MicroSeq system successfully identified 13 of the 20 isolates. Four and three isolates were misidentified at the genus and species level, respectively. Although the MicroSeq 500 16S rDNA bacterial identification system is better than three commercially available identification systems also evaluated, its database needs to be expanded for accurate identification of anaerobic Gram positive bacilli.