Automated methods for identification of bacteria from clinical specimens.

Automated methods for measuring enzyme activities of bacterial suspensions in saline are described. The methods were applied to bacteria cultured from urine specimens, and specific enzyme profiles characteristic for Escherichia coli, Klebsiella sp, Proteus sp, and Pseudomonas sp were established. Identification of 294 freshly isolated strains by automated and conventional methods were compared. Results from automated identification based on eight enzyme tests and assay of protein content, all performed on a bacterial suspension made from one colony in 1 ml of saline, agreed 100% with those obtained by conventional methods. Identification was achieved in 6 hours.     

Resistance mechanisms of gram-positive bacteria

The introduction and increasing use of antibiotics for antibacterial therapy has initiated a rapid development and expansion of antibiotic resistance in microorganisms, particularly in human pathogens. Additionally, a shift to an increase in number and severity of Gram-positive infections has been observed the last decades. Common to these pathogens is their tendency to accumulate multiple resistances under antibiotic pressure and selection. Methicillin-resistant Staphylococcus aureus (MRSA), that have acquired multiresistance to all classes of antibiotics, have become a serious nosocomial problem. Recently, the emergence of the first MRSA with reduced vancomycin susceptibility evoked the specter of a totally resistant S. aureus. Problems with multiresistance expand also to penicillin-resistant Streptococcus pneumoniae that are partially or totally resistant to multiple antibiotics, and to vancomycin-resistant Enterococcus ssp., completely resistant to all commonly used antibiotics. The rapid development of resistance is due to mutational events and/or gene transfer and acquisition of resistance determinants, allowing strains to survive antibiotic treatment.     

Performances of VITEK 2 colorimetric cards for identification of gram-positive and gram-negative bacteria

The purpose of this study was to evaluate the new VITEK 2 identification cards that use colorimetric reading to identify gram-positive and gram-negative bacteria (GP and GN cards, respectively) in comparison to fluorimetric cards (ID-GPC and ID-GNB, respectively). A total of 580 clinical isolates and stock collection strains belonging to 116 taxa were included in the study. Of the 249 gram-positive strains tested with both the ID-GPC and GP cards, 218 (87.5%) and 235 (94.4%) strains were correctly identified (to the genus and species level), respectively. Of the 331 gram-negative strains tested with the ID-GNB and GN cards, 295 (89.1%) and 321 (97%) strains were correctly identified, respectively. Another focus of the study was to apply the percentages of correct identifications obtained in this study to the list of bacteria isolated in our laboratory (32,739 isolates) in the year 2004. We obtained 97.9% correct identifications with the colorimetric cards and 93.9% with fluorescent cards.     

Methods for distinguishing gram-positive from gram-negative bacteria.

Lysis by KOH and hydrolysis of L-alanine-4-nitroanilide were compared with the Gram reaction of aerobic, microaerophilic, and anaerobic bacteria. Both tests correlated well with the Gram reaction with nonfermentative bacilli and Bacillus species, whereas they did not correlate with nonsporulating anaerobes. Only campylobacteria were KOH positive and L-alanine-4-nitroanilide and gram negative.     

Identification of gram-positive coccal and coccobacillary vancomycin-resistant bacteria.

A total of 84 of 150 vancomycin-resistant (defined as no inhibition of bacterial growth around a 30-micrograms vancomycin disk placed on 5% sheep blood-Trypticase soy agar [BBL Microbiology Systems, Cockeysville, Md.]) bacteria were definitively identified by determining the phenotypic criteria. The identity of representatives was also confirmed by DNA-DNA hybridizations. The following strains were identified: 1 Enterococcus faecium, 18 Leuconostoc mesenteroides, 15 Leuconostoc citreum, 9 Leuconostoc pseudomesenteroides, 2 Leuconostoc lactis, 20 Pediococcus acidilactici, 5 Pediococcus pentosaceus, and 14 Lactobacillus confusus. The remaining vancomycin-resistant strains were identified as probable Leuconostoc (6 strains), probable Pediococcus (1 strain), and probable lactobacilli (28 strains). A total of 32 strains of gram-positive coccobacillary bacteria remained unidentified. Tests used for the phenotypic identification of strains to the genus level included a Gram stain of bacteria grown in thioglycolate broth, gas production in Lactobacillus Mann, Rogosa, and Sharpe broth, hydrolysis of pyrrolidonyl-beta-naphthylamide, bile-esculin reaction, demonstration of streptococcal group D antigen, and growth at 10 and 45 degrees C and in 6.5% NaCl broth. Strains were identified to the species level by hydrolysis of esculin, reactions in litmus milk, slime production on 5% sucrose agar, acidification of maltose, melibiose, and raffinose broths, deamination of arginine, and growth at 42 degrees C and in 6.5% NaCl broth.     

Evaluation of the E test by using selected gram-positive bacteria.

The E test (AB Biodisk NA Inc.) was compared with standard reference methods using the National Committee for Clinical Laboratory Standards's recommendations for determining the MICs of four selected antibiotics against 208 clinical isolates of gram-positive bacteria. These bacteria included 32 strains of Streptococcus pneumoniae, 25 strains of Enterococcus faecium, 20 strains of oxacillin-sensitive Staphylococcus aureus (OSSA), 96 strains of oxacillin-resistant S. aureus (ORSA), and 35 strains of coagulase-negative staphylococci. Evaluation included MIC accuracy within 1 dilution, reproducibility testing, and cost analysis. There was 94% agreement between the E test and the reference method in testing S. pneumoniae and penicillin G. There was 92% agreement with ampicillin and 100% agreement with vancomycin in testing E. faecium isolates. Accuracy of the oxacillin E test with staphylococci was significantly improved by the use of salt-supplemented Mueller-Hinton agar, for an agreement of 100% with coagulase-negative staphylococci and oxacillin-sensitive S. aureus and that of 85% with oxacillin-resistant S. aureus, with no major discrepancies. The E test with American Type Culture Collection isolates and clinical strains gave excellent reproducibility and was less costly than microdilution panels when used to test fewer than three antibiotics. The E test is a simple, reliable, reproducible, and cost-effective method for MIC determination for gram-positive organisms.     

Automated systems for identification of microorganisms.

Automated instruments for the identification of microorganisms were introduced into clinical microbiology laboratories in the 1970s. During the past two decades, the capabilities and performance characteristics of automated identification systems have steadily progressed and improved. This article explores the development of the various automated identification systems available in the United States and reviews their performance for identification of microorganisms. Observations regarding deficiencies and suggested improvements for these systems are provided.     

Vancomycin-resistant gram-positive bacteria isolated from human sources

Recent reports of infections with vancomycin-resistant gram-positive bacteria prompted us to study vancomycin-resistant isolates from human sources to characterize the types of bacteria displaying this phenotype. Thirty-six vancomycin-resistant gram-positive isolates, 14 from clinical specimens and 22 from stool samples, were identified. These isolates were tentatively identified as Lactobacillus spp. (25 strains), Leuconostoc spp. (6 strains), and Pediococcus spp. (3 strains) on the basis of morphology and physiological tests. Two isolates of indeterminate morphology could not be unambiguously assigned to a genus. Four isolates of vancomycin-resistant lactobacilli from normally sterile body sites were considered to be clinically significant. Vancomycin-resistant gram-positive bacteria may represent an emerging class of nosocomial pathogens. Better methods for distinguishing the various genera in the clinical microbiology laboratory are needed.     

The laboratory identification of gram-positive anaerobic cocci

A collection of 256 clinical strains and 40 reference strains of gram-positive anaerobic cocci (GPAC) was studied, to characterise the recognised species more fully and to define groups of strains which might correspond to previously undescribed species. The methods used were: gas-liquid chromatography (GLC) for the detection of volatile fatty acids (VFAs); determination of the pre-formed enzyme profile with a commercially available kit, ATB 32A; microscopic appearance; colonial morphology; and antibiotic sensitivity tests. Strains were placed in one of five VFA groups according to their GLC profile; 96% of strains were further assigned to 12 groups by their enzyme profile. There was less than 99% agreement between the two methods. Of 111 clinical strains in the VFA-negative group, 110 gave one of three distinct enzyme profiles corresponding to Peptostreptococcus magnus, P. micros and P. heliotrinreducens. The assignment of strains to groups based on their microscopic appearance and colonial morphology agreed well with groupings according to enzyme profile. Identification of butyrate-producing GPAC was unsatisfactory because it relied heavily on the enzyme profile; testing for indole production was of limited discriminative value. Most strains of P. asaccharolyticus and P. indolicus were very similar in enzyme profile, microscopic appearance and colonial morphology, but a sub-group of P. asaccharolyticus could be distinguished. A further indole-positive group corresponding to Hare group III was also noted. Strains of P. prevotii and P. tetradius were very similar, but easily distinguished from other butyrate-producing GPAC. However, 45% of the butyrate-producing cocci could not be assigned to recognised species; most of these were assigned to one of two new groups, the ADH group and the bGAL group, by their enzyme profile, microscopic appearance and smell. Four strains that produced a terminal VFA peak of isovaleric acid formed a new group designated 'ivoricus'. Reliable features for the identification of P. anaerobius were GLC (all GPAC that produced isocaproic acid were identified as P. anaerobius), enzyme profile and sensitivity to SPS. Two clinical strains that produced caproci acid were identified as Hare group VIII; they were distinguished from Peptococcus niger by their enzyme profile and colonial morphology. A phenotypic classification based on GLC and enzyme profile is presented, with a method for the identification of most strains of GPAC within 48 h of primary isolation.     

Phoenix 100 versus Vitek 2 in the identification of gram-positive and gram-negative bacteria: a comprehensive meta-analysis

Phoenix 100 and Vitek 2 (operating with the current colorimetric cards) are commonly used in hospital laboratories for rapid identification of microorganisms. The present meta-analysis aims to evaluate and compare their performance on Gram-positive and Gram-negative bacteria. The MEDLINE database was searched up to October 2010 for the retrieval of relevant articles. Pooled correct identification rates were derived from random-effects models, using the arcsine transformation. Separate analyses were conducted at the genus and species levels; subanalyses and meta-regression were undertaken to reveal meaningful system- and study-related modifiers. A total of 29 (6,635 isolates) and 19 (4,363 isolates) articles were eligible for Phoenix and colorimetric Vitek 2, respectively. No significant differences were observed between Phoenix and Vitek 2 either at the genus (97.70% versus 97.59%, P = 0.919) or the species (92.51% versus 88.77%, P = 0.149) level. Studies conducted with conventional comparator methods tended to report significantly better results compared to those using molecular reference techniques. Speciation of Staphylococcus aureus was significantly more accurate in comparison to coagulase-negative staphylococci by both Phoenix (99.78% versus 88.42%, P < 0.00001) and Vitek 2 (98.22% versus 91.89%, P = 0.043). Vitek 2 also reached higher correct identification rates for Gram-negative fermenters versus nonfermenters at the genus (99.60% versus 95.90%, P = 0.004) and the species (97.42% versus 84.85%, P = 0.003) level. In conclusion, the accuracy of both systems seems modified by underlying sample- and comparator method-related parameters. Future simultaneous assessment of the instruments against molecular comparator procedures may facilitate interpretation of the current observations.     

Automated, rapid identification of bacteria by pattern analysis of growth inhibition profiles obtained with Autobac 1.

A scheme for identifying bacteria has been devised which utilizes the inhibition patterns obtained by Autobac 1 with routine and unusual antimicrobial agents and with other differentially inhibitory chemical compounds. Over 600 compounds were initially identified from the literature, and over 125 of these were selected for further testing on the basis of antibacterial activity most conducive to the instrument-generated differential scheme. Numerical growth index information derived by light scatter comparisons from the instrument were analyzed by computer, utilizing the quadratic discriminant function statistical technique. In comparison with conventional methods, accuracy for the 10 bacterial genera studied was 95% or greater. Results indicate a potential for both bacterial identification and antimicrobial agent susceptibility testing in the clinical laboratory within 3 to 5 h when using this automated approach.     

Evaluation of the automicrobic system gram-positive identification card for species identification of coagulase-negative staphylococci.

The AutoMicrobic system Gram-Positive Identification Card (Vitek Systems, Inc., Hazelwood, Mo.) was evaluated for identification of a group of 150 isolates of coagulase-negative staphylococci. Identifications obtained with the Gram-Positive Identification Card were compared with reference identifications derived from 15 conventional biochemical tests. The AutoMicrobic system correctly identified only 67.3% (101 of 150) of the test isolates. The greatest accuracy was achieved with Staphylococcus epidermidis isolates (95.7%), whereas Staphylococcus hominis isolates were least often correctly identified (26.7%).     

Activation of the alternative complement pathway by L-phase variants of gram-positive bacteria.

The present studies were performed to investigate the potential role of the alternative complement pathway in the host's defense against bacterial L-phase variants and to gain insight into the subcellular component of gram-positive bacteria responsible for activation of the alternative pathway. L-phase variants of Staphylococcus aureus and Streptococcus faecalis were able to activate the alternative pathway and consume C3 in C4-deficient guinea pig serum in amounts comparable to their respective bacterial-phase parent organisms. Activation of the complement system via the alternative pathway resulted in death of the L-phase variants. Membranes prepared from S. faecalis L-phase variants, by either osmotic lysis or mechanical disruption, retained their ability to activate the alternative pathway. Treatment of the membranes by three different methods (water washes, hot trichloroacetic acid, and cold trichloroacetic acid) resulted in a greatly diminished ability of the membranes to activate the alternative pathway. In addition, the extracts derived from the membranes by water washes and by cold-trichloroacetic acid treatment were able to activate the alternative pathway. These studies indicate that these L-phase variants can activate the alternative pathway and suggest that membrane-associated factors play a role in the alternative pathway activation by S. faecalis L-phase variants.     

Impermeability to quinolones in gram-positive and gram-negative bacteria

The initial step in the accumulation of fluoroquinolone antimicrobial agents is binding to cell surface components reduced by lowered pH and divalent cations. Uptake into gram-negative and gram-positive bacteria is by simple diffusion. Entry through the outer membrane occurs preferentially for most agents by the porin route but a second process using the self-promoted uptake pathway is active especially for more hydrophobic agents. Fluoroquinolones bind to vesicles of phospholipid which may be the initiating step in cross-cytoplasmic membrane diffusion. An active efflux system has been described inEscherichia coli with evidence supporting its presence in several other bacteria. Total uptake is not altered by a resistant gyrase. Resistant isolates associated with reduced total quinolone accumulation due to lowered uptake have been described for laboratory mutants and clinical isolates. Most but not all of these have had alterations in outer membrane proteins. A functionally dominant resistance gene has been cloned from resistantStaphylococcus aureus and codes for a highly hydrophobic protein most likely membrane associated. This gene is expressed inEscherichia coli and specifies resistance especially to hydrophilic quinolones, possibly by altered accumulation.     

Rapid identification of obligately anaerobic gram-positive cocci using high-performance liquid chromatography.

High-performance liquid chromatography was evaluated as a rapid means of identifying obligately anaerobic gram-positive cocci of medical interest. Isolates were inoculated into a defined chemical medium consisting primarily of amino acids and were incubated aerobically for 1 h at 35 degrees C. After removal of organisms, the supernatant fluids were derivatized for 1 min at room temperature by the addition of o-phthalaldehyde. The total time required to run a chromatogram was approximately 50 min. Standardized peak heights for each medium component and any new peaks formed were calculated for each isolate and compared with those for uninoculated control medium. Multiple isolates of various species of anaerobic gram-positive cocci gave consistent patterns of medium utilization that could be used for identification purposes. This method can easily be adapted for laboratory use and has the potential for automated microbial identification.     

Rapid method for the differentiation of gram-positive and gram-negative bacteria on membrane filters.

Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. We developed a rapid and sensitive method for the differentiation of gram-positive and gram-negative bacteria, utilizing a polycarbonate membrane filter, crystal violet, iodine, 95% ethanol, and 6% carbol fuchsin, that can be completed in 60 to 90 s. Gram reactions of 49 species belonging to 30 genera of bacteria were correctly determined by the filter-Gram stain. The sensitivities of the filter-Gram stain and conventional slide-Gram stain were compared by testing dilutions of Escherichia coli, Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae suspensions in the presence and absence of whole human blood. The filter-Gram stain was approximately 100-fold more sensitive than the slide-Gram stain. The filter-Gram stain detected 2 to 100 bacteria, whereas the slide-Gram stain failed to detect less than 1,000 bacteria. The sensitivities of the methods were not significantly altered by the addition of whole human blood to the dilutions of bacteria tested. The filter-Gram stain could be a useful tool for the examination of body fluids with very low numbers of bacteria.