Selection of human antibodies against cell surface-associated oligomeric anthrax protective antigen

The protective antigen (PA(83)) of Bacillus anthracis is the dominant antigen in natural and vaccine-induced immunity to anthrax infection. Three human single-chain variable fragments (scFvs) against cell bound PA were isolated from an antibody phage display library. Specifically, the antibodies were evaluated for their ability to bind to cell bound heptameric PA and ultimately protect against the cytotoxicity of lethal toxin. In total, all three scFvs possessed neutralizing activity against the cytotoxic effects of lethal toxin in a macrophage lysis assay. The K(d) values of the Fabs were determined, interestingly their protective effects did not parallel their affinities; hence, a simple binding argument alone to PA(63) cannot be used as the distinguishing feature for the prediction of their neutralization abilities. Immunofluorescent microscopy experiments were conducted and provided strong evidence for Fab binding to oligomeric PA on the cell surface and thus a plausible mechanism for the toxin neutralization activity that was observed. The results of this study presented herein suggest that our antibodies compete with LF-PA cell surface interactions, and thus may provide potential application of human antibodies as passive immunization prophylactics in cases of B. anthracis exposure and infection.     

Biochemical identification of citrobacteria in the clinical laboratory.

We biochemically identified 235 Citrobacter strains to the species level on the basis of the recently proposed taxonomic changes of Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993). Citrobacter isolates were initially identified as C. koseri or as members of the C. freundii complex or C. amalonaticus group on the basis of indole production, formation of H2S, malonate utilization, and acid production from D-arabitol and adonitol. On the basis of the results of these tests, 68% of the Citrobacter strains were identified as members of the C. freundii complex, 25% were C. koseri, and 8% were members of the C. amalonaticus group. By using a 15-test system recently proposed by Brenner et al. (D. J. Brenner, P. A. D. Grimont, A. G. Steigerwalt, G. R. Fanning, E. Ageron, and C. F. Riddle, Int. J. Syst. Bacteriol. 43:645-658, 1993) to help identify new species in the C. freundii complex and C. amalonaticus group, 81% of the C. freundii complex strains and 100% of the C. amalonaticus strains could be definitively assigned to one of the previously established or recently designated species or hybridization groups of the genus Citrobacter. Within the C. freundii complex, C. freundii predominated overall (37%), followed by C. youngae (24%), C. braakii (13%), and C. werkmanii (6%). Only one strain each of C. sedlakii and Citrobacter DNA group 11 was identified in this study. Among C. amalonaticus complex members, all were identified as C. amalonaticus with the singular exception of one fecal isolate of C. farmeri. C. freundii and C. koseri were the two Citrobacter species most commonly (80 of 93 [86%]) isolated from extraintestinal sources (genitourinary tract, wounds, blood).     

Biotyping of Aeromonas isolates as a correlate to delineating a species-associated disease spectrum.

A group of 147 Aeromonas isolates from diverse clinical and environmental sources was subjected to the biotyping scheme of Popoff and Veron. Of the 147 isolates biotyped, 137 (93%) could be identified, with Aeromonas hydrophila predominating (48%) and equal percentages (25 to 27%) of the other two species (Aeromonas sobria and Aeromonas caviae). A number of additional biochemical properties were found to be significantly associated with one or more of these three species. These included lysine decarboxylase activity, hemolysis of sheep erythrocytes, lecithinase production, staphylolytic activity, arbutin hydrolysis, and acid production from utilization of various carbohydrates. By incorporating these phenotypic properties into an extended biotyping system, 98% of the isolates were identified. Selective distribution of individual species with respect to certain body sites was noted.     

Multicenter evaluation of the new Vitek 2 Neisseria-Haemophilus identification card

The new Neisseria-Haemophilus identification (NH) card for Vitek 2 was compared with 16S rRNA gene sequencing (16S) as the reference method for accurate identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria. Testing was performed on the Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains tested 20 times over a minimum of 10 days at all three sites. A challenge set of 30 strains with known identifications and 371 recent fresh and frozen clinical isolates were also tested. Expected positive and negative biochemical reactions were also evaluated for substrate reproducibility. All microorganisms were tested on the NH card, and all clinical and stock isolates were saved for 16S testing. All reproducibility tests yielded expected results within a 95% confidence interval. For challenge microorganisms, there was 98% overall correct identification, including 8% low discrimination, 2% incorrect identification, and 0% unidentified. For clinical strains, there was 96.5% overall correct identification, including 10.2% low discrimination, 2.7% incorrect identification, and 0.8% unidentified. The 2.7% (10/371) of clinical isolates that gave an incorrect identification consisted of 7 isolates correct to genus and 3 strains incorrect to genus. There were an additional 27 strains (primarily Neisseria species) for which the 16S identification result was different from the NH card result. These were all unclaimed species by the system. The new NH card met all performance criteria within a 95% confidence interval compared to identification of clinical isolates by 16S.     

Prototypal diarrheagenic strains of Hafnia alvei are actually members of the genus Escherichia.

We analyzed five bacterial strains, designated 19982, 9194, 10457, 10790, and 12502, that were isolated from stool specimens of individuals with diarrheal illness by the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh (M. J. Albert, S. M. Faruque, M. Ansaruzzaman, M. M. Islam, K. Haider, K. Alam, I. Kabir, and R. Robins-Browne, J. Med. Microbiol. 37:310-314, 1992). The strains were initially identified as Hafnia alvei with a commercial identification system and were reported to contain the eae gene of enteropathogenic Escherichia coli. Results of conventional biochemical analyses, testing of susceptibility to cephalothin, lysis by a Hafnia-specific phage, and amplification of the outer membrane protein gene phoE with species-specific primers support the identification of these strains as members of the genus Escherichia rather than Hafnia alvei. These strains varied from typical E. coli strains by their inability to produce acid from lactose or D-sorbitol and failure to elaborate the enzyme beta-D-glucuronidase. PCR analysis confirmed previous findings that the strains were positive for the eae gene and negative for other virulence markers present among recognized categories of diarrheagenic E. coli. Our findings support the hypothesis that these strains are a new category of diarrheagenic isolates belonging to the genus Escherichia and illustrate the importance of using multiple methodologies when identifying new bacterial agents of diarrheal disease.