Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes

Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-D-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.     

The structure of the cell wall peptidoglycan of Bacillus cereus RSVF1, a strain closely related to Bacillus anthracis

The peptidoglycan of Bacillus cereus RSVF1, a close relative of Bacillus anthracis, has several distinguishing features: the overwhelming majority of cross-linked muropeptides are dimers, higher oligomers are only present in minute quantities; and virtually all muropeptides lack the N-acetyl group from glucosamine residues, thus explaining resistance of the cell walls to lysozyme.     

Administration of a highly attenuated, live respiratory syncytial virus vaccine to adults and children.

A highly attenuated respiratory syncytial virus (RSV) experimental vaccine, RSV ts-2, was sequentially evaluated in adults, seropositive children, and finally, fully susceptible seronegative children. The vaccine was administered intranasally in doses ranging from 10(5.2) to 10(6.3) PFU/ml. In both adults and children, the vaccine proved to be poorly infectious. Although poor infectivity would not have been predicted from tissue culture studies of RSV ts-2 growth, the human experience closely parallels the experience in a series of animal models, including the chimpanzee. The poor infectivity of this RSV vaccine virus preparation suggests that the postulated defect in the RSV ts-2 fusion protein may be important in determining in vivo infectivity of RSV.     

Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.     

Antibiotic susceptibility of isolates of Bacillus anthracis, a bacterial pathogen with the potential to be used in biowarfare

Bacillus anthracis is a bacterial species that could be used in a bioterrorist attack. We tested a collection of isolates with a range of relevant antimicrobial compounds. All isolates tested were susceptible to ciprofloxacin and doxycycline. Penicillin and amoxicillin, with or without clavulanate, showed in vitro activity against all B. anthracis isolates. Ceftriaxone demonstrated lower-level in vitro activity compared to penicillin-related compounds against B. anthracis . In vitro data from this study are in keeping with available guidelines.     

Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains

An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.     

A real-time PCR assay for the detection of varicella-zoster virus DNA and differentiation of vaccine, wild-type and control strains

Varicella-zoster vaccine is a live attenuated virus. It is, therefore, necessary to have a test to differentiate vaccine from wild-type varicella-zoster virus (VZV) strains for the investigation of varicella or zoster-like rash illness in individuals vaccinated previously. In addition, it is necessary to have a rapid VZV assay for use in the context of smallpox bioterrorism laboratory testing. Using specific primers and hybridization probes, a rapid method to differentiate vaccine strain VZV from wild-type VZV was developed based on the presence or absence of a Pst I restriction site within open reading frame (ORF) 38. Using this ORF 38 assay in conjunction with a similar previously described ORF 62 assay allows for further differentiation of vaccine strain, wild-type and a laboratory control strain (Ellen) VZV. This is accomplished because Ellen VZV is similar to wild-type VZV with respect to the ORF 38 assay but is similar to vaccine strain VZV with respect to the ORF 62 assay. The hybridization probes for each ORF are labeled with different fluorescent tags thus allowing both assays to be run simultaneously in a single tube. Both assays demonstrate a high degree of specificity for VZV and can reliably detect between 10 and 100 copies of VZV DNA. Thus, the real-time polymerase chain reaction (PCR) assay for VZV described below provides a rapid assay allowing the simultaneous differentiation of vaccine, wild-type and laboratory control strains of VZV.     

Construction and characterization of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203, a prototype live oral vaccine.

We engineered an oral Shigella vaccine prototype that can invade intestinal epithelial cells but cannot undergo extensive intracellular replication or extend to adjacent epithelial cells. Strain CVD 1203, derived from wild-type Shigella flexneri 2a by introducing deletions in chromosomal aroA and invasion plasmid virG, was highly attenuated in the Sereny test. Two 10(9)-CFU orogastric doses (2 weeks apart) stimulated production of secretory immunoglobulin A antibodies to S. flexneri 2a and protected against conjunctival sac challenge with virulent S. flexneri 2a.     

Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences

A community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain known as pulsed-field type USA300 (USA300) is epidemic in the United States. Previous comparative whole-genome sequencing studies demonstrated that there has been recent clonal emergence of a subset of USA300 isolates, which comprise the epidemic clone. Although the core genomes of these isolates are closely related, the level of diversity among USA300 plasmids was not resolved. Inasmuch as these plasmids might contribute to significant gene diversity among otherwise closely related USA300 isolates, we performed de novo sequencing of endogenous plasmids from 10 previously characterized USA300 clinical isolates obtained from different geographic locations in the United States. All isolates tested contained small (2- to 3-kb) and/or large (27- to 30-kb) plasmids. The large plasmids encoded heavy metal and/or antimicrobial resistance elements, including those that confer resistance to cadmium, bacitracin, macrolides, penicillin, kanamycin, and streptothricin, although all isolates were sensitive to minocycline, doxycycline, trimethoprim-sulfamethoxazole, vancomycin, teicoplanin, and linezolid. One of the USA300 isolates contained an archaic plasmid that encoded staphylococcal enterotoxins R, J, and P. Notably, the large plasmids (27 to 28 kb) from 8 USA300 isolates--those that comprise the epidemic USA300 clone--were virtually identical (99% identity) and similar to a large plasmid from strain USA300_TCH1516 (a previously sequenced USA300 strain from Houston, TX). These plasmids are largely divergent from the 37-kb plasmid of FPR3757, the first sequenced USA300 strain. The high level of plasmid sequence identity among the majority of closely related USA300 isolates is consistent with the recent clonal emergence hypothesis for USA300.     

Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers

We report the first community-based evaluation of Shigella flexneri 2a strain SC602, a live, oral vaccine strain attenuated by deletion of the icsA (virG) plasmid virulence gene, given at 10(4) CFU. The primary objectives of this trial were to determine the safety and immunogenicity of the vaccine and to determine the duration of colonization. Four of 34 volunteers experienced transient fevers, and three reported diarrhea during the first 3 days of the study. Half of the volunteers mounted a positive serum immunoglobulin A (IgA) response to S. flexneri lipopolysaccharide. All but one of the volunteers excreted the vaccine in their stools for 1 to 33 days, and this excretion was often intermittent. Data from the community-based study were supplemented with an inpatient trial in which three volunteers received 10(3) and nine received 10(4) CFU. All volunteers who received 10(3) CFU excreted SC602 and had an IgA antibody-secreting cell response. Two of these had a serum IgA response. Six of the nine volunteers who received 10(4) CFU excreted SC602. One vaccinee had a transient fever and two met the definition of diarrhea. Six volunteers that received 10(4) CFU had an antibody-secreting cell response, and four had a serum IgA response. SC602 has now been tested at 10(4) CFU in a total of 58 volunteers. The cumulative results of these clinical trials, reported here and previously (Coster et al., Infect. Immun. 67:3437-3443, 1999), have demonstrated that SC602 is a substantially attenuated candidate vaccine that can evoke protection against the most severe symptoms of shigellosis in a stringent human challenge model of disease.     

Subclinical chlamydial infection of the female mouse genital tract generates a potent protective immune response: implications for development of live attenuated chlamydial vaccine strains

Chlamydia trachomatis is a major cause of sexually transmitted disease (STD) for which a vaccine is needed. CD4(+) T-helper type 1 (Th1) cell-mediated immunity is an important component of protective immunity against murine chlamydial genital infection. Conventional vaccine approaches have not proven effective in eliciting chlamydial-specific CD4 Th1 immunity at the genital mucosa. Thus, it is possible that the development of a highly efficacious vaccine against genital infection will depend on the generation of a live attenuated C. trachomatis vaccine. Attenuated strains of C. trachomatis do not exist, so their potential utility as vaccines cannot be tested in animal models of infection. We have developed a surrogate model to study the effect of chlamydial attenuation on infection and immunity of the female genital tract by treating mice with a subchlamydiacidal concentration of oxytetracycline following vaginal infection. Compared to untreated control mice, antibiotic-treated mice shed significantly fewer infectious organisms (3 log(10)) from the cervico-vagina, produced a minimal inflammatory response in urogenital tissue, and did not experience infection-related sequelae. Antibiotic-treated mice generated levels of chlamydia-specific antibody and cell-mediated immunity equivalent to those of control mice. Importantly, antibiotic-treated mice were found to be as immune as control untreated mice when rechallenged vaginally. These findings demonstrate that subclinical chlamydial infection of the murine female genital tract is sufficient to stimulate a potent protective immune response. They also present indirect evidence supporting the possible use of live attenuated chlamydial organisms in the development of vaccines against chlamydial STDs.     

A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. A nanoPCR assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (PRV). Three pairs of primers for nanoPCR developed in this study were selected from conserved regions of PRV, producing specific amplicons of 431 bp (gB), 316 bp (gE), and 202 bp (gG). The sensitivity of this assay using purified plasmid constructs containing the specific gene fragments was 100–1000 fold higher than conventional PCR. The PRV nanoPCR assay did not amplify porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine teschovirus, or African swine fever virus but produced three bands of expected size with PRV and two bands of expected size with the gene-deleted PRV-Bartha-K61. Of 110 clinical samples collected from seven provinces in China, 53% and 48% were positive for wild-type PRV according to the nanoPCR assay and virus isolation, respectively.     

Enterobacterial repetitive intergenic consensus 1R PCR assay for detection of Raoultella sp. isolates among strains identified as Klebsiella oxytoca in the clinical laboratory

The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of Raoultella and the two genetic groups of Klebsiella oxytoca, was applied to 19 clinical isolates identified as K. oxytoca. By this method, as confirmed by species-specific gene sequencing, two Raoultella ornithinolytica and two unclassifiable K. oxytoca isolates were identified.     

Development of a heteroduplex mobility assay to identify field isolates of porcine reproductive and respiratory syndrome virus with nucleotide sequences closely related to those of modified live-attenuated vaccines

Porcine reproductive and respiratory syndrome has been devastating the swine industry since the late 1980s. The disease has been controlled, to some extent, through the use of modified live-attenuated (MLV) vaccines once available. However, such a practice periodically resulted in isolation or detection of vaccine-like viruses from pigs as determined by a partial genomic sequencing. In this study, we developed a heteroduplex mobility assay (HMA) for quickly identifying porcine reproductive and respiratory syndrome virus (PRRSV) isolates with significant nucleotide sequence identities (>/=98%) with the modified live-attenuated vaccines. The major envelope gene (ORF5) of 51 PRRSV field isolates recovered before and after the introduction of the vaccines was amplified, denatured, and reannealed with the HMA reference vaccine strains Ingelvac PRRS MLV and Ingelvac PRRS ATP, respectively. Nine of the 51 field isolates and the VR2332 parent virus of Ingelvac PRRS MLV, which were all highly related to Ingelvac PRRS MLV with 相似文献   

Molecular characterization of rare G12P[6] rotavirus isolates closely related to G12 strains from the United States, CAU 195 and CAU 214

Two human G12 rotaviruses, CAU 195 and CAU 214, were isolated from South Korea using cell culture and characterized on the basis of sequence divergence in the VP7, VP4, and NSP4 genes. Phylogenetic analysis of the VP7 gene sequences indicated that these strains clustered into lineage III and were most closely related to G12 rotaviruses isolated in the United States. The VP4 and NSP4 gene sequences showed that two strains belonged to the P[6]-Ia lineage and genotype [B]. This finding provides information that can be used to evaluate G12 strains and aid in the development of effective vaccines in the future.     

Safety, immunogenicity, and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG.

We evaluated the safety and immunogenicity of attenuated Shigella flexneri 2a vaccine candidate CVD 1203, which harbors precise deletions in the plasmid gene virG and in the chromosomal gene aroA. CVD 1203 invades epithelial cells but undergoes minimal intracellular proliferation and cell-to-cell spread. Fasting healthy volunteers, aged 18 to 40 years, were randomly allocated (double-blind design) to receive either CVD 1203 vaccine or placebo, along with sodium bicarbonate buffer, on days 0 and 14, as follows. At the time of the first inoculation, 10 subjects received placebo (group 1) and 22 subjects received either 1.5 x 10(8) (group 2; 11 subjects) or 1.5 x 10(9) (group 3; 11 subjects) CFU of CVD 1203. Fourteen days later, subjects from group 1 received 1.2 x 10(6) CFU of CVD 1203 and subjects from groups 2 and 3 received 1.2 x 10(8) vaccine organisms. Clinical tolerance was dose dependent. After a single dose of CVD 1203 at 10(6), 10(8), or 10(9) CFU, self-limited (<48-h duration) objective reactogenicity (fever, diarrhea, or dysentery) developed in 0, 18, and 72% of subjects, respectively, and in no placebo recipients. CVD 1203 induced immunoglobulin G seroconversion to S. flexneri 2a lipopolysaccharide (LPS) in 30, 45, and 36% of subjects from groups 1, 2, and 3, respectively, and stimulated immunoglobulin A-producing anti-LPS antibody-secreting cells in 60, 91, and 100% of subjects, respectively. After vaccination, significant rises in tumor necrosis factor alpha concentration in serum (groups 1, 2, and 3) and stool (group 2) samples were observed. We conclude that engineered deletions in virG and aroA markedly attenuate wild-type S. flexneri but preserve immunogenicity; however, less reactogenic vaccines are needed.     

Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2)

Nucleotide sequences have been obtained for RNA segments encoding the PB2, PB1, PA, NP, M1, M2, NS1, and NS2 proteins of the influenza A/Ann Arbor/6/60 (H2N2) wild-type (wt) virus and its cold-adapted (ca) derivative that has been used for preparing investigational live attenuated vaccines. Twenty-four nucleotide differences between the ca and wt viruses were detected, of which 11 were deduced to code for amino acid substitutions in the ca virus proteins. One amino acid substitution each was predicted for the PB2, M2, and NS1 proteins. Two amino acid substitutions were predicted for the NP and the PA proteins. Four substitutions were predicted for the PB1 protein. The biological significance of mutations in the PB2, PB1, PA, and M2 genes of the ca virus is suggested by currently available genetic data, a comparison with other available influenza gene sequences, and the nature of the predicted amino acid changes. In addition, the sequence data confirm the close evolutionary relationship between the genomes of influenza A (H2N2) and influenza A (H3N2) viruses.     

Randomized, controlled human challenge study of the safety, immunogenicity, and protective efficacy of a single dose of Peru-15, a live attenuated oral cholera vaccine

Peru-15 is a live attenuated oral vaccine derived from a Vibrio cholerae O1 El Tor Inaba strain by a series of deletions and modifications, including deletion of the entire CT genetic element. Peru-15 is also a stable, motility-defective strain and is unable to recombine with homologous DNA. We wished to determine whether a single oral dose of Peru-15 was safe and immunogenic and whether it would provide significant protection against moderate and severe diarrhea in a randomized, double-blind, placebo-controlled human volunteer cholera challenge model. A total of 59 volunteers were randomly allocated to groups to receive either 2 x 10(8) CFU of reconstituted, lyophilized Peru-15 vaccine diluted in CeraVacx buffer or placebo (CeraVacx buffer alone). Approximately 3 months after vaccination, 36 of these volunteers were challenged with approximately 10(5) CFU of virulent V. cholerae O1 El Tor Inaba strain N16961, prepared from a standardized frozen inoculum. Among vaccinees, 98% showed at least a fourfold increase in vibriocidal antibody titers. After challenge, 5 (42%) of the 12 placebo recipients and none (0%) of the 24 vaccinees had moderate or severe diarrhea (> or = 3,000 g of diarrheal stool) (P = 0.002; protective efficacy, 100%; lower one-sided 95% confidence limit, 75%). A total of 7 (58%) of the 12 placebo recipients and 1 (4%) of the 24 vaccinees had any diarrhea (P < 0.001; protective efficacy, 93%; lower one-sided 95% confidence limit, 62%). The total number of diarrheal stools, weight of diarrheal stools, incidence of fever, and peak stool V. cholerae excretion among vaccinees were all significantly lower than in placebo recipients. Peru-15 is a well-tolerated and immunogenic oral cholera vaccine that affords protective efficacy against life-threatening cholera diarrhea in a human volunteer challenge model. This vaccine may therefore be a safe and effective tool to prevent cholera in travelers and is a strong candidate for further evaluation to prevent cholera in an area where cholera is endemic.     

Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1

Two important components of control programs for Bovine herpesvirus type 1 (BoHV-1), a major pathogen of cattle, are the detection of outbreaks and vaccination with glycoprotein E (gE)-deleted marker vaccines. In addition to serology, rapid and accurate investigation of BoHV-1 and genetic differentiation of vaccine and wild-type strains are also important methods. Therefore, a triplex quantitative real-time polymerase chain reaction (qPCR) for testing BoHV-1 was developed. Apart from a BoHV-1 specific glycoprotein D (gD) assay, a gE-specific system for differentiation between wild-type BoHV-1 and gE-deleted vaccine strains was established. Finally, an internal control, based on the beta-actin gene was introduced successfully completing the multiplex system. The triplex BoHV-1-qPCR has an analytical sensitivity of less than 10 genome copies per reaction, and the diagnostic sensitivity was equal to or even greater than that of the 'gold standard' method of virus isolation in cell culture. A series of reference strains, including gE-deleted BoHV-1 and field isolates were detected reliably. For validation of the specificity of the test, nasal swabs, semen and different organ material from cattle, negative for BoHV-1, and genetically related herpesvirus strains were examined. The new multiplex BoHV-1-specific qPCR system allows highly sensitive and rapid genetic detection and differentiation of BoHV-1 and will be a valuable method for the control of BoHV-1 infection.