Jocelyn Rankin; obituary; in memoriam
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.
We identified three encapsulated Bacillus cereus strains, isolated from patients with severe pneumonia, in a collection of B. cereus isolates associated with human illness. We found that the extent of capsule expression was influenced by culturing conditions. Our findings highlight consequent clinical and laboratory diagnostic challenges posed by such isolates.
CDC; infectious diseases; history; commentary
Bacillus anthracis and Bacillus cereus can usually be distinguished by standard microbiological methods (e.g., motility, hemolysis, penicillin susceptibility and susceptibility to gamma phage) and PCR. However, we have identified 23 Bacillus spp. isolates that gave discrepant results when assayed by standard microbiological methods and PCR. We used multiple-locus variable-number tandem repeat analysis (MLVA), multiple-locus sequence typing (MLST), and phenotypic analysis to characterize these isolates, determine if they cluster phylogenetically and establish whether standard microbiological identification or PCR were associated with false positive/negative results.
Six isolates were LRN real-time PCR-positive but resistant to gamma phage; MLVA data supported the identification of these isolates as gamma phage-resistant B. anthracis. Seventeen isolates were LRN real-time PCR-negative but susceptible to gamma phage lysis; these isolates appear to be a group of unusual gamma phage-susceptible B. cereus isolates that are closely related to each other and to B. anthracis. All six B. anthracis MLVA chromosomal loci were amplified from one unusual gamma phage-susceptible, motile, B. cereus isolate (although the amplicons were atypical sizes), and when analyzed phylogenetically, clustered with B. anthracis by MLST.
MLVA and MLST aided in the identification of these isolates when standard microbiological methods and PCR could not definitely identify or rule out B. anthracis. This study emphasized the need to perform multiple tests when attempting to identify B. anthracis since relying on a single assay remains problematic due to the diverse nature of bacteria.
A molecular typing method based on the 16S rRNA sequence diversity was developed for Haemophilus influenzae isolates. A total of 330 H. influenzae isolates were analyzed, representing a diverse collection of U.S. isolates. We found a high level of 16S rRNA sequence heterogeneity (up to 2.73%) and observed an exclusive correlation between 16S types and serotypes (a to f); no 16S type was found in more than one serotype. Similarly, no multilocus sequence typing (MLST) sequence type (ST) was found in more than one serotype. Our 16S typing and MLST results are in agreement with those of previous studies showing that serotypable H. influenzae isolates behave as highly clonal populations and emphasize the lack of clonality of nontypable (NT) H. influenzae isolates. There was not a 1:1 correlation between 16S types and STs, but all H. influenzae serotypable isolates clustered similarly. This correlation was not observed for NT H. influenzae; the two methods clustered NT H. influenzae isolates differently. 16S rRNA gene sequencing alone provides a level of discrimination similar to that obtained with the analysis of seven genes for MLST. We demonstrated that 16S typing is an additional and complementary approach to MLST, particularly for NT H. influenzae isolates, and is potentially useful for outbreak investigation.
The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.
Neisseria meningitidis is infrequently reported as a laboratory-acquired infection. Prompted by two cases in the United States in 2000, we assessed this risk among laboratorians. We identified cases of meningococcal disease that were possibly acquired or suspected of being acquired in a laboratory by placing an information request on e-mail discussion groups of infectious disease, microbiology, and infection control professional organizations. A probable case of laboratory-acquired meningococcal disease was defined as illness meeting the case definition for meningococcal disease in a laboratorian who had occupational exposure to an N. meningitidis isolate of the same serogroup within 14 days of illness onset. Sixteen cases of probable laboratory-acquired meningococcal disease occurring worldwide between 1985 and 2001 were identified, including six U.S. cases between 1996 and 2000. Nine cases (56%) were serogroup B; seven (44%) were serogroup C. Eight cases (50%) were fatal. All cases occurred among clinical microbiologists. In 15 cases (94%), isolate manipulation was performed without respiratory protection. We estimated that an average of three microbiologists are exposed to the 3,000 meningococcal isolates seen in U.S. laboratories yearly and calculated an attack rate of 13/100,000 microbiologists between 1996 and 2001, compared to 0.2/100,000 among U.S. adults in general. The rate and case/fatality ratio of meningococcal disease among microbiologists are higher than those in the general U.S. population. Specific risk factors for laboratory-acquired infection are likely associated with exposure to droplets or aerosols containing N. meningitidis. Prevention should focus on the implementation of class II biological safety cabinets or additional respiratory protection during manipulation of suspected meningococcal isolates.
We used multiple-locus variable-number tandem repeat analysis (MLVA) and pagA sequencing to genotype a Bacillus anthracis isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.
We evaluated the API 20NE and the RapID NF Plus systems with 58 Burkholderia pseudomallei and 23 B. mallei strains for identification of these agents, but neither was reliable for confirmatory identification, with only 0 to 60% strains identified accurately. A greater diversity of strains in the system databases would be beneficial.
Members of the genus Brucella are categorized as biothreat agents and pose a hazard for both humans and animals. Current identification methods rely on biochemical tests that may require up to 7 days for results. We sequenced the 16S rRNA genes of 65 Brucella strains along with 17 related strains likely to present a differential diagnostic challenge. All Brucella 16S rRNA gene sequences were determined to be identical and were clearly different from the 17 related strains, suggesting that 16S rRNA gene sequencing is a reliable tool for rapid genus-level identification of Brucella spp. and their differentiation from closely related organisms.
Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia in children and young adults in the United States. Rapid and reliable identification of N. meningitidis serogroups is crucial for judicious and expedient response to cases of meningococcal disease, including decisions about vaccination campaigns. From 1997 to 2002, 1,298 N. meningitidis isolates, collected in the United States through the Active Bacterial Core surveillance (ABCs), were tested by slide agglutination serogrouping (SASG) at both the ABCs sites and the Centers for Disease Control and Prevention (CDC). For over 95% of isolates, SASG results were concordant, while discrepant results were reported for 58 isolates. To resolve these discrepancies, we repeated the SASG in a blinded fashion and employed ctrA and six serogroup-specific PCR assays (SGS-PCR) to determine the genetic capsule type. Seventy-eight percent of discrepancies were resolved, since results of the SGS-PCR and SASG blinded study agreed with each other and confirmed the SASG result at either state health laboratories or CDC. This study demonstrated the ability of SGS-PCR to efficiently resolve SASG discrepancies and identified the main cause of the discrepancies as overreporting of these isolates as nongroupable. It also reemphasized the importance of adherence to quality assurance procedures when performing SASG and prompted prospective monitoring for SASG discrepancies involving isolates collected through ABCs in the United States.
Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.
An outbreak of serogroup W-135 meningococcal disease occurred during the 2000 Hajj in Saudi Arabia. Disease was reported worldwide in Hajj pilgrims and their close contacts; however, most cases were identified in Saudi Arabia. Trends in Saudi meningococcal disease were evaluated and the epidemiology of Saudi cases from this outbreak described. Saudi national meningococcal disease incidence data for 1990 to 2000 were reviewed; cases from January 24 to June 5, 2000 were retrospectively reviewed. The 2000 Hajj outbreak consisted of distinct serogroup A and serogroup W-135 outbreaks. Of 253 identified cases in Saudi Arabia, 161 (64%) had serogroup identification; serogroups W-135 and A caused 93 (37%) and 60 (24%) cases with attack rates of 9 and 6 cases per 100,000 population, respectively. The 2000 Hajj outbreak was the first large serogroup W-135 meningococcal disease outbreak identified worldwide. Enhanced surveillance for serogroup W-135, especially in Africa, is essential to control this emerging epidemic disease.
Meningococcal infections; meningitis; meningococcal; Neisseria meningitides; epidemiology; disease outbreaks; Saudi Arabia; Africa; research
Adventure travel is becoming more popular, increasing the likelihood of contact with unusual pathogens. We investigated an outbreak of leptospirosis in “Eco-Challenge” multisport race athletes to determine illness etiology and implement public health measures. Of 304 athletes, we contacted 189 (62%) from the United States and 26 other countries. Eighty (42%) athletes met our case definition. Twenty-nine (36%) case-patients were hospitalized; none died. Logistic regression showed swimming in the Segama River (relative risk [RR]=2.0; 95% confidence interval [CI]=1.3 to 3.1) to be an independent risk factor. Twenty-six (68%) of 38 case-patients tested positive for leptospiral antibodies. Taking doxycycline before or during the race was protective (RR=0.4, 95% CI=0.2 to 1.2) for the 20 athletes who reported using it. Increased adventure travel may lead to more frequent exposure to leptospires, and preexposure chemoprophylaxis for leptospirosis (200 mg oral doxycycline/week) may decrease illness risk. Efforts are needed to inform adventure travel participants of unique infections such as leptospirosis.
leptospirosis; febrile illness; adventure travel immunoassay; doxycycline; research
A multilocus sequence typing (MLST) scheme has been developed for the unambiguous characterization of encapsulated and noncapsulated Haemophilus influenzae isolates. The sequences of internal fragments of seven housekeeping genes were determined for 131 isolates, comprising a diverse set of 104 serotype a, b, c, d, e, and f isolates and 27 noncapsulated isolates. Many of the encapsulated isolates had previously been characterized by multilocus enzyme electrophoresis (MLEE), and the validity of the MLST scheme was established by the very similar clustering of isolates obtained by these methods. Isolates of serotypes c, d, e, and f formed monophyletic groups on a dendrogram constructed from the differences in the allelic profiles of the isolates, whereas there were highly divergent lineages of both serotype a and b isolates. Noncapsulated isolates were distinct from encapsulated isolates and, with one exception, were within two highly divergent clusters. The relationships between the major lineages of encapsulated H. influenzae inferred from MLEE data could not be discerned on a dendrogram constructed from differences in the allelic profiles, but were apparent on a tree reconstructed from the concatenated nucleotide sequences. Recombination has not therefore completely eliminated phylogenetic signal, and in support of this, for encapsulated isolates, there was significant congruence between many of the trees reconstructed from the sequences of the seven individual loci. Congruence was less apparent for noncapsulated isolates, suggesting that the impact of recombination is greater among noncapsulated than encapsulated isolates. The H. influenzae MLST scheme is available at www.mlst.net, it allows any isolate to be compared with those in the MLST database, and (for encapsulated isolates) it assigns isolates to their phylogenetic lineage, via the Internet.
To resolve discrepancies in slide agglutination serotyping (SAST) results from state health departments and the Centers for Disease Control and Prevention (CDC), we characterized 141 of 751 invasive Haemophilus influenzae isolates that were identified in the United States from January 1998 to December 1999 through an active, laboratory-based, surveillance program coordinated by the CDC. We found discrepancies between the results of SAST performed at state health departments and those of PCR capsule typing performed at the CDC for 56 (40%) of the isolates characterized: 54 isolates that were identified as a particular serotype by SAST were shown to be unencapsulated by PCR, and two isolates that were reported as serotypes b and f were found to be serotypes f and e, respectively, by PCR. The laboratory error most likely to affect the perceived efficacy of the conjugate H. influenzae type b (Hib) vaccine was the misidentification of isolates as serotype b: of 40 isolates identified as serotype b by SAST, 27 (68%) did not contain the correlating capsule type genes. The frequency of errors fell substantially when standardized reagents and routine quality control of SAST were used during a study involving three laboratories. An overall 94% agreement between SAST and PCR results showed that slide agglutination could be a valid and reliable method for serotyping H. influenzae if the test was performed correctly, in accordance with standardized and recommended procedures. An ongoing prospective analysis of all H. influenzae surveillance isolates associated with invasive disease in children less than 5 years old will provide more accurate national figures for the burden of invasive disease caused by Hib and other H. influenzae serotypes.
We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.
We investigated the diversity of the primary sequences of 16S rRNA genes among Neisseria meningitidis strains (Men) and evaluated the use of this approach as a molecular subtyping tool. We aligned and compared a 1,417-bp fragment of the 16S rRNA gene from 264 Men strains of serogroups A, B, C, and Y (MenA, MenB, MenC, and MenY, respectively) isolated throughout the world over a 30-year period. Thirty-one positions of difference were found among 49 16S types: differences between types ranged from 1 to 14 positions (0.07 to 0.95%). 16S types and serogroups were highly associated; only 3 out 49 16S types were shared by two or more serogroups. We have identified 16S types that are exclusively associated with strains of certain hypervirulent clones: 16S type 5 with MenA subgroup III, 16S type 4 with the MenB electrophoretic type 5 (ET-5) complex, and 16S types 12 and 13 with MenC of the ET-37 complex. For MenC strains, 16S sequencing provided the highest sensitivity and specificity and the best overall association with the outbreak-related versus sporadic isolates when compared with pulsed-field gel electrophoresis, multilocus enzyme electrophoresis, and multilocus sequence typing. We demonstrated for the first time an unexpected diversity among 16S rRNA genes of Men strains, identified 16S types associated with well-defined hypervirulent clones, and showed the potential of this approach to rapidly identify virulent strains associated with outbreaks and/or an increased incidence of sporadic disease.
In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA generom these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene seqs fuences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.
Anthrax; Bacillus anthracis; Bioterrorism; 16S rRNA gene
On October 4, 2001, we confirmed the first bioterrorism-related anthrax case identified in the United States in a resident of Palm Beach County, Florida. Epidemiologic investigation indicated that exposure occurred at the workplace through intentionally contaminated mail. One additional case of inhalational anthrax was identified from the index patient’s workplace. Among 1,076 nasal cultures performed to assess exposure, Bacillus anthracis was isolated from a co-worker later confirmed as being infected, as well as from an asymptomatic mail-handler in the same workplace. Environmental cultures for B. anthracis showed contamination at the workplace and six county postal facilities. Environmental and nasal swab cultures were useful epidemiologic tools that helped direct the investigation towards the infection source and transmission vehicle. We identified 1,114 persons at risk and offered antimicrobial prophylaxis.
Anthrax; Bacillus anthracis; bioterrorism; nasal swab cultures; environmental cultures
On October 12, 2001, two envelopes containing Bacillus anthracis spores passed through a sorting machine in a postal facility in Washington, D.C. When anthrax infection was identified in postal workers 9 days later, the facility was closed. To determine if exposure to airborne B. anthracis spores continued to occur, we performed air sampling around the contaminated sorter. One CFU of B. anthracis was isolated from 990 L of air sampled before the machine was activated. Six CFUs were isolated during machine activation and processing of clean dummy mail. These data indicate that an employee working near this machine might inhale approximately 30 B. anthracis-containing particles during an 8-h work shift. What risk this may have represented to postal workers is not known, but the risk is approximately 20-fold less than estimates of sub-5 micron B. anthracis-containing particles routinely inhaled by asymptomatic, unvaccinated workers in a goat-hair mill.
Bacillus anthracis; anthrax; risk assessment; occupational exposure
Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying anthrax strains during the 2001 bioterrorism-associated outbreak. Because B. anthracis has a low level of genetic variability, only a few subtyping methods, with varying reliability, exist. We initially used multiple-locus variable-number tandem repeat analysis (MLVA) to subtype 135 B. anthracis isolates associated with the outbreak. All isolates were determined to be of genotype 62, the same as the Ames strain used in laboratories. We sequenced the protective antigen gene (pagA) from 42 representative outbreak isolates and determined they all had a pagA sequence indistinguishable from the Ames strain (PA genotype I). MLVA and pagA sequencing were also used on DNA from clinical specimens, making subtyping B. anthracis possible without an isolate. Use of high-resolution molecular subtyping determined that all outbreak isolates were indistinguishable by the methods used and probably originated from a single source. In addition, subtyping rapidly identified laboratory contaminants and non-outbreak–related isolates.
B. anthracis; subtyping; multiple-locus variable-number tandem repeat analysis (MLVA); pagA