Comparison of antibiotic susceptibility profiles and molecular typing patterns of clinical and environmental Salmonella enterica serotype Newport.

The genus Salmonella is composed of more than 2,400 serotypes, many of which cause enteric diseases in humans and animals. Several Salmonella serotypes are multidrug resistant, and there is evidence of the clonal spread of these strains from animals to humans. Salmonella enterica serotype Newport is one of the serotypes that increasingly present a multidrug-resistant phenotype. Source tracking and antibiotic resistance testing are important considerations for identifying the outbreak strain. The first goal of this study was to examine the antibiotic susceptibility patterns of clinical and environmental Salmonella Newport isolates from various geographic locations and to compare the discriminatory ability of two DNA fingerprinting techniques. The second goal was to determine whether the antibiotic resistance profiles and typing patterns correlated. Thirty Salmonella Newport isolates, including environmental and human clinical strains, were subjected to pulsed-field gel electrophoresis (PFGE), ribotyping, and antibiotic susceptibility testing. Eighty percent of the isolates showed total or intermediate resistance to one or more drugs; 75% of the isolates were multidrug resistant. Ribotyping with the EcoRI enzyme and PFGE with the XbaI enzyme each divided the isolates into 14 groups. Cluster analysis based on antibiotic susceptibility patterns generated 23 profiles. The susceptible and resistant isolates were not differentiated on the basis of either of the molecular typing techniques. Hence, no correlation was observed between the antibiotic resistance profiles and the DNA subtyping patterns. In conclusion, ribotyping is as discriminatory as PFGE and, when used in combination with antibiotic resistance profiles, provides a powerful tool for the source tracking of Salmonella Newport.

Bacillus acidiceler sp. nov., isolated from a forensic specimen, containing Bacillus anthracis pX02 genes.

Research at the Center for Biological Defense identified plasmid-borne forms of Bacillus anthracis pXO2 genes in a Gram-positive, endospore-forming rod, isolated from a forensic specimen considered a credible threat of harbouring anthrax. Conventional, commercial and molecular-based methods indicated that the isolate (CBD 119(T)) was not B. anthracis and considered not to be a member of the Bacillus cereus group. Based on the 16S rRNA gene sequence similarities, strain CBD 119(T) was most closely related to Bacillus luciferensis LMG 18422(T) (99.3 %). Phenotyping and fatty acid methyl ester analysis of the isolate were conducted alongside B. luciferensis JCM 12212(T). The major cellular fatty acids (anteiso-C(15 : 0), iso-C(15 : 0), and >7 iso or anteiso forms) supported inclusion of the isolate in the genus Bacillus. Strain CBD 119(T) was inconsistent with B. luciferensis JCM 12212(T) for 18 of 96 traits evaluated including motility, degree of endospore-driven swelling and pH optimum; the two were linked by fatty acid methyl ester analysis as separate but closely related species. DNA-DNA relatedness between strain CBD 119(T) and B. luciferensis JCM 12212(T) resulted in less than 20 % hybridization. The results of biochemical and physiological characterization, chemotaxonomic analysis and DNA-DNA hybridization differentiated strain CBD 119(T) both phenotypically and genotypically from the only species with validly published name with greater than 97 % 16S rRNA gene sequence similarity. The isolate has an accelerated doubling time when grown in aerated broth at pH 5.9 relative to that at pH 7.1. Therefore, it is proposed that strain CBD 119(T) represents a novel species, Bacillus acidiceler sp. nov. The type strain is strain CBD 119(T) (=NRRL B-41736(T)=DSM 18954(T)).

Bacillus anthracis virulent plasmid pX02 genes found in large plasmids of two other Bacillus species.

In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples.

Use of two selective media and a broth motility test can aid in identification or exclusion of Bacillus anthracis.

During the anthrax attack of 2001, the Florida Department of Health (FDOH) Bureau of Laboratories in Tampa received hundreds of isolates suspected of being Bacillus anthracis. None were confirmed to be B. anthracis since most isolates were motile and not even in the Bacillus cereus group. Although the sentinel laboratories now send fewer isolates to FDOH laboratories, should another attack occur the number of isolates submitted would likely increase dramatically, and this upsurge would seriously challenge personnel who are expected to be busy examining an increased number of environmental samples. We examined two selective and differential growth media and alternative motility methods that could be used to streamline the processing of suspicious isolates. Of 60 isolates previously sent to the FDOH laboratory, 56 were endospore-forming gram-positive rods and only 7 grew on mannitol-egg yolk-polymyxin B agar and/or the Anthracis chromogenic agar. Microscopic observation of early-log-phase growth (2 to 3 h) in a shaking broth was the best method to detect motility in 40 isolates that appeared nonmotile in the motility media investigated. One of these growth media and microscopic examination of shaken broth cultures can be used to show that an isolate is not B. anthracis before expensive molecular and antibody-based tests are performed. By doing so, costs could be reduced and analysis time shortened.