Rapid detection and simultaneous antibiotic susceptibility analysis of Yersinia pestis directly from clinical specimens by use of reporter phage.

Yersinia pestis is a tier 1 agent due to its contagious pneumopathogenicity, extremely rapid progression, and high mortality rate. As the disease is usually fatal without appropriate therapy, rapid detection from clinical matrices is critical to patient outcomes. We previously engineered the diagnostic phage ΦA1122 with luxAB to create a "light-tagged" reporter phage. ΦA1122::luxAB rapidly detects Y. pestis in pure culture and human serum by transducing a bioluminescent signal response. In this report, we assessed the analytical specificity of the reporter phage and investigated diagnostic utility (detection and antibiotic susceptibility analysis) directly from spiked whole blood. The bioreporter displayed 100% (n = 59) inclusivity for Y. pestis and consistent intraspecific signal transduction levels. False positives were not obtained from species typically associated with bacteremia or those relevant to plague diagnosis. However, some non-pestis Yersinia strains and Enterobacteriaceae did elicit signals, albeit at highly attenuated transduction levels. Diagnostic performance was assayed in simple broth-enriched blood samples and standard aerobic culture bottles. In blood, <10(2) CFU/ml was detected within 5 h. In addition, Y. pestis was identified directly from positive blood cultures within 20 to 45 min without further processing. Importantly, coincubation of blood samples with antibiotics facilitated simultaneous antimicrobial susceptibility profiling. Consequently, the reporter phage demonstrated rapid detection and antibiotic susceptibility profiling directly from clinical samples, features that may improve patient prognosis during plague outbreaks.

Characterisation of Yersinia pestis isolates from natural foci of plague in the Republic of Georgia, and their relationship to Y. pestis isolates from other countries.

Forty Yersinia pestis isolates from endemic foci of plague in the Republic of Georgia, and six Y. pestis isolates from neighbouring former Soviet Union countries, were analysed for their biochemical and phenotypic properties, and their genetic relatedness was compared with Y. pestis strains KIM and CO92 by pulsed-field gel electrophoresis (PFGE). In addition, 11 Y. pestis isolates from the USA, together with published nucleotide sequences from Y. pestis strains KIM, CO92 and 91001, were compared with the 46 isolates in the present collection using multilocus sequence typing (MLST), based on sequence data for the 16S rRNA, hsp60, glnA, gyrB, recA, manB, thrA and tmk loci. Four virulence gene loci (caf1, lcrV, psaA and pla) were also sequenced and analysed. Two sequence types (ST1 and ST2), which differed by a single nucleotide, were identified by MLST. With the exception of a single isolate (771G), all of the Georgian Y. pestis isolates belonged to ST2. PFGE also grouped the Georgian Y. pestis isolates separately from the non-Georgian isolates. Overall, PFGE discriminated the Y. pestis isolates more effectively than MLST. The sequences of three of the four virulence genes (lcrV, psaA and pla) were identical in all Georgian and non-Georgian isolates, but the caf1 locus was represented by two allele types, with caf1 NT1 being associated with the non-Georgian isolates and caf1 NT2 being associated with the Georgian isolates. These results suggest that Georgian Y. pestis isolates are of clonal origin.

The vibrio pathogenicity island of epidemic Vibrio cholerae forms precise extrachromosomal circular excision products.

The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. Like many pathogenicity islands, the VPI has at its termini a phage-like integrase gene (int), a transposase-like gene (vpiT), and phage-like attachment (att) sites, and is inserted at a tRNA-like locus (ssrA). We report that the VPI precisely excises from the chromosome and that its left and right ends join to form an extrachromosomal circular excision product (pVPI). Two-stage nested PCR analysis and DNA sequencing confirmed the int-att-vpiT junction and that the core attP of pVPI is identical to the chromosomal VPI attR site. Excision was independent of toxR and toxT. Excision was independent of recA, suggesting that it is mediated by site-specific recombination. Interestingly, while excision was detected in int and vpiT mutants, excision was abolished in a double (int vpiT) mutant and was restored by plasmids containing genes for either recombinase. Excision results in deletion of A361 in the ssrA locus, which flanks the right junction of the VPI. Since A361 encodes U70 in the critical G. U base pair in the acceptor stem of the ssrA RNA that is the determinant for aminoacylation with alanine, this deletion might have deleterious effects on ssrA function. Also, vpiT may have undergone interchromosomal translocation or may represent an independent integration event, as it was found downstream of hutA in some isolates. Our results provide new insight into the molecular biology of the VPI, and we propose that the process of excision and circularization is important in the emergence, pathogenesis, and persistence of epidemic V. cholerae.