Amber J. Schmidtke

The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.

Shiga toxin-producing Escherichia coli (STEC) are a common cause for food-borne diarrheal illness outbreaks and sporadic cases. Here, we report the availability of the draft genome sequences of 228 STEC strains representing 32 serotypes with known pulsed-field gel electrophoresis (PFGE) types and epidemiological relationships, as well as 12 strains representing other diarrheagenic E. coli pathotypes.

Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935–2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970–1990 (94%) but declined to ≈70% after 1991 and has remained constant. During 2006–2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.

AmpD indirectly regulates the production of AmpC β-lactamase via the cell wall recycling pathway. Recent publications have demonstrated the presence of multipleampDgenes inPseudomonas aeruginosaandEscherichia coli. In the prototypeP. aeruginosastrain, PAO1, the threeampDgenes (ampD,ampDh2, andampDh3) contribute to a stepwise regulation ofampCβ-lactamase and help explain the partial versus full derepression ofampC. In the present study, the roles of the threeampDhomologs in nine clinicalP. aeruginosaisolates with either partial or full derepression ofampCwere evaluated. In eight of nine isolates, decreased RNA expression of theampDgenes was not associated with an increase inampCexpression. Sequence analyses revealed that every derepressed isolate carried mutations inampD, and in two fully derepressed strains, onlyampDwas mutated. Furthermore, everyampDh2gene was of the wild type, and in some fully derepressed isolates,ampDh3was also of the wild type. Mutations inampDandampDh3were tested for their effect on function by using a plasmid model system, and the observed mutations resulted in nonfunctional AmpD proteins. Therefore, although the sequential deletion of theampDhomologs ofP. aeruginosacan explain partial and full derepression in PAO1, the same model does not explain the overproduction of AmpC observed in these clinical isolates. Overall, the findings of the present study indicate that there is still an unknown factor(s) that contributes toampCregulation inP. aeruginosa.

TwoPseudomonas aeruginosamutants exhibiting increased expression ofampCwere selected during exposure to ciprofloxacin. These mutants also exhibited significant increases inmexCD-oprJexpression, but further studies failed to show a link between the increased expression ofmexCD-oprJandampC. IncreasedampCexpression was not related to mutations withinampR, theampC-ampRintergenic region,ampD,ampDh2, orampDh3or to changes in the levels of expression of these amidase genes. However,ampDcomplementation restored wild-type levels ofampCexpression and ceftazidime susceptibility, suggesting alternative mechanisms ofampCregulation.

Mutations within the structural gene ofampDcan lead to AmpC overproduction and increases in β-lactam MICs in organisms with an inducibleampC. However, identification of mutations alone cannot predict the impact that those mutations have on AmpD function. Therefore, a model system was designed to determine the effect ofampDmutations on ceftazidime MICs using an AmpD⁻mutantEscherichia colistrain which produced an inducible plasmid-encoded AmpC.ampDgenes were amplified by PCR from strains ofE. coli,Citrobacter freundii, andPseudomonas aeruginosa. Also, carboxy-terminal truncations ofC. freundii ampDgenes were constructed representing deletions of 10, 21, or 25 codons. AmplifiedampDproducts were cloned into pACYC184 containing induciblebla_ACT-1-ampR. Plasmids were transformed intoE. colistrains JRG582 (AmpD⁻) and K-12 259 (AmpD⁺). The strains were evaluated for a derepressed phenotype using ceftazidime MICs. Some mutatedampDgenes, including theampDgene of a derepressedC. freundiiisolate, resulted in substantial decreases in ceftazidime MICs (from >256 μg/ml to 12 to 24 μg/ml) for the AmpD⁻strain, indicating no role for these mutations in derepressed phenotypes. However,ampDtruncation products andampDfrom a partially derepressedP. aeruginosastrain resulted in ceftazidime MICs of >256 μg/ml, indicating a role for these gene modifications in derepressed phenotypes. The use of this model system indicated that alternative mechanisms were involved in the derepressed phenotype observed in strains ofC. freundiiandP. aeruginosa. The alternative mechanism involved in the derepressed phenotype of theC. freundiiisolate was downregulation ofampDtranscription.