Jonathan G. Frye

CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database

The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI‐1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI‐1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non‐motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2‐propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co‐ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2‐propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co‐ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.

Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide ‘slime’. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one‐third of the functional genome. The large number of genes showing surface‐specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming‐specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface‐dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.

Co‐regulation of Salmonella enterica genes required for virulence and resistance to antimicrobial peptides by SlyA and PhoP/PhoQ

Analysis of the transcriptome of slyA mutant Salmonella enterica serovar Typhimurium revealed that many SlyA‐dependent genes, including pagC, pagD, ugtL, mig‐14, virK, phoN, pgtE, pipB2, sopD2, pagJ and pagK, are also controlled by the PhoP/PhoQ regulatory system. Many SlyA‐ and PhoP/PhoQ‐co‐regulated genes have functions associated with the bacterial envelope, and some have been directly implicated in virulence and resistance to antimicrobial peptides. Purified His‐tagged SlyA binds to the pagC and mig‐14 promoters in regions homologous to a previously proposed ‘SlyA‐box’. The pagC promoter lacks a consensus PhoP binding site and does not bind PhoP in vitro, suggesting that the effect of PhoP on pagC transcription is indirect. Stimulation of pagC expression by PhoP requires SlyA. Levels of SlyA protein and mRNA are not significantly changed under low‐magnesium PhoP‐inducing conditions in which pagC expression is profoundly elevated, however, indicating that the PhoP/PhoQ system does not activate pagC expression by altering SlyA protein concentration. Models are proposed in which PhoP may control SlyA activity via a soluble ligand or SlyA may function as an anti‐repressor to allow PhoP activation. The absence of almost all SlyA‐activated genes from the Escherichia coli K12 genome suggests that the functional linkage between the SlyA and PhoP/PhoQ regulatory systems arose as Salmonella evolved its distinctive pathogenic lifestyle.

Identification of New Flagellar Genes of Salmonella enterica Serovar Typhimurium

RNA levels of flagellar genes in eight different genetic backgrounds were compared to that of the wild type by DNA microarray analysis. Cluster analysis identified new, potential flagellar genes, three putative methyl-accepting chemotaxis proteins, STM3138 (McpA), STM3152 (McpB), and STM3216(McpC), and a CheV homolog, STM2314, in Salmonella, that are not found in Escherichia coli. Isolation and characterization of Mud-lac insertions in cheV, mcpB, mcpC, and the previously uncharacterized aer locus of S. enterica serovar Typhimurium revealed them to be controlled by sigma28-dependent flagellar class 3 promoters. In addition, the srfABC operon previously isolated as an SsrB-regulated operon clustered with the flagellar class 2 operon and was determined to be under FlhDC control. The previously unclassified fliB gene, encoding flagellin methylase, clustered as a class 2 gene, which was verified using reporter fusions, and the fliB transcriptional start site was identified by primer extension analysis. RNA levels of all flagellar genes were elevated in flgM or fliT null strains. RNA levels of class 3 flagellar genes were elevated in a fliS null strain, while deletion of the fliY, fliZ, or flk gene did not affect flagellar RNA levels relative to those of the wild type. The cafA (RNase G) and yhjH genes clustered with flagellar class 3 transcribed genes. Null alleles in cheV, mcpA, mcpB, mcpC, and srfB did not affect motility, while deletion of yhjH did result in reduced motility compared to that of the wild type.

Multiplex PCR-Based Method for Identification of Common Clinical Serotypes of Salmonella enterica subsp. enterica

ABSTRACT A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subsp. enterica encountered in Washington State and the United States in general. Six genetic loci from S. enterica serovar Typhimurium and four from S. enterica serovar Typhi were used to create an assay consisting of two five-plex PCRs. The assays gave reproducible results with 30 different serotypes that represent the most common clinical isolates of S. enterica subsp. enterica. Of these, 22 serotypes gave unique amplification patterns compared with each other and the other 8 serotypes were grouped into four pairs. These were further resolved by two additional PCRs. We compared the data from PCR serotyping with conventional serotyping and found that PCR serotyping was nearly as discriminatory as conventional serotyping was. The results from a blind test screening 111 clinical isolates revealed that 97% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 h and, in conjunction with pulsed-field gel electrophoresis, forms a very robust test method for the molecular subtyping of Salmonella enterica subsp. enterica.

Insights into the complex regulation of rpoS in Borrelia burgdorferi

Co‐ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (σS), as regulated by RpoN (σ54), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of σS‐dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a σ54‐dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2Δ123, a constitutively active form of Rrp2, activated σ54‐dependent transcription of rpoS/P‐lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT‐PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancer‐independent fashion; and finally, (iv) rpoN is required for cell density‐ and temperature‐dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the σS regulon in B. burgdorferi.

Salmonella enterica Serovar Typhimurium Requires the Lpf, Pef, and Tafi Fimbriae for Biofilm Formation on HEp-2 Tissue Culture Cells and Chicken Intestinal Epithelium

ABSTRACT Recent work has demonstrated that Salmonella enterica serovar Typhimurium forms biofilms on HEp-2 tissue culture cells in a type 1 fimbria-dependent manner. To investigate how biofilm growth of HEp-2 tissue culture cells affects gene expression in Salmonella, we compared global gene expression during planktonic growth and biofilm growth. Microarray results indicated that the transcription of ∼100 genes was substantially altered by growth in a biofilm. These genes encode proteins with a wide range of functions, including antibiotic resistance, central metabolism, conjugation, intracellular survival, membrane transport, regulation, and fimbrial biosynthesis. The identification of five fimbrial gene clusters was of particular interest, as we have demonstrated that type 1 fimbriae are required for biofilm formation on HEp-2 cells and murine intestinal epithelium. Mutations in each of these fimbriae were constructed in S. enterica serovar Typhimurium strain BJ2710, and the mutants were found to have various biofilm phenotypes on plastic, HEp-2 cells, and chicken intestinal tissue. The pef and csg mutants were defective for biofilm formation on each of the three surfaces tested, while the lpf mutant exhibited a complete loss of the ability to form a biofilm on chicken intestinal tissue but only an intermediate loss of the ability to form a biofilm on tissue culture cells and plastic surfaces. The bcf mutant displayed increased biofilm formation on both HEp-2 cells and chicken intestinal epithelium, while the sth mutant had no detectable biofilm defects. In all instances, the mutants could be restored to a wild-type phenotype by a plasmid carrying the functional genes. This is the first work to identify the genomic responses of Salmonella to biofilm formation on host cells, and this work highlights the importance of fimbriae in adhering to and adapting to a eukaryotic cell surface. An understanding of these interactions is likely to provide new insights for intervention strategies in Salmonella colonization and infection.

DNA Microarray-Based Typing of an Atypical Monophasic Salmonella enterica Serovar

ABSTRACT A multidrug-resistant fljB-lacking Salmonella enterica serovar [4,5,12:i:−] emerged in Spain in 1997. We analyzed the genome from four strains of this serovar using a microarray containing almost all the predicted protein coding regions of serovar Typhimurium strain LT2, including the pSLT plasmid. Only a few differences from serovar Typhimurium LT2 were observed, suggesting the serovar to be Typhimurium as well. Six regions of interest were identified from the microarray data. Cluster I was a deletion of 13 genes, corresponding to part of the regulon responsible for the anaerobic assimilation of allantoin. Clusters II and IV were associated with the absence of the Fels-1 and Fels-2 prophage. Cluster III was a small group of Gifsy-1 prophage-related genes that appeared to be deleted or replaced. Cluster V was a deletion of 16 genes, including iroB and the operon fljAB, which is reflected in the serovar designation. Region VI was the gene STM2240, which appears to have an additional homologue in these strains. The regions spanning the deletions involving the allantoin operon and the fljAB operon were PCR amplified and sequenced. PCR across these regions may be an effective marker for this particular emergent serovar. While the microarray data for all isolates of the new serovar were essentially identical for all LT2 chromosomal genes, the isolates differed in their similarity to pSLT, consistent with the heterogeneity in plasmid content among isolates of the new serovar. Recent isolates have acquired a more-complete subset of homologues to this virulence plasmid. In general, microarrays can provide useful complementary data to other typing methods.

Inc A/C plasmids are prevalent in multidrug-resistant Salmonella enterica isolates.

ABSTRACT Bacterial plasmids are fragments of extrachromosomal double-stranded DNA that can contain a variety of genes that are beneficial to the host organism, like those responsible for antimicrobial resistance. The objective of this study was to characterize a collection of 437 Salmonella enterica isolates from different animal sources for their antimicrobial resistance phenotypes and plasmid replicon types and, in some cases, by pulsed-field gel electrophoresis (PFGE) in an effort to learn more about the distribution of multidrug resistance in relation to replicon types. A PCR-based replicon typing assay consisting of three multiplex PCRs was used to detect 18 of the 26 known plasmid types in the Enterobacteriaceae based on their incompatibility (Inc) replicon types. Linkage analysis was completed with antibiograms, replicon types, serovars, and Inc A/C. Inc A/C plasmids were prevalent in multidrug-resistant isolates with the notable exception of Salmonella enterica serovar Typhimurium. Cluster analysis based on PFGE of a subset of 216 isolates showed 155 unique types, suggesting a variable population, but distinct clusters of isolates with Inc A/C plasmids were apparent. Significant linkage of serovar was also seen with Inc replicon types B/O, I1, Frep, and HI1. The present study showed that the combination of Salmonella, the Inc A/C plasmids, and multiple-drug-resistant genes is very old. Our results suggest that some strains, notably serovar Typhimurium and closely related types, may have once carried the plasmid but that the resistance genes were transferred to the chromosome with the subsequent loss of the plasmid.