Escherichia coli B-Strains Are Intrinsically Resistant to Colistin and Not Suitable for Characterization and Identification of mcr Genes.

Antimicrobial resistance is an increasing threat to human and animal health. Due to the rise of multi-, extensive, and pandrug resistance, last resort antibiotics, such as colistin, are extremely important in human medicine. While the distribution of colistin resistance genes can be tracked through sequencing methods, phenotypic characterization of putative antimicrobial resistance (AMR) genes is still important to confirm the phenotype conferred by different genes. While heterologous expression of AMR genes (e.g., in Escherichia coli) is a common approach, so far, no standard methods for heterologous expression and characterization of mcr genes exist. E. coli B-strains, designed for optimum protein expression, are frequently utilized. Here, we report that four E. coli B-strains are intrinsically resistant to colistin (MIC 8-16 µg/mL). The three tested B-strains that encode T7 RNA polymerase show growth defects when transformed with empty or mcr-expressing pET17b plasmids and grown in the presence of IPTG; K-12 or B-strains without T7 RNA polymerase do not show these growth defects. E. coli SHuffle T7 express carrying empty pET17b also skips wells in colistin MIC assays in the presence of IPTG. These phenotypes could explain why B-strains were erroneously reported as colistin susceptible. Analysis of existing genome data identified one nonsynonymous change in each pmrA and pmrB in all four E. coli B-strains; the E121K change in PmrB has previously been linked to intrinsic colistin resistance. We conclude that E. coli B-strains are not appropriate heterologous expression hosts for identification and characterization of mcr genes. IMPORTANCE Given the rise in multidrug, extensive drug, and pandrug resistance in bacteria and the increasing use of colistin to treat human infections, occurrence of mcr genes threatens human health, and characterization of these resistance genes becomes more important. We show that three commonly used heterologous expression strains are intrinsically resistant to colistin. This is important because these strains have previously been used to characterize and identify new mobile colistin resistance (mcr) genes. We also show that expression plasmids (i.e., pET17b) without inserts cause cell viability defects when carried by B-strains with T7 RNA polymerase and grown in the presence of IPTG. Our findings are important as they will facilitate improved selection of heterologous strains and plasmid combinations for characterizing AMR genes, which will be particularly important with a shift to Culture-independent diagnostic tests where bacterial isolates become increasingly less available for characterization.

Salmonella enterica serovar Cerro displays a phylogenetic structure and genomic features consistent with virulence attenuation and adaptation to cattle.

Salmonella enterica subsp. enterica (S.) serovar Cerro is rarely isolated from human clinical cases of salmonellosis but represents the most common serovar isolated from cattle without clinical signs of illness in the United States. In this study, using a large, diverse set of 316 isolates, we utilized genomic methods to further elucidate the evolutionary history of S. Cerro and to identify genomic features associated with its apparent virulence attenuation in humans. Phylogenetic analyses showed that within this polyphyletic serovar, 98.4% of isolates (311/316) represent a monophyletic clade within section Typhi and the remaining 1.6% of isolates (5/316) form a monophyletic clade within subspecies enterica Clade A1. Of the section Typhi S. Cerro isolates, 93.2% of isolates (290/311) clustered into a large clonal clade comprised of predominantly sequence type (ST) 367 cattle and environmental isolates, while the remaining 6.8% of isolates (21/311), primarily from human clinical sources, clustered outside of this clonal clade. A tip-dated phylogeny of S. Cerro ST367 identified two major clades (I and II), one of which overwhelmingly consisted of cattle isolates that share a most recent common ancestor that existed circa 1975. Gene presence/absence and rarefaction curve analyses suggested that the pangenome of section Typhi S. Cerro is open, potentially reflecting the gain/loss of prophage; human isolates contained the most open pangenome, while cattle isolates had the least open pangenome. Hypothetically disrupted coding sequences (HDCs) displayed clade-specific losses of intact speC and sopA virulence genes within the large clonal S. Cerro clade, while loss of intact vgrG, araH, and vapC occurred in all section Typhi S. Cerro isolates. Further phenotypic analysis suggested that the presence of a premature stop codon in speC does not abolish ornithine decarboxylase activity in S. Cerro, likely due to the activity of the second ornithine decarboxylase encoded by speF, which remained intact in all isolates. Overall, our study identifies specific genomic features associated with S. Cerro's infrequent isolation from humans and its apparent adaptation to cattle, which has broader implications for informing our understanding of the evolutionary events facilitating host adaptation in Salmonella.

Characterization of Basal Transcriptomes Identifies Potential Metabolic and Virulence-Associated Adaptations Among Diverse Nontyphoidal Salmonella enterica Serovars.

The zoonotic pathogen Salmonella enterica includes >2,600 serovars, which differ in the range of hosts they infect and the severity of disease they cause. To further elucidate the mechanisms behind these differences, we performed transcriptomic comparisons of nontyphoidal Salmonella (NTS) serovars with the model for NTS pathogenesis, S. Typhimurium. Specifically, we used RNA-seq to characterize the understudied NTS serovars S. Javiana and S. Cerro, representing a serovar frequently attributed to human infection via contact with amphibians and reptiles, and a serovar primarily associated with cattle, respectively. Whole-genome sequence (WGS) data were utilized to ensure that strains characterized with RNA-seq were representative of their respective serovars. RNA extracted from representative strains of each serovar grown to late exponential phase in Luria-Bertani (LB) broth showed that transcript abundances of core genes were significantly higher (p<0.001) than those of accessory genes for all three serovars. Inter-serovar comparisons identified that transcript abundances of genes in Salmonella Pathogenicity Island (SPI) 1 were significantly higher in both S. Javiana and S. Typhimurium compared to S. Cerro. Together, our data highlight potential transcriptional mechanisms that may facilitate S. Cerro and S. Javiana survival in and adaptation to their respective hosts and impact their ability to cause disease in others. Furthermore, our analyses demonstrate the utility of omics approaches in advancing our understanding of the diversity of metabolic and virulence mechanisms of different NTS serovars.

Development of Chemically Defined Media Reveals Citrate as Preferred Carbon Source for Liberibacter Growth.

Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus L. asiaticus, L. americanus, and L. africanus are causal agents of citrus greening disease, otherwise known as huanglongling (HLB). Candidatus L. solanacearum is responsible for potato Zebra chip disease. Cultures of L. crescens grow slowly on BM-7 complex medium, while attempts to culture the Ca. Liberibacter pathogens in BM-7 have failed. Developing a defined medium for the growth of L. crescens will be useful in the study of Liberibacter metabolism and will improve the prospects for culturing the Ca. Liberibacter pathogens. Here, M15 medium is presented and described as the first chemically defined medium for the growth of L. crescens cultures that approaches the growth rates obtained with BM-7. The development of M15 was a four step process including: (1) the identification of Hi-Graces Insect medium (Hi-GI) as an essential, yet undefined component in BM-7, for the growth of L. crescens, (2) metabolomic reconstruction of Hi-GI to create a defined medium for the growth of L. crescens cultures, and (3) the discovery of citrate as the preferred carbon and energy source for L. crescens growth. The composition of M15 medium includes inorganic salts as in the Hi-GI formula, amino acids derived from the metabolomic analyses of Hi-GI, and a 10-fold increase in vitamins compared to the Hi-GI formula, with exception choline chloride, which was increased 5000-fold in M15. Since genome comparisons of L. crescens and the Ca. Liberibacter pathogens show that they are very similar metabolically. Thus, these results imply citrate and other TCA cycle intermediates are main energy sources for these pathogens in their insect and plant hosts. Thus, strategies to reduce citrate levels in the habitats of these pathogens may be effective in reducing Ca. Liberibacter pathogen populations thereby reducing symptoms in the plant host.