Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S. Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5α under an IPTG (isopropyl-β-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3. Pairwise comparisons of the predicted protein structures associated with all nine mcr homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance. IMPORTANCE Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a “highest priority critically important antimicrobial for human medicine” (WHO, Critically Important Antimicrobials for Human Medicine, 5th revision, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne mcr genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

Listeria (L.) monocytogenes is an opportunistic pathogen causing life-threatening infections in diverse mammalian species including humans and ruminants. As little is known on the link between strains and clinicopathological phenotypes, we studied potential strain-associated virulence and organ tropism in L. monocytogenes isolates from well-defined ruminant cases of clinical infections and the farm environment. The phylogeny of isolates and their virulence-associated genes were analyzed by multilocus sequence typing (MLST) and sequence analysis of virulence-associated genes. Additionally, a panel of representative isolates was subjected to in vitro infection assays. Our data suggest the environmental exposure of ruminants to a broad range of strains and yet the strong association of sequence type (ST) 1 from clonal complex (CC) 1 with rhombencephalitis, suggesting increased neurotropism of ST1 in ruminants, which is possibly related to its hypervirulence. This study emphasizes the importance of considering clonal background of L. monocytogenes isolates in surveillance, epidemiological investigation and disease control.

Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n ‫؍‬ 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. V irulent phages have been shown to present a tremendous selective pressure on their bacterial host populations. Not only is phage predation a major driver of bacterial diversification (1, 2), but it may also select for hypermutators, which could increase the frequency of mutations in bacterial populations (3, 4). Whereas bacteria are limited to one cell division per generation, a single phage-infected cell can produce a burst ranging from less than 5 to over 1,000 progeny phages in a similar period of time (5-7). Phages consequently have the capability to rapidly outgrow their bacterial hosts and can significantly reduce or eliminate susceptible bacteria in the local environment (8, 9). Therefore, the potential for bacterial strains to persist in an environment containing lytic phages may be contingent upon that strain accumulating spontaneous mutations that grant resistance to phage infection (10). These phage-resistant mutant strains most typically resist phage infection through mechanisms of adsorption inhibition, i.e., alterations of the cell surface that affect phage attachment (11). However, one study reported that nearly all phage-resistant mutant strains of Streptococcus thermophilus had acquired CRISPR spacers that matched invading phage genomes (12); these S. thermophilus mutant strains would be expected to resist phage infection after the adsorption step. Phage-resistant mutant strains that resist infection through mechanisms of adsorption inh...

Cold shock-domain family proteins (Csps) are highly conserved nucleic acid binding proteins regulating the expression of various genes including those involved in stress resistance and virulence in bacteria. We show here that Csps are involved in virulence, cell aggregation and flagella-based extracellular motility of Listeria monocytogenes. A L. monocytogenes mutant deleted in all three csp genes (ΔcspABD) is attenuated with respect to human macrophage infection as well as virulence in a zebrafish infection model. Moreover, this mutant is incapable of aggregation and fails to express surface flagella or exhibit swarming motility. An evaluation of double csp gene deletion mutant (ΔcspBD, ΔcspAD and ΔcspAB) strains that produce single csp genes showed that there is redundancy as well as functional differences among the three L. monocytogenes Csps in their contributions to virulence, cellular aggregation, flagella production, and swarming motility. Protein and mRNA expression analysis further showed impaired expression of key virulence and motility genes in the csp mutants. Our observations at protein and mRNA level suggest Csp-dependent expression regulation of these genes at transcriptional and post-transcriptional levels. In a mutant lacking all csp genes (ΔcspABD) as well as those possessing single csp genes (ΔcspBD, ΔcspAD, and ΔcspAB) we detected reduced levels of proteins or activity as well as transcripts from the prfA, hly, mpl, and plcA genes suggesting a Csp-dependent transcriptional regulation of these genes. These csp mutants also had reduced or completely lacked ActA proteins and cell surface flagella but contained elevated actA and flaA mRNA levels compared to the parental wild type strain suggesting Csp involvement in post-transcriptional regulation of these genes. Overall, our results suggest that Csps contribute to the expression regulation of virulence and flagella-associated genes thereby promoting host pathogenicity, cell aggregation and flagella-based motility processes in L. monocytogenes.

Gram-positive bacteria are ubiquitous and diverse microorganisms that can survive and sometimes even thrive in continuously changing environments. The key to such resilience is the ability of members of a population to respond and adjust to dynamic conditions in the environment. In bacteria, such responses and adjustments are mediated, at least in part, through appropriate changes in the bacterial transcriptome in response to the conditions encountered. Resilience is important for bacterial survival in diverse, complex, and rapidly changing environments and requires coordinated networks that integrate individual, mechanistic responses to environmental cues to enable overall metabolic homeostasis. In many Gram-positive bacteria, a key transcriptional regulator of the response to changing environmental conditions is the alternative sigma factor B . B has been characterized in a subset of Gram-positive bacteria, including the genera Bacillus, Listeria, and Staphylococcus. Recent insight from nextgeneration-sequencing results indicates that B -dependent regulation of gene expression contributes to resilience, i.e., the coordination of complex networks responsive to environmental changes. This review explores contributions of B to resilience in Bacillus, Listeria, and Staphylococcus and illustrates recently described regulatory functions of B .