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.

Significance Many model organisms specify germ cells using maternally supplied germ-line determinants. In contrast, mice rely on embryonic cell–cell signaling to induce cells to become germ cells. Molecular evidence for inductive germ-line specification had previously been provided only for the mouse. Here we provide functional evidence for inductive germ cell specification in an invertebrate, by showing that bone morphogenetic protein (BMP) signaling, which induces mouse germ cell specification, is required for establishment of embryonic germ cells in a cricket. BMP pathway knockdown causes reduction or loss of germ cells, and elevated levels of BMP signaling cause supernumerary and ectopic germ cells. BMP-based germ cell induction in mice and crickets suggests that this may be a shared ancestral mechanism in animals.

The growth of Listeria monocytogenes on refrigerated, ready-to-eat food products is a major health and economic concern. The natural antimicrobial nisin targets the bacterial cell wall and can be used to inhibit L. monocytogenes growth on cheese. Cell wall composition and structure, and therefore the efficacy of cell wall acting control strategies, can be severely affected by environmental and stress conditions. The goal of this study was to determine the effect of a range of pH and temperatures on the efficacy of nisin against several strains of L. monocytogenes in a lab-scale, cheese model. Cheese was made with or without the addition of nisin at different pH and then inoculated with L. monocytogenes; L. monocytogenes numbers were quantified after 1, 7, and 14 days of incubation at 6, 14, or 22 • C. While our data show that nisin treatment is able to reduce L. monocytogenes numbers, at least initially, growth of this pathogen can occur even in the presence of nisin, especially when cheese is stored at higher temperatures. Several environmental factors were found to affect nisin efficacy against L. monocytogenes. For example, nisin is more effective when cheese is stored at lower temperatures. Nisin is also more effective when cheese is made at higher pH (6 and 6.5), compared to cheese made at pH 5.5, and this effect is at least partially due to the activity of cell envelope modification genes dltA and mprF. Serotype was also found to affect nisin efficacy against L. monocytogenes; serotype 4b strains showed lower susceptibility to nisin treatment compared to serotype 1/2 strains. Overall, our results highlight the importance of considering environmental conditions specific to a food matrix when developing and applying nisin-based intervention strategies against L. monocytogenes.

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The transcriptional activator Positive Regulatory Factor A (PrfA) regulates expression of genes essential for virulence in Listeria monocytogenes. To define the PrfA regulon, the 10403S wildtype (WT) strain, a constitutively active prfA* mutant, and an isogenic ∆prfA mutant were grown under PrfA-inducing conditions in a medium containing glucose-1-phosphate and pre-treated with 0.2% activated charcoal. RNA-seq-generated transcript levels were compared as follows: (i) prfA* and WT; (ii) WT and ∆prfA; and (iii) prfA* and ∆prfA. Significantly higher transcript levels in the induced WT or constitutively active PrfA* were identified for 18 genes and 2 ncRNAs in at least one of the three comparisons. These genes included: (i) 10/12 of the genes previously identified as directly PrfA-regulated; (ii) 2 genes previously identified as PrfA-regulated, albeit likely indirectly; and (iii) 6 genes newly identified as PrfA-regulated, including one (LMRG_02046) with a σA-dependent promoter and PrfA box located within an upstream open reading frame. LMRG_02046, which encodes a putative cyanate permease, is reported to be downregulated by a σB-dependent anti-sense RNA. This newly identified overlap between the σB and PrfA regulons highlights the complexity of regulatory networks important for fine-tuning bacterial gene expression in response to the rapidly changing environmental conditions associated with infection.

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 in a Salmonella enterica serotype Typhimurium (S. Typhimurium) genome using an in silico approach, followed by experimental functional analysis. The amino acid sequence of mcr-9, detected in a multidrug resistant (MDR) 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. The S. Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2 mg/L European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. To determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host, mcr-9 was cloned in colistin-susceptible Escherichia coli NEB5α under an IPTG-induced promoter. Expression of mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/L colistin, albeit at a lower level when compared to 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, and Mcr-7 share a high degree of similarity at the structural level. The results of our approach 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), 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.