A Single Residue within the MCR-1 Protein Confers Anticipatory Resilience.

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz., the two-component signal transduction system CpxRA (conjugative pilus expression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers.

Resolving colistin resistance and heteroresistance in Enterobacter species.

Species within the Enterobacter cloacae complex (ECC) include globally important nosocomial pathogens. A three-year study of ECC in Germany identified Enterobacter xiangfangensis as the most common species (65.5%) detected, a result replicated by examining a global pool of 3246 isolates. Antibiotic resistance profiling revealed widespread resistance and heteroresistance to the antibiotic colistin and detected the mobile colistin resistance (mcr)-9 gene in 19.2% of all isolates. We show that resistance and heteroresistance properties depend on the chromosomal arnBCADTEF gene cassette whose products catalyze transfer of L-Ara4N to lipid A. Using comparative genomics, mutational analysis, and quantitative lipid A profiling we demonstrate that intrinsic lipid A modification levels are genospecies-dependent and governed by allelic variations in phoPQ and mgrB, that encode a two-component sensor-activator system and specific inhibitor peptide. By generating phoPQ chimeras and combining them with mgrB alleles, we show that interactions at the pH-sensing interface of the sensory histidine kinase phoQ dictate arnBCADTEF expression levels. To minimize therapeutic failures, we developed an assay that accurately detects colistin resistance levels for any ECC isolate.

Genetic diversity and risk factor analysis of drug-resistant Escherichia coli recovered from broiler chicken farms.

A total of 38 Escherichia coli isolates were recovered from 120 samples collected from various sources of broiler chicken farms (n = 10 each) in Andhra Pradesh and Telangana states. Though the recovered E. coli isolates were found variably resistant to the tested antibiotics, all the tested isolates were susceptible to meropenem. Alarming multi-drug resistance (MDR) was observed (34/38) among the recovered isolates, wherein antibiotic-resistant genes (blaTEM, blaSHV, and tetA) were detected, except for blaCTX-M-9. The heatmap with cluster analysis exhibited that majority of the E. coli isolates recovered from different sources and regions clustered together based on their phenotypic resistance suggesting co-sharing of resistance. However, the pulsed-field gel electrophoresis (PFGE) typing revealed an extremely diverse genotypic profile. Further, a significant statistical association was not observed between hypothesized risk factors and recovered MDR- E. coli isolates from various sources, although a significant statistical association between antibiotic resistance with large flock size, poor biosecurity practices, poor workers' hygiene, and poor disinfection practices was noticed. Since the study highlighted an alarming level of drug resistance among the recovered E. coli isolates, further in-depth research in similar veins is required to ensure the prudent use of antimicrobials in the poultry sector and the implementation of an antimicrobial surveillance system.

Pulsed-field gel electrophoresis fingerprinting of Listeria monocytogenes isolates recovered from foods of animal origin and fishes in North-Eastern India.

L isteria monocytogenes is a pathogen of great concern to the food industry. The present study was aimed to explore the clonal relationships amongst L. monocytogenes strains isolated from foods of animal origin (milk, beef, chevon (goat meat), pork and chicken) and fish. Forty-seven L. monocytogenes strains were characterized by pulsed-field gel electrophoresis (PFGE). The PFGE analysis using ApaI and AscI enzymes revealed 37 pulsotypes, with Simpson's discriminatory index of 0.987. This study demonstrated the presence of a few similar L. monocytogenes pulsotypes in different foods of animal origin in different places and years of isolation and this indicates that some L. monocytogenes subtypes may be ubiquitous which are acclimatizing and persisting in different foods of animal origin. This also emphasizes the importance of cross-contamination in local wet markets. Thus, the understanding of genetic diversity will contribute to the development of rational and workable strategies to control this important zoonotic infection.

Ecology of Listeria monocytogenes and Listeria species in India: the occurrence, resistance to biocides, genomic landscape and biocontrol.

Listeria monocytogenes, the causative agent of listeriosis, has been implicated in increasing foodborne outbreaks worldwide. The disease is manifested in various forms ranging from severe sepsis in immune-compromised individuals, febrile gastroenteritis, still birth, abortions and meningoencephalitis. In India, data from studies on the detection and molecular epidemiological analysis of L. monocytogenes are only recently emerging. The presence of Listeria in different ecological niches has been recorded from India, including foods, soil, vegetables, mangrove swamps, seafood, freshwater fishes, clinical cases, and also insects. The organism has also been isolated from women with spontaneous abortions, miscarriage or recurrent obstetric history, aborted foetuses, animal clinical cases and wildlife samples. A novel species of Listeria has also been characterized. Listeria monocytogenes strains isolated from clinical, environmental, and foods showed biofilm-forming abilities. Listeria monocytogenes serotype 4b isolates of ST328, a predominant and unique ST observed in India, was repeatedly isolated from different sources, times, and geographical locations. Here, we reviewed the occurrence of Listeria in different sources in India, its resistance to biocides, and provide epidemiological analysis on its genomic landscape.

Enterobacter bugandensis: a novel enterobacterial species associated with severe clinical infection.

Nosocomial pathogens can cause life-threatening infections in neonates and immunocompromised patients. E. bugandensis (EB-247) is a recently described species of Enterobacter, associated with neonatal sepsis. Here we demonstrate that the extended spectrum ß-lactam (ESBL) producing isolate EB-247 is highly virulent in both Galleria mellonella and mouse models of infection. Infection studies in a streptomycin-treated mouse model showed that EB-247 is as efficient as Salmonella Typhimurium in inducing systemic infection and release of proinflammatory cytokines. Sequencing and analysis of the complete genome and plasmid revealed that virulence properties are associated with the chromosome, while antibiotic-resistance genes are exclusively present on a 299 kb IncHI plasmid. EB-247 grew in high concentrations of human serum indicating septicemic potential. Using whole genome-based transcriptome analysis we found 7% of the genome was mobilized for growth in serum. Upregulated genes include those involved in the iron uptake and storage as well as metabolism. The lasso peptide microcin J25 (MccJ25), an inhibitor of iron-uptake and RNA polymerase activity, inhibited EB-247 growth. Our studies indicate that Enterobacter bugandensis is a highly pathogenic species of the genus Enterobacter. Further studies on the colonization and virulence potential of E. bugandensis and its association with septicemic infection is now warranted.