A new variant of the colistin resistance gene MCR-1 with co-resistance to ß-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to ß-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to ß-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

Altered B cell immunoglobulin signature exhibits potential diagnostic values in human colorectal cancer.

Antibody-secreting B cells have long been considered the central element of gut homeostasis; however, tumor-associated B cells in human colorectal cancer (CRC) have not been well characterized. Here, we show that the clonotype, phenotype, and immunoglobulin subclasses of tumor-infiltrating B cells have changed compared to adjacent normal tissue B cells. Remarkably, the tumor-associated B cell immunoglobulin signature alteration can also be detected in the plasma of patients with CRC, suggesting that a distinct B cell response was also evoked in CRC. We compared the altered plasma immunoglobulin signature with the existing method of CRC diagnosis. Our diagnostic model exhibits improved sensitivity compared to the traditional biomarkers, CEA and CA19-9. These findings disclose the altered B cell immunoglobulin signature in human CRC and highlight the potential of using the plasma immunoglobulin signature as a non-invasive method for the assessment of CRC.

A CRISPR-guided mutagenic DNA polymerase strategy for the detection of antibiotic-resistant mutations in M. tuberculosis.

A sharp increase in multidrug-resistant tuberculosis (MDR-TB) threatens human health. Spontaneous mutation in essential gene confers an ability of Mycobacterium tuberculosis resistance to anti-TB drugs. However, conventional laboratory strategies for identification and prediction of the mutations in this slowly growing species remain challenging. Here, by combining XCas9 nickase and the error-prone DNA polymerase A from M. tuberculosis, we constructed a CRISPR-guided DNA polymerase system, CAMPER, for effective site-directed mutagenesis of drug-target genes in mycobacteria. CAMPER was able to generate mutagenesis of all nucleotides at user-defined loci, and its bidirectional mutagenesis at nick sites allowed editing windows with lengths up to 80 nucleotides. Mutagenesis of drug-targeted genes in Mycobacterium smegmatis and M. tuberculosis with this system significantly increased the fraction of the antibiotic-resistant bacterial population to a level approximately 60- to 120-fold higher than that in unedited cells. Moreover, this strategy could facilitate the discovery of the mutation conferring antibiotic resistance and enable a rapid verification of the growth phenotype-mutation genotype association. Our data demonstrate that CAMPER facilitates targeted mutagenesis of genomic loci and thus may be useful for broad functions such as resistance prediction and development of novel TB therapies.

Single-Fluorescence ATP Sensor Based on Fluorescence Resonance Energy Transfer Reveals Role of Antibiotic-Induced ATP Perturbation in Mycobacterial Killing.

The rapid emergence of multidrug-resistant/extensively drug-resistant tuberculosis (TB) is responsible for treatment failure in patients with TB and significantly endangers global public health. Recently, bioenergetics has become a new paradigm for anti-TB drug discovery and is based on the link between bacterial ATP levels and drug efficacy. A better understanding of the role of ATP fluctuations during antibiotic treatment may provide insight into antibiotic-mediated killing of mycobacteria. Here, we employed an advanced single-fluorescence FRET (fluorescence resonance energy transfer)-based ATP biosensor, ATPser, for the stable and convenient detection of intracellular ATP fluctuations in mycobacteria. This strategy correlated closely with the results obtained from conventional luminescence ATP assays, indicating the reliability of the system for bioenergetics analysis in mycobacteria. Moreover, the reporter strains expressing ATPser displayed obvious ATP changes when subjected to different stresses, such as starvation and ATP depletion. Interestingly, we observed that different antibiotics induced fluctuations in cellular ATP levels in individual cells of various magnitudes, revealing a strong connection between ATP fluctuations and drug efficacy. Furthermore, drug combinations accelerated ATP perturbation, resulting in increased cell death. We concluded that ATPser enabled real-time measurement of ATP at the single-cell level in mycobacteria, and monitoring ATP dynamics in drug-treated bacteria may shed light on novel treatment strategies. IMPORTANCE Bioenergetics has emerged as a new paradigm for antituberculosis (anti-TB) drug discovery, and the cellular ATP level is the core indicator reflecting bacterial metabolic homeostasis. Although several bulk assays have been designed for the measurement of cellular ATP content, a more convenient strategy is required for real-time ATP measurement of single viable cells. In this study, by combining the ε-subunit of Bacillus subtilis FoF1-ATP synthase with a circularly permuted green fluorescent protein [(cp)GFP], we constructed a FRET-based single-fluorescence ATP sensor, ATPser, for real-time single-cell ATP detection among a mycobacterial population. Using the ATPser, we designed different drug combinations containing components that have similar/opposite effects on ATP alternation. Our results demonstrated that increased cellular ATP fluctuations were associated with depletion of mycobacterial viability, while counteracting ATP fluctuations weakened the killing effect of the drug regime. Thus, potentially efficient drug combinations can be considered based on their similar effects on mycobacterial ATP levels, and ATPser may be a useful tool to study mycobacterial bioenergetics and to guide drug regime design.

MCR-1-dependent lipid remodelling compromises the viability of Gram-negative bacteria.

The global dissemination of the mobilized colistin resistance gene, mcr-1, threatens human health. Recent studies by our group and others have shown that the withdrawal of colistin as a feed additive dramatically reduced the prevalence of mcr-1. Although it is accepted that the rapid reduction in mcr-1 prevalence may have resulted, to some extent, from the toxic effects of MCR-1, the detailed mechanism remains unclear. Here, we found that MCR-1 damaged the outer membrane (OM) permeability in Escherichia coli and Klebsiella pneumonia and that this event was associated with MCR-1-mediated cell shrinkage and death during the stationary phase. Notably, the capacity of MCR-1-expressing cells for recovery from the stationary phase under improved conditions was reduced in a time-dependent manner. We also showed that mutations in the potential lipid-A-binding pocket of MCR-1, but not in the catalytic domain, restored OM permeability and cell viability. During the stationary phase, PbgA, a sensor of periplasmic lipid-A and LpxC production that performed the first step in lipid-A synthesis, was reduced after MCR-1 expression, suggesting that MCR-1 disrupted lipid homeostasis. Consistent with this, the overexpression of LpxC completely reversed the MCR-1-induced OM permeability defect. We propose that MCR-1 causes lipid remodelling that results in an OM permeability defect, thus compromising the viability of Gram-negative bacteria. These findings extended our understanding of the effect of MCR-1 on bacterial physiology and provided a potential strategy for eliminating drug-resistant bacteria.

Prediction of Antibiotic Resistance Evolution by Growth Measurement of All Proximal Mutants of Beta-Lactamase.

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum ß-lactamase gene blaCTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEARP and PEARR, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of blaCTX-M-14 evolution, respectively. Single to quintuple mutations of blaCTX-M-14 predicted to confer resistance by PEARP were significantly enriched among the clinical isolates harboring blaCTX-M-14 variants, and the PEARR scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies.

The Involvement of Mycobacterium Type III-A CRISPR-Cas System in Oxidative Stress.

Type I and type II CRISPR-Cas systems are employed to evade host immunity by targeting interference of bacteria's own genes. Although Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, possesses integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2∼5, cas10, cas6) were upregulated in Mycobacterium bovis BCG under oxidative stress treatment, indicating the role of type III-A CRISPR-Cas system in oxidative stress. To explore the functional role of type III-A CRISPR-Cas system, TCC (Type III-A CRISPR-Cas system, including cas6, cas10, and csm2-6) mutant was generated. Deletion of TCC results in increased sensitivity in response to hydrogen peroxide and reduced cell envelope integrity. Analysis of RNA-seq dataset revealed that TCC impacted on the oxidation-reduction process and the composition of cell wall which is essential for mycobacterial envelop integrity. Moreover, disrupting TCC led to poor intracellular survival in vivo and in vitro. Finally, we showed for the first time that TCC contributed to the regulation of regulatory T cell population, supporting a role of TCC in modulating host immunity. Our finding reveals the important role of TCC in cell envelop homeostasis. Our work also highlights type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, thus may be a potential target for therapy.

Involvement of Transcription Elongation Factor GreA in Mycobacterium Viability, Antibiotic Susceptibility, and Intracellular Fitness.

There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (ΔgreA) was generated in Mycobacterium smegmatis, Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardation and poor survival in response to adverse stress, besides rendering M. tuberculosis more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting greA results in the differential regulation of 195 genes in M. smegmatis with 167 being negatively regulated. Among these, KEGG pathways significantly enriched for differentially regulated genes included tryptophan metabolism, starch and sucrose metabolism, and carotenoid biosynthesis, supporting a role of GreA in the metabolic regulation of mycobacteria. Moreover, like Escherichia coli GreA, M. smegmatis GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of glgX, a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of glgX by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of greA. Our data indicates that GreA is an important factor for the survival and resistance establishment in Mycobacterium spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment.

Dynamics of mcr-1 prevalence and mcr-1-positive Escherichia coli after the cessation of colistin use as a feed additive for animals in China: a prospective cross-sectional and whole genome sequencing-based molecular epidemiological study.

BACKGROUND: The global dissemination of colistin resistance encoded by mcr-1 has been attributed to extensive use of colistin in livestock, threatening colistin efficacy in medicine. The emergence of mcr-1 in common pathogens, such as Escherichia coli, is of particular concern. China banned the use of colistin in animal feed from May 1, 2017. We investigated subsequent changes in mcr-1 prevalence in animals, humans, food, and the environment, and the genomic epidemiology of mcr-1-positive E coli (MCRPEC). METHODS: Sampling was done before (October to December, 2016) and after (October to December, 2017, and 2018, respectively) the colistin ban. 3675 non-duplicate pig faecal samples were collected from 14 provinces (66 farms) in China to measure intervention-related changes in mcr-1 prevalence. 15 193 samples were collected from pigs, healthy human volunteers, patients colonised or infected with Enterobacteriaceae who were admitted to hospital, food and the environment in Guangzhou, to characterise source-specific mcr-1 prevalence and the wider ecological effect of the ban. From these samples, 688 MCRPEC were analysed with whole genome sequencing, plasmid conjugation, and S1 pulsed-field gel electrophoresis with Southern blots to characterise associated genomic changes. FINDINGS: After the ban, mcr-1 prevalence decreased significantly in national pig farms, from 308 (45%) of 684 samples in 2016 to 274 (19%) of 1416 samples in 2018 (p<0·0001). A similar decrease occurred in samples from most sources in Guangzhou (959 [19%] of 5003 samples in 2016; 238 [5%] of 4489 samples in 2018; p<0·0001). The population structure of MCRPEC was diverse (23 sequence clusters); sequence type 10 clonal complex isolates were predominant (247 [36%] of 688). MCRPEC causing infection in patients admitted to hospital were genetically more distinct and appeared less affected by the ban. mcr-1 was predominantly found on plasmids (632 [92%] of 688). Common mcr-1 plasmid types included IncX4, IncI2, and IncHI2 (502 [76%] of 656); significant increases in IncI2-associated mcr-1 and a distinct lineage of mcr-1-associated IncHI2 were observed post ban. Changes in the frequency of mcr-1-associated flanking sequences (ISApl1-negative MCRPEC), 63 core genome single nucleotide polymorphisms, and 30 accessory genes were also significantly different after the ban (Benjamini-Hochberg-adjusted p<0·05), consistent with rapid genetic adaptation in response to changing selection pressures. INTERPRETATION: A rapid, ecosystem-wide, decline in mcr-1 was observed after the use of colistin in animal feed was banned, with associated genetic changes in MCRPEC. Withdrawal of antimicrobials from animal feed should be an important One Health measure contributing to the wider control of antimicrobial resistance globally. FUNDING: National Natural Science Foundation of China.