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Epidemiological surveys have shown that carbapenem resistance is mainly transmitted across species by carbapenemase genes located on conjugative plasmids. As chromosomal integration of carbapenemase genes has rarely been identified, only a few studies have investigated their advantages to the carbapenem-resistant bacterial community. Here, we confirmed the increased stability of bla IMP-6 on a chromosome-integrated plasmid in an Escherichia coli isolate compared with that on original plasmids in the absence of antibiotic pressure. Although plasmids carrying carbapenemase genes are supposedly lost in successive generations, we found that the complete plasmid backbone was retained in bacterial cells even after the occasional loss of their antibiotic-resistance cassettes. This backbone structure has been observed worldwide to carry various antimicrobial resistance genes. Although the chromosomally integrated plasmid carrying bla IMP-6 could not be transmitted by conjugation, we found that meropenem treatment for 1 wk allowed the plasmid to be released from the chromosome and spread among E. coli strains that were susceptible to meropenem. The copy number of bla IMP-6 on the plasmid was amplified eight times, resulting in enhanced resistance. Although the carbapenemase producers that carry chromosomal carbapenemase genes comprised of small subpopulations, they functioned as stable, long-term reservoirs of carbapenem resistance that could be disseminated via plasmids with amplified resistance upon meropenem stimulation. Although plasmids occasionally lose their resistance cassettes as a scaffold for the acquisition of another resistance gene, chromosomal integration may contribute to the effective sharing of carbapenem resistance within a population, complicating the development of a strategy to avoid the dissemination of antimicrobial resistance. IMPORTANCE Although carbapenem antibiotics are the last resort for combating multidrug-resistant organisms, global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens public health. Carbapenemases, which are enzymes responsible for carbapenem resistance, are mainly encoded by genes on plasmids that can be transmitted across bacterial species. Owing to the rarity of chromosomally encoded carbapenemase genes, studies investigating their properties in bacterial communities are lacking. In our study, we revealed the stability of carbapenemase genes on chromosomes compared with those on plasmids, which can be lost through the loss of antimicrobial resistance cassettes despite robust retention of plasmid backbones. Following exposure to meropenem, the carbapenemase gene integrated into the chromosome was released as a plasmid, restarting the dissemination of enhanced carbapenem resistance through amplified copy numbers of carbapenemase genes. Chromosomally encoded carbapenemase genes may function as a reservoir of resistance genes within the bacterial community and challenge infection control against CRE dissemination.

The spread of New Delhi metallo-β-lactamase (NDM)-producing Enterobacterales represents a public health risk. The horizontal transfer of plasmids encoding the NDM gene, bla NDM , usually mediates its spread to other bacteria within the family. In contrast, Enterobacterales with a chromosome-located bla . Escherichia coli M719 possessing both chromosomes- and plasmid-located NDM is rarely reported. The phenotypic differences between chromosome- and plasmid-located carbapenemase genes are poorly understood. To determine the significance in terms of the location of drug resistance genes, we examined carbapenemase activity and stability of chromosome- and plasmid-located bla NDM . Escherichia coli M719 possessing both chromosomes- and plasmid-located bla NDM genes was used as a wild-type strain (WT) for the construction of mutants, Δp bla NDM and Δc bla NDM , wherein chromosome- or plasmid-located bla NDM, was knocked out, respectively. The mutant Δp bla NDM showed lower hydrolyzing activity against imipenem and gene expression than the WT or Δc bla NDM mutant. The MICs of both mutant strains were still above the breakpoint of imipenem and meropenem. Moreover, the chromosome-located bla NDM gene was stable for at least 30 days in the absence of antimicrobial pressure, whereas the Δc bla NDM mutant lost bla NDM to 87% at 30 days compared to that of the initial inoculum. Organisms harboring the plasmid-located carbapenemase genes were found to provide a higher level of carbapenem resistance than those with chromosome-located genes. However, the latter organisms with chromosomal carbapenemase genes exhibited more stable carbapenem resistance than did the former ones. In summary, chromosomally located carbapenemase genes require further monitoring and more attention should be paid to them. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) carrying bla NDM have spread worldwide since they were first reported in 2009. Many studies using whole-genome sequencing have identified the genetic structures, plasmid scaffolds of bla NDM , and mechanisms of spread via horizontal transfer. Chromosome-located bla NDM and integration mechanisms from plasmids have rarely been reported, and their significance is not fully understood. Here, we showed that the chromosome-located bla NDM was associated with lower levels of carbapenem resistance and carbapenemase activity than the plasmid-located bla NDM . However, it conferred carbapenem resistance above the breakpoints and the loss of chromosome-located bla NDM was not observed in the absence of antibiotic pressure. This study suggests that CRE strains carrying chromosome-located bla NDM may persist in clinical and environmental settings for a long period even without antibiotic pressure and need to be monitored along with plasmid-located bla NDM .

Background: Carbapenemase-producing Enterobacterales (CPE) are spreading in hospitals, environment and retail foods in Yangon, Myanmar., Objectives: To investigate whether CPE colonize healthy individuals living in Yangon and whether clinical-related strains are spreading in the community., Methods: CPE was isolated from faecal samples obtained from healthy Japanese residents of Yangon with no history of hospitalization. Isolates were subjected to WGS using short- and long-read sequencers and compared with those previously isolated in Yangon., Results: Six Escherichia coli strains harbouring blaNDM-1 or blaNDM-5 belonging to five different STs-ST10, ST38, ST48, ST410 and ST8453-were isolated from 69 volunteers. The ST38 isolates were related to those previously isolated from retail food in Yangon. The ST410 and ST8453 isolates were highly related to previous Yangon isolates including those of clinical and food origins., Conclusions: The analysis suggested the acquisition of blaNDM-positive E. coli, which are disseminating in a clinical setting and through retail foods, by healthy residents in Yangon., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The emergence and global spread of extended-spectrum or AmpC β-lactamase (ESBL/AmpC)-producing Enterobacteriaceae in companion animals have led to the hypothesis that companion animals might be reservoirs for cross-species transmission because of their close contact with humans. However, current knowledge in this field is limited; therefore, the role of companion animals in cross-species transmission remains to be elucidated. Herein, we studied ESBL/AmpC-producing Enterobacteriaceae, Escherichia coli in particular, isolated from extraintestinal sites and feces of companion dogs. Whole-genome sequencing analysis revealed that (i) extraintestinal E. coli isolates were most closely related to those isolated from feces from the same dog, (ii) chromosomal sequences in the ST131/C1-M27 clade isolated from companion dogs were highly similar to those in the ST131/C1-M27 clade of human origin, (iii) certain plasmids, such as IncFII/pMLST F1:A2:B20/blaCTX-M-27, IncI1/pMLST16/blaCTX-M-15, or IncI1/blaCMY-2 from dog-derived E. coli isolates, shared high homology with those from several human-derived Enterobacteriaceae, (iv) chromosomal blaCTX-M-14 was identified in the ST38 isolate from a companion dog, and (v) eight out of 14 tested ESBL/AmpC-producing E. coli isolates (i.e., ST131, ST68, ST405, and ST998) belonged to the human extraintestinal pathogenic E. coli (ExPEC) group. All of the bla-coding plasmids that were sequenced genome-wide were capable of horizontal transfer. These results suggest that companion dogs can spread ESBL/AmpC-producing ExPEC via their feces. Furthermore, at least some ESBL/AmpC-producing ExPECs and bla-coding plasmids can be transmitted between humans and companion dogs. Thus, companion dogs can act as an important reservoir for ESBL/AmpC-producing E. coli in the community., Competing Interests: The authors have declared that no competing interests exist.

Background: Evolution optimizes a living system at both the genome and transcriptome levels. Few studies have investigated transcriptome evolution, whereas many studies have explored genome evolution in experimentally evolved cells. However, a comprehensive understanding of evolutionary mechanisms requires knowledge of how evolution shapes gene expression. Here, we analyzed Escherichia coli strains acquired during long-term thermal adaptive evolution., Results: Evolved and ancestor Escherichia coli cells were exponentially grown under normal and high temperatures for subsequent transcriptome analysis. We found that both the ancestor and evolved cells had comparable magnitudes of transcriptional change in response to heat shock, although the evolutionary progression of their expression patterns during exponential growth was different at either normal or high temperatures. We also identified inverse transcriptional changes that were mediated by differences in growth temperatures and genotypes, as well as negative epistasis between genotype-and heat shock-induced transcriptional changes. Principal component analysis revealed that transcriptome evolution neither approached the responsive state at the high temperature nor returned to the steady state at the regular temperature. We propose that the molecular mechanisms of thermal adaptive evolution involve the optimization of steady-state transcriptomes at high temperatures without disturbing the heat shock response., Conclusions: Our results suggest that transcriptome evolution works to maintain steady-state gene expression during constrained differentiation at various evolutionary stages, while also maintaining responsiveness to environmental stimuli and transcriptome homeostasis.

Background: Cell growth rate reflects an organism's physiological state and largely relies on the ability of gene expression to respond to the environment. The relationship between cellular growth rate and gene expression remains unknown., Results: Growth rate-coordinated changes in gene expression were discovered by analyzing exponentially growing Escherichia coli cells cultured under multiple defined environments, in which osmotic pressure, temperature and starvation status were varied. Gene expression analyses showed that all 3,740 genes in the genome could be simply divided into three clusters (C1, C2 and C3), which were accompanied by a generic trend in the growth rate that was coordinated with transcriptional changes. The direction of transcriptional change in C1 indicated environmental specificity, whereas those in C2 and C3 were correlated negatively and positively with growth rates, respectively. The three clusters exhibited differentiated gene functions and gene regulation task division., Conclusions: We identified three gene clusters, exhibiting differential gene functions and distinct directions in their correlations with growth rates. Reverses in the direction of the growth rate correlated transcriptional changes and the distinguished duties of the three clusters indicated how transcriptome homeostasis is maintained to balance the total expression cost for sustaining life in new habitats.

A synthetic dual-function operon with a bistable structure was designed and successfully integrated into the bacterial genome. Bistability was generated by the mutual inhibitory structure comprised of the promoters P(tet) and P(lac) and the repressors LacI and TetR. Dual function essential for cell growth was introduced by replacing the genes (i.e., hisC and leuB) encoding proteins involved in the biosynthesis of histidine and leucine from their native chromosomal locations to the synthetic operon. Both colony formation and population dynamics of the cells carrying this operon showed that the cells survived starvation and the newly formed population transited between the two stable states, representing the induced hisC and leuB levels, in accordance with the nutritional status. The results strongly suggested that the synthetic design of proto-operons sensitive to external perturbations is practical and functional in native cells.

The co-production of MCR and carbapenemase in Enterobacteriaceae has been previously reported. Here, we describe a clinical strain of Escherichia coli from Vietnam carrying both mcr-1 and bla NDM–1. Whole-genome sequencing showed that the genome of this strain consists of a 4,975,832-bp chromosome and four plasmids. The mcr-1 and bla NDM–1 genes are located on IncI2 and IncA/C2-type plasmids, respectively. Genetic analysis revealed the presence of a multidrug-resistant region with the structure of a novel complex class 1 integron including a class 1 integron region bearing two 5′ conserved segments and one 3′ conserved segment and two complete structures of IS CR1. The complex integron contains aminoglycoside resistance genes aadA2 , aadB , strA , strB , and aphA6 , quinolone resistance gene qnrA1 , extended-spectrum β-lactamase gene bla OXA–4, and a Tn 125 -like transposon bearing bla NDM–1. In addition, the dfrA12 - gcuF - aadA2 - cmlA1 - aadA1-qacH gene cassette array belonging to the sul3 -type integron was also identified, but the region found downstream of the gene cassette array is the IS 440 - tet(M) -IS 26 element instead of the sul3 gene. The results further support that Enterobacteriaceae isolates co-harboring mcr and bla NDM are widely being distributed. The structural characteristics of the complex integron reveal that IS CR1 elements play an important role in the mobilization of bla NDM–1 and the development of multidrug-resistant regions. [ABSTRACT FROM AUTHOR]

Distinctive survival strategies, specialized in regulation and in quality control, were observed in thermal adaptive evolution with a laboratory Escherichia coli strain. The two specialists carried a single mutation either within rpoH or upstream of gro ESL, which led to the activated global regulation by sigma factor 32 or an increased amount of Gro EL/ ES chaperonins, respectively. Although both specialists succeeded in thermal adaptation, the common winner of the evolution was the specialist in quality control, that is, the strategy of chaperonin-mediated protein folding. To understand this evolutionary consequence, multilevel analyses of cellular status, for example, transcriptome, protein and growth fitness, were carried out. The specialist in quality control showed less change in transcriptional reorganization responding to temperature increase, which was consistent with the finding of that the two specialists showed the biased expression of molecular chaperones. Such repressed changes in gene expression seemed to be advantageous for long-term sustainability because a specific increase in chaperonins not only facilitated the folding of essential gene products but also saved cost in gene expression compared with the overall transcriptional increase induced by rpoH regulation. Functional specialization offered two strategies for successful thermal adaptation, whereas the evolutionary advantageous was more at the points of cost-saving in gene expression and the essentiality in protein folding. [ABSTRACT FROM AUTHOR]