In recent years, subminimal inhibitory concentrations (sub-MIC) of antibiotics have been found to exert unexpected physiological effects on bacterial cells, beyond their common growth-inhibition properties. Our previous research demonstrated that sub-MIC ampicillin, combined with mild mechanical stimulation using glass balls, significantly promotes intercellular plasmid transformation (cell-to-cell plasmid transformation) in Escherichia coli within air-solid biofilms. In this study, we investigated whether other antibiotics with diverse mechanisms of action similarly enhance plasmid transformation. Our findings revealed that various antibiotics indeed promote cell-to-cell plasmid transformation, and this effect was observed under both aerobic and anaerobic conditions. Interestingly, anaerobic conditions resulted in higher frequencies of plasmid transformation compared to aerobic conditions. Supporting these results, we found that several single-gene knockouts of aerobic respiratory chain components under aerobic conditions also enhanced plasmid transformation. This suggests that the unavailability of aerobic respiration may favor the process of intercellular plasmid transfer. Collectively, our results indicate that a wide range of sub-MIC antibiotics can stimulate horizontal plasmid transfer and that anaerobic conditions are particularly conducive to this process. Based on these findings, we hypothesize that the anaerobic gut environment of antibiotic-treated animals or humans, characterized by biofilm-like high cell densities of antibiotic-exposed bacteria and regular peristaltic and segmental movements, could serve as a favorable niche for horizontal gene transfer via intercellular plasmid transformation.

Persisters represent a subset of cells that exhibit transient tolerance to antimicrobials. These persisters can withstand sudden exposure to antimicrobials, even as the majority of normal cells perish. In this study, we have demonstrated the capacity of ampicillin-tolerant and alkali-tolerant persisters to execute horizontal gene transfer via in situ transformation within biofilms. Air-solid biofilms, comprising two Escherichia coli populations each with a distinct plasmid, were formed on agar media. They were treated with lethal doses of ampicillin or NaOH for 24 h, followed by a 1-min glass-ball roll. This process led to a high frequency of horizontal plasmid transfer (10-- 7 -10- 6 per cell) from dead cells to surviving persisters within the biofilms. Plasmid transfer was DNasesensitive and also occurred by adding purified plasmid DNA to plasmid-free biofilms, demonstrating a transformation mechanism. This marks the first evidence of persisters' novel ability for horizontal gene transfer, via transformation.

Persister cells (PCs), a subpopulation occurring within normal cells, exhibit a transient tolerance to antibiotics because of their dormant state. PCs are categorized into two types: type I PCs, which emerge during the stationary phase, and type II PCs, which emerge during the logarithmic phase. Using the conventional colony-forming method, we previously demonstrated that type I PCs of Escherichia coli form more frequently in air–solid biofilm culture than in liquid culture. In the current study, we modified a cell filamentation method as a more efficient and rapid alternative for quantifying PCs. This modified method yielded results consistent with those of the conventional method with 103–104 times higher sensitivity and less detection time, within several hours, and further revealed the existence of multiple levels of type I PCs, including a substantial number of deeply dormant cells. This study also discovered a potential epigenetic memory mechanism, spanning several generations (four or six cell divisions), which influences type II PC formation based on prior biofilm experience in E. coli.

Horizontal gene transfer (HGT) is a bacterial evolution tool for improved survival. Although several environmental stimuli induce or promote HGT, the diversity and complexity of the environmental factors have not been sufficiently elucidated. In this study, we showed that the biofilm culture of Escherichia coli at the air-solid interface in the presence of a subminimal inhibitory concentration (sub-MIC) of ampicillin (∼0.5-4 µg/mL) and subsequent mechanical stimulation (rolling small glass balls, ø = 5-8 mm) cooperatively promoted horizontal plasmid transfer without the usual competence-inducing conditions. Either of the two treatments promoted plasmid transfer at a lower frequency than when the treatments were combined. The effect of several parameters on the two treatments was tested and then optimized, achieving a high frequency of plasmid transfer (up to 10-6 per cell) under optimal conditions. Plasmid transfer was DNase-sensitive for both treatments, demonstrating its mechanism of transformation. Plasmid transfer occurred using various E. coli strains, plasmids, ball materials, shaking conditions, and even the mechanical stimulation of brushing the biofilm with a toothbrush, indicating the conditional flexibility of this phenomenon. This is the first demonstration of the promoting effect of the combination of a sub-MIC antibiotic and mechanical stimulation on horizontal plasmid transfer between E. coli cells via transformation. Regarding environmental bacterial physiology, the aggregations or biofilms of bacterial cells may encounter both sub-MIC antibiotics and mechanical stimuli in some specific environments, therefore, this type of HGT could also occur naturally.

Persister cells, or persisters, are a fraction of bacterial cells that have become temporarily tolerant to antibiotics despite their lack of typical antibiotic-resistant genes. In a previous study, we found that colony–biofilm culture (i.e., biofilm formed at an air–solid interface) promoted the formation and phenotype memory retention of persisters of Escherichia coli and other bacteria. To assess whether these same effects are caused by other types of stimuli that bacterial cells encounter in the environment, we examined the effects of bovine serum on the formation and phenotype retention of ampicillin-tolerant persisters in E. coli liquid culture. Bovine serum did indeed exert these effects significantly, and its effects were negated by heat treatment. Similar effects were observed with bovine serum albumin, albeit weaker than those of BS. Given that serum is a component of blood and lymph and is thus a general substance within animal and human bodies, our findings suggest that bacteria encountering these body fluids may enhance their abilities for persister formation and phenotype memory retention to allow their longer survival in antibiotic-containing environments.

ABSTRACT The spread of multidrug-resistant bacteria, particularly those producing carbapenemases, has become a major public health concern. The presence of carbapenemase genes has primarily been reported in clinical samples, whereas the presence of these genes in commercially available foods has insufficiently been studied despite its growing importance. The present study aimed to detect and characterize carbapenemase genes ( bla NDM, bla IMP , bla KPC , and bla OXA-48-like ) on the surfaces of commercially available spices using PCR to amplify conserved regions of these genes. It was revealed that DNAs of these genes are commonly present (13 genes/29 samples) on spices derived from at least 9 different countries. This is the first detection of any carbapenemase gene on eight spices (black pepper, cumin, clove, cardamom, mustard, caraway, parsley, and rosemary) among these. This is also the first detection of the bla IMP and bla NDM as well as the broad detection of the bla OXA-48-like on spices. We also isolated complete, functional bla NDM-1 genes from three spices (black pepper, cumin, and clove) up to the present.

Escherichia coli is well known for its ability to undergo artificial transformation. This bacterium is not generally recognized as naturally transformable. However, several past studies have demonstrated that laboratory and natural strains of E. coli can express modest natural competence under feasible environmental conditions. Here we describe a protocol outlining natural transformation in E. coli in environmental media such as natural freshwater.

Bacteria continuously change their genetic characteristics to adapt to the changing environment by means of horizontal gene transfer. Although three conventional mechanisms of horizontal gene transfer are well known (transformation, transduction, and conjugation), new variations of these mechanisms have also been described. We previously reported that DNase-sensitive cell-to-cell transfer of non-conjugative plasmids, termed as "cell-to-cell transformation," occurs between the cells of two Escherichia coli strains in a co-culture. In this study, to further investigate the mechanism of cell-to-cell transformation, we constructed a new experimental system for cell-to-cell transformation. By using this system, we found that high temperatures of approximately 41ºC-45 °C significantly promote cell-to-cell plasmid transformation. This transfer was much more frequent in solid-air biofilms than in liquid culture, suggesting an importance of biofilm environment. Plasmid transfer frequency reached over 10-7/cell under the optimal strain-plasmid combination and conditions tested. DNase sensitivity test and plasmid isolation from the transformants confirmed the horizontal transfer of full-length plasmids via transformation. Comparative natural transformation experiments, which used similar strains and plasmids under equivalent culture conditions, revealed that cell-to-cell transformation occurs approximately 103 times more frequently than natural transformation, indicating the uniqueness and effectiveness of the cell-to-cell transformation mechanism. As temperatures of approximately 41ºC-45 °C are common in the avian intestines and under some other environmental situations, the phenomenon demonstrated here can occur efficiently in such locations. To the best of our knowledge, this is the first study to demonstrate the enhancing effect of high temperatures on cell-to-cell plasmid transformation in E. coli.

to a culture base, their cell-to-cell interactions become very tight, so that they cannot be dispersed perfectly by the convcntional techniques (e.g,, trypsin,iEDTA treatment),i'!) To resolve this problem, we tried to apply a colorimetric MTT assay to primary cultured hepatocytes. The MTT assay is a rapid colorimetric microtiter assay which was originally developed to measure the living cell number ef lyrnphocytes.3) This methed is based on t-he selective ability of mitochondrial dehydrogenases in living cells to reduce the yellow soluble salt MTT (3-(4.5-dirnethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromidc)toapurple insoluble formuzun precipitate, The number of viable cells is, therefore,

Persister cells, or persisters, are a specific subpopulation of bacterial cells that have acquired temporary antibiotic-resistant phenotypes. In this study, we showed that Escherichia coli produces many more persister cells in colony-biofilm culture than in the usual liquid culture and that these persisters can be maintained in higher numbers than those from liquid culture for up to 4 weeks at 37°C in a fresh, nutrient-rich, antibiotic-containing medium, even after complete withdrawal from the colony-biofilm culture. This suggests the presence of a long-retention effect, or "memory effect", in the persister cell state of E. coli cells. We also discovered that such increases in persisters during colony-biofilm culture and their memory effects are common, to a greater or lesser degree, in other bacterial species. This is true not only for gram-negative bacteria (Acinetobacter and Salmonella) but also for gram-positive bacteria (Staphylococcus and Bacillus). This is the first report to suggest the presence of a common memory mechanism for the persister cell state, which is inscribed during colony-biofilm culture, in a wide variety of bacteria.

Transformation is one mode of horizontal gene transfer (HGT) in bacteria, wherein extracellular naked DNA is taken up by cells that have developed genetic competence. Sensitivity to DNase, which degrades naked DNA, is the key to distinguishing transformation from the DNase-resistant HGT mechanisms. In general, Escherichia coli is not believed to be naturally transformable; it develops high competence only under artificial conditions, including exposure to high Ca2+ concentrations. However, E. coli can reportedly express modest competence under certain conditions that are feasible in natural environments outside laboratory. In addition, recent data suggest that environmental factors influence multiple routes of transformation. In this mini review, we (1) summarize our studies on transformation-based HGT using E. coli experimental systems and (2) discuss the possible occurrence of transformation via multiple mechanisms in the environment and its possible impact on the spread of antibiotic resistance genes.

Bacteria undergo horizontal gene transfer via various mechanisms. We recently reported that cell-to-cell transfer of nonconjugative plasmids occurs between strains of Escherichia coli in co-cultures, and that a specific strain (CAG18439) causes frequent plasmid transfer involving a DNase-sensitive mechanism, which we termed "cell-to-cell transformation". Here we found that CAG18439 is a type of P1 bacteriophage lysogen that continuously releases phages. We tested the ability of P1vir bacteriophage to induce horizontal plasmid transfer and demonstrated that such a horizontal plasmid transfer was caused by adding culture supernatants of P1vir-infected cells harboring plasmids to other plasmid-free cells. This plasmid transfer system also reproduced the major features of plasmid transfer involving CAG18439, suggesting that P1vir-induced plasmid transfer is equivalent or very similar to plasmid transfer involving CAG18439. We further revealed that approximately two-thirds of the P1vir-induced plasmid transfer was DNase-sensitive, but that complete abolition of plasmid transfer was observed when proteins were denatured or removed, despite the presence or absence of DNase. Therefore, we concluded that P1vir-induced plasmid transfer is largely due to the occurrence of cell-to-cell transformation, which involves the assistance of some proteinaceous factor, and partly due to the occurrence of plasmid transduction, which is mediated by phage virions. This is the first demonstration of the P1-phage-induced cell-to-cell transformation.

Horizontal gene transfer is a strong tool that allows bacteria to adapt to various environments. Although three conventional mechanisms of horizontal gene transfer (transformation, transduction, and conjugation) are well known, new variations of these mechanisms have also been observed. We recently reported that DNase-sensitive cell-to-cell transfer of nonconjugative plasmids occurs between laboratory strains of Escherichia coli in co-culture. We termed this phenomenon “cell-to-cell transformation.” In this report, we found that several combinations of Escherichia coli collection of reference (ECOR) strains, which were co-cultured in liquid media, resulted in DNase-sensitive cell-to-cell transfer of antibiotic resistance genes. Plasmid isolation of these new transformants demonstrated cell-to-cell plasmid transfer between the ECOR strains. Natural transformation experiments, using a combination of purified plasmid DNA and the same ECOR strains, revealed that cell-to-cell transformation occurs much more frequently than natural transformation under the same culture conditions. Thus, cell-to-cell transformation is both unique and effective. In conclusion, this study is the first to demonstrate cell-to-cell plasmid transformation in natural E. coli strains.