In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. ...Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.
•ColE-type plasmid with blaOXA-232 increased persister formation rate against ciprofloxacin and amikacin.•Plasmids with deletion of hypothetical gene, vbhA, and MOB decreased persister ...formation.•Particularly, vbhA-encoding antitoxin significantly effected persister formation against ciprofloxacin.
Bacterial persister cells are a sub-population of cells that are tolerant to high concentrations of antibiotics. In this study, we investigated the effect of plasmids bearing carbapenemase genes on persister cell formation.
Three plasmids, IncX3-type plasmid with blaNDM-1, IncN-type plasmid with blaKPC-2, and ColE-type plasmid with blaOXA-232, were transformed into Escherichia coli MG1655. For the ColE-type plasmid (pM5_OXA232), gene-deletion plasmids were constructed and transformed into the MG1655. Persister assays were performed against ciprofloxacin and amikacin, and expression levels of relA and spoT were measured for the wild-type E. coli and all transformants.
Unlike the other two plasmids, transformation of ColE-type plasmid (pM5_OXA232) caused a significant increase in the formation of persister cells. Compared with transformants that harboured intact pM5_OXA232, transformants that harboured plasmids with deletions of gene(s), vbhA, hypothetical gene, or a mobile gene cassette showed decreased persister cell formation. Expression levels of relA and spoT exhibited patterns similar to those of persister cell formation rates, particularly against ciprofloxacin.
In this study, we showed that a small ColE-type plasmid bearing blaOXA-232 has an effect on persister cell formation, possibly contributing to the dissemination of low-level carbapanemase.
Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of ...colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.
Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent in hospitals in many Asian countries. Recent emergence of community-associated (CA) MRSA worldwide has added another serious ...concern to the epidemiology of S. aureus infections. To understand the changing epidemiology of S. aureus infections in Asian countries, we performed a prospective, multinational surveillance study with molecular typing analysis.
We evaluated the prevalence of methicillin resistance in S. aureus isolates in CA and healthcare-associated (HA) infections, and performed molecular characterization and antimicrobial susceptibility tests of MRSA isolates.
MRSA accounted for 25.5% of CA S. aureus infections and 67.4% of HA infections. Predominant clones of CA-MRSA isolates were ST59-MRSA-SCCmec type IV-spa type t437, ST30-MRSA-SCCmec type IV-spa type t019 and ST72-MRSA-SCCmec type IV-spa type t324. Previously established nosocomial MRSA strains including sequence type (ST) 239 and ST5 clones were found among CA-MRSA isolates from patients without any risk factors for HA-MRSA infection. CA-MRSA clones such as ST59, ST30 and ST72 were also isolated from patients with HA infections.
Our findings confirmed that MRSA infections in the community have been increasing in Asian countries. Data also suggest that various MRSA clones have spread between the community and hospitals as well as between countries.
Abstract The characteristics of 218 Klebsiella pneumoniae isolates from patients with hospital-acquired pneumonia in nine Asian countries were investigated. In total, 92 isolates (42.2%) produced ...extended-spectrum β-lactamases (ESBLs), amongst which 67 (72.8%) possessed CTX-M ESBL genes; CTX-M-15 was the major ESBL (55 isolates; 59.8%). Multilocus sequence typing (MLST) and plasmid replicon typing were performed to investigate the genetic backgrounds of the 55 CTX-M-15-producing K. pneumoniae isolates. Twenty-five sequence types (STs) were identified. Clonal complex 11 (CC11) including ST11 was the most prevalent clone (20 isolates; 36.4%) and was distributed in all Asian countries except Taiwan. ST15 was the next most frequently identified clone (8 isolates; 14.5%). An IncFIIA-type plasmid was predominantly associated with blaCTX-M-15 (45 isolates; 81.8%). However, another plasmid type (IncA/C) was also identified and replicon types of seven isolates could not be determined. The high prevalence of CTX-M-15 amongst K. pneumoniae isolates in Asian countries may be due both to the acquisition of plasmids carrying blaCTX-M-15 and the spread of certain clones such as ST11 and ST15.
Five types of Escherichia coli strains were obtained and sequenced: colistin-susceptible (CL-S) strains, in vitro induced colistin-resistant (CL-IR) strains, mcr-1-negative colistin-resistant strains ...from livestock (CL-chrR), mcr-1-positive colistin-resistant strains (CL-mcrR), and mcr-1-transferred transconjugants (TC-mcr). Amino acid alterations of PmrAB, PhoPQ, and EptA were identified, and their mRNA expression was measured. Their growth rate was evaluated, and an in vitro competition assay was performed. Virulence was compared through serum resistance and survival in macrophages and Drosophila melanogaster. CL-IR and CL-chrR strains were colistin-resistant due to amino acid alterations in PmrAB, PhoPQ, or EptA, and their overexpression. All colistin-resistant strains did not show reduced growth rates compared with CL-S strains. CL-IR and CL-chrR strains were less competitive than the susceptible strain, but CL-mcrR strains were not. In addition, TC-mcr strains were also significantly more competitive than their respective parental susceptible strain. CL-IR strains had similar or decreased survival rates in human serum, macrophages, and fruit flies, compared with their parental, susceptible strains. CL-chrR strains were also less virulent than CL-S strains. Although CL-mcrR strains showed similar survival rates in human serum and fruit fly to CL-S strains, the survival rates of TC-mcr strains decreased significantly in human serum, macrophages, and fruit flies, compared with their susceptible recipient strain (J53). Chromosome-mediated, colistin-resistant E. coli strains have a fitness cost, but plasmids bearing mcr-1 do not increase the fitness burden of E. coli. Along with high usage of polymyxins, the no fitness cost of mcr-1-positive strains may facilitate rapid spread of colistin resistance.Five types of Escherichia coli strains were obtained and sequenced: colistin-susceptible (CL-S) strains, in vitro induced colistin-resistant (CL-IR) strains, mcr-1-negative colistin-resistant strains from livestock (CL-chrR), mcr-1-positive colistin-resistant strains (CL-mcrR), and mcr-1-transferred transconjugants (TC-mcr). Amino acid alterations of PmrAB, PhoPQ, and EptA were identified, and their mRNA expression was measured. Their growth rate was evaluated, and an in vitro competition assay was performed. Virulence was compared through serum resistance and survival in macrophages and Drosophila melanogaster. CL-IR and CL-chrR strains were colistin-resistant due to amino acid alterations in PmrAB, PhoPQ, or EptA, and their overexpression. All colistin-resistant strains did not show reduced growth rates compared with CL-S strains. CL-IR and CL-chrR strains were less competitive than the susceptible strain, but CL-mcrR strains were not. In addition, TC-mcr strains were also significantly more competitive than their respective parental susceptible strain. CL-IR strains had similar or decreased survival rates in human serum, macrophages, and fruit flies, compared with their parental, susceptible strains. CL-chrR strains were also less virulent than CL-S strains. Although CL-mcrR strains showed similar survival rates in human serum and fruit fly to CL-S strains, the survival rates of TC-mcr strains decreased significantly in human serum, macrophages, and fruit flies, compared with their susceptible recipient strain (J53). Chromosome-mediated, colistin-resistant E. coli strains have a fitness cost, but plasmids bearing mcr-1 do not increase the fitness burden of E. coli. Along with high usage of polymyxins, the no fitness cost of mcr-1-positive strains may facilitate rapid spread of colistin resistance.
Many mechanisms have been proposed to be involved in the formation of bacterial persister cells. In this study, we investigated the impact of
encoding DNA methylation on persister cell formation in ...Acinetobacter. We constructed plasmids overexpressing
encoding DNA-(adenine N6)-methyltransferase and four genes as possibly involved in persistence and introduced them into three A. baumannii strains. For persister cell formation assays, bacteria were exposed to ciprofloxacin, imipenem, cefotaxime, and rifampin, and the transcription levels of the genes were measured by qRT-PCR. In addition, growth curves of strains were determined. We found that all five genes were upregulated following antibiotic exposure. Dam overexpression increased persister cell formation rates and activated the four persister cell-involved genes. Among the four persister cell-involved genes, only RecC overexpression increase persister cell formation rates. While
-overexpressing strains showed higher growth rates,
-overexpressing strains showed decreased growth rates. In this study, we revealed that a DNA methyltransferase may regulate persister cell formation in A. baumannii, while RecC seems to mediate epigenetic regulation of persister cell formation. However, Dam and RecC may act at different persister cell formation states.
Bacterial persister cells are not killed by high concentration of antibiotics, despite its antibiotic susceptibility. It has been known that they may cause antibiotic treatment failure and contribute to the evolution of antibiotic resistance. Although many mechanisms have been suggested and verified for persister cell formation, many remains to be uncovered. In this study, we report that DNA methyltransferase leads to an increase in persister cell formation, through transcriptional activation of several regulatory genes. Our results suggest that DNA methyltransferases could be target proteins to prevent formation of persister cells.
Perovskite Solar CellsIn article number 2301927, Bumjoon J. Kim, Jongmin Choi and co‐workers develop perovskite solar cells integrated with an ambipolar polymer, which offer several advantages, such ...as comprehensive defect passivation, bidirectional charge transport, and suppression of lithium‐ion migration. These attributes contribute to an enhancement in both performance and stability.
Antibiotic-resistant Gram-negative bacteria remain a globally leading cause of bacterial infection-associated mortality, and it is imperative to identify novel therapeutic strategies. Recently, the ...advantage of using antibacterials selective against Gram-negative bacteria has been demonstrated with polymyxins that specifically target the lipopolysaccharides of Gram-negative bacteria. However, the severe cytotoxicity of polymyxins limits their clinical use. Here, we demonstrate that polymyxin B nonapeptide (PMBN), a polymyxin B derivative without the terminal amino acyl residue, can significantly enhance the effectiveness of commonly used antibiotics against only Gram-negative bacteria and their persister cells. We show that although PMBN itself does not exhibit antibacterial activity or cytotoxicity well above the 100-fold minimum inhibitory concentration of polymyxin B, PMBN can increase the potency of co-treated antibiotics. We also demonstrate that using PMBN in combination with other antibiotics significantly reduces the frequency of resistant mutant formation. Together, this work provides evidence of the utilities of PMBN as a novel potentiator for antibiotics against Gram-negative bacteria and insights for the eradication of bacterial persister cells during antibiotic treatment.
The significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.
Abstract Relationships between the PmrAB two-component system and colistin resistance were investigated in Acinetobacter baumannii . The sequences of pmrA , pmrB and pmrC in 26 colistin-susceptible ...(ColS) and 7 colistin-resistant (ColR) A. baumannii isolates were determined. In addition, 30 ColR mutants (colistin minimum inhibitory concentration >64 mg/L) were selected in vitro from 10 ColS strains and the pmrA , pmrB and pmrC sequences of the in-vitro-selected ColR mutants were also determined. Expression of pmrA and pmrB was compared between the ColR mutants and their parent ColS strains using a quantitative real-time polymerase chain reaction method. Elevated expression of pmrA and pmrB genes was evident both in wild-type and in in-vitro-selected ColR strains. However, no amino acid differences in the pmrA , pmrB and pmrC genes were found between wild-type ColR and ColS isolates. Although six kinds of amino acid alterations in pmrB were identified in in-vitro-selected ColR mutants, no changes were found in some of the mutants. These findings indicate that increased expression of the PmrAB system is essential for colistin resistance in A. baumannii but that amino acid alterations might be only partially responsible for resistance.
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