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ABSTRACT We present the identification and characterization of the complete genome of metronidazole (MTZ)-resistant Prevotella bivia strain TOH-2715 [minimum inhibitory concentration (MIC): 8 mg/L], isolated from the urine of an elderly Japanese woman, as well as details of its mobile genetic elements (MGEs) containing antimicrobial resistance (AMR) genes and its relationship with other bacterial species determined using whole-genome sequencing (WGS) data. TOH-2715 possessed two chromosomes with putative MGEs containing AMR genes. Two AMR-related MGE regions were present in chromosome 2. MGE-region 1 (7,821 bp) included Tn6456, where nimK was located, and MGE-region 2 (58.8 Kbp) included the integrative and conjugative element (ICE), where tet(Q) and ermF were located. The genetic structure of the ICE of TOH-2715 was similar to that of CTnDOT-family transposons, where ermF and tet(Q) are located. A search of public databases revealed that nimK was present in Prevotella spp., including P. bivia, and was partially composed of a Tn6456-like element lacking the efflux transporter gene qacE and the Crp/Fnr family transcriptional regulator gene in some cases. Core ICE gene analysis showed that ICEs similar to that of TOH-2715 were present in Prevotella spp. and Bacteroides spp., suggesting horizontal gene transfer among anaerobes. This is the report of WGS analysis of an MTZ-resistant clinical strain of P. bivia (TOH-2715) with Tn6456 encoding nimK. Other submitted genomes have described the presence of nimK, but none of them have described MTZ resistance. Additionally, we described putative MGE regions containing the AMR gene within the genus Prevotella and among anaerobes, raising concerns about the future spread of nimK among anaerobes.IMPORTANCEMetronidazole (MTZ) is an important antimicrobial agent in anaerobic infections and is widely used in clinical settings. The rate of MTZ resistance in anaerobic bacteria has been increasing in recent years, and the nim gene (nitro-imidazole reductase) is one of the resistance mechanisms. Prevotella bivia is found in humans in the urinary tract and vagina and is known to cause infections in some cases. One of the nim genes, nimK, has recently been discovered in this species of bacteria, but there are no reports of antimicrobial resistance (AMR)-related regions in its whole genome level. In this study, we analyzed the AMR region of nimK-positive P. bivia derived from clinical specimens based on comparisons with other anaerobic genomes. P. bivia was found to be engaged in horizontal gene transfer with other anaerobic bacteria, and the future spread of the nimK gene is a concern.

Abstract tRNA modifications play a crucial role in ensuring accurate codon recognition and optimizing translation levels. While the significance of these modifications in eukaryotic cells for maintaining cellular homeostasis and physiological functions is well-established, their physiological roles in bacterial cells, particularly in pathogenesis, remain relatively unexplored. The TusDCB protein complex, conserved in γ-proteobacteria like Escherichia coli, is involved in sulfur modification of specific tRNAs. This study focused on the role of TusDCB in the virulence of uropathogenic E. coli (UPEC), a bacterium causing urinary tract infections. The findings indicate that TusDCB is essential for optimal production of UPEC's virulence factors, including type 1 fimbriae and flagellum, impacting the bacterium's ability to aggregate in bladder epithelial cells. Deletion of tusDCB resulted in decreased virulence against urinary tract infection mice. Moreover, mutant TusDCB lacking sulfur transfer activity and tusE- and mnmA mutants revealed the indispensability of TusDCB's sulfur transfer activity for UPEC pathogenicity. The study extends its relevance to highly pathogenic, multidrug-resistant strains, where tusDCB deletion reduced virulence-associated bacterial aggregation. These insights not only deepen our understanding of the interplay between tRNA sulfur modification and bacterial pathogenesis but also highlight TusDCB as a potential therapeutic target against UPEC strains resistant to conventional antimicrobial agents.

To better understand the molecular genetics of the Shiga toxin type 2 subunit A gene (stx2A gene), we collected many subtypes of stx2A genes and performed detailed molecular evolutionary analyses of the gene. To achieve the aim of the study, we used several bioinformatics technologies, including time-scaled phylogenetic analyses, phylogenetic distance analyses, phylodynamics analyses, selective pressure analyses, and conformational epitope analyses. A time-scaled phylogeny showed that the common ancestor of the stx2A gene dated back to around 18,600 years ago. After that, the gene diverged into two major lineages (Lineage 1 and 2). Lineage 1 comprised the stx2a–2d subtypes, while Lineage 2 comprised the stx2e, 2g, 2h, and 2o subtypes. The evolutionary rates of the genes were relatively fast. Phylogenetic distances showed that the Lineage 2 strains had a wider genetic divergence than Lineage 1. Phylodynamics also indicated that the population size of the stx2A gene increased after the 1930s and spread globally. Moreover, negative selection sites were identified in the Stx2A proteins, and these sites were diffusely distributed throughout the protein. Two negative selection sites were located adjacent to an active site of the common Stx2A protein. Many conformational epitopes were also estimated in these proteins, while no conformational epitope was found adjacent to the active site. The results suggest that the stx2A gene has uniquely evolved and diverged over an extremely long time, resulting in many subtypes. The dominance of the strains belonging to Lineage 1 suggests that differences in virulence may be involved in the prosperity of the offspring. Furthermore, some subtypes of Stx2A proteins may be able to induce effective neutralizing antibodies against the proteins in humans.

Background: We performed molecular epidemiological analyses of Clostridioides difficile isolates in a university hospital in Japan to reveal the risk of C. difficile infection. Methods: Cultured isolates from 919 stool samples from 869 patients obtained from July 2015 to August 2016 were subjected to toxin gene detection, ribotyping, multilocus sequence typing, antimicrobial susceptibility testing, and quantitative real-time polymerase chain reaction testing for C. difficile toxin gene expression. Results: Of the 919 stool samples from 869 patients, C. difficile was isolated from 153 samples (16.6%), of which 49 (32%) and 104 (68%) were from patients with and without C. difficile infection, respectively. Analyses showed genetic diversity, with ST8 and ST17 strains of healthcare-associated infections, some of which caused C. difficile infections. There was no significant difference in the transcription levels of C. difficile toxin genes between isolates from patients with and without C. difficile infection. Conclusions: Major Japanese clonal strains, ST8 and ST17, have been in the hospital environment for a long time and cause healthcare-associated C. difficile infections. The C. difficile toxin genes were transcribed in the isolates from both patients with and without C. difficile infection but were no significant relationship with the development of C. difficile infection.

Uropathogenic Escherichia coli (UPEC) is a major causative agent of urinary tract infections. The bacteria internalize into the uroepithelial cells, where aggregate and form microcolonies. UPEC fimbriae and flagella are important for the formation of microcolonies in uroepithelial cells. PapB/FocB family proteins are small DNA-binding transcriptional regulators consisting of approximately 100 amino acids that have been reported to regulate the expression of various fimbriae, including P, F1C, and type 1 fimbriae, and adhesins. In this study, we show that TosR, a member of the PapB/FocB family is the activator of flagellar expression. The tosR mutant had similar expression levels of type 1, P and F1C fimbriae as the parent strain, but flagellar production was markedly lower than in the parent strain. Flagellin is a major component of flagella. The gene encoding flagellin, fliC, is transcriptionally activated by the sigma factor FliA. The fliA expression is induced by the flagellar master regulator FlhDC. The flhD and flhC genes form an operon. The promoter activity of fliC, fliA and flhD in the tosR mutant was significantly lower than in the parent strain. The purified recombinant TosR does not bind to fliC and fliA but to the upstream region of the flhD gene. TosR is known to bind to an AT-rich DNA sequence consisting of 29 nucleotides. The characteristic AT-rich sequence exists 550–578 bases upstream of the flhD gene. The DNA fragment lacking this sequence did not bind TosR. Furthermore, loss of the tosR gene reduced motility and the aggregation ability of UPEC in urothelial cells. These results indicate that TosR is a transcriptional activator that increases expression of the flhDC operon genes, contributing to flagellar expression and optimal virulence.

ABSTRACT Fosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused by Escherichia coli. In recent years, quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found that ackA and pta contribute to fosfomycin activity against E. coli. ackA and pta mutant E. coli had reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition, ackA and pta mutants had decreased expression of glpT that encodes one of the fosfomycin transporters. Expression of glpT is enhanced by a nucleoid-associated protein, Fis. We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta defective strains to be due to a decrease in Fis levels in these mutants. Furthermore, ackA and pta are conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli, and deletion of ackA and pta from these strains resulted in decreased susceptibility to fosfomycin. These results suggest that ackA and pta in E. coli contribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin. IMPORTANCE The spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of the ackA and pta genes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.

A total of seventy VanA-type vancomycin-resistant enterococci (VRE) isolates obtained in Taiwan in the early 2000s were retrospectively characterized. Forty isolates were obtained from human patients and thirty from livestock. Of these VRE isolates, twenty-three (57.5%) of the human VRE and thirty (100%) of the livestock VRE were Enterococcus faecalis, and the remaining seventeen (42.5%) of the human VRE were E. faecium. Of the 53 E. faecalis isolates, twenty-two (96%) of the human VRE and thirty (100%) of the livestock VRE exhibited a high level of resistance to vancomycin and sensitivity to teicoplanin. They also had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. The vancomycin resistance of all of the 22 human isolates, and 20 of the 30 livestock isolates, transferred to E. faecalis FA2-2 at a frequency of 10−5 to 10−3 per donor cell in broth. Each of the transconjugants responded to E. faecalis pheromone (i.e., E. faecalis FA2-2 culture filtrate), indicating that the conjugative plasmids were pheromone-responsive plasmids. Three of the conjugative plasmids originated from human isolates, and five plasmids from livestock isolates were corresponded and classified as type A plasmid. Two plasmids originated from human isolates and six plasmids from livestock isolates were corresponded and classified as type B plasmid. E. faecalis FA2-2 containing either the type A or type B plasmid responded to the synthetic pheromone cAD1. The type A and type B plasmids transferred between E. faecalis FA2-2 and JH2SS at a frequency of about 10−2 per donor cell and conferred vancomycin, bacitracin, and erythromycin resistances. The complete DNA sequence of the representative type A plasmid pTW9 (85,068 bp) showed that the plasmid carried a Tn1546-like element encoding vanA-type resistance, erythromycin resistance (ermB), and bacitracin resistance (bcrABDR). The plasmid contained the regulatory region found in the pheromone-responsive plasmid and encoded the genes traA, traD and iad1, which are the key negative regulatory elements, and traE1, a key positive regulator of plasmid pAD1, indicating that plasmid pTW9 was pAD1-type pheromone-responsive plasmid. PFGE analysis of SmaI-digested chromosomal DNAs showed that several E. faecalis strains harboring an identical type A pheromone-responsive plasmid were indistinguishable, and that these were identified both in human and livestock isolates, indicating the transmissions of the VRE strains between livestock and humans. These data showed that the multiple-drug-resistant pheromone-responsive conjugative plasmids have been widely spread in both human and livestock VRE, and there was high potential for transfers of VRE from food animals to humans in Taiwan in the early 2000s.

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common foodborne pathogens. However, no drug that prevents the severe complications caused by this bacterium has been approved yet. This study showed that a macroporous magnesium oxide (MgO)-templated carbon material (MgOC150) adsorbs Shiga toxins, and Type III secretory EspA/EspB proteins responsible for EHEC pathogenesis, and decreases the extracellular levels of these proteins. On the other hand, this material did not affect the growth of EHEC. Citrobacter rodentium traditionally used to estimate Type III secretion system-associated virulence in mice is highly virulent. The survival period of infected mice was prolonged when MgOC150 was administered. This adsorbent disturbed neither mammalian cells nor normal intestinal bacteria, such as Enterococcus hirae, Lactobacillus acidophilus, and Lactobacillus casei. In contrast, MgOC150 adsorbed antimicrobial agents, including β-lactams, quinolones, tetracyclines, and trimethoprim/sulfamethoxazole. However, fosfomycin and amikacin were not adsorbed. Thus, MgOC150 can be used with fosfomycin and amikacin to treat infections. MgOC150 is used for industrial purposes, such as an electrode catalyst, a bioelectrode, and enzyme immobilization. The study proposed another potential application of MgOC150, assisting anti-EHEC chemotherapy.

Pseudomonas aeruginosa is one of the most common pathogens isolated in clinical settings and produces a wide range of extracellular molecules that contributes to the virulence. Chemotherapy options to prevent and treat P. aeruginosa infections are limited because this pathogen is highly and innately resistant to some classes of conventional drugs. Alternative methods to conquer P. aeruginosa, including multidrug resistant strains, are being investigated. This study showed that a macroporous magnesium oxide (MgO)-templated carbon material (MgOC150) attenuates the toxicity of this bacterium in human epithelial cells. A proteomic analysis revealed that MgOC150 adsorbs some extracellular proteases, including elastase (LasB) and alkaline protease (AprA), required for the virulence of P. aeruginosa, which decreases the accumulation of these molecules. MgOC150 also adsorbed pyocyanin, which is another molecule involved in its pathogenesis, but is a nonprotein small-sized molecule. These results suggest a potency of MgOC150 that suppresses the virulence of P. aeruginosa. MgOC150 has been used for industrial purposes, as an electrode catalyst and a bioelectrode and for enzyme immobilization. Thus, MgOC150 could be beneficial for developing novel anti-Pseudomonas therapy.

Despite the increasing evidence of the clinical impact of Pseudomonas-derived cephalosporinase (PDC) sequence polymorphisms, the molecular evolution of its encoding gene, blaPDC, remains elusive. To elucidate this, we performed a comprehensive evolutionary analysis of blaPDC. A Bayesian Markov Chain Monte Carlo phylogenetic tree revealed that a common ancestor of blaPDC diverged approximately 4660 years ago, leading to the formation of eight clonal variants (clusters A–H). The phylogenetic distances within clusters A to G were short, whereas those within cluster H were relatively long. Two positive selection sites and many negative selection sites were estimated. Two PDC active sites overlapped with negative selection sites. In docking simulation models based on samples selected from clusters A and H, piperacillin was bound to the serine and the threonine residues of the PDC active sites, with the same binding mode for both models. These results suggest that, in P. aeruginosa, blaPDC is highly conserved, and PDC exhibits similar antibiotic resistance functionality regardless of its genotype.

DNA gyrase plays important roles in genome replication in various bacteria, including Pseudomonasaeruginosa. The gyrA gene encodes the gyrase subunit A protein (GyrA). Mutations in GyrA are associated with resistance to quinolone-based antibiotics. We performed a detailed molecular evolutionary analyses of the gyrA gene and associated resistance to the quinolone drug, ciprofloxacin, using bioinformatics techniques. We produced an evolutionary phylogenetic tree using the Bayesian Markov Chain Monte Carlo (MCMC) method. This tree indicated that a common ancestor of the gene was present over 760 years ago, and the offspring formed multiple clusters. Quinolone drug-resistance-associated amino-acid substitutions in GyrA, including T83I and D87N, emerged after the drug was used clinically. These substitutions appeared to be positive selection sites. The molecular affinity between ciprofloxacin and the GyrA protein containing T83I and/or D87N decreased significantly compared to that between the drug and GyrA protein, with no substitutions. The rate of evolution of the gene before quinolone drugs were first used in the clinic, in 1962, was significantly lower than that after the drug was used. These results suggest that the gyrA gene evolved to permit the bacterium to overcome quinolone treatment.

ABSTRACT Vancomycin-resistant enterococci pose a threat in the clinical setting and have been linked to hospital outbreaks worldwide. In 2017, a local spread of VanA-type vancomycin-resistant enterococci (VRE) occurred in Japan, and 25 enterococcal isolates, including 14 Enterococcus faecium, 8 E. raffinosus, and 3 E. casseliflavus isolates, were identified from four inpatients. Molecular analysis of the multispecies of VanA-type VRE revealed the involvement of both the dissemination of clonally related VRE strains between patients and the horizontal transfer of plasmids harboring the vanA gene cluster between Enterococcus spp. Pulsed-field gel electrophoresis showed that the plasmid DNAs without S1 nuclease treatment were able to migrate into the gel, suggesting that the topology of the plasmid was linear. Whole-genome sequencing revealed that this plasmid, designated pELF2, was 108,102 bp long and encoded multiple antimicrobial resistance genes, including ermA and ant(9). The amino acid sequences of putative replication- and transfer-related genes were highly conserved between pELF2 and pELF1, the latter of which was the first identified enterococcal conjugative linear plasmid. On comparing the genomic structure, pELF2 showed the presence of a backbone similar to that of pELF1, especially with respect to the nucleotide sequences of both terminal ends, indicating a hybrid-type linear plasmid, possessing two different terminal structures. pELF2 possessed a broad host range and high conjugation frequencies for enterococci. The easy transfer of pELF2 to different Enterococcus spp. in vitro might explain this local spread of multiple species, highlighting the clinical threat from the spread of antimicrobial resistance by an enterococcal linear plasmid. IMPORTANCE Increasing multidrug resistance, including vancomycin resistance, in enterococci is a major concern in clinical settings. Horizontal gene transfer, such as via plasmids, has been shown to play a crucial role in the acquisition of vancomycin resistance. Among vancomycin resistance types, the VanA type is one of the most prevalent, and outbreaks caused by VanA-type vancomycin-resistant enterococci (VRE) have occurred worldwide. Here, we describe an enterococcal linear plasmid responsible for multispecies local spread of VanA-type VRE. Such a study is important because although hospital outbreaks caused by mixed enterococcal species have been reported, this particular spread indicates plasmid transfer across species. This is a crucial finding because the high risk for such a spread of antimicrobial resistance calls for regular monitoring and surveillance.

AST-120 (Kremezin) is used to treat progressive chronic kidney disease (CKD) by adsorbing uremic toxin precursors produced by gut microbiota, such as indole and phenols. In this study, we propose that AST-120 reduces indole level, consequently suppresses indole effects on induction of drug tolerance and virulence in Escherichia coli including enterohaemorrhagic strains. In experiments, AST-120 adsorbed both indole and tryptophan, a precursor of indole production, and led to decreased expression of acrD and mdtEF which encode drug efflux pumps, and elevated glpT, which encodes a transporter for fosfomycin uptake and increases susceptibility to aztreonam, rhodamine 6G, and fosfomycin. AST-120 also decreased the production of EspB, which contributes to pathogenicity of enterohaemorrhagic E. coli (EHEC). Aztreonam, ciprofloxacin, minocycline, trimethoprim, and sulfamethoxazole were also adsorbed by AST-120. However, fosfomycin, in addition to rifampicin, colistin and amikacin were not adsorbed, thus AST-120 can be used together with these drugs for therapy to treat infections. These results suggest another benefit of AST-120, i.e., that it assists antibacterial chemotherapy.

The Tol/Pal system (also written as “The Tol-Pal system”) is a set of protein complexes produced by most Gram-negative bacteria. It comprises the inner membrane-associated and the outer membrane-anchored subunits composed of the TolA, TolQ, and TolR proteins and the TolB and Pal proteins, respectively. Although the Tol/Pal system was first defined as bacterial proteins involved in colicin uptake of Escherichia coli, its global roles have been characterized in several studies as mentioned in this article. Pathogenesis of many Gram-negative pathogens is sustained by the Tol/Pal system. It is also essential for cell growth and fitness in some pathogens. Therefore, the Tol/Pal system is proposed as a potential target for antimicrobial chemotherapy. Although the tol/pal mutants are low in virulence, they still have the ability to stimulate the immune system. The Pal protein is highly immunogenic and induces both adaptive and innate immune responses. Therefore, the tol/pal mutant strains and Pal proteins also have potential vaccine properties. For these reasons, the Tol/Pal system represents a promising research target in the development of antibacterial therapeutic strategies for refractory infections caused by multi-drug-resistant (MDR), Gram-negative pathogens. In this paper, we summarize studies on the Tol/Pal system associated with bacterial pathogenesis and vaccine development.

Abstract Background VanB-type vancomycin (VAN) resistance gene clusters confer VAN resistances on Enterococcus spp. over a wide range of MIC levels (MIC = 4–1000 mg/L). However, the epidemiology and the molecular characteristics of the VAN susceptible VanB-type Enterococcus still remain unclear. Results We characterized 19 isolates of VanB-type Enterococcus faecium that might colonize in the gut and were not phenotypically resistant to VAN (MIC = 3 mg/L). They were obtained from two hospitals in Japan between 2009 and 2010. These isolates had the identical vanB gene cluster and showed same multilocus sequence typing (MLST) (ST78) and the highly related profiles in pulsed-field gel electrophoresis (PFGE). The vanB gene cluster was located on a plasmid, and was transferable to E. faecium and E. faecalis. Notably, from these VanB-type VREs, VAN resistant (MIC≥16 mg/L) mutants could appear at a frequency of 10− 6–10− 7/parent cell in vitro. Most of these revertants acquired mutations in the vanS B gene, while the remainder of the revertants might have other mutations outside of the vanB gene cluster. All of the revertants we tested showed increases in the VAN-dependent expression of the vanB gene cluster, suggesting that the mutations affected the transcriptional activity and increased the VAN resistance. Targeted mutagenesis revealed that three unique nucleotide substitutions in the vanB gene cluster of these strains attenuated VAN resistance. Conclusions In summary, this study indicated that stealthy VanB-type E. faecium strains that have the potential ability to become resistance to VAN could exist in clinical settings.

Outer membrane proteins are commonly produced by gram-negative bacteria, and they have diverse functions. A subgroup of proteins, which includes OmpA, OmpW and OmpX, is often involved in bacterial pathogenesis. Here we show that OmpA, rather than OmpW or OmpX, contributes to the virulence of enterohemorrhagic Escherichia coli (EHEC) through its type III secretion system (T3SS). Deletion of ompA decreased secretion of the T3SS proteins EspA and EspB; however, the expression level of the LEE genes that encode a set of T3SS proteins did not decrease. The ompA mutant had less abilities to form A/E lesions in host epithelial cells and lyse human red blood cells than the parent strain. Moreover, the virulence of an ompA mutant of Citrobacter rodentium (traditionally used to estimate T3SS-associated virulence in mice) was attenuated. Mice infected with the ompA mutant survived longer than those infected with the parent strain. Furthermore, mice infected with ompA developed symptoms of diarrhea more slowly than mice infected with the parent strain. Altogether, these results suggest that OmpA sustains the activity of the T3SS and is required for optimal virulence in EHEC. This work expands the roles of outer membrane proteins in bacterial pathogenesis.

Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5′ end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.

Urinary tracts infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a common infectious disease. With the shortage of new antimicrobial agents, the increase in UPEC resistance to commonly used drugs, such as fluoroquinolones and β-lactams including carbapenems is a critical issue. UPEC invades urinary tract cells, where it aggregates, and subsequently, forms biofilm-like multicellular colonies termed intracellular bacterial communities (IBCs). This process allows the bacteria to establish infections and so may be a good potential target for new drugs to treat infections. Here, we show that deletion of the tolB gene, encoding a protein of the Tol-Pal system that was originally characterized as a protein complex for colicin uptake and maintenance of the outer membrane, decreases the level of bacterial internalization into and aggregation within cultured bladder epithelial cells and also inhibits the colonization of mice urinary tracts. The tolB mutant also exhibited defective motility because of impaired flagellum syntheses. The fliC and motA mutants, which are non-motile strains, also exhibited lower levels of bacterial internalization and aggregation than their wild-type parent. Additional deletion of tolB in the fliC mutant did not further decrease these, suggesting that the attenuated virulence of the tolB mutant is a result of defective motility. The tolA, tolQ, tolR, and pal mutants that lack other members of the Tol-Pal system also exhibited lower levels of motility and aggregation within bladder epithelial cells compared to their wild-type parent. These combined results suggest another role of the Tol-Pal system, i.e., that it is responsible for optimal internalization, aggregation followed by IBC formation within urinary tract cells, and bacterial motility.

AST-120 (Kremezin) is used to treat progressive chronic kidney disease by adsorbing uremic toxin precursors produced by the gut microbiota, such as indole and phenols. Previously, we found that AST-120 decreased drug tolerance and virulence in Escherichia coli by adsorbing indole. Here, we show that AST-120 adsorbs phenazine compounds, such as pyocyanin, produced by Pseudomonas aeruginosa including multidrug-resistant P. aeruginosa strains, and suppresses pyocyanin-associated toxicity in A-549 (alveolar adenocarcinoma) and Caco-2 (colon adenocarcinoma) cells. Addition of fosfomycin, colistin and amikacin, which are often used to treat P. aeruginosa, inhibited the bacterial growth, regardless of the presence or absence of AST-120. These results suggest a further benefit of AST-120 that supports anti-Pseudomonas chemotherapy in addition to that of E. coli and propose a novel method to treat P. aeruginosa infection.

Bacteria adapt to changes in their environment using a mechanism known as the two-component regulatory system (TCS) (also called “two-component signal transduction system” or “two-component system”). It comprises a pair of at least two proteins, namely the sensor kinase and the response regulator. The former senses external stimuli while the latter alters the expression profile of bacterial genes for survival and adaptation. Although the first TCS was discovered and characterized in a non-pathogenic laboratory strain of Escherichia coli, it has been recognized that all bacteria, including pathogens, use this mechanism. Some TCSs are essential for cell growth and fitness, while others are associated with the induction of virulence and drug resistance/tolerance. Therefore, the TCS is proposed as a potential target for antimicrobial chemotherapy. This concept is based on the inhibition of bacterial growth with the substances acting like conventional antibiotics in some cases. Alternatively, TCS targeting may reduce the burden of bacterial virulence and drug resistance/tolerance, without causing cell death. Therefore, this approach may aid in the development of antimicrobial therapeutic strategies for refractory infections caused by multi-drug resistant (MDR) pathogens. Herein, we review the progress of TCS inhibitors based on natural and synthetic compounds.

Fosfomycin is resurfacing as a “last resort drug” to treat infections caused by multidrug resistant pathogens. This drug has a remarkable benefit in that its activity increases under oxygen-limited conditions unlike other commonly used antimicrobials such as β-lactams, fluoroquinolones and aminoglycosides. Especially, utility of fosfomycin has being evaluated with particular interest to treat chronic biofilm infections caused by Pseudomonas aeruginosa because it often encounters anaerobic situations. Here, we showed that P. aeruginosa PAO1, commonly used in many laboratories, becomes more susceptible to fosfomycin when grown anaerobically, and studied on how fosfomycin increases its activity under anaerobic conditions. Results of transport assay and gene expression study indicated that PAO1 cells grown anaerobically exhibit a higher expression of glpT encoding a glycerol-3-phosphate transporter which is responsible for fosfomycin uptake, then lead to increased intracellular accumulation of the drug. Elevated expression of glpT in anaerobic cultures depended on ANR, a transcriptional regulator that is activated under anaerobic conditions. Purified ANR protein bound to the DNA fragment from glpT region upstream, suggesting it is an activator of glpT gene expression. We found that increased susceptibility to fosfomycin was also observed in a clinical isolate which has a promoted biofilm phenotype and its glpT and anr genes are highly conserved with those of PAO1. We conclude that increased antibacterial activity of fosfomycin to P. aeruginosa under anaerobic conditions is attributed to elevated expression of GlpT following activation of ANR, then leads to increased uptake of the drug.

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU, which encodes UTP-glucose- 1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis. Spontaneous mutants that developed resistance to the antimicrobial effects of BacL1 and BacA were revealed to carry a truncation deletion of the C-terminal amino acid (aa) region 288 to 298 of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis. This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL1. Interestingly, these mutants had reduced susceptibility to beta-lactams besides Bac41, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalis. [ABSTRACT FROM AUTHOR]