TusDCB, a sulfur transferase complex involved in tRNA modification, contributes to UPEC pathogenicity.

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.

[Research on mechanisms of drug resistance and virulence expression in pathogenic Escherichia coli].

Urinary tract infections (UTIs) are one of the most common infections. Uropathogenic Escherichia coli (UPEC) is the most common causative organism. Once UPEC enters the urinary tract, it infects the bladder and then ascends the urinary tract to the kidneys, where it causes pyelonephritis, a more severe form of the disease. While various virulence factors, including adhesions and cytotoxic factors to bladder epithelial cells, have been identified and their functions have been analyzed, the question remains, "How can UPEC, which is harmless in the intestinal tract, be induced to become pathogenic in the urinary tract?" and "How does UPEC ascend the urinary tract and infect the kidneys?" On the other hand, UPEC invades host cells and forms biofilm-like microcolonies that are resistant to various antimicrobial agents. We are working to solve this problem by identifying the factors responsible for the virulence of UPEC and the establishment of infection of the kidney, as well as the factors involved in microcolony formation and elucidating their functions. Here I outline the virulence expression of UPEC from bladder to kidney infection and the mechanism of UTI refractoriness, focusing on our studies.

The PapB/FocB family protein TosR acts as a positive regulator of flagellar expression and is required for optimal virulence of uropathogenic Escherichia coli.

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.

Exploration of the protein-dependent mechanism of Lactobacillus crispatus GAI98322 to prevent recurrent cystitis.

OBJECTIVES: To elucidate the mechanism of Lactobacillus crispatus (L. crispatus) suppositories to prevent patients from recurrent cystitis (RC), independent from viable-Lactobacilli-bacteria- and acid-dependent ones such as hydrogen peroxide and lactate. METHODS: We used the GAI98322 strain of L. crispatus in all experiments and pH-matched. cell-free culture supernatant of L. crispatus (CFCS) was collected. The growth inhibitory activity and the biofilm formation inhibitory activity of the CFCS against uropathogenic Escherichia coli (UPEC), Extended Spectrum beta (ß) Lactamase producing (ESBL+) UPEC, and Pseudomonas aeruginosa (P. aeruginosa) was assessed by agar-disk diffusion tests and crystal violet assay. Also, CFCS was subjected to mass spectrometry to specify ingredients. RESULTS: The CFCS suppressed the proliferation of E. coli, ESBL + E. coli, and P. aeruginosa. Also, the CFCS at a concentration of 40% significantly impeded the biofilm formation of these three bacteria. The aggregation-promoting factor and Lysin was detected from CFCS. CONCLUSIONS: The cell-free supernatant from the GAI98322 strain of L. crispatus inhibits the growth/biofilm formation of broad pathogens by aggregation promoting factor and lysin, which may prevent hosts from RC regardless of the antimicrobial resistance of the pathogens and even under pH modulation.

Inactivation of ackA and pta Genes Reduces GlpT Expression and Susceptibility to Fosfomycin in Escherichia coli.

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 ß-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.

Enterococcal Linear Plasmids Adapt to Enterococcus faecium and Spread within Multidrug-Resistant Clades.

Antimicrobial resistance (AMR) of bacterial pathogens, including enterococci, is a global concern, and plasmids are crucial for spreading and maintaining AMR genes. Plasmids with linear topology were identified recently in clinical multidrug-resistant enterococci. The enterococcal linear-form plasmids, such as pELF1, confer resistance to clinically important antimicrobials, including vancomycin; however, little information exists about their epidemiological and physiological effects. In this study, we identified several lineages of enterococcal linear plasmids that are structurally conserved and occur globally. pELF1-like linear plasmids show plasticity in acquiring and maintaining AMR genes, often via transposition with the mobile genetic element IS1216E. This linear plasmid family has several characteristics enabling long-term persistence in the bacterial population, including high horizontal self-transmissibility, low-level transcription of plasmid-carried genes, and a moderate effect on the Enterococcus faecium genome alleviating fitness cost and promoting vertical inheritance. Combining all of these factors, the linear plasmid is an important factor in the spread and maintenance of AMR genes among enterococci.

Adsorption of extracellular proteases and pyocyanin produced by Pseudomonas aeruginosa using a macroporous magnesium oxide-templated carbon decreases cytotoxicity.

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.

The Two-Component System CpxRA Represses Salmonella Pathogenicity Island 2 by Directly Acting on the ssrAB Regulatory Operon.

The acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The ssrAB bicistronic operon, located in SPI-2, encodes the SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the ssrAB regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of ssrA. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes. IMPORTANCE SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the ssrAB regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.

A Macroporous Magnesium Oxide-Templated Carbon Adsorbs Shiga Toxins and Type III Secretory Proteins in Enterohemorrhagic Escherichia coli, Which Attenuates Virulence.

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 ß-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.

Roles of the Tol/Pal System in Bacterial Pathogenesis and Its Application to Antibacterial Therapy.

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.

Elucidation of host diversity of the VanD-carrying genomic islands in enterococci and anaerobes.

BACKGROUND: VanD is a rare type of vancomycin resistance worldwide. However, the host diversity of the vanD gene cluster and the structural similarity of their genomic islands are not well understood. METHODS: Three VanD-type Enterococcus faecium strains (AA620, AA622 and AA624) isolated from a Japanese patient who underwent vancomycin treatment in 2017 were analysed. This study utilized WGS analysis to characterize the three VanD-type E. faecium strains and describes the diversity of hosts possessing VanD-carrying genomic islands. RESULTS: The three isolates exhibited variable MICs of vancomycin. In the relatively vancomycin-resistant AA620, mutations were identified in vanSD and ddl. The strains AA622 and AA624 had intact ddl and harboured two vanD gene clusters. qRT-PCR results revealed the ddl mutation to be a factor affecting the high vancomycin resistance range of AA620. WGS data showed the 155 kb and 185 kb genomic islands harbouring the vanD gene cluster inserted in the coding region of the lysS gene, located in the chromosome in AA620 and AA622/624, respectively. Comparing the VanD-carrying genomic islands to available sequences of other enterococci and enteric anaerobes revealed how the genomic islands of these organisms isolated worldwide shared similar core genes and backbones. These anaerobes belonged to various genera within the order Eubacteriales. The phylogenetic cluster of the genomic island core genome alignment did not correlate with the host-species lineage, indicating horizontal gene transfer in the gut microbiota. CONCLUSIONS: By horizontal gene transfer, various bacteria forming the gut microbiota maintain VanD-carrying genomic islands.

Roles of OmpA in Type III Secretion System-Mediated Virulence of Enterohemorrhagic Escherichia coli.

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.

cAMP Receptor Protein Positively Regulates the Expression of Genes Involved in the Biosynthesis of Klebsiella oxytoca Tilivalline Cytotoxin.

Klebsiella oxytoca is a resident of the human gut. However, certain K. oxytoca toxigenic strains exist that secrete the nonribosomal peptide tilivalline (TV) cytotoxin. TV is a pyrrolobenzodiazepine that causes antibiotic-associated hemorrhagic colitis (AAHC). The biosynthesis of TV is driven by enzymes encoded by the aroX and NRPS operons. In this study, we determined the effect of environmental signals such as carbon sources, osmolarity, and divalent cations on the transcription of both TV biosynthetic operons. Gene expression was enhanced when bacteria were cultivated in tryptone lactose broth. Glucose, high osmolarity, and depletion of calcium and magnesium diminished gene expression, whereas glycerol increased transcription of both TV biosynthetic operons. The cAMP receptor protein (CRP) is a major transcriptional regulator in bacteria that plays a key role in metabolic regulation. To investigate the role of CRP on the cytotoxicity of K. oxytoca, we compared levels of expression of TV biosynthetic operons and synthesis of TV in wild-type strain MIT 09-7231 and a Δcrp isogenic mutant. In summary, we found that CRP directly activates the transcription of the aroX and NRPS operons and that the absence of CRP reduced cytotoxicity of K. oxytoca on HeLa cells, due to a significant reduction in TV production. This study highlights the importance of the CRP protein in the regulation of virulence genes in enteric bacteria and broadens our knowledge on the regulatory mechanisms of the TV cytotoxin.

Adsorption of Phenazines Produced by Pseudomonas aeruginosa Using AST-120 Decreases Pyocyanin-Associated Cytotoxicity.

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.

Roles of OmpX, an Outer Membrane Protein, on Virulence and Flagellar Expression in Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI). This bacterium adheres to and internalizes within urinary tract cells, where it aggregates and subsequently forms biofilm-like multicellular colonies that protect UPEC from antimicrobial agents and the host's immune system. Here, we show that OmpX, an outer membrane protein, plays a role in the pathogenesis of UPEC in renal cells. Deletion of ompX decreased bacterial internalization and aggregation within kidney epithelial cells and also impaired the colonization of mouse urinary tracts, but the ompX mutant still adhered to the epithelial cells at a level similar to that of the parent strain. FlhD, the master regulator of flagellum-related genes, had a low expression level in the ompX mutant compared to the parent strain, and the ompX mutant exhibited defective motility due to lower flagellar production than the parent strain. The fliC mutant, which lacks flagella, exhibited lower levels of bacterial internalization and aggregation than the parent strain. Additional deletion of ompX in the fliC mutant did not further decrease bacterial internalization. These combined results suggest that OmpX contributes to flagellar production in UPEC and then sustains UPEC virulence associated with bacterial internalization and aggregation within urinary tract cells and colonization in the urinary tract.

Isolation of Serratia fonticola Producing FONA, a Minor Extended-Spectrum ß-Lactamase (ESBL), from Imported Chicken Meat in Japan.

Five novel strains of Serratia fonticola that produce FONA, a minor extended-spectrum beta-lactamase (ESBL), were isolated during routine surveillance of ESBL-producing Enterobacteriaceae in imported chicken meat in Japan in 2017 and 2018. These strains exhibited a clear ESBL phenotype in susceptibility tests carried out in the presence of clavulanic acid; however, all strains tested negative in a multiplex polymerase chain reaction assay used to detect TEM, SHV, and CTX-M ß-lactamase genes. After identification of the bacterial species as S. fonticola, full length blaFONA genes were amplified and the DNA sequences were determined. The blaFONA genes from all 5 strains were different from those previously reported (blaFONA-1 to blaFONA-6); they clustered close to one another but were distinct from previously reported blaFONA genes in a phylogenic analysis based on amino acid sequences.

Roles of the Tol-Pal system in the Type III secretion system and flagella-mediated virulence in enterohemorrhagic Escherichia coli.

The Tol-Pal system is a protein complex that is highly conserved in many gram-negative bacteria. We show here that the Tol-Pal system is associated with the enteric pathogenesis of enterohemorrhagic E. coli (EHEC). Deletion of tolB, which is required for the Tol-Pal system decreased motility, secretion of the Type III secretion system proteins EspA/B, and the ability of bacteria to adhere to and to form attaching and effacing (A/E) lesions in host cells, but the expression level of LEE genes, including espA/B that encode Type III secretion system proteins were not affected. The Citrobacter rodentium, tolB mutant, that is traditionally used to estimate Type III secretion system associated virulence in mice did not cause lethality in mice while it induced anti-bacterial immunity. We also found that the pal mutant, which lacks activity of the Tol-Pal system, exhibited lower motility and EspA/B secretion than the wild-type parent. These combined results indicate that the Tol-Pal system contributes to the virulence of EHEC associated with the Type III secretion system and flagellar activity for infection at enteric sites. This finding provides evidence that the Tol-Pal system may be an effective target for the treatment of infectious diseases caused by pathogenic E. coli.

Progress Overview of Bacterial Two-Component Regulatory Systems as Potential Targets for Antimicrobial Chemotherapy.

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.

First Report of the Local Spread of Vancomycin-Resistant Enterococci Ascribed to the Interspecies Transmission of a vanA Gene Cluster-Carrying Linear Plasmid.

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.