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.

First Isolation of Vancomycin-Resistant Enterococcus faecium Carrying Plasmid-Borne vanD1.

Vancomycin-resistant Enterococcus faecium carrying the vanD1 gene on plasmid pEF-D was isolated from a fecal sample of a hospitalized patient in Japan. The strain JH5687 showed moderate resistance to vancomycin (MIC, 16 µg/mL) but remained susceptible to teicoplanin (MIC, 1 µg/mL). The backbone gene organization of pEF-D was highly homologous to that of conjugative plasmid pMG1 or pHTß. The calculated conjugation frequency of JH5687 was 10-4 to 10-5 per donor cell.

Effect of Sub-MICs of Macrolides on the Sensitivity of Pseudomonas aeruginosa to Nitrosative Stress: Effectiveness against P. aeruginosa with and without Multidrug Resistance.

Sub-MICs of the 14-membered macrolides erythromycin (EM) and clarithromycin (CAM) decreased the growth of Pseudomonas aeruginosa PAO1 and increased its sensitivity to endogenous and exogenous nitrosative stress. However, a 16-membered macrolide, josamycin (JM), was not or less effective. In 9 of 13 non-multidrug-resistant P. aeruginosa (non-MDRP) and 9 of 27 MDRP ST235 strains, the sub-MIC of EM induced significant reductions in bacterial numbers following treatment with a nitric oxide donor.

Isolation of thymidine-dependent and extended-spectrum-ß-lactamase-producing Escherichia coli small-colony variant from urine of a septuagenarian female patient with recurrent cystitis: A case report with genetic investigation.

Thymidine-dependent small-colony variant (TD-SCV) of Escherichia coli was isolated from urine of a septuagenarian female patient on hemodialysis suffering from recurrent cystitis. The patient had been treated with frequent administrations of trimethoprim sulfamethoxazole (SXT), every time her cystitis symptoms developed. In the TD-SCV isolate, the deletion was detected in the thyA gene associated with thymidylate synthase. Interestingly, the isolate was found to produce extended-spectrum ß-lactamase (ESBL), and the experiment on conjugational transfer of the resistance trait was successful. By means of genetic analysis, the isolate was found to carry blaCTX-M-1 group. To the best of our knowledge, this is the first report of urinary tract infection caused by the transmissible ESBL-producing TD-SCV of E. coli. MICs of the TD-SCV were obtained only on the Mueller Hinton agar media supplemented with appropriate concentrations of thymidine, which might lead to the difficulty for proper chemotherapy in daily medicine. Furthermore, transmission of the ESBL gene via plasmid should be of concern.

Dissemination and genetic analysis of the stealthy vanB gene clusters of Enterococcus faecium clinical isolates in Japan.

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 vanSB 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.

First case report of bacteremia caused by Dysgonomonas mossii.

We here report a case of Dysgonomonas mossii bacteremia with cholangitis. An 85-year-old male patient with recurrent hepatitis B surface antigen-negative/anti-hepatitis C virus-negative hepatocellular carcinoma came to our hospital in poor physical condition. Two sets of blood cultures revealed a positive result for D. mossii. As matrix-assisted laser desorption/ionization time-of-flight mass spectrometry failed to identify D. mossii, analysis of 16S rRNA gene sequencing was performed; however, this gene is not specific enough to distinguish between D. mossii and D. oryzarvi. Finally, D. mossii infection was confirmed by gyrB and recA sequencing. To our knowledge, this is the first report of D. mossii causing human infection, which was identified in culture and confirmed using a combination of 16S rRNA, gyrB, and recA sequencing.

Identification of a second two-component signal transduction system that controls fosfomycin tolerance and glycerol-3-phosphate uptake.

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagic Escherichia coli (EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression of torR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression of glpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

Elevated Expression of GlpT and UhpT via FNR Activation Contributes to Increased Fosfomycin Susceptibility in Escherichia coli under Anaerobic Conditions.

Because a shortage of new antimicrobial agents is a critical issue at present, and with the spread of multidrug-resistant (MDR) pathogens, the use of fosfomycin to treat infections is being revisited as a "last-resort option." This drug offers a particular benefit in that it is more effective against bacteria growing under oxygen-limited conditions, unlike other commonly used antimicrobials, such as fluoroquinolones and aminoglycosides. In this study, we showed that Escherichia coli strains, including enterohemorrhagic E. coli (EHEC), were more susceptible to fosfomycin when grown anaerobically than when grown aerobically, and we investigated how the activity of this drug was enhanced during anaerobic growth of E. coli. Our quantitative PCR analysis and a transport assay showed that E. coli cells grown under anaerobic conditions had higher levels of expression of glpT and uhpT, encoding proteins that transport fosfomycin into cells with their native substrates, i.e., glycerol-3-phosphate and glucose-6-phosphate, and led to increased intracellular accumulation of the drug. Elevation of expression of these genes during anaerobic growth requires FNR, a global transcriptional regulator that is activated under anaerobic conditions. Purified FNR bound to DNA fragments from regions upstream of glpT and uhpT, suggesting that it is an activator of expression of glpT and uhpT during anaerobic growth. We concluded that the increased antibacterial activity of fosfomycin toward E. coli under anaerobic conditions can be attributed to elevated expression of GlpT and UhpT following activation of FNR, leading to increased uptake of the drug.

Role of the CpxAR two-component signal transduction system in control of fosfomycin resistance and carbon substrate uptake.

Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.

Nosocomial infection caused by vancomycin-susceptible multidrug-resistant Enterococcus faecalis over a long period in a university hospital in Japan.

Compared with other developed countries, vancomycin-resistant enterococci (VRE) are not widespread in clinical environments in Japan. There have been no VRE outbreaks and only a few VRE strains have sporadically been isolated in our university hospital in Gunma, Japan. To examine the drug susceptibility of Enterococcus faecalis and nosocomial infection caused by non-VRE strains, a retrospective surveillance was conducted in our university hospital. Molecular epidemiological analyses were performed on 1711 E. faecalis clinical isolates collected in our hospital over a 6-year period [1998-2003]. Of these isolates, 1241 (72.5%) were antibiotic resistant and 881 (51.5%) were resistant to two or more drugs. The incidence of multidrug resistant E. faecalis (MDR-Ef) isolates in the intensive care unit increased after enlargement and restructuring of the hospital. The major group of MDR-Ef strains consisted of 209 isolates (12.2%) resistant to the five drug combination tetracycline/erythromycin/kanamycin/streptomycin/gentamicin. Pulsed-field gel electrophoresis analysis of the major MDR-Ef isolates showed that nosocomial infections have been caused by MDR-Ef over a long period (more than 3 years). Multilocus sequence typing showed that these strains were mainly grouped into ST16 (CC58) or ST64 (CC8). Mating experiments suggested that the drug resistances were encoded on two conjugative transposons (integrative conjugative elements), one encoded tetracycline-resistance and the other erythromycin/kanamycin/streptomycin/gentamicin-resistance. To our knowledge, this is the first report of nosocomial infection caused by vancomycin-susceptible MDR-Ef strains over a long period in Japan.

Identification of novel genes responsible for overexpression of ampC in Pseudomonas aeruginosa PAO1.

The development of resistance to antipseudomonal penicillins and cephalosporins mediated by the chromosomal ampC gene in Pseudomonas aeruginosa is of clinical importance. We isolated piperacillin-resistant mutants derived from P. aeruginosa PAO1 and analyzed two mutants that had an insertion in mpl and nuoN. One mutant, YT1677, was resistant to piperacillin and ceftazidime and had an insertion in mpl, which encodes UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase. The other mutant, YT7988, showed increased MICs of piperacillin, ceftazidime, cefepime, and cefoperazone, and the insertion was mapped to nuoN, which encodes NADH dehydrogenase I chain N. Complementation experiments demonstrated that these mutations resulted in higher levels of resistance to ß-lactams. The expression of genes reported to be involved in ß-lactam resistance was examined by real-time PCR in YT1677 and YT7988 mutants. Overexpression was observed for only ampC, and other genes were expressed normally. Deletion of the ampR gene in YT1677 and YT7988 resulted in decreased expression of ampC, indicating that the mutations in YT1677 and YT7988 affected the expression of ampC through the function of AmpR.

Molecular epidemiological analyses of Clostridioides difficile isolates in a university hospital in Japan.

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.

Identification of VanN-type vancomycin resistance in an Enterococcus faecium isolate from chicken meat in Japan.

Five VanN-type vancomycin-resistant Enterococcus faecium strains were isolated from a sample of domestic chicken meat in Japan. All isolates showed low-level resistance to vancomycin (MIC, 12 mg/liter) and had the same pulsed-field gel electrophoresis profile. The vancomycin resistance was encoded on a large plasmid (160 kbp) and was expressed constitutively. The VanN-type resistance operon was identical to the first resistance operon to be reported, with the exception of a 1-bp deletion in vanT(N) and a 1-bp substitution in vanS(N).

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.

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.

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.

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.

Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate.

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.

Fluoroquinolone enhances the mutation frequency for meropenem-selected carbapenem resistance in Pseudomonas aeruginosa, but use of the high-potency drug doripenem inhibits mutant formation.

The mutation frequency for carbapenem resistance in Pseudomonas aeruginosa strains that were selected with carbapenems was enhanced in the presence of subinhibitory concentrations of fluoroquinolones. The mutants showed either a loss of OprD activity or increased mexAB-oprM expression. The highest mutant isolation frequency was obtained by selection with meropenem, while doripenem inhibited mutant growth.

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii.

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N-acetyl-L-Met, N-acetyl-L-Gln and N-acetyl-L-Leu, but had a lower hydrolytic activity towards N-acetyl-L-Phe. The kinetic parameters K(m) and k(cat) were determined to be 24.6 mm and 370 s(-1), respectively, for N-acetyl-l-Met at 90 degrees C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn(2+), Mn(2+) and Ni(2+)), and Mn(2+) addition caused an alteration in substrate specificity. Site-directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole-3-acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal-binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.