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.

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.

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.

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.

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.

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.

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.

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.

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.

Oxygen Limitation Enhances the Antimicrobial Activity of Fosfomycin in Pseudomonas aeruginosa Following Overexpression of glpT Which Encodes Glycerol-3-Phosphate/Fosfomycin Symporter.

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 ß-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.

Membrane-active antimicrobial poly(amino-modified alkyl) ß-cyclodextrins synthesized via click reactions.

The emergence of drug-resistant bacteria has led to the high demand for new antibiotics. In this report, we investigated membrane-active antimicrobial ß-cyclodextrins. These contain seven amino-modified alkyl groups on a molecule, which act as functional moieties to permeabilize bacterial cell membranes. The polyfunctionalization of cyclodextrins was achieved through a click reaction assisted by microwave irradiation. A survey using derivatives with systematically varied functionalities clarified the unique correlation of the antimicrobial activity of these compounds with their molecular structure and hydrophobicity/hydrophilicity balances. The optimum hydrophobicity for the compounds being membrane-active was specific to bacterial strains and animal cells; this led to specific compounds having selective toxicity against bacteria including multidrug-resistant pathogens. The results demonstrate that cyclodextrin is a versatile molecular scaffold for rationally designed structures and can be used for the development of new antibiotics.

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.

Genetic shifts in methicillin-resistant Staphylococcus aureus epidemic clones and toxin gene profiles in Japan: comparative analysis among pre-epidemic, epidemic and post-epidemic phases.

PURPOSE: The decline in methicillin-resistant Staphylococcus aureus (MRSA) isolation rates has become a general observation worldwide, including Japan. We hypothesized that some genetic shift in MRSA might cause this phenomenon, and therefore we investigated the genetic profiles among MRSA clinical isolates obtained from three different epidemic phases in Japan. METHODOLOGY: A total of 353 MRSA isolates were selected from 202 medical facilities in 1990 (pre-epidemic phase), 2004 (epidemic phase) and 2016 (post-epidemic phase). Molecular typing was performed by PCR detection of 22 genes using the polymerase chain reaction (PCR)-based ORF typing (POT) system, including an additional eight genes including small genomic islets and seven toxin genes. RESULTS: Isolates with a POT1 of score 93, identified as presumed clonal complex (pCC)5-staphylococcal cassette chromosome mec (SCCmec) type II including ST5-SCCmec type II New York/Japan clone, represented the major epidemic MRSA lineage in 1990 and 2004. In 2016, however, a marked decrease in isolates with a POT1 score of 93, along with changes in the epidemiology of toxin genes carried, was noted, where the carriers of tst genes including the tst-sec combination were markedly reduced, and those possessing the seb gene alone were markedly increased. Rather, isolates with a POT1 score of 106, including pCC1 or pCC8 among the isolates with SCCmec type IV, which often links to community-associated MRSA, were predominant. Interestingly, the pCC1 and pCC8 lineages were related to sea and tst-sec carriage, respectively. CONCLUSIONS: Over time, a transition in MRSA genetic profiles from a POT1 score of 93 in 1990 and 2004 to 106 in 2014 was found in Japan.

New colony multiplex PCR assays for the detection and discrimination of vancomycin-resistant enterococcal species.

New colony multiplex PCR assays for detection of seven types of vancomycin-resistance determinants and eight types of Enterococcus species were developed. For 135 enterococcal isolates examined in this study, these assays showed high sensitivity and specificity, and could provide the rapid and accurate detection of vancomycin-resistant determinants and Enterococcus spp.

Cooperative Actions of CRP-cAMP and FNR Increase the Fosfomycin Susceptibility of Enterohaemorrhagic Escherichia coli (EHEC) by Elevating the Expression of glpT and uhpT under Anaerobic Conditions.

Bacterial infections to anaerobic site are often hard to be treated because the activity of most of antimicrobials decreases under anaerobic conditions. However, fosfomycin rather provides a greater activity under anaerobic conditions than aerobic conditions. Previously, we found that expression of glpT and uhpT, fosfomycin symporters in enterohaemorrhagic Escherichia coli (EHEC) was upregulated by FNR, a global regulator during the anaerobiosis of the bacterium, which led to increased uptake and susceptibility to this drug. In this study, we showed that expression of glpT and uhpT is induced by CRP-cAMP, the regulator complex under both aerobic and anaerobic conditions. The activity of CRP-cAMP in EHEC was elevated under anaerobic conditions because levels of both CRP and cAMP were higher in the cells when grown anaerobically than those when grown aerobically. Results of expression study using mutants indicated that CRP-cAMP is indispensable for expression of glpT but not uhpT-whereas that of uhpT requires UhpA that is the response regulator composing of two-component system with the sensor kinase, UhpB. The CRP-cAMP protein bound to a region that overlaps RNA polymerase binding site for glpT and region upstream of UhpA binding site for uhpT. FNR bound to a region further upstream of CRP-cAMP binding site on region upstream of the glpT gene. These combined results suggested that increased antibacterial activity of fosfomycin to EHEC under anaerobic conditions is due to activation of FNR and increment of CRP-cAMP activity. Then, FNR enhances the expression of glpT activated by CRP-cAMP while CRP-cAMP and FNR cooperatively aids the action of UhpA to express uhpT to maximum level.

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.

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.