Pseudomonas paralcaligenes sp. nov., isolated from a hospitalized patient.

A Gram-stain-negative, aerobic, rod-shaped, non-endospore-forming bacterium, designated as strain MRCP1333T, was isolated from a faecal sample from a hospital patient in Japan. MRCP1333T grew at temperatures of 15-40 °C (optimum 25-35 °C), with 1.0-3.0 % (w/v, 171-513 mM) NaCl [optimum 1-2 % (w/v), 171-342 mM], and at pH 6.0-9.5 (optimum pH 7.0-8.0). The results of phylogenetic analysis based on the sequences of the 16S rRNA gene and the 53 genes encoding the bacterial ribosome protein subunits indicated that MRCP1333T represented a member of the Pseudomonas aeruginosa group, most closely related to Pseudomonas alcaligenes. Whole-genome comparisons, using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that MRCP1333T represented a distinct species in the P. aeruginosa group. Phenotypic characterization tests demonstrated utilization by this strain of citrate, glycerol, and d-malic acid, the ability to reduce nitrite to nitrogen and the ability of this strain to grow in the presence of minocycline and tetrazolium blue, distinguishing this strain from P. alcaligenes and other closely related species of the P. aeruginosa group. The major fatty acids of MRCP1333T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 38.4 %), summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 21.1 %) and C16 : 0 (20.6 %). The DNA G+C content of MRCP1333T was 66.5 mol%. Genetic and phenotypic evidence indicated that MRCP1333T should be classified as representing a novel species, for which the name Pseudomonas paralcaligenes sp. nov. is proposed. The type strain is MRCP1333T (=LMG 32254T,=JCM 34250T).

A transferrable IncL/M plasmid harboring a gene encoding IMP-1 metallo-ß-lactamase in clinical isolates of Enterobacteriaceae.

BACKGROUND: The worldwide spread of carbapenemase-producing Enterobacteriaceae (CPE) has reduced the clinical utility of carbapenems. Plasmids often play an important role in the spread of genes encoding drug-resistance factors, especially in the horizontal transfer of these genes among species of Enterobacteriaceae. This study describes a patient infected with three species of CPE carrying an identical transferrable IncL/M plasmid. METHODS: Clinical isolates of CPE were collected at St. Luke's International Hospital, Tokyo, Japan, from 2015 to 2019. Three species of CPE isolates, Enterobacter cloacae, Klebsiella aerogenes and Serratia marcescens, were isolated from a patient who developed severe gallstone pancreatitis associated with bloodstream infection, with all three isolates producing IMP-1 metallo-ß-lactamase. The complete sequences of the plasmids of the three isolates were determined by both MiSeq and MinION. The medical chart of this patient was retrospectively reviewed conducted to obtain relevant clinical information. RESULTS: The three CPE species carried an IncL/M plasmid, pSL264, which was 81,133 bp in size and harbored blaIMP-1. The genetic environment surrounding blaIMP-1 consisted of int1-blaIMP-1-aac(6')-IIc-qacL-qacEdelta1-sul1-istB-IS21. Conjugation experiments showed that S. marcescens could transmit the plasmid to E. cloacae and K. aerogenes. In contrast, pSL264 could not transfer from E. cloacae or K. aerogenes to S. marcescens. CONCLUSION: The IncL/M plasmid pSL264 harboring blaIMP-1 was able to transfer among different species of Enterobacteriaceae in a patient receiving long-term antimicrobial treatment. The worldwide emergence and spread of IncL/M plasmids harboring carbapenemase-encoding genes among species of Enterobacteriaceae is becoming a serious public health hazard.

Emergence of Carbapenem-Resistant Providencia rettgeri and Providencia stuartii Producing IMP-Type Metallo-ß-Lactamase in Japan.

Four Providencia rettgeri isolates and one Providencia stuartii isolate were obtained from urine samples of five patients in 2018 in Japan. All of the isolates were resistant to imipenem and meropenem, and three were highly resistant to both carbapenems, with MICs of 512 µg/ml. The three highly carbapenem-resistant isolates harbored blaIMP-70, encoding a variant of IMP-1 metallo-ß-lactamase with two amino acid substitutions (Val67Phe and Phe87Val), and the other two harbored blaIMP-1 and blaIMP-11, respectively. Whole-genome sequencing revealed that an isolate harbored two copies of blaIMP-1 on the chromosome and that the other four harbored a copy of blaIMP-11 or blaIMP-70 in a plasmid. Expression of blaIMP-70 conferred carbapenem resistance in Escherichia coli Recombinant IMP-70 and an IMP-1 variant with Val67Phe but without Phe87Val had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that an amino acid substitution of Val67Phe affects increased activities against meropenem in IMP-70. These results suggest that Providencia spp. become more highly resistant to carbapenems by acquisition of two copies of blaIMP-1 or by mutation of blaIMP genes with amino acid substitutions, such as blaIMP-70.

Pseudomonas yangonensis sp. nov., isolated from wound samples of patients in a hospital in Myanmar.

Strains of a Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, designated MY50T, MY63 and MY101, were isolated from wound samples of three hospitalized patients in Yangon, Myanmar. Strains MY50T, MY63 and MY101 grew at temperatures of 4-44 °C, in media containing 1.0-7.0 % (w/v) NaCl and at pH 6.0-9.5. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences showed that these strains belonged to the genus Pseudomonas and were part of the Pseudomonas oleovorans group and located close to Pseudomonas guguanensis and Pseudomonas mendocina. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization analyses, confirmed that strains MY50T, MY63 and MY101 were the same strain and they were a distinct species in the P. oleovorans group. Results of phenotypic characterization tests demonstrated that utilization of p-hydroxy-phenylacetic acid, glycerol, l-pyroglutamic acid and quinic acid could distinguish these strains from other species of the P. oleovorans group. These genetic and phenotypic characteristics suggest that they should be classified as representing a novel species, under the proposed name Pseudomonas yangonensis sp. nov. The type strain is MY50T (=LMG 31602T,=JCM 33396T), with a DNA G+C content of 62.82 mol%.

Emergence of carbapenem-resistant and colistin-susceptible Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 in Japan.

BACKGROUND: The spread of Enterobacteriaceae producing both carbapenemases and Mcr, encoded by plasmid-mediated colistin resistance genes, has become a serious public health problem worldwide. This study describes three clinical isolates of Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 that were resistant to carbapenem but susceptible to colistin. METHODS: Thirty-two clinical isolates of E. cloacae complex non-susceptible to carbapenems were obtained from patients at 14 hospitals in Japan. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods and E-tests. Their entire genomes were sequenced by MiSeq and MinION methods. Multilocus sequence types were determined and a phylogenetic tree constructed by single nucleotide polymorphism (SNP) alignment of whole genome sequencing data. RESULTS: All 32 isolates showed MICs of ≥2 µg/ml for imipenem and/or meropenem. Whole-genome analysis revealed that all these isolates harbored blaIMP-1, with three also harboring mcr-9. These three isolates showed low MICs of 0.125 µg/ml for colistin. In two of these isolates, blaIMP-1 and mcr-9 were present on two separate plasmids, of sizes 62 kb and 280/290 kb, respectively. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr-9. In the third isolate, however, both blaIMP-1 and mcr-9 were present on the chromosome. CONCLUSION: The mcr-9 is silently distributed among carbapenem-resistant E. cloacae complex isolates, of which are emerging in hospitals in Japan. To our knowledge, this is the first report of isolates of E. cloacae complex harboring both blaIMP-1 and mcr-9 in Japan.

Molecular Characterization of Multidrug-Resistant Pseudomonas aeruginosa Isolates in Hospitals in Myanmar.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR P. aeruginosa strains isolated from hospitals in Myanmar. Forty-five MDR P. aeruginosa isolates obtained from different patients in seven hospitals in Myanmar were screened using the broth microdilution method. The whole genomes of the MDR isolates were sequenced using a MiSeq platform (Illumina). Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced, and drug resistance genes were identified. Of the 45 isolates, 38 harbored genes encoding carbapenemases, including DIM-1, IMP-1, NDM-1, VIM-2, and VIM-5, and 9 isolates had genes encoding 16S rRNA methylases, including RmtB, RmtD3, RmtE, and RmtF2. Most MDR P. aeruginosa strains isolated in Myanmar belonged to sequence type 1047 (ST1047). This is the first molecular epidemiological analysis of MDR P. aeruginosa clinical isolates in Myanmar. These findings strongly suggest that P. aeruginosa ST1047 strains harboring carbapenemases, including DIM-, IMP-, NDM-, and VIM-type metallo-ß-lactamases, have been spreading throughout medical settings in Myanmar.

Emergence of Carbapenem-Resistant Pseudomonas asiatica Producing NDM-1 and VIM-2 Metallo-ß-Lactamases in Myanmar.

Pseudomonas asiatica is a recently proposed species of the genus Pseudomonas This study describes eight isolates of carbapenem-resistant P. asiatica harboring blaNDM-1 and blaVIM-2, genes encoding metallo-ß-lactamase (MBL). These isolates were obtained from urine samples of patients hospitalized in Myanmar. These isolates were resistant to carbapenems but susceptible to colistin. All eight isolates were positive for a carbapenemase inactivation method, CIMTrisII, and seven were positive on an immunochromatographic assay for NDM-type MBL. One isolate was highly resistant to aminoglycosides. Whole-genome sequencing showed that seven isolates harbored blaNDM-1 and one harbored blaVIM-2, with these genes located on the chromosome. One isolate harbored blaNDM-1 and rmtC, a gene encoding 16S rRNA methylase. Five types of genomic environments surrounding blaNDM-1 and blaVIM-2 were detected in these eight isolates, with four isolates having the same type. These data indicate that P. asiatica isolates harboring genes encoding carbapenemases, including blaNDM-1 and blaVIM-2, are spreading in medical settings in Myanmar.

Rifaximin prevents ethanol-induced liver injury in obese KK-Ay mice through modulation of small intestinal microbiota signature.

Exacerbation of alcoholic hepatitis (AH) with comorbid metabolic syndrome is an emerging clinical problem, where microbiota plays a profound role in the pathogenesis. Here, we investigated the effect of rifaximin (RFX) on liver injury following chronic-binge ethanol (EtOH) administration in KK-Ay mice, a rodent model of metabolic syndrome. Female, 8-wk-old KK-Ay mice were fed Lieber-DeCarli diet (5% EtOH) for 10 days, following a single EtOH gavage (4 g/kg body wt). Some mice were given RFX (0.1 g/L, in liquid diet) orally. Small intestinal contents were collected from mice without binge. Intestinal microbiota was quantified using aerobic and anaerobic culturing techniques and further analyzed by 16S rRNA sequencing in detail. EtOH feeding/binge caused hepatic steatosis, oxidative stress, and induction of inflammatory cytokines in KK-Ay mice, which were markedly prevented by RFX treatment. Hepatic mRNA levels for cluster of differentiation 14, Toll-like receptor (TLR) 4, TLR2, and NADPH oxidase 2 were increased following EtOH feeding/binge, and administration of RFX completely suppressed their increase. The net amount of small intestinal bacteria was increased over threefold after chronic EtOH feeding as expected; however, RFX did not prevent this net increase. Intriguingly, the profile of small intestinal microbiota was dramatically changed following EtOH feeding in the order level, where the Erysipelotrichales predominated in the relative abundance. In sharp contrast, RFX drastically blunted the EtOH-induced increases in the Erysipelotrichales almost completely, with increased proportion of the Bacteroidales. In conclusion, RFX prevents AH through modulation of small intestinal microbiota/innate immune responses in obese KK-Ay mice.NEW & NOTEWORTHY Here we demonstrated that rifaximin (RFX) prevents chronic-binge ethanol (EtOH)-induced steatohepatitis in KK-Ay mice. Chronic EtOH feeding caused small intestinal bacterial overgrowth, with drastic alteration in the microbiota profile predominating the order Erysipelotrichales. RFX minimized this EtOH induction in Erysipelotrichales with substitutive increases in Bacteroidales. RFX also prevented EtOH-induced increases in portal lipopolysaccharide, and hepatic cluster of differentiation 14, toll-like receptor (TLR) 2, and TLR4 mRNA levels, suggesting the potential involvement of microbiota-related innate immune responses.

Pseudomonas juntendi sp. nov., isolated from patients in Japan and Myanmar.

A Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, designated as strain BML3T, was isolated from a sputum sample of a hospital patient in Japan. Strain BML3T grew at temperatures from 4 to 40 °C, in 1.0-7.0 % (w/v) NaCl and at pH 6.0-9.0. Results of phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA gene and rpoB, rpoD and gyrB, showed that strain BML3T was part of the Pseudomonas putida group and located close to Pseudomonas asiatica, Pseudomonas monteiliiand P. putida . Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization, confirmed strain BML3T to be a distinct species among the P. putida group. Phenotypic characterization tests demonstrated that the utilization of phenylmercuric acetate could distinguish this strain from other closed species of the P. putida group. Based on genetic and phenotypic evidence, strain BML3T should be classified as a novel species, for which the name Pseudomonas juntendi sp. nov. is proposed. The type strain is BML3T (=DSM 109244T,=JCM 33395T), with a DNA G+C content of 62.66 mol %.

Pseudomonas asiatica sp. nov., isolated from hospitalized patients in Japan and Myanmar.

A novel Gram-negative, aerobic, rod-shaped, non-spore-forming bacterial strain, RYU5T, was isolated from a stool sample of an inpatient at a hospital in Okinawa, Japan. The optimal growth temperature of RYU5T was 30 °C. Phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA, rpoB, rpoD and gyrB genes, showed that RYU5T was a member of the Pseudomonas putida group and was located close to Pseudomonas monteilii and P. putida. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization, confirmed that strain RYU5T should be classified as a novel species of Pseudomonas. Phenotypic characterization tests showed that utilization of d-mannose, d-serine, l-arabinose and d-fructose could distinguish this strain from other related species of the genus Pseudomonas. Based on genetic and phenotypic evidence, strain RYU5T should be classified as a novel species, for which the name Pseudomonas asiatica sp. nov. is proposed. The type strain is RYU5T (=DSM 107182T, =JCM 32716T), with a DNA G+C content of 62.25 mol%.

Assessment of a newly developed immunochromatographic assay for NDM-type metallo-ß-lactamase producing Gram-negative pathogens in Myanmar.

BACKGROUND: To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-ß-lactamases (NDMs) was developed. METHODS: The immunochromatographic assay for New Delhi metallo-ß-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing. RESULTS: Of the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, - 4, - 5 and - 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs. CONCLUSIONS: The immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.

Development of a New Application for Comprehensive Viability Analysis Based on Microbiome Analysis by Next-Generation Sequencing: Insights into Staphylococcal Carriage in Human Nasal Cavities.

The nasal carriage rate of Staphylococcus aureus in human is 25 to 30%, and S. aureus sporadically causes severe infections. However, the mechanisms underlying staphylococcal carriage remain largely unknown. In the present study, we constructed an rpoB-based microbiome method for staphylococcal species discrimination. Based on a microbiome scheme targeting viable cell DNA using propidium monoazide (PMA) dye (PMA microbiome method), we also developed a new method to allow the comprehensive viability analysis of any bacterial taxon. To clarify the ecological distribution of staphylococci in the nasal microbiota, we applied these methods in 46 nasal specimens from healthy adults. PMA microbiome results showed that Staphylococcaceae and Corynebacteriaceae were the most predominant viable taxa (average relative abundance: 0.435262 and 0.375195, respectively), and Staphylococcus epidermidis exhibited the highest viability in the nasal microbiota. Staphylococcus aureus detection rates from nasal specimens by rpoB-based conventional and PMA microbiome methods were 84.8% (39 of 46) and 69.5% (32 of 46), respectively, which substantially exceeded the values obtained by a culture method using identical specimens (36.9%). Our results suggest that Staphylococcaceae species, especially S. epidermidis, adapted most successfully to human nasal cavity. High detection of S. aureus DNA by microbiome methods suggests that almost all healthy adults are consistently exposed to S. aureus in everyday life. Furthermore, the large difference in S. aureus detection rates between culture and microbiome methods suggests that S. aureus cells frequently exist in a viable but nonculturable state in nasal cavities. Our method and findings will contribute to a better understanding of the mechanisms underlying carriage of indigenous bacteria.IMPORTANCE Metagenomic analyses, such as 16S rRNA microbiome methods, have provided new insights in various research fields. However, conventional 16S rRNA microbiome methods do not permit taxonomic analysis of only the viable bacteria in a sample and have poor resolving power below the genus level. Our new schemes allowed for viable cell-specific analysis and species discrimination, and nasal microbiome data using these methods provided some interesting findings regarding staphylococcal nasal carriage. According to our comprehensive viability analysis, the high viability of Staphylococcus species, especially Staphylococcus epidermidis, in human nasal carriage suggests that this taxon has adapted most successfully to human nasal tissue. Also, a higher detection rate of S. aureus DNA by microbiome methods (84.8%) than by a culture method (36.9%) suggests that almost all healthy adults are consistently exposed to Staphylococcus aureus in the medium and long term. Our findings will contribute to a better understanding of the mechanisms underlying the carriage of indigenous bacteria.

Human Host Defense Cathelicidin Peptide LL-37 Enhances the Lipopolysaccharide Uptake by Liver Sinusoidal Endothelial Cells without Cell Activation.

The liver is a major organ that removes waste substances from the blood, and liver sinusoidal endothelial cells (LSECs) are professional scavenger cells, which incorporate and degrade various endogenous and exogenous molecules including pathogenic factor LPS. Mammalian cells express a number of peptide antibiotics that function as effectors in the innate host defense systems. LL-37, a human cathelicidin antimicrobial peptide, has a potent LPS-neutralizing activity and exhibits protective actions on various infection models. However, the effect of LL-37 on the LPS clearance has not been clarified. In this study, to further understand the host-protective mechanism of LL-37, we evaluated the effect of LL-37 on the LPS clearance in vitro. LL-37 enhanced the LPS uptake by human LSECs. Of interest, LL-37 was similarly incorporated into LSECs both in the presence and the absence of LPS, and the incorporated LPS and LL-37 were colocalized in LSECs. Importantly, the uptake of LPS and LL-37 was inhibited by endocytosis inhibitors, heparan sulfate proteoglycan analogs, and glycosaminoglycan lyase treatment of the cells. Moreover, the uptake of LL-37-LPS did not activate TLR4 signaling in both MyD88-dependent and -independent pathways. In addition, the incorporated LL-37-LPS was likely transported to the lysosomes in LSECs. Together these observations suggest that LL-37 enhances the LPS uptake by LSECs via endocytosis through the complex formation with LPS and the interaction with cell-surface heparan sulfate proteoglycans, thereby facilitating the intracellular incorporation and degradation of LPS without cell activation. In this article, we propose a novel function of LL-37 in enhancing LPS clearance.

Spleen-derived lipocalin-2 in the portal vein regulates Kupffer cells activation and attenuates the development of liver fibrosis in mice.

The liver has an immune tolerance against gut-derived products from the portal vein (PV). A disruption of the gut-liver axis leads to liver injury and fibrosis. The spleen is connected to the PV and regulates immune functions. However, possible splenic effects on liver fibrosis development are unclear. Lipocalin-2 (Lcn2) is an antimicrobial protein that regulates macrophage activation. To clarify the role of the spleen in liver fibrosis development, we induced liver fibrosis in mice after splenectomy, and investigated liver fibrosis development. Liver fibrosis resulted in significantly increased splenic Lcn2 levels, but all other measured cytokine levels were unchanged. Splenectomized mice showed enhanced liver fibrosis and inflammation accompanied by significantly decreased Lcn2 levels in PV. Lipopolysaccharide-stimulated primary Kupffer cells, resident liver macrophages, which were treated with recombinant Lcn2 (rLcn2) produced less tumor necrosis factor-α and Ccl2 and the activation of hepatic stellate cells, the effector cells for collagen production in the liver, was suppressed by co-culture with rLcn2-treated Kupffer cells. In addition, the involvement of gut-derived products in splenectomized mice was evaluated by gut sterilization. Interestingly, gut sterilization blocked the effect of splenectomy on liver fibrosis development. In conclusion, spleen deficiency accelerated liver fibrosis development and decreased PV Lcn2 levels. The mechanism of splenic protection against liver fibrosis development may involve the splenic Lcn2, triggered by gut-derived products that enter the liver through the PV, regulates Kupffer cells activated by the gut-liver axis. Thus, the splenic Lcn2 may have an important role in regulating the immune tolerance of the liver in liver fibrosis development.

A Modified Carbapenem Inactivation Method, CIMTris, for Carbapenemase Production in Acinetobacter and Pseudomonas Species.

The carbapenem inactivation method (CIM) and modified CIM (mCIM) are simple and economical phenotypic screening methods for detecting carbapenemase production in Gram-negative bacteria. Although the mCIM has been recommended by the Clinical and Laboratory Standards Institute, both the CIM and mCIM have limitations. This study describes another modified CIM, called CIMTris, in which carbapenemase was extracted from bacteria with 0.5 M Tris-HCl (pH 7.6) buffer. The ability of the CIMTris to detect carbapenemase production was examined in Acinetobacter and Pseudomonas species. The CIMTris had an overall sensitivity of 97.6% and an overall specificity of 92.6%, whereas the mCIM had a sensitivity of 45.1% and a specificity of 100% for the isolates tested. These findings indicate that the CIMTris is useful for detecting carbapenemase production in Acinetobacter and Pseudomonas species.

Antimicrobial cathelicidin peptide LL-37 inhibits the pyroptosis of macrophages and improves the survival of polybacterial septic mice.

LL-37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL-37 can modulate various inflammatory reactions. We previously revealed that LL-37 suppresses the LPS/ATP-induced pyroptosis of macrophages in vitro by both neutralizing the action of LPS and inhibiting the response of P2X7 (a nucleotide receptor) to ATP. Thus, in this study, we further evaluated the effect of LL-37 on pyroptosis in vivo using a cecal ligation and puncture (CLP) sepsis model. As a result, the intravenous administration of LL-37 improved the survival of the CLP septic mice. Interestingly, LL-37 inhibited the CLP-induced caspase-1 activation and pyroptosis of peritoneal macrophages. Moreover, LL-37 modulated the levels of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in both peritoneal fluids and sera, and suppressed the activation of peritoneal macrophages (as evidenced by the increase in the intracellular levels of IL-1ß, IL-6 and TNF-α). Finally, LL-37 reduced the bacterial burdens in both peritoneal fluids and blood samples. Together, these observations suggest that LL-37 improves the survival of CLP septic mice by possibly suppressing the pyroptosis of macrophages, and inflammatory cytokine production by activated macrophages and bacterial growth. Thus, the present findings imply that LL-37 can be a promising candidate for sepsis because of its many functions, such as the inhibition of pyroptosis, modulation of inflammatory cytokine production and antimicrobial activity.

"Slow VISA," a novel phenotype of vancomycin resistance, found in vitro in heterogeneous vancomycin-intermediate Staphylococcus aureus strain Mu3.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1×10(-6)). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated "slow VISA" (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase ß-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.

Emergence of carbapenem-resistant Pseudomonas alcaligenes and Pseudomonas paralcaligenes clinical isolates with plasmids harbouring bla IMP-1 in Japan.

Introduction. The emergence of carbapenem-resistant Pseudomonas species producing metallo-ß-lactamase (MBL) has become a serious medical problem worldwide. IMP-type MBL was firstly detected in 1991 in Japan. Since then, it has become one of the most prevalent types of MBLs.Hypothesis/Gap statement. Avirulent species of Pseudomonas, such as Pseudomonas alcaligenes, function as reservoirs of drug resistance-associated genes encoding carbapenemases in clinical settings.Methodology. Active surveillance for carbapenem-resistant Gram-negative pathogens was conducted in 2019 at a hospital in Tokyo, Japan. Of the 543 samples screened for carbapenem-resistant isolates, 2 were species of Pseudomonas. One was from a stool sample from a medical staff member, and the other was from a stool sample from a hospitalized patient.Results. Whole-genome sequencing showed that the former isolate was a strain of P. alcaligenes, and the latter was a strain of Pseudomonas paralcaligenes, a species close to P. alcaligenes. Both isolates were resistant to all carbapenems and harboured bla IMP-1 genes encoding IMP-1 MBL, which conferred resistance to carbapenems. The bla IMP-1 genes of P. alcaligenes and P. paralcaligenes were located on the plasmids, pMRCP2, 323125 bp in size, and pMRCP1333, 16592 bp in size, respectively. The sequence of 82 % of pMRCP2 was 92 % similar to the sequence of a plasmid of P. aeruginosa PA83, whereas the sequence of 79 % of pMRCP1333 was >95 % similar to the sequence of a plasmid of Achromobacter xylosoxidans FDAARGOS 162. The genomic environments surrounding the bla IMP-1 of pMRCP2 and pMRCP1333 differed completely from each other.Conclusions. These results indicate that the two isolates acquired bla IMP-1 from different sources and that P. alcaligenes and P. paralcaligenes function as vectors and reservoirs of carbapenem-resistant genes in hospitals.

Human anti-microbial cathelicidin peptide LL-37 suppresses the LPS-induced apoptosis of endothelial cells.

Sepsis is a systemic disease resulting from harmful host response to bacterial infections. During the exacerbation of severe sepsis or septic shock, apoptosis of endothelial cells is induced in susceptible organs such as the lung and liver and triggers microcirculatory disorder and organ dysfunction. LPS, an outer membrane component of Gram-negative bacteria, is one of the major virulence factors for the pathogenesis. We previously reported that LL-37, a human anti-microbial cathelicidin peptide, potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. However, the effect of LL-37 on the LPS-induced endothelial cell apoptosis remains to be clarified. In this study, to further elucidate the action of LL-37 on severe sepsis/endotoxin shock, we investigated the effects of LL-37 on the LPS-induced endothelial cell apoptosis in vitro and in vivo using lung-derived normal human microvascular blood vessel endothelial cells (HMVEC-LBls) and D-galactosamine hydrochloride (D-GalN)-sensitized murine endotoxin shock model. LL-37 suppressed the LPS-induced apoptosis of HMVEC-LBls. In addition, LL-37 inhibited the binding of LPS possibly to the LPS receptors (CD14 and toll-like receptor 4) expressed on the cells. Thus, LL-37 can suppress the LPS-induced apoptosis of HMVEC-LBls via the inhibition of LPS binding to the cells. Furthermore, LL-37 drastically suppressed the apoptosis of hepatic endothelial cells as well as hepatocytes in the liver of murine endotoxin shock model. Together, these observations suggest that LL-37 could suppress the LPS-induced apoptosis of endothelial cells, thereby attenuating lethal sepsis/endotoxin shock.