PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Role of Lipoteichoic Acid from the Genus Apilactobacillus in Inducing a Strong IgA Response.

Lactic acid bacterium-containing fermentates provide beneficial health effects by regulating the immune response. A naturally fermented vegetable beverage, a traditional Japanese food, reportedly provides health benefits; however, the beneficial function of its bacteria has not been clarified. Apilactobacillus kosoi is the predominant lactic acid bacterium in the beverage. Using murine Peyer's patch cells, we compared the immunoglobulin A (IgA)-inducing activity of A. kosoi 10HT to those of 29 other species of lactic acid bacteria and found that species belonging to the genus Apilactobacillus (A. kosoi 10HT, A. apinorum JCM30765T, and A. kunkeei JCM16173T) possessed significantly higher activity than the others. Thereafter, lipoteichoic acids (LTAs), important immunostimulatory molecules of Gram-positive bacteria, were purified from the three Apilactobacillus species, and their IgA-inducing activity was compared to those of LTAs from Lactiplantibacillus plantarum JCM1149T and a probiotic strain, Lacticaseibacillus rhamnosus GG. The results revealed that LTAs from Apilactobacillus species had significantly higher activity than others. We also compared the LTA structure of A. kosoi 10HT with that of L. plantarum JCM1149T and L. rhamnosus GG. Although d-alanine or both d-alanine and carbohydrate residues were substituents of free hydroxyl groups in the polyglycerol phosphate structure in LTAs from strains JCM1149T and GG, d-alanine residues were not found in LTA from strain 10HT by 1H nuclear magnetic resonance (NMR) analysis. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis of the glycolipid structure of LTA revealed that LTA from strain 10HT contained dihexosyl glycerol, whereas trihexosyl glycerol was detected in LTAs from other strains. These structural differences may be related to differences in IgA-inducing activity. IMPORTANCE The components of lactic acid bacteria that exert immunostimulatory effects are of increasing interest for therapeutic and prophylactic options, such as alternatives to antibiotics, cognitive enhancements, and vaccine adjuvants. LTAs act as immunostimulatory molecules in the host innate immune system by interacting with pattern recognition receptors. However, as LTA structures differ among species, detailed knowledge of the structure-function relationship for immunostimulatory effects is required. Comparisons of the IgA-inducing activity of LTAs have demonstrated that LTAs from the genus Apilactobacillus possess distinctive activities to stimulate mucosal immunity. The first analysis of the LTA structure from the genus Apilactobacillus suggests that it differs from structures of LTAs of related species of lactic acid bacteria. This knowledge is expected to aid in the development of functional foods containing lactic acid bacteria and pharmaceutical applications of immunostimulatory molecules from lactic acid bacteria.

Multiclonal Expansion and High Prevalence of ß-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones.

ß-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were ß-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

Contribution of Novel Amino Acid Alterations in PmrA or PmrB to Colistin Resistance in mcr-Negative Escherichia coli Clinical Isolates, Including Major Multidrug-Resistant Lineages O25b:H4-ST131-H30Rx and Non-x.

Colistin is a last-line drug for multidrug-resistant Gram-negative bacteria. We previously reported four plasmid-mediated colistin resistance (mcr) gene-negative colistin-resistant Escherichia coli clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-H30Rx (isolates SRE34 and SRE44; MIC for colistin = 16 mg/liter), non-x (SME296; MIC = 8 mg/liter), and O18-ST416 (SME222; MIC = 4 mg/liter). In this study, we investigated the colistin resistance mechanism and identified novel amino acid substitutions or deletions in the PmrAB two-component system that activates eptA (encoding a phosphoethanolamine transferase) and arnT (encoding an undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase) in all colistin-resistant isolates. SRE34 possessed deletion Δ27-45 (LISVFWLWHESTEQIQLFE) in PmrB, SRE44 possessed substitution L105P in PmrA, and both SME222 and SME296 included substitution G206D in PmrB. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that lipid A is modified with phosphoethanolamine in all four isolates. Deletion of pmrAB decreased colistin MICs to 0.5 mg/liter and lowered eptA and arnT expression. Chromosomal replacement of mutated pmrA or pmrB in colistin-susceptible O25b:H4-ST131 strain SME98 (colistin MIC = 0.5 mg/liter) increased the colistin MIC to that of the respective parent colistin-resistant isolate. In addition, SME98 mutants in which pmrAB was replaced with mutated pmrAB showed no significant differences in bacterial growth and competition culture from the parent strain, except for the mutant with L105P in PmrA, whose growth was significantly suppressed in the presence of the parent strain. In conclusion, some O25b:H4-ST131 strains appear to acquire colistin resistance via phosphoethanolamine modification of lipid A through amino acid changes in PmrAB, and the amino acid changes in PmrB do not influence bacterial growth.

Evaluation of consistency in quantification of gene copy number by real-time reverse transcription quantitative polymerase chain reaction and virus titer by plaque-forming assay for human respiratory syncytial virus.

The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

Whole genome analysis of a multidrug-resistant Streptococcus pneumoniae isolate from a patient with invasive pneumococcal infection developing disseminated intravascular coagulation.

Multidrug-resistant Streptococcus pneumoniae strains were isolated from blood and sputum of a patient with disseminated intravascular coagulation in Sapporo city, Japan. These antibiograms were only susceptible to vancomycin, linezolid, daptomycin, some carbapenems, and some fluoroquinolones. Identical antibiograms, serotypes (19F), and sequence types (ST10017) suggested a shared origin of these isolates. Only one ST10017 strain has been isolated in the same city in Japan previously (2014), and the 2014 isolate is still susceptible to macrolides. The whole genome of the blood-derived isolate was sequenced. The strain harbored resistance mutations in parC, gyrA, pbp1a, pbp2a, pbp2b, and pbp2x, and harbored the resistance genes, ermB and tetM. The nucleotide sequences of parC and pbp2x genes of strain MDRSPN001 were clearly different from those of other S. pneumoniae strains and were similar to those of oral streptococci strains. These findings suggest that strain MDRSPN001 has been rapidly and drastically evolving multidrug resistance by gene replacement and accumulation of genes originating from other strains, such as oral streptococci, Streptococcus mitis.

Tigecycline Nonsusceptibility Occurs Exclusively in Fluoroquinolone-Resistant Escherichia coli Clinical Isolates, Including the Major Multidrug-Resistant Lineages O25b:H4-ST131-H30R and O1-ST648.

Tigecycline (TGC) is a last-line drug for multidrug-resistant Enterobacteriaceae We investigated the mechanism(s) underlying TGC nonsusceptibility (TGC resistant/intermediate) in Escherichia coli clinical isolates. The MIC of TGC was determined for 277 fluoroquinolone-susceptible isolates (ciprofloxacin [CIP] MIC, <0.125 mg/liter) and 194 fluoroquinolone-resistant isolates (CIP MIC, >2 mg/liter). The MIC50 and MIC90 for TGC in fluoroquinolone-resistant isolates were 2-fold higher than those in fluoroquinolone-susceptible isolates (MIC50, 0.5 mg/liter versus 0.25 mg/liter; MIC90, 1 mg/liter versus 0.5 mg/liter, respectively). Two fluoroquinolone-resistant isolates (O25b:H4-ST131-H30R and O125:H37-ST48) were TGC resistant (MICs of 4 and 16 mg/liter, respectively), and four other isolates of O25b:H4-ST131-H30R and an isolate of O1-ST648 showed an intermediate interpretation (MIC, 2 mg/liter). No TGC-resistant/intermediate strains were found among the fluoroquinolone-susceptible isolates. The TGC-resistant/intermediate isolates expressed higher levels of acrA and acrB and had lower intracellular TGC concentrations than susceptible isolates, and they possessed mutations in acrR and/or marR The MICs of acrAB-deficient mutants were markedly lower (0.25 mg/liter) than those of the parental strain. After continuous stepwise exposure to CIP in vitro, six of eight TGC-susceptible isolates had reduced TGC susceptibility. Two of them acquired TGC resistance (TGC MIC, 4 mg/liter) and exhibited expression of acrA and acrB and mutations in acrR and/or marR In conclusion, a population of fluoroquinolone-resistant E. coli isolates, including major extraintestinal pathogenic lineages O25b:H4-ST131-H30R and O1-ST648, showed reduced susceptibility to TGC due to overexpression of the efflux pump AcrAB-TolC, leading to decreased intracellular concentrations of the antibiotics that may be associated with the development of fluoroquinolone resistance.

Mitochondrial proteins NIP-SNAP-1 and -2 are a target for the immunomodulatory activity of clarithromycin, which involves NF-κB-mediated cytokine production.

Macrolide antibiotics have immunomodulatory activities, including suppression of cytokine production, cell adhesion molecule expression, and mucin production. These immunomodulatory activities improve the symptoms of respiratory diseases associated with chronic inflammation. However, the underlying molecular mechanism(s) is not well understood yet. To address this, we prepared clarithromycin (CAM)-conjugated Sepharose and examined bound cellular proteins by proteome analysis. We identified mitochondrial proteins 4-nitrophenylphosphatase domain and non-neuronal synaptosomal associated protein 25-like protein homolog (NIP-SNAP)-1 and -2 and very long-chain acyl-CoA dehydrogenase (VLCAD) as CAM-binding proteins. Production of proinflammatory cytokines (IL-8 and IL-6) induced by lipopolysaccharides (LPSs) and Pam3-CSK4 in human epithelial cell lines BEAS-2B and T24 were suppressed by knockdown of NIP-SNAP-1 or -2, and partly by knockdown of VLCAD. Also, knockdown of NIP-SNAP-1 or -2 in various cell lines suppressed LPS-induced expression of IL-8 and IL-6 mRNA and NF-κB activity. Thus, CAM suppresses NF-κB-mediated proinflammatory cytokine production by interacting with mitochondrial proteins, NIP-SNAP-1 and -2.

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60.

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.

Adaptive Cross-Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa Induced by Topical Dosage of Neomycin.

BACKGROUND: Topical antimicrobial formulations containing neomycin are commonly used to prevent and treat burn infections. However, Pseudomonas aeruginosa shows rapid acquisition of adaptive resistance to neomycin. This study aimed to evaluate the survival of P. aeruginosa during exposure to neomycin at high concentrations comparable to those used in topical formulations, and to investigate the effect of adaptive resistance to neomycin on the susceptibility to other aminoglycosides. METHODS: Strain IID1130 [neomycin minimal inhibitory concentration (MIC) = 4 µg/ml] was incubated on an agar medium containing neomycin at high concentrations (8-4,096 µg/ml), and growing colonies were macroscopically observed. Acquisition of adaptive resistance was examined for 5 P. aeruginosa strains. Cells were sequentially passaged on agar medium containing neomycin with step-wise increased concentrations (8-2,048 µg/ml). To assess reversion of antibiotic susceptibility, the resulting colonies were repeatedly subcultured on antibiotic-free agar plates. RESULTS: Growing IID1130 colonies were macroscopically detected on a neomycin-containing (2,048 µg/ml) agar plate for 48 h. These cells showed increasing MIC for not only neomycin, but also gentamicin and amikacin; the MIC values were occasionally higher than the breakpoints. When the adapted cells were subcultured on antibiotic-free agar, several passages were required for reversion of susceptibility. CONCLUSIONS: Our findings suggest that P. aeruginosa can survive in the presence of neomycin with a concentration typically used in topical dosage forms, and that the acquired adaptive resistance is persistent and is accompanied by cross-resistance to other aminoglycosides.

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3.

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-ß and -λ production. Furthermore, IFN-ß promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

Intrafamilial, Preferentially Mother-to-Child and Intraspousal, Helicobacter pylori Infection in Japan Determined by Mutilocus Sequence Typing and Random Amplified Polymorphic DNA Fingerprinting.

BACKGROUND: The infection route of Helicobacter pylori has been recognized to be mainly intrafamilial, preferentially mother-to-child, especially in developed countries. To determine the transmission route, we examined whether multilocus sequence typing (MLST) was useful for analysis of intrafamilial infection. The possibility of intraspousal infection was also evaluated. MATERIALS AND METHODS: Clonal relationships between strains derived from 35 index Japanese pediatric patients, and their family members were analyzed by two genetic typing procedures, MLST and random amplified polymorphic DNA (RAPD) fingerprinting. RESULTS: Mostly coincident results were obtained by MLST and RAPD. By MLST, the allele of loci in the isolates mostly matched between the index child and both the father and mother for 9 (25.7%) of the 35 patients, between the index child and the mother for 25 (60.0%) of the 35 patients. CONCLUSIONS: MLST is useful for analyzing the infection route of H. pylori as a highly reproducible method. Intrafamilial, especially mother-to-children and sibling, infection is the dominant transmission route. Intraspousal infection is also thought to occur in about a quarter in the Japanese families.

Lipoteichoic acid composed of poly-glycerolphosphate containing l-lysine and involved in immunoglobulin A-inducing activity in Apilactobacillus genus.

Lipoteichoic acid (LTA) in the gram-positive bacterial cell wall acts as an immunomodulatory factor in host cells. The chemical structures vary among bacterial species and strains, and may be related to biological activities. In our previous work, much higher immunoglobulin A (IgA)-inducing activity was observed in cells of the Apilactobacillus genus (Apilactobacillus kosoi 10HT, Apilactobacillus apinorum JCM 30765T, and Apilactobacillus kunkeei JCM 16173T) than other lactic acid bacteria, and their LTA was responsible for the activity. In the present study, we elucidated the chemical structures of LTA from these Apilactobacillus strains to explore the structure-function relationship of the IgA-inducing activity. The 1H-nuclear magnetic resonance spectra suggested that their LTA structures were similar. All have a poly-glycerolphosphate main chain, which comprised 12 to 20 average number of the repeating units, with partial substitutions of glucose(α1-, glucosyl(α1-2)glucose(α1- (α-linked-kojibiose), and l-lysine at the C-2 hydroxy group of the glycerol residue. l-Lysine is a substituent never seen before in LTA, and is a probable characteristic of the Apilactobacillus genus. Removal of l-lysine residue from LTA by mild alkaline treatment decreased IgA induction in murine Peyer's patch experiments. The novel l-lysine residue in Apilactobacillus LTA plays a crucial role in the remarkably high IgA-inducing activity.

Characterization of a Lactobacillus gasseri JCM 1131T lipoteichoic acid with a novel glycolipid anchor structure.

We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131(T). The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.

Inhibitory Effect of the Glycerophosphate Moiety of Lipoteichoic Acid from Lactic Acid Bacteria on Dexamethasone-Induced Atrogin-1 Expression in C2C12 Myotubes.

Atrogin-1, which is an important regulator of ubiquitin-mediated protein degradation in skeletal muscle, is a major marker of muscle loss and disuse muscle atrophy. To investigate which components of lactic acid bacteria (LAB) suppress dexamethasone (DEX)-induced atrogin-1 expression, mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of components of LAB. Heat-killed cells and lipoteichoic acid (LTA) derived from five LAB strains significantly suppressed DEX-induced atrogin-1 expression. The glycerophosphate (GroP) fraction prepared from chemically-degraded LTA and sn-glycerol-1-phosphate suppressed DEX-induced atrogin-1 expression, whereas the glycolipid anchor fraction of LTA did not. Heat-killed cells obtained by culturing under low-Mn2+ conditions, which generated fewer poly-GroP polymers in LTA, displayed significantly lower inhibitory activity compared to heat-killed cells grown under normal conditions. These results suggested that LTA of LAB contributed to suppressing atrogin-1 expression and that the GroP moiety of LTA was responsible for its inhibitory activity.

Oligosaccharide Metabolism and Lipoteichoic Acid Production in Lactobacillus gasseri and Lactobacillus paragasseri.

Lactobacillus gasseri and Lactobacillus paragasseri are human commensal lactobacilli that are candidates for probiotic application. Knowledge of their oligosaccharide metabolic properties is valuable for synbiotic application. The present study characterized oligosaccharide metabolic systems and their impact on lipoteichoic acid (LTA) production in the two organisms, i.e., L. gasseri JCM 1131T and L. paragasseri JCM 11657. The two strains grew well in medium with glucose but poorly in medium with raffinose, and growth rates in medium with kestose differed between the strains. Oligosaccharide metabolism markedly influenced their LTA production, and apparent molecular size of LTA in electrophoresis recovered from cells cultured with glucose and kestose differed from that from cells cultured with raffinose in the strains. On the other hand, more than 15-fold more LTA was observed in the L. gasseri cells cultured with raffinose when compared with glucose or kestose after incubation for 15 h. Transcriptome analysis identified glycoside hydrolase family 32 enzyme as a potential kestose hydrolysis enzyme in the two strains. Transcriptomic levels of multiple genes in the dlt operon, involved in D-alanine substitution of LTA, were lower in cells cultured with raffinose than in those cultured with kestose or glucose. This suggested that the different sizes of LTA observed among the carbohydrates tested were partly due to different levels of alanylation of LTA. The present study indicates that available oligosaccharide has the impact on the LTA production of the industrially important lactobacilli, which might influence their probiotic properties.

Possible clinical outcomes using early enteral nutrition in individuals with allogeneic hematopoietic stem cell transplantation: A single-center retrospective study.

OBJECTIVES: Intensive nutritional support during allogeneic hematopoietic stem cell transplantation (allo-HSCT) yields improved clinical outcomes. However, the clinical implications of early enteral nutrition (EN) in allo-HSCT remain unclear. This retrospective study was conducted to determine the significance of early EN in individuals who underwent allo-HSCT, and the association between early nutritional intervention and clinical outcomes, including the status of the intestinal microbiome. METHODS: Thirty-one participants received EN before conditioning. The intestinal microbiota was examined by meta 16S rRNA gene sequencing of fecal samples. RESULTS: The median body mass variation was only -0.35 kg on day 60. The probability of 2-y overall survival was 61.1%. The cumulative incidence of treatment-related mortality was 17.4%, and those of acute graft-versus-host disease were 32.3% (grades II-IV) and 3.2% (grades III-IV). Chronic graft-versus-host disease was observed in four participants. Dysbiosis of the intestines and acute graft-versus-host disease occurred simultaneously, and Enterococcus species were abundant. CONCLUSIONS: Our results suggest that early nutritional support can improve the outcomes for individuals who have undergone allo-HSCT and can maintain homeostasis of their intestinal microbiome. Future prospective clinical trials are required to elucidate the role of EN in allo-HSCT and the association between the intestinal microbiome and EN.

Structural analysis of the lipoteichoic acid anchor glycolipid: Comparison of methods for degradation of the glycerophosphate backbone polymer.

Acetic acid treatment [98% (v/v), 100 °C, 3 h] was proposed as a new method for degrading the glycerophosphate polymer moiety of Gram-positive bacterial lipoteichoic acid. We demonstrated that this method resulted in partial O-acetylation on the carbohydrate residues of the anchor glycolipid. Hence, the acetic acid treatment is not suitable for the chemical structural analysis of lipoteichoic acid.

Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin.

Pseudomonas aeruginosa is an important etiological agent of opportunistic infections. Injectable colistin is available as a last-line treatment option for multidrug-resistant P. aeruginosa infections. When cells were inoculated at a high number, colistin-susceptible P. aeruginosa grew on agar medium containing colistin at a concentration 10-fold higher than the minimum inhibitory concentration without acquiring colistin resistance. This study examined the responsible mechanism for growth in the presence of a high concentration of colistin. Cell wash fluid derived from P. aeruginosa efficiently reduced colistin antimicrobial activity. This reduction was mediated by lipopolysaccharide (LPS) in the wash fluid. Extracellular LPS inhibited colistin activity more effectively than cell-bound LPS in fixed cells. Cell wash fluids from Escherichia coli and Acinetobacter baumannii also reduced colistin activity; however, they were less potent than those from P. aeruginosa. The amount of LPS in cell wash fluid from P. aeruginosa was approximately 10-fold higher than that in fluid from E. coli or A. baumannii. In conclusion, cell-free LPS derived from bacterial cells inhibited the antimicrobial activity of colistin, and this effect was greatest for P. aeruginosa. Thus, large amounts of broken and dead cells of P. aeruginosa at infection foci will reduce the effectiveness of colistin, even against cells that have not yet acquired resistance.