Enrichment culture evaluation and characterization of Streptococcus agalactiae among pregnant women in Japan.

Introduction. Maternal screening tests and prophylactic antibiotics are important to prevent neonatal and infant group B streptococcal (GBS) infections.Hypothesis/Gap Statement. The performance of enrichment broth media for GBS screening that are available in Japan is unclear. Whole-genome data of GBS isolates from pregnant women in Japan is lacking.Aim. The aim of this study was to compare the protocol performance of six enrichment broths and two subculture agar plates, which were all available in Japan, for GBS detection. In addition, we showed whole-genome data of GBS isolates from pregnant women in Japan.Methodology. We collected 133 vaginal-rectal swabs from pregnant women visiting clinics and hospitals in Nagasaki Prefecture, Japan, and compared the protocol performance of 6 enrichment broths and 2 subculture agar plates. All GBS isolates collected in this study were subjected to whole-genome sequencing analysis.Results. We obtained 133 vaginal-rectal swabs from pregnant women at 35-37 weeks of gestation from 8 private clinics and 2 local municipal hospitals within Nagasaki Prefecture, Japan. The detection rate of the protocol involving the six enrichment broths and subsequent subcultures varied between 95.5 and 100 %, depending on the specific choice of enrichment broth. The GBS carriage rate among pregnant women in this region was 18.8 %. All 25 isolates derived from the swabs were susceptible to penicillin, whereas 48 and 36 % of the isolates demonstrated resistance to erythromycin and clindamycin, respectively. The distribution of serotypes was highly diverse, encompassing seven distinct serotypes among the isolates, with the predominant serotype being serotype V (n = 8). Serotype V isolates displayed a tendency towards increased resistance to erythromycin and clindamycin, with all resistant isolates containing the ermB gene.Conclusion. There was no difference in performance among the culture protocols evaluated in this study. GBS strains isolated from pregnant women appeared to have greater genomic diversity than GBS strains detected in neonates/infants with invasive GBS infections. To confirm this result, further studies with larger sample sizes are needed.

Genomic analysis and identification of a novel superantigen, SargEY, in Staphylococcus argenteus isolated from atopic dermatitis lesions.

During surveillance of Staphylococcus aureus in lesions from patients with atopic dermatitis (AD), we isolated Staphylococcus argenteus, a species registered in 2011 as a new member of the genus Staphylococcus and previously considered a lineage of S. aureus. Genome sequence comparisons between S. argenteus isolates and representative S. aureus clinical isolates from various origins revealed that the S. argenteus genome from AD patients closely resembles that of S. aureus causing skin infections. We previously reported that 17%-22% of S. aureus isolated from skin infections produce staphylococcal enterotoxin Y (SEY), which predominantly induces T-cell proliferation via the T-cell receptor (TCR) Vα pathway. Complete genome sequencing of S. argenteus isolates revealed a gene encoding a protein similar to superantigen SEY, designated as SargEY, on its chromosome. Population structure analysis of S. argenteus revealed that these isolates are ST2250 lineage, which was the only lineage positive for the SEY-like gene among S. argenteus. Recombinant SargEY demonstrated immunological cross-reactivity with anti-SEY serum. SargEY could induce proliferation of human CD4+ and CD8+ T cells, as well as production of TNF-α and IFN-γ. SargEY showed emetic activity in a marmoset monkey model. SargEY and SET (a phylogenetically close but uncharacterized SE) revealed their dependency on TCR Vα in inducing human T-cell proliferation. Additionally, TCR sequencing revealed other previously undescribed Vα repertoires induced by SEH. SargEY and SEY may play roles in exacerbating the respective toxin-producing strains in AD. IMPORTANCE: Staphylococcus aureus is frequently isolated from active lesions of atopic dermatitis (AD) patients. We reported that 17%-22% of S. aureus isolated from AD patients produced a novel superantigen staphylococcal enterotoxin Y (SEY). Unlike many S. aureus superantigens that activate T cells via T-cell receptor (TCR) Vß, SEY activates T cells via TCR Vα and stimulates cytokine secretion. Staphylococcus argenteus was isolated from AD patients during the surveillance for S. aureus. Phylogenetic comparison of the genome indicated that the isolate was very similar to S. aureus causing skin infections. The isolate encoded a SEY-like protein, designated SargEY, which, like SEY, activated T cells via the TCR Vα. ST2250 is the only lineage positive for SargEY gene. ST2250 S. argenteus harboring a superantigen SargEY gene may be a novel staphylococcal clone that infects human skin and is involved in the exacerbation of AD.

In vitro activity of cefiderocol against carbapenemase-producing and meropenem-non-susceptible Gram-negative bacteria collected in the Japan Antimicrobial Resistant Bacterial Surveillance.

OBJECTIVES: The treatment options available for infections caused by multidrug-resistant Gram-negative pathogens are often limited. Cefiderocol (CFDC) is a novel siderophore cephalosporin that exhibits activity against these pathogens. Several studies have reported the in vitro activity of CFDC against isolates from Europe, the United States, and China, but the activity against carbapenem-resistant bacteria with IMP-type carbapenemase has not been extensively studied. We, therefore, studied the in vitro activities of CFDC against carbapenem-resistant bacteria with available genomic backgrounds based on whole-genome sequencing (WGS) in Japan, where the IMP-type is the predominant carbapenemase produced by Gram-negative rods. METHODS: We selected 603 isolates (528 Enterobacterales, 18 Pseudomonas aeruginosa, and 57 Acinetobacter spp.) from a collection of Gram-negative clinical isolates collected during a Japan Antimicrobial Resistance Bacterial Surveillance program and evaluated the antimicrobial activities of CFDC, ceftolozane/tazobactam (CTLZ/TAZ), imipenem-relebactam (IPM/REL), and ceftazidime/avibactam (CAZ/AVI) against carbapenemase-producing Enterobacterales, carbapenemase-non-producing meropenem-non-susceptible Enterobacterales, and carbapenemase-producing nonfermentative bacteria. RESULTS: Among these, 97.7% of carbapenemase-producing Enterobacterales (99.2% of IMP-type carbapenemase-producing Enterobacterales), 100% of carbapenemase-producing P. aeruginosa, and 91.2% of carbapenemase-producing Acinetobacter spp. were susceptible to CFDC, showing better antimicrobial activity than the other antimicrobial agents evaluated in this study. CFDC was highly effective against class A-, B-, and D ß-lactamase-harbouring isolates when compared to the other antimicrobial agents. In addition, the relationship between CFDC resistance and three genetic factors involved in resistance was discussed. CONCLUSIONS: This is the first large-scale study to systematically demonstrate the efficacy of CFDC against IMP-type carbapenemase-producing strains with known genomic backgrounds.

Exploration of Exopolysaccharide from Leuconostoc mesenteroides HDE-8: Unveiling Structure, Bioactivity, and Food Industry Applications.

A strain of Leuconostoc mesenteroides HDE-8 was isolated from homemade longan fermentation broth. The exopolysaccharide (EPS) yield of the strain was 25.1 g/L. The EPS was isolated and purified, and the structure was characterized using various techniques, including X-ray diffraction (XRD), nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, high-performance size exclusion chromatography (HPSEC), and scanning electron microscopy (SEM). The monosaccharide composition of the EPS was glucose, with a molecular weight (Mw) of 1.7 × 106 Da. NMR spectroscopy revealed that the composition of the HDE-8 EPS consisted of D-glucose pyranose linked by α-(1→4) and α-(1→6) bonds. The SEM analysis of the EPS showed an irregular sheet-like structure. Physicochemical analysis demonstrated that EPSs exhibit excellent thermal stability and high viscosity, making them suitable for fermentation in heat-processed and acidic foods. Additionally, milk coagulation tests showed that the presence of EPSs promotes milk coagulation when supplemented with sucrose. It suggests that EPSs have wide-ranging potential applications as food additives, improving the texture and taste of dairy products. This study provides practical guidance for the commercial use of HDE-8 EPSs in the food and related industries.

Complete sequence of carbapenem-resistant Ralstonia mannitolilytica clinical isolate co-producing novel class D ß-lactamase OXA-1176 and OXA-1177 in Japan.

In 2020, the Ralstonia mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in R. mannitolilytica, and referred to as the "megaplasmid" in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D ß-lactamase genes blaOXA-1176 and blaOXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli recombinant clone expressing blaOXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that blaOXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing blaOXA-1177 gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of blaOXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCERalstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including ß-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant R. mannitolilytica clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel blaOXA-1176 and blaOXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings.

National genomic surveillance integrating standardized quantitative susceptibility testing clarifies antimicrobial resistance in Enterobacterales.

Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the blaCTX-M extended-spectrum ß-lactamase genes, blaCTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC ß-lactamase genes were blaCMY-2 and blaDHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of blaCMY-2 and blaDHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

Emergence of colistin-resistant Enterobacter cloacae and Raoultella ornithinolytica carrying the phosphoethanolamine transferase gene, mcr-9, derived from vegetables in Japan.

IMPORTANCE: Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

Biosynthesis and characterization of antibacterial bacterial cellulose composite membrane composed of montmorillonite and exopolysaccharides.

Bacterial cellulose (BC), as a natural renewable polymer material, has the advantages of porous nanonetwork structure, high degree of polymerization, high purity, high crystallinity, excellent mechanical properties and biocompatibility. However, BC lacks antibacterial properties, which leads to the limitation of BC material in food packaging and medical materials. In this study, a new antibacterial material using the combination of montmorillonite (MMT), BC and exopolysaccharides (EPS) produced by Weissella confusa H2 was synthesized. Fourier infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) analysis showed that BC-EPS, BC-MMT and BC-EPS-MMT composite membranes conformed to the typical type I cellulose structure. Compared to BC membrane, scanning electron microscopy (SEM) showed that the porosity of BC-EPS, BC-MMT and BC-EPS-MMT composite membranes was low and compact. The physical properties of BC-EPS, BC-MTT and BC-EPS-MTT composite membranes showed lower water vapor transmittance. The BC-MTT and BC-EPS-MTT composite membranes exhibit a lower swelling ratio in 120 min. The thermal properties show that BC-EPS, BC-MTT and BC-EPS-MTT composite membranes have higher thermal stability (352 °C, 310 °C, 314 °C). Additionally, both BC-MMT and BC-EPS-MMT demonstrated strong inhibitory effects against various bacterial strains, including Staphylococcus aureus, Escherichia coli, Salmonella paratyphi A, and Bacillus subtilis. The exceptional properties exhibited by composite membranes establishes them as a highly promising option in the field of food packaging and medical material applications.

Municipal wastewater monitoring revealed the predominance of bla GES genes with diverse variants among carbapenemase-producing organisms: high occurrence and persistence of Aeromonas caviae harboring the new bla GES variant bla GES-48.

IMPORTANCE: The emergence and spread of carbapenemase-producing organisms (CPOs) represent a global health threat because they are associated with limited treatment options and poor clinical outcomes. Wastewater is considered a hotspot for the evolution and dissemination of antimicrobial resistance. Thus, analyses of municipal wastewater are critical for understanding the circulation of these CPOs and carbapenemase genes in local communities, which remains scarcely known in Japan. This study resulted in several key observations: (i) the vast majority of bla GES genes, including six new bla GES variants, and less frequent bla IMP genes were carbapenemase genes encountered exclusively in wastewater influent; (ii) the most dominant CPO species were Aeromonas spp., in which a remarkable diversity of new sequence types was observed; and (iii) CPOs were detected from combined sewer wastewater, but not from separate sewer wastewater, suggesting that the load of CPOs from unrecognized environmental sources could greatly contribute to their detection in influent wastewater.

Study of a Fluorescent System Based on the Naphthalene Derivative Fluorescent Probe Bound to Al3.

The naphthalene derivative fluorescent probe F6 was synthesized and a 1 × 10-3 mol/L solution of Al3+ and other metals to be tested was prepared for the subsequent experiments. The Al3+ fluorescence system of the naphthalene derivative fluorescent probe F6 was successfully constructed as demonstrated by fluorescence emission spectroscopy. The optimal time, temperature and pH of the reaction were investigated. The selectivity and anti-interference ability of the probe F6 for Al3+ were investigated by fluorescence spectroscopy in a methanol solution. The experiments showed that the probe has high selectivity and anti-interference ability for Al3+. The binding ratio of F6 to Al3+ was 2:1, and the binding constant was calculated to be 1.598 × 105 M-1. The possible mechanism of the binding of the two was speculated. Different concentrations of Al3+ were added to Panax Quinquefolium and Paeoniae Radix Alba. The results showed that the recoveries of Al3+ were 99.75-100.56% and 98.67-99.67%, respectively. The detection limit was 8.73 × 10-8 mol/L. The experiments demonstrated that the formed fluorescence system can be successfully adapted for the determination of Al3+ content in two Chinese herbal medicines, which has good practical application.

Purification, characterization and probiotic proliferation effect of exopolysaccharides produced by Lactiplantibacillus plantarum HDC-01 isolated from sauerkraut.

In this study, an exopolysaccharide (EPS)-producing strain of Lactiplantibacillus plantarum HDC-01 was isolated from sauerkraut, and the structure, properties and biological activity of the studied EPS were assessed. The molecular weight of the isolated EPS is 2.505 × 106 Da. Fourier transform infrared spectrometry (FT-IR) and nuclear magnetic resonance (NMR) results showed that the EPS was composed of glucose/glucopyranose subunits linked by an α-(1 → 6) glycosidic bond and contained an α-(1 → 3) branching structure. X-ray diffraction (XRD) analysis revealed the amorphous nature of the EPS. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that the isolated EPS had a smooth and compact surface with several protrusions of varying lengths and irregularly shaped material. Moreover, the studied EPS showed good thermal stability, water holding capacity, and milk coagulation ability and promoted the growth of probiotics. L. plantarum EPS may be used as prebiotics in the fields of food and medicine.

Preparation of g-C3N4/TCNQ Composite and Photocatalytic Degradation of Pefloxacin.

g-C3N4 and g-C3N4/TCNQ composites with different doping levels were prepared using the copolymerization thermal method with melamine as a precursor. XRD, FT-IR, SEM, TEM, DRS, PL, and I-T characterized them. The composites were successfully prepared in this study. The photocatalytic degradation of pefloxacin (PEF), enrofloxacin (ciprofloxacin), and ciprofloxacin (ciprofloxacin) under visible light (λ > 550 nm) showed that the composite material had the best degradation effect on PEF. When TCNQ doping is 20 mg and catalyst dosage is 50 mg, the catalytic effect is the best, and the degradation rate reaches 91.6%, k = 0.0111 min-1, which is four times that of g-C3N4. Repeated experiments found that the cyclic stability of the g-C3N4/TCNQ composite was good. The XRD images were almost unchanged after five reactions. The radical capture experiments revealed that ·O2- was the main active species in the g-C3N4/TCNQ catalytic system, and h+ also played a role in PEF degradation. And the possible mechanism for PEF degradation was speculated.

Characterization of Dextran Biosynthesized by Glucansucrase from Leuconostoc pseudomesenteroides and Their Potential Biotechnological Applications.

Glucansucrase was purified from Leuconostoc pseudomesenteroides. The glucansucrase exhibited maximum activity at pH 5.5 and 30 °C. Ca2+ significantly promoted enzyme activity. An exopolysaccharide (EPS) was synthesized by this glucansucrase in vitro and purified. The molecular weight of the EPS was 3.083 × 106 Da. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy showed that the main structure of glucan was 97.3% α-(1→6)-linked D-glucopyranose units, and α-(1→3) branched chain accounted for 2.7%. Scanning electron microscopy (SEM) observation of dextran showed that its surface was smooth and flaky. Atomic force microscopy (AFM) of dextran revealed a chain-like microstructure with many irregular protuberances in aqueous solution. The results showed that dextran had good thermal stability, water holding capacity, water solubility and emulsifying ability (EA), as well as good antioxidant activity; thus it has broad prospects for development in the fields of food, biomedicine, and medicine.

New Molecular Mechanism of Superbiofilm Elaboration in a Staphylococcus aureus Clinical Strain.

Previously, we reported a novel regulator of biofilm (rob) with a nonsense mutation in the superbiofilm-elaborating strain JP080. Intriguingly, the complementation of JP080 with wild-type rob did not completely abolish its superbiofilm-elaborating phenotype. Therefore, we searched for other possible mutation(s) using complete genome sequence data and found a missense mutation in the gene icaR, which altered its 35th amino acid (Ala35Thr). To further study the mechanism of superbiofilm elaboration in JP080, we reconstructed the same mutations of rob and icaR in the strain FK300 and analyzed the phenotypes. The mutation of rob (A331T) increased biofilm elaboration, as previously demonstrated; similarly, an icaR mutation increased poly-N-acetylglucosamine and biofilm production in strain FK300. Furthermore, our analyses indicated that the double mutant of rob and icaR produced significantly more biofilms than the single mutants. Additionally, gel shift analysis revealed that the icaR from JP080 lost its ability to bind to the ica promoter region. These findings suggest that the icaR mutation in JP080 may result in a nonfunctional protein. We compared ica operon expression in an icaR single mutant, rob single mutant, and rob and icaR double mutant to the wild type. The rob and icaR mutants showed increased ica operon transcription by approximately 19- and 79-fold, respectively. However, the rob and icaR double mutant showed an approximately 350-fold increase, indicating the synergistic effects of icaR and rob on JP080 biofilm elaboration. Consequently, we concluded that the double mutations rob and icaR synergistically increased ica operon transcription, resulting in a superbiofilm phenotype in Staphylococcus aureus. IMPORTANCE Poly-N-acetylglucosamine (PNAG) is a major component of S. aureus biofilm. PNAG production is mediated by the products of four genes, icaADBC encoded in the ica operon, and the major negative regulator of this operon is IcaR encoded just upstream of icaADBC. Previously, we reported another negative regulator, Rob, through gene expression analysis of clinically isolated superbiofilm-elaborating strain JP080. The rob gene is encoded at different loci distant from the ica operon. Here, we report that JP080 also carried a mutation in icaR and demonstrated that IcaR and Rob synergistically regulate PNAG production. We successfully reconstructed these mutations in a wild type, and the double mutant resulted in superbiofilm-elaborating phenotype. We clearly show that loss of function of both IcaR and Rob is the very reason that JP080 is showing the superbiofilm-elaborating phenotype. This study clearly demonstrated there are at least two independent regulators synergistically fine-tuning PNAG production and suggested the complex regulatory mechanism of biofilm production.

Genomic landscape of blaGES-5- and blaGES-24-harboring Gram-negative bacteria from hospital wastewater: emergence of class 3 integron-associated blaGES-24 genes.

OBJECTIVES: This study aimed to characterize Gram negative bacteria carrying blaGES carbapenemase genes detected in wastewater from a hospital with no history of detection of clinical isolates producing GES carbapenemases. METHODS: Six hospital effluent samples were screened for carbapenemase-producing organisms (CPO) using CHROMagar mSuperCARBA and MacConkey agar with 1 µg/mL imipenem. Polymerase chain reaction (PCR) amplification and sequencing of carbapenemase genes, multilocus sequence typing, antimicrobial susceptibility testing, and whole-genome sequencing were performed. RESULTS: Among 21 CPO isolates, 11 Klebsiella spp. and 5 Enterobacter kobei isolates carried blaGES-24, and 4 E. roggenkampii and 1 Pseudomonas aeruginosa isolates carried blaGES-5. Genomic analysis of 8 representative isolates comprising 6 blaGES-24-positive and 2 blaGES-5-positive revealed that class 3 integrons with complete or defective Tn402-like transposition modules were predominantly associated with two tandem copies of blaGES-24. Furthermore, a total of 5 new class 3 integrons, In3-18 to In3-22, were identified among 5 blaGES-24 and 1 blaGES-5 plasmids. One strain each of K. pneumoniae subsp. pneumoniae and K. quasipneumoniae subsp. similipneumoniae harboring blaGES-24 plasmids also carried a rare blaVEB-1-positive class 1 integron on a non-typeable plasmid, where these blaVEB-1 plasmids had high sequence similarity. Virulence gene profiles differed between Klebsiella spp. and Enterobacter spp.; the former harbored type III fimbriae cluster, salmochelin, and T6SS type i2 gene clusters, while the latter had curli pili operon, aerobactin, T2SS gene clusters, and T6SS type i3 gene clusters. CONCLUSION: Our findings confirmed the linkage of blaGES-24 with rare Tn402-like class 3 integrons and the structural diversity of their gene cassette arrays.

Complete genome sequences of two Escherichia coli clinical isolates from Egypt carrying mcr-1 on IncP and IncX4 plasmids.

Colistin is a last-resort antibiotic used in the treatment of multidrug resistant Gram-negative bacteria. However, the activity and efficacy of colistin has been compromised by the worldwide spread of the mobile colistin resistance genes (mcr-1 to mcr-10). In this study, two clinical Escherichia coli strains, named EcCAI51, and EcCAI73, harbored mcr-1, showed multidrug-resistant phenotypes (with colistin MIC = 4 µg/ml), and belonged to phylogroup D: multilocus sequence type 1011 (ST1011) and phylogroup A: ST744, respectively. Findings revealed the existence of mcr-1 gene on two conjugable plasmids, pAMS-51-MCR1 (∼122 kb IncP) and pAMS-73-MCR1 (∼33 kb IncX4), in EcCAI51, and EcCAI73, respectively. The mcr-1-pap2 element was detected in the two plasmids. Additionally, the composite transposon (ISApl1-IS5D-pap2-mcr-1-ISApl1) was identified only in pAMS-51-MCR1 suggesting the potential for horizontal gene transfer. The two strains carried from 16 to 18 different multiple acquired antimicrobial resistance genes (ARGs). Additionally, two different multireplicon virulence plasmids (∼117 kb pAMS-51-Vr and ∼226 kb pAMS-73-Vr) carrying the sit operon, the Salmochelin siderophore iroBCDE operon and other several virulence genes were identified from the two strains. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of EcCAI73, and EcCAI51 with global E. coli lineages at HC levels of 50 (HC50) to 100 (HC100) core genome allelic differences. To the best of our knowledge, this study presented the first complete genomic sequences of mcr-1-carrying IncP and IncX4 plasmids from human clinical E. coli isolates in Egypt. In addition, the study illustrated the mcr-1 broad dissemination in diverse plasmids and dissimilar E. coli clones.

Characterization of exopolysaccharide produced by Levilactobacillus brevis HDE-9 and evaluation of its potential use in dairy products.

The bacterial strain HDE-9 was isolated from sauerkraut and identified as Levilactobacillus brevis. An exopolysaccharide (EPS) was isolated and purified from L. brevis HDE-9, and a preliminary investigation of its structural characteristics and biological activity was conducted. The molecular weight of the EPS was >1.0 × 106 Da. Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy revealed that the EPS was composed of α-(1 â†’ 6) linked d-glucopyranose units. X-ray diffraction (XRD) data on the EPS revealed its non-crystalline amorphous structure. Scanning electron microscopy (SEM) of the EPS revealed a smooth surface with sheet structures. The EPS exhibited the high value in thermal stability, water solubility, water holding capacity (WHC), and emulsification activity (EA). The water contact angle of the EPS revealed relatively high hydrophobicity in the presence of sucrose. The EPS also showed a strong milk solidification capacity in a dose-dependent manner. The EPS could significantly improve the texture of yoghurt, indicating its potential application as a functional starter in the production of fermented dairy products.

Genomic epidemiology and temperature dependency of hypermucoviscous Klebsiella pneumoniae in Japan.

Klebsiella pneumoniae (Kp) has emerged as a global life-threatening pathogen owing to its multidrug resistance and hypervirulence phenotype. Several fatal outbreaks of carbapenem-resistant hypervirulent Kp have been reported recently. Hypermucoviscosity (HMV) is a phenotype commonly associated with hypervirulence of Kp, which is usually regulated by rmpA or rmpA2 (regulators of the mucoid phenotype). Here, we found that temperature was important in the HMV phenotype of Kp, and the impact of temperature on HMV was not uniform among strains. We investigated the HMV phenotype at 37 °C and room temperature (20-25 °C) in 170 clinically isolated hypermucoviscous Kp strains in Japan and analysed the association between the HMV phenotype, virulence genes and antimicrobial resistance (AMR) genes. String length distribution at different temperatures was correlated with the genomic population of Kp. The strains carrying rmpA/rmpA2 frequently showed the HMV phenotype at 37 °C, while the strains negative for these genes tended to show the HMV phenotype at room temperature. Hypervirulent Kp clusters carrying rmpA/rmpA2 without extended-spectrum beta-lactamases (ESBL)/carbapenemases produced higher string lengths at 37 °C than at room temperature, and were mostly isolated from the respiratory tract. Other HMV strains showed distinct characteristics of not carrying rmpA/rmpA2 but were positive for ESBL/carbapenemases, with a higher string length at room temperature than at 37 °C, and were frequently isolated from bloodstream infections. In total, 21 (13.5 %) HMV isolates carried ESBL and carbapenemases, among which five isolates were carbapenem-resistant hypervirulent Kp with a pLVPK-like plasmid (an epidemic virulence plasmid) and a pKPI-6-like plasmid (an epidemic blaIMP-6-bearing plasmid in Japan), suggesting the convergence of worldwide hypervirulence and epidemic AMR in Japan.