Chicken Juice Enhances C. jejuni NCTC 11168 Biofilm Formation with Distinct Morphological Features and Altered Protein Expression.

Campylobacter jejuni is the foodborne pathogen causing most gastrointestinal infections. Understanding its ability to form biofilms is crucial for devising effective control strategies in food processing environments. In this study, we investigated the growth dynamics and biofilm formation of C. jejuni NCTC 11168 in various culture media, including chicken juice (CJ), brain heart infusion (BHI), and Mueller Hinton (MH) broth. Our results demonstrated that C. jejuni exhibited a higher growth rate and enhanced biofilm formation in CJ and in 1:1 mixtures of CJ with BHI or MH broth compared to these measures in BHI or MH broth alone. Electron microscopy unveiled distinct morphological attributes of late-stage biofilm cells in CJ, including the presence of elongated spiral-shaped cells, thinner stretched structures compared to regular cells, and extended thread-like structures within the biofilms. Proteomic analysis identified significant alterations in protein expression profiles in C. jejuni biofilms, with a predominance of downregulated proteins associated with vital functions like metabolism, energy production, and amino acid and protein biosynthesis. Additionally, a significant proportion of proteins linked to biofilm formation, virulence, and iron uptake were suppressed. This shift toward a predominantly coccoid morphology echoed the reduced energy demands of these biofilm communities. Our study unlocks valuable insights into C. jejuni's biofilm in CJ, demonstrating its adaptation and survival.

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

Whole-Genome Sequence Analysis of Antibiotic Resistance, Virulence, and Plasmid Dynamics in Multidrug-Resistant E. coli Isolates from Imported Shrimp.

We analyzed antimicrobial resistance and virulence traits in multidrug-resistant (MDR) E. coli isolates obtained from imported shrimp using whole-genome sequences (WGSs). Antibiotic resistance profiles were determined phenotypically. WGSs identified key characteristics, including their multilocus sequence type (MLST), serotype, virulence factors, antibiotic resistance genes, and mobile elements. Most of the isolates exhibited resistance to gentamicin, streptomycin, ampicillin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, and trimethoprim/sulfamethoxazole. Multilocus sequence type (MLST), serotype, average nucleotide identity (ANI), and pangenome analysis showed high genomic similarity among isolates, except for EC15 and ECV01. The EC119 plasmid contained a variety of efflux pump genes, including those encoding the acid resistance transcriptional activators (gadE, gadW, and gadX), resistance-nodulation-division-type efflux pumps (mdtE and mdtF), and a metabolite, H1 symporter (MHS) family major facilitator superfamily transporter (MNZ41_23075). Virulence genes displayed diversity, particularly EC15, whose plasmids carried genes for adherence (faeA and faeC-I), invasion (ipaH and virB), and capsule (caf1A and caf1M). This comprehensive analysis illuminates antimicrobial resistance, virulence, and plasmid dynamics in E. coli from imported shrimp and has profound implications for public health, emphasizing the need for continued surveillance and research into the evolution of these important bacterial pathogens.

Complete genome sequence of Delftia tsuruhatensis strain HA60 isolated from a commercial hydroxyapatite nano-particle product (nano-hydroxyapatite).

Delftia tsuruhatensis is a Gram-negative rod-shaped aerobic bacterium with environmental remediation functions. D. tsuruhatensis strain HA60 was isolated from a commercial nano-particle product, nano-hydroxyapatite. We report that the genome of D. tsuruhatensis strain HA60 has a circular genome of 6,922,195 base pairs with a G+C content of 66.45%.

Unraveling the molecular dynamics of Pseudomonas aeruginosa biofilms at the air-liquid interface.

Aim: The aim of this study was to probe the dynamics of Pseudomonas aeruginosa PA14 air-liquid interface (ALI) biofilms over time through global proteomic analysis. Materials & methods: P. aeruginosa PA14 ALI biofilm samples, collected over 48-144 h, underwent differential expression analysis to identify varying proteins at each time point. Results: A consistent set of 778 proteins was identified, with variable expression over time. Upregulated proteins were mainly linked to 'amino acid transport and metabolism'. Biofilm-related pathways, including cAMP/Vfr and QS, underwent significant changes. Flagella were more influential than pili, especially in early biofilm development. Proteins associated with virulence, transporters and iron showed differential expression throughout. Conclusion: The findings enhance our understanding of ALI biofilm development.

This study looks at how the bacteria Pseudomonas aeruginosa forms a community called a biofilm at the air­liquid interface (ALI), an important environment for bacterial growth. Biofilms at the ALI are resistant to external forces and contribute to antibiotic resistance. Over 48­144 h, we observed a noticeable increase in biofilm thickness. Our data suggested that the flagella, a sort of propeller of the bacterium, plays a crucial role, especially in the initial stages of ALI biofilm formation. Proteins associated with virulence, transporters and iron also showed their significance in ALI biofilms. These findings offer valuable insights into the protein changes and functions involved in P. aeruginosa ALI biofilms, improving our understanding of biofilm development.

Genome sequence of Clostridium septicum strain WW106, isolated from influent wastewater at a research center with multiple-species research animal facilities.

Clostridium septicum is an anaerobic Gram-positive rod-shaped bacterial pathogen known as a lethal causative agent of progressive gas gangrene in animals and humans. We report the 3.43-Mbp genome sequence of C. septicum strain WW106, isolated from influent wastewater at a research center with multiple-species laboratory animal facilities.

Genome sequence of Gordonia alkaliphila strain WW102, isolated from influent wastewater at a research center with multiple-species research animal facilities.

Gordonia alkaliphila is a little known mesophilic Gram-positive rod-shaped bacterium. We report the 3.85-Mbp genome sequence of G. alkaliphila strain WW102, isolated from wastewater at a research center with multiple-species laboratory animal facilities. The genome predicted FadD32 gene clusters that are involved in the biosynthesis of mycolic acids as found in Mycobacterium tuberculosis.

Synthesis of Antibacterial Copper Oxide Nanoparticles by Pulsed Laser Ablation in Liquids: Potential Application against Foodborne Pathogens.

Spherical copper oxide nanoparticles (CuO/Cu2O NPs) were synthesized by pulsed laser ablation in liquids (PLAL). The copper target was totally submerged in deionized (DI) water and irradiated by an infrared laser beam at 1064 nm for 30 min. The NPs were then characterized by dynamic light scattering (DLS) and atomic emission spectroscopy (AES) to determine their size distribution and concentration, respectively. The phases of copper oxide were identified by Raman spectroscopy. Then, the antibacterial activity of CuO/Cu2O NPs against foodborne pathogens, such as Salmonella enterica subsp. enterica serotype Typhimurium DT7, Escherichia coli O157:H7, Shigella sonnei ATCC 9290, Yersinia enterocolitica ATCC 27729, Vibrio parahaemolyticus ATCC 49398, Bacillus cereus ATCC 11778, and Listeria monocytogenes EGD, was tested. At a 3 ppm concentration, the CuO/Cu2O NPs exhibited an outstanding antimicrobial effect by killing most bacteria after 5 h incubation at 25 °C. Field emission scanning electron microscope (FESEM) confirmed that the CuO/Cu2O NPs destructed the bacterial cell wall.

Concentration-Dependent Global Quantitative Proteome Response of Staphylococcus epidermidis RP62A Biofilms to Subinhibitory Tigecycline.

Staphylococcus epidermidis is a leading cause of biofilm-associated infections on implanted medical devices. During the treatment of an infection, bacterial cells inside biofilms may be exposed to sublethal concentrations of the antimicrobial agents. In the present study, the effect of subinhibitory concentrations of tigecycline (TC) on biofilms formed by S. epidermidis strain RP62A was investigated using a quantitative global proteomic technique. Sublethal concentrations of TC [1/8 (T1) and 1/4 minimum inhibitory concentration (MIC) (T2)] promoted biofilm production in strain RP62A, but 1/2 MIC TC (T3) significantly inhibited biofilm production. Overall, 413, 429, and 518 proteins were differentially expressed in biofilms grown with 1/8 (T1), 1/4 (T2), and 1/2 (T3) MIC of TC, respectively. As the TC concentration increased, the number of induced proteins in each Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway increased. The TC concentration dependence of the proteome response highlights the diverse mechanisms of adaptive responses in strain RP62A biofilms. In both COG and KEGG functional analyses, most upregulated proteins belong to the metabolism pathway, suggesting that it may play an important role in the defense of strain RP62A biofilm cells against TC stress. Sub-MIC TC treatment of strain RP62A biofilms led to significant changes of protein expression related to biofilm formation, antimicrobial resistance, virulence, quorum sensing, ABC transporters, protein export, purine/pyrimidine biosynthesis, ribosomes, and essential proteins. Interestingly, in addition to tetracycline resistance, proteins involved in resistance of various antibiotics, including aminoglycosides, antimicrobial peptides, ß-lactams, erythromycin, fluoroquinolones, fusidic acid, glycopeptides, lipopeptides, mupirocin, rifampicin and trimethoprim were differentially expressed. Our study demonstrates that global protein expression profiling of biofilm cells to antibiotic pressure may improve our understanding of the mechanisms of antibiotic resistance in biofilms.

Complete Genome Sequence of Bacillus toyonensis Strain HA0190, Isolated from a Commercial Hydroxyapatite Product Extensively Used as a Synthetic Bone Graft Substitute.

Bacillus toyonensis is a member of the Bacillus cereus group and is used as a probiotic in animal feeds and biological applications. We report the 5.8-Mbp genome sequence of strain HA0190, an isolate from a commercial hydroxyapatite nanoparticle product. The genome contains a circular chromosome and two plasmids, pBT001 and pBT002.

Draft Genome Sequences of 12 Panton-Valentine Leucocidin-Positive and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Strains Isolated from an Intensive Care Unit in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for difficult-to-treat staphylococcal infections due to multidrug resistance. Twelve Panton-Valentine leucocidin (PVL)-positive and multidrug-resistant clinical MRSA isolates from hospitals in Pakistan were sequenced and annotated to investigate genetic markers associated with antimicrobial resistance, virulence, and biofilm formation.

Generation of a B2M homozygous knockout human somatic cell nuclear transfer-derived embryonic stem cell line using the CRISPR/Cas9 system.

Beta2-microglobulin (B2M) is a subunit of human leukocyte antigen class-I (HLA-I) heterodimer that mediates immune rejection through activation of cytotoxic T cells. B2M binding to HLA-I proteins is essential for functional HLA-I on the cell surface. Here, we generated a B2M homozygous knockout somatic cell nuclear transfer-induced embryonic stem cell (SCNT-ESC) line using CRISPR/Cas9-mediated gene targeting. B2M KO cell line, which does not express HLA-I molecules on cell surface, has pluripotency and differentiation ability to three germ layers. This cell line provides a useful cell source for investigating immunogenicity of allogeneic ESCs and their derivatives for tissue regeneration.

Draft Genome Sequences of Eight Community-Associated Methicillin-Resistant Staphylococcus aureus Strains.

Here, we report the draft genome sequences of eight community-associated methicillin-resistant Staphylococcus aureus strains that were resistant to cefoxitin, ampicillin, and erythromycin. Three isolates, i.e., CAR1, CAR2, and CAR8, were sequence type 8 (ST8) with staphylococcal cassette chromosome mec (SCCmec) type IVa and were Panton-Valentine leukocidin (PVL) positive, which has been known as a predominant clone in the United States.

Draft Genome Sequence of the Multidrug-Resistant Strain Stenotrophomonas maltophilia N0320, Isolated from a Commercial Nanoparticle Product.

Stenotrophomonas maltophilia is an emerging opportunistic pathogen that is frequently associated with hospital infections. We report the 4.8-Mbp draft genome sequence of the oxidase-positive S. maltophilia strain N0320, an isolate from a commercial hydroxyapatite nanoparticle product.

Dynamic Adaptive Response of Pseudomonas aeruginosa to Clindamycin/Rifampicin-Impregnated Catheters.

Pseudomonas aeruginosa is the most common Gram-negative pathogen causing nosocomial multidrug resistant infections. It is a good biofilm producer and has the potential for contaminating medical devices. Despite the widespread use of antibacterial-impregnated catheters, little is known about the impacts of antibacterial coating on the pathogenesis of P. aeruginosa. In this study, we investigated the adaptive resistance potential of P. aeruginosa strain PAO1 in response to continuous antibiotic exposure from clindamycin/rifampicin-impregnated catheters (CR-IC). During exposure for 144 h to clindamycin and rifampicin released from CR-IC, strain PAO1 formed biofilms featuring elongated and swollen cells. There were 545 and 372 differentially expressed proteins (DEPs) identified in the planktonic and biofilm cells, respectively, by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Both Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the planktonic cells responded to the released antibiotics more actively than the biofilm cells, with metabolism and ribosomal biosynthesis-associated proteins being significantly over-expressed. Exposure to CR-IC increased the invasion capability of P. aeruginosa for Hela cells and upregulated the expression of certain groups of virulence proteins in both planktonic and biofilm cells, including the outer membrane associated (flagella, type IV pili and type III secretion system) and extracellular (pyoverdine) virulence proteins. Continuous exposure of P. aeruginosa to CR-IC also induced the overexpression of antibiotic resistance proteins, including porins, efflux pumps, translation and transcription proteins. However, these upregulations did not change phenotypic minimum inhibitory concentration (MIC) during the experimental timeframe. The concerning association between CR-IC and overexpression of virulence factors in P. aeruginosa suggests the need for additional investigation to determine if it results in adverse clinical outcomes.

ß-Lactam resistance development affects binding of penicillin-binding proteins (PBPs) of Clostridium perfringens to the fluorescent penicillin, BOCILLIN FL.

Alteration in the binding of bacterial penicillin-binding proteins (PBPs) to ß-lactams is important in the development of drug resistance. The PBPs of wild type Clostridium perfringens ATCC 13124 and three ß-lactam-resistant mutants were compared for the ability to bind to a fluorescent penicillin, BOCILLIN FL. The binding of the high molecular weight protein PBP1, a transpeptidase, to BOCILLIN FL was reduced in all of the resistant strains. In contrast, the binding of BOCILLIN FL to a low molecular weight protein, PBP6, a D-alanyl-d-alanine carboxypeptidase that was more abundant in all three resistant strains, was substantially increased. A competition assay with ß-lactams reduced the binding of all of the PBPs, including PBP6, to BOCILLIN FL. ß-Lactams enhanced transcription of the putative gene for PBP6 in both wild type and resistant strains. This is the first report showing that mutations in a high molecular weight PBP and overexpression of a low molecular weight PBP in resistant C. perfringens strains affected their binding to ß-lactams.

Voltage Dependent N Type Calcium Channel in Mouse Egg Fertilization.

Repetitive changes in the intracellular calcium concentration ([Ca2+]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca2+]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca2+ ion elevation during [Ca2+]i oscillations are Ca2+ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca2+ ion influx through Ca2+ channel on the plasma membrane. Ca2+ channels have been characterized into voltage-dependent Ca2+ channels (VDCCs), ligand-gated Ca2+ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca2+]i oscillation was observed in a Ca2+ contained medium with sperm factor or adenophostin A injection but disappeared in Ca2+ free medium. Ca2+ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca2+]i oscillation profiles in SrCl2 treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca2+ influx is essential for [Ca2+]i oscillation during mammalian fertilization. This Ca2+ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

STK3/4 Expression Is Regulated in Uterine Endometrial Cells during the Estrous Cycle.

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

ST2 blockade mitigates peritoneal fibrosis induced by TGF-ß and high glucose.

Peritoneal fibrosis (PF) is an intractable complication of peritoneal dialysis (PD) that leads to peritoneal membrane failure. This study investigated the role of suppression of tumorigenicity (ST)2 in PF using patient samples along with mouse and cell-based models. Baseline dialysate soluble (s)ST2 level in patients measured 1 month after PD initiation was 2063.4 ± 2457.8 pg/mL; patients who switched to haemodialysis had elevated sST2 levels in peritoneal effluent (1576.2 ± 199.9 pg/mL, P = .03), which was associated with PD failure (P = .04). Baseline sST2 showed good performance in predicting PD failure (area under the receiver operating characteristic curve = 0.780, P = .001). In mice with chlorhexidine gluconate-induced PF, ST2 was expressed in fibroblasts and mesothelial cells within submesothelial zones. In primary cultured human peritoneal mesothelial cells (HPMCs), transforming growth factor-ß treatment increased ST2, fibronectin, ß-galactosidase and Snail protein levels and decreased E-cadherin level. Anti-ST2 antibody administration reversed the up-regulation of ST2 and fibronectin expression; it also reduced fibrosis induced by high glucose (100 mmol/L) in HPMCs. Thus, high ST2 level in dialysate is a marker for fibrosis and inflammation during peritoneal injury, and blocking ST2 may be an effective therapeutic strategy for renal preservation.

Effects of nano-hydroxyapatite on the formation of biofilms by Streptococcus mutans in two different media.

OBJECTIVES: The aim of this study was to examine the effect of nano-hydroxyapatite (nHA) on biofilm formation by Streptococcus mutans, which is actively involved in the initiation of dental caries. DESIGN: The effects of nHA on growth and biofilm formation by S. mutans were investigated in two media: a saliva analog medium, basal medium mucin (BMM); and a nutrient-rich medium, brain heart infusion (BHI); in the presence and absence of sucrose. RESULTS: Sucrose enhanced the growth of S. mutans in both media. In the presence of sucrose, nHA enhanced bacterial growth and biofilm formation more in BMM medium than in BHI. nHA also affected the transcription of glucosyltransferase (gtf) genes and production of polysaccharide differently in the two media. In BHI medium, the transcription of all three gtf genes, coding for enzymes that synthesize soluble and insoluble glucans from sucrose, was increased more than 3-fold by nHA. However, in BMM medium, only the transcription of gtfB and gtfC, coding for insoluble glucans, was substantially enhanced by nHA. CONCLUSIONS: nHA appeared to enhance biofilm formation by increasing glucosyltransferase transcription, which resulted in an increase in production of insoluble glucans. This effect was influenced by the growth conditions.