Serum CCDC25 Levels as a Potential Marker for Metabolic Syndrome.

BACKGROUND/AIM: Metabolic syndrome (MetS) stands as a significant risk for developing various severe health problems. Therefore, the discovery of biomarkers capable of predicting the progression of metabolic conditions is crucial for improving overall health outcomes. Recently, we reported that coiled-coil domain containing 25 (CCDC25) might be associated with key proteins involved in metabolic pathways, by bioinformatics analysis. Thus, we assumed that serum CCDC25 levels might have an association with MetS status. PATIENTS AND METHODS: In this study, based on the modified National Cholesterol Education Program-Adult Treatment Panel III (modified NCEP-ATP III) criteria, the participants who had three or more of abnormal criteria were defined as MetS, and those who had 1 or 2 abnormal criteria as pre-MetS groups; those who had no abnormal criteria were classified as the healthy control (HC) group. Serum CCDC25 levels were measured using the dot blot assay. RESULTS: The results showed that serum CCDC25 levels of the MetS group (0.072±0.026 ng/µl) were significantly higher (p<0.001) than that of pre-MetS (0.031±0.011 ng/µl) or HC groups (0.018±0.007 ng/µl). We can discern a consistent trend indicating that serum CCDC25 level is well correlated with the number of abnormal criteria of MetS of each participant. Although serum CCDC25 levels correlated with the distribution of all 5 MetS criteria, the highest correlation was seen in serum CCDC25 levels and triglyceride (TG) levels, with r=0.563, followed by systolic blood pressure (SBP) levels (r=0.557) and high-density lipoprotein-cholesterol (HDL-C) levels (r=-0.545). CONCLUSION: CCDC25 showed correlations with all MetS parameters, particularly with TG, SBP, and HDL-C. This prompts speculation that heightened CCDC25 levels may indicate the development and/or progression of those MetS-associated diseases.

1H NMR metabolic profiling of Staphylococcus pseudintermedius isolated from canine uroliths.

Staphylococcus pseudintermedius is a urease-producing bacteria which is a major cause of magnesium ammonium phosphate (MAP) urolithiasis in canine. A positive urolith culture is an important risk factor for MAP urolithiasis in canine. The mechanism underlying the metabolic changes of S. pseudintermedius after crystallization in artificial urine (AU) needs more defined baseline metabolic information. Therefore, we extensively investigated the metabolic changes of S. pseudintermedius extensively after crystallization in AU. A high urease activity and positive biofilm formation strain, entitled the S. pseudintermedius (SPMAP09) strain, was isolated from canine MAP uroliths, and analyzed using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics. The molecular mechanism-specific metabolic phenotypes were clearly observed after crystallization in AU at day 3. The crystals induced by SPMAP09 were also confirmed and the major chemical composition identified as struvite. Interestingly, our findings demonstrated that a total of 11 identified metabolites were significantly changed. The levels of formate, homocarnosine, tyrosine, cis-aconitate, glycolate, ethyl malonate, valine and acetate level were significantly higher, accompanied with decreased levels of inosine, glucose, and threonine at day 3 compared with the initial time-point (day 0). In addition, our results exhibited that the glyoxylate and dicarboxylate metabolism was significantly related to the SPMAP09 strain at day 3 in AU. Thus, metabolic changes of the SPMAP09 strain after crystallization in AU potentially helps to explain the preliminary molecular mechanism for the crystals induced by S. pseudintermedius.

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Rapid Detection of OXA-48-like Carbapenemase Genes in Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) possessing various carbapenemases, particularly the OXA-48 group, are now rapidly spreading and becoming a major public health concern worldwide. Phenotypic detection of OXA-48-like carbapenemases is still suboptimal due to their weak carbapenemase activity, whereas highly sensitive and specific polymerase chain reaction (PCR)-based methods take at least 3-4 h. We, therefore, developed a recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip assay for the rapid detection of blaOXA-48-like in Enterobacterales. A total of 131 clinical isolates including 61 blaOXA-48-like-carrying Enterobacterales isolates and 70 Gram-negative bacilli isolates containing other bla genes were subjected to the RPA method performed under isothermal conditions at 37 °C within 10 min and visually inspected by LF strip within 5 min. The RPA-LF assay provided 100% sensitivity (95% confidence interval, 92.6-100%) and 100% specificity (93.5-100%) for detecting blaOXA-48-like genes from bacterial colonies. Its detection limit was 100 times less than that of the PCR method. This assay is rapid, easy to perform, and provides excellent performance without any special equipment. It may be applied for directly identifying the blaOXA-48-like genes in Enterobacterales obtained from blood culture. Rapid identification of carbapenemase types is essential for selecting appropriate antimicrobial options, particularly the ß-lactams combined with novel ß-lactamase inhibitors.

Effects of Escherichia coli and Proteus mirabilis on the Growth and Aggregation of Calcium Oxalate Crystal under Microaerobic Conditions.

Escherichia coli and Proteus mirabilis are common single- and polymicrobial urinary tract infections which can survive under various oxygen levels, including inside of stone matrices. Therefore, we aimed to investigate and compare the calcium oxalate monohydrate (COM) lithogenic activities including COM crystal growth and aggregation under microaerobic conditions of E. coli and P. mirabilis isolated from the same stone matrix. The crystal growth was analyzed at the delta crystal area while the crystal aggregation was analyzed as the number of crystal aggregates. The results showed that compared to blank control, E. coli, P. mirabilis and the co-culture of E. coli and P. mirabilis were able to significantly promote COM crystal growth under microaerobic conditions. Interestingly, the delta crystal area in the co-culture under microaerobic conditions was larger than that of E. coli alone and P. mirabilis alone. In addition, only P. mirabilis alone and the co-culture were able to significantly increase COM aggregates. This study demonstrated that single- and co-culture of E. coli and P. mirabilis could promote COM crystal growth and aggregation under microaerobic conditions. The co-culture of E. coli and P. mirabilis may provide the combination effect on COM crystal interactions. The bacterial surfaces and the important effects on bacteria-crystal interactions should be further evaluated.

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.

Anti-Proteus Activity, Anti-Struvite Crystal, and Phytochemical Analysis of Sida acuta Burm. F. Ethanolic Leaf Extract.

Proteus mirabilis is a significant cause of urinary tract infection that may contribute to struvite stones. Anti-infection of this bacterium and anti-struvite formation must be considered. Sida acuta Burm. F. (SA) has been used for the treatment of diseases related to kidneys. Therefore, we investigated the effects of the SA leaf ethanolic extract (SAEE) on growth and on virulent factors (swarming motility and urease activity) of Proteusmirabilis isolated from kidney stone formers. We also evaluated anti-struvite crystal formation and phytochemical constituents of SAEE. The minimum inhibitory concentrations (MICs) of SAEE against three clinical P. mirabilis isolates were 8 mg/mL. Intriguingly, the 1/2MIC of SAEE had significant inhibitory effects on the swarming motility and urease activity of clinical P. mirabilis isolates when compared with the condition without SAEE. The SAEE at the various concentrations significantly inhibited the average weights of struvite crystals in a dose-dependent manner, compared with the control. The phytochemical analysis revealed that SAEE contained catechin, chlorogenic acid, rutin, and ferulic acid. This study indicated that SAEE has anti-P. mirabilis and anti-struvite crystal activities via its bioactive compounds. For this reason, SAEE may be developed as a new agent for the treatment of struvite stone induced by P. mirabilis.

Risk factors for canine magnesium ammonium phosphate urolithiasis associated with bacterial infection.

BACKGROUND: With limited information available, the association among urinary tract infections, urease-producing bacteria and the presence of magnesium ammonium phosphate (MAP) urolithiasis in canines in Thailand requires more study. OBJECTIVES: This study aimed to investigate the association between demographic characteristics of canines and the presence of MAP urolithiasis in canines, and to evaluate antimicrobial susceptibility patterns of bacteria isolated from canine uroliths. METHODS: A total of 56 canines admitted for treatment with surgical removal of uroliths were recruited. Demographic characteristics and clinical chemistry data were recorded. Bacteria isolated from the removed uroliths were identified. Chemical compositions of the uroliths were analyzed by Fourier transform infrared spectrometer. Potential risk factors were determined with univariable and multivariable logistic regression analyses. RESULTS: Of 56 canine urolithiasis, bacteria were isolated from uroliths of 38 canines (27 MAP and 11 non-MAP) but not from uroliths of 18 canines (5 MAP and 13 non-MAP). The most common bacteria found in nidus of MAP uroliths was Staphylococcus pseudintermedius (approximately 51%). An antimicrobial resistance was frequently found in Staphylococci isolates (42.86%). Multivariate logistic regression analysis showed that the predictors of MAP urolith in canine urolithiasis were being female (p = 0.044; adjusted odds ratio [OR], 10.22; 95% confidence interval [CI], 1.06-98.24) and the positive urolith culture (p = 0.012; adjusted OR, 8.60; 95% CI, 1.60-46.30). CONCLUSIONS: Our results indicate that S. pseudintermedius (a urease-producing bacterium) is the major causative bacteria of MAP uroliths. A positive urolith culture and being female are risk factors of MAP urolithiasis in canines.

Rapid detection of Enterococcus and vancomycin resistance using recombinase polymerase amplification.

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

Inhibition of Bacterial Efflux Pumps by Crude Extracts and Essential Oil from Myristica fragrans Houtt. (Nutmeg) Seeds against Methicillin-Resistant Staphylococcus aureus.

Myristicafragrans Houtt. (Nutmeg) is a widely known folk medicine across several parts of Asia, particularly used in antimicrobial treatment. Bacterial resistance involves the expression of efflux pump systems (chromosomal norA and mepA) in methicillin-resistant Staphylococcus aureus (MRSA). Crude extract (CE) and essential oil (EO) obtained from nutmeg were applied as efflux pump inhibitors (EPIs), thereby enhancing the antimicrobial activity of the drugs they were used in. The major substances in CE and EO, which function as EPIs, in a descending order of % peak area include elemicin, myristicin, methoxyeugenol, myristicin, and asarone. Here, we investigated whether the low amount of CE and EO used as EPIs was sufficient to sensitize MRSA killing using the antibiotic ciprofloxacin, which acts as an efflux system. Interestingly, synergy between ciprofloxacin and CE or EO revealed the most significant viability of MRSA, depending on norA and mepA, the latter being responsible for EPI function of EO. Therefore, CE and EO obtained from nutmeg can act as EPIs in combination with substances that act as efflux systems, thereby ensuring that the MRSA strain is susceptible to antibiotic treatment.

Synergistic effect of vancomycin combined with cefotaxime, imipenem, or meropenem against Staphylococcus aureus with reduced susceptibility to vancomycin

Background/aim: We investigated the synergistic effect between vancomycin and ß-lactams against vancomycin-susceptible (VSSA) and nonsusceptible MRSA isolates [heterogeneous vancomycin-intermediate S. aureus (hVISA) and VISA]. Materials and methods: A total of 29 MRSA, including 6 VISA, 14 hVISA, and 9 VSSA isolates, were subjected to a microbroth dilu- tion-minimum inhibitory concentration (MIC) checkerboard using vancomycin combined with cefotaxime, imipenem, or meropenem. To confirm synergistic activity, the representative strains of VISA, hVISA, and VSSA were then selected for the time-kill curve method. Results: The combination of vancomycin with imipenem, meropenem, or cefotaxime exhibited synergistic effects against 17 (2 VISA, 9 hVISA, and 6 VSSA), 14 (3 VISA, 9 hVISA and 2 VSSA), and 5 (3 VISA and 2 hVISA) isolates, respectively. Additive and indifferent effects were found in the remaining isolates, but no antagonistic effect was observed. Using time-kill assay, the vancomycin combined with either imipenem or cefotaxime demonstrated synergism against both VISA and hVISA isolates, while the synergistic effect with meropenem was obtained only in the VISA isolates. Conclusion: This study demonstrated in vitro enhanced antibacterial activity of vancomycin plus ß-lactams against clinical hVISA or VISA isolates. These combinations may be an alternative treatment for MRSA infections in clinical practice.

Anti-metastatic Effects of Cationic KT2 Peptide (a Lysine/Tryptophan-rich Peptide) on Human Melanoma A375.S2 Cells.

BACKGROUND/AIM: KT2 is a lysine/tryptophan-rich peptide modified from Crocodylus siamensis Leucrocin I. In this study, we examined the cell toxicity, cellular uptake, anti-migration and anti-invasion activities of KT2 in A375.S2 human melanoma cells. MATERIALS AND METHODS: A375.S2 cells were treated with KT2 peptide and then we performed MTT assay, study of cellular uptake by a confocal microscope, wound healing assay, transwell migration/invasion assay, and evaluation of the expression of metastasis-associated proteins. RESULTS: KT2 can be internalized through the plasma membrane and can slightly alter cell morphology, decrease the percentage of viable cells and inhibit cell migration and invasion of A375.S2 cells in a dose-dependent manner. This peptide suppressed MMP-2 activity, as measured by gelatine zymography assay. The protein level of MMP-2 was decreased by KT2. KT2 also down-regulated metastasis pathway-related molecules, including FAK, RhoA, ROCK1, GRB2, SOS-1, p-JNK, p-c-Jun, PI3K, p-AKT (Thr308), p-AKT (Ser473), p-p38, MMP-9, NF-kB, and uPA. CONCLUSION: These results indicate that KT2 inhibits the migration and invasion of human melanoma cells by decreasing MMP-2 and MMP-9 expression through inhibition of FAK, uPA, MAPK, PI3K/AKT NF-kB, and RhoA-ROCK signalling pathways. These findings suggest that KT2 deserves further investigation as an anti-metastatic agent for human melanoma.

PmrB mutations including a novel 10-amino acid repeat sequence insertion associated with low-level colistin resistance in carbapenem-resistant Acinetobacter baumannii.

The global emergence of colistin resistance in carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates is a serious public health concern. We therefore aimed to investigate colistin resistance mechanisms in 5 colistin-resistant (COL-R) CRAB isolates collected from Thai patients in 2016 by whole genome sequencing (WGS) compared with those of 5 colistin-intermediate (COL-I) CRAB isolates from the same period. All isolates were subjected to antimicrobial susceptibility testing, efflux pump inhibitor-based test and WGS. Mutations in known genes associated with colistin resistance were analyzed and deleterious mutations were then predicted by PROVEAN tool. The 10 CRAB isolates carried blaOXA-23 with the addition of blaOXA-58 in 1 isolate. All COL-R isolates exhibited colistin MICs of 4 µg/mL except for 1 isolate with that of 16 µg/mL. They belonged to ST2, ST16, ST23, ST164 and ST215, whereas the COL-I isolates with colistin MICs of ≤0.25-1 µg/mL were ST2, ST164 and ST215. Neither increased efflux pump activity nor mcr gene was found in any COL-R isolate. Three COL-R isolates contained different PmrB variants: a novel 10-amino acid (aa) repeat sequence insertion, VILGCILIFS between positions 27 and 28 (S27_A28insVILGCILIFS) in transmembrane domain (TM); a 1-aa insertion, alanine between positions 162 and 163 (A162_I163insA) in TM; and a 1-aa substitution, A226T in histidine kinase domain. One COL-R isolate possessed PmrA variant with A80V substitution. These alterations were predicted as deleterious. Mechanisms of colistin resistance in the remaining COL-R isolate were still unknown. In conclusion, the alterations in both PmrB and PmrA were predicted and suggested as initial mutations responsible for low-level colistin resistance in our CRAB isolates. Under selective pressure, these isolates may exhibit higher level colistin resistance by the additional mutations, leading to more therapeutic difficulties.

Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein A in Positive Blood Culture Samples.

Bloodstream infection (BSI) is a major cause of mortality in hospitalized patients worldwide. Staphylococcus aureus is one of the most common pathogens found in BSI. The conventional workflow is time consuming. Therefore, we developed a lateral flow immunoassay (LFIA) for rapid detection of S. aureus-protein A in positive blood culture samples. A total of 90 clinical isolates including 58 S. aureus and 32 non-S. aureus were spiked in simulated blood samples. The antigens were extracted by a simple boiling method and diluted before being tested using the developed LFIA strips. The results were readable by naked eye within 15 min. The sensitivity of the developed LFIA was 87.9% (51/58) and the specificity was 93.8% (30/32). When bacterial colonies were used in the test, the LFIA provided higher sensitivity and specificity (94.8% and 100%, respectively). The detection limit of the LFIA was 107 CFU/mL. Initial evaluation of the LFIA in 20 positive blood culture bottles from hospitals showed 95% agreement with the routine methods. The LFIA is a rapid, simple and highly sensitive method. No sophisticated equipment is required. It has potential for routine detection particularly in low resource settings, contributing an early diagnosis that facilitates effective treatment and reduces disease progression.

Direct detection of methicillin-resistant in Staphylococcus spp. in positive blood culture by isothermal recombinase polymerase amplification combined with lateral flow dipstick assay.

Methicillin-resistant staphylococci (MRS) are important antimicrobial-resistant pathogens in sepsis. Conventional blood cultures take 24-72 h. The polymerase chain reaction (PCR)-based methods give faster results (2-3 h) but need expensive thermal cyclers. We therefore developed an isothermal recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) assay for rapid detection of MRS in spiked blood culture samples. Fifty-six clinical isolates including 38 mecA-carrying staphylococci and 18 non-mecA-carrying organisms as confirmed by PCR methods were studied. RPA primer set and probe specific for mecA gene (encoding penicillin-binding protein 2a) were designed. RPA reaction was carried out under isothermal condition (45 °C) within 20 min and read by LFD in 5 min. The RPA-LFD provided 92.1% (35/38) sensitivity for identifying MRS in positive blood culture samples, and no cross-amplification was found (100% specificity). This test failed to detect three mecA-carrying S.sciuri isolates. The detection limits of RPA-LFD method for identifying MRS were equal to those of PCR method. The RPA-LFD is simple, fast, and user-friendly. This method could detect the mecA gene directly from the positive blood culture samples without requirement for special equipment. This method would be useful for appropriate antibiotic therapy and infection control, particularly in a low-resource setting.

Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital.

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5-2 µg/mL, with MIC50 and MIC90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3-8 µg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC50 and MIC90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried blaOXA-23-like. The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

Analysis of xanthyletin and secondary metabolites from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 against Pythium insidiosum.

BACKGROUND: Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy. RESULTS: Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57-1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups. CONCLUSIONS: Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.

Elongation factor Tu on Escherichia coli isolated from urine of kidney stone patients promotes calcium oxalate crystal growth and aggregation.

Escherichia coli is the most common bacterium isolated from urine and stone matrix of calcium oxalate (CaOx) stone formers. Whether it has pathogenic role(s) in kidney stone formation or is only entrapped inside the stone remains unclear. We thus evaluated differences between E. coli isolated from urine of patients with kidney stone (EUK) and that from patients with urinary tract infection (UTI) without stone (EUU). From 100 stone formers and 200 UTI patients, only four pairs of EUK/EUU isolates had identical antimicrobial susceptibility patterns. Proteomic analysis revealed nine common differentially expressed proteins. Among these, the greater level of elongation factor Tu (EF-Tu) in EUK was validated by Western blotting. Outer membrane vesicles (OMVs) derived from EUK had greater promoting activities on CaOx crystallization, crystal growth and aggregation as compared to those derived from EUU. Neutralizing the OMVs of EUK with monoclonal anti-EF-Tu antibody, not with an isotype antibody, significantly reduced all these OMVs-induced promoting effects. Moreover, immunofluorescence staining of EF-Tu on bacterial cell surface confirmed the greater expression of surface EF-Tu on EUK (vs. EUU). Our data indicate that surface EF-Tu and OMVs play significant roles in promoting activities of E. coli on CaOx crystallization, crystal growth and aggregation.

Differential colony size, cell length, and cellular proteome of Escherichia coli isolated from urine vs. stone nidus of kidney stone patients.

BACKGROUND: Escherichia coli is associated with kidney stone disease, as a cause or an effect (secondary or recurrent urinary tract infection, UTI). Defining phenotypic or functional differences between E. coli inside stone nidus (ECS, associated with infection-induced stone) and outside the stone (i.e. from urine) (ECU, represented secondary infection) would be helpful to better understand bacterial involvement in this disease. METHODS: ECS and ECU were isolated from 100 stone formers and subjected to antimicrobial susceptibility test, ERIC-PCR genotyping, determination of biofilm formation, bacterial colony size on agar plate and cell length in broth, 2-DE, nanoLC-MS/MS, protein network analysis, and pyruvate dehydrogenase (PDH) activity assay. RESULTS: From 100 stone formers, 36 had positive bacterial culture, of which 5 pairs had identical antimicrobial susceptibility patterns and comparable ERIC-PCR genotypes. ECS had smaller colony size and longer cell length than ECU. 2-DE proteomic analysis revealed significantly differential levels of proteins involved in carbohydrate metabolism, stress response, and RNA/protein metabolism. Functional validation demonstrated lower PDH activity in ECS. CONCLUSIONS: All these differential phenotypic and cellular proteome findings might be adaptive response of E. coli from remote infection to survive within the stone matrix that subsequently caused recurrent UTI in kidney stone patients.

Phenotypic characteristics and comparative proteomics of Staphylococcus aureus strains with different vancomycin-resistance levels.

Reduced vancomycin susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide problem. Unfortunately, its genetic marker and molecular mechanisms remained unknown. This study investigated differential phenotypic characteristic and protein expression profiles among three groups of MRSA isolates, including vancomycin-susceptible S. aureus (VSSA), heterogeneous vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) (n = 7 isolates/group). Phenotypic characteristic revealed significant greater number of isolates with non-spreading colony in VISA as compared to both VSSA and hVISA groups. 2-DE followed by nanoLC-MS/MS analyses revealed increased glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both hVISA and VISA, whereas 50S ribosomal protein L14 (RplN) and DNA-binding protein II (Hup) were increased only in VISA. The non-spreading colony and GAPDH level of MRSA may be used as the markers for differentiation of VSSA, hVISA and VISA.

Salivary Myeloperoxidase, Assessed by 3,3'-Diaminobenzidine Colorimetry, Can Differentiate Periodontal Patients from Nonperiodontal Subjects.

Periodontal diseases, which result from inflammation of tooth supporting tissues, are highly prevalent worldwide. Myeloperoxidase (MPO), from certain white blood cells in saliva, is a biomarker for inflammation. We report our study on the salivary MPO activity and its association with severity of periodontal diseases among Thai patients. Periodontally healthy subjects (n = 11) and gingivitis (n = 32) and periodontitis patients (n = 19) were enrolled. Assessments of clinically periodontal parameters were reported as percentages for gingival bleeding index (GI) and bleeding on probing (BOP), whereas pocket depth (PD) and clinical attachment loss (CAL) were measured in millimeters and then made to index scores. Salivary MPO activity was measured by colorimetry using 3,3'-diaminobenzidine as substrate. The results showed that salivary MPO activity in periodontitis patients was significantly higher than in healthy subjects (p = 0.003) and higher than in gingivitis patients (p = 0.059). No difference was found between gingivitis and healthy groups (p = 0.181). Significant correlations were observed (p < 0.01) between salivary MPO activity and GI (r = 0.632, p < 0.001), BOP (r = 0.599, p < 0.001), PD (r = 0.179, p = 0.164), and CAL (r = 0.357, p = 0.004) index scores. Sensitivity (94.12%), specificity (54.55%), and positive (90.57%) and negative (66.67%) predictive values indicate that salivary MPO activity has potential use as a screening marker for oral health of the Thai community.