Selective pks+ Escherichia coli strains induce cell cycle arrest and apoptosis in colon cancer cell line.

pks+ Escherichia coli (E. coli) triggers genomic instability in normal colon cells which leads to colorectal cancer (CRC) tumorigenesis. Previously, we reported a significant presentation of pks+ E. coli strains in CRC patients' biopsies as compared to healthy cohorts. In this work, using an in vitro infection model, we further explored the ability of these strains in modulating cell cycle arrest and activation of apoptotic mediators in both primary colon epithelial cells (PCE) and CRC cells (HCT-116). Sixteen strains, of which eight tumours and the matching non-malignant tissues, respectively, from eight pks+ E. coli CRC patients were subjected to BrDU staining and cell cycle analysis via flow cytometry, while a subset of these strains underwent analysis of apoptotic mediators including caspase proteins, cellular reactive oxygen species (cROS) and mitochondrial membrane potential (MMP) via spectrophotometry as well as proinflammatory cytokines via flow cytometry. Data revealed that all strains exerted S-phase cell cycle blockade in both cells and G2/M phase in PCE cells only. Moreover, more significant upregulation of Caspase 9, cROS, proinflammatory cytokines and prominent downregulation of MMP were detected in HCT-116 cells indicating the potential role of pks related bacterial toxin as anticancer agent as compared to PCE cells which undergo cellular senescence leading to cell death without apparent upregulation of apoptotic mediators. These findings suggest the existence of discrepancies underlying the mechanism of action of pks+ E. coli on both cancer and normal cell lines. This work propounds the rationale to further understand the mechanism underlying pks+ E. coli-mediated CRC tumorigenesis and cancer killing.

Release of pro-inflammatory cytokines TNF-α, IFN-γ and IL-6 by Burkholderia pseudomallei- stimulated peripheral blood mononucleocytes of acute myeloid leukemia patients.

Acute myeloid leukemia (AML) is a malignant disease progressed from abnormal production of immature myeloid cells, which is often associated with concurrent infections after diagnosis. It was widely established that infections are the major contributors to mortality in this group due to the prevalency of neutropenia. Gram-negative Burkholderia pseudomallei is the causative agent of melioidosis. This disease had been reported in several neutropenic cancer patients undergoing chemotherapy resulting in severe clinical presentations and high mortalities which is in need of critical attention. Studies show that cytokines are important mediators of melioidosis progression and low neutrophil counts are associated with progression of its severity. However, to date, there are no reports on cytokine production in neutropenic cancer patients who are prone to melioidosis. Hence, here we assessed the cytokine production in neutropenic AML patients by introducing B. pseudomallei to their peripheral blood mononuclear cell (PBMC) culture in vitro. We observed that inflammatory response related cytokines namely TNF-α, IFN-γ IL-6 and IL-10 were highly circulated in infected PBMCs suggesting that these cytokines may play important roles in the progression of severity in melioidosis infected neutropenic patients.

Antibacterial biocompatible arginine functionalized mono-layer graphene: No more risk of silver toxicity.

Antibacterial ability is vital in biological approaches as well as functional biomaterials. Besides, cytocompatibility aspect of biologic media, tissue and organs is always concern for appropriate synthesis. From the past, metallic/oxide phases of silver (Ag) material in various macro, micro or nano configurations have been widely used for antibacterial targets. While, background of Ag toxicity within particle, film and composites is posing gradual ion release affected by molecular bounding. Recent researches conducted to control, optimize and neutralize Ag limitations finding the benefits of ideal (∼ 100%) mediation against both Gram-negative and Gram-positive bacteria. Whereas, non-degradable releases history is still a challenge and its longer accumulation may cause to disrupt biostructures and disease risk. Thus, facile development of large-area organic materials with switchable bacteria toxicity and normal cell compatibility function is interesting for concerned approaches. Here, smart positively-charged stable arginine amino acid incorporated mono layer graphene (Arg-EMGr) nanobiocomposite introduced as useful antibacterial and safe bactericidal agent competitive with Ag direct. The immunity characteristic versus Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) comparably assessed with graphene oxide (GO) and different concentrations GO-AgNPs morphology. As cell viability matter, 1,3,5,7-days vitro culture assay shown attachment proliferation and cytotoxicity due to short interaction.

Toward improved mechanical, tribological, corrosion and in-vitro bioactivity properties of mixed oxide nanotubes on Ti-6Al-7Nb implant using multi-objective PSO.

Recently, the robust optimization and prediction models have been highly noticed in district of surface engineering and coating techniques to obtain the highest possible output values through least trial and error experiments. Besides, due to necessity of finding the optimum value of dependent variables, the multi-objective metaheuristic models have been proposed to optimize various processes. Herein, oriented mixed oxide nanotubular arrays were grown on Ti-6Al-7Nb (Ti67) implant using physical vapor deposition magnetron sputtering (PVDMS) designed by Taguchi and following electrochemical anodization. The obtained adhesion strength and hardness of Ti67/Nb were modeled by particle swarm optimization (PSO) to predict the outputs performance. According to developed models, multi-objective PSO (MOPSO) run aimed at finding PVDMS inputs to maximize current outputs simultaneously. The provided sputtering parameters were applied as validation experiment and resulted in higher adhesion strength and hardness of interfaced layer with Ti67. The as-deposited Nb layer before and after optimization were anodized in fluoride-base electrolyte for 300min. To crystallize the coatings, the anodically grown mixed oxide TiO2-Nb2O5-Al2O3 nanotubes were annealed at 440°C for 30min. From the FESEM observations, the optimized adhesive Nb interlayer led to further homogeneity of mixed nanotube arrays. As a result of this surface modification, the anodized sample after annealing showed the highest mechanical, tribological, corrosion resistant and in-vitro bioactivity properties, where a thick bone-like apatite layer was formed on the mixed oxide nanotubes surface within 10 days immersion in simulated body fluid (SBF) after applied MOPSO. The novel results of this study can be effective in optimizing a variety of the surface properties of the nanostructured implants.

Molecular analysis of vibrio cholerae strains isolated in Malaysia: public health implications.

The genetic diversity or clonality among Vibrio cholerae O1, O139 and non-O1/ non-O139 of clinical and environmental origin using ribotyping and PFGE was performed in order to ascertain the public health implications of the different genotypes circulating within the Malaysian environment. Using an in-house typing scheme, of the 214 strains included, 202 strains were isolated locally between 1992 and 1998, seven were obtained from Bangladesh and five were reference strains. Amongst the 176 El Tor O1 strains, 152 clinical strains demonstrated five ribotypes--E1a, E1b, E2a, E3 and E1c. E1b was the most predominant ribotype demonstrated by 84% of the El Tor O1 strains and was present in all years demonstrating that this strain was intrinsic to Malaysia. PFGE analysis of these strains demonstrated minimal variation amongst the 15 PFGE profiles obtained. Ribotpye E2a amongst five clinical and two environmental O1 strains, were from one location and had previously been reported in Indonesia and the Philippines, thus demonstrating strong evidence that these strains may have been imported into Malaysia. Among Vibrio cholerae O139 strains, 91.7% were of ribotype A1a similar to the original O139, while two others were of ribotype A1b and one of A1e, corresponding to ribotypes 1, 2 and 3 of Dalsgaard and colleagues' scheme for O139 strains. PFGE analysis demonstrated that 89% of ribotype A1a could be differentiated into three PFGE genotypes which were very closely related. The eight non-O1/non-O139 serogroup strains were heterogeneous in both ribotype and PFGE patterns.

GEP-based method to formulate adhesion strength and hardness of Nb PVD coated on Ti-6Al-7Nb aimed at developing mixed oxide nanotubular arrays.

PVD process as a thin film coating method is highly applicable for both metallic and ceramic materials, which is faced with the necessity of choosing the correct parameters to achieve optimal results. In the present study, a GEP-based model for the first time was proposed as a safe and accurate method to predict the adhesion strength and hardness of the Nb PVD coated aimed at growing the mixed oxide nanotubular arrays on Ti67. Here, the training and testing analysis were executed for both adhesion strength and hardness. The optimum parameter combination for the scratch adhesion strength and micro hardness was determined by the maximum mean S/N ratio, which was 350W, 20 sccm, and a DC bias of 90V. Results showed that the values calculated in the training and testing in GEP model were very close to the actual experiments designed by Taguchi. The as-sputtered Nb coating with highest adhesion strength and microhardness was electrochemically anodized at 20V for 4h. From the FESEM images and EDS results of the annealed sample, a thick layer of bone-like apatite was formed on the sample surface after soaking in SBF for 10 days, which can be connected to the development of a highly ordered nanotube arrays. This novel approach provides an outline for the future design of nanostructured coatings for a wide range of applications.

Dormant phages of Helicobacter pylori reveal distinct populations in Europe.

Prophages of Helicobacter pylori, a bacterium known to co-evolve in the stomach of its human host, were recently identified. However, their role in the diversity of H. pylori strains is unknown. We demonstrate here and for the first time that the diversity of the prophage genes offers the ability to distinguish between European populations, and that H. pylori prophages and their host bacteria share a complex evolutionary history. By comparing the phylogenetic trees of two prophage genes (integrase and holin) and the multilocus sequence typing (MLST)-based data obtained for seven housekeeping genes, we observed that the majority of the strains belong to the same phylogeographic group in both trees. Furthermore, we found that the Bayesian analysis of the population structure of the prophage genes identified two H. pylori European populations, hpNEurope and hpSWEurope, while the MLST sequences identified one European population, hpEurope. The population structure analysis of H. pylori prophages was even more discriminative than the traditional MLST-based method for the European population. Prophages are new players to be considered not only to show the diversity of H. pylori strains but also to more sharply define human populations.

Diagnostic and prognostic value of an immunofluorescent assay for melioidosis.

Melioidosis caused by Burkholderia pseudomallei is endemic in southeast Asia. The clinical manifestations range from wound infections to acute septicemia. In some cases, recurrence can also occur following complete recovery. Case fatality rates are high and a major factor is the delay in the culture and identification of the bacterium. An immunofluorescent assay (IFAT) using whole-cell antigen for the detection of total antibodies to B. pseudomallei was tested with 650 sera. Using a cut-off value of 1:80, 66 sera from culture-confirmed cases were positive with titers > or = 320. In another 523 sera from patients in which no other etiology could be found, 149 (23.4%) were positive. To monitor disease activity, persistence of antibody levels was investigated on 61 serial sera samples collected from 14 other confirmed cases on follow-up visits while on oral maintenance therapy. The IFAT demonstrated a reduction in titers in cases of localized infections, suggesting that either the infection was being resolved or arrested while septicemic patients maintained high IFAT titers on follow-up, suggesting the possibility of continuous sequestration of antigen from an intracellular source.

Short report: Electron microscopic demonstration of extracellular structure of Burkholderia pseudomallei.

The occurrence of latency and relapse in human melioidosis suggests adaptations by Burkholderia pseudomallei that help to avoid the human immune response. Ruthenium red-stained preparations of bacterial cultures viewed by electron microscopy revealed three morphologically distinct variants; one with a very marked and another with a less electron-dense layer surrounding the cell wall, and a third variety devoid of such a structure. This structure may be attributable to a layer of polysaccharide, suggesting the presence of a glycocalyx that may aid in the survival of the organism during latency.

Detection of immunoglobulins M and G using culture filtrate antigen of Burkholderia pseudomallei.

IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.

Exotoxin profiles of clinical isolates of Aeromonas hydrophila.

Eighty-six clinical isolates of Aeromonas hydrophila were studied for their ability to produce four exotoxins: a haemolysin active against rabbit erythrocytes, cytotoxin and enterotoxin detectable with Vero cell cultures, and the cholera toxin-like factor detected by an enzyme-linked immunosorbent assay. At least one exotoxin was produced by 80% of enteric and 96% of non-enteric isolates. The exotoxin profiles of non-enteric isolates were more restricted than those of enteric isolates, with haemolysin and cytotoxin producers preponderant. Although haemolysin and cytotoxin were produced by isolates from all sources, the enterotoxin and cholera toxin-like factor were more common amongst enteric isolates. The production of haemolysin and cytotoxin were closely related but the association between the enterotoxin and the cholera toxin-like factor was not significant.

Distribution of immunoglobulin classes and IgG subclasses against a culture filtrate antigen of Burkholderia pseudomallei in melioidosis patients.

The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups. The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups. However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7. The IgA response was intermediate with GMTIs ranging from 119.2 to 170. The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections. As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts. None of the sera from the control groups had any significant titres of antibodies.

Possible virulence factors involved in bacteraemia caused by Aeromonas hydrophila.

Eighteen strains of Aeromonas hydrophila from patients with bacteraemia were investigated for possible virulence factors. Cytotoxin and haemolysin were produced by all strains, whereas cholera toxin-like factor was produced by 33% of strains only. Enterotoxin production was not detected. Haemagglutination of guinea-pig, fowl and rabbit erythrocytes was demonstrated by 83%, 67% and 61% of strains, respectively. Fucose- and mannose-sensitive haemagglutinins were predominant. None of the strains agglutinated sheep erythrocytes. Extrachromosomal DNA was detected in 17 strains, 16 of which had a plasmid (3.6-5.1 MDa), the majority being between 4.6 and 5.1 MDa.

Comparison of five assays for the heat-labile enterotoxin of Escherichia coli.

Enzyme-linked immunosorbent assay (ELISA), DNA-DNA hybridisation, Vero cell assay, the Biken test and a new membrane-filter method were compared in the detection of heat-labile enterotoxin (LT) of Escherichia coli. Six subcultures of each of 50 strains of E. coli from the Biken collection were evaluated "blind" in the laboratory. The combined results of the most reproducible tests (ELISA and DNA-DNA hybridisation) were used to calculate the sensitivity and specificity of the other assays. The Vero-cell assay had a high sensitivity (98%) but a lower specificity (91%). The Biken and membrane-filter assays had sensitivities of 58-71% and 77-84% respectively, depending on the type of antiserum used. Only one false positive result was obtained with the Biken test; specificity of the membrane-filter assay was 94-95%. The membrane-filter assay, with anti-cholera toxin, is specific and reasonably sensitive. It has particular advantages over DNA-DNA hybridisation and the Biken test, and it may prove suitable for screening large numbers of E. coli isolates in epidemiological studies in developing countries.

Ribotyping and DNA macrorestriction analysis of isolates of Burkholderia pseudomallei from cases of melioidosis in Malaysia.

Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which accounted for 90% of all isolates. PFGE revealed a number of different strains within these ribotypes, but some pairs of isolates from unrelated cases gave closely similar DNA profiles. These results are in agreement with Australian studies which showed a high prevalence of a few ribotypes of B. pseudomallei which are further divisible by genotyping, in areas where melioidosis is endemic.

An immunohistochemical method for the diagnosis of melioidosis.

In order to assess the usefulness of immunohistochemistry in the diagnosis of melioidosis, an infection by Burkholderia pseudomallei, polyclonal antibodies were applied to tissues from known cases of melioidosis and to other infected tissues. Formalin-fixed, paraffin-embedded tissues were stained by a modified immunoperoxidase technique. In autopsy tissues with inflammatory lesions of melioidosis, the cytoplasm of phagocytes and intact bacilli, both intra- and extracellular, were stained very strongly positive. Relatively more focal positive staining was observed in some but not all surgical biopsies from proven cases of melioidosis. In granulomas staining was mainly found in the central necrotic areas, with little staining of peripheral phagocytes. All control materials stained negative. Immunohistochemistry appears to be a useful diagnostic tool in melioidosis.

Epidemiology and molecular characterization of nosocomially transmitted multidrug-resistant Klebsiella pneumoniae.

OBJECTIVES: To describe the epidemiology, antimicrobial susceptibility, genomic profiles, and control of a nosocomial outbreak of multidrug-resistant Klebsiella pneumoniae (MRKP) that occurred in the pediatric oncology unit of the University of Malaya Medical Centre in Kuala Lumpur. MATERIALS AND METHODS: A prospective epidemiologic and microbiologic study was conducted of MRKP isolated from the blood and wound of a boy with necrotizing fasciitis after a 7-day course of ceftazidime and amikacin. In the following 2 weeks, phenotypically similar MRKP were isolated from the blood cultures of four other patients and rectal swabs of another three patients and two liquid soap samples located in the same ward. RESULTS: Antimicrobial profiles demonstrated that all the isolates were resistant to ceftazidime, sensitive to imipenem and ciprofloxacin, and confirmed to be extended-spectrum beta-lactamase producers. Plasmids of varying molecular weights were present in all isolates. In eight of these isolates, which included four from blood, there were common large molecular weight plasmids ranging from 80 kb to 100 kb. Pulsed-field gel electrophoresis analysis using XbaI demonstrated six different DNA profiles, A to F. Profile A was shared by two blood culture isolates and were related by 91%. Profile B was found in one rectal swab isolate and one isolate from liquid soap and were related by 94%. Profile C was shared by one blood isolate and one liquid soap isolate and showed 100% relatedness. Profiles D, E, and F each were demonstrated by one blood isolate and two rectal swab isolates, respectively. These showed only 65% relatedness. CONCLUSIONS: The MRKP strains in this outbreak were not clonal in origin. The decline of the outbreak after 4 weeks was attributed to the reemphasis of standard infection control procedures and the implementation of a program that addressed sites of environmental contamination.

Colonisation factors amongst clinical isolates of enterotoxigenic Escherichia coli.

The production of heat-labile (LT) and heat-stable (ST) enterotoxins, colonisation factor antigens (CFAs) and haemagglutinins was investigated amongst 310 Escherichia coli (E. coli) isolates obtained from 62 children under the age of five, with diarrhoea. Twenty-one isolates were found to produce enterotoxins, of which fifteen (71%) isolates produced ST only, 2 (10%) produced LT only and 4 (19%) produced both LT and ST. However, none of the isolates demonstrated any of the common CFAs identified to date, but 8 out of the 21 isolates demonstrated haemagglutination with rabbit, sheep or human group A erythrocytes, suggesting the presence of putative CFAs, yet unidentified.

Comparison of two assays for the detection of haemolysins of Aeromonas species.

The haemolysins produced by Aeromonas species were detected and compared by two assay methods--a modified blood agar plate assay and the rabbit erythrocyte haemolysin method. Both assays showed a high level of agreement (86%). The titres of the rabbit erythrocyte haemolysin assay correlated with the haemolytic zone diameter of the ox blood agar assay. In addition the agar haemolysin assay had simple media requirements, was easy to perform and results were well defined.

Acute respiratory failure in melioidosis.

BACKGROUND: In melioidosis caused by Burkholderia pseudomallei, although every organ in the body may be involved, the highest mortality of 73% occurs when the respiratory system is affected. These patients invariably die of acute respiratory failure. Most of them also have underlying predisposing factors like diabetes mellitus. AIM OF STUDY: A retrospective study of six such cases was carried out in order to elicit the possible causes and mechanisms of acute respiratory failure in patients with melioidosis. METHOD: Patients' records were reviewed for demographic, clinical, laboratory, radiological and histopathological data. RESULTS: The rapidity of onset of respiratory failure was remarkable and was accompanied by relentless hypoxaemia that was refractory to treatment despite the application of high positive end expiratory pressure and other supportive measures. All had bilateral opacities on frontal chest radiographs, focal and diffuse necrotizing pneumonia and presence of hyaline membranes in lung tissues seen histologically, supporting the accepted criteria for ALI/ARDS. CONCLUSION: Patients with sepsis due to B. pseudomallei develop ALI/ARDS very rapidly resulting in high mortality rates. Possible mechanisms involved are discussed. Awareness of the disease in endemic areas, the development of rapid diagnostic methods and appropriate management procedures are urgently needed for the prevention of ARDS and subsequent reduction in mortality in such cases.