Biological characteristics associated with virulence in Clostridioides difficile ribotype 002 in Hong Kong.

Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea and can sometimes lead to pseudo-membranous colitis and toxic megacolon. We previously reported that the PCR ribotype 002 was a common C. difficile ribotype in Hong Kong that was associated with increased mortality. In this study, we assessed in vitro bacteriological characteristics and in vivo virulence of ribotype 002 compared to other common ribotypes, including ribotypes 012, 014 and 046. We observed significantly higher toxin A (p < 0.05) and toxin B (p < 0.05) production, sporulation (p < 0.001) and germination rates (p < 0.0001) in ribotype 002 than other common ribotypes. In a murine model of C. difficile infection, ribotype 002 caused significantly more weight loss (p < 0.001) and histological damage (p < 0.001) than other common ribotypes. These findings may have contributed to the higher prevalence and mortality observed, and provided mechanistic insights that can help public surveillance and develop novel therapeutics to combat against this infection.

Clostridium difficile toxin B induces autophagic cell death in colonocytes.

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3-II, formation of LC3+ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB-induced autophagy was also accompanied by the repression of phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy-related genes Beclin 1, Atg5 and Atg7 attenuated TcdB-induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro-death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.

Surveillance of antibiotic resistance among common Clostridium difficile ribotypes in Hong Kong.

Incidence of Clostridium difficile infection (CDI) is rapidly increasing and it poses a major health burden globally. However, data regarding the epidemiology of CDI in Asia are limited. We aimed to characterize the antimicrobial susceptibility patterns of common ribotypes of toxigenic C. difficile in Hong Kong. Fifty-three PCR ribotypes were identified among 284 toxigenic C. difficile clinical isolates. The five most prevalent ribotypes were 002 (13%), 017 (12%), 014 (10%), 012 (9.2%), and 020 (9.5%). All tested C. difficile strains remained susceptible to metronidazole, vancomycin, meropenem and piperacillin/tazobactam, but highly resistant to cephalosporins. Of the fluoroquinolones, highest resistance to ciprofloxacin was observed (99%), followed by levofloxacin (43%) and moxifloxacin (23%). The two newly emerged PCR ribotypes, 017 and 002, demonstrated high levels of co-resistance towards clindamycin, tetracycline, erythromycin and moxifloxacin. PCR ribotypes 017 and 002 with multi-drug resistance are rapidly emerging and continuous surveillance is important to monitor the epidemiology of C. difficile to prevent outbreaks of CDI.

Unraveling methylation changes of host macrophages in Mycobacterium tuberculosis infection.

OBJECTIVES: To characterize at high resolution DNA methylation changes of cytokines which occur in the genome of macrophages in association with Mycobacterium tuberculosis (MTB) infection METHODS: We studied the methylation profiles of THP-1 derived macrophage cells infected with clinical MTB strains [Beijing/W & non-Beijing/W lineage, sensitive (INH(S), RIF(S)) & resistant (INH(R), RIF(R)) strains] and of host macrophages from MTB infected cohorts (active & latent patients) with the human methylation CpG islands microarrays. RESULTS: Methylated modification on the promoter sequences of cytokines and their receptors were found to be associated with MTB infection in a strain- and host-dependent manner. Our epigenetic analyses revealed that infection with Beijing/W MTB strains enhanced IL6R, IL4R and IL17R hyper-methylations in infected macrophages. Validation of IL6R methylated sequence confirmed that MTB infection induced DNA methylation of CpG67 region in the IL6R promoter. In addition, studies on the human macrophage methylation profiles from the patient cohorts indicated that the methylation rate of IL17 family members and related genes were significantly altered in patients with active MTB infections. CONCLUSIONS: Our study offered novel insights into the epigenetic changes in the interaction of host macrophages in MTB infections and warrant further explorations into these changes in modulating the immune response in active and latent MTB infections.

Differential MicroRNA Expression in Human Macrophages with Mycobacterium tuberculosis Infection of Beijing/W and Non-Beijing/W Strain Types.

OBJECTIVES: The role of microRNAs in association with Mycobacterium tuberculosis (MTB) infection and the immunology regulated by microRNAs upon MTB infection have not been fully unravelled. We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of Beijing/W and non-Beijing/W clinical strains. We also studied the microRNA profiles of the host macrophages by microarray in a small cohort with active MTB disease, latent infection (LTBI), and from healthy controls. RESULTS: The results revealed that 14 microRNAs differentiated infections of Beijing/W from non-Beijing/W strains (P<0.05). A unique signature of 11 microRNAs in human macrophages was identified to differentiate active MTB disease from LTBI and healthy controls. Pathway analyses of these differentially expressed miRNAs suggest that the immune-regulatory interactions involving TGF-ß signalling pathway take part in the dysregulation of critical TB processes in the macrophages, resulting in active expression of both cell communication and signalling transduction systems. CONCLUSION: We showed for the first time that the Beijing/W TB strains repressed a number of miRNAs expressions which may reflect their virulence characteristics in altering the host response. The unique signatures of 11 microRNAs may deserve further evaluation as candidates for biomarkers in the diagnosis of MTB and Beijing/W infections.

Sleep quality in efavirenz-treated Chinese HIV patients - comparing between GT and GG genotype of CYP2B6-516 G/T polymorphisms.

Seventy-two adult Chinese HIV-positive treatment-naïve patients were recruited in a study to evaluate prospectively the associations between CYP2B6 516 G/T polymorphisms and sleep quality following treatment with an efavirenz-based regimen. Overall, the patients gave an allelic frequency of 0.3 for CYP2B6 516 T, and a genotype frequency of 9.4% for TT. Compared to GG, GT gave a higher median value of plasma efavirenz level at four weeks (3.77 mg/L vs 2.59 mg/L, p < 0.001) and 12 months (3.57 mg/L vs 2.97 mg/L, p = 0.026). Using generalised estimating equations analysis to track the variance over time, there was poorer Pittsburgh Sleep Quality Index in GT compared to GG, while GT was associated with a higher efavirenz level of >4 mg/L. There was however no difference in the component sleep scores nor was there direct association between sleep quality and plasma efavirenz levels. The results suggested that CYP2B6 genotype was associated with different patterns of sleep problems, further investigation of which is warranted with the objective of optimizing therapy with efavirenz-based regimens.

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development.

This study aimed at elucidating the physiological basis of bacterial antibiotic tolerance. By use of a combined phenotypic and gene knockout approach, exogenous nutrient composition was identified as a crucial environmental factor which could mediate progressive development of tolerance with markedly varied drug specificity and sustainability. Deprivation of amino acids was a prerequisite for tolerance formation, conferring condition-specific phenotypes against inhibitors of cell wall synthesis and DNA replication (ampicillin and ofloxacin, respectively), according to the relative abundances of ammonium salts, phosphate, and nucleobases. Upon further depletion of glucose, this variable phase consistently evolved into a sustainable mode, along with enhanced capacity to withstand the effect of the protein synthesis inhibitor gentamicin. Nevertheless, all phenotypes produced during spontaneous nutrient depletion lacked the sustainable, multidrug-tolerant features exhibited by the stationary-phase population and were attributed to complex interaction between starvation-mediated metabolic and stress protection responses on the basis of the following reasons: (i) the nutrition-dependent tolerance characteristics observed suggested that adaptive biosynthetic mechanisms could suppress but not fully avert tolerance under transient starvation conditions; (ii) formation of specific phenotypes could be inhibited by suppressing protein synthesis prior to nutrient depletion; (iii) bacteriostatic drugs produced only weak tolerance in the absence of starvation signals; and (iv) the attenuation of the stringent and SOS responses, as well as the functionality of other putative tolerance determinants, including rpoS, hipA, glpD, and phoU, could alter the induction requirement and drug specificity of the resultant phenotypes. These data reveal the common physiological grounds characteristic of starvation responses and the onset of antibiotic tolerance in bacteria.

Application of denaturing HPLC to rapidly identify rifampicin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas.

OBJECTIVES: Since the emergence of multidrug-resistant and extensively drug-resistant tuberculosis, there has been a call for a rapid assay to detect rifampicin-resistant strains that can be implemented into a routine service to analyse all strains in a specific geographical location. Denaturing HPLC (dHPLC) is a rapid screening test that can detect mutations in PCR amplicons. The aim of this study was to evaluate the dHPLC analysis of rifampicin-resistant Mycobacterium tuberculosis isolates using an extensive strain collection from Hong Kong and the UK and a collection of 84 consecutive clinical isolates. METHODS: DNA from 51 rifampicin-resistant M. tuberculosis strains from the UK and Hong Kong identified from 1996 to 2005 was extracted and each mutation was defined by capillary electrophoresis. A 400 bp PCR product was amplified from each strain, heteroduplexed with a known susceptible control (H37Rv) and analysed by dHPLC at 67.0 degrees C. RESULTS: Forty-five out of 51 (88.2%) rifampicin-resistant strains with known DNA mutations were detected by dHPLC. Two out of 84 clinical isolates were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates. dHPLC detected a mutation in 1 out of 82 phenotypically rifampicin-susceptible isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all strains and isolates, mutation detection by dHPLC analysis exhibited 88.2% sensitivity and 98.8% specificity. CONCLUSIONS: This study shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service.

Search for antimycobacterial constituents from a Tibetan medicinal plant, Gentianopsis paludosa.

From the whole plant of Gentianopsis paludosa seven compounds were isolated and identified to be 1,7-dihydroxy-3,8-dimethoxyxanthone (1), 1,7,8-trihydroxy-3-methoxyxanthone (2), 1-hydroxy-3,7,8-trimethoxyxanthone (3), 1,8-dihydroxy-2,6-dimethoxyxanthone (4), oleanolic acid (5), 4',5,7-trihydroxyflavone (6) and luteolin-7-O-glucoside (7). Compounds 1, 2, 3 and 5 showed modest inhibitory effect against the growth of Mycobacterium smegmatis and M. tuberculosis.

The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria.

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8-31 mug ml(-1)) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2-4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9(+) Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9 x 10(8) organisms ml(-1). Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials.

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong.

OBJECTIVES: To characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA). METHODS: The MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products. RESULTS: Resistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci. CONCLUSIONS: These findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

High-level gentamicin resistance mediated by a Tn4001-like transposon in seven nonclonal hospital isolates of Streptococcus pasteurianus.

We report on the first occurrence of high-level gentamicin resistance (MICs > or = 512 microg/ml) in seven clinical isolates of Streptococcus pasteurianus from Hong Kong. These seven isolates were confirmed to be the species S. pasteurianus on the basis of nucleotide sequencing of the superoxide dismutase (sodA) gene. Epidemiological data as well as the results of pulse-field gel electrophoresis analysis suggested that the seven S. pasteurianus isolates did not belong to the same clone. Molecular characterization showed that they carried a chromosomal, transposon-borne resistance gene [aac(6')Ie-aph(2'')Ia] which was known to encode a bifunctional aminoglycoside-modifying enzyme. The genetic arrangement of this transposon was similar to that of Tn4001, a transposon previously recovered from Staphylococcus aureus and other gram-positive isolates. Genetic linkage with other resistance elements, such as the ermB gene for erythromycin resistance, was not evident. On the basis of these findings, we suggest that routine screening for high-level gentamicin resistance should be recommended for all clinically significant blood culture isolates. This is to avoid the inadvertent use of short-course combination therapy with penicillin and gentamicin, which may lead to the failure of treatment for endocarditis, the selection of drug-resistant Streptococcus pasteurianus and other gram-positive organisms, as well as the unnecessary usage of gentamicin, a drug with potential toxicity.

Disinfection of Legionella pneumophila by photocatalytic oxidation.

Photocatalytic oxidation (PCO) was proven to be efficacious in the inactivation of Legionella pneumophila serogroup 1 Strains 977, 1009, 1014 and ATCC 33153. The local (Strains 997, 1009 and 1014) and ATCC (Strain 33153) strains showed sensitivity differences towards PCO. The inactivation mechanisms of PCO were investigated by transmission and scanning electron microscopy by which PCO was found to disintegrate the cells eventually. Before the disintegration, there was lipid peroxidation of outer and cytoplasmic membrane causing holes formation and leading to the entry of OH into the cells to oxidize the intracellular components. Fatty acid profile analysis found that the amount of saturated, 16-carbon branched-chain fatty acid, which is predominant in Legionella, decreased in the surviving populations from PCO. A relationship between the amount of this fatty acid and the PCO sensitivity of the tested strains was also observed. Mineralization of cells by PCO was proven by total organic carbon analysis.

Persistent Staphylococcus capitis septicemia in a preterm infant.

A preterm infant had persistent Staphylococcus capitis septicemia with 11 consecutive positive blood cultures over a period of 33 days. The clinical evidence suggested that the source of infection probably originated from the gastrointestinal tract. The combination of rifampin and linezolid treatment, together with prolonged stoppage of enteral feeding, successfully terminated the infection. Rifampin and linezolid should be considered as alternative antimicrobial agents when glycopeptides fail to eradicate Gram-positive pathogens from the host.

Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.

A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains /==" BORDER="0"> 16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains

An in vitro study of ceftazidime and vancomycin concentrations in various fluid media: implications for use in treating endophthalmitis.

PURPOSE: To investigate the precipitation process of a mixture of vancomycin and ceftazidime by equilibrium dialysis and determine its subsequent effect on the level of free antibiotics for treatment of endophthalmitis. METHODS: Concentrations of vancomycin and ceftazidime in an equilibrium dialysis chamber were measured during the equilibrium process by high-performance liquid chromatography. Normal saline (NS), balanced salt solution (BSS), and vitreous were used separately as the medium of dialysis. RESULTS: Precipitation of ceftazidime occurred at 37 degrees C but not at room temperature and did not affect the pH of the medium. It formed precipitate on its own or when mixed with vancomycin in all the three media of NS, BSS, and vitreous. More precipitation was formed if ceftazidime was initially prepared in BSS than in NS. After 168 hours in the dialysis chambers, ceftazidime prepared in NS precipitated to 54% of that in vitreous, compared with 88% if prepared in BSS. At 48 hours, ceftazidime prepared in NS decreased from an initial concentration of 137.5 to 73.4 microg/mL in vitreous medium and to 6.3 microg/mL if prepared in BSS. Precipitation of vancomycin was negligible. CONCLUSIONS: Based on this in vitro investigation, ceftazidime precipitates in vitreous at body temperature, regardless of the presence of vancomycin. NS is preferred to BSS as a preparation medium for antibiotics for intravitreal injection, because the extent of ceftazidime precipitation is less. However, due to precipitation, the concentration of free ceftazidime in vitreous may not be sufficiently high for antibacterial activity against most common organisms.