Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia.

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase, while defects in divisome accessory proteins increased susceptibility to multiple ß-lactams. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and ß-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.IMPORTANCEThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to ß-lactams in a multidrug-resistant bacterium.

The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals. Here, we present the results of a genome-wide screen for cell envelope-associated resistance and susceptibility determinants in a Burkholderia cenocepacia clinical isolate. For this purpose, we construct a high-density, randomly-barcoded transposon mutant library and expose it to 19 cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profile over a hundred functional associations and identify mediators of antibiotic susceptibility in the Bcc cell envelope. We reveal connections between ß-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism. The synergy of the ß-lactam/ß-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. In comparison with ceftazidime, avibactam more strongly potentiates the activity of aztreonam and meropenem in a panel of Bcc clinical isolates. Finally, we characterize in Bcc the iron and receptor-dependent activity of the siderophore-cephalosporin antibiotic, cefiderocol. Our work has implications for antibiotic target prioritization, and for using additional combinations of ß-lactam/ß-lactamase inhibitors that can extend the utility of current antibacterial therapies.

A machine learning model trained on a high-throughput antibacterial screen increases the hit rate of drug discovery.

Screening for novel antibacterial compounds in small molecule libraries has a low success rate. We applied machine learning (ML)-based virtual screening for antibacterial activity and evaluated its predictive power by experimental validation. We first binarized 29,537 compounds according to their growth inhibitory activity (hit rate 0.87%) against the antibiotic-resistant bacterium Burkholderia cenocepacia and described their molecular features with a directed-message passing neural network (D-MPNN). Then, we used the data to train an ML model that achieved a receiver operating characteristic (ROC) score of 0.823 on the test set. Finally, we predicted antibacterial activity in virtual libraries corresponding to 1,614 compounds from the Food and Drug Administration (FDA)-approved list and 224,205 natural products. Hit rates of 26% and 12%, respectively, were obtained when we tested the top-ranked predicted compounds for growth inhibitory activity against B. cenocepacia, which represents at least a 14-fold increase from the previous hit rate. In addition, more than 51% of the predicted antibacterial natural compounds inhibited ESKAPE pathogens showing that predictions expand beyond the organism-specific dataset to a broad range of bacteria. Overall, the developed ML approach can be used for compound prioritization before screening, increasing the typical hit rate of drug discovery.

Induced dormancy in Indian meal moth Plodia interpunctella (Hübner) and its impact on the quality improvement for mass rearing in parasitoid Habrobracon hebetor (Say).

A steady supply of hosts at the susceptible stage for parasitism is a major component of mass rearing parasitoids for biological control programs. Here we describe the effects of storing 5th instar Plodia interpunctella larvae in dormancy on subsequent host development in the context of host colony maintenance and effects of the duration of host dormancy on the development of Habrobracon hebetor parasitoids reared from dormant hosts. We induced dormancy with a combination of short daylength (12L:12D) and lower temperature (15°C), conditions known to induce diapause in this species, and held 5th instar larvae of P. interpunctella for a series of dormancy durations ranging from 15 to 105 days. Extended storage of dormant 5th instar larvae had no significant impacts on survival, development, or reproductive potential of P. interpunctella, reinforcing that dormant hosts have a substantial shelf life. This ability to store hosts in dormancy for more than 3 months at a time without strong negative consequences reinforces the promise of using dormancy to maintain host colonies. The proportion of hosts parasitized by H. hebetor did not vary significantly between non-dormant host larvae and dormant host larvae stored for periods as long as 105 days. Concordant with a prior study, H. hebetor adult progeny production from dormant host larvae was higher than the number of progeny produced on non-dormant host larvae. There were no differences in size, sex ratio, or reproductive output of parasitoids reared on dormant hosts compared to non-dormant hosts stored for up to 105 days. Larval development times of H. hebetor were however longer when reared on dormant hosts compared to non-dormant hosts. Our results agree with other studies showing using dormant hosts can improve parasitoid mass rearing, and we show benefits for parasitoid rearing even after 3 months of host dormancy.

Facile extraction and characterization of calcium hydroxide from paper mill waste sludge of Bangladesh.

Herein, paper mill waste sludge (PMS) from two different sources has been investigated to extract calcium hydroxide, Ca(OH)2 by a facile and inexpensive extraction process. PMS samples, collected from local paper mill plants of Bangladesh, were the main precursors wherein HCl and NaOH were used for chemical treatment. The as-synthesized products were analysed by a variety of characterization tools including X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) elemental analyses. Our studies confirm that the extracted product contains Ca(OH)2 as a major content, albeit it also includes CaCO3 phase owing to the inescapable carbonation process from the surrounding environment. The particle size of the synthesized products is in the range of 450-500 nm estimated from SEM micrographs. The crystallite domain size of the same estimated from XRD analyses and was found to be approximately 47 and 31 nm respectively for product-A and product-B considering major (101) Bragg peak of Ca(OH)2. The yield percentage of the isolated products is about 65% for samples collected from both sources.

Asymmetry Matters: Diffusion Tensor Tractography of the Uncinate Fasciculus in Children with Verbal Memory Deficits.

BACKGROUND AND PURPOSE: Verbal declarative memory performance relies on frontotemporal connectivity. The uncinate fasciculus is a major association tract connecting the frontal and temporal lobes. Hemispheric asymmetries contribute to various cognitive and neurobehavioral abilities. Here we investigated microstructural alterations and hemispheric asymmetry of the uncinate fasciculus and their possible correlation to memory performance of children with learning disorders attributed to verbal memory deficits. MATERIALS AND METHODS: Two groups of right-handed children with learning disorders attributed to verbal memory deficits and typically developing children (n = 20 and 22, respectively) underwent DTI on a 1.5T scanner. Tractography of the uncinate fasciculus in both hemispheres was performed, and fractional anisotropy and diffusivity indices (radial diffusivity, axial diffusivity, and trace) were obtained. The asymmetry index was calculated. Verbal memory was assessed using subsets of the Stanford Binet Intelligence Scale, 4th edition, a dyslexia assessment test, and the Illinois test of Psycholinguistic Abilities. Correlation between diffusion metrics and verbal memory performance was investigated in the learning disorders group. Also, hemispheric differences in each group were tested, and between-group comparisons were performed. RESULTS: Children with learning disorders showed absence of the normal left-greater-than-right asymmetry of fractional anisotropy and the normal right-greater-than-left asymmetry of radial diffusivity seen in typically developing children. Correlation with verbal memory subsets revealed that the higher the fractional anisotropy and asymmetry index, the better the rapid naming performance (P <.05) was. CONCLUSIONS: These findings demonstrated microstructural aberrations with reduction of hemispheric asymmetry of the uncinate fasciculus, which could disrupt the normal frontotemporal connectivity in children with learning disorders attributed to verbal memory deficits. This outcome gives more understanding of pathologic mechanisms underlying this disorder.

Deep learning-driven prediction of drug mechanism of action from large-scale chemical-genetic interaction profiles.

MOTIVATION: Chemical-genetic interaction profiling is a genetic approach that quantifies the susceptibility of a set of mutants depleted in specific gene product(s) to a set of chemical compounds. With the recent advances in artificial intelligence, chemical-genetic interaction profiles (CGIPs) can be leveraged to predict mechanism of action of compounds. This can be achieved by using machine learning, where the data from a CGIP is fed into the machine learning platform along with the chemical descriptors to develop a chemogenetically trained model. As small molecules can be considered non-structural data, graph convolutional neural networks, which can learn from the chemical structures directly, can be used to successfully predict molecular properties. Clustering analysis, on the other hand, is a critical approach to get insights into the underlying biological relationships between the gene products in the high-dimensional chemical-genetic data. METHODS AND RESULTS: In this study, we proposed a comprehensive framework based on the large-scale chemical-genetics dataset built in Mycobacterium tuberculosis for predicting CGIPs using graph-based deep learning models. Our approach is structured into three parts. First, by matching M. tuberculosis genes with homologous genes in Escherichia coli (E. coli) according to their gene products, we grouped the genes into clusters with distinct biological functions. Second, we employed a directed message passing neural network to predict growth inhibition against M. tuberculosis gene clusters using a collection of 50,000 chemicals with the profile. We compared the performance of different baseline models and implemented multi-label tasks in binary classification frameworks. Lastly, we applied the trained model to an externally curated drug set that had experimental results against M. tuberculosis genes to examine the effectiveness of our method. Overall, we demonstrate that our approach effectively created M. tuberculosis gene clusters, and the trained classifier is able to predict activity against essential M. tuberculosis targets with high accuracy. CONCLUSION: This work provides an analytical framework for modeling large-scale chemical-genetic datasets for predicting CGIPs and generating hypothesis about mechanism of action of novel drugs. In addition, this work highlights the importance of graph-based deep neural networks in drug discovery.

Identification of putative essential protein domains from high-density transposon insertion sequencing.

A first clue to gene function can be obtained by examining whether a gene is required for life in certain standard conditions, that is, whether a gene is essential. In bacteria, essential genes are usually identified by high-density transposon mutagenesis followed by sequencing of insertion sites (Tn-seq). These studies assign the term "essential" to whole genes rather than the protein domain sequences that encode the essential functions. However, genes can code for multiple protein domains that evolve their functions independently. Therefore, when essential genes code for more than one protein domain, only one of them could be essential. In this study, we defined this subset of genes as "essential domain-containing" (EDC) genes. Using a Tn-seq data set built-in Burkholderia cenocepacia K56-2, we developed an in silico pipeline to identify EDC genes and the essential protein domains they encode. We found forty candidate EDC genes and demonstrated growth defect phenotypes using CRISPR interference (CRISPRi). This analysis included two knockdowns of genes encoding the protein domains of unknown function DUF2213 and DUF4148. These putative essential domains are conserved in more than two hundred bacterial species, including human and plant pathogens. Together, our study suggests that essentiality should be assigned to individual protein domains rather than genes, contributing to a first functional characterization of protein domains of unknown function.

Evaluating the Respiratory Support Pattern and Relationship with Initial Oxygen Saturation of the COVID Isolation Unit Patients in Sylhet.

COVID-19 is the devastating pandemic of the century predominantly fatal due to its respiratory failure nature. Severe and critical patients need oxygen supplementation in different forms. This cross-sectional study was conducted in four tertiary hospitals of Sylhet, Bangladesh from November 2020 to March 2021. All the patients admitted in the COVID-19 isolation units and fulfill the selection criterion were enrolled in this study. The primary objective of this study was to evaluate different types of respiratory supports and its relationship with initial oxygen saturation (SpO2). Total 481 patients were enrolled. There was a male predominance (65.00%) in the participants. Highest number of participants was from 61-70 years age group. Number of ventilated patients were significantly high (p<0.001) in the COVID-19 patient group. The initial SpO2 and hospital staying period of COVID-19 positive and negative group did not show any significant difference but these two parameters showed significant difference among died and survived group (p<0.001). Nearly one fourth patients (24.94%) of total patients were treated in ICU with high flow nasal cannula (HFNC), non-invasive ventilation (NIV) and mechanical ventilation. Among the ICU admitted patients nearly one-fourth (24.16%) patients were treated with mechanical ventilation. Mortality rate was 62.00% for ventilated patients, 70.60% for NIV patients and 15.80% for the HFNC patients.

Development and diapause induction of the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) at different photoperiods.

Diapause concerns the fascinating phenomenon in the biology of insect development which allows better understanding the local adaptation and phenotypic plasticity to seasonal variations in environment. There is lot of reasons to carry out the research on diapause both for fundamental and applied sciences. Photoperiod is one of the main environmental cues followed by insects to predict the forthcoming seasonal changes and to adapt these changes in their life-history traits. Thus, the effect of different photoperiod regimes on development and diapause induction of larvae of the Indian meal moth Plodia interpunctella (Hübner) was evaluated at a constant temperature of 17 °C. Development was significantly faster at a photoperiod of 12:12 light:darkness (L:D) than at 8:16, 10:14, 14:10 and 16:8 L:D. A photoperiod of 12:12 (L:D) induced most larvae (≥ 71%) to enter diapause, while this percentage was slightly lower (60%) at both shorter(8 h) and longer (16 h) day lengths (50%). The different photoperiod regimes did not affect the percentage of adult emergence. Fat and protein composition of the diapausing larvae differed significantly among treatments as well as between diapausing and non-diapausing larvae. Larvae developing from 8:16 (L:D) contained the maximum amount of protein (36.8%) compared to other regimes, while the minimum amount (21.0%) was noted in larvae that developed at 16:8 (L:D). Six types of fatty acids were detected in the larvae: myristic acid (methyl tetradecenoate), palmitoleic acid (9-hexadecenoic acid, methyl ester), palmitic acid (hexadecenoic acid, methyl ester), linoleic acid (9, 12-Octadecadienoic acid (Z, Z), methyl ester), oleic acid [9-octadecenoic acid, methyl ester (E)] and stearic acid (octadecanoic acid, methyl ester). The results also reveal that the percent of fatty acids detected in the diapausing larvae varies significantly and the same trends imply in the interaction of fatty acid and photoperiod regimes. Moreover, three quarters of the total variance was accounted for by the Principal Component Analysis (PCA) of the fatty acids. Different proportions of fatty acids were noted among treatments, suggesting that photoperiod influences a number of key biological traits in P. interpunctella, much more than the percentage of the diapausing larvae per se.

A Broad-Host-Range CRISPRi Toolkit for Silencing Gene Expression in Burkholderia.

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans, and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne gRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi toolkit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderia's pathogenic arsenal.

Molecular analysis of hemagglutinin, neuraminidase, matrix genes provide insight into the genetic diversity of seasonal H3N2 human influenza a viruses in Bangladesh during July-August, 2012.

Influenza A virus subtype H3 is a threat to public health and it is important to understand the evolution of the viruses for the surveillance and the selection of vaccine strains. Comparative analysis of four Bangladeshi isolates with isolates circulating other parts of the world based on three candidate genes hemagglutinin (HA), neuraminidase (NA), matrix protein (MA) showed no evidence of significant distinct subclade of viruses circulating in the country over the period of study. Despite these findings, we found N161S substitution in all four H3N2 influenza stains resulting in the gain of NSS160-162 glycosylation site. All H3N2 Influenza subtypes in the study had amino acid substitution at position 31 on the M2 protein (Aspartic acid to Asparagine) which is known to be responsible for amantadine drug resistance.

Evaluation of Behavior Change Communication Campaigns to Promote Modern Cookstove Purchase and Use in Lower Middle Income Countries.

Nearly three billion people worldwide burn solid fuels and kerosene in open fires and inefficient stoves to cook, light, and heat their homes. Cleaner-burning stoves reduce emissions and can have positive health, climate, and women's empowerment benefits. This article reports on the protocol and baseline data from the evaluation of four behavior change communication (BCC) campaigns carried out in lower to middle income countries aimed at promoting the sale and use of cleaner-burning stoves. Interventions implemented in Bangladesh, Kenya, and Nigeria are using a range of BCC methods including mass media, digital media, outdoor advertising, and inter-personal communication. The mixed methods evaluation comprises three large-scale surveys: one pre-BCC and two follow-ups, along with smaller scale assessments of stove uptake and patterns of use. Baseline results revealed varying levels of awareness of previous promotions and positive attitudes and beliefs about modern (i.e., relatively clean-burning) cookstoves. Differences in cookstove preferences and behaviors by gender, socio-demographics, media use, and country/region were observed that may affect outcomes. Across all three countries, cost (lack of funds) a key perceived barrier to buying a cleaner-burning stove. Future multivariate analyses will examine potential dose-response effects of BCC on cookstove uptake and patterns of use. BCC campaigns have the potential to promote modern cookstoves at scale. More research on campaign effectiveness is needed, and on how to optimize messages and channels. This evaluation builds on a limited evidence base in the field.

Nationwide prevalence of Rickettsia felis infections in patients with febrile illness in Bangladesh.

From July 2015 to December 2016, the presence of rickettsial pathogens was investigated for 414 patients with unknown fever in eight places in all the divisions of Bangladesh. Rickettsia felis was identified in blood samples from all the regions (overall detection rate, 19.6%), suggesting nationwide prevalence of R. felis infections.

User involvement in service delivery predicts outcomes of assistive technology use: a cross-sectional study in Bangladesh.

BACKGROUND: Knowledge about the relation between user involvement in the provision of assistive technology and outcomes of assistive technology use is a prerequisite for the development of efficient service delivery strategies. However, current knowledge is limited, particularly from low-income countries where affordability is an issue. The objective was therefore to explore the relation between outcomes of assistive technology use and user involvement in the service delivery process in Bangladesh. METHODS: Using structured interviews, data from 136 users of hearing aids and 149 users of manual wheelchairs were collected. Outcomes were measured using the International Outcome Inventory for Hearing Aids (IOI-HA), which was adapted for wheelchair users. Predictors of user involvement included preference, measurement and training. RESULTS: Users reported outcomes comparable to those found in other high- and low-income countries. User involvement increased the likelihood for reporting better outcomes except for measurement among hearing aid users. CONCLUSIONS: The findings support the provision of assistive technology as a strategy to improve the participation of people with disabilities in society. They also support current policies and guidelines for user-involvement in the service delivery process. Simplified strategies for provision of hearing aids may be explored.

Low-dose beta-carotene supplementation and deworming improve serum vitamin A and beta-carotene concentrations in preschool children of Bangladesh.

Despite the national vitamin A and antihelminthic prophylaxis programmes, both intestinal geohelminths and subclinical vitamin A deficiency continue to be prevalent among children in developing countries. Studies on potential synergistic effects of vitamin A supplementation and deworming on retinol status have inconsistent results. The purpose of the present study was to investigate the impacts of low-dose beta-carotene supplementation and antihelminthic therapy on serum retinol and beta-carotene concentrations in preschool children of Bangladesh. Two hundred and forty-four children, known to be infected with Ascaris lumbricoides, were randomized into four treatment groups: I-IV. Group I and II received two oral doses of 400 mg of albendazole each, the first dose at baseline and the second dose after four months; Group III and IV received placebo in place of albendazole. In addition, Group I and III received 1.2 mg of beta-carotene powder in capsule daily for six months, and Group II and IV received placebo in place of beta-carotene. Serum retinol and beta-carotene levels were measured before and after six months of the interventions. Serum retinol and beta-carotene increased significantly in Group I where both antihelminthic therapy and daily beta-carotene supplementation were given (p<0.05 and p<0.001 respectively). Antihelminthic therapy alone only improved serum beta-carotene concentration (p<0.0001). Low-dose beta-carotene supplementation, along with an antihelminthic therapy, synergistically improved vitamin A status. This finding has public-health implications for improving vitamin A status of children in developing countries.

Bangladesh moves from being a low-prevalence nation for HIV to one with a concentrated epidemic in injecting drug users.

Bangladesh has been conducting annual serological surveillance for HIV and syphilis since 1998 among most at-risk populations including sex workers, males having sex with males, injecting drug users (IDUs) and heroin smokers. During the seventh round conducted between January and June 2006, 10,368 people were sampled and the overall HIV prevalence was 0.9%. The highest HIV rate was recorded in male IDUs from the capital city Dhaka (7%), and the rates have risen significantly over the rounds (P < 0.001). In Dhaka, most of the HIV-positive IDUs (10.5%) were localized in one neighbourhood, while in the remaining neighbourhoods 1% were positive (P < 0.001). In all other groups, HIV prevalence was <1%. Active syphilis rates were highest in female IDUs (9.9%) followed by female street-based sex workers (8.6%). However, rates in female sex workers in Dhaka declined significantly over the years (P < 0.001). Bangladesh has to act urgently to prevent escalation of the epidemic.

Molecular evidence for polyphyletic origin of human immunodeficiency virus type 1 subtype C in Bangladesh.

HIV-1 positive blood samples were collected between 1999 and 2005 from population groups most at risk of HIV infection in Bangladesh through the national surveillance, from clients of the Voluntary Counseling and Testing (VCT) Unit for HIV at ICDDR,B and a survey of HIV in patients with tuberculosis. Partial sequences of the gag gene were used for subtyping the HIV strains by nested PCR using selective primers. Of the 198 HIV strains tested, subtype C (41.4%) was the commonest strain identified. Phylogenetic analysis of Bangladeshi subtype C strains showed that they clustered in polyphyletic branches representing HIV strains from different parts of the world. Most of the strains from injecting drug users (IDU) clustered together and were similar to Indian strains. The VCT strains however were very heterogeneous and clustered with strains from India, Myanmar, Ethiopia and Zimbabwe. Data suggest that there have been few introductions into the IDU population where the epidemic is driven by indigenous transmission. On the other hand there have been many and regular introductions of subtype C viruses through migrant workers in the VCT group. Very little overlap was observed in the strains obtained from IDU and those from other population groups.

Investigation of Hong Kong doctors' current knowledge, beliefs, attitudes, confidence and practices: implications for the treatment of tobacco dependency.

BACKGROUND: Physicians play a crucial role in promoting smoking cessation. However, there are lack of data on Chinese doctors' knowledge, beliefs, attitudes, confidence and usual practices in relation to smoking cessation. Understanding of these indicators is important in the design of any effective intervention program targeting doctors. METHODS: To assess Chinese doctors' knowledge, beliefs, attitudes, confidence and usual practices in relation to smoking cessation, a mailed questionnaire survey was conducted among 4,000 doctors registered with the Hong Kong Medical Association (HKMA) in 2002. RESULTS: Of the 757 respondents (18.9% response rate), 78% were male, 94% were non-smokers and 50% had received no basic training on smoking cessation. More than half of the doctors did not hold adequate knowledge (53%) or favorable attitudes (55%) towards smoking cessation; 44% were less confident in their smoking cessation skills. About 77% of the doctors obtained information on their patients' smoking status and recorded it in their medical record, and 29% advised all smoking patients to quit. Doctors who gave smoking cessation advice were more likely to be aged above 50 years, with more than 30 years' practice experience, working in the private sector, non- or ex-smokers, with more positive beliefs towards smoking cessation, and with higher confidence in smoking cessation skills (p < 0.001). Different factors associated with establishing and recording smoking status, arranging follow-up sessions, acquiring more knowledge and developing a more favorable attitude and greater confidence on smoking cessation-related matters were also identified. CONCLUSION: The survey has shown that existing smoking cessation service provision in Hong Kong for patients who smoke is inadequate, and has identified a lack of smoking cessation skills among doctors. Action should be taken to train doctors in smoking cessation skills and encourage them to routinely establish the smoking status of their patients and to advise all smokers to quit smoking.