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Background Burkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China. Methods Burkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test. Results A phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (1012 PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3–12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage. Conclusions As the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis. Graphical Abstract

This study was designed to investigate the molecular epidemiology of mobile colistin resistance (mcr) using a “One-Health” approach in Laos and to predict whether any dominant plasmid backbone and/or strain type influences the dissemination of mcr. We collected 673 samples from humans (rectal normal flora), poultry, and the environment (water, flies, birds, etc.) in Vientiane, Lao People's Democratic Republic (Laos), from May to September 2018. A total of 238 Escherichia coli (E. coli) isolated from non-duplicative samples, consisting of 98 MCR-positive E. coli (MCRPEC) (“mcr” denotes the gene encoding mobile colistin resistance, and “MCR” denotes the subsequent protein encoded by mcr) and 140 MCR-negative E. coli (MCRNEC), were characterized by phenotype and Illumina sequencing. A subset of MCRPEC was selected for MinION sequencing, conjugation assay, plasmid stability, and growth kinetics in vitro. The prevalence of MCRPEC was found to be 14.6% (98/673), with the highest prevalence in human rectal swabs (45.9% (45/98), p < 0.0001, odds ratio (OR): 0.125, 95% confidence interval (CI): 0.077–0.202). The percentages of MCRPEC from other samples were 14.3% (2/14) in dog feces, 12.0% (24/200) in flies, 11.0% (11/100) in chicken meat, 8.9% (8/90) in chicken cloacal, 8.0% (4/50) in chicken caeca, and 7.5% (4/53) in wastewater. MCRPEC was significantly more resistant to co-amoxiclav, sulfamethoxazole-trimethoprim, levofloxacin, ciprofloxacin, and gentamicin than MCRNEC (p < 0.05). Genomic analysis revealed the distribution of MCRPEC among diverse clonal types. The putative plasmid Inc types associated with mcr-1 were IncX4, IncHI2, IncP1, IncI2, and IncFIA, and those associated with mcr-3 were IncFII, IncFIA, IncFIB, IncP1, and IncR. Recovery of highly similar plasmids from both flies and other sampling sectors implied the role of flies in the dissemination of mcr-1. mcr-positive plasmids were shown to be conjugative, and a significantly high transfer rate into a hypervirulent clone ST1193 was observed. Plasmids containing mcr irrespective of Inc type were highly stable and invariably did not exert a fitness effect upon introduction into a new host. These findings signify the urgent need for a standard infection control program to radically decontaminate the source of resistance.

Melioidosis is a tropical infection caused by the soil bacterium Burkholderia pseudomallei. Despite the substantial impact of this often overlooked pathogen on both the health-care systems and economies of numerous low-income and middle-income countries around the world, melioidosis is not officially classified as a neglected tropical disease (NTD) by WHO. Melioidosis causes a higher estimated disease burden and mortality than many other recognised NTDs, with deaths primarily occurring among rural poor populations in low-income and middle-income countries. Fortunately, the impact of melioidosis in a region can be reduced once awareness is established of its known or suspected endemicity. In this Personal View, we provide evidence in support of official recognition of melioidosis as an NTD. We urge member states to request that WHO revisit their NTD list and appeal to government and philanthropic organisations to establish programmes in endemic countries to control melioidosis in order to reduce its global health burden.

Melioidosis is thought to be endemic, although underdiagnosed, in Africa. We identified 5 autochthonous cases of Burkholderia pseudomallei infection in a case series in Kenya. Incidence of B. pseudomallei bacteremia in Kenya’s Kilifi County is low, at 1.5 cases per million person-years, but this result might be an underestimate.

OBJECTIVES: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms. METHODS: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs. RESULTS: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28). CONCLUSIONS: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype., Cryptococcus neoformans is an opportunistic fungal pathogen which primarily affects people with immune defects including those living with HIV. Here, the authors sequence and analyze genomes of 699 isolates, and identify recent population expansion driven by three phylogenetic groups.

BackgroundAntimicrobial resistance (AMR) is highly prevalent in low- and middle-income countries (LMIC). International travel contributes substantially to the global spread of intestinal multidrug-resistant gram-negative (MDR-GN) bacteria. Of the 100 million annual visitors to LMIC, 30–70% become colonized by MDR-GN bacteria. The phenomenon has been well documented, but since sampling has only been conducted after travelers’ return home, data on the actual colonization process are scarce. We aimed to characterize colonization dynamics by exploring stool samples abroad on a daily basis while visiting LMIC.MethodsA group of 20 European volunteers visiting Lao People’s Democratic Republic for three weeks provided daily stool samples and filled in daily questionnaires. Acquisition of extended-spectrum beta-lactamase-producing gram-negative bacteria (ESBL-GN) was examined by selective stool cultures followed by whole-genome sequencing (WGS) of isolates.ResultsWhile colonization rates were 70% at the end of the study, daily sampling revealed that all participants had acquired ESBL-GN at some time point during their overseas stay, the colonization status varying day by day. WGS analysis ascribed the transient pattern of colonization to sequential acquisition of new strains, resulting in a loss of detectable colonization by the initial MDR-GN strains. All but one participant acquired multiple strains (n=2–7). Of the total of 83 unique strains identified (53 E. coli, 10 Klebsiella, 20 other ESBL-GN species), some were shared by as many as four subjects.ConclusionsThis is the first study to characterize in real time the dynamics of acquiring MDR-GN during travel. Our data show multiple transient colonization events indicative of constant microbial competition.

Background Melioidosis is a frequently fatal disease requiring specific treatment. The yield of Burkholderia pseudomallei from sites with a normal flora is increased by culture using selective, differential media such as Ashdown’s agar and selective broth. However, since melioidosis mainly affects people in resource-poor countries, the cost effectiveness of selective culture has been questioned. We therefore retrospectively evaluated this in two laboratories in southeast Asia. Methodology/Principal findings The results of all cultures in the microbiology laboratories of Mahosot Hospital, Vientiane, Laos and Angkor Hospital for Children, Siem Reap, Cambodia, in 2017 were reviewed. We identified patients with melioidosis who were only diagnosed as a result of culture of non-sterile sites and established the total number of such samples cultured using selective media and the associated costs in each laboratory. We then conducted a rudimentary cost-effectiveness analysis by determining the incremental cost-effectiveness ratio (ICER) per DALY averted and compared this against the 2017 GDP per capita in each country. Overall, 29 patients in Vientiane and 9 in Siem Reap (20% and 16.9% of all culture-positive patients respectively) would not have been diagnosed without the use of selective media, the majority of whom (18 and 8 respectively) were diagnosed by throat swab culture. The cost per additional patient detected by selective culture was approximately $100 in Vientiane and $39 in Siem Reap. Despite the different patient populations (all ages in Vientiane vs. only children in Siem Reap) and testing strategies (all samples in Vientiane vs. based on clinical suspicion in Siem Reap), selective B. pseudomallei culture proved highly cost effective in both settings, with an ICER of ~$170 and ~$28 in Vientiane and Siem Reap, respectively. Conclusions/Significance Selective culture for B. pseudomallei should be considered by all laboratories in melioidosis-endemic areas. However, the appropriate strategy for implementation should be decided locally., Author summary Melioidosis is a frequently fatal disease caused by a soil bacterium called Burkholderia pseudomallei, that is widespread in the rural tropics. Because staff are often not familiar with it and because it may be hidden if it is outgrown by other bacteria, special culture media can help laboratories diagnose the disease. However, this costs more money so it is not always done even in areas where the disease is known to be present. We have looked at the results of a year’s bacterial cultures in two different laboratories in southeast Asia to identify how many patients were only identified using these special culture techniques, how much it cost, and whether the investment was considered worthwhile in terms of the gain in healthy life years in these patients who might otherwise have died had the disease not been diagnosed. Even though the laboratories adopted very different strategies for using the special media and served very different populations, in both places the use of the special techniques was very cost effective in terms not just of lives saved, but on purely financial grounds when compared with the GDP of each country.

We investigated the impact of breakpoint discrepancies between CLSI and EUCAST on susceptibility interpretation of clinical isolates at the Microbiology Laboratory, Mahosot Hospital, Vientiane, Laos and performed a literature search to compare our findings to published reports. Zone diameters for first-line antimicrobial agents tested against non-duplicate clinical isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in 2017 were interpreted separately using EUCAST 2018 and CLSI 2018 breakpoints and greement measured. Applying EUCAST instead of CLSI breakpoints to 428 E. coli, 208 K. pneumoniae and 78 P. aeruginosa isolates would have increased rates of ciprofloxacin resistance (59.1% vs 46.5% in E. coli, 37.5% vs 13.9% in K. pneumoniae, 28.2% vs 10.3% in P. aeruginosa) and amoxicillinclavulanic acid resistance (52.3% vs 19.9% in E. coli, 35.6% vs 22.1% in K. pneumoniae). Our results are supported by a literature search which identified 20 articles whose main objective was comparing susceptibility interpretation between CLSI and EUCAST. 19/20 articles reported significant discrepancies in one or more pathogen-antimicrobial combinations, nearly always due to a reduction in susceptibility rates and/or increase in resistance rates when applying more restrictive EUCAST breakpoints. We conclude that breakpoint discrepancies between CLSI and EUCAST have a significant impact on susceptibility interpretation of clinical isolates and AMR surveillance initiatives, and highlight the need for globally harmonized clinical breakpoints.

Melioidosis is a tropical infection, first described in Myanmar but now rarely diagnosed there, which is widespread in Southeast Asia. The infection is predominantly acquired by people and animals through contact with soil or water. This study aimed to detect the causative organism, Burkholderia pseudomallei, in environmental samples from farms in Thanlyin and Hmawbi townships near Yangon, Myanmar. One hundred and twenty soil samples and 12 water samples were collected and processed using standard microbiological methods.Burkholderia species were isolated from 50 of the 120 (42%) soil samples but none of the water samples. Arabinose assimilation was tested to differentiate between B. pseudomallei and the nonpathogenic Burkholderia thailandensis, and seven of 50 isolates (14%) were negative. These were all confirmed as B. pseudomallei by a species-specific multiplex polymerase chain reaction (PCR). This is the first study to detect environmental B. pseudomallei in Myanmar and confirms that melioidosis is still endemic in the Yangon area.

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging. Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language. We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address. Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.

INTRODUCTION: Pneumococcal conjugate vaccines (PCVs) prevent disease through both direct protection of vaccinated individuals and indirect protection of unvaccinated individuals by reducing nasopharyngeal (NP) carriage and transmission of vaccine-type (VT) pneumococci. While the indirect effects of PCV vaccination are well described, the PCV coverage required to achieve the indirect effects is unknown. We will investigate the relationship between PCV coverage and VT carriage among undervaccinated children using hospital-based NP pneumococcal carriage surveillance at three sites in Asia and the Pacific. METHODS AND ANALYSIS: We are recruiting cases, defined as children aged 2-59 months admitted to participating hospitals with acute respiratory infection in Lao People's Democratic Republic, Mongolia and Papua New Guinea. Thirteen-valent PCV status is obtained from written records. NP swabs are collected according to standard methods, screened using lytA qPCR and serotyped by microarray. Village-level vaccination coverage, for the resident communities of the recruited cases, is determined using administrative data or community survey. Our analysis will investigate the relationship between VT carriage among undervaccinated cases (indirect effects) and vaccine coverage using generalised estimating equations. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the relevant ethics committees at participating sites. The results are intended for publication in open-access peer-reviewed journals and will demonstrate methods suitable for low- and middle-income countries to monitor vaccine impact and inform vaccine policy makers about the PCV coverage required to achieve indirect protection.

Melioidosis is clearly highly endemic in Laos, although the disease has only been diagnosed regularly in humans (1359 cases) since 1999, and only a single animal case has been microbiologically confirmed. Burkholderia pseudomallei is extensively and abundantly present in soil and surface water in central and southern Laos, but the true distribution of the disease across the country remains to be determined. Surveillance is almost non-existent and diagnostic microbiology services are not yet well established, whilst awareness of melioidosis is low amongst policy-makers, healthcare providers, and the public. It is hoped that this situation will improve over the next decade as the country rapidly develops, especially as this is likely to be accompanied by a further increase in the prevalence of diabetes, meaning that more people in this predominantly agricultural population will be at risk of contracting melioidosis.

The environmental bacterium $\textit{Burkholderia pseudomallei}$ causes an estimated 165,000 cases of human melioidosis per year worldwide and is also classified as a biothreat agent. We used whole genome sequences of 469 $\textit{B. pseudomallei}$isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia and East Asia. Repeated reintroductions were observed within the Malay Peninsula and between countries bordered by the Mekong River. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those over-represented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted.

Infectious diarrhoea is a major public health concern worldwide. Bacteria, the focus of this review, are responsible for 20-40% of diarrhoeal episodes, contributing to high rates of childhood mortality in developing regions, and substantial morbidity and economic losses in developed regions. The epidemiology is changing with salmonellosis decreasing in industrialized countries and diarrhoeagenic Escherichia coli contributing to an increasing burden of disease worldwide. Molecular diagnostics have improved our understanding of the epidemiology, aetiology and pathogenesis of bacterial gastroenteritis, and have revealed new pathogenic agents, although widespread introduction of such diagnostics into clinical practice will require careful cost-benefit analyses. The development of antimicrobial resistance in gastrointestinal pathogens has implications for treatment options. We review the epidemiology of infectious diarrhoea, the principal aetiological agents and their clinical features, and the diagnosis, treatment and prevention of bacterial gastroenteritis; we also propose an investigation and management algorithm. © 2013 Elsevier Ltd. All rights reserved.

Introduction of pneumococcal conjugate and polysaccharide vaccines into the United Kingdom's routine immunization programmes is expected to change the epidemiology of invasive pneumococcal disease (IPD). We have documented the epidemiology of IPD in an English region (South West) with high-quality surveillance data before these programmes were established. We analysed data on isolates of Streptococcus pneumoniae from blood and CSF between 1996 and 2005 from microbiology laboratories in the South West that were reported and/or referred for serotyping to the Health Protection Agency Centre for Infections. The mean annual incidence of IPD increased from 11.2/100 000 in 1996 to 13.6/100 000 in 2005 (P

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.