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Unsafe water supplies continue to raise public health concerns, especially in urban areas in low resource countries. To understand the extent of public health risk attributed to supply water in Dhaka city, Bangladesh, Escherichia coli isolated from tap water samples collected from different locations of the city were characterized for their antibiotic resistance, pathogenic properties and genetic diversity. A total of 233 E. coli isolates obtained from 175 tap water samples were analysed for susceptibility to 16 different antibiotics and for the presence of genes associated with virulence and antibiotic resistance. Nearly 36% (n = 84) of the isolates were multi-drug(≥ 3 classes of antibiotics) resistant (MDR) and 26% (n = 22) of these were positive for extended spectrum β-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for bla CTX-M-15, 7 for bla OXA-1-group (all had bla OXA-47) and 2 for bla CMY-2. Quinolone resistance genes, qnrS and qnrB were detected in 6 and 2 isolates, respectively. Around 7% (n = 16) of the isolates carried virulence gene(s) characteristic of pathogenic E. coli; 11 of these contained lt and/or st and thus belonged to enterotoxigenic E. coli and 5 contained bfp and eae and thus belonged to enteropathogenic E. coli. All MDR isolates carried multiple plasmids (2 to 8) of varying sizes ranging from 1.2 to >120 MDa. Ampicillin and ceftriaxone resistance were co-transferred in conjugative plasmids of 70 to 100 MDa in size, while ampicillin, trimethoprim-sulfamethoxazole and tetracycline resistance were co-transferred in conjugative plasmids of 50 to 90 MDa. Pulsed-field gel electrophoresis analysis revealed diverse genetic fingerprints of pathogenic isolates. Multi-drug resistant E. coli are wide spread in public water supply in Dhaka city, Bangladesh. Transmission of resistant bacteria and plasmids through supply water pose serious threats to public health in urban areas.

In the early 1900s, with mortality of ∼30%, typhoid and paratyphoid (caused by Salmonella Typhi and Paratyphi A) ravaged parts of the world; with improved water, sanitation, and hygiene in resource-rich countries and the advent of antimicrobials, mortality dwindled to <1%. Today, the burden rests disproportionately on South Asia, where the primary means for combatting the disease is antimicrobials. The rising prevalence of antimicrobial resistance in Salmonella enterica serovars Typhi and Paratyphi A, causative agents of typhoid and paratyphoid, have led to fears of untreatable infections. Of specific concern is the emerging resistance against azithromycin, the only remaining oral drug to treat extensively drug resistant (XDR) typhoid. Since the first report of azithromycin resistance from Bangladesh in 2019, cases have been reported from Nepal, India, and Pakistan. The genetic basis of this resistance is a single point mutation in the efflux pump AcrB (R717Q/L). Here, we report 38 additional cases of azithromycin-resistant (AzmR) Salmonella Typhi and Paratyphi A isolated in Bangladesh between 2016 and 2018. Using genomic analysis of 56 AzmR isolates from South Asia with AcrB-R717Q/L, we confirm that this mutation has spontaneously emerged in different Salmonella Typhi and Paratyphi A genotypes. The largest cluster of AzmR Typhi belonged to genotype 4.3.1.1; Bayesian analysis predicts the mutation to have emerged sometime in 2010. A travel-related Typhi isolate with AcrB-R717Q belonging to 4.3.1.1 was isolated in the United Kingdom, increasing fears of global spread. For real-time detection of AcrB-R717Q/L, we developed an extraction-free, rapid, and low-cost mismatch amplification mutation assay (MAMA). Validation of MAMA using 113 AzmR and non-AzmR isolates yielded >98% specificity and sensitivity versus phenotypic and whole-genome sequencing assays currently used for azithromycin resistance detection. With increasing azithromycin use, AcrB-R717Q/L is likely to be acquired by XDR strains. The proposed tool for active detection and surveillance of this mutation may detect pan-oral drug resistance early, giving us a window to intervene. IMPORTANCE In the early 1900s, with mortality of ∼30%, typhoid and paratyphoid ravaged parts of the world; with improved water, sanitation, and hygiene in resource-rich countries and the advent of antimicrobials, mortality dwindled to <1%. Today, the burden rests disproportionately on South Asia, where the primary means for combatting the disease is antimicrobials. However, prevalence of antimicrobial resistance is rising and, in 2016, an extensively drug resistant Typhi strain triggered an ongoing outbreak in Pakistan, leaving only one oral drug, azithromycin, to treat it. Since the description of emergence of azithromycin resistance, conferred by a point mutation in acrB (AcrB-R717Q/L) in 2019, there have been increasing numbers of reports. Using genomics and Bayesian analysis, we illustrate that this mutation emerged in approximately 2010 and has spontaneously arisen multiple times. Emergence of pan-oral drug resistant Salmonella Typhi is imminent. We developed a low-cost, rapid PCR tool to facilitate real-time detection and prevention policies.

Rising antimicrobial resistance (AMR) in Salmonella Typhi restricts typhoid treatment options, heightening concerns for pan-oral drug-resistant outbreaks. However, lack of long-term temporal surveillance data on AMR in countries with high burden like Bangladesh is scarce. Our study explores the AMR trends of Salmonella Typhi isolates from Bangladesh, drawing comparisons with antibiotic consumption to optimize antibiotic stewardship strategies for the country. The typhoid fever surveillance from 1999 to 2022 included two pediatric hospitals and three private clinics in Dhaka, Bangladesh. Blood cultures were performed at treating physicians' discretion; cases were confirmed by microbiological, serological, and biochemical tests. Antibiotic susceptibility was determined following CLSI guidelines. National antibiotic consumption data for cotrimoxazole, ciprofloxacin, and azithromycin was obtained from IQVIA-MIDAS database for comparison. Over the 24 years of surveillance, we recorded 12,435 culture-confirmed typhoid cases and observed declining resistance to first-line drugs (amoxicillin, chloramphenicol, and cotrimoxazole); multidrug resistance (MDR) decreased from 38% in 1999 to 17% in 2022. Cotrimoxazole consumption dropped from 0.8 to 0.1 Daily defined doses (DDD)/1000/day (1999-2020). Ciprofloxacin non-susceptibility persisted at >90% with unchanged consumption (1.1-1.3 DDD/1000/day, 2002-2020). Low ceftriaxone resistance (<1%) was observed, with slightly rising MIC (0.03 to 0.12 mg/L, 1999-2019). Azithromycin consumption increased (0.1 to 3.8 DDD/1000/day, 1999-2020), but resistance remained ≤4%. Our study highlights declining MDR amongst Salmonella Typhi in Bangladesh; first-line antimicrobials could be reintroduced as empirical treatment options for typhoid fever if MDR rates further drops below 5%. The analysis also provides baseline data for monitoring the impact of future interventions like typhoid conjugate vaccines on typhoid burden and associated AMR.

Typhoid fever, caused by Salmonella enterica serovar Typhi, is responsible for an estimated burden of approximately 17 million new episodes per year worldwide. Adequate and timely antimicrobial treatment invariably cures typhoid fever. The increasing antimicrobial resistance (AMR) of S . Typhi severely limits the treatment options. We studied whole-genome sequences (WGS) of 536 S . Typhi isolates collected in Bangladesh between 1999 and 2013 and compared those sequences with data from a recent outbreak in Pakistan and a laboratory surveillance in Nepal. The analysis suggests that multiple ancestral origins of resistance against ciprofloxacin and ceftriaxone are present in three countries. Such independent genetic events and subsequent dissemination could enhance the risk of a rapid global spread of these highly resistant clones. Given the current treatment challenges, vaccination seems to be the most appropriate short-term intervention to reduce the disease burden of typhoid fever at a time of increasing AMR. Typhoid fever, caused by Salmonella enterica serovar Typhi, is a global public health concern due to increasing antimicrobial resistance (AMR). Characterization of S . Typhi genomes for AMR and the evolution of different lineages, especially in countries where typhoid fever is endemic such as Bangladesh, will help public health professionals to better design and implement appropriate preventive measures. We studied whole-genome sequences (WGS) of 536 S . Typhi isolates collected in Bangladesh during 1999 to 2013 and compared those sequences with data from a recent outbreak in Pakistan reported previously by E. J. Klemm, S. Shakoor, A. J. Page, F. N. Qamar, et al. (mBio 9:e00105-18, 2018, https://doi.org/10.1128/mBio.00105-18 ), and a laboratory surveillance in Nepal reported previously by C. D. Britto, Z. A. Dyson, S. Duchene, M. J. Carter, et al. [PLoS Negl. Trop. Dis. 12(4):e0006408, 2018, https://doi.org/10.1371/journal.pntd.0006408 ]. WGS had high sensitivity and specificity for prediction of ampicillin, chloramphenicol, co-trimoxazole, and ceftriaxone AMR phenotypes but needs further improvement for prediction of ciprofloxacin resistance. We detected a new local lineage of genotype 4.3.1 (named lineage Bd) which recently diverged into a sublineage (named Bdq) containing qnr genes associated with high-level ciprofloxacin resistance. We found a ceftriaxone-resistant isolate with the bla CTX-M-15 gene and a genotype distinct from the genotypes of extensively drug-resistant (XDR) isolates from Pakistan. This result suggests a different source and geographical origin of AMR. Genotype 4.3.1 was dominant in all three countries but formed country-specific clusters in the maximum likelihood phylogenetic tree. Thus, multiple independent genetic events leading to ciprofloxacin and ceftriaxone resistance took place in these neighboring regions of Pakistan, Nepal, and Bangladesh. These independent mutational events may enhance the risk of global spread of these highly resistant clones. A short-term global intervention plan is urgently needed. IMPORTANCE Typhoid fever, caused by Salmonella enterica serovar Typhi, is responsible for an estimated burden of approximately 17 million new episodes per year worldwide. Adequate and timely antimicrobial treatment invariably cures typhoid fever. The increasing antimicrobial resistance (AMR) of S . Typhi severely limits the treatment options. We studied whole-genome sequences (WGS) of 536 S . Typhi isolates collected in Bangladesh between 1999 and 2013 and compared those sequences with data from a recent outbreak in Pakistan and a laboratory surveillance in Nepal. The analysis suggests that multiple ancestral origins of resistance against ciprofloxacin and ceftriaxone are present in three countries. Such independent genetic events and subsequent dissemination could enhance the risk of a rapid global spread of these highly resistant clones. Given the current treatment challenges, vaccination seems to be the most appropriate short-term intervention to reduce the disease burden of typhoid fever at a time of increasing AMR.

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.

Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

IntroductionThe newly identified SARS-CoV-2 can cause serious acute respiratory infections such as pneumonia. In France, mortality rate in the general population was approximately 10% and could reach higher levels at the hospital. In the current context of high incidence rates of SARS-CoV-2 in the community, a significant increase in the rate of nosocomial transmission is expected. The risk of nosocomial transmission could even be higher in low-income countries that have fragile healthcare systems. This protocol is intended to estimate the prevalence and incidence of suspected or confirmed cases of nosocomial SARS-CoV-2 infection, the clinical spectrum and the determinants (risk factors/protective) at participating hospitals.Methods and analysisThis will be an international multicentre prospective, observational, hospital-based study in adults and children. It will include volunteer patients and healthcare professionals in France and hospitals affiliated with the GABRIEL network. Demographic and clinical data will be collected using case report forms designed especially for the purpose of the project. A nasopharyngeal swab will be collected and tested for SARS-CoV-2 by reverse-transcriptase PCR. Characteristics of the study participants, the proportion of confirmed nosocomial SARS-CoV-2 infections relative to all patients with syndromes suggestive of SARS-CoV-2 infection, will be analysed. Appropriate multivariate modelling will be used to identify the determinants associated with nosocomial onset.Ethics and disseminationThis study was approved by the clinical research and committee of all participating countries. The findings will be submitted to peer-reviewed journal for publication and shared with national health authorities.Trial registration numberNCT04290780.

Background Typhoid fever, caused by Salmonella Typhi, follows a fecal-oral transmission route and is a major global public health concern, especially in developing countries like Bangladesh. Increasing emergence of antimicrobial resistance (AMR) is a serious issue; the list of treatments for typhoid fever is ever-decreasing. In addition to IncHI1-type plasmids, Salmonella genomic island (SGI) 11 has been reported to carry AMR genes. Although reports suggest a recent reduction in multidrug resistance (MDR) in the Indian subcontinent, the corresponding genomic changes in the background are unknown. Results Here, we assembled and annotated complete closed chromosomes and plasmids for 73  S. Typhi isolates using short-length Illumina reads. S. Typhi had an open pan-genome, and the core genome was smaller than previously reported. Considering AMR genes, we identified five variants of SGI11, including the previously reported reference sequence. Five plasmids were identified, including the new plasmids pK91 and pK43; pK43and pHCM2 were not related to AMR. The pHCM1, pPRJEB21992 and pK91 plasmids carried AMR genes and, along with the SGI11 variants, were responsible for resistance phenotypes. pK91 also contained qnr genes, conferred high ciprofloxacin resistance and was related to the H58-sublineage Bdq, which shows the same phenotype. The presence of plasmids (pHCM1 and pK91) and SGI11 were linked to two H58-lineages, Ia and Bd. Loss of plasmids and integration of resistance genes in genomic islands could contribute to the fitness advantage of lineage Ia isolates. Conclusions Such events may explain why lineage Ia is globally widespread, while the Bd lineage is locally restricted. Further studies are required to understand how these S. Typhi AMR elements spread and generate new variants. Preventive measures such as vaccination programs should also be considered in endemic countries; such initiatives could potentially reduce the spread of AMR.

The epidemiology, clinical characteristics, management and outcome of Guillain–Barré syndrome (GBS) differ between low-income and middle-income countries (LMIC) and high-income countries (HIC). At present, limited data are available on GBS in LMIC and the true incidence of GBS in many LMIC remains unknown. Increased understanding of GBS in LMIC is needed because poor hygiene and high exposure to infections render populations in LMIC vulnerable to GBS outbreaks. Furthermore, insufficient diagnostic and health-care facilities in LMIC contribute to delayed diagnosis in patients with severe presentations of GBS. In addition, the lack of national clinical guidelines and absence of affordable, effective treatments contribute to worse outcomes and higher mortality in LMIC than HIC. Systematic population-based surveillance studies, cohort and case–control studies are required to understand the incidence and risk factors for GBS. Novel, targeted and cost-effective treatment strategies need to be developed in the context of health system challenges in LMIC. To ensure integrative rehabilitation services in LMIC, existing prognostic models must be validated, and responsive outcome measures that are cross-culturally applicable must be developed. Therefore, fundamental and applied research to improve the clinical management of GBS in LMIC should become a critical focus of future research programmes.The incidence and severity of Guillain–Barré syndrome (GBS) are increased in low-income and middle-income countries (LMIC) by distinct geographic, economic and environmental factors. Here, Papri and colleagues highlight the most important challenges and knowledge gaps relating to GBS in LMIC.

Background: Campylobacter jejuni is the predominant antecedent infection in Guillain-Barré syndrome (GBS). Molecular mimicry and cross-reactive immune responses to C. jejuni lipo-oligosaccharides (LOS) precipitate the development of GBS, although this mechanism has not been established in patients from developing countries. We determined the carbohydrate mimicry between C. jejuni LOS and gangliosides, and the cross-reactive antibody response in patients with GBS in Bangladesh. Methodology: Sera from 97 GBS patients, and 120 neurological and family controls were tested for antibody reactivity against LOS from C. jejuni isolates from GBS patients in Bangladesh (BD-07, BD-39, BD-10, BD-67 and BD-94) by enzyme-linked immunosorbent assay (ELISA). Cross-reactivity to LOS was determined by ELISA. The LOS outer core structures of C. jejuni strains associated with GBS/MFS were determined by mass spectrometry. Principle Findings: IgG antibodies to LOS from C. jejuni BD-07, BD-39, BD-10, and BD-67 IgG antibodies were found in serum from 56%, 58%, 14% and 15% of GBS patients respectively, as compared to very low frequency (<3%) in controls (p<0.001). Monoclonal antibodies specific for GM1 and GD1a reacted strongly with LOS from the C. jejuni strains (BD-07 and BD-39). Mass spectrometry analysis confirmed the presence of GM1 and GD1a carbohydrate mimics in the LOS from C. jejuni BD-07 and BD-39. Both BD-10 and BD-67 express the same LOS outer core, which appears to be a novel structure displaying GA2 and GD3 mimicry. Up to 90-100% of serum reactivity to gangliosides in two patients (DK-07 and DK-39) was inhibited by 50 μg/ml of LOS from the autologous C. jejuni isolates. However, patient DK-07 developed an anti-GD1a immune response while patient DK-39 developed an anti-GM1 immune response. Conclusion: Carbohydrate mimicry between C. jejuni LOS and gangliosides, and cross-reactive serum antibody precipitate the majority of GBS cases in Bangladesh.