Linkage of Whole Genome Sequencing, Epidemiological, and Clinical Data to Understand the Genetic Diversity and Clinical Outcomes of Shigella flexneri among Men Who Have Sex with Men in England.

The public health value of whole genome sequencing (WGS) for Shigella spp. in England has been limited by a lack of information on sexual identity and behavior. We combined WGS data with other data sources to better understand Shigella flexneri transmission in men who have sex with men (MSM). WGS data for all S. flexneri isolates referred to the national reference laboratory were linked to i) clinical and behavioral data collected in seven of 21 health regions in England using a standardized exposure questionnaire and, ii) national HIV surveillance data. We included 926 S. flexneri isolates, of which 43.0% (n = 398) fell phylogenetically within two domestically circulating clades associated with genotypic markers of azithromycin resistance. Approximately one third of isolates in these clades were from people living with HIV, primarily acquired through sex between men. 182 (19.7%) isolates had linked questionnaire data; 88% (84/95) of MSM isolates fell phylogenetically within the domestically circulating clades, while 92% (72/78) of isolates from other cases fell within lineages linked with travel to high-risk regions. There was no evidence of sustained transmission between networks of MSM and the wider community. MSM were more likely to be admitted to hospital and receive antimicrobials. Our study emphasizes the importance of sex between men as a major route of transmission for S. flexneri. Combined WGS, epidemiological and clinical data provide unique insights that can inform contact tracing, clinical management and the delivery of targeted prevention activities. Future studies should investigate why MSM experience more severe clinical outcomes. IMPORTANCE Within the last 2 decades there have been an increasing number of Shigella spp. outbreaks among men who have sex with men (MSM) worldwide. In 2015, Public Health England (PHE) introduced routine whole genome sequencing (WGS) for the national surveillance of Shigella spp. However, the lack of information on sexual identity and behavior has hindered interpretation. Our study illustrates the power of linking WGS data with epidemiological, behavioral, and clinical data. We provide unique population-level insights into different transmission networks that can inform the delivery of appropriate public health interventions and patient management. Furthermore, we describe and compare clinical characteristics and outcomes of S. flexneri infection in MSM and other exposure groups. We found that MSM were more likely to be admitted to hospital and receive antimicrobials, indicating that their infections were potentially more severe. The exact reasons for this are unclear and require further exploration.

Ultrasensitive pathogen detection with a rolling circle amplification-empowered multiplex electrochemical DNA sensor.

We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.

Emergence of novel strains of Shigella flexneri associated with sexual transmission in adult men in England, 2019-2020.

National surveillance of shigellosis in England revealed an increase in sexually transmitted Shigella flexneri in adult males in 2019 that persisted throughout 2020. We observed a resurgence of azithromycin-resistant S. flexneri serotype 3a, and the emergence of two novel multidrug-resistant clades of S. flexneri 2a and S. flexneri 1b.

Associated factors, post infection child growth, and household cost of invasive enteritis among under 5 children in Bangladesh.

Both Campylobacter- and Shigella-induced invasive enteritis are common in under-5 Bangladeshi children. Our study aimed to determine the factors associated with Campylobacter and Shigella enteritis among under-5 children, the post-infection worsening growth, and the household cost of invasive enteritis. Data of children having Shigella (591/803) and Campylobacter (246/1148) isolated from the fecal specimen in Bangladesh were extracted from the Global Enteric Multicenter Study (GEMS) for the period December 2007 to March 2011. In multiple logistic regression analysis, fever was observed more frequently among shigellosis cases [adjusted OR 2.21; (95% CI 1.58, 3.09)]. Breastfeeding [aOR 0.55; (95% CI 0.37, 0.81)] was found to be protective against Shigella. The generalized estimating equations multivariable model identified a negative association between Shigella and weight-for-height z score [aOR - 0.11; (95% CI - 0.21, - 0.001)]; a positive association between symptomatic Campylobacter and weight-for-age z score [aOR 0.22; (95% CI 0.06, 0.37)] and weight-for-height z score [aOR 0.22; (95% CI 0.08, 0.37)]. Total costs incurred by households were more in shigellosis children than Campylobacter-induced enteritis ($4.27 vs. $3.49). Households with low-level maternal education tended to incur less cost in case of their shigellosis children. Our findings underscore the need for preventive strategies targeting Shigella infection, which could potentially reduce the disease burden, associated household costs, and child growth faltering.

Prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw foods in Peshawar, Pakistan.

This study was designed to investigate the prevalence and associated risk factors of Shigella flexneri isolated from drinking water and retail raw food samples in Peshawar, Pakistan. A total of 1,020 different samples were collected from various areas of Peshawar between January 2016 and May 2017, followed by identification of S. flexneri through biochemical, serological, and 16S rRNA gene sequencing. Potential risk factors associated with the development and spreading of S. flexneri infection were also investigated. Overall, 45 (4.41%) samples were positive for Shigella species. Among these samples, the predominant species was S. flexneri (n = 44) followed by S. boydii (n = 1). Interestingly, S. sonnei and S. dysenteriae isolates were not found in any sample. The isolation rate of S. flexneri in drinking water samples, market raw milk, and fruits/vegetables from Peshawar were 6.47%, 3.5%, and 2.9%, respectively. The phylogenetic reconstruction showed genetic diversity among three clades, as clades I and II have isolates of S. flexneri that were circulating within the drinking water, milk, fruits/vegetables, while clade III isolates were recovered from milk samples. Most of S. flexneri were detected in June to September. Potential risk factors of S. flexneri were water sources contaminated by toilet wastes (p = 0.04), surface water drainage (p = 0.0002), hospital wastes (p = 0.01), unhygienic handling (p < 0.05), and transportation of raw food (p = 0.04). In conclusion, S. flexneri isolates of closely related lineage originating from non-clinical samples might be associated with an increased human risk to shigellosis in Pakistan, as significant numbers of S. flexneri were observed in the drinking water and retail raw food samples. PRACTICAL APPLICATION: This study demonstrated the presence of S. flexneri in drinking water and retail raw food samples which seem to possess a serious threat to public health. Potential sources of food and water contamination should properly be monitored by public health authorities to reduce cases of shigellosis.

Correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri and the sul genes.

ABSTRACT: The aim of this study was to discuss the correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri (S. flexneri) and the antibiotic resistance genes sul1, sul2, and sul3 and SXT element.From May 2013 to October 2018, 102 isolates of S. flexneri were collected from the clinical samples in Jinan. The Kirby-Bauer (K-B) test was employed to determine the antibiotic susceptibility of the S. flexneri isolates. The antibiotic resistance rate was analyzed with the WHONET5.4 software. The isolates were subject to the PCR amplification of the sul genes (sul1, sul2, and sul3) and the SXT element. On the basis of the sequencing results, the correlation between the sulfamethoxazole-trimethoprim resistance of the S. flexneri isolates and the sul genes was analyzed.The antibiotic resistance rates of the 102 S. flexneri isolates to ampicillin, streptomycin, chloramphenicol, tetracycline, and sulfamethoxazole-trimethoprim were 90.2%, 90.2%, 88.2%, 88.2%, and 62.7%, respectively. The antibiotic resistance rates of these isolates to cefotaxime, ceftazidime, and ciprofloxacin varied between 20% and 35%. However, these isolates were 100% susceptible to cefoxitin. Positive fragments were amplified from 59.8% (61/102) of the 102 S. flexneri isolates, the sizes of the sul1 and sul2 genes being 338 bp and 286 bp, respectively. The sequence alignment revealed the presence of the sul1 and sul2 genes encoding for dihydrofolate synthase. The carrying rate of the sul1 gene was 13.7% (14/102), and that of the sul2 gene was 48.0% (49/102). No target gene fragments were amplified from the 3 isolates resistant to sulfamethoxazole-trimethoprim. The sul3 gene and SXT element were not amplified from any of the isolates. The testing and statistical analysis showed that the resistance of the S. flexneri isolates to sulfamethoxazole-trimethoprim correlated to the sul1 and sul2 genes.The acquired antibiotic resistance genes sul1 and sul2 were closely associated with the resistance of the 102 S. flexneri isolates to sulfamethoxazole-trimethoprim.

Selection and Characterization of Cell Surface Specific Aptamer and Development of Fluorescence Assay for Detection of Shigella flexneri from Water Samples.

The present study demonstrates, development of ssDNA aptamers against whole cell of S. flexneri employing a whole bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX). After ten rounds of SELEX, cell surface specific aptamer pool was cloned, sequenced and divided based on sequence similarities and secondary structure. Binding affinity of FITC labelled aptamer from different group were carried out by flow cytometry analysis. The dissociation constant (Kd) values for specific and higher binder were evaluated to range from 144 to 329 nM. Six high binding aptamers with lower dissociation constant was chosen for selectivity study. Aptamer SHI 23, SHI 37 and SHI 42 showed higher selectivity towards S. flexneri in comparison with other related bacteria. Further applicability of selected aptamer was proven by fluorescence assay for convenience detection of target cell from spiked water sample and natural contaminated water samples. Altogether, aptamer generated in this study can be alternative DNA ligands for detection of S. flexneri compared to available ligands.

Outbreak of multi-drug-resistant (MDR) Shigella flexneri in northern Australia due to an endemic regional clone acquiring an IncFII plasmid.

Epidemiological surveillance of Shigella spp. in Australia is conducted to inform public health response. Multi-drug resistance has recently emerged as a contributing factor to sustained local transmission of Shigella spp. All data were collected as part of routine public health surveillance, and strains were whole-genome sequenced for further molecular characterisation. 108 patients with an endemic regional Shigella flexneri strain were identified between 2016 and 2019. The S. flexneri phylogroup 3 strain endemic to northern Australia acquired a multi-drug resistance conferring blaDHA plasmid, which has an IncFII plasmid backbone with virulence and resistance elements typically found in IncR plasmids. This is the first report of multi-drug resistance in Shigella sp. in Australia that is not associated with men who have sex with men. This strain caused an outbreak of multi-drug-resistant S. flexneri in northern Australia that disproportionality affects Aboriginal and Torres Strait Islander children. Community controlled public health action is recommended.

Association of Serotype With Antimicrobial Resistance Patterns Among Shigella flexneri Isolates From Pakistan: The Importance of Serotype 2b.

BACKGROUND: Shigella flexneri is an emerging threat in low socioeconomic countries including Pakistan. No previous data is available on the association between S. flexneri serotypes and antimicrobial resistance in Pakistan. OBJECTIVES: The objective of the present study was to assess the association between serotypes and antimicrobial resistance patterns among S. flexneri isolated from clinical and nonclinical samples. METHODS: A total of 199 S. flexneri isolates were subjected to molecular serotyping and antibiotic resistance. RESULTS: The most prevalent S. flexneri serotype was 2b (38%) followed by 1b (24%), 7a (20%), 2a (11%), 1d (5%) and Y (2%). The phylogenetic reconstruction showed 12 clades among which the clades II, III, V, VIII, IX and XI have consisted of serotypes that were found both in human population and environment samples. A high level of multidrug resistance (MDR) was observed in serotype 2b (37.68%) followed by 1b (19.5%) and 7a (19.5%), 2a (11.5%), 1d (5%) and Y (2%). All isolates of serotype 2b showed high level of resistance to amoxicillin/clavulanic acid (100%) followed by quinolone (74.6%) and trimethoprim-sulfamethoxazole (54.6%). Interestingly, none of the serotype was resistant to piperacillin-tazobactam, imipenem and amikacin. The most frequently detected resistance genes among serotype 2b were blaOXA (100%) followed by qnrS (88%), cat (81%) and sul2 (63%). CONCLUSION: The most frequent S. flexneri serotype was 2b while 1d and Y was first time reported in Pakistan. High frequency of MDR serotypes of S. flexneri is a serious threat in diarrhea endemic regions and thus require urgent strategies for its continuous monitoring and prevention.

A high number of multidrug-resistant and predominant genetically related cluster of Shigella flexneri strains isolated over 34 years in Brazil.

Shigella flexneri has been a major public health problem in developing countries. This work analyzed the frequency of 16 virulence genes, the genotypic diversity, and the antimicrobial resistance profiles of 130 S. flexneri strains isolated in Brazil. The ipaH gene was found in all the 130 strains. The frequencies of the other genes were variable ial (88.5%), sigA (82.3%), iuc (74.6%), virA (73%), pic (72.3%), virF (57.7%), sat (48.5%), ipaBCD (37%), sen (36%), set1A (35.4%), sepA (30%), set1B (30%), virB (14%), icsA (10%), and ipgD (5.4%). A total of 57 (43.8%) strains were multidrug-resistant. ERIC-PCR grouped 96 of the strains into a single cluster with ≥ 70.4% of similarity, 75 of these strains presented a similarity ≥ 80.9%. PFGE grouped 120 of the strains into a single cluster with 57.4% of similarity and 82 of these strains presented a similarity ≥ 70.6%. In conclusion, the high frequency of some virulence genes reinforces the pathogenic potential of the strains studied. The high rates of MDR strains are alarming once it may lead to failure when antimicrobial treatment is necessary. Genotype techniques reveled a major cluster with high genetic similarity including S. flexneri strains from the different Brazilian states and distinct years of isolation, showing that they probably emerged from a common ancestor.

FTIR-assisted MALDI-TOF MS for the identification and typing of bacteria.

MALDI-TOF MS is well-recognized for microbial identification and widely used in research and clinical fields due to its specificity, speed of analysis, and low cost of consumables. However, the classification or identification accuracy is poor for E. coli and Shigella. In addition, FTIR is a promising tool for bacterial typing. In this study, 14 strains of E. coli and 9 strains of Shigella were typed by both MALDI-TOF MS and FTIR techniques. Alternatively, a data fusion strategy using these two approaches was attempted to achieve better typing accuracy. Hierarchical clustering analysis (HCA) revealed that the typing accuracies for selected E. coli and Shigella from blood agar were 65.2%, 78.3%, and 100% for MALDI-TOF MS, FTIR, and FTIR combined with MALDI-TOF MS, respectively. Based on these results, a library constructed of the MS-IR fusion data was employed to identify individual bacteria at the strain level, and the library-based typing accuracies for 207 test spectra of sample strains from three different culture media yielded 97.6% accuracy for distinguishing between E. coli and Shigella at genus level. For species level and strain level, it yielded 95.2% and 92.3% typing accuracy, respectively. These results indicate that FTIR is a better technique for typing selected E. coli and Shigella than MALDI-TOF MS, and the accuracy is increased by combining the data from MALDI-TOF MS and FTIR spectroscopy. FTIR could be used to supplement MALDI-TOF MS for the identification and typing of taxonomic microorganisms.

A propósito de un nuevo caso de shigelosis por una cepa multirresistente no importada / No disponible

No disponible

Shigella flexneri: an emerging pathogen.

Shigella flexneri is a leading etiologic agent of diarrhea in low socioeconomic countries. Notably, various serotypes in S. flexneri are reported from different regions of the world. The precise approximations of illness and death owing to shigellosis are missing in low socioeconomic countries, although it is widespread in different regions. The inadequate statistics available reveal S. flexneri to be a significant food and waterborne pathogen. All over the world, different antibiotic-resistant strains of S. flexneri serotypes have been emerged especially multidrug-resistant strains. Recently, increased resistance was observed in cephalosporins (3rd generation), azithromycin, and fluoroquinolones. There is a need for a continuous surveillance study on antibiotic resistance that will be helpful in the update of the antibiogram. The shigellosis burden can be reduced by adopting preventive measures like delivery of safe drinking water, suitable sanitation, and development of an effective and inexpensive multivalent vaccine. This review attempts to provide the recent findings of S. flexneri related to epidemiology and the emergence of multidrug resistance.

Molecular epidemiology of Shigella flexneri isolated from pediatrics in a diarrhea-endemic area of Khyber Pakhtunkhwa, Pakistan.

Shigella flexneri is considered as an important causative agent of Shigellosis causing diarrhea in the countries with a low socioeconomic status. No study has been carried out on the molecular prevalence of S. flexneri in Khyber Pakhtunkhwa, Pakistan. So this study was designed to evaluate the molecular prevalence of S. flexneri and their associated risk factors. A total of 2014 diarrheal stool samples were collected from January 2016 to May 2017 from pediatrics patients of Khyber Pakhtunkhwa followed by identification of S. flexneri through biochemical, serological, and molecular methods. The overall prevalence of Shigella species was found to be 7.9% (n = 160). The predominant Shigella specie was S. flexneri (n = 155, 96.8%) followed by S. boydii (n = 5, 3.1%). Interestingly, no sample was found positive for S. sonnei and S. dysenteriae. The majority of Shigellosis cases occurred from June to September. Potential risk factors related with Shigellosis were unhygienic latrine usage, bad hand washing, and consumption of unhygienic food and water, and pipe leakage in the sewage system. In this study, we have observed a high number of Shigellosis cases especially those caused by S. flexneri. It is suggested that effective health awareness programs should be organized by the regional health authorities to minimize the magnitude of pediatrics Shigellosis.

Development of quinolone resistance and prevalence of different virulence genes among Shigella flexneri and Shigella dysenteriae in environmental water samples.

The purpose of this study was to find out the mechanism of quinolone resistance in Shigella sp. isolated from environmental water samples from various parts of Kolkata, India. Out of 196 Shigella sp. isolated from 2014 to 2017, we selected 32 Shigella isolates for antimicrobial susceptibility tests. The minimum inhibitory concentrations (MIC) for quinolones ranged from 30 to 50 µg ml-1 for ofloxacin, 5-20 µg ml-1 for ciprofloxacin and 20-30 µg ml-1 for norfloxacin. A few amino acid changes were found in quinolone resistance determining region (QRDR) of gyrA. Mutations in gyrA lead to a higher increment of MIC of quinolones. Among the plasmid-mediated (PMQR) quinolone resistance genes investigated, qnrB and aac(6')-lb-cr genes were found in all isolates. qnrA and qnrS were found in 25% and 62% of the isolates, respectively. ipaH gene was found in all of the isolates followed by the presence of other virulence genes ial, sen and stx1. Almost all the isolates having high MICs showed efflux pump activity in drug accumulation assay. All the mechanisms may or may not be present in a single strain. Several types of efflux pumps, presence of PMQR genes and mutations in drug target site of QRDR region may play the crucial role for resistance in our isolates.

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria.

BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.

Dissemination of Multidrug-Resistant Shigella flexneri and Shigella sonnei with Class 1, Class 2, and Atypical Class 1 Integrons in China.

Background: Emergence of multidrug-resistant Shigella, a major causative agent of bacterial dysentery, has generated many concerns not only in China but also worldwide. However, the prevalence of Shigella resistance caused by integron in the nonpopular season of diarrhea is not clear. Materials and Methods: Thirty-one Shigella flexneri and 22 Shigella sonnei samples collected in December 2010 from 10 cities of China were characterized for antimicrobial susceptibility, gene cassettes, widespread of integrons, and pulsed-field gel electrophoresis (PFGE) profile. Results: Multidrug resistance (MDR) was detected in 29 (93.5%) S. flexneri and 20 (90.9%) S. sonnei isolates. Class 1 integrons were detected in 25 (80.6%) S. flexneri and in 13 (59.1%) S. sonnei isolates; class 2 integrons were detected in 26 (83.9%) S. flexneri and in 19 (86.4%) S. sonnei isolates. Interestingly, the atypical class 1 integrons were mostly detected in S. flexneri (45.2%) isolates, whereas in only 1 (4.5%) S. sonnei isolate. DNA sequencing revealed two novel cassette arrays, dfrA5 and aacA4-cmlA, of class 1 integrons in S. flexneri, and dfrA17-aadA5 in S. sonnei isolates. The cassette arrays, dfrA1-sat1-aadA1 of class 2 integron and blaoxa-30-aadA1 of atypical class 1 integron, were also identified. PFGE profiles demonstrated A6 subtype of S. flexneri strains prevalent in Shanghai, Changchun, Jinan, and Changsha; and F6 subtype of S. sonnei prevalent in Jinan, Changchun, and Shanghai. Conclusion: The dissemination of MDR Shigella strains with integrons makes it an increasing public health problem in China. Increased surveillance and the development of adequate prevention strategies are warranted.

Biomimetic Octopus-like Particles for Ultraspecific Capture and Detection of Pathogens.

Infectious diseases caused by pathogenic bacteria (such as sepsis and meningitis) seriously threaten public health; therefore, rapid and accurate identification of the target bacteria is urgently needed to prevent and treat bacterial infections. Although technologies including plate-counting and polymerase chain reaction have been established to detect the pathogenic bacteria, they are either time-consuming or sophisticated. Herein, a biomimetic octopus-like structure integrating merits of multiarm and multivalent interaction is designed for ultraspecific capture and detection of pathogens. The flexible polymeric arms and multivalent ligands work together to mimic the arm-sucker coordination of an octopus to effectively grasp the target pathogens, leading to remarkably high capacity and specificity for the target capture (above 98%, 10 CFU mL-1) without a nonspecific absorption of background pathogens. The captured bacteria can be identified as a point of care by the surface-enhanced Raman spectroscopy method with a detection limit of 10 cells mL-1.