ABSTRACT
BACKGROUND: The purpose of this study was to look into the presence of plasmid-mediated quinolone resistance (PMQR) genes and biofilm formation in several species of clinical Shigella isolates that were resistant to quinolones. METHODS: The stool samples of 150 patients (younger than 10 years) with diarrhea were collected in this cross-sectional study (November 2020 to December 2021). After cultivation of samples on Hektoen Enteric agar and xylose lysine deoxycholate agar, standard microbiology tests, VITEK 2 system, and polymerase chain reaction (PCR) were utilized to identify Shigella isolates. The broth microdilution method was used to determine antibiotic susceptibility. PMQR genes including qnrA, qnrB, qnrC, qnrD, qnrE, qnrS, qnrVC, qepA, oqxAB, aac(6')-Ib-cr, and crpP and biofilm formation were investigated in quinolone-resistant isolates by PCR and microtiter plate method, respectively. An enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used to determine the clonal relatedness of quinolone-resistant isolates. RESULTS: A total of 95 Shigella isolates including S. sonnei (53, 55.8%), S. flexneri (39, 41.1%), and S. boydii (3, 3.2%) were identified. The highest resistance rates of the isolates were against ampicillin (92.6%, n = 88/95). Overall, 42 of 95 (44.2%) isolates were simultaneously resistant against two or more quinolones including 26 (61.9%) S. sonnei and 16 (38.1%) S. flexneri. All isolates were multidrug-resistant (resistance to more than 3 antibiotics). The occurrence of PMQR genes was as follows: qnrS (52.4%), qnrA and aac(6')-Ib-cr (33.3%), and qnrB (19.0%). The prevalence in species was as follows: 61.5% and 37.5% (qnrS), 19.2% and 56.3% (qnrA), 38.5% and 25.0 (aac(6')-Ib-cr), and 19.2% and 18.8% (qnrB) for S. sonnei and S. flexneri, respectively. The other PMQR genes were not detected. In total, 52.8% (28/53) of quinolone-susceptible and 64.3% (27/42) of quinolone-resistant isolates were biofilm producers. Biofilm formation was not significantly different between quinolone-resistant and quinolone-susceptible isolates (P-value = 0.299). Quinolone-resistant isolates showed a high genetic diversity according to the ERIC-PCR. CONCLUSION: It seems that qnrS, qnrA, and aac(6')-Ib-cr play a significant role in the quinolone resistance among Shigella isolates in our region. Also the quinolone-resistant S. flexneri and S. sonnei isolates had a high genetic diversity. Hence, antibiotic therapy needs to be routinely revised based on the surveillance findings.
Subject(s)
Anti-Bacterial Agents , Biofilms , Microbial Sensitivity Tests , Plasmids , Quinolones , Shigella , Humans , Biofilms/drug effects , Biofilms/growth & development , Cross-Sectional Studies , Quinolones/pharmacology , Shigella/genetics , Shigella/drug effects , Shigella/isolation & purification , Plasmids/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Prevalence , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/epidemiology , Dysentery, Bacillary/drug therapy , FemaleABSTRACT
OBJECTIVE: The current research aimed to investigate the physicochemical and bacteriological quality status of the Kalte River in Wolaita Sodo Town, southern Ethiopia. METHODS: A total of 42 water samples were collected using sterile glass bottles from three different river sites: Damota (upstream), Kera (midstream), and Gututo (downstream). All the water samples were examined for the presence of heterotrophic bacteria, total coliform and fecal coliform using direct plate count method and membrane filtration method. Standard methods suggested by American water works association were used to analysis the physicochemical parameters of the water samples. RESULTS: The results revealed that the total heterotrophic bacteria, total coliform, and fecal coliform count ranged from 8.9 to 12.6 × 104 cfu/ml, 7.5-11.3 × 102 cfu/ml and 5.7-9.7 × 104 cfu/ml, respectively. The bacterial count results indicated that the river water crossed the WHO-recommended limit of potable water. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella, and Shigella species were the common bacterial pathogens isolated from river water samples. The results of the physicochemical analysis revealed that some of the parameters Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), and turbidity exceeded the maximum permissible limits of WHO and other parameters were below the WHO permissible limits. CONCLUSION: Therefore, the presence of bacterial pathogens, fecal coliform indicators, and some physicochemical parameters of the Kalte River exceeding the recommended limits may expose users of the river water to the risk of infection.
Subject(s)
Rivers , Water Microbiology , Ethiopia , Rivers/microbiology , Rivers/chemistry , Bacteria/isolation & purification , Bacteria/classification , Enterobacteriaceae/isolation & purification , Escherichia coli/isolation & purification , Water Quality , Staphylococcus aureus/isolation & purification , Pseudomonas aeruginosa/isolation & purification , Salmonella/isolation & purification , Shigella/isolation & purification , Environmental Monitoring/methods , Drinking Water/microbiologyABSTRACT
Foodborne illnesses, caused by harmful microorganisms in food, are a significant global health issue. Current methods for identifying these pathogens are both labor-intensive and time-consuming. In this research, we devised a swift and precise detection technique using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) for three foodborne pathogens found in meat. By employing a dedicated detection device, RPA-LFD allows for the rapid analysis of DNA from Escherichia coli O157 (E. coli O157), Salmonella, and Shigella-pathogens that are prohibited in food. The detection thresholds for E. coli O157, Salmonella, and Shigella are 0.168 fg/µl (1.04 CFU/ml), 0.72 fg/µl (27.49 CFU/ml), and 1.25 fg/µl (48.84 CFU/ml), respectively. This method provides a short detection window, operates at low temperatures, follows simple procedures, and exhibits high sensitivity. Our study establishes the RPA-LFD method for simultaneously identifying the nucleic acid of three foodborne pathogens, offering an efficient solution for quickly identifying multiple contaminants.
Subject(s)
Escherichia coli O157 , Food Contamination , Food Microbiology , Nucleic Acid Amplification Techniques , Recombinases , Salmonella , Shigella , Escherichia coli O157/isolation & purification , Escherichia coli O157/genetics , Salmonella/isolation & purification , Salmonella/genetics , Nucleic Acid Amplification Techniques/methods , Food Microbiology/methods , Recombinases/metabolism , Shigella/isolation & purification , Shigella/genetics , Food Contamination/analysis , Meat/microbiology , DNA, Bacterial/genetics , Animals , Sensitivity and Specificity , Foodborne Diseases/microbiologyABSTRACT
Shigella bacteria utilize the type III secretion system (T3SS) to invade host cells and establish local infection. Invasion plasmid antigen D (IpaD), a component of Shigella T3SS, has garnered extensive interest as a vaccine target, primarily due to its pivotal role in the Shigella invasion, immunogenic property, and a high degree of conservation across Shigella species and serotypes. Currently, we are developing an epitope- and structure-based multivalent vaccine against shigellosis and require functional epitope antigens of key Shigella virulence determinants including IpaD. However, individual IpaD B-cell epitopes, their contributions to the overall immunogenicity, and functional activities attributing to bacteria invasion have not been fully characterized. In this study, we predicted continuous B-cell epitopes in silico and fused each epitope to a carrier protein. Then, we immunized mice intramuscularly with each epitope fusion protein, examined the IpaD-specific antibody responses, and measured antibodies from each epitope fusion for the activity against Shigella invasion in vitro. Data showed that all epitope fusion proteins induced similar levels of anti-IpaD IgG antibodies in mice, and differences were noted for antibody inhibition activity against Shigella invasion. IpaD epitope 1 (SPGGNDGNSV), IpaD epitope 2 (LGGNGEVVLDNA), and IpaD epitope 5 (SPNNTNGSSTET) induced antibodies significantly better in inhibiting invasion from Shigella flexneri 2a, and epitopes 1 and 5 elicited antibodies more effectively at preventing invasion of Shigella sonnei. These results suggest that IpaD epitopes 1 and 5 can be the IpaD representative antigens for epitope-based polyvalent protein construction and protein-based cross-protective Shigella vaccine development.IMPORTANCEShigella is a leading cause of diarrhea in children younger than 5 years in developing countries (children's diarrhea) and continues to be a major threat to public health. No licensed vaccines are currently available against the heterogeneous Shigella species and serotype strains. Aiming to develop a cross-protective multivalent vaccine against shigellosis and dysentery, we applied novel multiepitope fusion antigen (MEFA) technology to construct a broadly immunogenic polyvalent protein antigen, by presenting functional epitopes of multiple Shigella virulence determinants on a backbone protein. The functional IpaD epitopes identified from this study will essentially allow us to construct an optimal polyvalent Shigella immunogen, leading to the development of a cross-protective vaccine against shigellosis (and dysentery) and the improvement of global health. In addition, identifying functional epitopes from heterogeneous virulence determinants and using them as antigenic representatives for the development of cross-protective multivalent vaccines can be applied generally in vaccine development.
Subject(s)
Antigens, Bacterial , Epitopes, B-Lymphocyte , Shigella flexneri , Antigens, Bacterial/immunology , Antigens, Bacterial/genetics , Animals , Mice , Shigella flexneri/immunology , Shigella flexneri/genetics , Epitopes, B-Lymphocyte/immunology , Shigella Vaccines/immunology , Shigella Vaccines/administration & dosage , Shigella Vaccines/genetics , Dysentery, Bacillary/prevention & control , Dysentery, Bacillary/immunology , Dysentery, Bacillary/microbiology , Mice, Inbred BALB C , Epitope Mapping , Female , Shigella/immunology , Shigella/genetics , Bacterial Proteins/immunology , Bacterial Proteins/genetics , Antibodies, Bacterial/immunology , Antibodies, Bacterial/blood , Shigella sonnei/immunology , Shigella sonnei/genetics , Type III Secretion Systems/immunology , Type III Secretion Systems/geneticsABSTRACT
INTRODUCTION: Shigellosis is a gastrointestinal disease causes high morbidity and mortality worldwide, however, there is no anti-Shigella vaccine. The use of antibiotics in shigellosis treatment exacerbates antibiotic resistance. Antibodies, particularly egg yolk antibody (IgY), offer a promising approach to address this challenge. This study aimed to investigate the prophylactic effect of IgY produced against a recombinant chimeric protein containing the immunogens IpaD, IpaB, StxB, and VirG from Shigella. METHODS: The chimeric protein, comprising IpaD, IpaB, StxB, and VirG, was expressed in E. coli BL21 and purified using the Ni-NTA column. Following immunization of chickens, IgY was extracted from egg yolk using the PEG-6000 method and analyzed through SDS-PAGE and ELISA techniques. Subsequently, the prophylactic efficacy of IgY was assessed by challenging of mice with 10 LD50 of S. dysenteriae and administering different concentrations of IgY (1.25, 2.5, 5, and 10â¯mg/kg) under various time conditions. RESULTS: The recombinant protein, weighing 82â¯kDa, was purified and confirmed by western blotting. The IgY concentration was determined as 9.5â¯mg/ml of egg yolk and the purity of the extracted IgY was over 90â¯%. The results of the ELISA showed that at least 19â¯ng of pure antibody identified recombinant protein and reacts with it. The challenge test employing IgY and Shigella demonstrated a direct correlation between the survival rate and antibody concentration, with increased concentrations leading to decreased mortality rates. Treatment of mice with 10â¯mg/kg IgY leads to 80â¯% survival of the mice against 10 LD50 S. dysenteriae. CONCLUSION: Our findings suggest that IgY may offer therapeutic potential in treating Shigella infections and combating antibiotic resistance.
Subject(s)
Chickens , Dysentery, Bacillary , Egg Yolk , Immunoglobulins , Animals , Immunoglobulins/immunology , Mice , Egg Yolk/immunology , Dysentery, Bacillary/prevention & control , Dysentery, Bacillary/immunology , Shigella/immunology , Bacterial Proteins/immunology , Recombinant Proteins/immunology , Female , Antibodies, Bacterial/immunology , Mice, Inbred BALB C , Antigens, Bacterial/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacologyABSTRACT
Diarrheal diseases are important causes of morbidity and mortality, worldwide. The occurrence of multiple pathogens in stool samples of symptomatic and asymptomatic individuals in resource-limited countries have been repeatedly described. In this study, we assessed the differentiated effects of combined pathogen detections on recorded symptoms. A case-control study was conducted among 620 under-five-year-old children in rural northeastern Tanzania with emphasis of multiple detection. The median age of children was 11 months (IQR = 7, 20), and 52.1% were male. Cases (50.2%, n = 157) were less likely than controls (64.5%, n = 198) to have multiple colonization with gastrointestinal tract (GIT) pathogens. The children's age was positively associated with the likelihood of harboring multiple GIT pathogens [OR, 1.02, 95% CI = 1.01, 1.04]. Shigella spp./enteroinvasive Escherichia coli (EIEC) [OR = 2.80, 95% CI 1.62, 4.83] and norovirus [OR = 2.04, 95% CI 1.23, 3.39] were more common in cases and were strongly associated with diarrhea, while enteroaggregative E. coli (EAEC) [OR = 0.23, 95%CI 0.17-0.33] were more common in controls. Diarrheal diseases in under-five children from rural Tanzania are likely to be due to infections with Shigella spp./EIEC, and norovirus with strongly age-dependent associations.
Subject(s)
Diarrhea , Rural Population , Humans , Tanzania/epidemiology , Male , Female , Case-Control Studies , Diarrhea/epidemiology , Diarrhea/microbiology , Infant , Child, Preschool , Rural Population/statistics & numerical data , Shigella/isolation & purification , Feces/microbiology , Gastrointestinal Tract/microbiology , Norovirus/isolation & purification , Escherichia coli/isolation & purificationABSTRACT
Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain-mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains' residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a closed and open D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell-matrix adhesion.
Subject(s)
Cell Adhesion , Focal Adhesions , Vinculin , Vinculin/metabolism , Vinculin/genetics , Humans , Focal Adhesions/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Mutation , Host-Pathogen Interactions , HeLa Cells , Protein Binding , Shigella/metabolism , Shigella/genetics , Antigens, Bacterial/metabolism , Antigens, Bacterial/genetics , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/metabolismABSTRACT
Shigella spp. are Gram-negative gastrointestinal bacterial pathogens that cause bacillary dysentery or shigellosis in humans. Isolation of Shigella from outbreak-associated foods is often problematic due to the lack of selectivity of cultural enrichment broths. To facilitate Shigella recovery from foods, we have developed strain-specific enrichment media based on the genomically-predicted antimicrobial resistance (AMR) features of an outbreak-associated Shigella sonnei strain harboring resistance genes for streptomycin (STR) and trimethoprim (TMP). To assess performance of the method, baby carrots were artificially contaminated with the S. sonnei strain at low (2.4 CFU), medium (23.5 CFU), and high levels (235 CFU) along with 10-fold higher levels of a Shigella-inhibiting Escherichia coli strain. The target S. sonnei strain was successfully recovered from artificially-contaminated baby carrots when enriched in modified Tryptone Soya Broth (mTSB) supplemented with TMP, whereas Shigella was not recovered from Shigella broth (SB) or SB supplemented with STR. Quantitative PCR analysis indicated that supplementation of the enrichment broths with TMP or STR increased the relative proportion of S. sonnei in enrichment cultures, except at the lowest inoculation level for STR. Microbiome profiling of the baby carrot enrichment cultures conducted by 16S rRNA gene sequencing indicated that both SB-STR and mTSB-TMP repressed the growth of competing Enterobacteriaceae in the enrichment cultures, relative to SB without supplementation. Overall, improved Shigella recovery was achieved with the addition of the appropriate custom selective agent during cultural enrichments demonstrating that genomically informed custom selective enrichment of Shigella could be a valuable tool for supporting future foodborne shigellosis outbreak investigations.
Subject(s)
Daucus carota , Food Microbiology , Shigella sonnei , Humans , Shigella sonnei/drug effects , Shigella sonnei/genetics , Daucus carota/microbiology , Anti-Bacterial Agents/pharmacology , Food Safety , Shigella/drug effects , Shigella/genetics , Dysentery, Bacillary/microbiology , Drug Resistance, Bacterial , Drug Resistance, Microbial , Food Contamination/analysisABSTRACT
We meta-analyzed the diagnostic accuracy of rapid diagnostic tests (dipsticks) and loop-mediated isothermal amplification (LAMP) method to detect Shigella species. We searched MEDLINE, Embase, Web of Science and Google Scholar from inception to 2023 for studies reporting on the performance of Shigella dipstick and LAMP tests compared with culture or polymerase chain reaction (PCR). Our search identified 2618 studies, of which fourteen met the inclusion criteria for the systematic review. Ten studies covering 4056 tests (from twelve countries) were included in the meta-analysis. The overall pooled sensitivity and specificity were 98% (95% CI: 94-100) and 97% (95% CI: 92-99), respectively. Pooled sensitivity and specificity of dipsticks were 95% and 98%, respectively. In contrast, LAMP showed higher pooled sensitivity (100%) and diagnostic odds ratio (431752), but similar specificity (97%). LAMP and dipstick tests exhibited promising performance, suggesting that they could be useful for assisting in the diagnosis of shigellosis.
Subject(s)
Dysentery, Bacillary , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Sensitivity and Specificity , Shigella , Humans , Nucleic Acid Amplification Techniques/methods , Shigella/isolation & purification , Shigella/genetics , Dysentery, Bacillary/diagnosis , Dysentery, Bacillary/microbiology , Molecular Diagnostic Techniques/methods , Diagnostic Tests, Routine/methods , Rapid Diagnostic TestsABSTRACT
BACKGROUND: Diarrhea caused by Salmonella and Shigella species are the leading cause of illness especially in developing countries. These infections are considered as the main public health problems in children, including Ethiopia. This study aimed to assess the prevalence, associated factors, and antimicrobial susceptibility patterns of Salmonella and Shigella species in Sheik Hassan Yabere Referral Hospital Jigjiga, Eastern Ethiopia from August 05 to November 15, 2022. METHOD: A cross-sectional study was conducted among 239 under-five children with diarrhea selected through a convenient sampling technique. A structured questionnaire was used to collect associated factors. A stool sample was collected and processed for the identification of Salmonella and Shigella species using MacConkey adar, Xylose Lysine Deoxycholate agar (Oxoid Ltd) and Biochemical tests. The antimicrobial susceptibility pattern of isolates was performed using the Kirby-Bauer disc diffusion technique. The data was entered into Epi-data version 4.6 and exported to the statistical package of social science version 22 for analysis. The association between outcome and independent variables was assessed using bivariate, multivariable, and chi-square and P-value < 0.05 was considered as statistical significance. RESULT: Overall prevalence of Salmonella and Shigella species was 6.3% (95% CI, 5.7-6.9%), of which 3.8% (95 CI, 3.2-4.4%) were Salmonella species and 2.5% (95% CI, 1.95-3%) were Shigella species. Unimproved water source (AOR = 5.08, 95% CI = 1.45, 17.25), open field (AOR = 2.3, 95% CI = 1.3, 5.03), rural residence (AOR = 1.8, 95% CI = 1.4, 7.5), Hand-washing practice (p = 0.001), and raw meat consumption (p = 0.002) were associated with occurrence of Salmonella and Shigella species. Salmonella and Shigella isolates were resistant to Ampicilin (100%). However, Salmonella isolates was sensitive to Norfloxacin (100%). About 22.2% and 16.7% of Salmonella and Shigella isolates were multi-drug resistant, respectively. CONCLUSION: Prevalence of Salmonella and Shigella species were lower than most studies done in Ethiopia. Hand-washing habit, water source type, Open field waste disposal habit, raw meat consumption and rural residence were associated with Salmonellosis and shigellosis. All isolated Salmonella were sensitive to norfloxacin. The evidence from this study underscores the need for improved water, sanitation and hygiene (WASH) system and the imperative to implement drug susceptibility tests for the treatment of Salmonella and Shigella infection.
Subject(s)
Diarrhea , Dysentery, Bacillary , Microbial Sensitivity Tests , Salmonella , Shigella , Humans , Ethiopia/epidemiology , Cross-Sectional Studies , Child, Preschool , Female , Salmonella/isolation & purification , Salmonella/drug effects , Male , Prevalence , Shigella/drug effects , Shigella/isolation & purification , Infant , Diarrhea/microbiology , Diarrhea/epidemiology , Dysentery, Bacillary/epidemiology , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/drug therapy , Salmonella Infections/epidemiology , Salmonella Infections/microbiology , Salmonella Infections/drug therapy , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Risk Factors , Feces/microbiology , Drug Resistance, BacterialABSTRACT
Background: Food-borne infections continue to be a major public health problem at the international level. The issue becomes more serious in developing countries like Ethiopia. Objective: This study aimed to examine the prevalence of Salmonella and Shigella species and intestinal parasites, as well as antimicrobial resistance patterns and associated factors among food handlers at the University of Gondar cafeteria in northwest Ethiopia. Methods: An institutional-based cross-sectional study was conducted from February to June 2021 in the University of Gondar cafeterias. Data related to the socio-demographic characteristics and hygienic practices of study participants were collected using structured questionnaires. A total of 290 stool samples were collected from food handlers. Culture and conventional biochemical tests were used to isolate the Salmonella and the Shigella species. Wet mount, Formol-ether concentration, and Kato Katz techniques were applied to identify intestinal parasites. Additionally, drug susceptibility tests were performed using the disk diffusion method. Statistical analysis was done using SPSS version 26. Results: Of 290 food handlers' stool samples analyzed, Twenty-seven 27 (9.3%) were positive for both Salmonella and Shigella species. The prevalence of Salmonella and Shigella species was 16 (5.5%) and 11 (3.8%), respectively. Most of the isolated pathogens were resistant to tetracycline 19 (70.4%), and trimethoprim/sulphamethoxazole 19 (70.4%). The overall rate of multi-drug resistant Shigella and Salmonella isolate was 59.3%. Besides, Fifty-seven 57 (19.7%) of the participants were positive for one or more intestinal parasites. The most prevalent intestinal Parasitosis was E. histolytica/dispar 22 (7.6%), followed by G. lamblia 13 (4.5%), and Ascaris lumbricoides 11 (3.8) not washing hands after using the toilet (AOR: 4.42, 95% CI: 1.57, 10.56), and consuming unpasteurized milk (AOR: 3.14, 95% CI: 1.65, 3.96), were factors significantly associated with the prevalence of Salmonella, and Shigella infection. Similarly, not washing hands after using the toilet (AOR: 2.19, 95% CI: 1.0, 1.4), and consuming unpasteurized milk (AOR: 10.4, 95% CI: 3.8, 28.8), were factors significantly associated with the prevalence of intestinal parasites infection. Conclusion: The prevalence of intestinal parasites, Salmonella, and Shigella species was high. Therefore, it is imperative to implement a public health policy that includes ongoing microbiological surveillance.
Subject(s)
Feces , Food Handling , Salmonella , Shigella , Humans , Ethiopia/epidemiology , Cross-Sectional Studies , Female , Male , Salmonella/isolation & purification , Prevalence , Shigella/isolation & purification , Adult , Feces/microbiology , Feces/parasitology , Intestinal Diseases, Parasitic/epidemiology , Universities , Young Adult , Adolescent , Surveys and Questionnaires , Salmonella Infections/epidemiology , Middle Aged , Food Services/statistics & numerical dataABSTRACT
Fish sold at retail markets are often contaminated with harmful bacterial pathogens, posing significant health risks. Despite the growing aquaculture industry in Bangladesh to meet high demand, little attention has been paid to ensuring the safety of fish. The objective of this study was to evaluate the microbiological quality of tilapia and pangas fish sold in retail markets across Dhaka city, Bangladesh. Specifically, the study aimed to compare the quality of fish from traditional wet markets and modern supermarkets, as well as fish samples collected during morning and evening hours. A total of 500 raw cut-fish samples (250 tilapia and 250 pangas) were collected at the point of sale from 32 wet markets and 25 supermarkets. All samples were tested for Escherichia coli, extended-spectrum ß-lactamase-producing E. coli (ESBL-Ec), along with the foodborne pathogens Salmonella, Shigella, Vibrio, and Cryptosporidium spp. Bacterial isolates were characterized using antibiotic susceptibility tests (AST) and the presence of common virulence and antibiotic-resistant genes. Fish samples from retail markets had higher prevalence of tested bacteria including E. coli (92 %), V. cholerae (62 %), ESBL-Ec (48 %), and Salmonella spp. (24 %). There was a significant difference in the prevalence of E. coli (97 % vs. 71 %), ESBL-Ec (58 % vs. 8 %) and Salmonella spp. (28 % vs. 8 %) on the wet market samples compared to supermarket samples (p < 0.005). The mean concentration of E. coli on fish from the wet market was 3.0 ± 0.9 log10 CFU/g, while that from supermarkets was 1.6 ± 0.9 log10 CFU/g. The mean concentration of ESBL-Ec in fish from wet markets and supermarkets were 2.3 ± 0.8 log10 CFU/g and 1.6 ± 0.5 log10 CFU/g, respectively. AST revealed that 46 % of E. coli isolates were multi-drug resistant (MDR), while 4 %, 2 % and 5 % of E. coli, Salmonella spp. and Vibrio spp. isolates, respectively, were resistant to carbapenems. At least 3 % of total E. coli isolates were found to be diarrheagenic, while 40 % of Salmonella isolates harbored pathogenic genes (stn, bcfC, ssaQ, avrA and sodC1), and none of the V. cholerae isolates harbored ctxA and tcpA. Our research shows that raw-cut fish samples from retail markets are contaminated with pathogenic and antibiotic-resistant bacteria, which could be a significant food safety concern. Public health interventions should be implemented to improve food safety and hygiene practices in the retail fish markets.
Subject(s)
Drug Resistance, Bacterial , Seafood , Tilapia , Animals , Tilapia/microbiology , Bangladesh/epidemiology , Seafood/microbiology , Anti-Bacterial Agents/pharmacology , Microbial Sensitivity Tests , Escherichia coli/isolation & purification , Escherichia coli/drug effects , Escherichia coli/genetics , Prevalence , Salmonella/isolation & purification , Salmonella/drug effects , Salmonella/genetics , Food Microbiology , Food Contamination/analysis , Cryptosporidium/isolation & purification , Cryptosporidium/genetics , Bacteria/isolation & purification , Bacteria/drug effects , Bacteria/genetics , Bacteria/classification , Vibrio/isolation & purification , Vibrio/genetics , Vibrio/drug effects , Fishes/microbiology , Shigella/isolation & purification , Shigella/genetics , Shigella/drug effectsABSTRACT
This study examined the relative proportion of enteric pathogens associated with severe gastroenteritis (GE) among children younger than 2 years in a phase III efficacy trial of the ROTASIIL® vaccine in India, evaluated the impact of co-infections on vaccine efficacy (VE), and characterized the association between specific pathogens and the clinical profile of severe GE. Stored stool samples collected from cases of severe GE in the phase III trial were tested by quantitative polymerase chain reaction using TaqMan™ Array Cards. Etiology was attributed by calculating the adjusted attributable fraction (AF) for each pathogen. A test-negative design was used to estimate VE. The pathogens with the highest AFs for severe diarrhea were rotavirus (23.5%), adenovirus 40/41 (17.0%), Shigella spp./enteroinvasive Escherichia coli, norovirus GII, enterotoxigenic E. coli, and Cryptosporidium spp. A considerable proportion of the disease in these children could not be explained by the pathogens tested. Severe GE cases associated with rotavirus and Shigella spp. were more likely to have a longer duration of vomiting and diarrhea, respectively. Cases attributed to Cryptosporidium spp. were more severe and required hospitalization. In the intention-to-treat population, VE was estimated to be 43.9% before and 46.5% after adjustment for co-infections; in the per-protocol population, VE was 46.7% before and 49.1% after adjustments. Rotavirus continued to be the leading cause of severe GE in this age group. The adjusted VE estimates obtained did not support co-infections as a major cause of lower vaccine performance in low- and middle-income countries.
Subject(s)
Coinfection , Diarrhea , Gastroenteritis , Rotavirus Infections , Rotavirus Vaccines , Rotavirus , Humans , Rotavirus Vaccines/therapeutic use , Rotavirus Vaccines/immunology , Rotavirus Vaccines/administration & dosage , Infant , Gastroenteritis/virology , Gastroenteritis/microbiology , Gastroenteritis/prevention & control , Rotavirus Infections/prevention & control , Rotavirus Infections/epidemiology , Diarrhea/virology , Diarrhea/microbiology , Diarrhea/prevention & control , Diarrhea/epidemiology , Coinfection/microbiology , Coinfection/virology , Rotavirus/immunology , Female , Vaccine Efficacy , Shigella/immunology , Male , India/epidemiology , Feces/virology , Feces/microbiology , Vaccines, Attenuated , Norovirus/immunology , Enterotoxigenic Escherichia coli/immunologyABSTRACT
The urgent need for comprehensive and systematic analyses of Shigella as the key pathogen led us to meticulously explore the epidemiology and molecular attributes of Shigella isolates. Accordingly, we procured 24 isolates (10 from Xinjiang and 14 from Wuhan, China) and performed serotype identification and antimicrobial susceptibility testing. Resistance gene detection and homology analysis by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE), respectively, were performed for genetic diversity analysis. All isolates were identified as Shigella flexneri, with 70% (35.4-91.9%) and 30% (8.1-64.6%) of the Xinjiang isolates and 85.7% (56.2-97.5%) and 14.3% (2/14, 2.5-43.9%) of the Wuhan isolates belonging to serotype 2a and serotype 2b, respectively. All isolates displayed resistance to at least two antibiotics and complete resistance to ampicillin. Multidrug resistance (MDR) was recorded in 70.8% (48.8-86.6%) of isolates, with Xinjiang isolates exhibiting relatively higher resistance to ampicillin-sulbactam, piperacillin, ceftriaxone, and aztreonam. Conversely, Wuhan isolates displayed higher MDR and resistance to tetracycline, ciprofloxacin, levofloxacin, and cefepime relative to Xinjiang isolates. Molecular scrutiny of antibiotic-resistance determinants revealed that blaTEM was the main mechanism of ampicillin resistance, blaCTX-M was the main gene for resistance to third- and fourth-generation cephalosporins, and tetB was the predominant gene associated with tetracycline resistance. Four Xinjiang and seven Wuhan isolates shared T1-clone types (>85%), and two Xinjiang and one Wuhan isolates were derived from the T6 clone with a high similarity of 87%. Six PFGE patterns (T1, T2, T5, T6-3, T8, and T10) of S. flexneri were associated with MDR. Thus, there is a critical need for robust surveillance and control strategies in managing Shigella infections, along with the development of targeted interventions and antimicrobial stewardship programs tailored to the distinct characteristics of Shigella isolates in different regions of China.
Subject(s)
Anti-Bacterial Agents , Drug Resistance, Multiple, Bacterial , Dysentery, Bacillary , Electrophoresis, Gel, Pulsed-Field , Genetic Variation , Microbial Sensitivity Tests , Shigella flexneri , China/epidemiology , Anti-Bacterial Agents/pharmacology , Humans , Dysentery, Bacillary/microbiology , Dysentery, Bacillary/epidemiology , Drug Resistance, Multiple, Bacterial/genetics , Shigella flexneri/drug effects , Shigella flexneri/genetics , Shigella flexneri/isolation & purification , Shigella flexneri/classification , Shigella/genetics , Shigella/drug effects , Shigella/isolation & purification , Shigella/classification , Serogroup , Polymerase Chain ReactionABSTRACT
Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.
Subject(s)
Dysentery, Bacillary , Shigella Vaccines , Shigella , Humans , Shigella Vaccines/immunology , Shigella Vaccines/administration & dosage , Dysentery, Bacillary/prevention & control , Dysentery, Bacillary/immunology , Animals , Shigella/immunology , Shigella/pathogenicity , Vaccines, Subunit/immunology , Vaccine Development , Vaccines, Attenuated/immunologyABSTRACT
The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.
Subject(s)
Enterotoxigenic Escherichia coli , Escherichia coli Infections , Shigella , Child , Humans , Child, Preschool , Enterotoxigenic Escherichia coli/genetics , Escherichia coli Infections/diagnosis , Escherichia coli Infections/epidemiology , Rapid Diagnostic Tests , Shigella/genetics , Diarrhea/diagnosis , Diarrhea/epidemiologyABSTRACT
Escape from the bacterial-containing vacuole (BCV) is a key step of Shigella host cell invasion. Rab GTPases subverted to in situ-formed macropinosomes in the vicinity of the BCV have been shown to promote its rupture. The involvement of the BCV itself has remained unclear. We demonstrate that Rab35 is non-canonically entrapped at the BCV. Stimulated emission depletion imaging localizes Rab35 directly on the BCV membranes before vacuolar rupture. The bacterial effector IcsB, a lysine Nε-fatty acylase, is a key regulator of Rab35-BCV recruitment, and we show post-translational acylation of Rab35 by IcsB in its polybasic region. While Rab35 and IcsB are dispensable for the first step of BCV breakage, they are needed for the unwrapping of damaged BCV remnants from Shigella. This provides a framework for understanding Shigella invasion implicating re-localization of a Rab GTPase via its bacteria-dependent post-translational modification to support the mechanical unpeeling of the BCV.
Subject(s)
Bacterial Proteins , Protein Processing, Post-Translational , Shigella , Vacuoles , rab GTP-Binding Proteins , rab GTP-Binding Proteins/metabolism , Humans , Shigella/metabolism , Bacterial Proteins/metabolism , Vacuoles/metabolism , Vacuoles/microbiology , HeLa CellsABSTRACT
Escherichia coli serves as a proxy indicator of fecal contamination in aquatic ecosystems. However, its identification using traditional culturing methods can take up to 24 h. The application of DNA markers, such as conserved signature proteins (CSPs) genes (unique to all species/strains of a specific taxon), can form the foundation for novel polymerase chain reaction (PCR) tests that unambiguously identify and detect targeted bacterial taxa of interest. This paper reports the identification of three new highly-conserved CSPs (genes), namely YahL, YdjO, and YjfZ, which are exclusive to E. coli/Shigella. Using PCR primers based on highly conserved regions within these CSPs, we have developed quantitative PCR (qPCR) assays for the evaluation of E. coli/Shigella species in water ecosystems. Both in-silico and experimental PCR testing confirmed the absence of sequence match when tested against other bacteria, thereby confirming 100% specificity of the tested CSPs for E. coli/Shigella. The qPCR assays for each of the three CSPs provided reliable quantification for all tested enterohaemorrhagic and environmental E. coli strains, a requirement for water testing. For recreational water samples, CSP-based quantification showed a high correlation (r > 7, p < 0.01) with conventional viable E. coli enumeration. This indicates that novel CSP-based qPCR assays for E. coli can serve as robust tools for monitoring water ecosystems and other critical areas, including food monitoring.
Subject(s)
Escherichia coli , Water Microbiology , Water Quality , Escherichia coli/genetics , Escherichia coli/classification , Escherichia coli Proteins/genetics , Real-Time Polymerase Chain Reaction/methods , Shigella/genetics , Shigella/classification , Shigella/isolation & purification , Conserved Sequence , Environmental Monitoring/methods , Polymerase Chain Reaction/methods , Feces/microbiologyABSTRACT
Introduction: Shigella is the etiologic agent of a bacillary dysentery known as shigellosis, which causes millions of infections and thousands of deaths worldwide each year due to Shigella's unique lifestyle within intestinal epithelial cells. Cell adhesion/invasion assays have been extensively used not only to identify targets mediating host-pathogen interaction, but also to evaluate the ability of Shigella-specific antibodies to reduce virulence. However, these assays are time-consuming and labor-intensive and fail to assess differences at the single-cell level. Objectives and methods: Here, we developed a simple, fast and high-content method named visual Adhesion/Invasion Inhibition Assay (vAIA) to measure the ability of anti-Shigellaantibodies to inhibit bacterial adhesion to and invasion of epithelial cells by using the confocal microscope Opera Phenix. Results: We showed that vAIA performed well with a pooled human serum from subjects challenged with S. sonnei and that a specific anti-IpaD monoclonal antibody effectively reduced bacterial virulence in a dose-dependent manner. Discussion: vAIA can therefore inform on the functionality of polyclonal and monoclonal responses thereby supporting the discovery of pathogenicity mechanisms and the development of candidate vaccines and immunotherapies. Lastly, this assay is very versatile and may be easily applied to other Shigella species or serotypes and to different pathogens.