O1 Strains Research Articles

Environmental Vibrio cholerae Strains Harboring Cholera Toxin and Vibrio Pathogenicity Island 1, Nigeria, 2008-2015.

Analysis of clinical and environmental Vibrio cholerae O1 strains obtained during 2008-2015 in Nigeria showed that lineages Afr9 and Afr12 carrying cholera toxin and Vibrio pathogenicity island 1 can be isolated from water. Our findings raise concerns about the role of the environment in maintenance and emergence of cholera outbreaks in Nigeria.

Growth response of Vibrio cholerae O1 and V. cholerae non O1/non O139 strains to algae extracts from stream water in far north Cameroon

Background and Objectives: Vibrio cholerae O1 or non O1/non O139 is found in water ecosystems where it colonizes phytoplankton and has different lifestyle. This study aimed to investigate the impact of some algae extracts on the survival/ growth of both V. cholerae strains. Materials and Methods: Algae extracts consisting of three fractions, F1 containing chlorophyll-a, F2 containing chloro- phyll-b, and F3 containing carotenoids, and raw extract (RAE) were obtained from the algal bloom collected in the Kaliao stream (Maroua, Cameroon). The survival and growth of V. cholerae O1 and V. cholerae non O1/non O139, in microcosms consisting of sterile saline with these extracts and peptone (PEP) respectively added at concentrations of 0.01, 0.05 and 0.1 mg/L, and 50/50 mixtures F1+F2, F2+F3, and F2+PEP at a concentration 0.05 mg/L, were compared during a 24h experi- ment. Results: The microcosms F2 and RAE did not support the growth of O1 strain; V. cholerae non O1/non O139 count in all algae extract microcosms ranging from 3.97 log (CFU/mL) to 5.2 log (CFU/mL). In all PEP microcosms, the counts of both strains reached an uncountable value. Microcosms F1+F2 and F2+F3 supported the growth of V. cholerae O1 and V. cholerae non-O1/nonO139 strains. Conclusion: The algae compounds showed strain-specific effect on the growth of V. cholerae.

Ten-year comparison of the efficiency of legal regulations of foodborne pathogens in broilers in Türkiye

Broiler meats are the primary source of foodborne pathogens, such as Campylobacter jejuni and Salmonella spp., and E. coli O157. The illegal use of antibiotics for preventive purposes or the habit of using broad-spectrum antibiotics in farms causes antibiotic resistance in these pathogens. This study compared C. jejuni, Salmonella spp. and E. coli O157 and their antibiotic resistance patterns in broiler meats in 2011-2012 and 2021-2022. The research we conducted in 2011-2012 to observe the effectiveness of the legal regulations enacted in Türkiye in 2014 and 2018 for controlling pathogens and monitoring the use of antibiotics was also repeated with a similar sample set in 2021-2022. The results showed the prevalence of C. jejuni, Salmonella spp., and E. coli O157 in 2011-2012 and 2021-2022 with 68.0%, 30.4%, 0.8% and 10.08%, 7.77%, 0.21%, respectively. The antimicrobial susceptibility tests against gentamicin, streptomycin, erythromycin, ciprofloxacin, enrofloxacin, tetracycline and oxytetracycline were performed by agar disk diffusion method. According to results in 2011-2012, all C. jejuni and Salmonella spp. were resistant to at least one tested antibiotic, with 31.57% Salmonella spp. and 16.47% C. jejuni being multidrug-resistant. In addition, one of the E. coli O157 strains was resistant to one antibiotic, and the other was resistant to two antibiotics. In 2021-2022, 81.3% C. jejuni, 46% Salmonella spp., and only one E. coli O157 were resistant to at least one tested antibiotic. Isolated strains of 8.33% C. jejuni and 21.62% Salmonella spp. were multidrug-resistant. In the past, antibiotics were used for preventative purposes, as indicated by the survey results. Today, it is evident that these rates have decreased to low levels as a result of legal regulations and monitoring.

Development of a simple allele-specific PCR for the detection of pathogenic Vibrio cholerae O1 and O139 in seafood

This study developed a simple allele specific (AS) PCR assay for the detection of pathogenic Vibrio cholerae strains in seafood. A pair of primers, (1) an AS primer targeting the mutation at site G450 in the hapR2 allele and a (2) common forward primer, were designed to detect the pathogenic O1 and O139 strains. An artificial mismatch was also added to the AS primer to increase its specificity to the target allele. The PCR assays using different DNA polymerases of varying efficiencies were then optimized. The application of the AS primers in both conventional and real-time PCR assays was facilitated with enriched seafood samples spiked with known concentration of pathogenic V. cholerae. The designed AS primers showed high specificity and sensitivity to the hapR2 allele for both conventional and real-time PCR assays. Enrichment of seafood samples for 8 h is recommended to detect viable pathogenic V. cholerae presence in seafood. In this study, we developed an AS-PCR that is simple, rapid, and a low-cost alternative for routine detection of pathogenic V. cholerae in different seafood samples thus aiding in the surveillance and implementation of food safety measures at the seafood post-harvest level.

Outer Membrane Vesicles Formed by Clinical Proteus mirabilis Strains May Be Incorporated into the Outer Membrane of Other P. mirabilis Cells and Demonstrate Lytic Properties.

Outer membrane vesicles (OMVs) are extracellular structures, ranging in size from 10 to 300 nm, produced by Gram-negative bacteria. They can be incorporated into the outer membrane of a recipient's cells, which may enable the transfer of substances with lytic properties. Due to the scarce information regarding the OMVs produced by Proteus mirabilis, the aim of this study was to test the blebbing abilities of the clinical P. mirabilis O77 and O78 strains and to determine the blebs' interactions with bacterial cells, including their possible bactericidal activities. The production of OMVs was visualised by Transmission electron microscopy (TEM). The presence of OMVs in the obtained samples as well as the phenomenon of OMV fusion to recipient cells were confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting assays. The bacteriolytic activity of the OMVs was examined against P. mirabilis clinical strains and reference Staphylococcus aureus and Escherichia coli strains. It was shown that each of the two tested P. mirabilis strains could produce OMVs which were able to fuse into the cells of the other strain. The lytic properties of the O78 OMVs against another P. mirabilis O78 strain were also demonstrated. This promising result may help in the future to better understand the mechanisms of the pathogenesis and to treat the infections caused by P. mirabilis.

Commercial bacteriophage preparations for the control of Listeria monocytogenes and Shiga toxin-producing Escherichia coli in raw and pasteurized milk

Listeria monocytogenes was the etiologic agent in nearly all recent outbreaks in North America attributed to pasteurized dairy products, whereas Escherichia coli O157 infections were responsible for most of the rare, yet serious complications from outbreaks involving unpasteurized dairy. This study determined the susceptibility of selected strains of L. monocytogenes and Shiga toxin-producing E. coli (STEC) to commercial phage preparations and their ability to control these pathogens in pasteurized and raw milk during 7-day storage at 7 °C. Both phage products demonstrated high lytic efficiency against 17 strains of L. monocytogenes whereas the efficiency of E. coli phages was more variable against 11 strains of O157 and non-O157 STEC. Broth microdilution assays identified effective endpoint multiplicities of infection (MOI) ranging from log 2.53 to 5.13, which differed between strains of L. monocytogenes and phage products. Mean log MOIs of E. coli phages against STEC also varied within and between products from 0.43 to 7.05. Despite these observations, the change in counts over time of three L. monocytogenes strains exposed to phage in pasteurized milk (log MOI 6) was similar with counts ∼ 4 log CFU/mL lower than control at day 7. Results for STEC O157 varied by strain but counts were lower than control in all cases by 72 h thorough day 7. Titers on isolates of both pathogens isolated from pasteurized milk indicated that the surviving populations were less susceptible to phage. The addition of a phage preparation to raw milk did not reduce populations of either pathogen or affect the change in counts of any strain over time when compared to control. Reduced efficacy in raw milk may be attributed to reduced phage binding as titers in raw milk decreased steadily (∼2 log PFU/mL) during storage. Commercial phage products may be a promising pathogen control intervention for pasteurized dairy products, warranting further investigation.

Diversity and Complexity of CTXΦ and Pre-CTXΦ Families in Vibrio cholerae from Seventh Pandemic.

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTXETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic.

Hydrolysis probe assays for the detection of pathogenic Enterohemorrhagic Escherichia coli: Multi-Country validation study

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens frequently associated with outbreaks linked to red meat, posing significant global food safety concerns. While several commercial assays exist for detecting EHEC strains belonging to serogroups O26, O103, O111, and O121, they often fail to distinguish pathogenic strains from non-pathogenic strains lacking virulence genes. These non-pathogenic E. coli interfere with the accuracy of existing assays, resulting in false-positives rates between 81 – 100 % for beef products. This study aimed to standardize and validate four hydrolysis probe assays for the specific detection of pathogenic strains of E. coli O26, O103, O111, and O121. Primers and probes were designed to target single nucleotide polymorphisms conserved among pathogenic strains of each target serogroup. The standardized assays were validated using a comprehensive collection of pure culture and DNA samples (n = 248) from Canada, France, Switzerland, and the United States; laboratory-inoculated beef and spinach (n = 132), fractionally inoculated ground beef (n = 390), and samples from the federal red meat surveillance program (n = 166). The results demonstrated an overall accuracy of 94.8 % in predicting the virulence profile of pure culture strains. These four assays are a single-step PCR screening tool that can determine the presence of virulent strains. These assays provide food testing laboratories worldwide with a set of reliable methods for identifying pathogenic O26, O103, O111, and O121 strains. Thus, this assay will help to reduce product waste and financial losses caused by misleading positive results caused by presence of non-pathogenic strains.

Eco-friendly synthesis and antibacterial potential of chitosan crosslinked-EDTA silver nanocomposite (CCESN)

This study presents a novel and eco-friendly approach for synthesizing silver nanocomposite at room temperature. The method utilizes chitosan derived from snail (Archachatina marginata) shell waste crosslinked with EDTA as a combined reducing and capping agent. The existence of silver nanoparticles in the composite was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffractometry (XRD), energy dispersive X-ray (EDX), energy dispersive X-ray fluorescence (EDXRF) and thermogravimetric analysis (TGA). The TEM, SEM, XRD, and analyses revealed that the silver nanoparticle has a face-centered cubic structure with an average size of 45.30 nm respectively. EDX and EDXRF showed characteristic silver peaks confirming the formation of silver nanoparticles in the composite while TGA indicated that silver nanoparticles contributed to good thermal stability of the composite. The formation of silver nanoparticles was indicated by a brown color transformation and an ultraviolet visible (UV Vis) absorption peak at 435 nm. The synthesized nanocomposite demonstrated promising antibacterial activity against both Staphylococcus saprophyticus DSM 18669 and Escherichia coli O157 strains, with S. saprophyticus showing higher susceptibility. This highlights the potential of chitosan-EDTA silver nanocomposites as alternative antimicrobial agents.

Lipopolysaccharide Core Truncation in Invasive Escherichia coli O157:H7 ATCC 43895 Impairs Flagella and Curli Biosynthesis and Reduces Cell Invasion Ability.

Escherichia coli O157:H7 (E. coli O157) is known for causing severe foodborne illnesses such as hemorrhagic colitis and hemolytic uremic syndrome. Although E. coli O157 is typically regarded as an extracellular pathogen and a weak biofilm producer, some E. coli O157 strains, including a clinical strain ATCC 43895, exhibit a notable ability to invade bovine crypt cells and other epithelial cells, as well as to form robust biofilm. This invasive strain persists in the bovine host significantly longer than non-invasive strains. Various surface-associated factors, including lipopolysaccharides (LPS), flagella, and other adhesins, likely contribute to this enhanced invasiveness and biofilm formation. In this study, we constructed a series of LPS-core deletion mutations (waaI, waaG, waaF, and waaC) in E. coli O157 ATCC 43895, resulting in stepwise truncations of the LPS. This approach enabled us to investigate the effects on the biosynthesis of key surface factors, such as flagella and curli, and the ability of this invasive strain to invade host cells. We confirmed the LPS structure and found that all LPS-core mutants failed to form biofilms, highlighting the crucial role of core oligosaccharides in biofilm formation. Additionally, the LPS inner-core mutants ΔwaaF and ΔwaaC lost the ability to produce flagella and curli. Furthermore, these inner-core mutants exhibited a dramatic reduction in adherence to and invasion of epithelial cells (MAC-T), showing an approximately 100-fold decrease in cell invasion compared with the outer-core mutants (waaI and waaG) and the wild type. These findings underscore the critical role of LPS-core truncation in impairing flagella and curli biosynthesis, thereby reducing the invasion capability of E. coli O157 ATCC 43895.

Exploiting membrane vesicles derived from avian pathogenic Escherichia coli as a cross-protective subunit vaccine candidate against avian colibacillosis

Avian pathogenic Escherichia coli (APEC) is a notable pathogen that frequently leads to avian colibacillosis, posing a substantial risk to both the poultry industry and public health. The commercial vaccines against avian colibacillosis are primarily inactivated vaccines, but their effectiveness is limited to specific serotypes. Recent advances have highlighted bacterial membrane vesicles (MV) as a promising candidate in vaccine research. How to produce bacterial MVs vaccines on a large scale is a significant challenge for the industrialization of MVs. The msbB gene encodes an acyltransferase and has been implicated in altering the acylation pattern of lipid A, leading to a decrease in lipid A content in lipopolysaccharides (LPS). Here, we evaluated the immunoprotective efficacy of MVs derived from the LPS low-expressed APEC strain FY26ΔmsbB, which was an APEC mutant strain with a deletion of the msbB gene. The nitrogen cavitation technique was employed to extract APEC MVs, with results indicating a significant increase in MVs yield compared to that obtained under natural culture. The immunization effectiveness was assessed, revealing that FY26ΔmsbB MVs elicited an antibody response of laying hens and facilitated bacterial clearance. Protective efficacy studies demonstrated that immunization with FY26ΔmsbB MVs conferred the immune protection in chickens challenged with the wild-type APEC strain FY26. Notably, LPS low-carried MVs recovered from the mutant FY26ΔmsbB also displayed cross-protective capabilities, and effectively safeguarding against infections caused by O1, O7, O45, O78, and O101 serotypes virulent APEC strains. These findings suggest that MVs generated from the LPS low-expressed APEC strain FY26ΔmsbB represent a novel and empirically validated subunit vaccine for the prevention and control of infections by various APEC serotypes.

Applications of Escherichia Coli Esterases for Bioremediation and Treatment of Wastewater Organic Chemical Pollutants

This study used computational techniques, including 3D enzyme structural modeling and molecular docking, to gain insight into the bioremediation of organic wastewater contaminants using E. coli esterase enzymes. Furthermore, a total of 24 wastewater organic chemicals belonging to different categories, such as pharmaceuticals, artificial sweeteners, pesticides/herbicides, endocrine disrupting chemicals, and persistent organic pollutants, were identified through the toxicology database. Comparative genetics and reported literature. Furthermore, 3D PDB and AlphaFold structures of 194 esterase enzymes from E. coli were retrieved by first identifying a common domain (Alpha Beta hydrolase domain) using the InterPro database. Molecular docking of esterase enzymes and pollutants was used, resulting in the best binding enzymes to their respective organic wastewater pollutants, including bezafibrate which showed the best binding with all enzymes ranging from -6.33 kcal/mol to -9.87 kcal/mol . Subsequently, the majority of the ligands (organic wastewater pollutants) reacted with enzymes such as the ORFC-like enzymes, which were computationally annotated in this study for the first time, yuaR (strain K12), menH (strain ETI89/UPEC), and menH (strain O157). :H7) has significant binding affinities and consists of a common Alpha Beta hydrolasefold-1 domain. This suggests that esterase enzymes containing the Alpha Beta hydrolasefold-1 domain may be involved in the efficient degradation of organic wastewater pollutants.

Strain variation and anomalous climate synergistically influence cholera pandemics.

Explanations for the genesis and propagation of cholera pandemics since 1817 have remained elusive. Evolutionary pathogen change is presumed to have been a dominant factor behind the 7th "El Tor" pandemic, but little is known to support this hypothesis for preceding pandemics. The role of anomalous climate in facilitating strain replacements has never been assessed. The question is of relevance to guide the understanding of infectious disease emergence today and in the context of climate change. We investigate the roles of climate and putative strain variation for the 6th cholera pandemic (1899-1923) using newly assembled historical records for climate variables and cholera deaths in provinces of former British India. We compare this historical pandemic with the 7th (El Tor) one and with the temporary emergence of the O139 strain in Bangladesh and globally. With statistical methods for nonlinear time series analysis, we examine the regional synchrony of outbreaks and associations of the disease with regional temperature and rainfall, and with the El Niño Southern Oscillation (ENSO). To establish future expectations and evaluate climate anomalies accompanying historical strain replacements, climate projections are generated with multi-model climate simulations for different 50-year periods. The 6th cholera pandemic featured the striking synchronisation of cholera outbreaks over Bengal during the El Niño event of 1904-07, following the invasion of the Bombay Presidency with a delay of a few years. Accompanying anomalous weather conditions are similar to those related to ENSO during strain replacements and pandemic expansions into Africa and South America in the late 20th century. Rainfall anomalies of 1904-05 at the beginning of the large cholera anomaly fall in the 99th percentile of simulated changes for the regional climate. Evolutionary pathogen change can act synergistically with climatic conditions in the emergence and propagation of cholera strains. Increased climate variability and extremes under global warming provide windows of opportunity for emerging pathogens.

Variability in the acid adaptation of ten different O157:H7 and non-O157 Escherichia coli strains in orange juice and the impact on UV radiation resistance

This study aimed to assess the impact of adaptation of ten strains of O157:H7 and non-O157 Escherichia coli to low pH (acid shock or slow acidification) and the effects of this exposure or not on the resistance of E. coli strains to UV radiation in orange juice (pH 3.5). The acid-shocked cells were obtained through culture in tryptic soy broth (TSB) with a final pH of 4.8, which was adjusted by hydrochloric, lactic, or citric acid and subsequently inoculated in orange juice at 4 °C for 30 days. No significant differences (p > 0.05) in survival in orange juice were observed between the serotypes O157:H7 and non-O157:H7 for acid-shocked experiments. After slow acidification, where the cells were cultured in TSB supplemented with glucose 1% (TSB + G), a significant increase (p < 0.05) in survival was observed for all strains evaluated. The D-values (radiation dose (J/cm2) necessary to decrease the microbial population by 90%) were determined as the inverse of the slopes of the regressions (k) obtained by plotting log (N/N0). The results show that among the strains tested, E. coli O157:H7 (303/00) and O26:H11 were the most resistant and sensitive strains, respectively. According to our results, the method of acid adaptation contributes to increasing the UV resistance for most of the strains tested.

Genomic insights into the 2022–2023Vibrio cholerae outbreak in Malawi

Malawi experienced its deadliest Vibrio cholerae (Vc) outbreak following devastating cyclones, with >58,000 cases and >1700 deaths reported between March 2022 and May 2023. Here, we use population genomics to investigate the attributes and origin of the Malawi 2022–2023 Vc outbreak isolates. Our results demonstrate the predominance of ST69 clone, also known as the seventh cholera pandemic El Tor (7PET) lineage, expressing O1 Ogawa (~ 80%) serotype followed by Inaba (~ 16%) and sporadic non-O1/non-7PET serogroups (~ 4%). Phylogenetic reconstruction revealed that the Malawi outbreak strains correspond to a recent importation from Asia into Africa (sublineage AFR15). These isolates harboured known antimicrobial resistance and virulence elements, notably the ICEGEN/ICEVchHai1/ICEVchind5 SXT/R391-like integrative conjugative elements and a CTXφ prophage with the ctxB7 genotype compared to historical Malawian Vc isolates. These data suggest that the devastating cyclones coupled with the recent importation of 7PET serogroup O1 strains, may explain the magnitude of the 2022–2023 cholera outbreak in Malawi.

62. Selection by infrared spectroscopy of E. coli O78 strains for the production of autovaccines in Muscovy ducks

62. Selection by infrared spectroscopy of E. coli O78 strains for the production of autovaccines in Muscovy ducks

Enteroaggregative Escherichia coli: Frequent, yet underdiagnosed pathotype among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic

Enteroaggregative Escherichia coli (EAEC) strains including those of serogroup O111 are important causes of diarrhea in children. In the Czech Republic, no information is available on the etiological role of EAEC in pediatric diarrhea due to the lack of their targeted surveillance. To fill this gap, we determined the proportion of EAEC among E. coli O111 isolates from children with gastrointestinal disorders ≤ 2 years of age submitted to the National Reference Laboratory for E. coli and Shigella during 2013–2022. EAEC accounted for 177 of 384 (46.1 %) E. coli O111 isolates, being the second most frequent E. coli O111 pathotype. Most of them (75.7 %) were typical EAEC that carried aggR, usually with aaiC and aatA marker genes; the remaining 24.3 % were atypical EAEC that lacked aggR but carried aaiC and/or aatA. Whole genome sequencing of 11 typical and two atypical EAEC O111 strains demonstrated differences in serotypes, sequence types (ST), virulence gene profiles, and the core genomes between these two groups. Typical EAEC O111:H21/ST40 strains resembled by their virulence profiles including the presence of the aggregative adherence fimbriae V (AAF/V)-encoding cluster to such strains from other countries and clustered with them in the core genome multilocus sequence typing (cgMLST). Atypical EAEC O111:H12/ST10 strains lacked virulence genes of typical EAEC and differed from them in cgMLST. All tested EAEC O111 strains displayed stacked-brick aggregative adherence to human intestinal epithelial cells. The AAF/V-encoding cluster was located on a plasmid of 95,749 bp or 93,286 bp (pAAO111) which also carried aggR, aap, aar, sepA, and aat cluster. EAEC O111 strains were resistant to antibiotics, in particular to aminopenicillins and cephalosporins; 88.3 % produced AmpC β-lactamase, and 4.1 % extended spectrum β-lactamase. We conclude that EAEC are frequent among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic. To reliably assess the etiological role of EAEC in pediatric diarrhea, a serotype-independent, PCR-based pathotype surveillance system needs to be implemented in the future.

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

A Rapid and Robust DNA Extraction Method for PCR-Based Diagnosis of V. cholerae

Cholera, caused by _V. cholerae_, needs rapid diagnosis because of its threat for rapid spread. Recent molecular diagnosis by PCR assay is more advantageous than the conventional methods. The bottleneck in PCR diagnosis lies in the delay in template DNA extraction of _V. cholerae_, which is the basic requirement. This study describes a simple, less expensive, and rapid template DNA extraction method to lessen the total time for PCR diagnosis of _V. cholerae._ To obtain template DNA, our method involves boiling of _V. cholerae_ suspension in distilled water (takes 18-24 hours) instead of boiling the suspension in broth medium, which is a lengthy process (takes 72 hours). The validation of our template DNA was conducted using 40 _V. cholerae_ O1 strains that were confirmed previously, and its usefulness was checked on 20 clinical strains of _V. cholerae_ O1 isolated from acute diarrhea patients; results were compared with those of conventional template DNA. The analysis of our template DNA in simplex and quadruplex PCR assays revealed the presence of _ctxA_, _tcpA _(El Tor), _rfb _(O1), _ToxR_, _ctxB_, _zot_, _ace,rst _, and _OmpW _genes in _V. cholerae _O1. The comparison of the results of PCR assays using the template DNA from both sources showed equal results and matched correctly. The sensitivity of our template DNA was 1.5x103 CFU per assay. Our template DNA extraction method is simple, less expensive, and rapid, and it can be employed for early diagnosis of _V. cholerae_ during outbreaks, in research laboratories, and in hospital setups where infrastructures are available.

Qualitative detection of E. coli in distributed drinking water using real-time reverse transcription PCR targeting 16S rRNA: Validation and practical experiences

Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140–2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43–1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on “in silico” analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories. The application of RT-PCR to confirm the hygienic quality of distributed drinking water after actions to repair or maintain distribution pipes was compared with the culture method on 8076 routine samples, analyzed by the drinking water laboratories in the Netherlands. This comparison study showed a 96.4 % agreement between RT-PCR and culture. In 3.3 % of the samples E. coli was detected with RT-PCR and not with the culture method and in 0.1 % of the samples E. coli was only detected by culture confirming either a higher sensitivity for RT-PCR or the detection of RNA from uncultivable cells. Finally, the application of RT-PCR was highlighted during a contamination event in Belgium where we demonstrate the potency of RT-PCR as a tool to rapidly monitor the spread of microbial contamination and to monitor the effect of measures to remove the contamination This is the first fully validated rapid nucleic based method for detection of E. coli in distributed drinking water. These results demonstrate that this RT-PCR method can be used as a rapid alternative to the culture method to monitor E. coli in distributed drinking water. However, it should be emphasized that nucleic acid based detection methods rely on highly different detection principles (detection of captured nucleic acids present in a sample) than culture base methods (presence of cells cultivable on a selective medium) resulting in occasional different analysis results. Varying treatment and disinfection steps (UV, chlorine, monochloramine, Ozone) or environmental factors (decay) can influence the results and cause differences between RT-PCR and culture methods.