O1 Strains Research Articles

Novel reusable animal model for comparative evaluation of in vivo growth and protein-expression of Escherichia coli O157 strains in the bovine rumen.

Previously, we had demonstrated that Escherichia coli O157:H7 (O157) strain 86–24 expresses proteins involved in survival rather than virulence in vitro in rumen fluid from dairy cattle limit fed a maintenance diet. Here, we verified if this observation would be true for different O157 strains grown in vitro in rumen fluid from, and in vivo in the rumen of, animals on contrasting maintenance (high fiber) and lactation (high energy-protein) diets usually limit fed to dairy cattle. For the in vivo studies, an economical, novel, reusable and non-terminal rumen-fistulated animal model permitting simultaneous evaluation of multiple bacterial strains in the bovine rumen was developed. All experiments were conducted in duplicate using different animals to account for host-related variations. The O157 strains included, 86–24, EDL933 and the super shed SS-17. E. coli NalR (#5735), derived from a bovine intestinal commensal E. coli, was included as a control. As expected, diet influenced ruminal pH and volatile fatty acid (VFA) composition. The pH ranged from 6.2–7.0 and total VFA concentrations from 109–141 μM/ml, in animals fed the maintenance diet. In comparison, animals fed the lactation diet had a ruminal pH ranging between 5.18–6.0, and total VFA of 125–219 μM/ml. Strain dependent differences in O157 recovery from the rumen fluid of cattle fed either diet was observed, both in vitro and in vivo, with O157 strains 86–24 and EDL933 demonstrating similar survival patterns. Analysis of the O157 proteomes expressed in the rumen fluid/rumen verified previous observations of adaptive responses. Any difference in the adaptive response was mainly influenced by the animal’s diet and growth conditions (in vitro and in vivo) and not the O157 strain. These new insights into the O157 responses could help formulate modalities to control O157 across strains in cattle at all stages of husbandry.

High-pressure processing treatment of beef burgers: Effect on Escherichia coli O157 inactivation evaluated by plate count and PMA-qPCR.

Propidium monoazide coupled to real time PCR (PMA-qPCR) is a novel methodology proposed for the quantification of viable bacteria in food after microbial inactivation treatments. The aim of this work was to assess the effectiveness of different pressure levels on the lethality of a pool of Escherichia coli O157 strains in beef burgers by plate count and PMA-qPCR using uidA as target gene. Also, the effect on native microbiota counts, E. coli O157 counts, and physiochemical parameters of beef burgers during storage in refrigeration and frozen conditions were assessed. The treatment at 600MPa for 5min was the most lethal and was selected for the evaluation of bacteria behavior under storage conditions. Native microbiota and E. coli O157 were not recovered during refrigerated and frozen storage (4°C for 7 days and -18°C for 35 days). Cooking weight loss, pH, chromatic parameters, and texture were affected by HPP. PRACTICAL APPLICATION: Practical Application: PMA-qPCR can be used as an alternative to assess microbial inactivation by different high pressure processing (HPP) conditions (pressure level, holding time and temperature) more rapidly than conventional plate counts. In addition, it has the benefit of being able to quantify viable but nonculturable bacteria from contaminated beef burgers after HPP. Moreover, this novel technique generates less pathogenic residues, which minimizes workers' exposure to human biohazards.

Characterization and application of bacteriophages for the biocontrol of Shiga-toxin producing Escherichia coli in Romaine lettuce

Shiga-toxin-producing Escherichia coli (STEC) have been implicated with numerous outbreaks associated with contaminated fresh produce. Bacteriophages (phages) are viruses that specifically target and utilize bacterial hosts for replication. Although phage products for biocontrol are currently available, the risk of potential bacterial resistance mandates further characterization of new strains for future formulations.Previously isolated STEC phages (n = 15) were probed for their host ranges and genome composition. Phage (n = 13) phylogenetic origins were explored and annotated for the identification of potential integrase genes and virulence genes. Phages (n = 3) were then selected based on their ease of propagation and ability to target E. coli O157 for further analysis. Select phages were imaged by transmission electron microscopy and tested for their burst size, latent period, pH and temperature stability, as well as efficacy against four strains of E. coli O157 both in broth culture and on fresh Romaine lettuce at a temperature of 10 °C.Whole-genome nucleotide alignment revealed that the 13 phages grouped into three distinct clusters and two singletons. The lack of integrase and virulence genes demonstrated their suitability for the food industry. Phages VE04, VE05 and VE07 demonstrated increasing stability with increasing pH and remained stable at temperatures of −20 °C, 4 °C, and 22 °C. Phage VE04 was shown to have the shortest latent period and a burst size of 50 ± 5. All phages were equally effective in significantly reducing E. coli O157 populations in liquid culture (P < 0.05). Likewise, phages VE04, VE05 and VE07 were effective against the tested E. coli O157 strains on Romaine lettuce. Compared to control groups, log-reduction ranged from 2.6 log colony forming units (CFU)/cm2 to approximately 6 log CFU/cm2 after 3 days of storage at a temperature of 10 °C (P < 0.05). Phages remained stable and persisted despite the absence of a STEC host in broth culture and on Romaine lettuce.The discovery of new phages that inactivate E. coli O157 supports the formulation of products with reduced potential of bacterial resistance and the development of future biocontrol methods.

Phenotypic whole-cell screening identifies a protective carbohydrate epitope on Klebsiella pneumoniae

The increasing occurrence of recalcitrant multi-drug resistant (MDR) Klebsiella pneumoniae infections coupled with a diminishing pipeline of new antibioticswarrants the investigation of alternative antimicrobial therapies. We employed a target-agnostic phage display approach using live K. pneumoniaebacteria with the aim of isolating therapeutic monoclonal antibodies (mAbs) targetingconserved epitopes among clinically relevant strains. mAb targets were explored using ELISA and biolayer interferometry, and a high-throughput opsonophagocytic killing assay was developed to determine functional activity. Fluorescence-activated cell sorting was used to screen a global panel of clinical isolates, and high-content imaging further explored binding and functional activity. One mAb was tested in vivousing a lethal murine model of pneumonia. mAbs binding to carbohydrate epitopes were isolated in phage display selections enriched on wild-type and capsule-deficient strains. mAbs binding O1 lipopolysaccharide (LPS) and cross-binding O1/O2 LPS were identified. mAbs were shown to promote opsonophagocytic killing by human monocyte-derived macrophages, and clearance of macrophage-associated bacteria. One mAb, named B39, protected mice against MDR O1 and O2 strains when dosed therapeutically in a murine pneumonia model. Binding to a panel of O1 and O2 clinical isolates suggests B39 binds to both d-galactan-I and d-galactan-II of the LPS. With the rise of antimicrobial resistance among enteric pathogens, the discovery of a novel therapeutic mAb targeting the most prevalent K. pneumoniaeserotypes demonstrates a significant advancement in the field, and showcases the potential of alternative antimicrobial therapies for the treatment of MDR infections.

Evaluation of cell-mediated immunity of E.coli nanovaccines in chickens

Nanovaccine is a revolutionary type of immunizations for various diseases that is simple to manufacture and administer. As a result, we are working to develop innovative nanovaccines against E. coli, which is capable of causing disease both inside and outside of its predilection sites, causing respiratory and systemic disease (colibacillosis).Colibacillosis is a global disease that significantly affects poultry production. The present study aims to evaluate in vivo cell-mediated immunity against a chitosan-nanovaccine from E. coli serogroups O1 and O78 to aid in limiting colibacillosis in chicken. Two hundred specific pathogen-free (SPF) three weeks old broiler chickens were used and divided into five groups: the first group inoculated with the outer membrane and flagellar antigen (OF), the second group inoculated with chitosan capsulated-outer membrane protein-flagellar antigen (CSC-O-F), the third group inoculated with chitosan loaded-outer membrane protein-flagellar antigen (CSL-O-F), the fourth group was vaccinated with (CSL-O-F-M) adjuvanted with Montanide ISA 71 RVG, and the fifth group was left as unvaccinated control. The immune response was measured by ELISA, lymphocyte proliferation test, and challenge test. The duration of immunity was also studied. The CSL-O-F-M had the highest antibody titer in an ELISA test using the O1 strain, and the CSC-O-F had the highest antibody titer in an ELISA test using the O78 strain. For both O1 and O78 strains, the CSL-O-F-M had the strongest cell-mediated immune response, which was validated by the challenge test and duration study. We recommend producing nanovaccines (CSL-O-F-M) from E.coli O1 and O78 strains as a new manufacturing vaccine based on the demonstrated results. Because it produces highly effective humoral and cell-mediated immune responses, this novel vaccine may be useful in reducing the risk of colibacillosis.

Etiological and epidemiological characteristics of Vibrio cholerae in Beijing, 2015-2021

Objective: To analyze the etiological and epidemiological characteristics of Vibrio cholerae in Beijing during 2015-2021 and provide evidence for the prevention and control of cholera. Methods: The V. cholerae strains isolated in Beijing during 2015-2021 were analyzed by serotyping and virulence genes detection. Pulsed field gel electrophoresis (PFGE) was performed for the molecular typing of the strains. Based on the collected epidemiological and clinical data of cholera cases,the epidemiological characteristics of cholera were analyzed by descriptive epidemiology method. Results: A total of 76 Vibrio cholerae O1 strains were isolated in Beijing during 2015-2021, including 61 strains from human, 10 strains from environment and 5 strains from seafood. The 76 strains consisted of 68 Ogawa strains and 8 Inaba strains. Six Ogawa strains isolated from sporadic cases carried ctxAB. After NotⅠ digestion, 76 strains were divided into 33 PFGE patterns. From 2015 to 2021, a total of 38 cholera epidemics were reported in Beijing, most of them were sporadic ones, accounting for 92.11% (35/38). A total of 45 cases were reported, and the cases occurred during June-September accounted for 97.78% (44/45). Cholera cases occurred in 9 districts of Beijing, and the cases reported in Chaoyang district accounted for 42.22% (19/45) and in Changping district accounted for 31.11% (14/45). The age of the cholera cases ranged from 19 to 63 years. Except for one case with unknown clinical symptoms, 44 cases had diarrhea symptoms with 84.09% (37/44) of the cases reporting diarrhea (3-9 times/day), followed by yellow watery stool (95.45%, 42/44), abdominal pain (68.18%, 30/44), nausea and vomiting (40.91%, 18/44) and fever (36.36%, 16/44). Conclusion: Vibrio cholerae strains isolated in Beijing during 2015-2021 were mainly O1 serotype Ogawa,most of which were non-toxigenic. The PFGE of the strains varied. Cholera epidemics occurred in 9 districts of Beijing, but most were sporadic ones with incidence peak during June-September.

Elucidating the correlation between the number of TTTTGAT heptamer repeats and cholera toxin promoter activity in Vibrio cholerae O1 pandemic strains.

A complex virulence-regulatory cascade controls expression of the cholera toxin genes (ctxAB) in Vibrio cholerae, which eventually leads to the production and secretion of choleragen (CT), responsible for rice watery diarrhoea in infected individuals. The cholera toxin promoter (PctxAB) contains a series of heptad repeats (5'-TTTTGAT-3'), which has previously been shown to play a crucial role in transcriptional regulation of ctxAB by recruiting the transcriptional activators ToxT, ToxR and the nucleoid-associated protein H-NS along the ctx promoter. The number of these repeats differs not only between the two biotypes of V. cholerae O1 strains, but also among the strains belonging to the same biotype. In this study, we examined if regulation of PctxAB is influenced in any way by the number of these repeats. Based on our observations, we posit that ctx activation indeed depends on the number of TTTTGAT heptad repeats within PctxAB, and occupation of the distal repeats by H-NS could prevent transcriptional activation of the ctx genes in V. cholerae O1 pandemic isolates. Our results suggest that ToxT-dependent transcriptional activation may not require entire displacement of H-NS and supports a recently described revised model of ToxT and H-NS mediated PctxAB transcriptional regulation.

Multiplex polymerase chain reaction typing scheme based on Escherichia coli O157:H7 Sakai prophage (Sp)-associated genes

ObjectivesEscherichia coli strains of the O157 serogroup include significant foodborne pathogens: enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli, which are responsible for a considerable number of hospitalizations and deaths worldwide each year. There is a constant need for rapid, reliable, and easy-to-use methods for their identification, typing, and phylogenetic classification. In this study, we proposed a new multiplex polymerase chain reaction (PCR)–based typing system for pathogenic E. coli, focusing on the O157 serogroup. MethodsWe designed primers targeting 12 lambdoid prophage regions carried by the prototypic polylysogenic strain of EHEC, the O157:H7 Sakai strain. The reactions were tested in vitro as well as in silico with the PubMLST database. ResultsThe PCR assays can be grouped into four multiplex reactions, and their results can be given as a four-digit code. In vitro and in silico testing showed that these Sakai prophage regions are prevalent not only in E. coli O157 strains but also in Shiga toxigenic E. coli non-O157 strains and the method provides appropriate resolution. ConclusionsThe proposed method could be a valuable tool in epidemiologic tracing and preliminary phylogenetic grouping of this diverse group of pathogens.

Characterization of the O-antigen gene clusters and development of a molecular serotyping method for Vibrio fluvialis

Vibrio fluvialis is an emerging foodborne pathogen that causes severe infections. Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens such as V. fluvialis. For example, variation of the O-antigen, which is highly polymorphic and is responsible for the majority of antigenic variability on the bacterial cell surface, provides the basis for serotyping of Gram-negative bacteria. Currently, there has been no analysis of the O-antigen gene clusters in V. fluvialis. In this study, the putative O-antigen gene clusters of 18 V. fluvialis serogroups (O1–O18), which exhibit a high level diversity, were analyzed by whole-genome sequencing. A microsphere-based suspension array (MSA) based on O-serogroup-specific genes was developed for identification of V. fluvialis strains O1–O18 and evaluated for specificity and sensitivity in double-blind tests. Furthermore, analysis of 62 publicly available V. fluvialis genomes identified 13 new O-antigen gene cluster types. The detection sensitivity was determined to be 10−2 ng for genomic DNA and 103 CFU for pure cultures. When testing simulated samples in an oyster background, 2 to 20 CFU per gram inoculated could be detected after enrichment using this method. Our work provides an efficient tool for rapid detection and identification of V. fluvialis serogroups from clinical and environmental samples, with the potential for use in epidemiological investigations and food safety applications.

Characterisation of new anti-O157 bacteriophages of bovine origin representing three genera

Shiga-toxin-producing Escherichia coli (STEC) strains of the serogroup O157 are foodborne pathogens associated with severe clinical disease. As antibiotics are counter-indicated for treatment of these infections, they represent prime candidates for targeted application of bacteriophages to reduce infection burden. In this study, we characterised lytic bacteriophages representing three phage genera for activity against E. coli O157 strains. The phages vb_EcoM_bov9_1 (Tequatrovirus), vb_EcoM_bov11CS3 (Vequintavirus), and vb_EcoS_bov25_1D (Dhillonvirus) showed effective lysis of enterohaemorrhagic E. coli EHEC O157:H7 strains, while also exhibiting activity against other strains of the O157 serogroup, as well as of the ‘big six’ (STEC) serogroups, albeit with lower efficiency. They had a burst size of 293, 127 and 18 per cell and a latent period of 35, 5 and 30 min, respectively. In situ challenge experiments using the O157 Sakai strain on minced beef showed a reduction by 2–3-fold when treated with phages at a 0.1 MOI (multiplicity of infection), and approximately 1 log reduction when exposed to MOI values of 10 and 100. A cocktail of the phages, applied at 10 × and 100 × MOI showed 2 to 3 log reduction when samples were treated at room temperature, and all treatments at 37 °C with 100 × MOI resulted in a 5 to 6 log reduction in cell count. Our results indicate that the phages vb_EcoM_bov9_1 and vb_EcoM_bov11CS3, which have higher burst sizes, are promising candidates for biocontrol experiments aimed at the eradication of E. coli O157 strains in animals or foodstuff.

The Importance of Shiga Toxin-Producing Escherichia coli O145:NM[H28]/H28 Infections in Argentina, 1998-2020.

Shiga toxin-producing Escherichia coli (STEC) is known as a pathogen associated with food-borne diseases. The STEC O145 serogroup has been related with acute watery diarrhea, bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Argentina has the highest rate of HUS worldwide with 70% of the cases associated with STEC infections. We aimed to describe the epidemiology and genetic diversity of STEC O145 strains isolated across Argentina between 1998–2020. The strains isolated from 543 cases of human disease and four cattle, were pheno-genotipically characterized. Sequencing of five strains was performed. The strains were serotyped as O145:NM[H28]/H28, O145:H25, and O145:HNT, and mainly characterized as O145:NM[H28]/stx2a/eae/ehxA (98.1%). The results obtained by sequencing were consistent with those obtained by traditional methods and additional genes involved in different mechanisms of the pathogen were observed. In this study, we confirmed that STEC O145 strains are the second serogroup after O157 and represent 20.3% of HUS cases in Argentina. The frequency of STEC O145 and other significant serogroups is of utmost importance for public health in the country. This study encourages the improvement of the surveillance system to prevent severe cases of human disease.

Large-Scale Phylogenetic Analysis Reveals a New Genetic Clade among Escherichia coli O26 Strains.

ABSTRACTShiga toxin-producing Escherichia coli (STEC) O26 is the predominant non-O157 serogroup causing hemolytic uremic syndrome worldwide. Moreover, the serogroup is highly dynamic and harbors several pathogenic clones. Here, we investigated the phylogenetic relationship of STEC O26 at a global level based on 1,367 strains from 20 countries deposited in NCBI and Enterobase databases. The whole-genome-based analysis identified a new genetic clade, called ST29C4. The new clade was unique in terms of multilocus sequence type (ST29), CRISPR (group Ia), and dominant plasmid gene profile (ehxA+/katP-/espP-/etpD-). Moreover, the combination of multiple typing methods (core genome single nucleotide polymorphism [SNP] typing, CRISPR typing, and virulence genes analysis) demonstrated that this new lineage ST29C4 was in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains. Besides, we observed that ST29C4 harbored extraintestinal pathogenic E. coli (ExPEC)-related virulence gene (VG), tsh, and STEC-associated VG, stx2a, suggesting the emergence of a hybrid pathogen. The ST29C4 strains also exhibited high similarity in stx2a-prophage and integrase with the O104:H4 strain, further demonstrating its potential risk to human health. Collectively, the large-scale phylogenetic analysis extends the understanding of the clonal structure of O26 strains and provides new insights for O26 strain microevolution.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) O26 is the second prevalent STEC serogroup only to O157, which can cause a series of diseases ranging from mild diarrhea to life-threatening hemolytic uremic syndrome (HUS). The serogroup is highly diverse and multiple clones are characterized, including ST29C1-C3 and ST21C1-C2. However, the phylogenetic relationship of these clones remains fully unclear. In this study, we revealed a new genetic clade among O26 strains, ST29C4, which was unique in terms of CRISPR, multilocus sequence type (MLST), and plasmid gene profile (PGP). Moreover, the combination of multiple typing methods demonstrated that this new clone was located in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains (i.e., ST29C1-C2 and ST21C1-C2). Overall, the large-scale phylogenetic analysis extends our current understanding of O26 microevolution.

Test-System for Identification of Typical and Genetically Altered Variants of Cholera Vibrios, Biovar El Tor, Using Real-Time PCR

The aim of the work was to analyze and assess the results of using PCR test-system “V. cholerae variant ctxB-rtxC FL genes” for identification of Vibrio cholerae O1 with differentiation between typical toxigenic and genetically modified variants in a multiplex format with a real-time hybridization-fluorescent recording of results. Materials and methods. To achieve this goal, a set of reagents for the PCR test-system “V. cholerae O1 variant ctxB-rtxC FL genes”, as well as a method for identifying V. cholerae O1 with differentiation between typical toxigenic and genetically altered variants were utilized. The specificity, specific activity and sensitivity of the developed test-system by the example of 35 V. cholerae O1 strains, 6 V. cholerae non-O1 strains, 5 heterologous strains (Shigella zonnei – 2 strains, one Salmonella typhimurium strain, S. enteritidis, Escherichia coli) were investigated. Results and discussion. The panel of reagents for the PCR test system “V. cholerae variant ctxB-rtxC FL genes” detects DNA fragments of the ctxBCl, ctxBEl, rtxC genes in the genome of toxigenic V. cholerae О1 (has specific activity, analytical sensitivity 1·103 GE/ml) and does not detect these genes in non-toxigenic V. cholerae O1 and non-O1, as well as in heterologous strains of microorganisms (100 % specificity). RF Patent No. 2732448 was granted for the PCR test-system “V. cholerae variant ctxB-rtxC FL genes”. It can be used to increase the efficiency of the epidemiological surveillance system and to carry out a justified scope of anti-epidemic measures in the event of cholera importation into the territory of the Russian Federation.

Условия и сроки хранения лиофилизированных поли- и моноклональных антитоксических пероксидазных конъюгатов

The article discusses the selection of optimal conditions for the lyophilization of poly- and monoclonal peroxidase conjugates used in the direct version of the enzyme immunoassay intended for the cholera toxin detection in Vibrio cholerae O1, O139 strains. Effective stabilizers included in protective media comprise 1 % sucrose, 1 % sodium thiosulfate, 0.5 % egg albumin, or 0.5 % bovine serum albumin. Their use increases the shelf life, increases stability, and contributes to the preservation of high sensitivity and specificity of poly- and monoclonal peroxidase conjugates. Evaluation of the serological activity of antitoxic conjugates in ELISA after lyophilization in a protective medium with stabilizers showed that it remains at the initial level. The results of testing lyophilized conjugates during storage allow us to speak about the possibility of their use for two years without changing the main indicators.

Two multistate outbreaks of a reoccurring Shiga toxin-producing Escherichia coli strain associated with romaine lettuce: USA, 2018-2019.

Leafy green vegetables are a common source of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) foodborne illness outbreaks. Ruminant animals, primarily cattle, are the major reservoir of STEC O157. Epidemiological, traceback and field investigations were conducted to identify potential outbreak sources. Product and environmental samples were tested for STEC. A reoccurring strain of STEC O157 caused two multistate outbreaks linked to romaine lettuce in 2018 and 2019, resulting in 234 illnesses in 33 states. Over 80% of patients interviewed consumed romaine lettuce before illness. The romaine lettuce was sourced from two California growing regions: Santa Maria and Salinas Valley in 2018 and Salinas Valley in 2019. The outbreak strain was isolated from environmental samples collected at sites >90 miles apart across growing regions, as well as from romaine-containing products in 2019. Although the definitive route of romaine contamination was undetermined, use of a contaminated agricultural water reservoir in 2018 and contamination from cattle grazing on adjacent land in 2019 were suspected as possible factors. Preventing lettuce contamination from growth to consumption is imperative to preventing illness. These outbreaks highlight the need to further understand mechanisms of romaine contamination, including the role of environmental or animal reservoirs for STEC O157.

Spontaneous Phage Resistance in Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens affecting poultry worldwide. The emergence of multidrug-resistant pathogens has renewed the interest in the therapeutic use of bacteriophages (phages). However, a major concern for the successful implementation of phage therapy is the emergence of phage-resistant mutants. The understanding of the phage-host interactions, as well as underlying mechanisms of resistance, have shown to be essential for the development of a successful phage therapy. Here, we demonstrate that the strictly lytic Escherichia phage vB_EcoM-P10 rapidly selected for resistance in the APEC ST95 O1 strain AM621. Whole-genome sequence analysis of 109 spontaneous phage-resistant mutant strains revealed 41 mutants with single-nucleotide polymorphisms (SNPs) in their core genome. In 32 of these, a single SNP was detected while two SNPs were identified in a total of nine strains. In total, 34 unique SNPs were detected. In 42 strains, including 18 strains with SNP(s), gene losses spanning 17 different genes were detected. Affected by genetic changes were genes known to be involved in phage resistance (outer membrane protein A, lipopolysaccharide-, O- antigen-, or cell wall-related genes) as well as genes not previously linked to phage resistance, including two hypothetical genes. In several strains, we did not detect any genetic changes. Infecting phages were not able to overcome the phage resistance in host strains. However, interestingly the initial infection was shown to have a great fitness cost for several mutant strains, with up to ∼65% decrease in overall growth. In conclusion, this study provides valuable insights into the phage-host interaction and phage resistance in APEC. Although acquired resistance to phages is frequently observed in pathogenic E. coli, it may be associated with loss of fitness, which could be exploited in phage therapy.

Passive immunization with anti- chimeric protein PilQ/PilA –DSL region IgY does not protect against mortality associated with Pseudomonas aeruginosa sepsis in a rabbit model

BackgroundPseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. MethodsSpecific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. ResultsWe found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. ConclusionAlthough produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

Prevalence of genotypic antimicrobial resistance in clinical Shiga toxin-producing Escherichia coli in Norway, 2018 to 2020.

IntroductionShiga toxin-producing Escherichia coli (STEC) can cause severe to fatal disease in humans. Antimicrobial treatment is sometimes necessary, but contraindicated due to undesirable clinical outcome. However, recent studies have shown promising outcomes following antimicrobial treatment. Before the establishment of a possible antimicrobial treatment strategy for STEC infections, the prevalence of antimicrobial resistance in STEC needs to be determined.Gap statementThe resistance status of Norwegian clinical STEC is not known and should be assessed.AimWe aim to characterize genotypic antimicrobial resistance determinants in clinical STEC in Norway, and determine the prevalence of genotypic resistance in order to inform possible antimicrobial treatment options for STEC infections.MethodologyWe included all clinical STEC submitted to the Norwegian Reference Laboratory from March 2018 to April 2020. All samples were whole-genome sequenced and screened for genotypic antimicrobial resistance,virulence determinants and plasmid incompatibility groups. We performed phylogenetic clustering of STEC by core-genome multi-locus sequence typing, and statistical association analyses between isolate characteristics and genotypic resistance.ResultsA total of 459 STEC were analysed. For 385 (83.9 %) STEC we did not identify any antimicrobial resistance determinants. Seventy-four STEC (16.1 %) harboured antimicrobial resistance determinants against one or more antimicrobial classes. The most frequent genotypic resistance was identified against aminoglycosides (10.5 %). Thirty-nine STEC (8.5 %) had a multi-drug resistance (MDR) genotype. Genotypic resistance was more prevalent in non-O157 than O157 STEC (P=0.02). A positive association was seen between genotypic resistance and the low-virulent STEC O117:H7 phylogenetic cluster (no. 14) (P<0.001). Genotypic resistance was not significantly associated to high-virulent STEC. STEC O146:H28 and isolates harbouring the plasmid replicon type IncQ1 were positively associated with MDR.ConclusionThe overall prevalence of genotypic resistance in clinical STEC in Norway is low (16.1 %). Genotypic resistance is more prevalent in non-O157 strains compared to O157 strains, and not significantly associated to high-virulent STEC. Resistance to antimicrobials suggested for treatment, especially azithromycin is low and may present an empiric treatment alternative for severe STEC infections.

Environmental and Epidemiological Aspects of V. cholerae O1 Contamination of Water Bodies in the Republic of Kalmykia

Background: Within the framework of cholera surveillance in the Russian Federation, annual isolation of nontoxigenic V. cholerae O1 strains from water bodies is registered in the Republic of Kalmykia. This fact is the rationale for the present study and analysis of data on contamination of water bodies with V. cholerae O1 with account for some environmental and quality indicators of water bodies used for recreational purposes. Objective: To study contamination of water bodies with V. cholerae O1 as a component of cholera control surveillance in the Republic of Kalmykia. Materials and methods: We used notifications issued by Offices of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Rospotrebnadzor) in the constituent entities of the Russian Federation, passports for V. cholerae O1 strains, and information from “Cholera vibrios. Russia” database for 1991–2019. Results: In the 1970s, 1980s and 1990s, several outbreaks, sporadic and single imported cases of cholera were registered in the republic. The total of 446 strains of V. cholerae O1 El Tor were isolated during the analyzed period. The PCR analysis identified them as V. cholerae O1 ctxA–tcpA– and V. cholerae O1 ctxA– tcpA+. We observed an increasing trend in the number of isolated strains with the approximation coefficient of 0.374 against its general decrease in the country in 1991–2019. Strains were isolated annually, mainly from the Elista River, Zayachy Pond and other water bodies with poor microbiological and chemical water quality parameters. V. cholerae O1 ctxA– tcpA+ were isolated mainly at the wastewater discharge sites, which indicated their imported origin, along with V. cholerae O1 ctxA– tcpA– with InDel and PCR genotypes not previously found in this region. We consider the contamination of water bodies with V. cholerae O1 ctxA– tcpA– and V. cholerae O1 ctxA– tcpA+ as prerequisites for possible deterioration of the local epidemiological situation due to cholera importation from endemic areas. Conclusion: When analyzing the pollution of water bodies in the Republic of Kalmykia with V. cholerae O1, we established a long-term annual isolation of cholera vibrios from some water bodies in the city of Elista, including Elistinka river, Zayachy and Kolonsky ponds, notorious for poor microbiological and chemical water quality indicators. The contamination was mainly attributed to domestic wastewater discharge and poorly treated effluents of sewage treatment plants into the Elistinka river within the city boundaries, as well as watercourses from hollows. This proves the imported origin of the bacterium confirmed by isolation of V. cholerae O1 ctxA– tcpA– with InDel- and PCR-genotypes not previously found in this region.

Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification

BackgroundStarch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase.ResultsIn this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%.ConclusionsOur study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.