Escherichia coli phage Eco_BIFF was isolated from several laboratory stocks of E. coli K-12 MG1655 derivatives. The source of the contamination is unknown. Eco_BIFF is a lytic phage that shows effective growth inhibition of E. coli K-12. Here, we announce the complete genome sequence of Eco_BIFF, and major findings from its genome annotation.

Escherichia coli or E. coli is a member of the fecal coliform group and is a more specific indicator of fecal contamination than other fecal coliform species, its presence indicate possibly presence of harmful bacteria which will cause diseases and it also suggests the extent as well as the nature of the contaminants. E. coli bacteria able to survive in water for 4 – 12 weeks and at present, it appears as an indicator to provide the accurate bacterial contamination of fecal matter in drinking water, because of the availability of simple, affordable, fast, sensitive and exact detection techniques. According to the laboratory experiment based techniques, 24 - 48 hours are required for the bacterial concentration to be reported. So, there is a necessity for continuous monitoring. Techniques for detection of many pathogenic bacterial strains are not yet available, sometimes days to weeks are required to get the results. To overcome the difficulties, expensive and time-consuming techniques are required to detect, count and identify the presence of specific bacterial strain. Public health relies on online monitoring of water quality that depends majorly on examination of fecal indicator bacteria, thus protection of health requires fecal pollution indicator so that it is not required to analyze drinking water to overcome the problems associated with waterborne diseases. This paper will brief the classification, sources, survival of E. coli bacteria and its correlation with basic water quality parameters in water sources.```

The human gut microbiome includes beneficial, commensal and pathogenic bacteria that possess antimicrobial resistance (AMR) genes and exchange these predominantly through conjugative plasmids. Escherichia coli is a significant component of the gastrointestinal microbiome and is typically non-pathogenic in this niche. In contrast, extra-intestinal pathogenic E. coli (ExPEC) including ST131 may occupy other environments like the urinary tract or bloodstream where they express genes enabling AMR and host adhesion like type 1 fimbriae. The extent to which non-pathogenic gut E. coli and infectious ST131 share AMR genes and key associated plasmids remains understudied at a genomic level. Here, we examined AMR gene sharing between gut E. coli and ST131 to discover an extensive shared preterm infant resistome. In addition, individual ST131 show extensive AMR gene diversity highlighting that analyses restricted to the core genome may be limiting and could miss AMR gene transfer patterns. We show that pEK499-like segments are ancestral to most ST131 Clade C isolates, contrasting with a minority with substantial pEK204-like regions encoding a type IV fimbriae operon. Moreover, ST131 possess extensive diversity at genes encoding type 1, type IV, P and F17-like fimbriae, particular within subclade C2. The type, structure and composition of AMR genes, plasmids and fimbriae varies widely in ST131 and this may mediate pathogenicity and infection outcomes.

Abstract: Escherichia coli are remarkably versatile microorganisms and important members of the normal intestinal microbiota of humans and animals. This harmless commensal organism can acquire a mixture of comprehensive mobile genetic elements that contain genes encoding viru-lence factors, becoming an emerging human pathogen capable of causing a broad spectrum of intestinal and extraintestinal diseases. Nine definite enteric E. coli pathotypes have been well characterized, causing diseases ranging from various gastrointestinal disorders to urinary tract infections. These pathotypes employ many virulence factors and effectors subverting the func-tions of host cells to mediate its virulence and pathogenesis. This review summarizes new de-velopments in our understanding of diverse virulence factors associated encoding genes used by different pathotypes of enteric pathogenic E. coli to cause intestinal and extraintestinal diseases in humans.

Background: Most drug-resistant Escherichia coli isolates in dogs come from diseased dogs. Prior to this study, the prevalence and risk factors of fecal carriage drug-resistant E. coli and epidemic clone sequence type (ST) 131 (including subtypes) isolates in dogs were unknown. Methods: Rectal swabs were used for E. coli isolation from 299 dogs in a veterinary teaching hospital in Taiwan. Antibiotic resistance and multiplex PCR analyses of E. coli for major STs were performed. Result: There were 43.1% cefazolin-resistant, 22.1% fluoroquinolone-resistant, and 9.4% extended-spectrum beta-lactamase-producing E. coli in our cohort. In the phylogenetic study, B2 was the predominant group (30.1%). The cefazolin-resistant group and ciprofloxacin-resistant group had greater antibiotic exposure in the last 14 days (P < 0.05). The age, sex, and dietary habits of the antibiotic-resistant and -susceptible groups were similar. In the seven isolates of ST131 in fecal colonization, the most predominant subtypes were FimH41 and FimH22. Conclusion: Recent antibiotic exposure was related to the fecal carriage of antibiotic-resistant E. coli isolates. Three major subtypes (FimH41, H22, and H30) of ST131 can thus be found in fecal carriage in dogs in Taiwan.

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a serious-global public health issue. A total of 292 E. coli isolates obtained from fecal samples of pigs in Central (n = 103) and Northeastern (n = 189) provinces of Thailand were included in this study. Eighty-six E. coli isolates were phenotypically confirmed to be β-lactamase producers (29.5%) and screened for the presence of β-lactamase genes. The genes in CTX-M family was most frequently found (90.7%). The blaCTX-M-15 gene (59.3%) was predominantly identified CTX-M genotype, followed by blaCTX-M-14 (31.4%) and blaCTX-M-4 (25.6%). The blaTEM-1 gene was prevalent (75.6%). The blaCTX-M-4 and blaCTX-M-14 genes were located on conjugative plasmid. The results highlight healthy pigs as reservoirs of ESBL-producing E. coli carrying ESBL genes that could be horizontally transferred.

Experimental evolution was utilized to produce 5 magnetite nanoparticle-resistant (FeNP1-5) populations of Escherichia coli. The control populations were not exposed to magnetite nanoparticles. The 24-hour growth of these replicates was evaluated in the presence of increasing concentrations magnetite NPs as well as other ionic metals (gallium III, iron II, iron III, silver I) and antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, tetracycline). Scanning electron microscope was utilized to determine cell size and shape in response to magnetite nanoparticle selection. Whole genome sequencing was carried out to determine if any genomic changes that resulted from magnetite nanoparticle resistance. After 25 days of selection magnetite resistance was evident in the FeNP treatment. The FeNP populations also showed a highly significantly (p < 0.0001) greater 24-growth as measured by optical density in metals (Fe (II), Fe (III), Ga (III), Ag and Cu II); as well as antibiotics (ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline). The FeNP resistant populations also showed a significantly greater cell length compared to controls (p < 0.001). Genomic analysis of FeNP identified both polymorphisms and hard selective sweeps in the RNA polymerase genes rpoA, rpoB, and rpoC. Collectively, our results show that E. coli can rapidly evolve resistance to magnetite nanoparticles and that this result is correlated resistances to other metals and antibiotics. There were also changes in cell morphology resulting from adaptation to magnetite NPs. Thus, the various applications of magnetite nanoparticles could result in unanticipated changes in resistance to both metal and antibiotics.

Under one-health perspective and the worldwide antimicrobial resistance concern, we investigate extraintestinal pathogenic Escherichia coli (ExPEC), uropathogenic E. coli (UPEC), and multidrug resistant (MDR) E. coli from 197 isolates recovered from healthy dogs in Spain between 2013 and 2017. Ninety-one (46.2%) isolates were classified as ExPEC and/or UPEC including 50 clones, among which (i) four clones were dominant (B2-CH14-180-ST127, B2-CH52-14-ST141, B2-CH103-9-ST372 and F-CH4-58-ST64815) and (ii) 15 had been shown to be displayed by previously published isolates causing extraintestinal infections in humans. Twenty-eight (14.2%) isolates were classified as MDR, associated with B1, D and E phylogroups and included 24 clones, of which eight had also been identified among human isolates causing infections. We selected 23 ST372 strains, 21 healthy dogs faecal isolates and two human clinical isolates for whole genome sequencing and built a SNP-tree with these 23 genomes and 174 genomes (128 from canine strains and 46 from human strains) obtained from public databases. The analysis of these 197 genomes allowed to identify six clusters. Cluster 1 comprised 74.6% of the strain genomes that were mostly composed of canine strain genomes (P < 0.00001). Clusters 4 and 6 also included canine strain genomes, while clusters 2, 3 and 5 were significantly associated with human strain genomes. All these findings suggest that dogs are reservoirs of ExPEC, UPEC and MDR E. coli isolates with zoonotic potential.

.The emergence and spread of antimicrobial resistance genes and resistant bacteria does not recognized animal, human or geographic borders. Addressing this threat requires a combination of multidisciplinary approach involving human, animal and environmental health (One Health). Because antimicrobial agents used in veterinary medicine maybe the same or like those in human medicine. Extended-spectrum beta lactamase (ESBL) E. coli is a growing public health problem worldwide, and the Agri-Food industry is constantly becoming sources of clinically important ESBL bacteria. Accordingly, the aim of this study was to investigate the occurrence and characteristics of ESBL-producing E. coli from dairy cattle, milk, and the farm environment. E. coli isolates were identified by their 16sRNA and their ESBL production was confirmed by ESBL-CHROMagar media and double disk diffusion method. Genotypes of ESBL producers were characterised using mPCR assay. Among the examined samples, 18(4.8 %) were positive for ESBL-producing E. coli. Of these, 66.7% were from milk, 27.8% and 5.5% were from farm environment and faecal samples respectively. Predominant ESBL Genotype identified were a combination of both TEM and CTX-M in eight out of 18 (44.4%) isolates. Four (22.2%) isolates produced CTX-M gene, two (11.1%) isolates produced TEM gene and four (22.2%) remaining isolates produced ESBL genes other than TEM, SHV, CTX-M and OXA. The SHV and OXA gene were not detected in all 18 isolates. The occurrence of these genotype in indicator organisms from dairy cattle, milk, and farm environment further re-enforced the potentials of food-animals as sources of infection for humans via the food chain. Thus, consolidating the need for the adoption of tripartite One Health approach in surveillance and monitoring antimicrobial resistance

Background: Infections caused by E. coli cause considerable disease burden and range from frequently occurring and relatively innocent urinary tract infection (UTI) to severe bloodstream infection (BSI). The incidence of infections caused by ESBL-producing E. coli (ESBL-PEc) is increasing, justifying surveillance and development of preventive strategies in several domains. Faecal carriage is universal and believed to be the most important reservoir for E. coli from which infections can originate. It is currently unknown to what extent Dutch E. coli carriage strains in the community reflect isolates causing disease. In this study, we will perform comparative genomics to infer the population structures of human-derived ESBL-PEc from community- and hospital-acquired infections and from community-based faecal carriage samples in the Netherlands. Furthermore, we will describe the molecular epidemiology of E. coli isolates causing invasive disease (BSI). Methods: This study uses four different microbiological data sources: 1) ESBL-PEc from patients with community-acquired UTI tested in primary care between May and November 2017, 2) ESBL-PEc from urine cultures obtained from patients hospitalized between January 2014 and December 2016, 3) E. coli from blood cultures obtained from patients hospitalized between January 2014 and December 2016, and 4) ESBL-PEc from faecal samples collected in a national population- prevalence study performed between January 2014 and January 2017. Clinical epidemiological data was collected from all patients and all isolates were subjected to whole genome sequencing. Discussion: The EPIGENEC study (EPIdemiology and GENetics of E. coli) will describe the molecular epidemiology of E. coli BSI and assess the genomic population structure of ESBL-PEc strains from community-acquired and nosocomial infections, and of ESBL-PEc reflecting community-based faecal carriage. Information from these studies may assist in optimizing surveillance strategies and determining targets and potential impact of future new preventive measures.

Enteropathogenic microorganisms like Escherichia coli cause severe intestinal problems by disrupting the gut homeostasis. The live microorganisms, when given in adequate quantities provide several beneficial effects to the host are known as probiotics. One of the pronounced benefits conferred by probiotic is to antagonize the growth of enteropathogens competing for adherence to the intestinal epithelium. Bifidobacterium is the major genera of human especially infant are intestinal microbiota. In current study, Bifidobacterium bifidum was isolated from the infant stools and probiotic potential was assessed using prescribed tolerance tests against low pH, gastric juices and bile salts. Anti-infectious activity of probiotic Bifidobacterium bifidum against enteropathogenic E. coli was checked both in vitro and in vivo using agar well diffusion assay and mice model respectively. Mice feces were evaluated for both Bifidobacterium bifidum and E. coli counts in all groups and analyzed statistically. In vitro results showed Bifidobacterium bifidum possess marked antibacterial activity against E. coli. There was significant decrease in enteropathogenic E. coli burden in the mice group fed with Bifidobacterium bifidum before and after challenge. In conclusion, the endogenous Bifidobacterium bifidum have excellent probiotic potential and can be used prophylactic and treatment option against enteropathogens.

Escherichia coli is known as one of the most important foodborne pathogens in humans, and contaminated chicken meat is an important source of foodborne infection with this bacterium. The occurrence of extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-Ec), in particular, in chicken meat is considered a global health problem. This study aimed to determine the magnitude of E. coli, with special emphasis on ESBL-Ec, along with their phenotypic antimicrobial resistance pattern in frozen chicken meat. The study also focused on the determination of ESBL-encoding genes in E. coli. A total of 113 frozen chicken meat samples were purchased from 40 outlets of nine branded supershops in five megacities in Bangladesh. Isolation and identification of E. coli were done based on cultural and biochemical properties, as well as PCR assay. The resistance pattern was determined by the disc diffusion method. ESBL-encoding genes were determined by multiplex PCR. The results showed that 76.1% of samples were positive for E. coli, of which 86% were ESBL producers. All the isolates were multidrug-resistant (MDR). Resistance to 9–11 and 12–13 antimicrobial classes was observed in 38.4% and 17.4% isolates, respectively, while only 11.6% were resistant to 3–5 classes. Possible extensive drug resistance (pXDR) was found in 2.3% of isolates. High single resistance was observed for oxytetracycline (93%) and amoxicillin (91.9%), followed by ampicillin (89.5%), trimethoprim–sulfamethoxazole, and pefloxacin (88.4%), and tetracycline (84.9%). Most importantly, 89.6% of isolates were resistant to carbapenems. All the isolates were positive for the blaTEM gene. However, the blaSHV and blaCTX-M-2 genes were identified in two ESBL-non producer isolates. None of the isolates carried the blaCTX-M-1 gene. This study provided evidence of the existence of MDR and pXDR ESBL-Ec in frozen chicken meat in Bangladesh, which may pose a risk to human health if the meat is not properly cooked or pickled raw only. This emphasizes the importance of the implementation of good slaughtering and processing practices by the processors.

The emergence of multi-drug resistant E. coli is an important matter of increasing considerable concern to global public health. The aim of this study was to investigate the incidence, antibiotic resistance pattern, phylogroups and genetic variation of E. coli isolates from raw milk, vegetable salad and ground meat samples. Methods: Culture-based techniques, Kirby-Bauer disk diffusion susceptibility testing, PCR and RAPD assays were used to determine the incidence rate, antimicrobial resistance pattern, phylogenetic groups and genetic diversity of the E. coli isolates. Results: E. coli isolates were highly resistant to amoxicillin (79.16%), trime-thoprim-sulfamethoxazole (70.83%), amoxicillin-clavulanic acid (62.50%), tetracycline (54.16%), chloramphenicol (54.16%), nitrofurantoin (54.16%), ampicillin (45.83%), streptomycin (45.83%), and kanamycin (33.33%); and completely susceptible to norfloxacin and azithromycin. 70.83% of the isolates were multi-drug resistant. Most E. coli isolates (46%) belonged to phylogroup A. RAPD with UBC245 primer categorized the isolates into 11 clusters. A high level of genetic di-versity was found among the isolates; however, 33.3% of the isolates were grouped in a major cluster (R5). Conclusions: Antibiotic resistance patterns are randomly distributed among the ge-netic clusters. Novel, practical, efficient food safety control and surveillance systems of multi-drug resistant foodborne pathogens are required to control the foodborne pathogen contamina-tion.

United States is the largest producer and the second largest exporter of broiler meat in the world. In the U.S, broiler production is largely converting to antibiotic-free programs which has caused an increase in morbidity and mortality within broiler farms. Escherichia coli and Clostridium perfringens are two important pathogenic bacteria readily found in the broiler environment and result in annual billion-dollar losses from colibacillosis, gangrenous dermatitis, and necrotic enteritis. Broiler industry is in search of non-antibiotic alternatives including novel vaccines, prebiotics, probiotics, and housing management strategies to mitigate production losses due to these diseases. This review provides an overview of the broiler industry and antibiotic free production, current challenges, and emerging research on antibiotic alternatives to reduce pathogenic microbial presence and improve bird health.

The carbapenemase OXA-244 is a derivate of OXA-48, and its detection is very difficult in laboratories. Here we report the identification and genomic analysis of an Escherichia coli isolate (28Eco12) harbouring the bla_OXA-244 gene identified in Colombia, South America. The 28Eco12 isolate was identified during a retrospective study and it was recovered from a patient treated in Colombia. The complete nucleotide sequence was established using the PacBio platform. A comparative genomics analysis with other bla_OXA-244–harbouring Escherichia coli strains was performed. The 28Eco12 isolate belonged to sequence type (ST) 38 and its genome was composed of two molecules, a chromosome of 5,343,367 bp and a plasmid of 92,027 bp, which belonged to the incompatibility group IncY and did not harbour resistance genes. The bla_OXA-244 gene was chromosomally-encoded and mobilized by an ISR1-related Tn6237 composite transposon. Notably, this transposon was inserted and located within a new genomic island. For our knowledge this is the first report of a bla_OXA-244–harbouring Escherichia coli isolate in American continent.Our results suggest that the introduction of the OXA-244-producing E. coli isolate was through clonal expansion of the ST38 pandemic clone. Other isolates producing OXA-244 could be circulating silently on the American continent.

In bioprocess development, the host and the genetic construct for a new biomanufacturing process are selected in the early developmental stages. This decision, made at the screening scale with very limited information about the performance of the selected cell factory in larger reactors, has a major influence on the performance of the final process. To overcome this, scaledown approaches are essential to run screenings that show the real cell factory performance at industrial like conditions. We present a fully automated robotic facility with 24 parallel mini-bioreactors that is operated by a model based adaptive input design framework for the characterization of clone libraries under scale-down conditions. The cultivation operation strategies are computed and continuously refined based on a macro-kinetic growth model that is continuously re-fitted to the available experimental data. The added value of the approach is demonstrated with 24 parallel fed-batch cultivations in a mini-bioreactor system with eight different Escherichia coli strains in triplicate. The 24 fed-batches ran under the desired conditions generating sufficient information to define the fastest growing strain in an environment with varying glucose concentrations similar to industrial scale bioreactors.

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from E. coli to S. cerevisiae. We demonstrated that there was no substantial decrease in conjugation frequency as plasmid size increased—up to 138.5 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids

In this work, a xylan-based antimicrobial additive agent was prepared and aimed for uses in paper products against Escherichia coli bacteria. The derived Cationic-Xylan-grafted-PHGH (CX-g-PHGH) was successfully synthesized by graft copolymerization of cationic-xylan with guanidine polymer (PHGH) using ceric ammonium nitrate as initiator. The obtained CX-g-PHGH had maximum PHGH grafting ratio of 18.45% and efficiency of 58.45%, and showed good viscosity and thermal stability. Furthermore, the paper samples prepared in this work were reinforced obviously with the addition of CX-g-PHGH by improved mechanical properties. Compared to the reference paper without any of the xylan-derivatives, the index of tensile, tear, burst and folding endurance of the paper had increases up to 20.07%, 25.31%, 30.20% and 77.78%, respectively. Moreover, the prepared CX-g-PHGH paper exhibited an efficient antimicrobial activity against E. coli bacterial, by which a lot of applications based on the new xylan-derived additive agent obtained in this work could be found, especially in field of antimicrobial paper products against E. Coli bacteria from contaminated food.

Background: Phylogroup B2 Escherichia coli have been associated with Ulcerative Colitis (UC). In this study, we aimed to compare colonization with the UC-associated E. coli p19A in different mice strains, to investigate the role of alpha hemolysin in a UC mouse model. Methods: In this study, Sigirr -/- and C57BL/6 mice were chosen, and UC was induced by adding Dextran Sulfate Sodium (DSS) to the drinking water. The mice were pre-treated with ciprofloxacin. p19A expressing luminescence and GFP, alpha-hemolysin knock out p19A-∆hlyI II, and non-pathogenic lab E. coli DH10B were cultured in LB broth, and orally gavaged into the mice. Colonization with p19A WT was visualized using an in-vivo imaging system. Results: p19A WT colonized the colon, ileum, Peyer’s patches, liver, and spleen of infected C57BL/6 and Sigirr -/- mice. A total of 99% of the p19A WT infected C57BL/6 mice and 29% of the p19A WT infected Sigirr -/- mice survived to the 4th post infection day. Conclusion: UC-associated E. coli p19A WT colonized the intestines of DSS-treated mice and caused extra-intestinal infection. Hemolysin is an important factor in this pathogenesis, since isogenic hemolysin mutants did not cause the same inflammation.

Escherichia coli and Shigella are common diarrhea-causing pathogens in children and adults. Enteroinvasive Escherichia coli (EIEC) shares a similar pathogenic mechanism with Shigella. However, EIEC are less virulent than Shigella. The aim of this work was to get a better understanding of the virulence differences between EIEC and S. flexneri. We investigated i) the bacterial gene co-expression networks (GCNs) and ii) the the transcriptional modules (WGCNA) of Caco-2 cells infected with EIEC or with S. flexneri during a three-hour period of bacterial infection. The GCN analysis showed that EIEC and S. flexneri networks presented different topologies. Additionally, the EIEC network revealed that pINV genes are not connected with chromosomal genes. WGCNA and eigengene analysis showed enterocyte gene expression variation along the three-hour bacterial post-infection period. Additionally, at one-hour post-infection EIEC induced a higher number of gene expression changes in Caco-2 cells than S. flexneri. Several of these genes are involved in autophagy. This study showed that the lower virulence of EIEC is associated with a lack of functional cooperation between pINV and chromosomal genes, differently from what was observed in S. flexneri. Consequently, EIEC becomes less efficient in subverting host-cell bacterial recognition as well as defense mechanisms such as autophagy.

Enteroaggregative Escherichia coli (EAEC) are highly heterogeneous in virulence; we wanted to understand the pathogenic potential of EAEC isolated various clinical and non-clinical sources in an animal model. We infected male BALB/c mice in six mice/group with 50 EAEC isolates, isolated from clinical and non-clinical sources. We studied colonization, weight loss, stool shedding, inflammatory markers and its relationship with 21 virulence genes and phylogroups, EAEC organ burden and histopathological changes. We detected significantly more inflammatory changes and fecal lactoferrin and calprotectin levels in mice infected with EAEC isolated from symptomatic cases. In clinical EAEC isolates, the presence of chromosomal genes {(aap (46%), aaiC (23.3%), and SPATEs (pet (13.3%), sat (20%), sigA and pic (6.6%)}, adhesive variants {(agg4A (53.3%), aggA (53.3%), aafA (36.6%), agg3A (40%)} of EAEC, and master regulator gene aggR (66.6%) were associated with higher levels of lactoferrin and calprotectin. Also, 70% (9/13) of EAEC isolated from acute diarrheal cases bearing chuA (70%) in our study were assigned to group B2 (4 isolates) and D (5 isolates). Real-time PCR analysis revealed that colonization by EAEC strains from different clinical and non-clinical sources occurs up to 10-15 days of life. Even from non-diarrhoeal stools and non-clinical sources, had the potential to cause prolonged colonization, weight loss, and inflammation in the intestine though the degree varied. Moreover, better understanding of EAEC pathogenic pathways is desperately needed in different clinical scenarios.

The enterohemorrhagic Escherichia coli (EHEC) O157: H7 EspF is known to be a multifunctional effector that triggers several damage processes in the host cells. However, in the process of EHEC O157: H7 infection, the interactions between EspF, its N- or C-terminus, and host proteins are still unclear. In this work, we use co-immunoprecipitation combined with mass spectrometry (CoIP-MS) to screen the interactions between EspF/EspF-N/EspF-C terminus and host proteins. A total of 311 host proteins are analyzed. The N-terminus of EspF is found to interact with 192 proteins, whereas 205 proteins interact with the C-terminus. These proteins are mainly involved in RNA splicing, endoplasmic reticulum stress, and a variety of metabolic signaling pathways. Surprisingly, MS results reveal that EspF can also phosphorylate H2AX, suggesting that EspF may directly mediate DNA damage. Here, by western blot and immunofluorescence (IF), we verified that EspF can cause phosphorylation of H2AX and mediates cell multi-nuclearation and cell hypertrophy. Furthermore, we verify here for the first time that SMC1 interacts with EspF -C-terminus, and provide evidence that EspF increases p-SMC1 levels. p-SMC1 is known to influence S-phase cell cycle arrest and usually increases during periods of DNA damage. Our work revealed a novel interaction between EspF and the host protein SMC1 and lays a foundation for further research on EspF-mediated host DNA damage, apoptosis, and even colorectal carcinogenesis.

Aims: The lack of information about the inter variability of the test results obtained by CLSI and EUCAST requires further clarifications to interpret antimicrobial susceptibility patterns better. This study aimed to compare the CLSI and EUCAST interpretations of the antimicrobial susceptibility test results of the ESBL–producing uropathogenic Escherichia coli and Klebsiella pneumonia strains. Methods: After obtaining 157 ESBL-producing E. coli and 95, ESBL-producing K. pneumonia isolates from the urine specimens of the patients, Kirby-Bauer’s disc diffusion method was used for conducting antimicrobial susceptibility test. The test procedures and the interpretation of the results were carried out according to the year 2017 versions of both of the two guidelines. For the statistical comparison of concordance between the two guidelines, the Kappa coefficients and the concordance rates were calculated. Results: The results were graded in the range from perfect to poor agreement. For E. coli, interpretations of the AST results revealed a moderate to perfect agreement between both methods. Weighted Kappa agreement scores in the range from 0.42 to 1. The agreement for AMC, TPZ30/6, ceftazidime 10, meropenem, and aztreonam was poor without any inconsistencies. For Klebsiella, the kappa agreement score was in the range from 0.25 to 1. It was incompatible with AMC, TPZ 30/6, ceftazidime 10, aztreonam; there was poor agreement for cefepime, amikacin and ertapenem. Conclusions: Our results showed agreement between the two guidelines for uropathogenic extended-spectrum ß-lactamase producing Escherichia coli and Klebsiella pneumoniae but also showed inconsistencies between two guidelines. Therefore, the results of our study contribute to the comparison of these guidelines for interpreting antibiotic susceptibilities.

Different surveillance studies (2005-2015) on the presence of ESBL-producing E. coli in the northwest Spain revealed that eae-positive isolates of serotype O153:H10 were periodically detected in meat (of beef, chicken and pork), and also implicated in human diarrhea. This study aimed: i) to characterize the degree of relatedness between human and animal isolates; ii) to know if this was a geographically restricted or disseminated genetic lineage. Thirty-two isolates were conventionally typified as O153:H10-A-ST10 fimH54, fimAvMT78, traT and eae-beta1, being 21 of those CTX-M-32 or SHV-12 producers. PFGE comparison of their macrorestriction profiles showed high similarity (>85%). The plasmidome analysis revealed a stable combination of IncF (F2:A-:B-), IncI1 (STunknown) and IncX1 plasmid types, together with non-conjugative Col-like. Besides, the core genome investigation based on the cgMLST scheme from Enterobase, proved close relatedness between isolates of human and animal origin. Our results demonstrate that a hybrid MDR aEPEC/ExPEC of clonal group O153:H10-A-ST10 (CH11-54) would be playing a successful role in spreading ESBLs (CTX-M-32) in our region within different hosts, including wildlife. It would be potentially implicated in human diarrhea via food (meat) transmission. Importantly, we proved genomic evidence of a related hybrid aEPEC/ExPEC in other countries.

Bacterial antibiotic resistance has become a major global health concern. One of the main reasons for the development of multi-drug resistance properties in bacteria is due to the bacterial efflux pump systems. They are important transport proteins, mainly involved in the removal of toxic substrates like antibiotics from inner cell environment. These pumps are responsible for the intrinsic ability of bacteria to get resistant to the antibiotic. Various types of efflux pumps are present in the Gram-positive and Gram-negative bacteria. Plant-derived products like Capsaicin, Olympicin A, and Indirubicin were found to be inhibitors of an efflux pump in Staphylococcus aureus similarly Ursolic acid derivatives; Daidzein and Lanatoside C were plant-derived inhibitors of an efflux pump in Escherichia coli. In this review detail information have been provided about efflux pump inhibitors that have been found to be effective in the Gram-positive bacteria and Gram-negative bacteria. The aim of this review is to focus on the role of plant-derived compounds as effective efflux pumps inhibitors with reference to mainly Staphylococcus aureus and Escherichia coli.

Diarrhoea caused by enterotoxigenic Escherichia coli is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhoea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed eleven single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched Lacto-N-neohexaose (Galbeta4GlcNAcbeta6[Galbeta4GlcNAcbeta3]Galbeta4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity.

Studies have previously described the occurrence of multidrug-resistant (MDR) Escherichia coli in human and veterinary medical settings, livestock, and, to a lesser extent, in the environment and food. While they mostly analyzed foodborne E. coli regarding phenotypic and sometimes genotypic antibiotic resistance and basic phylogenetic classification, we have limited understanding of the in vitro and in vivo virulence characteristics and global phylogenetic contexts of these bacteria. Here, we investigated an E. coli strain (PBIO3502) isolated from a pork sausage in Germany in 2021 in-depth. Whole-genome sequence analysis revealed sequence type (ST)58, which is an emerging international high-risk clonal lineage. In addition to its MDR phenotype that mostly matched the genotype, PBIO3502 demonstrated pronounced virulence features, including in vitro biofilm formation, siderophore secretion, serum resilience, and in vivo mortality in Galleria mellonella larvae. Along with the genomic analysis indicating close phylogenetic relatedness of our strain with publicly available, clinically relevant representatives of the same ST, these results suggest the zoonotic and pathogenic character of PBIO3502 with the potential to cause infection in humans and animals. Also, our study highlights the necessity of the One Health approach while integrating human, animal, and environmental health, as well as the role of meat products and food chains in the putative transmission of MDR pathogens.

Water borne pathogens present a threat to human health and their disinfection from water poses a challenge, prompting search for newer methods and newer materials. Disinfection of Gram-negative bacterium Escherichia coli and Gram-positive coccal bacterium Staphylococcus aureus in aqueous matrix was achieved within 60 and 90 minutes respectively at 35⁰C using solar-photocatalysis mediated by sonochemically synthesized Ag@ZnO core-shell nanoparticles. The efficiency of the process increased with increase in temperature and at 55⁰C the disinfection could be achieved in 45 and 60 min respectively for the two bacteria. A new ultrasound assisted chemical precipitation technique was used for the synthesis of Ag@ZnO core-shell nanoparticles. The characteristics of the synthesized material were established using physical techniques. The material remained stable even at 400o C. Disinfection efficiency of the Ag@ZnO core-shell nanoparticles was confirmed in case of real world water samples from pond, river, municipal tap and was found to be better than that of pure ZnO and TiO2 (Degussa P25). When the nanoparticle based catalyst was recycled and reused for subsequent disinfection experiments, its efficiency did not change remarkably even after three cycles. The sonochemically synthesized Ag@ZnO core-shell nanoparticles have a good potential for application in solar photocatalytic disinfection of water borne pathogens.

In this work, we pursued the biological characterization of the venom of Montivipera born-muelleri, a viper from the Lebanese mountains. In relativity to its antibacterial potential, the in-hibitory effect of this venom on the F1F0-ATPase enzymes of Gram-positive Staphylocoocus epider-midis and Gram-negative Escherichia coli bacteria was examined. In order to determine the de-gree of cytotoxicity of the venom on the HCT116 human colon cancer cell lines, the biological MTT proliferation and cell viability test were implemented. After validation of the enzymatic F1F0-ATPase model by the spectrophotometric method, using quercetin as the reference ligand, re-sults revealed that M. bornmuelleri venom is able to inhibit the activity of the enzyme of these two bacteria with a concentration of the order of 100-150 µg/mL. In addition, a venom concentration of 10 µg/mL was sufficient to kill the totality of HCT116 cell lines cultivated in vitro. These data show that M. bornmuelleri venom is a mixture of diverse molecules presenting activities of interest and is a potential source to explore in order to discover new drug candidates.

Recently the polyvalent bacteriophage FP01, isolated from wastewater in Valparaiso, Chile, was described to have lytic activity across species against Escherichia coli and Salmonella enterica serovars. Due to it polyvalent nature the bacteriophage FP01 could have potential application in food and agri-industry. Also, fundamental aspects of polyvalent bacteriophage biology are not well known. In this study we sequenced and describe the complete genome of the polyvalent phage FP01 (MH745368) using the nanopore technology. The bacteriophage FP01 genome has a 44,900 bp, double-stranded DNA with an average G+C content of 49.41% and 90 coding sequences (CDSs). We found that the phage FP01 critically depends on host factors for replication and transcription. Also, it has a critical lysogenic repressor pseudogene. Phylogenetic analyses indicated that the phage FP01 is closely related to phages lambda and P22. These results suggest that the phage FP01 could be a lytic variant of a lysogenic phage or acquired genes from lysogenic phages during host infection.

Cysteine cathepsins, a class of proteinaceous enzymes, regulate a wide variety of metabolic processes in human including protein breakdown and turnover and immune functions. Eleven cysteine cathepsins have been identified so far and a wide array of studies related to identifying their specific functions, regulation and distribution patterns in tissues have been conducted. However, in recent past, the association of cysteine cathepsins in occurrence and progression of cancers have been identified and this has caused unrest in scientists triggering them to investigate the physiology, biochemical pathways and interactions of these cathepsins in cancer metastasis and therefore has become a noteworthy topic of intensive research. This review focusses and collects together the published work on molecular functional and structural characterization studies that have been done so far on in vitro expression of genes encoding for cysteine cathepsins in the Escherichia coli bacterial expression system. Accordingly, it was found out that all cathepsins except for cathepsins K, C, H, X and W have been expressed this way and the majority of them were found to be expressed in E. coli BL21(DE3) pLysS expression host via pET3 expression vector. In addition, it was also noted that in most of the expression studies, the substrate that was used to validate the enzymatic activity of the recombinant enzyme that was produced was a cysteine residue along with a benzyloxy-carbonyl salt. Through this review, the authors suggest that there is a very high need that all cysteine cathepsins need to be characterized both structurally and functionally on a molecular platform to better understand their interactions including the biochemical pathways. It is also momentous that the mass production of the recombinant forms of these enzymes are facilitated via expression in such bacterial expression systems and in turn, would also provide a strong platform for the development and progression of studies related to human physiology including oncological studies such as cancer metastasis. Moreover, as per biochemical features of the enzymes that could be identified, the production of efficient inhibitors or inducers as per the necessity to improve health and promote wellbeing among the mankind could be facilitated.

This study evaluated the microbial safety of vended boiled, pasteurized and UHT milk sold in Nakawa, Kampala-Uganda. 15 milk samples were analyzed; 2 samples had Salmonella, 5 had S. aureus with a count of 1.66 0.02 log₁₀CFU/ml. E. coli was detected in 8 samples with 1.0 0.02 to 3.0 0.01log₁₀CFU/ml count. A high load of 3.0 CFU/ml was obtained in 3 samples with E. coli. Four E. coli positive samples had a contamination load of 2.0 0.015log₁₀ CFU/ml of which one was pasteurized milk. Only a pasteurized milk showed a low E. coli load at 1.0 0.02log₁₀ CFU/ml. All UHT milk had no microbial contamination. Both boiled and pasteurized milk had Salmonella, S. aureus and E. coli in levels above the set threshold limits. Milk consumers in Nakawa stand a potential public health risk of food poisoning reflected by presence of Salmonella, S. aureus and E. coli in some milk sold in the area.

Bacteria play a key role in both human health and disease. One of the most prevalent harmful bacteria is E. coli which is responsible for several illnesses ranging from diarrhea, stomach cramp and fever. In this work we explore the application of moderate electric or magnetic fields for treating deionized water that is contaminated with E. coli. We found that moderate alternating electric (AC) fields (10 V/cm to 1kV/cm) and moderate static magnetic fields (10 to 65 mT) can significantly inactivate E. coli by up to 90% or more. This provides the possibilities of developing a low cost and a practical bacteria inactivation technique using electric or magnetic fields.

Markets from Maputo City have been reported to have poorly hygienic premises and sellers unaware of the safest food handling practices. This study aimed to analyze associations between some lettuce (Lactuca sativa) handling features and the levels of total, fecal coliforms and Escherichia coli. Cabbage was purchased in the 3 markets. Just before the acquisition, the researchers recorded the source of water used to wash it, if they were sold in booths or on the floor, if they were sold mixed with other products, and if the sellers were wearing gloves. The cabbage heads (42) were analyzed through the technique of multiple tubes to estimate the most probable number. Average levels were 14.43 MPN/g for total coliforms, 13.9 MPN/g for fecal coliforms and 12.02 MPN/g for E. coli. The highest levels of contamination seemed associated with the use of well water, the food sold on the floor, the lettuce sold together with other products and the lack of gloves. The mixture of products only showed a major impact on the level of E. coli (p < 0.001). Yet, all levels were within the satisfactory range, according to the guidelines from The International Commission on Microbiological Specifications for Foods (ICMSF).

Genome-scale metabolic models (GEMs) have been widely used for phenotypic prediction of microorganisms. However, the lack of other constraints in the stoichiometric model often leads to a large metabolic solution space inaccessible. Inspired by previous studies that take allocation of macromolecule resources into account, we developed a simplified Python-based workflow for constructing enzymatic constrained metabolic network model (ECMpy) and constructed an enzyme-constrained model for Escherichia coli (eciML1515) by directly adding a total enzyme amount constraint in the latest version of GEM for E. coli (iML1515), considering the protein subunit composition in the reaction, and automated calibration of enzyme kinetic parameters. Using eciML1515, we predicted the overflow metabolism of E. coli and revealed that redox balance was the key reason for the difference between E. coli and Saccharomyces cerevisiae in overflow metabolism. The growth rate predictions on 24 single-carbon sources were improved significantly when compared with other enzyme-constrained models of E. coli. Finally, we revealed the tradeoff between enzyme usage efficiency and biomass yield by exploring the metabolic behaviors under different substrate consumption rates. Enzyme-constrained models can improve simulation accuracy and thus can predict cellular phenotypes under various genetic perturbations more precisely, providing reliable guidance for metabolic engineering.

Pasteurized milks are still causing food borne illness. Milk contamination can occur at any stage from its way from cow to our tables. Usually milk is pure and sterile when produced in udder of a healthy cow. Like humans, cow are reservoirs of bacteria which are harmless to humans and some cows can harbour few bacteria that are harmful to humans even though they are not harmful to the cow. Milk can be contaminated during or after milking. Also, cow feeds can be contaminated with mycotoxins such as aflatoxins produced by the fungi, Aspergillus flavus. Four types of aflatoxins are known which are; aflatoxin B1, B2, G1, G2. Cows comsuming feeds contaminated with aflatoxin B1 leads to secretion in the milk of aflatoxin M1 and M2 causing aflatoxicosis. Microbial contamination of milk and dairy products is a universal problem and foodborne infections accounting for 20 million cases annually in the world have been identified as an important public health and economic problem in developed as well as developing nations. The main objective of this study was to determine milk microbial quality in Kicukiro district. The specific objectives are to identify bacteria pathogens in milk collected in Kicukiro district, to compare milk quality among sectors of Kicukiro district, to compare milk processed by industries and home-processed milk. The methodology employed in this research was cross-sectional and experimental as the study began with collection of raw data and went through laboratory analysis from July–August, 2018. The findings showed that 59.56% of the milk fell within Grade I – Grade III (< 200,000 ≤ 2,000,000 cfu/ml) and 40.42 % of the milk samples were not within the acceptable limit of total count quality as per COMESA and EAS, non-lactobacilli and fungi were present in most samples as examined through microscope and no Staphylococcus aureus was present in any sample as examined by catalase and coagulase tests.

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

To evaluate the behaviour of the relevant microbial populations during stretched cheese production, the quantitative microbiological analysis was performed during the critical steps of the preparation. The obtained data distributions proved statistically significant increases in all indicators, on average by 4.55 ± 0.64 log CFU/g of presumptive lactococci counts, 4.06 ±0.61 of lactobacilli, 1.53 ± 0.57 log CFU/g of coliforms, 2.42 ± 0.67 log CFU/g of Escherichia coli, 1.53 ± 0.75 log CFU/g of yeasts and moulds, and 0.99 ± 0.27 log CFU/g of presumptive Staphylococcus aureus, from the early stage of milk coagulation until curd ripening (0–24 h). The following steaming/stretching process caused reductions in viable counts with the most significant inactivation effect on coliform bacteria, including E. coli (-4.0 ± 1.0 log CFU/g). Total viable counts and yeasts and moulds showed 2 and almost 3 log reduction (-2.2 ± 1.1 log CFU/g and -2.6 ± 0.9 log CFU/g), respectively. The lowest decreases in presumptive S. aureus counts were estimated at the level of -1.50 ± 0.64 log CFU/g. The counts of yeasts and moulds showed the best indicatory function during the entire storage period of vacuum-packaged cheeses at 6 °C.

The widespread use of antibiotics has led to a gradual increase in drug-resistant bacterial infections, which severely weakens the clinical efficacy of antibacterial therapies. In recent decades, stilbenes aroused great interest because of their high bioavailability, as well as for their manifold biological activity. Our research efforts are focused on synthetic heteroaromatic stilbene deriva-tives as they represent a potentially new type of antibiotic with a wide antibacterial spectrum. Herein, a preliminary molecular modeling study and a versatile synthetic scheme allowed us to define eight heteroaromatic stilbene derivatives with potential antimicrobial activity. In order to evaluate our compound’s activity spectrum and antibacterial ability, Minimum Inhibitory Con-centration (MIC) and Minimum Bactericidal Concentration (MBC) tests have been performed on Gram-positive and Gram-negative ATCC strains. Compounds PB4, PB5, PB7 and PB8 showed the best values in terms of MIC and were also evaluated for MBC, which however was found to be greater than MIC, confirming a bacteriostatic activity. For all compounds, we evaluated toxici-ty on colon-rectal adenocarcinoma cells tumor cells (CaCo2), once established that the whole se-lected set was more active than 5-Fluorouracil in reducing CaCo-2 cells viability. To the best of our knowledge, the biological assays have shown for these derivatives an excellent bacteriostatic activity, compared to similar molecular structures previously reported, thus paving the way for a new class of antibiotic compounds.

A New monogalactosyldiacylglycerol (MGDG), a known monogalactosylmonoacylglycerol (MGMG) and a known polyunsaturated fatty acid methyl ester (PUFAME) were isolated from the marine dinoflagellate Karenia mikimotoi. The planar structure of the glycolipids was elucidated using MS and NMR spectroscopic analyses and comparisons to the known glycolipid to confirm its structure. The isolation of PUFAME strongly supports the polyunsaturated fatty acid fragment of these glycolipids. The relative configuration of the sugar was deduced by comparisons of 3JHH values and proton chemical shifts with those of known glycolipids. All isolated compounds MGDG, MGMG and PUFAME (1-3) were evaluated for their antimicrobial and anti-inflammatory activity. All compounds modulated macrophage responses, with compound 3 exhibiting the greatest anti-inflammatory activity.

Ribosomes are macromolecular complexes important to protein translation, and thus, essential to life. Comprising an ensemble of ribosomal proteins and RNA molecules, ribosomes are conserved in structure and function across all domains of life, but recent structural studies have revealed differentiated structures of ribosomes from bacterial, archaea and eukaryotes. Additionally, unique ribosomal protein mass fingerprints have been found for individual species; thereby, indicating that ribosomes are differentiated in structure amongst different species. Given that structure defines function, differentiated function likely exists amongst ribosomes of different species, which could manifest as differences in translation efficiency that could impact on cell growth rate. But ribosomal proteins also hold phylogenetic significance in informing the evolutionary trajectory of each species. Such ribosomal proteins are thus not highly conserved and offers sufficient sequence space for the evolution of differentiated structure and function in different species. Using ribosomal proteins that hold phylogenetic significance as templates, this study sought to understand the mutational and conformational limits that define functional ribosomes. Specifically, ribosomal proteins in Bacillus subtilis that hold phylogenetic cues would be mutated through error-prone polymerase chain reaction to generate variants that are subsequently transformed into Escherichia coli. To help assess the functional properties of the heterologous ribosomal proteins, endogenous ribosomal protein genes would be inactivated by multiplex CRISPR interference. Since variants in ribosomal proteins would likely impact on ribosome function and translation efficiency, live/dead screening on LB agar would be effective as a preliminary screen for functional mutants. These mutants would subsequently be inoculated into liquid LB medium in 96 well plates to quantify relative growth rates between different strains harbouring different heterologous variants of ribosomal proteins. Plasmids containing different ribosomal protein mutants would be extracted from each functional strain and subjected to Sanger sequencing for determining the specific mutations involved. Collection of such mutations would provide a comprehensive mutational map that define the limits of ribosomal protein sequence space important to ribosome function. Furthermore, biochemical isolation of ribosomal proteins and their structural characterization by X-ray crystallography or cryo-electron microscopy would further illuminate the structural significance of each mutation on ribosome structure and function; thereby, elucidating the structural tolerance space for functional ribosomes. Overall, generating a diverse pool of mutant ribosomal proteins in viability assays followed by sequencing and structural characterization would help define the mutational and conformational limits of a functional and efficient ribosome.

Natural products have long played a major role in medicine and science. The garden snail Cornu aspersa is a rich source of biologically active natural substances which might be an important source for new drugs to treat human disease. Based on our previous studies seven fractions containing compounds with Mw &lt;3 kDa, &lt;10 kDa, &lt;20 kDa, &gt;20 kDa, and between 3-5 kDa, 5-10 kDa, and 10-30 kDa were purified from the mucus of C. aspersa and analyzed by tandem mass spectrometry (MALDI-TOF/TOF). Seventeen novel peptides with potential antibacterial activity have been identified by de novo MS/MS sequencing using tandem mass spectrometry. The different fractions were tested for antibacterial activity against Gram─ (Pseudomonas aureofaciens and Escherichia coli) and Gram+ (Brevibacillus laterosporus) bacterial strains as well anaerobic bacterium Clostridium perfringens. These results revealed that the peptide fractions exhibit a predominant antibacterial activity against B. laterosporus, the fraction with Mw 10 – 30 kDa against E. coli, another peptide fraction &lt;20 kDa against P. aureofaciens, and the protein fraction &gt;20 kDa against the bacterial strain C. perfringens. The discovery of new antimicrobial peptides (AMPs) from natural sources is of great importance for public health due to their effective antimicrobial activities and low resistance rates.