Antibiotic resistance, virulence genes, and phylogenetic groups of bacteria isolated from wild passerine birds in Iran.

Wild passerine birds may serve as environmental reservoirs and as vectors for the long-distance dispersal of microorganisms and resistance determinants. However, there is no much knowledge on pathogenic bacteria in wild birds in Iran. The present study aimed to analyze antibiotic resistance in wild passerine birds collected from the northeast region of Iran as the rich breeding bird fauna with a special focus on Escherichia coli virulence, integron, and phylogenetic groups. A total of 326 isolates were collected and identified from the cloaca of wild birds using a swab. The results showed a high percentage of resistance to tetracycline (45.8%) and ampicillin (26.7%). The resistance genes, tet(A), tet(B), tet(M), and tet(L) were detected in tetracycline-resistant isolates, while the blaTEM gene was the most prevalent in ampicillin-resistant isolates (38.6%). Out of the 129 E. coli isolates examined, 99 isolates were found to have virulence gene, with the highest prevalence of the fimbriae (fimH) gene (22.4%). Additionally, the E. coli strains were most often classified into phylogenetic groups B1 (48.8%) followed by B2 (19.3%). Also, the highest average frequency of class 1 integron was detected among our isolates. Results indicated that wild birds are reservoirs of multidrug resistance and virulence genes that may have the potential to be transferred to other organisms, including humans.

Evaluation of virulence determinants and cell surface properties associated with biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) Escherichia coli from livestock and poultry origin.

Antibiotic resistance poses a persistent threat to modern medicine due to the emergence of novel antibiotic-resistant strains. Therefore, a timely understanding of antibiotic resistance and the virulence biology of pathogenic bacteria, particularly those of public health significance, is crucial for implementing effective mitigation strategies. This study aimed to investigate the virulence profiles of ten S. aureus isolates (NDa to NDj) and ten E. coli isolates (ND1 to ND10) originating from livestock and poultry, and to assess how various cell surface properties and biofilm formation abilities influence antibiotic resistance phenotypes. Antibiotic resistance profiling through phenotypic (AST) and genotypic methods (PCR) confirmed that NDa to NDe were methicillin-resistant S. aureus (MRSA) and ND1 to ND5 were extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Virulence properties such as hemolytic activity, coagulase activity, and nuclease activity were found to be independent of the antibiotic resistance phenotype in S. aureus. In contrast, biofilm formation phenotype was observed to influence antibiotic resistance phenotypes, with MRSA and ESBL E. coli isolates demonstrating higher biofilm formation potency. Chemical and enzymatic analysis of S. aureus and E. coli biofilms revealed proteins and polysaccharides as major components, followed by nucleic acids. Furthermore, cell surface properties such as auto-aggregation and hydrophobicity were notably higher in isolates with strong to medium biofilm-forming capabilities (ESBL and MRSA isolates), corroborated by genomic confirmation of various genes associated with biofilm, adhesion, and colonization. In conclusion, this study highlights that surface hydrophobicity and biofilm formation ability of MRSA (NDa to NDe) and ESBL E. coli (ND1 to ND5) isolates may influence antibiotic resistance phenotypes.

The Role of Propionate-Induced Rearrangement of Membrane Proteins in the Formation of the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Adhesive-invasive E. coli has been suggested to be associated with the development of Crohn's disease (CD). It is assumed that they can provoke the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells and then, due to survival inside macrophages and dendritic cells, stimulate chronic inflammation. In previous reports, we have shown that passage of the CD isolate ZvL2 on minimal medium M9 supplemented with sodium propionate (PA) as a carbon source stimulates and inhibits the adherent-invasive properties and the ability to survive in macrophages. This effect was reversible and not observed for the laboratory strain K12 MG1655. We were able to compare the isogenic strain AIEC in two phenotypes-virulent (ZvL2-PA) and non-virulent (ZvL2-GLU). Unlike ZvL2-GLU, ZvL2-PA activates the production of ROS and cytokines when interacting with neutrophils. The laboratory strain does not cause a similar effect. To activate neutrophils, bacterial opsonization is necessary. Differences in neutrophil NADH oxidase activation and ζ-potential for ZvL2-GLU and ZvL2-PA are associated with changes in membrane protein abundance, as demonstrated by differential 2D electrophoresis and LC-MS. The increase in ROS and cytokine production during the interaction of ZvL2-PA with neutrophils is associated with a rearrangement of the abundance of membrane proteins, which leads to the activation of Rcs and PhoP/Q signaling pathways and changes in the composition and/or modification of LPS. Certain isoforms of OmpA may play a role in the formation of the virulent phenotype of ZvL2-PA and participate in the activation of NADPH oxidase in neutrophils.

Genomic Dissection of an Enteroaggregative Escherichia coli Strain Isolated from Bacteremia Reveals Insights into Its Hybrid Pathogenic Potential.

Escherichia coli is a frequent pathogen isolated from bloodstream infections. This study aimed to characterize the genetic features of EC092, an E. coli strain isolated from bacteremia that harbors enteroaggregative E. coli (EAEC) genetic markers, indicating its hybrid pathogenic potential. Whole-genome sequencing showed that EC092 belongs to phylogroup B1, ST278, and serotype O165:H4. Genes encoding virulence factors such as fimbriae, toxins, iron-uptake systems, autotransporter proteins (Pet, Pic, Sat, and SepA), and secretion systems were detected, as well as EAEC virulence genes (aggR, aatA, aaiC, and aap). EC092 was found to be closely related to the other EAEC prototype strains and highly similar in terms of virulence to three EAEC strains isolated from diarrhea. The genomic neighborhood of pet, pic, sat, sepA, and the EAEC virulence genes of EC092 and its three genetically related fecal EAEC strains showed an identical genomic organization and nucleotide sequences. Also, EC092 produced and secreted Pet, Pic, Sat, and SepA in the culture supernatant and resisted the bactericidal activity of normal human serum. Our results demonstrate that the strain EC092, isolated from bacteremia, is a hybrid pathogenic extraintestinal E. coli (ExPEC)/EAEC with virulence features that could mediate both extraintestinal and intestinal infections.

An expanded database and analytical toolkit for identifying bacterial virulence factors and their associations with chronic diseases.

Virulence factor genes (VFGs) play pivotal roles in bacterial infections and have been identified within the human gut microbiota. However, their involvement in chronic diseases remains poorly understood. Here, we establish an expanded VFG database (VFDB 2.0) consisting of 62,332 nonredundant orthologues and alleles of VFGs using species-specific average nucleotide identity ( https://github.com/Wanting-Dong/MetaVF_toolkit/tree/main/databases ). We further develop the MetaVF toolkit, facilitating the precise identification of pathobiont-carried VFGs at the species level. A thorough characterization of VFGs for 5452 commensal isolates from healthy individuals reveals that only 11 of 301 species harbour these factors. Further analyses of VFGs within the gut microbiomes of nine chronic diseases reveal both common and disease-specific VFG features. Notably, in type 2 diabetes patients, long HiFi sequencing confirms that shared VF features are carried by pathobiont strains of Escherichia coli and Klebsiella pneumoniae. These findings underscore the critical importance of identifying and understanding VFGs in microbiome-associated diseases.

The multifaceted role of TolA protein in promoting survival, biofilm formation and virulence of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) can spread beyond the intestines and cause systemic infections, leading to various clinical manifestations, including airsacculitis, pericarditis, perihepatitis and colisepticemia. The mechanisms facilitating this extraintestinal infections are not fully understood. In this study, we investigate how the tolA gene affects APEC virulence by encoding a protein involved in maintaining outer membrane integrity. We constructed a tolA deletion mutant of APEC strain E058 and evaluated its growth and survival in various environments, including in vitro cultures and in vivo infection models in chickens. We found that the motility-defective ΔtolA mutant exhibits reduced biofilm formation ability and weakened resistance to the environmental stresses, suggesting an important role for TolA in APEC's survival. The lack of tolA gene affects the bacterial ability to resist the host's immune system, such as complement-mediated serum killing or phagocytosis, as shown by the serum killing and macrophage phagocytosis assays. Additionally, in vivo infection studies using chickens demonstrated that the ΔtolA mutant displayed attenuated virulence, evidenced by reduced mortality and lower tissue bacterial burden. Reverse transcription quantitative real-time PCR (RT-qPCR) analysis revealed that inactivation of tolA led to downregulation of virulence genes associated with serum resistance (traT) and flagellar biosynthesis (fliR). Taken together, our findings demonstrate the multifaceted role of TolA protein in promoting the survival, immune evasion, biofilm formation, and virulence of APEC E058. This suggests that targeting TolA could potentially offer new strategies for combating APEC infections.

Detection of pathogenic, heteropathogenic and hybrid Escherichia coli strains in psittacines from zoos and breeders in the state of Ceará, Brazil.

The current study aimed to detect virulence, hetero-pathogenicity, and hybridization genes in Escherichia coli strains, previously isolated from cloacal swabs in commercial breeding psittacines and zoological collections, via multiplex PCR. A total of 68 strains of E. coli, previously isolated from psittacines in zoos and commercial breeding facilities in Ceará, Brazil, were assessed for the presence of the following genes and/or probes: eae, bfpA (EPEC - Enteropathogenic E. coli), CVD432 (EAEC - Enteroaggregative E. coli); LT gene and ST gene (ETEC - Enterotoxigenic E. coli); ipaH (EIEC - Enteroinvasive E. coli); stx1 and stx2 (STEC - Shiga toxin-producing E. coli); iroN, ompT, hlyF, iss, and iutA (APEC - Avian pathogenic E. coli). Of the 68 E. coli strains analyzed, 61 (98.7 %) were positive for the following genes and/or probes: Stx1 (61/98.7 %), ST gene (54/79.4 %), CVD432 (49/72 %), bfpA (44/64.7 %), eae (42/61.8 %), Stx2 (41/60.3 %), ipaH (34/50 %), LT gene (33/48.5 %), iroN (21/30.9 %), hlyF (11/6.2 %), iss (06/8.8 %) and iutA (06/8.8 %). The following diarrheagenic pathotypes were identified: 66 (97 %) from STEC, 49 (72 %) from EAEC, 35 (52 %) from EIEC, 25 (37 %) from ETEC, and one (1.5 %) from EPEC. Regarding hetero-pathogenicity, 50 (74 %) heterogeneous strains were identified. Positivity for APEC was seen in four (6 %) strains, all characterized as pathogenic hybrids. This study describes significant associations of virulence factors in E. coli strains DEC/DEC and DEC/APEC, which were isolated from psittacines and may be potentially harmful to One Health.

Prevalence of specific serogroups, antibiotic resistance and virulence factors of avian pathogenic Escherichia coli (APEC) isolated from clinical cases: A systematic review and meta-analysis.

Pathogenic strains of Escherichia coli infecting poultry, commonly called avian pathogenic E. coli (APEC) present significant risks, to the health of both poultry and the general public. This systematic review aimed to examine the prevalence of APEC serotypes, sequence types (ST), phylogenetic groups, virulence factors and antibiotic resistance patterns based on 189 research papers sourced from PubMed, Web of Science, and ProQuest. Then, data were extracted from the selected studies and analyzed to assess the global distribution and characteristics of APEC strains. The metaprop codes in the Meta and Metafor packages of R as implemented in RStudio were then used to conduct meta-analysis. Among APEC strains identified from these different research reports serogroup O78 had the highest overall prevalence (16 %), followed by serogroups O2 (10 %), and O117 (8 %). The most common ST profiles were ST117 (20 %), ST140 (15 %), ST95 (12 %), and ST131 (9 %). ST117 and ST140 are known reservoirs for pathogenic E. coli in humans. Moreover, phylogenetic assessment highlighted the prevalence of phylogroups A, A1, F, D, and B2 among APEC strains indicating diversity in phylogenetic origin within poultry populations. The presence of antimicrobial resistance was notable among APEC strains against antibiotics such as tetracyclines, penicillins, and cephalosporins. This resistance may be linked to use of antimicrobials in poultry production in certain regions presenting challenges for both animal health management and human infection control. Analysis of sequences linked to adherence or virulence indicated that genes encoding adhesins (csg, fimC), iron/metal uptake (sitB, sitC, iroD) and cytotoxicity (estB, hlyF), and serum resistance (traT, iss) were highly prevalent. These factors have been reported to contribute to APEC host colonization and virulence in poultry. In summary, this overview of the characteristics of APEC highlights the pressing importance of monitoring and implementing management approaches to reduce antimicrobial resistance considering that a phylogenetic diversity of E. coli strains causes infections in both poultry and humans and represents a risk to both animal and public health. Further, determining the major conserved aspects and predominant mechanisms of virulence of APEC is critical for improving diagnostics and developing preventative measures to reduce the burden of infection caused by pathogenic E. coli in poultry and lower risks associated with foodborne transmission of E. coli to humans through poultry and poultry products.

Unbiased identification of risk factors for invasive Escherichia coli disease using machine learning.

BACKGROUND: Invasive Escherichia coli disease (IED), also known as invasive extraintestinal pathogenic E. coli disease, is a leading cause of sepsis and bacteremia in older adults that can result in hospitalization and sometimes death and is frequently associated with antimicrobial resistance. Moreover, certain patient characteristics may increase the risk of developing IED. This study aimed to validate a machine learning approach for the unbiased identification of potential risk factors that correlate with an increased risk for IED. METHODS: Using electronic health records from 6.5 million people, an XGBoost model was trained to predict IED from 663 distinct patient features, and the most predictive features were identified as potential risk factors. Using Shapley Additive predictive values, the specific relationships between features and the outcome of developing IED were characterized. RESULTS: The model independently predicted that older age, a known risk factor for IED, increased the chance of developing IED. The model also predicted that a history of ≥ 1 urinary tract infection, as well as more frequent and/or more recent urinary tract infections, and ≥ 1 emergency department or inpatient visit increased the risk for IED. Outcomes were used to calculate risk ratios in selected subpopulations, demonstrating the impact of individual or combinations of features on the incidence of IED. CONCLUSION: This study illustrates the viability and validity of using large electronic health records datasets and machine learning to identify correlating features and potential risk factors for infectious diseases, including IED. The next step is the independent validation of potential risk factors using conventional methods.

Analysis of in vitro expression of virulence genes related to antibiotic and disinfectant resistance in Escherichia coli as an emerging periodontal pathogen.

The collective involvement of virulence markers of Escherichia coli as an emerging pathogen associated with periodontitis remains unexplained. This study aimed to implement an in vitro model of infection using a human epithelial cell line to determine the virulome expression related to the antibiotic and disinfectant resistance genotype and pulse field gel electrophoresis (PFGE) type in E. coli strains isolated from patients with periodontal diseases. We studied 100 strains of E. coli isolated from patients with gingivitis (n = 12), moderate periodontitis (n = 59), and chronic periodontitis (n = 29). The identification of E. coli and antibiotic and disinfectant resistance genes was performed through PCR. To promote the expression of virulence genes in the strains, an in vitro infection model was used in the human epithelial cell line A549. RNA was extracted using the QIAcube robotic equipment and reverse transcription to cDNA was performed using the QuantiTect reverse transcription kit (Qiagen). The determination of virulence gene expression was performed through real-time PCR. Overall, the most frequently expressed adhesion genes among the isolated strains of gingivitis, moderate periodontitis, and chronic periodontitis were fimH (48%), iha (37%), and papA (18%); those for toxins were usp (33%); those for iron acquisition were feoB (84%), fyuA (62%), irp-2 (61%), and iroN (35%); those for protectins were traT (50%), KpsMT (35%), and ompT (28%); and those for pathogenicity islands were malX (45%). The most common antibiotic and disinfectant resistance genes among gingivitis, moderate periodontitis, and chronic periodontitis strains were sul-2 (43%), blaSHV (47%), blaTEM (45%), tet(A) (41%), dfrA1 (32%), marR-marO (57%), and qacEA1 (79%). The findings revealed the existence of a wide distribution of virulome expression profiles related to the antibiotic and disinfectant resistance genotype and PFGE type in periodontal strains of E. coli. These findings may contribute toward improving the prevention and treatment measures for periodontal diseases associated with E. coli.

Genomic characterization of a high pathogenic Escherichia coli causing bacterial meningitis in a newborn in Italy, 2022: E. coli neonatal meningitis.

Here, we characterize the complete genome sequence of Escherichia coli isolated from a newborn affected by bacterial meningitis in Italy. Genome of E. coli strain 1455 harbored a circular chromosome and two plasmids of 167.740-bp and 4.073-bp in length, respectively. E. coli 1455 belonged to the ST3, serotype O17:H18 and carried different determinants including resistance to B-lactams, tetracyclines, and quinolones. In addition, genome of E. coli strain 1455 harbored 5 integrated pro-phage regions mainly located in the chromosome, while most of the virulence factors associated to the invasiveness and clinical severity and different antimicrobial resistance determinants (blaTEM-1, tet(A) and qnrS1) were located in the 167-Kb plasmid. Taken together, our findings suggest a possible widespread of a virulence factors-carrying plasmid worldwide and highlight the importance of genomic characterization in the diffusion of public health threats.

Gut colonization with antibiotic-resistant Escherichia coli pathobionts leads to disease severity in ulcerative colitis.

BACKGROUND: Escherichia coli is a Gram-negative commensal of human gut. Surprisingly, the role of E. coli in the pathogenesis of ulcerative colitis (UC) has not been explored until now. METHODS: Human gut microbiota composition and meta-gut resistome were evaluated using metagenomics. Antibiotic susceptibility of E. coli isolates against different class of antibiotics was investigated. Further, the genome sequence analysis of E. coli isolates was performed to gain insight into the antimicrobial resistance (AMR) mechanism and virulence factors. Gut proteome of UC and non-UC was examined to understand the effect of resistant bacteria on host physiology. RESULTS: In UC patients, meta-gut resistome was found to be dominated by AMR genes (829) compared to healthy controls (HC) [518]. The metagenome study revealed a higher prevalence of AMR genes in the rural population (378 in HC; 607 in UC) compared to the urban (340 in HC; 578 in UC). Approximately, 40% of all E. coli isolates were multi-drug resistant (MDR), with higher prevalence in UC (43.75%) compared to HC (33.33%). Up-regulated expression of antimicrobial human proteins (lactotransferrin, azurocidin, cathepsin G, neutrophil elastase, and neutrophil defensin 3) and inflammatory mediator (Protein S100-A9 and Protein S100-A8) suggest microbial infection in UC gut. CONCLUSIONS: In addition to the conventional culturomics method, a multi-omics strategy provides deeper insights into the disease etiology, emergence of MDR pathobionts, and their roles in the disruption of the healthy gut environment in UC patients.

In situ targeted base editing of bacteria in the mouse gut.

Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health1. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells2, we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a ß-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.

Effects of pH on the Pathogenicity of Escherichia coli and Klebsiella pneumoniae on the Kidney: In Vitro and In Vivo Studies.

Urine pH reflects the functional integrity of the body and may influence the virulence of uropathogenic Escherichia coli and Klebsiella pneumoniae, the main causes of urinary tract infections (UTIs). This study evaluated the effects of acidic pH on the pathogenicity of uropathogenic E. coli and K. pneumoniae strains, in vitro and in vivo. Four uropathogenic E. coli and four K. pneumoniae strains were used. Biofilm formation, growth competition indices, motility, and adhesion and invasion of human renal cells were analyzed in media with acidic, neutral, and alkaline pH. A murine lower UTI model was used, with urine adjusted to acidic, neutral, or alkaline pH. At acidic pH, E. coli and K. pneumoniae exhibited higher bacterial concentrations in the kidneys and systemic symptoms, including bacteremia. Alkaline urine pH did not affect bacterial concentrations of any strain. In mice with UTIs caused by E. coli Nu14 and K. pneumoniae HUVR42 and acidic urine pH, histopathological studies of the kidneys showed acute inflammation affecting the urothelium and renal parenchyma, which are traits of acute pyelonephritis. These results indicate that acidic pH could increase the pathogenicity of E. coli and K. pneumoniae in murine models of lower UTI, promoting renal infection and acute inflammation.

The application of organoids in cancers associated with pathogenic infections.

Cancers associated with pathogen infections are gradually becoming important threats to human health globally, and it is of great significance to study the mechanisms of pathogen carcinogenesis. Current mechanistic studies rely on animal and two-dimensional (2D) cell culture models, but traditional methods have been proven insufficient for the rapid modeling of diseases caused by new pathogens. Therefore, research focus has shifted to organoid models, which can replicate the structural and genetic characteristics of the target tissues or organs in vitro, providing new platforms for the study of pathogen-induced oncogenic mechanisms. This review summarizes the application of organoid technology in the studies of four pathogen-associated cancers: gastric cancer linked to Helicobacter pylori, liver cancer associated with hepatitis B virus or hepatitis C virus, colorectal cancer caused by Escherichia coli, and cervical cancer related to human papillomavirus. This review also proposes several limitations of organoid technology to optimize organoid models and advance the treatment of cancer associated with pathogen infections in the future.

Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand.

Extraintestinal pathogenic Escherichia coli (ExPEC), especially uropathogenic E. coli (UPEC) are responsible for urinary tract infections (UTIs), while diarrheagenic E. coli (DEC) cause foodborne illnesses. These pathogenic E. coli are a serious threat to human health and a public concern worldwide. However, the evidence on pork E. coli (PEC) harboring UPEC virulence-associated genes is currently limited. Therefore, this study aimed to determine the phylogroups, virulence genes, and their association between PEC and UPEC from UTI patients. In this study, 330 E. coli were obtained from archived stock culture isolated from pork (PEC; n = 165) and urine of patients with UTIs (UPEC; n = 165) during 2014-2022. Phylogroups, UPEC- and diarrheagenic E. coli (DEC) associated virulence genes were assessed using PCR assays. The results showed that phylogroups A (50.3%), and B1 (32.1%) were commonly found among PEC whereas phylogroups B2 (41.8%), and C (25.5%) were commonly detected in the UPEC. PEC and UPEC carried similar virulence-associated genes with different percentages. The most frequent UPEC virulence-associated gene among UPEC, and PEC strains was fimH, (93.3%, and 92.1%), followed by iucC (55.2%, and 12.7%), papC (21.8%, and 4.2%), afaC (22.4%, and 0%), hlyCA (17%, and 0.6%), cnf (16.4%, and 0.6%), and sfa/focDE (8.5%, and 4.8%). Additionally, 6 of 27 UPEC virulence-associated gene patterns were found in both PEC and UPEC strains regardless of phylogroups. Furthermore, the DEC virulence-associated genes were found in only 3 strains, one from PEC harboring eae, and two from UPEC carried fimH-bfpA or afaC-CVD432 indicating hybrid strains. Cluster analysis showed a relationship between PEC and UPEC strains and demonstrated that PEC harboring UPEC virulence-associated genes in pork may be associated with UPEC in humans. Food safety and hygiene practices during pork production chain are important procedures for minimizing cross-contamination of these strains that could be transmitted to the consumers.

Infection of mice by the enteroaggregative E. coli strain 042 and two mutant derivatives overexpressing virulence factors: impact on disease markers, gut microbiota and concentration of SCFAs in feces.

Several pathogenic Escherichia coli strains cause diarrhea. Enteroaggregative E. coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC cells form a "stacked-brick" arrangement over the intestinal epithelial cells. EAEC isolates express, among other virulence determinants, the AggR transcriptional activator and the aggregative adherence fimbriae (AAF). Overexpression of the aggR gene results in increased expression of virulence factors such as the aff genes, as well as several genes involved in specific metabolic pathways such as fatty acid degradation (fad) and arginine degradation (ast). To support the hypothesis that induction of the expression of some of these pathways may play a role in EAEC virulence, in this study we used a murine infection model to evaluate the impact of the expression of these pathways on infection parameters. Mice infected with a mutant derivative of the EAEC strain 042, characterized by overexpression of the aggR gene, showed increased disease symptoms compared to those exhibited by mice infected with the wild type (wt) strain 042. Several of these symptoms were not increased when the infecting mutant, which overexpressed aggR, lacked the fad and ast pathways. Therefore, our results support the hypothesis that different metabolic pathways contribute to EAEC virulence.

Prevalence and molecular characteristics of intestinal pathogenic Escherichia coli isolated from diarrheal pigs in Southern China.

Intestinal pathogenic Escherichia coli (InPEC) is one of the most common causes of bacterial diarrhea in farm animals, including profuse neonatal diarrhea and post weaning diarrhea (PWD) in piglets. In this study, we investigated the prevalence of InPEC and associated primary virulence factors among 543 non-duplicate E. coli isolates from diarrheal pigs from 15 swine farms in southern China. Six major virulence genes associated with InPEC were identified among 69 (12.71 %) E. coli isolates and included est (6.62 %), K88 (4.79 %), elt (3.68 %), eae (1.47 %), stx2 (0.92 %) and F18 (0.55 %). Three pathotypes of InPEC were identified including ETEC (8.10 %), EPEC (1.29 %) and STEC/ETEC (0.92 %). In particular, K88 was only found in ETEC from breeding farms, whereas F18 was only present in STEC/ETEC hybrid from finishing farms. Whole genome sequence analysis of 37 E. coli isolates revealed that InPEC strains frequently co-carried multiple antibiotic resistance gene (ARG). est, elt and F18 were also found to co-locate with ARGs on a single IncFIB/IncFII plasmid. InPEC isolates from different pathotypes also possessed different profiles of virulence genes and antimicrobial resistance genes. Population structure analysis demonstrated that InPEC isolates from different pathotypes were highly heterogeneous whereas those of the same pathotype were extremely similar. Plasmid analysis revealed that K88 and/or est/elt were found on pGX18-2-like/pGX203-2-like and pGX203-1-like IncFII plasmids, while F18 and elt/est, as well as diverse ARGs were found to co-locate on IncFII/IncFIB plasmids with a non-typical backbone. Moreover, these key virulence genes were flanked by or adjacent to IS elements. Our findings indicated that both clonal expansion and horizontal spread of epidemic IncFII plasmids contributed to the prevalence of InPEC and the specific virulence genes (F4, F18, elt and est) in the tested swine farms.

Correlation analysis of whole genome sequencing of a pathogenic Escherichia coli strain of Inner Mongolian origin.

Anal swabs of 1-month-old Holstein calves with diarrhea were collected from an intensive cattle farm, and a highly pathogenic Escherichia coli strain was obtained by isolation and purification. To study the virulence and resistance genes of pathogenic E. coli that cause diarrhea in calves, a strain of E. coli E12 isolated from calf diarrhea samples was used as experimental material in this experiment, and the virulence of the E12 strain were identified by the mouse infection test, and the whole genome map of the E12 strain were obtained by whole-genome sequencing and analyzed for genome characterization. The results showed that the lethality of strain E12 was 100%, the total length of E12-encoded genes was 4,294,530 bp, Cluster of Orthologous Groups of proteins (COG) annotated to 4,194 functional genes, and the virulence genes of sequenced strain E12 were compared with the virulence genes of sequenced strain E12 from the Virulence Factors of Pathogenic Bacteria (VFDB), which contained a total of 366 virulence genes in sequenced strain E12. The analysis of virulence genes of E12 revealed a total of 52 virulence genes in the iron transferrin system, 56 virulence genes in the secretory system, 41 virulence genes in bacterial toxins, and a total of 217 virulence genes in the Adhesin and Invasins group. The antibiotic resistance genes of sequenced strain E12 were identified through the Antibiotic Resistance Genes Database (ARDB) and Comprehensive Antibiotic Research Database, and it was found that its chromosome and plasmid included a total of 127 antibiotic resistance genes in four classes, and that E12 carried 71 genes related to the antibiotic efflux pumps, 36 genes related to antibiotic inactivation, and 14 antibiotic target alteration and reduced penetration into antibiotics, and 6 antibiotic resistance genes, and the resistance phenotypes were consistent with the genotypes. The pathogenic E. coli that causes diarrhea in calves on this ranch contains a large number of virulence and resistance genes. The results provide a theoretical basis for the prevention and treatment of diarrhea and other diseases caused by E. coli disease.

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

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