Genomic analysis of a novel phage vB_SenS_ST1UNAM with lytic activity against Salmonella enterica serotypes.

In this study, we present the complete annotated genome of a novel Salmonella phage, vB_SenS_ST1UNAM. This phage exhibits lytic activity against several Salmonella enterica serotypes, such as S. Typhi, S. Enteritidis, and S. Typhimurium strains, which are major causes of foodborne illness worldwide. Its genome consists of a linear, double-stranded DNA of 47,877 bp with an average G+C content of 46.6%. A total of 85 coding regions (CDS) were predicted, of which only 43 CDS were functionally assigned. Neither genes involved in the regulation of lysogeny, nor antibiotic resistance genes were identified. This phage harbors a lytic cassette that encodes a type II-holin and a Rz/Rz1-like spanin complex, along with a restriction-modification evasion system and a depolymerase that degrades Salmonella exopolysaccharide. Moreover, the comparative analysis with closely related phage genomes revealed that vB_SenS_ST1UNAM represents a novel genus, for which the genus "Gomezvirus" within the subfamily "ST1UNAM-like" is proposed.

Importance of Microbiome of Fecal Samples Obtained from Adolescents with Different Weight Conditions on Resistance Gene Transfer.

Antimicrobial resistance (AMR) is a relevant public health problem worldwide, and microbiome bacteria may contribute to the horizontal gene transfer associated with antimicrobial resistance. The microbiome of fecal samples from Mexican adolescents were analyzed and correlated with eating habits, and the presence of AMR genes on bacteria in the microbiome was evaluated. Fecal samples from adolescents were collected and processed to extract genomic DNA. An Illumina HiSeq 1500 system was used to determine resistance genes and the microbiome of adolescents through the amplification of gene resistance and the V3-V4 regions of RNA, respectively. Analysis of the microbiome from fecal samples taken from 18 obese, overweight, and normal-weight adolescents revealed that the Firmicutes was the most frequent phylum, followed by Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia. The following species were detected as the most frequent in the samples: F. prausnitzii, P. cori, B. adolescentis, E. coli and A. muciniphila. The presence of Bacteroides, Prevotella and Ruminococcus was used to establish the enterotype; enterotype 1 was more common in women and enterotype 2 was more common in men. Twenty-nine AMR genes were found for ß-lactamases, fluoroquinolones, aminoglycosides, macrolide, lincosamides, streptogramin (MLS), tetracyclines and sulfonamides. The presence of microorganisms in fecal samples that harbor AMR genes that work against antimicrobials frequently used for the treatment of microbial infections such as b-lactams, macrolides, aminoglycosides, MLS, and tetracyclines is of great concern, as these organisms may be an important reservoir for horizontal AMR gene transfer.

Molecular typification of Escherichia coli from community-acquired urinary tract infections in Mexico.

One hundred and five uropathogenic Escherichia coli (UPEC) strains from patients with community-acquired urinary tract infections were characterized according to phylogenetic group, virulence factors, serogroup, antibiotic resistance, and genotype. The pathogenic phylogenetic groups (B2, D, and F) were found in 71.4% of the tested strains. Among them, the main uropathogenic serogroups were O8, O25, and O75, in which 97.1% of the strains had a multidrug-resistant profile. Sixteen virulence genes were analysed using a combination of polymerase chain reaction (PCR) assays, with the fimH, irp-2, iutA, aer, iucC, PAI, sat, iroN, usp, and cnf1 genes being mainly found in pathogenic phylogroups. The E. coli O25b-ST131 clone was identified in 32% of the strains assigned to the pathogenic phylogroup B2. These findings demonstrate that virulence genes encoding adhesin components, iron-acquisition systems, toxins, and pathogenicity-associated islands were highly prevalent among the pathogenic phylogroup of UPEC strains.

Genomic characterization of two bacteriophages (vB_EcoS-phiEc3 and vB_EcoS-phiEc4) belonging to the genus Kagunavirus with lytic activity against uropathogenic Escherichia coli.

In this study, the genomes of two lytic bacteriophages, vB_EcoS-phiEc3 and vB_EcoS-phiEc4, were sequenced and characterized using bioinformatics approaches. Whole-genome analysis showed that both phages belonged to the Kagunavirus genus, Guernseyvirinae subfamily and Siphoviridae family. Moreover, their genomes had 45, 288 bp and 44,540 bp, and G + C content of 48.42% and 50.04%, respectively. The genome of vB_EcoS-phiEc3 harbored 80 protein coding sequences (CDSs), whereas vB_EcoS-phiEc4 harbored 75 CDSs. Among them, 50 CDSs in vB_EcoS-phiEc3 and 44 CDSs in vB_EcoS-phiEc4 were considered as functional genes. Their lytic activity against multidrug-resistant uropathogenic Escherichia coli (UPEC) strains, as well as the absence of antibiotic resistance genes, lysogenic and virulence genes, enable vB_EcoS-phiEc3 and vB_EcoS-phiEc4 as a safe therapy option against UPEC infections.

Patrones de adherencia en células HEp-2 de cepas de Escherichia coli aisladas de ríos contaminados de La Habana / Adherence patterns in HEp-2 cells of Escherichia coli strains isolated from contaminated rivers in Havana

Introducción: El proceso de adherencia es fundamental en el desarrollo de la mayoría de las infecciones ocasionadas por Escherichia coli. Para los patotipos de esta especie se describen tres patrones de adherencia diferentes: adherencia localizada, adherencia difusa y adherencia agregativa, los cuales se relacionan con los procesos patogénicos específicos que ocasiona en la clínica. Sin embargo, son pocos los estudios en relación con los fenotipos de adherencia in vitro de E. coli aisladas del ambiente. Objetivo: Determinar los fenotipos de adherencia de cepas de E. coli aisladas de ecosistemas dulceacuícolas de La Habana. Métodos: Se analizaron 108 cepas de E. coli aisladas de los ríos Almendares, Quibú y Luyanó de La Habana. Se determinó el patrón de adherencia mediante ensayos de adherencia en cultivo celular de la línea HEp-2 así como el serotipo de cada cepa. Resultados: El 25 por ciento de las cepas de E. coli aisladas fueron adherentes y el 75 por ciento fueron no adherentes. Veintidós cepas mostraron el típico patrón de adherencia difusa y cinco cepas mostraron una adherencia agregativa. Se encontraron cepas de los dos patrones de adherencia en los tres ríos evaluados. Las cepas presentaron 24 serotipos diferentes. Conclusiones: Se demostró que las cepas de E. coli ambientales circulantes en estos ecosistemas presentan características adherentes, cuya patogenicidad implica un riesgo potencial para la salud humana, especialmente en edades pediátricas(AU)

Introduction: Adherence is crucial to the development of most Escherichia coli infections. Three different adherence patterns have been described for pathotypes of this species: localized, diffuse and aggregative adherence, based on the specific clinical pathogenic processes they bring about. However, few studies have been conducted about in vitro adherence phenotypes of E. coli isolated from the environment. Objective: Determine the adherence phenotypes of E. coli strains isolated from freshwater ecosystems in Havana. Methods: An analysis was conducted of 108 E. coli strains isolated from the rivers Almendares, Quibú and Luyanó in Havana. Determination was made of the adherence pattern by adherence assays in HEp-2 cell line cultures, as well as of the serotype for each strain. Results: Of the E. coli strains isolated, 25 percent were adherent and 75 percent were not. Twenty-two strains displayed the typical diffusely adherent pattern and five displayed aggregative adherence. Strains exhibiting the two adherence patterns were found in the three rivers evaluated. The strains contained 24 different serotypes. Conclusions: The environmental E. coli strains circulating in these ecosystems were found to have adherent characteristics whose pathogenicity implies a potential risk to human health, particularly in childhood(AU)

|Isolation and characterization of novel bacteriophages as a potential therapeutic option for Escherichia coli urinary tract infections.

Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC), whose impact can be exacerbated by multidrug-resistant (MDR) strains. Effective control strategies are, therefore, urgently needed. Among them, phage therapy represents a suitable alternative. Here, we describe the isolation and characterization of novel phages from wastewater samples, as well as their lytic activity against biofilm and adherence of UPEC to HEp-2 cells. The results demonstrated that phage vB_EcoM-phiEc1 (ϕEc1) belongs to Myoviridae family, whereas vB_EcoS-phiEc3 (ϕEc3) and vB_EcoS-phiEc4 (ϕEc4) belong to Siphoviridae family. Phages showed lytic activity against UPEC and gut commensal strains. Phage ϕEc1 lysed UPEC serogroups, whereas phages ϕEc3 and ϕEc4 lysed only UTI strains with higher prevalence toward the O25 serogroup. Moreover, phages ϕEc1 and ϕEc3 decreased both biofilm formation and adherence, whereas ϕEc4 was able to decrease adherence but not biofilm formation. In conclusion, these novel phages showed the ability to decrease biofilm and bacterial adherence, making them promising candidates for effective adjuvant treatment against UTIs caused by MDR UPEC strains. KEY POINTS: Phage with lytic activity against MDR UPEC strains were isolated and characterized under in vitro conditions. A novel method was proposed to evaluate phage activity against bacterial adherence in HEp-2 cell.. Phages represent a suitable strategy to control infections caused by MDR bacteria.

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1ß, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1ß and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1ß and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria.

BACKGROUND: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. AIM: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. METHODS: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. RESULTS: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. CONCLUSION: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

Association of phylogenetic distribution and presence of integrons with multidrug resistance in Escherichia coli clinical isolates from children with diarrhoea.

BACKGROUND: Escherichia coli strains include both commensal and virulent clones distributed in different phylogenetic groups. Antimicrobial resistance is an increasingly serious public health threat at the global level and integrons are important mobile genetic elements involved in resistance dissemination. This paper aims to determine the phylogenetic groups and presence of class 1 (intl1) and 2 (intl2) integrons in E. coli clinical isolates from children with diarrhoea, and to associate these characteristics with their antimicrobial resistance. METHODS: Phylogeny and presence of integrons (intl1 and intl2) were analysed by PCR and amplicon sequencing in 70 E. coli isolates from children with and without diarrhoea (35 of each group) from Sinaloa, Mexico; these variables were analysed for correlation with the antimicrobial resistance profile of the isolates. RESULTS: The most frequent phylogroups were A (42.9%) and B2 (15.7%). The E. coli isolates from children with diarrhoea were distributed in all phylogroups; while strains from children without diarrhoea were absent from phylogroups C, E, and clade I. The 17.1% of the isolates carried integrons (15.7% intI1 and 1.4% intI2); 28.6% of the isolates from children with diarrhoea showed the class 1 integron. Strains of phylogroup A showed the highest frequency of integrons (33.3%). The association of multidrug resistance and the presence of integrons was identified in 58.3% of strains isolated from children with diarrhoea included in phylogroups A and B2. The sequence analysis of intl1 and intl2 showed silent point mutations and similarities with plasmids of some APEC and AIEC strains. CONCLUSION: Commensal E. coli strains are potential disseminators of antimicrobial resistance, and the improvement in the use of antimicrobials to treat childhood diarrhoea is essential for the control of such resistance.

Persistencia, internalización y translocación de Escherichia coli O157:H7, O157:H16 y O105ab en plantas y frutos de tomate (Solanum lycopersicum L.) / Persistence, internalization and translocation of Escherichia coli O157:H7, O157:H16 and O105ab in plants and tomato fruits (Solanum lycopersicum L.)

La presencia de bacterias patógenas, como Escherichia coli, afecta la calidad e inocuidad de las hortalizas que se consumen en fresco y se relaciona con graves problemas de salud. El objetivo de este trabajo fue determinar si 3 cepas diferentes de E. coli tienen la capacidad de penetrar y permanecer en plantas y frutos de tomate. Se siguió un diseño experimental completamente al azar, para lo cual se estableció un cultivo de tomate (variedad «Cid¼) en condiciones de invernadero y se evaluaron 3 tratamientos, T1 (E. coli O157: H7), T2 (E. coli de cultivo de tomate -#91;EcT-#93; O157: H16), T3 (E. coli de cultivo de espinaca -#91;EcH-#93; EcH O105ab) y un testigo T4, con 100 plantas cada uno y 4 formas de inoculación: en el sustrato, en el tallo, en el pecíolo y en el pedúnculo. Se realizaron muestreos en etapa vegetativa, floración, fructificación y madurez fisiológica para cuantificar en placa las UFC/g y saber si las bacterias lograban moverse y recuperarse en la raíz, el tallo, la flor y el fruto. Los grupos filogenéticos a los que correspondieron las bacterias recuperadas fueron confirmados mediante pruebas bioquímicas, serotipificación y PCR. A los 120 días la recuperación de bacterias en la planta fue del 23% (E. coli O157: H7), 28% (EcT O157: H16) y 55% (EcH O105ab) con la inoculación al sustrato, mientras que con la inoculación por punción la recuperación fue (en igual orden) del 5%, 3% y 4% a los 30 días; del 37%, 35% y 30% a los 90 días; y del 42%, 39% y 13% a los 65 días. Las cepas utilizadas mostraron la capacidad de entrar en la planta de tomate y de permanecer en ella y transportarse hasta llegar al fruto, sin producir síntomas que indiquen su presencia.

The presence of pathogenic bacteria, such as Escherichia coli affects the quality and safety of vegetables that are consumed fresh and is associated with serious health problems. The objective of this study was to determine if three different strains of E. coli can penetrate and remain in plants and tomato fruits. A completely randomized experimental design was followed for which a tomato crop ("Cid" variety) was established under greenhouse conditions and three treatments were evaluated, T1 (E. coli O157: H7), T2 (E. coli from tomato cultivation -#91;EcT-#93; O157: H16), T3 (E. coli from spinach cultivation -#91;EcH-#93; O105ab) and a T4 control, with 100 plants each and four forms of inoculation: in the substrate, steam, petiole and the peduncle. Samples were carried out in vegetative stage, flowering, fruiting and physiological maturity to quantify in petri dish CFU/g and know if the bacteria managed to move around and recover in root, stem, flower and fruit. The phylogenetic groups that corresponded to the bacteria recovered were confirmed by biochemical tests, serotyping and PCR. At 120 days the recovery of bacteria in the plant was 23% (E. coli O157: H7), 28% (EcT O157: H16) and 55% (EcH O105ab) whit inoculation to the substrate while the inoculation by puncture the recovery was (in the same order) of 5%, 3%, and 4% at 30 days; 37%, 35% and 30% at 90 days; and 42%, 39% and 13% at 65 days. The strains submit the ability to enter the tomato plant and to stay in it and transported to the fruit, without producing that indicate their presence.

[Persistence, internalization and translocation of Escherichia coli O157:H7, O157:H16 and O105ab in plants and tomato fruits (Solanum lycopersicum L.)]. / Persistencia, internalización y translocación de Escherichia coli O157:H7, O157:H16 y O105ab en plantas y frutos de tomate (Solanum lycopersicum L.).

The presence of pathogenic bacteria, such as Escherichia coli affects the quality and safety of vegetables that are consumed fresh and is associated with serious health problems. The objective of this study was to determine if three different strains of E. coli can penetrate and remain in plants and tomato fruits. A completely randomized experimental design was followed for which a tomato crop ("Cid" variety) was established under greenhouse conditions and three treatments were evaluated, T1 (E. coli O157:H7), T2 (E. coli from tomato cultivation [EcT] O157:H16), T3 (E. coli from spinach cultivation [EcH] O105ab) and a T4 control, with 100 plants each and four forms of inoculation: in the substrate, steam, petiole and the peduncle. Samples were carried out in vegetative stage, flowering, fruiting and physiological maturity to quantify in petri dish CFU/g and know if the bacteria managed to move around and recover in root, stem, flower and fruit. The phylogenetic groups that corresponded to the bacteria recovered were confirmed by biochemical tests, serotyping and PCR. At 120 days the recovery of bacteria in the plant was 23% (E. coli O157:H7), 28% (EcT O157:H16) and 55% (EcH O105ab) whit inoculation to the substrate while the inoculation by puncture the recovery was (in the same order) of 5%, 3%, and 4% at 30 days; 37%, 35% and 30% at 90 days; and 42%, 39% and 13% at 65 days. The strains submit the ability to enter the tomato plant and to stay in it and transported to the fruit, without producing that indicate their presence.

High prevalence of multidrug-resistant Escherichia coli isolates from children with and without diarrhoea and their susceptibility to the antibacterial activity of extracts/fractions of fruits native to Mexico.

PURPOSE: This paper aims to evaluate the antimicrobial resistance of Esherichia coli isolates from children under 5 years old, with and without diarrhoea, who were hospital outpatients in Culiacan, Sinaloa, Mexico. It also looks at the antimicrobial activity of fruit extracts against selected multidrug-resistant (MDR) E. coli strains. METHODOLOGY: A total of 205 E. coli isolates from stool samples were collected from 94 children under 5 years old who were outpatients from two hospitals in the city of Culiacan, Sinaloa, Mexico, during the autumn/winter of 2003/04; their resistance profiles to 19 commercial antimicrobials were investigated using the Kirby-Bauer method. The antibacterial activities of extracts/fractions of fruits (i.e. uvalama, Vitex mollis; ayale, Crescentia alata; and arrayan, Psidium sartorianum) were evaluated using the broth microdilution method. RESULTS: All E. coli isolates were susceptible to amikacin, nitrofurantoin and meropenem, and approximately 96 % were resistant to at least one antimicrobial, especially carbenicillin (93.2 %), cefuroxime sodium (53.7 %), ampicillin (40 %) and trimethoprim/sulfamethoxazole (35.1 %). Likewise, the frequency of MDR strains (44.9 %) was high, and no significant association with diarrhoea symptoms was found. Remarkably, all fruit extracts/fractions showed antibacterial activity against some, but not all, MDR isolates. The lowest minimal inhibitory concentration values were for the hexane fraction of arrayan (0.25 mg ml-1). CONCLUSION: A high number of antimicrobial-resistant E. coli (especially to ß-lactams and sulfonamides) and MDR isolates were detected in children under 5 years old, irrespective of diarrhoea symptoms; this is novel information for Culiacan, Sinaloa, Mexico. Moreover, our results showed that the studied fruit extracts/fractions are potential alternative or complementary treatments for MDR E. coli strains.

Genome Sequence of Enterotoxigenic Escherichia coli Strain FMU073332.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of bacterial diarrheal illness, affecting practically every population worldwide, and was estimated to cause 120,800 deaths in 2010. Here, we report the genome sequence of ETEC strain FMU073332, isolated from a 25-month-old girl from Tlaltizapán, Morelos, México.

Corrigendum: Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

[This corrects the article on p. 1201 in vol. 7, PMID: 27536289.].

Microbiological Quality of Fresh Nopal Juice.

The consumption of fresh nopal cactus juice is widely popular among health-conscious consumers in Mexico. The juice is prepared from fresh cladodes that have only been rinsed with tap water and are not subjected to a pasteurization or terminal bacterial reduction process. The aim of this study was to evaluate the microbial quality of commercially available fresh juices (n = 162) made with nopal in Texcoco, State of Mexico, during the summer and spring season. Standard microbiological methods, the PCR technique and the serological method were used for isolation and identification of bacteria. All samples contained total coliforms and 91% were positive for Escherichia coli. Although total coliforms and E. coli were detected throughout the study, their populations were significantly lower (p < 0.05) in winter and spring, respectively. Citrobacter youngae was found in 20% of the samples, an unidentified species of Citrobacter in 10%, C. freundii and Proteus mirabilis in 3%, and Salmonella Javiana in 1%. The presence of these microorganisms, especially Salmonella, in the nopal juices is unacceptable due to its health significance. The information generated in this study is relevant for human health risk assessment associated with the consumption of unpasteurized nopal juices and potential interventions to minimize pathogen contamination.

Data on enterobacteria activity on biofilm formation at surface mango fruit (Mangifera indica L.) cv Ataulfo.

Abiotic factors influenced the capacity of the strains to form biofilms. Classification of the adhesion type is related with the optical density measured on the biofilm formation of tested strains. The relationship between the biofilm formation in real values with theoretical values of the strains was used to determine the mechanism involved during mixed cultures.

Immunogenic peptide mimotopes from an epitope of Escherichia coli O157 LPS.

Escherichia coli O157:H7 is a subtype of Shiga toxin-producing E. coli that is associated with haemorrhagic colitis and haemolytic uremic syndrome (HUS). Studies of populations in endemic areas have reported that the presence of specific antibodies against the O157 lipopolysaccharide (LPS) is associated with a lower incidence of diarrhoea and HUS. Phage display and IgG anti-O157 LPS antibodies were used in the present study to select peptide mimotopes of O157 LPS expressed in protein III of the M13 phage. Synthetic peptides (SP) were designed using the derived amino acid sequences obtained from DNA nucleotides of 63 selected phagotopes. The LxP/YP/SxL motif was identified in five of the phagotope amino acid sequences. Antibody responses against the phagotopes and their corresponding SPs were evaluated. SP12, one of the designed SP, induced the production of antibodies against the homologous peptide (1:800) and O157 LPS (1:200). The specificity of anti-SP12 antiserum was confirmed by analyzing its response to SP3, an SP with a different amino acid sequence than that of SP12, as well as against an E. coli LPS different from O157. Competitive studies with SP12 and O157 LPS showed a significant decrease in anti-SP12 and anti-LPS O157 antiserum responses against SP12 and O157 LPS, respectively. Eighteen (82%) of the 22 human serum samples with positive reactivity against E coli O157 LPS reacted with SP12 SP (cut-off >0.4). These results support the idea that SP12 is an immunogenic mimotope of O157 LPS.

Effects of lng Mutations on LngA Expression, Processing, and CS21 Assembly in Enterotoxigenic Escherichia coli E9034A.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity in children under 5 years of age in low- and middle-income countries and a leading cause of traveler's diarrhea worldwide. The ability of ETEC to colonize the intestinal epithelium is mediated by fimbrial adhesins, such as CS21 (Longus). This adhesin is a type IVb pilus involved in adherence to intestinal cells in vitro and bacterial self-aggregation. Fourteen open reading frames have been proposed to be involved in CS21 assembly, hitherto only the lngA and lngB genes, coding for the major (LngA) and minor (LngB) structural subunit, have been characterized. In this study, we investigated the role of the LngA, LngB, LngC, LngD, LngH, and LngP proteins in the assembly of CS21 in ETEC strain E9034A. The deletion of the lngA, lngB, lngC, lngD, lngH, or lngP genes, abolished CS21 assembly in ETEC strain E9034A and the adherence to HT-29 cells was reduced 90%, compared to wild-type strain. Subcellular localization prediction of CS21 proteins was similar to other well-known type IV pili homologs. We showed that LngP is the prepilin peptidase of LngA, and that ETEC strain E9034A has another peptidase capable of processing LngA, although with less efficiency. Additionally, we present immuno-electron microscopy images to show that the LngB protein could be localized at the tip of CS21. In conclusion, our results demonstrate that the LngA, LngB, LngC, LngD, LngH, and LngP proteins are essential for CS21 assembly, as well as for bacterial aggregation and adherence to HT-29 cells.