Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples.

BACKGROUND: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream infections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. METHODS: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. RESULTS: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E-115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains exhibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). CONCLUSIONS: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile.

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Actinobacillus seminis GroEL-homologous protein agglutinates sheep erythrocytes.

Actinobacillus seminis, a commensal of ovine and caprine reproductive organs, is able to induce epididymitis in the small ruminants that it infects. In this work, we characterised two protein bands of approximately 150 kDa and 65 kDa. These proteins cross-reacted with a polyclonal serum against Gallibacterium anatis hemagglutinin and with a polyclonal serum from sheep with epididymitis, indicating that the proteins are expressed in vivo; the two proteins also interacted with biotin-labeled sheep fibrinogen and fibronectin, suggesting that they may function as adhesins. The participation of these proteins as adhesins was confirmed by a cultured human bladder cell-A. seminis adhesion assay and adherence inhibition by preincubation of A. seminis with polyclonal antiserum to the 150 kDa protein. Both proteins presented sequence identity with an A. seminis GroEL protein by mass spectrometry analysis and agglutinated glutaraldehyde-fixed sheep red blood cells. Immunogold labeling was observed by transmission electron microscopy on bacterial cells that were negatively stained, and a peroxidase reaction was detected in A. seminis biofilms, when an anti-A. seminis 150 kDa protein serum was used, indicating the presence of this protein on the surface of A. seminis and in biofilms. The A. seminis GroEL-homologue is a multifunctional protein that likely acts as a hemagglutinin.

Characterization of Escherichia coli Isolates from an Urban Lake Receiving Water from a Wastewater Treatment Plant in Mexico City: Fecal Pollution and Antibiotic Resistance.

The presence of enteric bacteria in water bodies is a cause of public health concerns, either by directly causing water- and food-borne diseases, or acting as reservoirs for antibiotic resistance determinants. Water is used for crop irrigation; and sediments and aquatic plants are used as fertilizing supplements and soil conditioners. In this work, the bacterial load of several micro-environments of the urban lake of Xochimilco, in Mexico City, was characterized. We found a differential distribution of enteric bacteria between the water column, sediment, and the rhizoplane of aquatic plants, with human fecal bacteria concentrating in the sediment, pointing to the need to assess such bacterial load for each micro-environment, for regulatory agricultural purposes, instead of only the one of the water, as is currently done. Resistance to tetracycline, ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole was common among Escherichia coli isolates, but was also differentially distributed, being again higher in sediment isolates. A distinct distribution of chloramphenicol minimum inhibitory concentrations (MIC) among these isolates suggests the presence of a local selective pressure favoring lower MICs than those of isolates from treated water. Fecal bacteria of human origin, living in water bodies along with their antibiotic resistance genes, could be much more common than typically considered, and pose a higher health risk, if assessments are only made on the water column of such bodies.

Specific Detection of Uropathogenic Escherichia coli via Fourier Transform Infrared Spectroscopy Using an Optical Biosensor.

Urinary tract infections (UTIs) are caused mainly by uropathogenic Escherichia coli (UPEC), accounting for both uncomplicated (75%) and complicated (65%) UTIs. Detecting UPEC in a specific, rapid, and timely manner is essential for eradication, and optical biosensors may be useful tools for detecting UPEC. Recently, biosensors have been developed for the selective detection of antigen-antibody-specific interactions. In this study, a methodology based on the principle of an optical biosensor was developed to identify specific biomolecules, such as the PapG protein, which is located at the tip of P fimbriae and promotes the interaction of UPEC with the uroepithelium of the human kidney during a UTI. For biosensor construction, recombinant PapG protein was generated and polyclonal anti-PapG antibodies were obtained. The biosensor was fabricated in silicon supports because its surface and anchor biomolecules can be modified through its various properties. The fabrication process was carried out using self-assembled monolayers (SAMs) and an immobilized bioreceptor (anti-PapG) to detect the PapG protein. Each stage of biosensor development was evaluated by Fourier transform infrared (FTIR) spectroscopy. The infrared spectra showed bands corresponding to the C-H, C=O, and amide II bonds, revealing the presence of the PapG protein. Then, the spectra of the second derivative were obtained from 1600 to 1700 cm-1 to specifically determine the interactions that occur in the secondary structures between the biological recognition element (anti-PapG antibodies) and the analyte (PapG protein) complex. The analyzed secondary structure showed ß-sheets and ß-turns during the detection of the PapG protein. Our data suggest that the PapG protein can be detected through an optical biosensor and that the biosensor exhibited high specificity for the detection of UPEC strains. Furthermore, these studies provide initial support for the development of more specific biosensors that can be applied in the future for the detection of clinical UPEC samples associated with ITUs.

A bioinformatic approach to identify confirmed and probable CRISPR-Cas systems in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex genomes.

Introduction: The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes. Methods: Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter. Results: A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes. Discussion: This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.

Intrauterine Transmission of Zika and Vertical Transfer of Neutralizing Antibodies Detected Immediately at Birth in Oaxaca, Mexico: An Analysis in the Context of Microcephaly.

Zika virus (ZIKV) can cause neurological issues in infants. To provide protection, neutralizing antibodies should be transferred from the mother to the infant. We conducted a study at the Hospital General de Pochutla, Oaxaca, Mexico. Samples were collected from mothers (blood and breast milk) and infants (saliva and dried blood spots) within the first 12 postnatal hours (December 2017 to February 2018) and tested for ZIKV total and neutralizing antibodies as well as ZIKV-PCR. Microcephaly was evaluated according to INTERGROWTH-21st standards. Maternal IgG seroprevalence was 28.4% with 10.4% active infection, while infant IgG seroprevalence was 5.5% with 2.4% active infection. There were two cases of virolactia, and 6.3% of the infant saliva samples tested positive for ZIKV. Additionally, 18.3% of the infants were in a cephalic perimeter percentile lower than 10 and had an association between microcephaly and serology or a PCR between 8.6 and 60.9%. The infant blood samples had neutralizing antibodies, indicating intrauterine protection. Microcephaly was correlated with serology or PCR, but in our study population, non-ZIKV factors may be involved as well. Low ZIKV infection values in breast milk mean that breastfeeding is safe in most of the mothers and infants of the endemic area studied.

Molecular analysis and distribution of multidrug-resistant Enterococcus faecium isolates belonging to clonal complex 17 in a tertiary care center in Mexico City.

BACKGROUND: Enterococcus faecium has recently emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. A high rate of resistance to different antibiotics has been associated with virulent clonal complex 17 isolates carrying the esp and hyl genes and the purK1 allele. RESULTS: Twelve clinical vancomycin-resistant Enterococcus faecium (VREF) isolates were obtained from pediatric patients at the Hospital Infantil de México Federico Gómez (HIMFG). Among these VREF isolates, 58.3% (7/12) were recovered from urine, while 41.7% (5/12) were recovered from the bloodstream. The VREF isolates showed a 100% rate of resistance to ampicillin, amoxicillin-clavulanate, ciprofloxacin, clindamycin, chloramphenicol, streptomycin, gentamicin, rifampicin, erythromycin and teicoplanin. In addition, 16.7% (2/12) of the isolates were resistant to linezolid, and 66.7% (8/12) were resistant to tetracycline and doxycycline. PCR analysis revealed the presence of the vanA gene in all 12 VREF isolates, esp in 83.3% (10/12) of the isolates and hyl in 50% (6/12) of the isolates. Phylogenetic analysis via molecular typing was performed using pulsed-field gel electrophoresis (PFGE) and demonstrated 44% similarity among the VREF isolates. MLST analysis identified four different sequence types (ST412, ST757, ST203 and ST612). CONCLUSION: This study provides the first report of multidrug-resistant VREF isolates belonging to clonal complex 17 from a tertiary care center in Mexico City. Multidrug resistance and genetic determinants of virulence confer advantages among VREF in the colonization of their host. Therefore, the prevention and control of the spread of nosocomial infections caused by VREF is crucial for identifying new emergent subclones that could be challenging to treat in subsequent years.

Recombinant Escherichia coli BL21 with LngA Variants from ETEC E9034A Promotes Adherence to HT-29 Cells.

The CS21 pilus produced by enterotoxigenic Escherichia coli (ETEC) is involved in adherence to HT-29 intestinal cells. The CS21 pilus assembles proteins encoded by 14 genes clustered into the lng operon. AIM: This study aimed to determine whether E. coli BL21 (ECBL) transformed with the lng operon lacking the lngA gene (pE9034AΔlngA) and complemented in trans with lngA variants of ETEC clinical strains, as well as point substitutions, exhibited modified adherence to HT-29 cells. METHODS: A kanamycin cassette was used to replace the lngA gene in the lng operon of the E9034A strain, and the construct was transformed into the ECBL strain. The pJET1.2 vector carrying lngA genes with allelic variants was transformed into ECBLpE9034AΔlngA (ECBLΔlngA). The point substitutions were performed in the pJETlngAFMU073332 vector. RESULTS: Bioinformatic alignment analysis of the LngA proteins showed hypervariable regions and clustered the clinical ETEC strains into three groups. Variations in amino acid residues affect the adherence percentages of recombinant ECBL strains with lngA variants and site-specific mutations with HT-29 cells. CONCLUSION: In this study, ECBL carrying the lng operon harboring lngA variants of six clinical ETEC strains, as well as point substitutions, exerted an effect on the adherence of ECBL to HT-29 cells, thereby confirming the importance of the CS21 pilus in adherence.

Epstein­Barr virus as a promoter of tumorigenesis in the tumor microenvironment of breast cancer (Review).

Epstein­Barr virus (EBV) is an oncovirus associated with various neoplasms, including breast cancer (BC). EBV­associated oncogenesis requires the action of several viral molecules, such as EBV nuclear antigen 3C, latent membrane protein 1, microRNAs and long non­coding RNAs, which are able of manipulating the cellular machinery, inducing an evasion of the immune system, blocking apoptosis processes, promoting cell survival and metastasis. The risk of developing cancer is associated with epigenetic alterations and alterations in various signaling pathways. The activation of all these molecules can modify the expression of EBV proteins with oncogenic activity, influencing the oncogenic process. It is clear that BC, being multifactorial, presents a greater complexity; in numerous cases, the infection associated with EBV may be crucial for this neoplasia, if particular conditions for both the virus and host are present. In the present review, all these variables are analyzed in an aim to improve the understanding of the participation of EBV in BC.

Screening of Antibiotic and Virulence Genes from Whole Genome Sequenced Cronobacter sakazakii Isolated from Food and Milk-Producing Environments.

The objective of this study was to use whole-genome sequencing (WGS) to screen for genes encoding for antibiotic resistance, fitness and virulence in Cronobacter sakazakii strains that had been isolated from food and powdered-milk-producing environments. Virulence (VGs) and antibiotic-resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder and PlasmidFinder tools. Susceptibility testing was performed using disk diffusion. Fifteen presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal-MLST. Nine C. sakazakii strains were found in the meningitic pathovar ST4: two were ST83 and one was ST1. The C. sakazakii ST4 strains were further distinguished using core genome MLST based on 3678 loci. Almost all (93%) strains were resistant to cephalotin and 33% were resistant to ampicillin. In addition, 20 ARGs, mainly involved in regulatory and efflux antibiotics, were detected. Ninety-nine VGs were detected that encoded for OmpA, siderophores and genes involved in metabolism and stress. The IncFIB (pCTU3) plasmid was detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52 and ISEhe3. The C. sakazakii isolates analyzed in this study harbored ARGs and VGs, which could have contributed to their persistence in powdered-milk-producing environments, and increase the risk of infection in susceptible population groups.

Characterization of Cronobacter sakazakii and Cronobacter malonaticus Strains Isolated from Powdered Dairy Products Intended for Consumption by Adults and Older Adults.

The objective of this study was to characterize Cronobacter spp. and related organisms isolated from powder dairy products intended for consumption by adults and older adults using whole-genome sequencing (WGS), and to identify genes and traits that encode antibiotic resistance and virulence. Virulence (VGs) and antibiotic resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder, and MOB-suite tools. Susceptibility testing was performed using disk diffusion. Five presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal MLST. Three C. sakazakii strains were of the clinical pathovar ST1, one was ST31, and the remaining isolate was C. malonaticus ST60. In addition, Franconibacter helveticus ST345 was identified. The C. sakazakii ST1 strains were further distinguished using core genome MLST based on 2831 loci. Moreover, 100% of the strains were resistant to cefalotin, 75% to ampicillin, and 50% to amikacin. The C. sakazakii ST1 strains were multiresistant (MDR) to four antibiotics. Additionally, all the strains adhered to the N1E-115 cell line, and two invaded it. Eighteen ARGs mainly involved in antibiotic target alteration and antibiotic efflux were detected. Thirty VGs were detected and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, and genes involved in metabolism and stress. The pESA3, pSP291-1, and pCMA1 plasmids were detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52, and IS26. The isolates of C. sakazakii and C. malonaticus exhibited multiresistance to antibiotics, harbored genes encoding various antibiotic resistance proteins, and various virulence factors. Consequently, these contaminated powdered dairy products pose a risk to the health of hypersensitive adults.

Comparative genomic analysis of uropathogenic Escherichia coli strains from women with recurrent urinary tract infection.

Introduction: Recurrent urinary tract infections (RUTIs) caused by uropathogenic Escherichia coli are costly public health problems impacting patients' quality of life. Aim: In this work, a comparative genomics analysis of three clinical RUTI strains isolated from bladder biopsy specimens was performed. Materials and methods: One hundred seventy-two whole genomes of urinary tract E. coli strains were selected from the NCBI database. The search for virulence factors, fitness genes, regions of interest, and genetic elements associated with resistance was manually carried out. The phenotypic characterization of antibiotic resistance, haemolysis, motility, and biofilm formation was performed. Moreover, adherence and invasion assays with human bladder HTB-5 cells, and transmission electron microscopy (TEM) were performed. Results: The UTI-1_774U and UTI-3_455U/ST1193 strains were associated with the extraintestinal pathotypes, and the UTI-2_245U/ST295 strain was associated with the intestinal pathotype, according to a phylogenetic analysis of 172 E. coli urinary strains. The three RUTI strains were of clinical, epidemiological, and zoonotic relevance. Several resistance genes were found within the plasmids of these strains, and a multidrug resistance phenotype was revealed. Other virulence genes associated with CFT073 were not identified in the three RUTI strains (genes for type 1 and P fimbriae, haemolysin hlyA, and sat toxin). Quantitative adherence analysis showed that UTI-1_774U was significantly (p < 0.0001) more adherent to human bladder HTB-5 cells. Quantitative invasion analysis showed that UTI-2_245U was significantly more invasive than the control strains. No haemolysis or biofilm activity was detected in the three RUTI strains. The TEM micrographs showed the presence of short and thin fimbriae only in the UTI-2_245U strain. Conclusion: The high variability and genetic diversity of the RUTI strains indicate that are a mosaic of virulence, resistance, and fitness genes that could promote recurrence in susceptible patients.

Features of antibody responses after SARS-COV-2 infection in healthcare workers in the first wave of COVID-19 pandemic in Mexico City.

OBJECTIVE: To investigate the antibody response to SARS-CoV-2 and identify associated factors in frontline and second-line healthcare workers (HCWs) at a large hospital in Mexico City during the first wave of COVID-19 pandemic. METHODS: This was a cross-sectional study of HCWs returning to work following mandatory isolation after recovering from COVID-19. Immunoglobulin (Ig) M and IgG antibodies elicited by SARS-CoV-2 were semiquantitatively measured using densitometric analysis of band intensities in lateral flow assay (LFA) devices. The mean pixel intensity (dots-per-inch [dpi]) of each band on the LFA was considered a measure of antibody titre. RESULTS: Of the 111 HCWs involved in the study, antibody responses were detected in 73/111 (66%) participants. Severe COVID symptoms was associated with old age. No differences in IgM intensity were observed between men and women, but IgG intensity was significantly higher in men than in women. Second-line HCWs produced a higher IgG intensity than firstline HCWs. The IgG intensity was high in severe cases. CONCLUSIONS: For HCWs who may acquire SARS-CoV-2 infection, it is necessary to establish a routine program for detection of the virus to avoid risk of infection and spread of COVID-19.

Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile.

This study characterized five Cronobacter spp. and six Salmonella spp. strains that had been isolated from 155 samples of powdered infant formula (PIF) sold in Chile and manufactured in Chile and Mexico in 2018-2020. Two strains of Cronobacter sakazakii sequence type (ST) ST1 and ST31 (serotypes O:1 and O:2) and one strain of Cronobacter malonaticus ST60 (O:1) were identified. All Salmonella strains were identified as Salmonella Typhimurium ST19 (serotype O:4) by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST (cgMLST). The C. sakazakii and C. malonaticus isolates were resistant to cephalothin, whereas the Salmonella isolates were resistant to oxacillin and ampicillin. Nineteen antibiotic resistance genes were detected in the C. sakazakii and C. malonaticus isolates; the most prevalent were mcr-9.1, blaCSA , and blaCMA . In Salmonella, 30 genes encoding for aminoglycoside and cephalosporin resistance were identified, including aac(6')-Iaa, ß-lactamases ampH, ampC1, and marA. In the Cronobacter isolates, 32 virulence-associated genes were detected by WGS and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, invasion, plasminogen activator, colonization, transcriptional regulator, survival in macrophages, use of sialic acid, and toxin-antitoxin genes. In the Salmonella strains, 120 virulence associated genes were detected, adherence, magnesium uptake, resistance to antimicrobial peptides, secretion system, stress protein, toxin, resistance to complement killing, and eight pathogenicity islands. The C. sakazakii and C. malonaticus strains harbored I-E and I-F CRISPR-Cas systems and carried Col(pHHAD28) and IncFIB(pCTU1) plasmids, respectively. The Salmonella strains harbored type I-E CRISPR-Cas systems and carried IncFII(S) plasmids. The presence of C. sakazakii and Salmonella in PIF is a health risk for infants aged less than 6 months. For this reason, sanitary practices should be reinforced for its production and retail surveillance.

Pheromone Activity after Stimulation with Ampicillin in a Plasmid-Free Enterococcus faecalis Strain.

Enterococci exhibit clumping under the selective pressure of antibiotics. The aim of this study was to analyze the effect of supernatants from a plasmid-free clone (C29) of Enterococcus faecalis subjected to 0.25×, 0.5×, and 0.75× of the minimal inhibitory concentration (MIC) of ampicillin on the expression of an aggregation substance (AS) by a donor plasmid clone (1390R). A clumping assay was performed. The relative expression of prgB (gene that encodes AS) was determined and semiquantified in 1390R, and iad1 expression was determined and semiquantified in C29. AS expression was analyzed in the stimulated 1390R cells by confocal microscopy, flow cytometry, and ELISA. Adherence was also measured. Maximal clumping was observed with the pheromone medium 0.25×. Only the 1390R strain stimulated with the C29 supernatant without ampicillin and with 0.25× was able to express prgB. No expression of prgB was observed at 0.5× and 0.75×. The difference in relative expression (RE) of 1390R without ampicillin and with 0.25× was 0.5-fold. AS expression in 1390R showed the greatest increase upon stimulation with 0.25×. When 1390R was stimulated with 0.5× and 0.75×, AS expression was also observed but was significantly lower. Ampicillin stimulated C29 switch-off pheromone expression in recipient cells, which in turn switched off AS expression in donor cells. We observed that although prgB was switched off after 0.5× stimulation in C29, the supernatants induced expression in certain 1390R strains. In conclusion, ampicillin was able to modulate pheromone expression in free plasmid clones which, in turn, modulated AS expression in plasmid donor cells. The fact that PrgB gene expression was switched off after the ampicillin stimulus at 0.5× MIC, whereas AS proteins were present on the surface of the bacteria, suggested that a mechanism of rescue associated with mechanism pheromone sensing may be involved.

Profiling the Virulence and Antibiotic Resistance Genes of Cronobacter sakazakii Strains Isolated From Powdered and Dairy Formulas by Whole-Genome Sequencing.

Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enterocolitis in preterm infants and newborns with a mortality rate of 15 to 80%. Powdered and dairy formulas (P-DF) have been implicated as major transmission vehicles and subsequently the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to use whole genome sequencing (WGS) and laboratory studies to characterize Cronobacter strains from the contaminated products. Seven strains were identified as C. sakazakii, and the remaining strain was Franconibacter helveticus. All C. sakazakii strains adhered to a neuroblastoma cell line, and 31 virulence genes were predicted by WGS. The antibiograms varied between strains. and included mcr-9.1 and bla CSA genes, conferring resistance to colistin and cephalothin, respectively. The C. sakazakii strains encoded I-E and I-F CRISPR-Cas systems, and carried IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. In summary, WGS enabled the identification of C. sakazakii strains and revealed multiple antibiotic resistance and virulence genes. These findings support the decision to recall the contaminated powdered and dairy formulas from the Chilean market in 2017.

Flagella, Type I Fimbriae and Curli of Uropathogenic Escherichia coli Promote the Release of Proinflammatory Cytokines in a Coculture System.

BACKGROUND: Urinary tract infections (UTIs) are a public health problem in Mexico, and uropathogenic Escherichia coli (UPEC) is one of the main etiological agents. Flagella, type I fimbriae, and curli promote the ability of these bacteria to successfully colonize its host. AIM: This study aimed to determine whether flagella-, type I fimbriae-, and curli-expressing UPEC induces the release of proinflammatory cytokines in an established coculture system. METHODS: The fliC, fimH, and csgA genes by UPEC strain were disrupted by allelic replacement. Flagella, type I fimbriae, and curli were visualized by transmission electron microscopy (TEM). HTB-5 (upper chamber) and HMC-1 (lower chamber) cells cocultured in Transwell® plates were infected with these UPEC strains and purified proteins. There was adherence to HTB-5 cells treated with different UPEC strains and they were quantified as colony-forming units (CFU)/mL. RESULTS: High concentrations of IL-6 and IL-8 were induced by the FimH and FliC proteins; however, these cytokines were detected in low concentrations in presence of CsgA. Compared with UPEC CFT073, CFT073ΔfimH, CFT073ΔfimHΔfliC, and CFT073ΔcsgAΔfimH strains significantly reduced the adherence to HTB-5 cells. CONCLUSION: The FimH and FliC proteins are involved in IL-6 and IL-8 release in a coculture model of HTB-5 and HMC-1 cells.

Molecular Epidemiology of Multidrug-Resistant Uropathogenic Escherichia coli O25b Strains Associated with Complicated Urinary Tract Infection in Children.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) has increased the incidence of urinary tract infection (UTI). It is the cause of more than 80% of community-acquired cystitis cases and more than 70% of uncomplicated acute pyelonephritis cases. AIM: The present study describes the molecular epidemiology of UPEC O25b clinical strains based on their resistance profiles, virulence genes, and genetic diversity. METHODS: Resistance profiles were identified using the Kirby-Bauer method, including the phenotypic production of extended-spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs). The UPEC serogroups, phylogenetic groups, virulence genes, and integrons were determined via multiplex PCR. Genetic diversity was established using pulsed-field gel electrophoresis (PFGE), and sequence type (ST) was determined via multilocus sequence typing (MLST). RESULTS: UPEC strains (n = 126) from hospitalized children with complicated UTIs (cUTIs) were identified as O25b, of which 41.27% were multidrug resistant (MDR) and 15.87% were extensively drug resistant (XDR). The O25b strains harbored the fimH (95.23%), csgA (91.26%), papGII (80.95%), chuA (95.23%), iutD (88.09%), satA (84.92%), and intl1 (47.61%) genes. Moreover, 64.28% were producers of ESBLs and had high genetic diversity. ST131 (63.63%) was associated primarily with phylogenetic group B2, and ST69 (100%) was associated primarily with phylogenetic group D. CONCLUSION: UPEC O25b/ST131 harbors a wide genetic diversity of virulence and resistance genes, which contribute to cUTIs in pediatrics.

Virulence and Antibiotic Resistance Genes in Listeria monocytogenes Strains Isolated From Ready-to-Eat Foods in Chile.

Listeria monocytogenes is causing listeriosis, a rare but severe foodborne infection. Listeriosis affects pregnant women, newborns, older adults, and immunocompromised individuals. Ready-to-eat (RTE) foods are the most common sources of transmission of the pathogen This study explored the virulence factors and antibiotic resistance in L. monocytogenes strains isolated from ready-to-eat (RTE) foods through in vitro and in silico testing by whole-genome sequencing (WGS). The overall positivity of L. monocytogenes in RTE food samples was 3.1% and 14 strains were isolated. L. monocytogenes ST8, ST2763, ST1, ST3, ST5, ST7, ST9, ST14, ST193, and ST451 sequence types were identified by average nucleotide identity, ribosomal multilocus sequence typing (rMLST), and core genome MLST. Seven isolates had serotype 1/2a, five 1/2b, one 4b, and one 1/2c. Three strains exhibited in vitro resistance to ampicillin and 100% of the strains carried the fosX, lin, norB, mprF, tetA, and tetC resistance genes. In addition, the arsBC, bcrBC, and clpL genes were detected, which conferred resistance to stress and disinfectants. All strains harbored hlyA, prfA, and inlA genes almost thirty-two the showed the bsh, clpCEP, hly, hpt, iap/cwhA, inlA, inlB, ipeA, lspA, mpl, plcA, pclB, oat, pdgA, and prfA genes. One isolate exhibited a type 11 premature stop codon (PMSC) in the inlA gene and another isolate a new mutation (deletion of A in position 819). The Inc18(rep25), Inc18(rep26), and N1011A plasmids and MGEs were found in nine isolates. Ten isolates showed CAS-Type II-B systems; in addition, Anti-CRISPR AcrIIA1 and AcrIIA3 phage-associated systems were detected in three genomes. These virulence and antibiotic resistance traits in the strains isolated in the RTE foods indicate a potential public health risk for consumers.