The nucleoid protein HU positively regulates the expression of type VI secretion systems in Enterobacter cloacae.

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria. IMPORTANCE: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

Antimicrobial resistance in food-associated Escherichia coli in Mexico and Latin America.

The World Health Organization (WHO) considers antimicrobial resistance to be one of the critical global public health priorities to address. Escherichia coli is a commensal bacterium of the gut microbiota in humans and animals; however, some strains cause infections and are resistant to antibiotics. One of the most common ways of acquiring pathogenic E. coli strains is through food. This review analyzes multidrug-resistant E. coli isolated from food, emphasizing Latin America and Mexico, and the mobile genetic elements (MGEs) responsible for spreading antibiotic resistance determinants among bacteria in different environments and hosts. We conducted a systematic search of the literature published from 2015 to 2022 in open access databases and electronic repositories. The prevalence of 11 E. coli pathotypes was described, with diarrheagenic E. coli pathotypes being the most frequently associated with foodborne illness in different Latin American countries, highlighting the presence of different antibiotic resistance genes mostly carried by IncF-type plasmids or class 1 integrons. Although the global incidence of foodborne illness is high, there have been few studies in Mexico and Latin America, which highlights the need to generate updated epidemiological data from the "One Health" approach, which allows monitoring of the multidrug-resistance phenomenon in E. coli from a common perspective in the interaction of human, veterinary, and environmental health.

The Enolase of the Haemophilus influenzae Mediates Binding to Collagens: An Extracellular Matrix Component.

Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis.

Targeting the Human Influenza a Virus: The Methods, Limitations, and Pitfalls of Virtual Screening for Drug-like Candidates Including Scaffold Hopping and Compound Profiling.

In this study, we describe the input data and processing steps to find antiviral lead compounds by a virtual screen. Two-dimensional and three-dimensional filters were designed based on the X-ray crystallographic structures of viral neuraminidase co-crystallized with substrate sialic acid, substrate-like DANA, and four inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir). As a result, ligand-receptor interactions were modeled, and those necessary for binding were utilized as screen filters. Prospective virtual screening (VS) was carried out in a virtual chemical library of over half a million small organic substances. Orderly filtered moieties were investigated based on 2D- and 3D-predicted binding fingerprints disregarding the "rule-of-five" for drug likeness, and followed by docking and ADMET profiling. Two-dimensional and three-dimensional screening were supervised after enriching the dataset with known reference drugs and decoys. All 2D, 3D, and 4D procedures were calibrated before execution, and were then validated. Presently, two top-ranked substances underwent successful patent filing. In addition, the study demonstrates how to work around reported VS pitfalls in detail.

GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Introduction: Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. Methods: To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. Results and discussion: We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Epigenetic mechanisms of particulate matter exposure: air pollution and hazards on human health.

Environmental pollution nowadays has not only a direct correlation with human health changes but a direct social impact. Epidemiological studies have evidenced the increased damage to human health on a daily basis because of damage to the ecological niche. Rapid urban growth and industrialized societies importantly compromise air quality, which can be assessed by a notable accumulation of air pollutants in both the gas and the particle phases. Of them, particulate matter (PM) represents a highly complex mixture of organic and inorganic compounds of the most variable size, composition, and origin. PM being one of the most complex environmental pollutants, its accumulation also varies in a temporal and spatial manner, which challenges current analytical techniques used to investigate PM interactions. Nevertheless, the characterization of the chemical composition of PM is a reliable indicator of the composition of the atmosphere, the quality of breathed air in urbanized societies, industrial zones and consequently gives support for pertinent measures to avoid serious health damage. Epigenomic damage is one of the most promising biological mechanisms of air pollution-derived carcinogenesis. Therefore, this review aims to highlight the implication of PM exposure in diverse molecular mechanisms driving human diseases by altered epigenetic regulation. The presented findings in the context of pan-organic cancer, fibrosis, neurodegeneration and metabolic diseases may provide valuable insights into the toxicity effects of PM components at the epigenomic level and may serve as biomarkers of early detection for novel targeted therapies.

The Flp type IV pilus operon of Mycobacterium tuberculosis is expressed upon interaction with macrophages and alveolar epithelial cells.

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.

The Fis Nucleoid Protein Negatively Regulates the Phase Variation fimS Switch of the Type 1 Pilus Operon in Enteropathogenic Escherichia coli.

In Escherichia coli the expression of type 1 pili (T1P) is determined by the site-specific inversion of the fimS ON-OFF switch located immediately upstream of major fimbrial subunit gene fimA. Here we investigated the role of virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the fimS switch in the human enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the fimS switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆fis mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the fimA, fimB, and fimE promoters using lacZ transcriptional fusions indicated that only PfimA activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the fimS switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of E. coli.

The Transcription of Flagella of Enteropathogenic Escherichia coli O127:H6 Is Activated in Response to Environmental and Nutritional Signals.

The flagella of enteropathogenic Escherichia coli (EPEC) O127:H6 E2348/69 mediate adherence to host proteins and epithelial cells. What environmental and nutritional signals trigger or down-regulate flagella expression in EPEC are largely unknown. In this study, we analyzed the influence of pH, oxygen tension, cationic and anionic salts (including bile salt), carbon and nitrogen sources, and catecholamines on the expression of the flagellin gene (fliC) of E2348/69. We found that sodium bicarbonate, which has been shown to induce the expression of type III secretion effectors, down-regulated flagella expression, explaining why E2348/69 shows reduced motility and flagellation when growing in Dulbecco's Minimal Essential Medium (DMEM). Further, growth under a 5% carbon dioxide atmosphere, in DMEM adjusted to pH 8.2, in M9 minimal medium supplemented with 80 mM glucose or sucrose, and in DMEM containing 150 mM sodium chloride, 0.1% sodium deoxycholate, or 30 µM epinephrine significantly enhanced fliC transcription to different levels in comparison to growth in DMEM alone. When EPEC was grown in the presence of HeLa cells or in supernatants of cultured HeLa cells, high levels (4-fold increase) of fliC transcription were detected in comparison to growth in DMEM alone. Our data suggest that nutritional and host signals that EPEC may encounter in the intestinal niche activate fliC expression in order to favor motility and host colonization.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli.

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

Structural Characterization of Haemophilus influenzae Enolase and Its Interaction with Human Plasminogen by In Silico and In Vitro Assays.

Haemophilus influenzae is the causal agent of invasive pediatric diseases, such as meningitis, epiglottitis, pneumonia, septic arthritis, pericarditis, cellulitis, and bacteremia (serotype b). Non-typeable H. influenzae (NTHi) strains are associated with localized infections, such as otitis media, conjunctivitis, sinusitis, bronchitis, and pneumonia, and can cause invasive diseases, such as as meningitis and sepsis in immunocompromised hosts. Enolase is a multifunctional protein and can act as a receptor for plasminogen, promoting its activation to plasmin, which leads to the degradation of components of the extracellular matrix, favoring host tissue invasion. In this study, using molecular docking, three important residues involved in plasminogen interaction through the plasminogen-binding motif (251EFYNKENGMYE262) were identified in non-typeable H. influenzae enolase (NTHiENO). Interaction with the human plasminogen kringle domains is conformationally stable due to the formation of four hydrogen bonds corresponding to enoTYR253-plgGLU1 (K2), enoTYR253-plgGLY310 (K3), and enoLYS255-plgARG471/enoGLU251-plgLYS468 (K5). On the other hand, in vitro assays, such as ELISA and far-western blot, showed that NTHiENO is a plasminogen-binding protein. The inhibition of this interaction using polyclonal anti-NTHiENO antibodies was significant. With these results, we can propose that NTHiENO-plasminogen interaction could be one of the mechanisms used by H. influenzae to adhere to and invade host cells.

Effectiveness, tolerability and safety of Direct Acting Antivirals in Mexican individuals with Hepatitis C virus genotype-1 and previous pegylated interferon and ribavirin therapy.

BACKGROUND: Direct Acting Antivirals (DAAs) represent a large improvement in the treatment of chronic hepatitis C, resulting in <90% sustained virological response (SVR). There are no reports on the real-world DAA response for Mexico and few reports exist for Latin America. The aim of the study was to report SVR, and immediate benefits with the DAA treatments sofosbuvir, ledispavir, with/without ribavirin (SOF/LDV ± RBV) and ombitasvir, paritaprevir, ritonavir, dasabuvir with/without RBV (OBV/PTV/r/DSV ± RBV) in patients with viral genotype 1a or 1b, and who did not respond to previous peginterferon/ribavirin (PegIFNα2a+RBV) therapy. METHODS: A descriptive, ambispective, longitudinal study was conducted. A cohort of 261 adult patients received PegIFNα2a+RBV therapy before 2014; 167 (64%) did not respond, 83 of these were subsequently treated with SOF/LDV ± RBV or OBV/PTV/r/DSV ± RBV. Child-Pugh-Score (CPS), Fibrosis-4 (FIB-4), and AST to Platelet Ratio Index (APRI) were evaluated before and after treatment. RESULTS: SVR with PegIFNα2a+RBV was 36%, and 97.5% with DAAs. CPS, FIB-4 and APRI improved significantly after DAA treatment, mainly because of liver transaminase reduction. CONCLUSIONS: DAA treatment showed excellent SVR rates in Mexican patients who had not responded to PegIFNα2a+RBV therapy. Improvement in CPS, FIB-4 and APRI without improvement in fibrosis was observed in cirrhotic and non-cirrhotic patients, as well as considerable reduction in liver transaminases, which suggests a reduction in hepatic necroinflammation.

Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase.

Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post-H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif (252FYNKENGMY260) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae. Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae, important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated.

Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin.

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together In Vivo at neuromuscular junctions and in motor neurons.

The transcriptional activator of the bfp operon in EPEC (PerA) interacts with the RNA polymerase alpha subunit.

Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.

Location of OprD porin in Pseudomonas aeruginosa clinical isolates.

Multidrug-resistant Pseudomonas aeruginosa is one of the main opportunistic pathogens causing severe infection. One of the mechanisms involved in the resistance to imipenem in clinical isolates is the loss of the OprD porin. Changes like substitutions, deletions, insertions, or mutations in the oprD gene can modify the conformation of OprD porin or inhibit its presence and generate resistance to carbapenems. The aim of this work was to obtain anti-OprD polyclonal antibodies and to determine by both immunofluorescence microscopy (IFI) and Western blot assays, the presence of the OprD porin in resistant-carbapenem P. aeruginosa strains with different changes in the oprD gene. Changes in the gene oprD were identified in clinical isolates of P. aeruginosa. When proteins were translated, several polymorphisms were found; however, these did not affect the presence of OprD porin (PCM25, PCM36, and PCM78). Also it was detected an insertion sequence ISPa1328 (PCM52) and a premature stop codon (PCM91), which inhibited the presence of the OprD porin. This study shows how changes in the oprD gene of P. aeruginosa clinical isolates affect the presence of the OprD porin detected by Western blot and indirect immunofluorescence assays using specific polyclonal anti-OprD antibodies generated in this work.

Coliform bacteria in San Pedro Lake, western Mexico.

Urbanization, livestock activities, and rainfall are factors that contribute to the contamination of inland water. This study aimed to determine the spatial and temporal variability of total coliforms (TCs) and fecal coliforms (FCs) in the surface water of San Pedro Lake as well as the gills and skin of Nile tilapia (Oreochromis niloticus) cultivated in the lake. The study consisted of seasonal sampling during an annual cycle. Using the multiple-tube fermentation technique, we quantified the microbial load of TCs in the lake and fish. The median of the TC and FC groups in surface water showed differences during the seasonal cycle, in which a significant correlation was observed between rainfall and bacterial load in the lake surface water. There was a significant seasonal difference between FCs and TCs in the gills as well as in skin FCs. Anthropogenic activities in the watershed combined with rainfall influence the bacterial load of San Pedro Lake. However, the water quality is still classified as excellent and uncontaminated according to Mexican regulations with lower FC values acceptable for higher FC values. In addition, the bacterial load in tilapia from San Pedro Lake does not pose a risk to human health. PRACTITIONER POINTS: Watershed livestock activities combined with rainfall increase fecal matter pollution in specific areas of the lake. San Pedro Lake displays satisfactory quality for aquatic life. The median fecal coliform population in lake fish (gills and skin) differs by season.

Glycogene expression profiles from a HaCaT cell line stably transfected with HPV16 E5 oncogene.

The altered expression of glycan antigens has been reported during cervix transformation, demonstrating increased mRNA levels of certain glycogenes. Human papillomavirus (HPV) is the aetiological agent of cervical cancer. High risk HPV E5 is considered an oncogene and has been implicated in cell transformation. E6 and E7 HPV oncoproteins modify the expression of certain glycogenes. The role of the E5 HPV protein in glycogene expression changes has not yet been reported. The aim of the present study was to determine the effects of HPV16 E5 oncoprotein on glycogene expression. For these, a microarray assay was performed using the HaCaT cell line and altered glycogenes were identified. The mRNA levels of certain glycogenes were determined via reverse transcription­quantitative PCR (RT­qPCR). Using in silico analysis, the present study identified that glycosylation pathways were altered by E5. Microarray analysis revealed alterations in certain glycogenes, including the upregulation of ST6GAL1, ST3GAL3, CHST2 and MANBA, and the downregulation of UGT2B15, GALNT11, NDST2 and UGT1A10. Increased mRNA levels were confirmed via RT­qPCR for sialyltransferases genes. Additionally, in silico analysis was performed to identify glycosylation networks altered in the presence of the E5 oncoprotein. The analysis revealed that E5 could modify glycan sialylation, the N­glycosylation pathway, keratan sulfate and glycosaminoglycan synthesis. To the best of our knowledge, the current study was the first to determine the role of the HPV16 E5 oncoprotein in glycogene expression changes. The results indicated that increased sialyltransferase mRNA levels reported in pre­malignant and malignant cervical tissues could be the result of E5 oncoprotein expression. The results provide a possible role of HPV infection on glycosylation changes reported during cervix transformation.

Isolation and Identification of Two Brucella Species from a Volcanic Lake in Mexico.

Brucellosis is a zoonosis caused by bacteria of the Brucella genus. Any source of contamination that could be infectious must be monitored to reduce the risk of exposure to brucellosis, so the purpose of this work was to determine the presence of Brucella spp. on surface water and tilapia (Oreochromis niloticus) skin from a volcanic lake in Mexico. A seasonal sampling during 2016-2017 was carried out at fifteen specific sites for water sampling and five sites for the collection of tilapia fish. From all water and fish samples tested, we found only three isolates of Brucella species. We isolated and identified B. abortus from surface water through bacteriological and molecular techniques, and B. abortus and B. suis from the same tilapia skin sample. The isolated strains likely came from breeding animals that are common to the region, such as infected pigs or cattle with Brucella abortus or B. suis, respectively. A similar finding has not been reported in a water from volcanic lake or tilapia fish in Mexico. We concluded that B. abortus and B. suis are present on the surface water of the volcanic lake and tilapia skin as possible contaminants derived from biological material from cows and pigs carrying this bacterium.

Virulence and Resistance Determinants of Uropathogenic Escherichia coli Strains Isolated from Pregnant and Non-Pregnant Women from Two States in Mexico.

BACKGROUND/PURPOSE: Uropathogenic E. coli (UPEC) is the main cause of urinary tract infection (UTI) and it is known that pregnant women have a higher risk for UTI. UPEC has a variety of virulence and antibiotic resistance factors that facilitate its pathogenic success and it is crucial to know which are the susceptibility patterns, Extended-Spectrum-ß-Lactamase (ESBL) production, virulence genes, pathogenicity islands (PAI), phylogenetic groups and serotypes among strains isolated from pregnant and non-pregnant women. METHODS: One hundred fifty UPEC strains were isolated from pregnant and non-pregnant women from two different Mexican states (Sonora and Puebla). Strains were analyzed using the Kirby-Bauer method for the determination of antibiotic susceptibility and ESBL. Virulence genes, PAIs and phylogenetic groups were determined using a multiplex PCR. Strains were serotyped by an agglutination assay. Blood agar and CAS agar were used for phenotypic assays. RESULTS: 92.7% of UPEC strains showed multidrug-resistant (MDR), 6.7% extremely-resistant (XDR) and 0.6% pandrug-resistant (PDR). The highest resistance was determined to be for ß-lactam antibiotics (>72% in both states) and 44.5% of the UPEC strains were ESBL+. The predominant virulence genes found were fimH (100%), iucD (85%) and iha (60%). The strains isolated from pregnant women from Puebla presented a large percentage of genes associated with upper urinary tract infections. PAIs were found in 51% and 68% of the strains from Sonora and Puebla, respectively. All the strains were siderophores producers and 41.5% produced hemolysis. The serotypes found were diverse and belonged to phylogroups A, B2 and C. CONCLUSION: The UPEC strains from this study are MDR with tendency to XDR or PDR, they can cause upper UTIs and are serotypically and phylogenetically diverse, which supports the need to develop new strategies for UTI treatment in pregnant and non-pregnant Mexican women.