Characterization of Staphylococcus epidermidis clinical isolates from hospitalized patients with bloodstream infection obtained in two time periods.

Background: In recent years Staphylococcus epidermidis has been considered an important and frequent causative agent of health care-associated infections (HAIs), increasing the costs of hospitalization, morbidity, and mortality. Antibiotic resistance and biofilm formation are the most important obstacles in the treatment of infections caused by this microorganism. The aim of this work was to determine the most prevalent STs, as well as the antibiotic resistance profile and biofilm formation of S. epidermidis clinical isolates obtained from hospitalized patients in two hospitals in Acapulco, Guerrero in two time periods. Methods: Twenty methicillin-resistant S. epidermidis strains isolated from patients with bacteremia in two hospitals in two time periods were analyzed. Identification and antibiotic susceptibility were performed using the Vitek automated system. Molecular confirmation of the identification and methicillin resistance was performed by duplex PCR of the mecA and nuc genes. Biofilm production was analyzed, and the clonal origin was determined by multilocus sequence typing (MLST). Results: We identified 14 antibiotic resistance profiles as well as 13 sequence types (ST), including the new ST761. We also found that ST2 and ST23 were the most prevalent and, together with ST59, were found in both time periods. Seventeen of our clinical isolates were multidrug-resistant, but all of them were sensitive to linezolid and vancomycin, and this was not related to biofilm production. Additionally, we standardized a duplex PCR to identify methicillin-resistant S. epidermidis strains. In conclusion, S. epidermidis STs 2, 23, and 59 were found in both time periods. This study is the first report of S. epidermidis ST761. The clinical isolates obtained in this work showed a high multidrug resistance that is apparently not related to biofilm production.

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.

Enteroaggregative Escherichia coli is associated with antibiotic resistance and urinary tract infection symptomatology.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is the causative agent of uncomplicated urinary tract infections (UTIs) in ambulatory patients. However, enteroaggregative E. coli (EAEC), an emergent bacterial pathogen that causes persistent diarrhoea, has recently been associated with UTIs. The aim of this study was to determine the frequency of EAEC virulence genes, antibiotic resistance, as well as biofilm production of UPEC isolates obtained from ambulatory patients with non-complicated UTIs that attended to the ISSSTE clinic in Chilpancingo, Guerrero, Mexico, and correlate these with the patients' urinary tract infection symptomatology. METHODS: One hundred clinical isolates were obtained. The identification of clinical isolates, antimicrobial susceptibility testing, and extended spectrum beta-lactamases (ESBLs) production were performed using the Vitek automated system. Assignment of E. coli phylogenetic groups was performed using the quadruplex phylo-group assignment PCR assay. UPEC virulence genes (hlyA, fimH, papC, iutA, and cnf1) and EAEC virulence genes (aap, aggR, and aatA) were detected by multiple PCR. RESULTS: We found that 22% of the isolates carried the aggR gene and were classified as UPEC/EAEC. The main phylogenetic group was B2 (44.1% were UPEC and 77.27% UPEC/EAEC isolates, respectively). Over half of the UPEC/EAEC isolates (63.64%) were obtained from symptomatic patients, however the aatA gene was the only one found to be associated with the risk of developing pyelonephritis (OR = 5.15, p = 0.038). A total of 77.71% of the UPEC/EAEC isolates were ESBL producers and 90.91% multidrug-resistant (MDR). In conclusion, UPEC/EAEC isolates are more frequent in symptomatic patients and the aatA gene was associated with a higher risk of developing pyelonephritis, along with UPEC genes hlyA and cfn1. UPEC/EAEC isolates obtained from UTI showed ESBL production and MDR.

Helicobacter pylori prevalence in healthy Mexican children: comparison between two non-invasive methods.

BACKGROUND: Helicobacter pylori detection in asymptomatic children with suspected infection or with symptoms that suggest gastric pathology is problematic, since most of the methods depend on the endoscopic study, an invasive and expensive method. Non-invasive methods can be a feasible alternative but must be validated. The purpose of this study was to evaluate the concordance between H. pylori DNA detection in saliva and dental plaque by PCR, with antigen detection in stool by immunochromatography, among asymptomatic children in the state of Guerrero, Mexico. METHODS: Dental plaque, saliva, and stool samples were obtained from 171 children between 6 and 12 years old. H. pylori detection in saliva and dental plaque was performed by PCR using specific primers for the 16S rRNA gene, while the detection in stool samples was performed by immunochromatography using the CerTest kit. RESULTS: We found an overall H. pylori prevalence of 59.6% (102/171). Of the H. pylori positive children 18% (20/111) were positive in saliva samples, 28.1% (34/121) in dental plaque samples, and 50.4% (71/141) in stool samples. A higher prevalence was found in girls (64.7%, p = 0.002). Although some of the children declared some dyspeptic symptoms, these were no related to H. pylori. In conclusion, we found a high prevalence of H. pylori in asymptomatic children and the highest proportion was detected by stool antigen test, which was the most feasible method to detect H. pylori infection.

CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis.

BACKGROUND: Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric mucosa in humans. One of the main virulence factors of H. pylori is the cag pathogenicity island (cagPAI), which encodes a type 4-secretion system (T4SS) and the cytotoxin CagA. Translocation of CagA through the T4SS triggers host-signaling pathways. One of the T4SS proteins is CagL, which is necessary for CagA translocation. CagL is a 26-kDa protein that contains a hypervariable motif, which spans residues 58 to 62. Several polymorphisms in this region have been associated with different disease outcomes, e.g. in Mexico, N58 is associated with a higher risk of gastric cancer. The aim of this work is to analyze the sequence of the hypervariable motif (residues 58 to 62) of clinical isolates from Mexican patients with chronic gastritis, and to correlate these polymorphisms with the vacA genotype. RESULTS: Of the 164 biopsies analyzed, only 30.5% (50/164) were positive for H. pylori. Thirty-six of the 50 clinical isolates (72%) were cagA positive, and 40 (80%) had the most virulent vacA genotype (s1/m1). Of the cagA positive strains, 94.4% were vacA s1/m1. All the cagA + strains contained the cagL gene. The most prevalent sequence in the polymorphic region (residues 58-62) was DKMGE (75.8%, 25/33), followed by NKMGQ and NEIGQ (6.1%, 2/33), and DEIGQ, NKMGE, DKIGE, and DKIGK (3%, 1/33). Regarding polymorphisms in positions 58 and 59, the most common were D58/K59 (81.8%, 27/33), followed by N58/K59 (9.1%, 3/33), and D58/E59 (3%, 1/33). Only two isolates (6.1%) contained residues N58/E59, which correspond to those found in H. pylori strain ATCC 26695. 92.6% of the clinical isolates having polymorphism D58/K59 had the genotype vacA s1/m1, considered to be the most virulent, while 7.4% had the genotypes vacA s1/m2 and s2/m2. CONCLUSIONS: In Mexican patients, CagL polymorphisms D58, K59, M60, E62, K122, and I134 are more common in patients with chronic gastritis.

vacA s1m1 genotype and cagA EPIYA-ABC pattern are predominant among Helicobacter pylori strains isolated from Mexican patients with chronic gastritis.

PURPOSE: Virulent genotypes of Helicobacter pylori vacA s1m1/cagA+/babA2+ have been associated with severe gastric diseases. VacA, CagA and BabA are polymorphic proteins, and their association with the disease is allele-dependent. The aims of this work were: (i) to determine the prevalence of H. pylori by type of chronic gastritis; (ii) to describe the frequency of cagA, babA2 and vacA genotypes in strains from patients with different types of chronic gastritis; (iii) to characterize the variable region of cagA alleles. METHODOLOGY: A total of 164 patients with chronic gastritis were studied. Altogether, 50 H. pylori strains were isolated, and the status of cagA, babA2 and vacA genotypes was examined by PCR. cagA EPIYA segment identification was performed using PCR and sequencing of cagA fragments of six randomly selected strains.Results/Key findings. The overall prevalence of H. pylori was 30.5 %. Eighty percent of the isolated strains were vacA s1m1, and the cagA and babA2 genes were detected in 74 and 32 % of the strains, respectively. The most frequent genotypes were vacA s1m1/cagA+/babA2- and vacA s1m1/cagA+/babA2+, with 40 % (20/50) and 28 % (14/50), respectively. In cagA+, the most frequent EPIYA motif was -ABC (78.4 %), and EPIYA-ABCC and -ABCCC motifs were found in 10.8 % of the strains. A modified EPIYT-B motif was found in 66.6 % of the sequenced strains. CONCLUSION: H. pylori strains carrying vacA s1m1, cagA+ and babA2- genotypes were the most prevalent in patients with chronic gastritis from the south of Mexico. In the cagA+ strains, the EPIYA-ABC motif was the most common.

Obesidad y análisis proteómico: su utilidad en la búsqueda de biomarcadores y blancos terapéuticos de enfermedades metabólicas / Utility of proteomic analysis in the search for biomarkers and therapeutic targets in metabolic alterations associated with obesity

La obesidad es una enfermedad compleja y multifactorial, caracterizada por un aumento de grasa corporal que puede ser ocasionado por un desequilibrio entre la ingesta de alimentos y el gasto energético. En el proceso de aumento de peso intervienen factores como la susceptibilidad genética, factores ambientales y el estilo de vida. Está bien documentado que la obesidad aumenta el riesgo de padecer numerosas enfermedades y trastornos metabólicos como la resistencia a la insulina, diabetes tipo 2, hipercolesterolemia, enfermedades cardiovasculares, hígado graso, inflamación de bajo grado, algunos tipos de cáncer y trastornos sicológicos. Debido al incremento de la obesidad y sus comorbilidades en los últimos años en la población mundial, los gastos médicos erogados para su tratamiento representan un problema grave para los sistemas de salud pública. El análisis proteómico a gran escala, es una herramienta potente y prometedora para el descubrimiento de biomarcadores tempranos y para la comprensión de los mecanismos moleculares que subyacen a las alteraciones metabólicas asociadas con la obesidad. No obstante, es imprescindible considerar las limitaciones técnicas y el análisis e interpretación de los hallazgos proteómicos, para avanzar en la comprensión funcional integral de la dinámica del proteoma ligado a la obesidad. Adicionalmente, los abordajes con un enfoque proteómico, permitirán el desarrollo de nuevas terapias preventivas, así como el descubrimiento de agentes terapéuticos potenciales en el tratamiento de disfunciones metabólicas. El objetivo de esta revisión es analizar las contribuciones más recientes de la proteómica en la búsqueda de biomarcadores relacionados con la obesidad.

Obesity is a complex and multifactorial disease characterized by an increase in body fat that can be caused by an imbalance between food intake and energy expenditure. In the process of weight gain, factors such as genetic susceptibility, environmental factors and lifestyle are involved. It is well documented that obesity increases the risk of numerous diseases and metabolic disorders such as insulin resistance, type 2 diabetes, hypercholesterolemia, cardiovascular disease, fatty liver, low grade inflammation, some types of cancer and psychological disorders. Due to the increase in obesity and its comorbidities in recent years at the global level, medical expenses incurred for its treatment represent a serious problem for public health systems. Large-scale proteomic analysis is a powerful and promising tool for the discovery of early biomarkers and for the understanding of the molecular mechanisms underlying the metabolic alterations associated with obesity. Nevertheless, it is essential to consider the technical limitations and the analysis and interpretation of the proteomic findings, to advance in the integral functional understanding of the dynamics of the proteome linked to obesity. In addition, approaches with a proteomic viewpoint will allow the development of new preventive therapies, as well as the discovery of potential therapeutic agents in the treatment of metabolic dysfunctions. The aim of this review is to analyze the most recent contributions of proteomics in the search for biomarkers related to obesity.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Characterization of Escherichia coli clinical isolates causing urinary tract infections in the community of Chilpancingo, Mexico

Escherichia coli is the main cause of urinary tract infections (UTI) in ambulatory patients, especially strains belonging to the B2 phylogenetic group and ST131 clonal group. Antibiotic treatment is usually administered empirically; however, it is not always effective due to bacterial multidrug resistance and the production of extended spectrum β-lactamases (ESBLs). The aim of this study was to characterize E. coli clinical isolates from patients with UTI in a community of the State of Guerrero, Mexico. From January to August 2014, 134 clinical isolates of E. coli were recovered. Strain identification and antibiotic susceptibility were performed using the Vitek automated system. Phylogenetic and O25b-ST13 groups were determined by multiple PCR. Identification of the blaCTX-M, blaTEM, and blaSHV genes was performed by conventional PCR. We found that over 50% of the isolates were resistant to betalactams and quinolones, while 0 to 33% were resistant to aminoglycosides and nitrofurans, and 56.49% of the strains were ESBL producers. B2 phylogenetic group was the most predominant (43%) compared to the other groups. The prevalence of bla genes was: blaCTX-M 64.3%, blaSHV 41.4%, and blaTEM 54.3%. These results show a high percentage (55%) of multidrug-resistant strains isolated from UTI patients from the community in the city of Chilpancingo, Guerrero, Mexico (AU)

No disponible

Type Three Secretion System in Attaching and Effacing Pathogens.

Enteropathogenic Escherichia coli and enterohemorrhagic E. coli are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen Citrobacter rodentium, belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens.

Characterization of Escherichia coli clinical isolates causing urinary tract infections in the community of Chilpancingo, Mexico.

Escherichia coli is the main cause of urinary tract infections (UTI) in ambulatory patients, especially strains belonging to the B2 phylogenetic group and ST131 clonal group. Antibiotic treatment is usually administered empirically; however, it is not always effective due to bacterial multidrug resistance and the production of extended spectrum ß-lactamases (ESBLs). The aim of this study was to characterize E. coli clinical isolates from patients with UTI in a community of the State of Guerrero, Mexico. From January to August 2014, 134 clinical isolates of E. coli were recovered. Strain identification and antibiotic susceptibility were performed using the Vitek automated system. Phylogenetic and O25b-ST13 groups were determined by multiple PCR. Identification of the blaCTX-M, blaTEM, and blaSHV genes was performed by conventional PCR. We found that over 50% of the isolates were resistant to betalactams and quinolones, while 0 to 33% were resistant to aminoglycosides and nitrofurans, and 56.49% of the strains were ESBL producers. B2 phylogenetic group was the most predominant (43%) compared to the other groups. The prevalence of bla genes was: blaCTX-M 64.3%, blaSHV 41.4%, and blaTEM 54.3%. These results show a high percentage (55%) of multidrug-resistant strains isolated from UTI patients from the community in the city of Chilpancingo, Guerrero, Mexico. [Int Microbiol 19(4): 209-215 (2016)].

Identification and regulation of a novel Citrobacter rodentium gut colonization fimbria (Gcf).

UNLABELLED: The Gram-negative enteric bacterium Citrobacter rodentium is a natural mouse pathogen that has been extensively used as a surrogate model for studying the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli. All three pathogens produce similar attaching and effacing (A/E) lesions in the intestinal epithelium. During infection, these bacteria employ surface structures called fimbriae to adhere and colonize the host intestinal epithelium. For C. rodentium, the roles of only a small number of its genome-carried fimbrial operons have been evaluated. Here, we report the identification of a novel C. rodentium colonization factor, called gut colonization fimbria (Gcf), which is encoded by a chaperone-usher fimbrial operon. A gcfA mutant shows a severe colonization defect within the first 10 days of infection. The gcf promoter is not active in C. rodentium under several in vitro growth conditions; however, it is readily expressed in a C. rodentium Δhns1 mutant lacking the closest ortholog of the Escherichia coli histone-like nucleoid structuring protein (H-NS) but not in mutants with deletion of the other four genes encoding H-NS homologs. H-NS binds to the regulatory region of gcf, further supporting its direct role as a repressor of the gcf promoter that starts transcription 158 bp upstream of the start codon of its first open reading frame. The gcf operon possesses interesting novel traits that open future opportunities to expand our knowledge of the structure, regulation, and function during infection of these important bacterial structures. IMPORTANCE: Fimbriae are surface bacterial structures implicated in a variety of biological processes. Some have been shown to play a critical role during host colonization and thus in disease. Pathogenic bacteria possess the genetic information for an assortment of fimbriae, but their function and regulation and the interplay between them have not been studied in detail. This work provides new insights into the function and regulation of a novel fimbria called Gcf that is important for early establishment of a successful infection by C. rodentium in mice, despite being poorly expressed under in vitro growth conditions. This discovery offers an opportunity to better understand the individual role and the regulatory mechanisms controlling the expression of specific fimbrial operons that are critical during infection.

A distinct regulatory sequence is essential for the expression of a subset of nle genes in attaching and effacing Escherichia coli.

Enteropathogenic Escherichia coli uses a type III secretion system (T3SS), encoded in the locus of enterocyte effacement (LEE) pathogenicity island, to translocate a wide repertoire of effector proteins into the host cell in order to subvert cell signaling cascades and promote bacterial colonization and survival. Genes encoding type III-secreted effectors are located in the LEE and scattered throughout the chromosome. While LEE gene regulation is better understood, the conditions and factors involved in the expression of effectors encoded outside the LEE are just starting to be elucidated. Here, we identified a highly conserved sequence containing a 13-bp inverted repeat (IR), located upstream of a subset of genes coding for different non-LEE-encoded effectors in A/E pathogens. Site-directed mutagenesis and deletion analysis of the nleH1 and nleB2 regulatory regions revealed that this IR is essential for the transcriptional activation of both genes. Growth conditions that favor the expression of LEE genes also facilitate the activation of nleH1 and nleB2; however, their expression is independent of the LEE-encoded positive regulators Ler and GrlA but is repressed by GrlR and the global regulator H-NS. In contrast, GrlA and Ler are required for nleA expression, while H-NS silences it. Consistent with their role in the regulation of nleA, purified Ler and H-NS bound to the regulatory region of nleA upstream of its promoter. This work shows that at least two modes of regulation control the expression of effector genes in attaching and effacing (A/E) pathogens, suggesting that a subset of effector functions may be coordinately expressed in a particular niche or time during infection.

Transcriptional regulation of the ecp operon by EcpR, IHF, and H-NS in attaching and effacing Escherichia coli.

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli are clinically important diarrheagenic pathogens that adhere to the intestinal epithelial surface. The E. coli common pili (ECP), or meningitis-associated and temperature-regulated (MAT) fimbriae, are ubiquitous among both commensal and pathogenic E. coli strains and play a role as colonization factors by promoting the interaction between bacteria and host epithelial cells and favoring interbacterial interactions in biofilm communities. The first gene of the ecp operon encodes EcpR (also known as MatA), a proposed regulatory protein containing a LuxR-like C-terminal helix-turn-helix (HTH) DNA-binding motif. In this work, we analyzed the transcriptional regulation of the ecp genes and the role of EcpR as a transcriptional regulator. EHEC and EPEC ecpR mutants produce less ECP, while plasmids expressing EcpR increase considerably the expression of EcpA and production of ECP. The ecp genes are transcribed as an operon from a promoter located 121 bp upstream of the start codon of ecpR. EcpR positively regulates this promoter by binding to two TTCCT boxes distantly located upstream of the ecp promoter, thus enhancing expression of downstream ecp genes, leading to ECP production. EcpR mutants in the putative HTH DNA-binding domain are no longer able to activate ecp expression or bind to the TTCCT boxes. EcpR-mediated activation is aided by integration host factor (IHF), which is essential for counteracting the repression exerted by histone-like nucleoid-structuring protein (H-NS) on the ecp promoter. This work demonstrates evidence about the interplay between a novel member of a diverse family of regulatory proteins and global regulators in the regulation of a fimbrial operon.

Structural insights into the biogenesis and biofilm formation by the Escherichia coli common pilus.

Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.