Genomic and phenotypic characterization of Pseudomonas sp. GOM7, a novel marine bacterial species with antimicrobial activity against multidrug-resistant Staphylococcus aureus.

Antimicrobial resistance (AMR) represents a serious threat to global health. The development of new drugs to combat infections caused by bacteria resistant to multiple or even all available antibiotics is urgent. Most antibiotics used up to date have been identified from soil microorganisms. The marine environment represents an alternative source with great potential for the identification of microorganisms that produce bioactive molecules, including antibiotics. In this study, we analyzed the antibacterial activity of a collection of 82 bacterial strains isolated from marine water and sediment samples collected from the Southwestern Gulf of Mexico. Eight of the marine isolates inhibited the growth of different pathogenic bacteria, seven of which were identified as presumptive Pseudomonas aeruginosa. Interestingly, genome sequencing and phylogenetic analysis revealed that the remaining marine isolate showing antibacterial activity is a novel Pseudomonas species that we denominated Pseudomonas sp. GOM7, which was not pathogenic in the Galleria mellonella infection model in the conditions tested. Notably, Pseudomonas sp. GOM7 inhibited the growth of multidrug and methicillin-resistant strains of the priority pathogen Staphylococcus aureus. Our results show that the anti-S. aureus compound(s) produced by Pseudomonas sp. GOM7 can be extracted from the culture supernatant of this bacterium with the organic solvent ethyl acetate. Annotation of the Pseudomonas sp. GOM7 genome revealed the presence of several biosynthetic gene clusters predicted to code for possible antimicrobial compounds. Our results further highlight the potential of bacteria from the Gulf of Mexico as a source of novel antimicrobials.

Molecular Characterization of Staphylococcus aureus Strains Isolated from Mobile Phones.

The widespread use of mobile phones (MP) among healthcare personnel might be considered as an important source of contamination. One of the most pathogenic bacteria to humans is Staphylococcus aureus, which can be transmitted through the constant use of MP. Nevertheless, which specific type of strains are transmitted and which are their sources have not been sufficiently studied. The aim of this study is to determine the source of contamination of MP and characterize the corresponding genotypic and phenotypic properties of the strains found. Nose, pharynx, and MP samples were taken from a group of health science students. We were able to determinate the clonality of the isolated strains by pulsed-field gel electrophoresis (PFGE) and spa gene typing (spa-type). Adhesin and toxin genes were detected, and the capacity of biofilm formation was determined. Several of the MP exhibited strains of S. aureus present in the nose and/or pharynx of their owners. methicillin-susceptible Staphylococcus aureus (MSSA), hospital-acquired methicillin-resistant S. aureus (HA-MRSA), and community-acquired methicillin-resistant S. aureus (CA-MRSA) strains were found, which indicated a variety of genotypes. This study concludes that MP can be contaminated with the strains of S. aureus present in the nose and/or pharynx of the owners; these strains can be of different types and there is no dominant genotype.

Frequency and expression of genes involved in adhesion and biofilm formation in Staphylococcus aureus strains isolated from periodontal lesions.

BACKGROUND/PURPOSE: The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an in vitro model, its biofilm formation, and its resistance to antibiotics. METHODS: S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells in vitro. RESULTS: S. aureus was identified in 18.6% (50/268) of the samples. All strains (n = 50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n = 48) possessed clfB (clumping factor B), and 88% (n = 44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified. CONCLUSION: Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease.

Cytotoxic Activity of Cu/TiO2 Nanoparticles on Uterine-Cervical Cancer Cells.

Nanoparticles based on metal oxides serve as carrier matrices for molecules of biological interest. In this work, we used different copper complexes that were coupled to TiO2 nanoparticles. Nanoparticles were prepared with the sol-gel method. The Cu/TiO2 nanoparticles were characterized through ultraviolet-visible and Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, nitrogen physisorption analysis, and scanning electron microscopy. Their biological activity was determined through DNA degradation and their cytotoxic effect on HeLa cells. The Cu/TiO2 nanoparticles presented a pore size between 2 and 6 nm, the size of nanoparticles agglomerates was between 100 and 500 nm. The nanoparticles of Cu/TiO2 degraded DNA starting at 15 min. The half maximal inhibitory concentration in HeLa cells depends on the used cooper complexes, the kinetics of cell death is of first order. Results revealed that these nanoparticles could be applied in uterine-cervical cancer treatment.

Virulence gene transcription, phylogroups, and antibiotic resistance of cervico-vaginal pathogenic E. coli in Mexico.

The pathogenicity of Escherichia coli strains that cause cervico-vaginal infections (CVI) is due to the presence of several virulence genes. The objective of this study was to define the variability regarding the genotype of antibiotic resistance, the transcription profiles of virulence genes after in vitro infection of the vaginal cell line A431 and the phylogroup composition of a group of cervico-vaginal E. coli strains (CVEC). A total of 200 E. coli strains isolated from Mexican women with CVI from two medical units of the Mexican Institute of Social Security were analysed. E. coli strains and antibiotic resistance genes were identified using conventional polymerase chain reaction (PCR), and phylogroups were identified using multiplex PCR. Virulence gene transcription was measured through reverse-transcriptase real-time PCR after infection of the vaginal cell line A431. The most common antibiotic resistance genes among the CVEC strains were aac(3)II, TEM, dfrA1, sul1, and qnrA. The predominant phylogroup was B2. The genes most frequently transcribed in these strains were fimH, papC, irp2, iroN, kpsMTII, cnf1, and ompT, mainly in CVEC strains isolated from chronic and occasional vaginal infections. The strains showed a large diversity of transcription of the virulence genes phenotype and antibiotic resistance genotype, especially in the strains of phylogroups, B2, A, and D. The strains formed 2 large clusters, which contained several subclusters. The genetic diversity of CVEC strains was high. These strains have a large number of transcription patterns of virulence genes, and one-third of them carry three to seven antibiotic resistance genes.

Use of silver nanoparticles to control Vibrio fluvialis in cultured angelfish Pterophyllum scalare.

Nanoparticles have multiple applications, among which is their use as antimicrobial agents in aquaculture. The objective of this work was to determine the antibacterial effect of silver nanoparticles (AgNPs) against Vibrio fluvialis in cultured angelfish Pterophyllum scalare. AgNPs were synthetized through chemical reduction and characterized by UV-visible and infrared spectroscopy. Particle size ranged from 60 to 170.8 nm, and scanning electron microscopy revealed cubic and spherical forms. A minimal inhibitory concentration of 222.5 ppm was determined, as well as inhibition halos between 8.66 and 14.3 mm. Inhibition of V. fluvialis growth was observed upon contact with AgNPs. An 88% survival of infected fish was obtained when treated with AgNPs, in contrast to 100% mortality of fish that were not treated. No damage to internal or external organs was observed in fish exposed to AgNPs. We conclude that AgNPs exert an antimicrobial effect against V. fluvialis, and thus represent a new alternative to control diseases caused by this microorganism in P. scalare culture.

Frecuencia y caracterización de Staphylococcus spp. en la mucosa bucal de pacientes diabéticos y no diabéticos / Frequency and characterization of Staphylococcus spp. in the buccal mucosa of diabetic and non-di

Antecedentes: Dentro de la gran diversidad de microorganismos en la microbiota oral, el género Staphylococcus es causante de una gran variedad de infecciones, desde leves hasta diseminadas que pueden causar la muerte. Los portadores de estas bacterias tienen más riesgo de presentar infecciones por estas mismas. Un grupo vulnerable de sufrir infecciones por estos microorganismos son los pacientes diabéticos por sus características sistémicas propias de la enfermedad, deterioro inmunológico y locales bucales, por lo que es importante conocer si son portadoras de este grupo de bacterias. Objetivo: Determinar la frecuencia de colonización por Staphylococcus spp. en aislamientos obtenidos de la mucosa bucal de pacientes diabéticos y sin diabetes. Material y métodos: Se tomó un raspado superficial de la mucosa bucal de personas diabéticas y sin diabetes para cultivo y análisis microbiológico. Se sembró en agar sal manitol y los aislados se identificaron por galerías API Staph. La concentración de glucosa se determinó con equipo Accu-Chek. El análisis fue descriptivo, las diferencias y asociaciones se investigaron con χ2 y T Student. Se consideró estadísticamente significativo cuando el valor de p < 0.05. Resultados: La colonización por Staphylococcus spp. total fue de 73.7%, no hubo diferencia significativa entre diabéticos y no diabéticos (p = 0.946). S. epidermidis se identificó en 69% y S. aureus en 17.6%, sin diferencia entre ambos grupos con p = 0.556 y p = 0.428 respectivamente. Setenta y seis por ciento de los pacientes portadores de prótesis bucales estaban colonizados con Staphylococcus spp. Conclusiones: No se encontró que los pacientes diabéticos tuvieran porcentajes significativamente mayores de colonización por Staphylococcus spp. a pesar de sus condiciones particulares inmunológicas, glucemia anormal y disminución de flujo salival en la cavidad bucal (AU)

Background: Within the great diversity of microorganisms in the buccal microbiota, the genus Staphylococcus is the cause of a great variety of infections ranging from mild to disseminated, which can cause death. The carriers of these bacteria are more at risk of developing infections by themselves. A vulnerable group to suffer infections by these microorganisms are diabetic patients due to their systemic characteristics of the disease, immunological deterioration and local buccal, so it is important to know if they are carriers of this group of bacteria. Objective: The objective was determined the frequency of colonization by Staphylococcus spp. in isolates obtained from the oral mucosa of diabetic and without diabetes patients. Material and methods: A superficial scraping of the buccal mucosa of diabetic and without diabetes people was taken for culture and microbiological analysis. It was seeded in sal manitol agar and the isolates were identified by API Staph galleries. The glucose concentration was determined with Accu-Chek equipment. The analysis was descriptive, differences and associations were investigated with χ2 and Student T. It was considered statistically significant when the value of p < 0.05. Results: Total colonization by Staphylococcus spp. was 73.7%, there was no significant difference between diabetics and nondiabetics (p = 0.946). S. epidermidis was identified in 69% and S. aureus in 17.6%, without difference between both groups with p = 0.556 and p = 0.428 respectively. 76% of patients with oral prostheses were colonized with Staphylococcus spp. Conclusions: Diabetic patients were not found to have significantly higher rates of colonization by Staphylococcus spp. despite their particular immunological conditions, abnormal glycemia and decreased salivary flow in the oral cavity (AU)

Community-acquired methicillin-resistant Staphylococcus aureus can persist in the throat.

Colonization by Staphylococcus aureus is an important factor in infections caused by this microorganism. Among the colonization niches of staphylococci are the nose, skin, intestinal tract, and, recently, the throat has been given relevance. Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) can be fatal. Persistence of S. aureus is an important process in the pathogenesis of this microorganism and must be studied. The aim of this study was to determine the persistence of S. aureus in the throat, and characterized the strains. We studied the persistence of S. aureus for 6 years in the throat of apparently healthy people. The isolated strains from the persistent carriers were characterized through PFGE, spa-typing, SCCmec typing, resistance to methicillin, presence of virulence genes (adhesins and toxins), and the formation of biofilm. We found persistent and intermittent carriers of S. aureus in the throat, with methicillin-sensitive (MSSA), methicillin-resistant (MRSA) strains, and confirmed for the first time that CA-MRSA colonizes this niche. These strains can colonize persistently the throat for four years or more. Typification of strains through PFGE and spa-typing revealed that some carriers present the same strain, whereas others present different strains along the period of persistence. Almost all strains induced a strong biofilm formation. All strains presented adhesin and toxin genes, but no shared genotype was found. We conclude that S. aureus, including CA-MRSA strains, can remain persistently in the throat, finding a wide variability among the persistent strains.

Cytolethal Distending Toxin From Campylobacter jejuni Requires the Cytoskeleton for Toxic Activity.

BACKGROUND: Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. OBJECTIVES: The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus. METHODS: Campylobacter jejuni ATCC 33291 and seven isolates donated from Instituto de Biotecnologia were used in this study. The presence of CDT genes in C. jejuni strains was determined by PCR. To evaluate the effect of CDT, HeLa cells were treated with bacterial lysate, and the damage and morphological changes were analyzed by microscopy, immunofluorescence staining, and flow cytometry. To evaluate the role of the cytoskeleton, HeLa cells were treated with either latrunculin A or by nocodazole and analyzed by microscopy, flow cytometry, and immunoquantification (ELISA). RESULTS: The results obtained showed that the eight strains of C. jejuni, including the reference strain, had the ability to produce the toxin. Usage of latrunculin A and nocodazole, two cytoskeletal inhibitors, blocked the toxic effect in cells treated with the toxin. This phenomenon was evident in flow cytometry analysis and immunoquantification of Cdc2-phosphorylated. CONCLUSIONS: This work showed that the cytotoxic activity of the C. jejuni CDT is dependent on its endocytosis. The alteration in the microtubules and actin filaments caused a blockage transit of the toxin, preventing it from reaching the nucleus of the cell, as well as preventing DNA fragmentation and alteration of the cell cycle. The CDT toxin appears to be an important element for the pathogenesis of campylobacteriosis, since all clinical isolates showed the presence of cdtA, cdtB and cdtC genes.

Genotypic characterization of methicillin-resistant Staphylococcus aureus strains isolated from the anterior nares and catheter of ambulatory hemodialysis patients in Mexico.

Methicillin-resistant Staphylococcus aureus (MRSA) is the causal agent of multiple nosocomial infections worldwide, including catheter-associated bacteremia in hemodialysis patients. The purposes of this work were to genetically characterize a group of MRSA isolates from catheter-related infections of ambulatory Mexican hemodialysis patients and to determine whether the strains are the same as those carried by the patients in their anterior nares. Sixteen pairs of MRSA isolates from the catheter (cat) and anterior nares (N) of hemodialysis patients were compared using pulsed-field gel electrophoresis (PFGE), PCR detection of adhesion genes and other virulence markers, and an antibiogram. Three pairs of N/cat MRSA isolates (18.7 %) with identical resistograms also showed the same combination of PCR-detected markers and PFGE pattern; one additional pair showed only an identical electrophoretic PFGE pattern. Of the MRSA isolates, 75 % (n = 24) were resistant to ≥ 7 antibiotics, 4 isolates were resistant to 11 antibiotics, and 7 isolates were resistant to the 12 antibiotics tested. The most frequent virulence marker combination found was spa, clfA, clfB, cna, bbp, ebps, map/eap, sdrC, sdrD, sdrE, ica, agr (65.6 %, n = 21). The SCCmec alleles of the 32 MRSA isolates were IV (n = 20), I (n = 7), II (n = 4), and V (n = 1), and no SCCmec type III MRSA was found. The genotypic characterization of the MRSA isolates studied in this work will contribute to a better understanding of the virulence gene makeup of catheter-colonizing S. aureus strains and will help to lower the infection risk in these patients.

Isolation of community-acquired methicillin-resistant Staphylococcus aureus in healthy carriers in a Mexican community.

OBJECTIVES: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) clones are spreading rapidly among the population in many regions worldwide. Little information is available on CA-MRSA in Mexico. The aim of this study was to identify CA-MRSA strains in the nose and throat of healthy people in a Mexican community. METHODS: A total of 131 MRSA strains from the nose and throat obtained from healthy people in Mexico City were characterized. The genes mecA, lukS-PV/lukF-PV, and ACME-arcA were detected by PCR. Staphylococcal cassette chromosome mec (SCCmec), pulsed-field gel electrophoresis (PFGE), and spa typing were performed. RESULTS: Bacteria that had a Panton-Valentine leukocidin (PVL)-positive gene and SCCmec type IV or V were designated as CA-MRSA strains. We found that 21.4% of MRSA strains were CA-MRSA and that the percentage of CA-MRSA strains was similar in the nose and the throat. A great diversity of profiles was found in the strains identified by PFGE pattern and spa typing. Only one strain similar to the USA300 genotype was found; this strain carried the ACME-arcA gene. CONCLUSIONS: CA-MRSA strains were detected in the nose and throat of healthy people. We identified a high level of genetic diversity among CA-MRSA strains in healthy people of Mexico City, which were different from the USA and pandemic clone profiles.

Characterization and persistence of Staphylococcus aureus strains isolated from the anterior nares and throats of healthy carriers in a Mexican community.

Healthy carriers of Staphylococcus aureus strains have an important role in the dissemination of this bacterium. To investigate the presence of S. aureus in the throat and anterior nares, samples from 1,243 healthy volunteers in a Mexican community were examined. The percentage of healthy carriers was 59.8%. Results showed that colonization of the throat occurred more frequently than that of the nares (46.5% versus 37.1%, P<0.0001). Of the S. aureus carriers, 22.2% were exclusive nasal carriers and 38% were exclusive throat carriers. A total of 1,039 strains were isolated; 12.6% were shown to be methicillin-resistant S. aureus (MRSA). Of MRSA strains, 32.1% were isolated from exclusive throat carriers. Most of the strains isolated from the anterior nares and throat of the same carriers were the same or related; however, some were different. Pulsed-field gel electrophoresis (PFGE) pattern analysis of the MRSA strains isolated from the exclusive nasal carriers or exclusive throat carriers showed that they belong to different clusters. A 6-year prospective study was performed to investigate the persistence of S. aureus in the throat. Results showed that 13% of subjects were persistent carriers. Most of them were colonized with the same clone of S. aureus throughout the time of the study, and just three had different clones. Antimicrobial susceptibility testing showed that 91.1% of the strains were penicillin resistant. The presence of mecA and nucA genes (in order to confirm methicillin resistance) and of thermostable nuclease of S. aureus was examined. This study showed that some strains of S. aureus regularly colonized the throats of healthy people and could persist for years.