Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments.

Pathogens that infect the gastrointestinal and respiratory tracts are subjected to intense pressure due to the environmental conditions of the surroundings. This pressure has led to the development of mechanisms of bacterial tolerance or persistence which enable microorganisms to survive in these locations. In this review, we analyze the general stress response (RpoS mediated), reactive oxygen species (ROS) tolerance, energy metabolism, drug efflux pumps, SOS response, quorum sensing (QS) bacterial communication, (p)ppGpp signaling, and toxin-antitoxin (TA) systems of pathogens, such as Escherichia coli, Salmonella spp., Vibrio spp., Helicobacter spp., Campylobacter jejuni, Enterococcus spp., Shigella spp., Yersinia spp., and Clostridium difficile, all of which inhabit the gastrointestinal tract. The following respiratory tract pathogens are also considered: Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cenocepacia, and Mycobacterium tuberculosis Knowledge of the molecular mechanisms regulating the bacterial tolerance and persistence phenotypes is essential in the fight against multiresistant pathogens, as it will enable the identification of new targets for developing innovative anti-infective treatments.

Mapping the thickness changes on retinal layers segmented by spectral-domain optical coherence tomography using the posterior pole program in glaucoma. / Mapeo de los cambios de grosor en el glaucoma de las capas retinianas maculares segmentadas usando el programa de polo posterior de la tomografía de coherencia óptica de dominio espectral.

OBJECTIVES: To evaluate changes in retinal layers of the macula (mRLs) using OCT posterior pole program (PPP) in primary open-angle glaucoma (POAG). MATERIAL AND METHODS: The study included 128 patients with POAG and 103 healthy controls who had PPP maps (macular grid 8×8) drawn by SD-OCT. Only one eye per patient was studied. The 9 mRLs were automatically segmented by prototype software, obtaining: a macular retinal nerve fibre layer (mRNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform+nuclear layer, photoreceptor layer, retinal pigment epithelium (RPE), outer retina and RPE+outer retina. Thickness values were obtained on 64 cells of the grid for each mRL, and mean thickness of superior and inferior hemispheres were calculated. Comparisons of mean thickness of these hemispheres and thickness of each cell between groups were determined. Differences in the cell by cell comparisons were represented quantitatively by heat maps for each mRL. RESULTS: Photoreceptors and RPE were found in POAG group when comparing thickness of hemispheres, thinning of mRNFL, GCL, IPL, and thickening of INL. Heat maps showed symmetrical thinning patters between superior and inferior hemispheres in inner retinal layers (except for INL) and asymmetrical thickening patters in outer retinal layers in GPAA group. CONCLUSIONS: There are thickness changes in all mRLs in POAG, when studied by PPP. Thinning of inner layers (except for INL), and thickening of outer layers in POAG show different symmetry patterns in relation to horizontal meridian.

Sperm chemorepulsion, a supplementary mechanism to regulate fertilization.

STUDY QUESTION: Are human spermatozoa able of chemorepulsive behaviour? SUMMARY ANSWER: Capacitated human spermatozoa are able to be chemorepelled by synthetic Progesterone Receptor Ligands (sPRL, known as contraceptives) and zinc (a cation released by the oocyte upon fertilization). WHAT IS KNOWN ALREADY: Moving cells can be oriented towards or against a molecular gradient, processes called chemoattraction and chemorepulsion, respectively, which have been described in unicellular organisms such as amoebas and bacteria, to organismic cells such macrophages and developmental cells. In the case of spermatozoa, chemoattraction may help the finding of an oocyte and has been widely studied in various invertebrate and mammalian species; however, chemorepulsion has not yet been verified in spermatozoa. STUDY DESIGN, SIZE, DURATION: This is an in vitro study involving human, rabbit and mouse spermatozoa which were used to perform 3-30 experiments per treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were obtained by masturbation from healthy donors who gave written consent. Only those samples exhibiting normal semen parameters according to current WHO criteria were included in the study. Rabbit spermatozoa were obtained by artificial vagina whereas mice spermatozoa were obtained from epididymis. The sperm selection assay (SSA), originally designed to evaluate sperm chemoattraction towards progesterone (P), and a video-microscopy and computer motion analysis system were used to test sperm chemorepulsion. Additional kinetic parameters were also determined by video-microscopy and computer motion analysis. In some experiments, the level of induced acrosome-reacted spermatozoa was determined. Rabbit mating manipulation was achieved to perform the sperm-oocyte co-incubation assay. MAIN RESULTS AND THE ROLE OF CHANCE: Sperm accumulation in the well containing 100 pg/ml of sPRL was lower than the culture medium negative control (P < 0.05). The percentage of sperm persistence against the well containing 100 pg/ml ulipristal acetate (UPA) (P = 0.001), and the percentage of sperm showing a repulsive pattern of movement (a linear trajectory followed by a transitional one after turning against the UPA), were higher than the culture medium negative control (P = 0.049). Sperm accumulation was diminished when spermatozoa where exposed to a homogeneous distribution of 100 pg/ml sPRL combined with a chemotactic gradient of progesterone (P), with respect to the culture medium negative control (P < 0.05). These results were reverted when non-capacitated spermatozoa were used to perform the same experimental settings. The accumulation of spermatozoa against 100 pg/ml sPRL was lower than the culture medium negative control also in rabbits and mice (P < 0.05). The relative number of rabbit spermatozoa arriving to the vicinity of the oocyte was diminished under the presence of 100 pg/ml UPA (P = 0.004). Sperm accumulation in the well containing zinc was decreased compared to the culture medium negative control (P < 0.05). A homogeneous distribution of zinc combined with a gradient of 10 pM P, was lower than the culture medium negative control (P = 0.016). The results were quite reproducible with two different methodologies (accumulation assay and video-microscopy combined with computer motion analysis), in three mammalian species. LIMITATIONS REASONS FOR CAUTION: The experiments were performed in vitro. Even though a quite complete characterization of sperm chemorepulsion was provided, the molecular mechanism that governs sperm repulsion is currently under investigation. WIDER IMPLICATIONS OF THE FINDINGS: Since the chemorepelled spermatozoa are those physiologically ready to fertilize the oocyte, these findings may have both biological and clinical implications, preventing either polyspermy under natural conditions or fertilization under pharmacological treatment with sPRL. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by the Universidad Nacional de Cordoba (Argentina). The authors declare that they do not have competing financial interests. TRIAL REGISTRATION NUMBER: N/A.

[Pulmonary co-infection due to Nocardia cyriacigeorgica and Aspergillus fumigatus]. / Coinfección pulmonar por Nocardia cyriacigeorgica y Aspergillus fumigatuss.

OBJECTIVE: Pulmonary nocardiosis is an uncommon pulmonary infection caused by aerobic gram-positive bacteria of the genus Nocardia. Nocardia sp. are environmental organisms spread worldwide. Approximately 50 Nocardia species have been described to date, about 30 of which are known to cause human disease. Nocardia cyriacigeorgica was first reported in 2001. CASE REPORT: We report a case of infection caused by N. cyriacigeorgica in a patient with B-cells non-Hodgkin lymphoma and diabetes mellitus. The microbiological findings reflect a possible co-infection by N. cyriacigeorgica and Aspergillus fumigatus. CONCLUSIONS: Patient's background and information related to risk factors are essential to detect the growth of Nocardia sp. in the laboratory. Furthermore, diagnosis of invasive pulmonary aspergillosis is particularly controversial, especially in intensive care units patients. Taking everything into account, we will discuss a possible co-infection by N. cyriacigeorgica and A. fumigatus in a critically ill patient.

The genotypic study of Mycobacterium tuberculosis complex resistant to isoniazid: Galicia, Spain (2008-2013).

Incorporation of rapid detection systems to identify mutations in M. tuberculosis complex that confer resistance to isoniazid and rifampicin has potentiated the knowledge of their distribution, given the geographical variability. We performed antibiograms of the 2,993 strains isolated in Galicia, Spain (2008-2013). In the strains resistant to isoniazid, a concentration of 0.4 mg/mL and MTBDRplus Genotype test (Hain Lifescience, Germany) were used. We found that 3.64 % of strains were resistant to isoniazid, while 0.43 % were resistant to isoniazid and rifampicin (multidrug resistant, MDR). The MTBDRplus test showed an overall sensitivity of 72.48 %, with 62.5 % sensitivity for non MDR isoniazid-resistant strains and 100 % sensitivity for MDR strains. The katG gene mutation was detected at codon 315 in 38.53 % of strains. The S315T mutation appeared in 61.54 % of MDR strains and 34.38 % of non-MDR strains. The 28.44 % had mutations in inhA, (93.55 % in C15T), and 38.46 % of MDR strains were mutated. In non-MDR strains, 37.50 % were wild-type, 35.42 % and 27.08 % had mutations in katG and inhA, respectively. The most frequent mutation in rpoß was S531L (46.15 %). The 38.71 % and 41.9 % of strains with resistance to isoniazid and streptomycin had mutations in katG and inhA, respectively (2 strains with mutations in T8C and T8A). The distribution pattern of resistance among strains with high and low concentrations of isoniazid showed statistically significant differences in relation to the mutation in katG and wild-type. The sensitivity of the Genotype MTBDRplus test for non-MDR strains in our area was at the lower threshold described.

[Prevalence and distribution of hepatitis B virus genotype D in Galicia (northwest of Spain): influence of age, sex and origin]. / Prevalencia y distribución del genotipo D del virus de la hepatitis B en Galicia (noroeste de España): influencia de la edad, el sexo y la procedencia.

OBJECTIVE: Phylogenetically, hepatitis B virus (HBV) is classified into genotypes and subgenotypes used for epidemiological studies. The aim of this study is to know the distribution of HBV subgenotypes D in our environment. METHODS: From 401 patients HBV surface antigen positive, HBV DNA-positive, partial HBV-DNA S gene was amplified, sequenced and analysed using geno2pheno (hbv) (Max-Planck Institute) on line application. RESULTS: We found 259 (64.6%) patients with HBV genotype D: 53 not subgenotypable, 9 (4%) D1, 61 (30%) D2, 15 (7%) D3 and 121 (59%) D4. Patients with D1 subgenotype were, on average, 23 years younger (p = 0.0001), with a higher proportion of women (p < 0.05). CONCLUSIONS: HBV subgenotype D4 was the most prevalent in our area. Patients with D1 subgenotype came from abroad were younger than the other subgenotypes and mostly women. These results show the interest of conducting studies at HBV subgenotype level.

Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study.

OBJECTIVES: Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. METHODS: The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. RESULTS: A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. CONCLUSIONS: The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care.

Evaluation of two commercial nucleic acid amplification kits for detecting Mycobacterium tuberculosis in saliva samples.

OBJECTIVE: The objective of this study was to evaluate the efficacy of two commercial nucleic acid amplification techniques to identify Mycobacterium tuberculosis in saliva. SUBJECTS AND METHODS: Fifty-two participants were recruited, 32 patients with a clinical and microbiological diagnosis of pulmonary tuberculosis and 20 healthy volunteers as controls. Three sputum samples were collected from each participant and were examined by direct bacilloscopy, cultured in liquid and solid media, and processed using the Mycobacterium tuberculosis direct (MTD) test for rRNA detection. One saliva sample was collected from each participant using conventional methods and was examined by direct bacilloscopy, cultured, and processed using the MTD test for rRNA detection and the FluoroType Mycobacterium tuberculosis assay for DNA detection. RESULTS: In saliva samples, the sensitivity, specificity, and positive and negative predictive values of the MTD test were 71.8%, 95%, 95.8%, and 67.8%, respectively. The values obtained with the FluoroType assay were 56.2%, 90%, 90%, and 56.2%, respectively. CONCLUSIONS: Our results indicate that when a sufficient volume of sputum cannot be obtained, saliva could be an alternative biological sample for the rapid diagnosis of pulmonary tuberculosis using commercial nucleic acid amplification techniques.

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF.

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.

Multidrug-resistant tuberculosis in a low-incidence region shows a high rate of transmission.

OBJECTIVE: To describe the characteristics of patients with multidrug-resistant tuberculosis (MDR-TB), a descriptive prospective study was carried out applying a combination of exhaustive conventional epidemiology with molecular genotyping. SETTING: All patients diagnosed with MDR-TB in Galicia, Spain, between 1998 and 2004 were included in the study. DESIGN: Of 9895 diagnosed cases of TB, 58 were MDR-TB (0.59%). The site of disease was pulmonary in 56 cases and 46 were smear-positive. Only two cases were co-infected with the human immunodeficiency virus (HIV) and seven were immigrants. Twenty-five (43%) had received previous TB treatment. These cases presented more risk factors for treatment default and a lower frequency of contact with cases of MDR-TB. RESULTS: Genotyping analysis was performed in 57 patients, showing evidence of four clusters (30 patients, 52.6%), each with identical genetic patterns. The patients included in the clusters were younger, and most had primary forms or had had contact with another case of MDR-TB, especially in hospital. Neither the Beijing/W nor the B strain was identified. CONCLUSION: There is a low prevalence of MDR-TB in Galicia. Unlike previous studies, there was a high rate of transmissibility, including nosocomial transmission. Transmission is not associated with HIV or previously reported strains with a high capacity for transmission.

Use of ligase chain reaction for the rapid diagnosis of lymph node tuberculosis.

Ligase chain reaction amplification (LCx Abbott Laboratories) was used to detect the presence of M. tuberculosis in 101 adenopathy specimens obtained from 98 patients. A total of 30 cases of lymph node tuberculosis were diagnosed, and the data were compared with results obtained using conventional techniques. The sensitivity of auramine staining and culture were 50.0% and 66.7%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value of the LCx assay were 80.0, 98.6, 96.0, and 92.1% respectively. The results confirm the reliability of ligase chain reaction amplification for the detection of lymph node tuberculosis.

[Tuberculosis in Santiago de Compostela from 1999 to 2002. An epidemiological study]. / Tuberculosis en el área de Santiago de Compostela durante los años 1999, 2000, 2001 y 2002. Un estudio epidemiológico.

OBJECTIVE: The purpose of this study was to determine the epidemiological characteristics of tuberculosis (TB) in the Public Health System District of Santiago de Compostela (population : 386125) from 1999 to 2002. METHODS: Inclusion criteria were: 1). microbiological and/or pathological diagnosis of TB in any specimen, and 2). patient younger 35 years old with recent medical history of TB. Mantoux test positive, and pleural effusion with linfocitosis and adenosine deaminase >47 IU/ml. RESULTS: 946 patients were included (568 men and 378 women), with ages ranging from 2 months to 96 years. The incidence of TB was 60.9/100000 in 1999, 67.6/100000 in 2000, 61.9/100000 in 2001 and 54.6/100000 in 2002. The incidence rate of tuberculous meningitis was 1.03/100000 in 1999 and 2000, 0.77/100000 in 2001 and 0.51/100000 in 2002. The percentage of cases associated with HIV was 3.4% in 1999, 1.9% in 2000, 2.4% in 2001 and 2002. We found an increase in the rate of males over 55 years of age; with incidence per 100000 inhabitants of 122.4 in 1999, 142.8 in 2000, 115 in 2001 and 119 in 2002, whereas in females the incidence was 40.6 in 1999, 60.9 in 2000, 54.1 in 2001 and 39.1 in 2002. CONCLUSIONS: In last four years the incidence of tuberculosis has decreased but remains high in males over 55 years old.

[Resistant tuberculosis by drugs]. / Tuberculosis resistente a fármacos.

Among all infectious diseases, tuberculosis has probably been the most frequent cause of death and morbidity in the history of humanity. On a world-wide basis, the tuberculosis (TB) represents a serious public health problem, existing great differences between developed and developing countries in terms of controlling the disease. One of the problems inherent in this disease is the resistance of Mycobacterium tuberculosis to drugs, specially the multi-resistance (defined as resistance to isoniziade and rifampicine), it represents a problem of global health. Faced with clinical suspect, its diagnosis must be immediately done and treatment must be started as soon as possible. For this purpose, we have at our disposal such effective diagnosis and therapeutic methods. However, the most important the most important action is that all these patients must be subjected to a close evolved control carried by specialized units.

[Tuberculosis in elderly patients. Presentation forms]. / Tuberculosis en pacientes ancianos. Formas de presentación.

OBJECTIVE: To analyse the distribution of the forms of presentation of tuberculosis (TPF) in elderly patients. MATERIAL AND METHODS: The medical records of patients diagnosed with tuberculosis attending the Tuberculosis Prevention and Control Unit of the Santiago Health District were reviewed over of six years period. The classification of TPF was: pulmonary forms (P), disease confined to the lung; extrapulmonary forms (EF), disease outside the lung; mixed forms (MF), the presence of both pulmonary and extrapulmonary tuberculosis; disseminated forms (DF), the presence of two or more extrapulmonary locations; and miliary TB, which was defined by a diffuse pulmonary radiographic pattern or diagnosis was undertaken by necropsy. RESULTS: A total of 278 tuberculosis infected patients were observed, 156 (56.2%) were men and 122 (43.8%) women, their mean age was 75.3 years (range 65-95). The distribution of TPF was: 155 (55.8%) P forms; 66 (23.7%) EF, of which 27 (41.0%) were ganglionary location, 12 (18.2%) bone and joint, 8 (12.0%) intestinal, 6 (9.1%) peritoneal, 5 (7.6%) meningeal, and other locations 8 (12.1%); MF 47 cases (16.9%); miliary TB 7 cases (2.5%) and. DF 3 cases (1.1%). None case was observed of HIV infected patient. CONCLUSIONS: Our findings confirm high incidence of extrapulmonary TB in elderly patients. Our experience shows a modification to the classical presentation of the disease, and thus the need for sensitivity in locating the disease.

[Detection of the cutaneous melanomas, their metastasis and relapses by scintigraphies with 99MTC-HMPAO]. / Detección de melanomas cutáneos, sus metástasis y recidivas mediante gammagrafías con 99mTc-HMPAO.

A pilot study was carried out in the Nuclear Medicine Department of the National Institute of Oncology and Radiobiology to learn the diagnostic efficacy of the 99mTc-HMPAO scintigraphy in the detection of cutaneous melanoma, distant and local metastases, in patients with a confirmed histological diagnosis. The planar and/or SPECT scintigraphy was performed in 17 patients and revealed 90% sensitivity for the detection of the disease. We calculated the tumor/background index in all the images obtained and found a value greater than 1 in every case. Thus, we can affirm that the 99mTc-HMPAO accumulation in tumors was higher than the background in all cases. We recommend that this procedure be considered in the follow up of these patients as a new diagnostic method which is very useful in the determination of the patient stage and prognostic evaluation.