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.

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.

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.

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.

[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.

[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.

Mycobacterium chelonae keratitis: resolution after debridement and presoaked collagen shields.

We report a case of Mycobacterium chelonae keratitis following corneal injury by a foreign body. Diagnosis was made by Ziehl-Neelsen staining and Löwenstein-Jensen culture of corneal scrapings. On the basis of the in vitro susceptibility testing, the patient was treated with topical fortified amikacin. Given the lack of response to this therapy, we decided to carry out a debridement of the infiltrative areas to eliminate infected tissue, and to use an amikacin-soaked collagen shield supplemented every 4 h with topical fortified amikacin to promote healing of the debrided area and to potentiate the effects of the antibiotic therapy. After this treatment, clinical resolution was observed and a further acid-fast stain and culture for mycobacterium were negative. Debridement of the infiltrative areas could be used in cases of mycobacterium keratitis when early diagnosis is made and before the corneal infection has become widespread.

[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.

[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.

[Correlation between CD4 lymphocytes and viral load in patients with HIV and tuberculosis]. / Correlación entre linfocitos CD4 y carga viral en pacientes infectados por el virus de la inmunodeficiencia humana con tuberculosis.

OBJECTIVE: A retrospective study of the correlation between CD4 lymphocytes and the viral load in 16 HIV-patients with tuberculosis. MATERIAL AND METHODS: The clinical forms of presentation of tuberculosis were classified according to the location/s of the disease into: pulmonary, distinguishing between typical pulmonary and atypical according to the radiological pattern; extrapulmonary; mixed forms: pulmonary and extrapulmonary; and miliary tuberculosis. RESULTS: Tuberculosis was exclusively pulmonary in 7 cases (44%), all were atypical; extrapulmonary 6 (38%); mixed 2 (12%); and miliary 1 (6%). The mean CD4 lymphocyte count was 111.1 (range 5-360), in 11 (69%) the counts were below 200 cells/mm3. The lowest CD4 count was in the mixed forms with a mean of 45 cells/mm3 whilst the highest was obtained in pulmonary forms with a mean of 128.3 cells/mm3. The mean viral load was 4.82 log (range, 0-5.93), the highest load was for mixed forms with a mean 5.69 log, whereas the lowest load was for pulmonary forms with a mean of 4.19 log. No significant correlation was observed between CD4 lymphocytes and viral load (correlation coefficient--0.1163). CONCLUSIONS: Though no significant correlation was observed, a high CD4 was associated to a low viral load and inversely a low CD4 with a high viral load.

[Epidemiological study of tuberculosis in the health area of Santiago de Compostella in 1992, 1993 and 1994]. / Estudio epidemiológico de la tuberculosis en el área sanitaria de Santiago de Compostela durante 1992, 1993 y 1994.

An exhaustive search for the clinical records of patients diagnosed with tuberculous disease was done in the hospitals of the area under study, which involves 392,000 population. During the years 1992, 1993 and 1994. There were included: 1) patients who had positive bacilloscopy and/or positive Lowenstein's culture in any specimen: 2) patients younger than 35-years-old who had pleural effusion, significant Mantoux and adenosine deaminase (ADA) over 47 U/I in the pleural effusion. In total 814 patients remained in the study with an average age of 38.39(19.39 DE) in 1992, 39.02 (20.04 DE) in 1993, and 34.1 years-old (19.2 DE) in 1994, with extreme ages of 2 months and 87 years-old. The incidence/100,000 H was: in 1992: 67.86, in 1993: 66.58 and in 1994: 73.2. The contagious forms incidence/100,000 H was: 1.5 in 1992 and 1993; and 1.79 in 1994. The hospital mortality incidence/100,000 H was 2.04 in 1992, 2.30 in 1993 and 2.6 in 1994. We conclude that tuberculosis is endemic in our area with moderately high and stationary incidence.

[Tuberculosis and HIV infection. Clinical characteristics and diagnosis]. / Tuberculosis e infección por el virus de la inmunodeficiencia humana. Presentación clínica y diagnóstico.

OBJECTIVE: To analyze the clinical characteristics and diagnose of tuberculosis in patients infected by human immunodeficiency virus in our setting. METHODS: We have revised the clinical, microbiological and histological characteristics of 92 cases diagnosed tuberculosis in seropositive patients, during a span time of 128 months. RESULTS: Of them, 71 were male and most of them (81.5%) were intravenous drugs users. The most (93.4%) were sintomatic when tuberculosis was diagnosed mainly fever and general and respiratory sintomatology. The most common signs were the presence of lymphadenopathies and hepatomegaly. The tuberculosis affected mainly pulmonary and ganglionary system. 34 cases had only pulmonary pathology, 24 extrapulmonary, 25 pulmonary and extrapulmonary and 9 miliary. The tuberculosis diagnose was based in microbiologic criteris. The most frequent source, as histologic critery, has been the ganglionary tissue. CONCLUSIONS: The only or associated pulmonary forms are more frequent. The most commonly extrapulmonary form is the ganglionary localization. The most of diagnose methods were obtained from respiratory tract and lymphadenopathy samples.

[An epidemiological study of tuberculosis in the health area of Santiago de Compostela during the years 1989, 1990 and 1991]. / Estudio epidemiológico de la tuberculosis en el área sanitaria de Santiago de Compostela durante los años 1989, 1990 y 1991.

In order to assess the incidence of tuberculous disease in our health area, we reviewed the clinical records of patients from the three hospitals of the area under study during the years 1989, 1990 and 1991, who had positive bacilloscopy, positive Lowenstein's culture in any specimen and/or compatible anatomopathologic report. After excluding 26 patients because they belonged to other health areas, 885 patients remained in the study, out of which 251 (64% men and 36% women) were from the year 1989, 270 (64% men and 36% women) from the year 1990 and 364 (62% men and 38% women) from the year 1991. The mean age was 38.4 (SD 20.5). Fifty-one percent of the patients were between 20 and 35 years old. The rate of new cases was 65.87 per 100.000 population in 1989, 71.05 in 1990 and 95.53 in 1991. Seventy-four cases were HIV-positive (8%). Tuberculous meningitis was present in 12 patients. The highest mortality was 1.79 per 100.000 population in 1990. We conclude that tuberculosis presents a medium-high incidence in our health area.

[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.

[Resistance of Streptococcus pneumoniae in Galicia]. / Resistencias de Streptococcus pneumoniae en Galicia.

We studied the antibiotic sensitivity of 130 Streptococcus pneumonia strains isolated from respiratory samples (96 cases), blood (13 cases), CSF (6 cases), ocular secretions (5 cases) and other origins (10 cases). High-level penicillin-resistance was found in 13.08% of strains, and intermediate-level penicillin resistance was also found in 22.3% of strains. A multiple-antibiotic resistance pattern was shown by 80.44% of all penicillin-resistant strains. Resistance rates found against antibiotics were as follows: tetracycline, 41.53%; chloramphenicol 17.69%; cotrimoxazole, 16.92% and erythromycin 6.92%. All isolated strains were sensitive to rifampin and vancomycin.

[Usefulness of polymerase chain reaction (PCR) in the diagnosis of meningococcal meningitis]. / Utilidad de la reacción en cadena de la polimerasa (PCR) en el diagnóstico de la meningitis meningocócica.

BACKGROUND: The aim of this work was to evaluate the applicability of polymerase chain reaction (PCR) in the microbiological diagnostic of meningococcal meningitis as compared with the conventional methods (Gram stain and culture). METHODS: One hundred and fifteen cerebrospinalis fluid samples from 115 patients with suspicious symptoms of meningitis were studied. 47 of them belonged to patients suspicious for meningococcal disease; 28 to patients with bacterial meningitis of other infectious etiologies; 10 to patients with meningitis showing lymphocytic pleocytosis and 30 to patients with an unconfirmed meningitis. The cerebrospinalis fluid samples were processed for culture by standard procedures and by PCR according to the method described by Newcombe et al for peripheral blood samples. RESULTS: Thirty five out of 39 patients suspicious of meningococcal meningitis were microbiologically confirmed, being 22 culture and PCR positive, 3 microscopically and PCR positive, 1 only microscopically positive, and 9 positive only by PCR. By using PCR methodology, the number of confirmed diagnostics of meningococcal meningitis increased in a 23% as compared to those obtained by microscopic observation and culture. Sensitivity, specificity, predictive positive value and predictive negative value were 87.1, 98.7, 97.1 and 94.1 respectively for the PCR method. CONCLUSIONS: Our results indicate that PCR can be used on routine basis as a complementary technique to the standard laboratory procedures for diagnosis of meningococcal meningitis.

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.