Mycobacterium marinum lymphocutaneous infection.

Mycobacterium marinum is a nontuberculous mycobacteria with worldwide distribution that lives in fresh or salt water and is responsible for infections in fish, and sometimes in humans. Human disease consists mainly of cutaneous nodules, but deep structure involvement may also occur. Diagnosis of M. marinum infection remains a challenge, with a considerable time delay between onset of symptoms and diagnosis. We present a 33-year-old man with no immunosuppressive history who was seen in our department with skin nodules over his hand and forearm, distributed in a sporotrichoid pattern. His hobbies included maintaining an aquarium of tropical fish. Histological examination of the patient's skin biopsy was compatible with the diagnosis of mycobacterial infection, and the Ziehl-Neelsen staining revealed acid-fast bacilli. Molecular techniques confirmed the suspicion of M. marinum infection. A necropsy was performed on one of the patient's fish, more specifically, a Poecilia reticulata, and resulted in identification of M. marinum from its gut. The patient was treated with clarithromycin, ethambutol, and rifampicin for 9 months, with clearance of infection.

Participatory implementation of an antibiotic stewardship programme supported by an innovative surveillance and clinical decision-support system.

BACKGROUND: Antibiotic resistance will cause about 10 million deaths per year by 2050. Fighting antimicrobial resistance is a health priority. Interventions aimed to reduce antimicrobial resistance, such as antibiotic stewardship programmes (ASPs), must be implemented. To be effective, those interventions, and the implementation process, should be matched with social-cultural context. The complexity of ASPs can no longer be developed without considering both organizational and information systems. AIM: To support ASPs through the co-design and implementation, in collaboration with healthcare workers, of a surveillance and clinical decision-support system to monitor antibiotic resistance and improve antibiotic prescription. METHODS: The surveillance and clinical decision-support system was designed and implemented in three Portuguese hospitals, using a participatory approach between researchers and healthcare workers following the Design Science Research Methodology. FINDINGS: Based on healthcare workers' requirements, we developed HAITooL, a real-time surveillance and clinical decision-support system that integrates visualizations of patient, microbiology, and pharmacy data, facilitating clinical decision. HAITooL monitors antibiotic usage and rates of antibiotic-resistant bacteria, allowing early identification of outbreaks. It is a clinical decision-support tool that integrates evidence-based algorithms to support proper antibiotic prescription. HAITooL was considered valuable to support monitoring of antibiotic resistant infections and an important tool for ASP sustainability. CONCLUSION: ASP implementation can be leveraged through a surveillance and clinical decision-support system such as HAITooL that allows antibiotic resistance monitoring and supports antibiotic prescription, once it has been adapted to the context and specific needs of healthcare workers and hospitals.

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.

Time of Dropout From the Liver Transplant List in Patients With Hepatocellular Carcinoma: Clinical Behavior According to Tumor Characteristics and Severity of Liver Disease.

BACKGROUND: Prolonged time on the waiting list affects post-transplant survival of patients with hepatocellular carcinoma (HCC). However, it is not yet known which patients will be at higher risk for early dropout from the list. We investigate specific risk factors for early waiting list dropout in patients with HCC. METHODS: This was a single-center, intention-to-treat analysis of adults with HCC, within the Milan criteria, from July 2006 through September 2013. Patients were divided into groups according to waiting list time. The main end point was dropout from the list. RESULTS: The dropout rates of the study cohort at 3, 6, and 12-months were 6.4%, 12.4%, and 17.7%, respectively. Patients who dropped out from the list tended to be older, with blood types A and O, and with higher Child-Pugh and Model for End-Stage Liver Disease (MELD) scores. They also had larger nodules, responded poorly to trans-arterial chemo-embolization (TACE), and had a higher alpha-fetoprotein. Those with blood types B and AB appeared to be protected for dropout (odds ratio [OR] = 0.21, P = .02). Patients who responded to TACE were also protected (OR = 0.22, P < .001). When we looked into time to dropout, the only baseline characteristic that stood out was a higher MELD score (13 for those dropping out up to 90 days vs 10 for those dropping out after 180 days, P = .0025). CONCLUSIONS: We conclude that patients who drop out early from the list are primarily driven by the severity of liver disease. Patients who had progressive HCC had a high tumor load and poor response to loco-regional therapies, dropping out from the list after 180 days of inclusion.

Screening for fragile X syndrome in males from specialized institutions in the northeast region of Brazil.

The objective of this study was to perform a study of fragile X syndrome (FXS) in São Luís, Maranhão, in males residing in five specialized institutions. Two hundred thirty-eight males with intel-lectual disability of unknown etiology participated in this study. Blood samples were processed and stored until DNA extraction. Screening for FMR1 gene mutations was performed using non-isotopic polymerase chain reaction amplification and DNA sequencing using an ABI Prism 3130 automated sequencer. Two individuals (0.84%) were positive for FMR1 mutations. One had a mutation due to expansion of the CGG repeat beyond normal levels and the other had a deletion in exon 1 of the FMR1 gene, which was confirmed by sequencing. Both probands were over 18 years old, which demonstrates the late diagnosis of the condition in these individuals and reinforces the need to implement ef-fective programs for early diagnosis of FXS in the state of Maranhão. We found that FXS might be transmitted in the families of the two indi-viduals bearing the mutation, and that it is important to understand the mutation dynamics to provide better counseling to the family members of these two individuals.

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.

GidB mutation as a phylogenetic marker for Q1 cluster Mycobacterium tuberculosis isolates and intermediate-level streptomycin resistance determinant in Lisbon, Portugal.

Development of streptomycin resistance in Mycobacterium tuberculosis is usually associated with mutations in rpsL and rrs genes, although up to 50% of clinical streptomycin-resistant isolates may present no mutation in either of these genes. In the present report we investigate the role of gidB gene mutations in streptomycin resistance. We have analyzed 52 streptomycin-resistant and 30 streptomycin-susceptible Mycobacterium tuberculosis clinical isolates by sequencing and endonuclease analysis of the gidB and rpsL genes. All clinical isolates were genotyped by 12-loci MIRU-VNTR. The gidB gene of 18 streptomycin-resistant isolates was sequenced and four missense mutations were found: F12L (1/18), L16R (18/18), A80P (4/18) and S100F (18/18). The remaining isolates were screened by endonuclease analysis for mutations A80P in the gidB gene and K43R in the rpsL gene. Overall, mutation A80P in the gidB gene was found in eight streptomycin-resistant isolates and 11 streptomycin-susceptible multidrug-resistant isolates. Also noteworthy, is the fact that gidB mutations were only present in isolates without rpsL and rrs mutations, all from genetic cluster Q1. Streptomycin quantitative drug susceptibility testing showed that isolates carrying the gidB A80P mutation were streptomycin intermediate-level resistant and that standard drug susceptibility testing yielded inconsistent results, probably due to borderline resistance. We conclude that gidB mutations may explain the high number of streptomycin-resistant strains with no mutation in rpsL or rrs. These mutations might occasionally confer low-level streptomycin resistance that will go undetected in standard susceptibility testing.

Antibacterial properties of compounds isolated from Carpobrotus edulis.

Several compounds isolated from the plant Carpobrotus edulis were evaluated for their activity against multidrug-resistant (MDR) bacteria and their efflux pump systems. Amongst the compounds isolated, six compounds were tested, namely uvaol, ß-amyrin, oleanolic acid, catechin, epicatechin and monogalactosyldiacylglycerol. Oleanolic acid presented high antibacterial activity against a large number of bacterial strains. The triterpene uvaol was the most active compound for modulation of efflux activity by MDR Gram-positive strains.

Non-antibiotics reverse resistance of bacteria to antibiotics.

BACKGROUND: Most clinical isolates that exhibit a multi-drug resistant phenotype owe that resistance to over-expressed efflux pumps. Compounds that are efflux pump inhibitors (EPIs) reduce or reverse resistance to antibiotics to which the bacterial strain is initially resistant. We have evaluated non-antibiotics to reduce resistance of commonly encountered bacterial pathogens to antibiotics. MATERIALS AND METHODS: The effect of non-antibiotics on the susceptibility of bacteria to antibiotics was conducted by minimum inhibition concentration determinations of the antibiotic in the absence and presence of the non-antibiotic. RESULTS: Non-antibiotics such as chlorpromazine, amitryptiline and trans-chlorprothixene are shown to reduce or reverse resistance of a variety of bacteria to antibiotics. CONCLUSION: The results suggest that non-antibiotics may serve as adjuncts to conventional antibiotics for the therapy of problematic antibiotic infections caused by bacteria that owe their resistance to over-expressed efflux pumps.

Identification of the plasmid-encoded qacA efflux pump gene in meticillin-resistant Staphylococcus aureus (MRSA) strain HPV107, a representative of the MRSA Iberian clone.

Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial bacterium for which prevention and control measures consist mainly of the application of biocides with antiseptic and disinfectant activity. In this study, we demonstrated the presence of the plasmid-located efflux pump gene qacA in MRSA strain HPV107, a clinical isolate representative of the MRSA Iberian clone. The existence of efflux activity in strain HPV107 due to the QacA pump was also established and this QacA efflux activity was linked with a phenotype of reduced susceptibility towards several biocide compounds. No association could be made with antibiotic resistance. This work emphasises the potential of QacA pump activity in the maintenance and dissemination of important MRSA strains in the hospital setting and, increasingly, in the community.

Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

Constituents of Carpobrotus edulis inhibit P-glycoprotein of MDR1-transfected mouse lymphoma cells.

A bioassay-guided separation protocol, including the testing of the extracts, fractions and pure compounds for their ability to inhibit P-glycoprotein (the efflux pump responsible for the multidrug resistance of the used cell line) of mouse lymphoma cells containing the human efflux pump gene MDR1, led to the isolation of seven compounds from the chloroform and ethyl acetate soluble fractions of the methanolic extract of Carpobrotus edulis. The compounds were identified by 1D, 2D NMR and MS investigations as triterpens (beta-amyrin, uvaol and oleanolic acid), monogalactosyldiacylglycerol, catechin, epicatechin and procyanidin B5. Uvaol was the most effective and promising compound in the reversal of multidrug resistance in MDR mouse lymphoma cell line.

Identification of nontuberculous mycobacteria in clinical samples using molecular methods: a 3-year study.

Nontuberculous mycobacteria (NTM) are being increasingly isolated in clinical laboratories and present technical and therapeutic challenges. In the present study, we report our experience with the identification of NTM received from 12 Lisbon hospitals over a 3-year period using GenoType Mycobacterium (CM/AS) assays (HAIN Lifescience GmbH, Nehren, Germany). Together, the two kits identified 96.6% of all NTM isolates tested. Among the 18 NTM species identified, Mycobacterium avium complex was the most frequent, although it accounted for only 34% of all NTM. Introducing these methods for the rapid identification of NTM highlights the importance of NTM as potential pathogens and assisted the selection of adequate therapy.

An AcrAB-mediated multidrug-resistant phenotype is maintained following restoration of wild-type activities by efflux pump genes and their regulators.

In this study, we aimed to answer the following question: 'How does a bacterium become so resistant to a given antibiotic even though the levels of antibiotic to which it has become resistant remained constant in the patient?'Escherichia coli AG100 strain induced to high-level resistance due to overexpression of an AcrAB efflux pump was serially cultured in 10mg/L tetracycline for 60 passages. Between each passage it became increasingly resistant to tetracycline, beta-lactams and quinolones with concomitant restoration of wild-type AcrAB activity. Because the multidrug-resistant phenotype could not be reversed with transfer to drug-free medium or with efflux pump inhibitors, it may have resulted from activation of a 'mutator gene' system that reduced the 'energy consumption' associated with an overexpressed efflux pump system.

Targeting human macrophages for enhanced killing of intracellular XDR-TB and MDR-TB.

Although many compounds have been described to inhibit the replication of drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis, most of these studies only evaluate their in vitro activity. There is a lack of studies that show whether any of these agents can kill these organisms at the site where they normally reside post infection, namely, the macrophage of the lung parenchyma. It is the aim of this mini-review to identify agents that have been shown to enhance the killing of intracellular drug-susceptible, multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains by non-killing macrophages. Because these agents appear to promote their activity by affecting the transport of K(+) and Ca(2+) from the phagolysosome containing the bacteria, and thereby promoting its acidification and activation of hydrolases that will eventually kill the organism, the authors suggest that compounds that are known to affect the transport of K(+) and Ca(2+) should be considered for possible activity against intracellular MDR- and XDR-TB.

New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps.

We have developed a number of methods that identify efflux pump mediated multi-drug resistant bacteria, characterize efflux systems and screen for inhibitors of efflux pumps. These approaches were complemented by the quantification of the expression of genes that regulate and code for constituents of efflux pumps. The methods described are easy to use, reproducible and for the most part, require instrumentation normally present in a clinical bacteriology laboratory. Because each method provides good reproducibility, they lend themselves for inter-laboratory use.

Promising therapy of XDR-TB/MDR-TB with thioridazine an inhibitor of bacterial efflux pumps.

Global rates of pulmonary tuberculosis (TB) continue to increase. Moreover, resistance of the causative organism Mycobacterium tuberculosis to the two most effective anti-TB medications continue to rise. Now, multi-drug resistant TB (MDR-TB) has progressed to extensively drug resistant TB (XDR-TB) - a M. tuberculosis organism that is resistant to the most effective second line drugs available for the treatment of TB. This review provides detailed, significant evidence that supports the use of an old neuroleptic compound, thioridazine (TZ), for the management of MDR-TB and XDR-TB infections and which has been shown to inhibit efflux pumps of bacteria. The argument has been previously presented but no one seems to be listening - and the disease continues unabated when there is a very good probability that the suggested drug will prove to be effective. When the prognosis is poor, available therapy predictably ineffective and death is inevitable, compassionate therapy with TZ should be contemplated. The risks are small and the rewards great.