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Showing papers in "The Open Microbiology Journal in 2013"


Journal ArticleDOI
TL;DR: The current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections is reviewed and particular emphasis is given to the potential role that S. a Aureus MDRefflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains.
Abstract: The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

288 citations


Journal ArticleDOI
TL;DR: Advances in the path of EPI discovery are summarized, potential avenues of E PI implementation and development are discussed, and the need for highly informative and comprehensive translational approaches is underlined.
Abstract: Conventional antimicrobials are increasingly ineffective due to the emergence of multidrug-resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered exploration for novel and unconventional approaches to controlling microbial infections. Multidrug efflux systems (MES) have been a profound obstacle in the successful deployment of antimicrobials. The discovery of small molecule efflux system blockers has been an active and rapidly expanding research discipline. A major theme in this platform involves efflux pump inhibitors (EPIs) from natural sources. The discovery methodologies and the available number of natural EPI-chemotypes are increasing. Advances in our understanding of microbial physiology have shed light on a series of pathways and phenotypes where the role of efflux systems is pivotal. Complementing existing antimicrobial discovery platforms such as photodynamic therapy (PDT) with efflux inhibition is a subject under investigation. This core information is a stepping stone in the challenge of highlighting an effective drug development path for EPIs since the puzzle of clinical implementation remains unsolved. This review summarizes advances in the path of EPI discovery, discusses potential avenues of EPI implementation and development, and underlines the need for highly informative and comprehensive translational approaches.

134 citations


Journal ArticleDOI
TL;DR: This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account.
Abstract: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bac- teria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed.

113 citations


Journal ArticleDOI
TL;DR: The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.
Abstract: It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rap- idly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bro- mide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, in- strument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bac- terial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resis- tant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram- positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determina- tion of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

88 citations


Journal ArticleDOI
TL;DR: The transport of antibiotics through the OmpF/C general porins and the TolC-like channels is reviewed with regards to recent data on their structure, function, assembly, regulation and contribution to bacterial resistance.
Abstract: Antibiotic translocation across membranes of Gram-negative bacteria is a key step for the activity on their spe- cific intracellular targets. Resistant bacteria control their membrane permeability as a first line of defense to protect them- selves against external toxic compounds such as antibiotics and biocides. On one hand, resistance to small hydrophilic an- tibiotics such as s-lactams and fluoroquinolones frequently results from the « closing » of their way in: the general outer membrane porins. On the other hand, an effective way out for a wide range of antibiotics is provided by TolC-like pro- teins, which are outer membrane components of multidrug efflux pumps. Accordingly, altered membrane permeability, including porin modifications and/or efflux pumps' overexpression, is always associated to multidrug resistance (MDR) in a number of clinical isolates. Several recent studies have highlighted our current understanding of porins/TolC structures and functions in Enterobacte- riaceae. Here, we review the transport of antibiotics through the OmpF/C general porins and the TolC-like channels with regards to recent data on their structure, function, assembly, regulation and contribution to bacterial resistance. Because MDR strains have evolved global strategies to identify and fight our antibiotic arsenal, it is important to con- stantly update our global knowledge on antibiotic transport.

84 citations


Journal ArticleDOI
TL;DR: A new perspective is provided on the possible ways by which resistance is acquired by the bacterial strains within the patient, with a special emphasis on the adaptive response of the infecting bacteria to the administered antibiotic.
Abstract: Acquisition of resistance is one of the major causes of failure in therapy of bacterial infections. According to the World Health Organization (WHO), thousands of deaths caused by Salmonella sp., Escherichia coli, Staphylococcus aureus or Mycobacteria tuberculosis are due to failure in therapy caused by resistance to the chemotherapeutic agents. Understanding the mechanisms of resistance acquisition by the bacterial strains is therefore essential to prevent and over- come resistance. However, it is very difficult to extrapolate from in vitro studies, where the variables are far less and un- der constant control, as compared to what happens in vivo where the chosen chemotherapeutic, its effective dose, and the patient's immune system are variables that differ substantially case-by-case. The aim of this review is to provide a new perspective on the possible ways by which resistance is acquired by the bacterial strains within the patient, with a special emphasis on the adaptive response of the infecting bacteria to the administered antibiotic.

33 citations


Journal ArticleDOI
TL;DR: The findings of this study increase the understanding of the diversity of E. coli in the environment which will ultimately help in the assessment of this organism and its role in public health.
Abstract: A common member of the intestinal microbiota in humans and animals is Escherichia coli. Based on the pres- ence of virulence factors, E. coli can be potentially pathogenic. The focus of this study was to isolate E. coli from un- treated surface waters (37 sites) in Illinois and Missouri and determine phenotypic and genotypic diversity among isolates. Water samples positive for fecal coliforms based on the Colisure ® test were streaked directly onto Eosin Methylene Blue (EMB) agar (37°C) or transferred to EC broth (44.5°C). EC broth cultures producing gas were then streaked onto EMB agar. Forty-five isolates were identified as E. coli using API 20E and Enterotube II identification systems, and some phe- notypic variation was observed in metabolism and fermentation. Antibiotic susceptibility of each isolate was also deter- mined using the Kirby-Bauer Method. Differential responses to 10 antimicrobial agents were seen with 7, 16, 2, and 9 of the isolates resistant to ampicillin, cephalothin, tetracycline, and triple sulfonamide, respectively. All of the isolates were susceptible or intermediate to amoxicillin, ciprofloxacin, polymyxin B, gentamicin, imipenem, and nalidixic acid. Geno- typic variation was assessed through multiplex Polymerase Chain Reaction for four virulence genes (stx1 and stx2 (shiga toxin), eaeA (intimin); and hlyA (enterohemolysin)) and one housekeeping gene (uidA (-D-glucuronidase)). Genotypic variation was observed with two of the isolates possessing the virulence gene (eaeA) for intimin. These findings increase our understanding of the diversity of E. coli in the environment which will ultimately help in the assessment of this organ- ism and its role in public health.

32 citations


Journal ArticleDOI
TL;DR: By forming their own reference database and using alternative analysis methods, this work could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli and even succeeded to distinguish Shigella sonnei from Escherichia coli and Salmonella enterica spp.
Abstract: Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli.

29 citations


Journal ArticleDOI
TL;DR: Genetic analysis using ERIC-PCR showed that closely related clones are responsible for the occurrence of extended-spectrum cephalosporins resistant Salmonella infection in Tehran.
Abstract: Background and Objectives: Salmonella is an important food-borne pathogen responsible for disease in humans and animals. The aim of this study was to investigate the genetic relationship among third generation cephalosporin- resistant Salmonella enterica strains by Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR. Methods: The study included all Salmonella isolates obtained from clinical cases in a pediatric hospital in Tehran, Iran during 2006 to 2009. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Stand- ards Institute. The genetic relationship between third generation cephalosporins-resistant Salmonella enterica strains was determined using ERIC-PCR. Results: Of 136 Salmonella enterica isolates recovered from pediatric patients, six isolates including four Salmonella enterica serotype Infantis and two Salmonella enterica serotype Enteritidis showed an extended-spectrum cephalosporins resistant phenotype. ERIC-PCR differentiated Salmonella enterica serotypes Infantis and Enteritidis into 2 distinct clus- ters arbitrarily named as E1 and E2. Profile E1 was found in two Salmonella enterica serotype Enteritidis isolates, and profile E2 was found in four Salmonella enterica serotype Infantis isolates. Conclusion: Extended-spectrum cephalosporins resistant Salmonella could be attributed to a few predominant serotypes including Enteritidis and Infantis in this study. Genetic analysis using ERIC-PCR showed that closely related clones are responsible for the occurrence of extended-spectrum cephalosporins resistant Salmonella infection in Tehran.

23 citations


Journal ArticleDOI
TL;DR: The results show that B. abortus aggregates and produces biofilms, which tolerate desiccation better than planktonic cells do, adhere and displace even in the absence of the lipopolysaccharide-O antigen, flagella, the transcriptional regulator VjbR, or the enzymes that synthesize, transport, and modify cyclic β (1,2) glucan.
Abstract: Brucella abortus causes brucellosis mainly in cattle. The infection is transmitted to humans by ingestion of animal products or direct contact with infected material. While the intracellular lifestyle of Brucella is well characterized, its extracellular survival is poorly understood. In nature, bacterial persistence is associated with biofilms, where aggre- gated cells are protected from adversity. The inability of Brucella abortus to aggregate under aerobiosis and that fact that the replicative niche of Brucella is characterized by microaerobic conditions prompted us to investigate the capacity of this pathogen to aggregate and grow in biofilms under microaerobiotic conditions. The results show that B. abortus ag- gregates and produces biofilms. The aggregates tolerate desiccation better than planktonic cells do, adhere and displace even in the absence of the lipopolysaccharide-O antigen, flagella, the transcriptional regulator VjbR, or the enzymes that synthesize, transport, and modify cyclic β (1,2) glucan.

15 citations


Journal ArticleDOI
TL;DR: In this paper, the ability of the neuroleptic drug pimozide to inhibit the Escherichia coli AcrAB-TolC efflux pump, whose overproduction confers resistance to various antimicrobial agents, was investigated.
Abstract: Efflux pump inhibitors (EPIs) are attractive compounds to reverse multidrug-resistance in clinically relevant bacterial pathogens. In this study we tested the ability of the neuroleptic drug pimozide to inhibit the Escherichia coli AcrAB-TolC efflux pump, whose overproduction confers resistance to various antimicrobial agents. A real-time Nile red efflux assay in the AcrAB – overproducing strain 3-AG100 revealed that pimozide was capable of full inhibition of this pump at a concentration of 100 µM, which is far below its intrinsic MIC (>1mM). However, MIC assay demonstrated very little effect of pimozide with regard to reduction in MICs of various antimicrobial compounds. Only oxacillin MICs were reduced twofold in the presence of pimozide at 100 and 200 µM. Since pimozide did considerably enhance accumulation of ethidium bromide in a fluorescence assay, ethidium bromide MIC assays in the presence and absence of this putative EPI were performed. They revealed that pimozide was able to reduce the MICs of ethidium bromide by 4-fold. In line with previous reports we suggest that the capability of EPIs to restore the susceptibility to antimicrobial agents can be highly substrate-specific due to different substrate binding sites.

Journal ArticleDOI
TL;DR: Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods.
Abstract: Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods.

Journal ArticleDOI
TL;DR: The high activity and long lasting residual effect of a safe, non-toxic organic food grade surface cleaner was confirmed and the antibacterial activity of 2% Biosecur® against Vibrio vulnificus was shown to be equivalent to that of tetracycline.
Abstract: This study evaluated the antibacterial activity of Biosecur ® citrus extract surface cleaner against Vibrio vulnifi- cus using plate count method. Two concentrations, 0.5% and 2% of Biosecur ® surface cleaner were plated on Vibrio vul- nificus Agar (VVA) and tested for reduction of Vibrio vulnificus. In order to investigate the lasting residual activity of Biosecur ® , antibacterial activity tests were also performed at time intervals up to 2.5 h after Biosecur ® was plated on VVA. Biosecur ® showed 6-log reduction of Vibrio vulnificus at 2%, and 3-log reduction of Vibrio vulnificus at 0.5%. The antibacterial activity of 2% Biosecur ® against Vibrio vulnificus was shown to be equivalent to that of tetracycline. The re- sidual activity of 2% Biosecur ® was shown to maintain for at least 2.5 h after application. This study confirmed the high activity and long lasting residual effect of a safe, non-toxic organic food grade surface cleaner.

Journal ArticleDOI
TL;DR: PFGE analysis revealed that patients harbouring S. aureus both in the nasal cavity and the leg ulcer had the same bacterial type at both sites.
Abstract: The aim of this study was to investigate the occurrence of bacterial cross-contamination between the nasal cavity and leg ulcers. Sixteen patients were included in the study. Staphylococcus aureus was isolated from the leg ulcer of 13 patients and 6 of these patients also harboured S. aureus in the nasal cavity. Klebsiella oxytoca was found in the ulcer and the nasal cavity of one patient. PFGE analysis revealed that patients harbouring S. aureus both in the nasal cavity and the leg ulcer had the same bacterial type at both sites. None of the S. aureus isolates were methicillin resistant.

Journal ArticleDOI
TL;DR: The microbial populations of a riparian buffer zone located downslope of a pasture irrigated with swine lagoon effluent were characterized and spatial relationships between soil series, site location, and gene abundance identified, which could be used to infer both incomplete and total DEA rates.
Abstract: Riparian buffer zones are important for both natural and developed ecosystems throughout the world because of their ability to retain nutrients, prevent soil erosion, protect aquatic environments from excessive sedimentation, and filter pollutants. Despite their importance, the microbial community structures of riparian buffer zones remains poorly defined. Our objectives for this study were twofold: first, to characterize the microbial populations found in riparian buffer zone soils; and second, to determine if microbial community structure could be linked to denitrification enzyme activity (DEA). To achieve these objectives, we investigated the microbial populations of a riparian buffer zone located downslope of a pasture irrigated with swine lagoon effluent, utilizing DNA sequencing of the 16S rDNA, DEA, and quantitative PCR (qPCR) of the denitrification genes nirK, nirS, and nosZ. Clone libraries of the 16S rDNA gene were generated from each of twelve sites across the riparian buffer with a total of 986 partial sequences grouped into 654 operational taxonomic units (OTUs). The Proteobacteria were the dominant group (49.8% of all OTUs), with the Acidobacteria also well represented (19.57% of all OTUs). Analysis of qPCR results identified spatial relationships between soil series, site location, and gene abundance, which could be used to infer both incomplete and total DEA rates.

Journal ArticleDOI
TL;DR: MspA has been successfully isolated from the harvested Mycobacterium smegmatis using aqueous nOPOE (n-octyloligooxyethylene) at 65°C and a maximum of 840µg of pure MspA per liter of the optimized simple growth medium has been obtained.
Abstract: The adaptation of the organism to a simple and cost-effective growth medium is mandatory in developing a process for large scale production of the octamericporinMspA, which is isolated from Mycobacterium smegmatis. A fermentation optimization with the minimal nutrients required for growth has been performed. During the fermentation, the iron- and ammonium chloride concentrations in the medium were varied to determine their impact on the observed growth rates and cell mass yields. Common antibiotics to control contamination were eliminated in favor of copper sulfate to reduce costs. MspA has been successfully isolated from the harvested M. smegmatisusing aqueous nOPOE (n-octyloligooxyethylene) at 65°C. Because of the extraordinary stability of MspA, it is possible to denature and precipitate virtually all other proteins and contaminants by following this approach. To further purify the product, acetone is used for precipitation. Gel electrophoresis confirmed the presence and purity of MspA. A maximum of 840µg (via Bradford assay) of pure MspA per liter of the optimized simple growth medium has been obtained. This is a 40% increase with respect to the previously reported culture medium for MspA.

Journal ArticleDOI
TL;DR: The cat-scratch disease should be considered in differential diagnosis of patients presenting prolonged-fever associated with multiple lymphadenopathies and weight loss, and the azithromycin would be an alternative therapeutic issue for this pathology in case of confirmed efficacy by studies in a large patient population.
Abstract: We report a 19-year-old patient with a Cat-scratch disease presenting three months continuous alteration of the general condition, including prolonged-fever, anorexia, asthenia, weight loss associated with adenitis and multiple thoracic-abdominal adenopathies, leukocytosis with neutrophil polynuclear predominance, and increased of C-reactive protein. The serologies of toxoplasmosis, infectious mononucleosis, human immunodeficiency virus, Brucellosis, Bartonellosis and the tuberculosis research by tuberculin reaction test and Ziehl acid-alcohol resistant bacilli direct examination were negatives. The cytomegalovirus and Epstein-Barr virus serologies were positives only for immunoglobulin-G. The Bartonella henselae diagnosis was made with the analysis of histopathological specimens. The clinical and biological symptoms regressed following eight weeks of azithromycin's treatment. According to this observation, the cat-scratch disease should be considered in differential diagnosis of patients presenting prolonged-fever associated with multiple lymphadenopathies and weight loss. The azithromycin would be an alternative therapeutic issue for this pathology in case of confirmed efficacy by studies in a large patient population.

Journal ArticleDOI
TL;DR: PATS compares excellently with Pulsed-Field Gel Electrophoresis (PFGE) in that both methods cluster geographically diverse O157 isolates similarly and attests to the discriminating power and applicability of PATS to epidemiological/nosocomial situations.
Abstract: Polymorphic Amplified Typing Sequences (PATS) is a PCR-based Escherichia coli O157 (O157) strain typing system. Here, we show that PATS compares excellently with Pulsed-Field Gel Electrophoresis (PFGE) in that both meth- ods cluster geographically diverse O157 isolates similarly. Comparative analysis of the results obtained in this simulated "blind" study attests to the discriminating power and applicability of PATS to epidemiological/nosocomial situations.

Journal ArticleDOI
TL;DR: In this paper, the half-life of gfp(m) (2+) mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP(m), was determined using quantitative PCR and fluorescence spectrophotometry, respectively.
Abstract: The present study was designed to determine the half-life of gfp(m) (2+) mRNA, which encodes mycobacterial codon-optimised highly fluorescent GFP(m) (2+) protein, and to find out whether mycobacterial promoter activity can be quantitated more accurately using the mRNA levels of the reporter gene, gfp(m) (2+), than the fluorescence intensity of the GFP(m) (2+) protein. Quantitative PCR of gfp(m) (2+) mRNA in the pulse-chased samples of the rifampicin-treated Mycobacterium smeg-matis/gfpm(2+) transformant showed the half-life of gfp(m) (2+) mRNA to be 4.081 min. The levels of the gfp(m) (2+) mRNA and the fluorescence intensity of the GFP(m) (2+) protein, which were expressed by the promoters of Mycobacterium tuberculosis cell division gene, ftsZ (MtftsZ), were determined using quantitative PCR and fluorescence spectrophotometry, respectively. The data revealed that quantification of mycobacterial promoter activity by determining the gfp(m) (2+) mRNA levels is more accurate and statistically significant than the measurement of GFP(m) (2+) fluorescence intensity, especially for weak promoters.