scholarly journals Legionella antimicrobial sensitivity testing: comparison of microbroth dilution with BCYE and LASARUS solid media

2020 ◽  
Author(s):  
Edward Portal ◽  
Kirsty Sands ◽  
Artjoms Portnojs ◽  
Victoria J Chalker ◽  
Owen B Spiller
Keyword(s):  
Legionella Pneumophila ◽  
Solid Medium ◽  
Bacterial Species ◽  
Serial Dilution ◽  
Sensitivity Testing ◽  
Antimicrobial Sensitivity ◽  
Solid Media ◽  
Agar Dilution ◽  
Microbroth Dilution ◽  
Serial Dilutions

Abstract Objectives There is a lack of international unification for AST methodology for Legionella pneumophila. Current literature contains multiple possible methods and this study compares each of them to determine methodological concordance. Methods Antibiotic susceptibility of 50 L. pneumophila strains was determined using broth microdilution (BMD), serial antimicrobial dilution in traditional buffered charcoal yeast extract (BCYE) agar (as well as comparison with gradient strip overlay on BCYE) and in a novel charcoal-free agar (LASARUS) for rifampicin, azithromycin, levofloxacin and doxycycline. Results The deviation of tested media relative to BMD highlighted the overall similarity of BMD and LASARUS across all antimicrobials tested (within one serial dilution). BCYE agar dilution showed an increased MIC of up to five serial dilutions relative to BMD, while MICs by gradient strip overlay on BCYE were elevated by two to three serial dilutions, with the exception of doxycycline, which was decreased by three serial dilutions relative to MIC values determined by BMD. The MIC range for azithromycin was wider than for other antimicrobials tested and found to be caused by the presence or absence of the lpeAB gene. Conclusions BMD-based antimicrobial susceptibility testing (AST) methodology should be the internationally agreed gold standard for Legionella spp. AST, as is common for other bacterial species. Traditional BCYE gave significantly elevated MIC results and its use should be discontinued for Legionella spp., while MIC determination using LASARUS solid medium gave results concordant (within one serial dilution) with BMD for all antimicrobials tested. To the best of our knowledge, this study is the first to identify the lpeAB gene in UK isolates.

scholarly journals Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

2021 ◽  
Author(s):  
Edward Portal ◽  
Ghislaine Descours ◽  
Christophe Ginevra ◽  
Massimo Mentasti ◽  
Baharak Afshar ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Legionella Pneumophila ◽  
Susceptibility Testing ◽  
Solid Medium ◽  
Community Acquired Pneumonia ◽  
High Morbidity ◽  
Testing Methods ◽  
Severe Community Acquired Pneumonia ◽  
Microbroth Dilution ◽  
Reference Strains

Abstract Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire’s disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

scholarly journals Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

2004 ◽  
Vol 72 (10) ◽  
pp. 5983-5992 ◽  
Author(s):  
Jessica A. Sexton ◽  
Jennifer L. Miller ◽  
Aki Yoneda ◽  
Thomas E. Kehl-Fie ◽  
Joseph P. Vogel
Keyword(s):  
Cell Surface ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Host Cells ◽  
Growth Defect ◽  
Intracellular Growth ◽  
Homologous Proteins ◽  
Type Iv ◽  
Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

scholarly journals A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nicole A. Ellis ◽  
Byoungkwan Kim ◽  
Jessica Tung ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Polymerase Activity ◽  
Growth Defect ◽  
Great Promise ◽  
Macrophage Infection ◽  
Crispr Interference ◽  
Crispr Array ◽  
Virulence Protein

AbstractCatalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

scholarly journals STUDIES ON THE BACTERIOPHAGE OF D'HERELLE

1925 ◽  
Vol 42 (4) ◽  
pp. 483-497 ◽  
Author(s):  
Jacques J. Bronfenbrenner ◽  
Charles Korb
Keyword(s):  
Quantitative Evaluation ◽  
Bacterial Species ◽  
Relative Concentration ◽  
Serial Dilution ◽  
The Other ◽  
Resistant Bacteria ◽  
Dilution Method ◽  
High Concentration ◽  
Average Size ◽  
Broth Dilution

The experiments reported above confirm the fact that lytic principle is distributed in active solution in a state of indivisible units. This permits its quantitative evaluation by serial dilution, as well as by plating on agar. The latter method, however, often gives readings considerably lower than those obtained by the broth dilution method of titration. By varying the concentration of agar it has been possible to show that the discrepancy is due to adsorption of the lytic agent on agar. When the concentration of the latter is increased from 0.3 per cent to 2.5 per cent the number of plaques of lysis is reduced more than 100 times. At the same time the average size of the plaques also decreases approximately to one-tenth of the original. The size, as well as the number of plaques, has been found to depend also on the condition of the culture employed in titration. Thus, when the culture exposed to the action of lytic agent is composed of young susceptible bacteria, the greater the concentration of bacteria, the smaller the plaques. When the culture is composed partly of young and partly of old susceptible bacteria, both the size and the number of the plaques are diminished with the increase in the relative concentration of old bacteria. On the other hand, presence in the culture of resistant bacteria does not affect either the size or the number of the plaques so long as the relative concentration of susceptible bacteria in the culture is sufficient to allow formation of them. The plaques appearing in the presence of a high concentration of resistant variants in the culture are relatively indistinct owing to overgrowth. Under carefully controlled conditions the size of plaques is found to be determined by the character of the lytic filtrate. Thus in the case of lytic agents which act upon more than one bacterial species the size of the plaques remains constant, irrespective of the bacterial substratum used for the production of the active filtrate.

scholarly journals Survival of virulentLegionella pneumophilain aerosols

1983 ◽  
Vol 90 (3) ◽  
pp. 451-460 ◽  
Author(s):  
P. Hambleton ◽  
M. G. Broster ◽  
P. J. Dennis ◽  
R. Henstridge ◽  
R. Fitzgeorge ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Respiratory Disease ◽  
Legionella Pneumophila ◽  
Solid Medium ◽  
Exponential Phase ◽  
Metabolic Status ◽  
Aqueous Suspensions

SUMMARYAqueous suspensions of virulentLegionellapneumophilagrown on solid medium retained virulence and aerosol survival characteristics for several months. Significant numbers of viable organisms were recovered from aerosols held at various relative humidities (r.h.) for up to 2 h. The organisms survived best at 65% r.h. and were least stable at 55% r.h.Exponential phase broth-grown organisms survived poorly in aerosols in comparison with stationary phase broth cultures or organisms grown on solid medium, suggesting that the metabolic status ofLegionella pneumophilaorganisms may be an important factor affecting their ability to survive in aerosols and cause respiratory disease.

Isolation and Identification of Multidrug-Resistant Isolates of Uropathogenic Klebsiella spp in Dogs

2021 ◽  
Vol 3 (1) ◽  
pp. 6-12
Author(s):  
M Mustapha ◽  
P Goel
Keyword(s):  
Antimicrobial Agents ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Urine Samples ◽  
Penicillin G ◽  
Isolation And Identification ◽  
Sensitivity Testing ◽  
Bacterial Organism ◽  
Tract Infections ◽  
Klebsiella Spp

The most widespread ailments in dogs are urinary tract infections (UTIs) caused by bacterial species. It is necessary to recognize the prevailing bacterial pathogens and their susceptibility to antimicrobial agents to effectively treat UTIs. The present study aimed to classify the bacterial organism that causes UTIs in dogs and their patterns of antimicrobial resistance. A total of 141 urine samples were collected from diseased dogs in Veterinary Clinical Complex LUVAS in Hisar, India. Culture, biochemical and sensitivity testing were performed for each of the urine samples based on standard method. Of the total 141 urine samples from dogs, 21 (14.9%) isolates were identified as Klebsiella spp. The isolates were found to be highly resistant to ampicillin (100%), penicillin G (100%), oxytetracycline (100%), enrofloxacin (85.7%), chloramphenicol (80.6%), ceftriaxone (76.2%) and cloxacillin (71.4%), while susceptibility was observed against gentamicin (100%), amikacin (100%) and neomycin (90.5%). In the current study, 19 out of 21 identified isolates were found to be multidrug-resistant. This study indicates that dogs in the study area are found to harbor highly resistant Klebsiella spp. Therefore, when deciding on the antibiotic regimen for UTIs cases, Vets should consider resistance profile of chosen antibacterial agents before usage in order to discourage dissemination of resistant organisms in the study area.

Influence factors and adjusting method of refractive indices of solid media

2018 ◽  
Vol 32 (32) ◽  
pp. 1850391 ◽  
Author(s):  
Tao Zhang
Keyword(s):  
Refractive Index ◽  
Solid Medium ◽  
Influence Factors ◽  
Covalent Bond ◽  
High Refractive Index ◽  
Covalent Bonds ◽  
Molecular Density ◽  
Nephelauxetic Effect ◽  
Solid Media ◽  
Bond Property

It is of great significance to explore changing behavior and adjustment method of the refractive index. Expression of the refractive index, which was derived from the electron-cloud conductor model, contains the equivalent volume V of the electron clouds and the molecular density [Formula: see text]. The expression shows that the refractive index increases with V and [Formula: see text]. Based on this expression, main factors influencing the refractive index of solid medium are analyzed and summarized: the Nephelauxetic Effect, number of bonding electrons in the molecule, chemical bond property, number of nonbonding electrons in outermost layer of the molecule, and molecular density of the medium (electromagnetic wave factor is not considered). The relationship between the refractive index and the Nephelauxetic Effect is established. The calculations show that the Nephelauxetic Effect helps to increase the refractive index. Some viewpoints related to the Nephelauxetic Effect are supplemented. Characteristics of the solid medium that has a high refractive index are proposed: (1) Covalent bond component is high, and number of covalent bonds in one molecule is large. (2) Periodic number is as large as possible without increasing the ionic bond component. (3) Molecular density is high. According to these characteristics, method of adjusting the refractive index is discussed, and the results are consistent with the real situations.

scholarly journals Is Antimicrobial Resistance Pattern of Enteric Fever Changing in Kathmandu Valley?

2013 ◽  
Vol 32 (3) ◽  
pp. 221-228
Author(s):  
Ganesh Kumar Rai ◽  
Subhana Karki ◽  
Bina Prajapati
Keyword(s):  
Antimicrobial Resistance ◽  
Nalidixic Acid ◽  
Enteric Fever ◽  
Public Health Problem ◽  
Salmonella Typhi ◽  
Resistance Pattern ◽  
Kathmandu Valley ◽  
Sensitivity Testing ◽  
Antimicrobial Sensitivity ◽  
Sensitivity Pattern

Introduction: Enteric fever is a public health problem in developing countries including Nepal. Antimicrobial sensitivity pattern of Salmonella sp causing enteric fever is changing over time and also differs according to geographical location. Periodic surveillance of antimicrobial resistance of Salmonella sp is mandatory for management of enteric fever rationally. The objective of this study was to analyze the resistance pattern of Salmonella sp to commonly used antimicrobials. Materials and Methods: This was a retrospective study undertaken reviewing the records of blood isolates of Salmonella sp over one year period at Kanti Children’s Hospital, Nepal. Antimicrobial sensitivity testing was done following Kirby Bauer’s disk diffusion technique using Muller Hinton agar. Results: Salmonella typhi was found to be 100% resistance to ampicillin and resistance to nalidixic acid and ciprofloxacin was also high. The resistance to chloramphenicol and ofloxacin was low. No resistance was found to amoxicillin, ceftriaxone, cefotaxime and amikacin. However, among the sensitive isolates of Salmonella typhi intermediate sensitivity to ceftriaxone and ceftazidime was reported to be high. Among the isolates of S paratyphi A, the resistance to nalidixic acid was 100% followed by 75% to ciprofloxacin. No resistance was found to chloramphenicol, ceftriaxone, cefixime, amikacin and low resistance to amoxicillin and ofloxacin. Among the sensitive isolates of S paratyphi A, majority of them were only moderately sensitive to cefotaxime and ceftazidime and about 1/3 of the organisms had only intermediate sensitivity to ceftriaxone. Conclusiion: Commonly used parenteral third generation cephalosporins, first line drugs like chloramphenicol and amoxicillin and ofloxacin among fluoroquinolones were found to be effective in vitro in treating enteric fever. However, Salmonella typhi was found to be highly resistant to the most frequently used drugs like ciprofloxacin and cefixime and Salmonella paratyphi A to ciprofloxacin. A comparative chart of antimicrobial sensitivity of enteric fever in children over 10 year period from different hospitals of Kathmandu valley is also presented in the study. DOI: http://dx.doi.org/10.3126/jnps.v32i3.6625 J. Nepal Paediatr. SocVol.32(3) 2012 221-228

scholarly journals Cd and Zn interactions and toxicity in ectomycorrhizal basidiomycetes in axenic culture

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4478 ◽  
Author(s):  
Vinicius H. De Oliveira ◽  
Mark Tibbett
Keyword(s):  
Radial Growth ◽  
Solid Medium ◽  
Axenic Culture ◽  
Dry Weight ◽  
Zinc Toxicity ◽  
Solid Media ◽  
Wide Range ◽  
Ecm Fungi ◽  
Ectomycorrhizal Basidiomycetes ◽  
Tolerance Indices

BackgroundMetal contamination in soils affects both above- and belowground communities, including soil microorganisms. Ectomycorrhizal (ECM) fungi are an important component in belowground community and tolerant strains have great potential in enhancing plant-based remediation techniques. We assessed cadmium and zinc toxicity in five ECM species in liquid media (Hebeloma subsaponaceum;H. cylindrosporum;H. crustuliniforme;Sclerodermasp.;Austroboletus occidentalis) and investigated the potential of Zn to alleviate Cd toxicity. Due to highly divergent results reported in the literature, liquid and solid media were compared experimentally for the first time in terms of differential toxicity thresholds in Cd and Zn interactions.MethodsA wide range of Cd and Zn concentrations were applied to ectomycorrhizal fungi in axenic cultures (in mg L−1): 0; 1; 3; 9; 27; 81; 243 for the Cd treatments, and 0; 1; 30; 90; 270; 810; 2,430 for Zn. Combined Zn and Cd treatments were also applied toH. subsaponaceumandSclerodermasp. Dry weight was recorded after 30 days, and in case of solid medium treatments, radial growth was also measured.Results and DiscussionAll species were adversely affected by high levels of Cd and Zn, andA. occidentaliswas the most sensitive, with considerable biomass decrease at 1 mg L−1Cd, whileSclerodermasp. andH. subsaponaceumwere the most tolerant, which are species commonly found in highly contaminated sites. Cd was generally 10 times more toxic than Zn, which may explain why Zn had little impact in alleviating Cd effects. In some cases, Cd and Zn interactions led to a synergistic toxicity, depending on the concentrations applied and type of media used. Increased tolerance patterns were detected in fungi grown in solid medium and may be the cause of divergent toxicity thresholds found in the literature. Furthermore, solid medium allows measuring radial growth/mycelial density as endpoints which are informative and in this case appeared be related to the high tolerance indices found inH. subsaponaceum.

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