scholarly journals The Resistant-Population Cutoff (RCOFF): a New Concept for Improved Characterization of Antimicrobial Susceptibility Patterns of Non-Wild-Type Bacterial Populations

2015 ◽  
Vol 53 (6) ◽  
pp. 1806-1811 ◽  
Author(s):  
Giorgia Valsesia ◽  
Michael Hombach ◽  
Florian P. Maurer ◽  
Patrice Courvalin ◽  
Malgorzata Roos ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Resistance Mechanisms ◽  
Clinical Strain ◽  
Wild Type ◽  
Bacterial Populations ◽  
Content Type ◽  
Resistant Population ◽  
Susceptibility Patterns

This study aimed to determine resistant-population cutoffs (RCOFFs) to allow for improved characterization of antimicrobial susceptibility patterns in bacterial populations. RCOFFs can complement epidemiological cutoff (ECOFF)-based settings of clinical breakpoints (CBPs) by systematically describing the correlation between non-wild-type and wild-type populations. We illustrate this concept by describing three paradigmatic examples of wild-type and non-wild-typeEscherichia colipopulations from our clinical strain database of disk diffusion diameters. The statistical determination of RCOFFs and ECOFFs and their standardized applications in antimicrobial susceptibility testing (AST) facilitates the assignment of isolates to wild-type or non-wild-type populations. This should improve the correlation ofin vitroAST data and distinct antibiotic resistance mechanisms with clinical outcome facilitating the setting and validation of CBPs.

scholarly journals A Statistical Approach for Determination of Disk Diffusion-Based Cutoff Values for Systematic Characterization of Wild-Type and Non-Wild-Type Bacterial Populations in Antimicrobial Susceptibility Testing

2015 ◽  
Vol 53 (6) ◽  
pp. 1812-1822 ◽  
Author(s):  
Giorgia Valsesia ◽  
Malgorzata Roos ◽  
Erik C. Böttger ◽  
Michael Hombach
Keyword(s):  
Resistance Mechanisms ◽  
Beta Lactamase ◽  
Wild Type ◽  
Beta Lactam ◽  
Bacterial Populations ◽  
Content Type ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli

In this study, we introduce a new approach for determination of epidemiologic cutoffs (ECOFFs) and resistant-population cutoffs (RCOFFs) based on receiver operating characteristic (ROC) curves. As an example, the method was applied for determination of ECOFFs for seven different beta-lactam antibiotics and wild-type populations ofEscherichia coli,Klebsiella pneumoniae, andEnterobacter cloacae. In addition, RCOFFs were determined for bacterial populations with defined resistance mechanisms (“resistotypes”), i.e., extended-spectrum beta-lactamase (ESBL)-positiveE. coli, ESBL-positiveK. pneumoniae, and ESBL-positiveE. cloacae; AmpC cephalosporinase-positiveE. coliand AmpC-positiveK. pneumoniae; and broad-spectrum beta-lactamase (BSBL)-positiveE. coli. RCOFFs and ECOFFs are instrumental for a systematic characterization of associations between resistotypes and wild-type populations.

scholarly journals Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

2015 ◽  
Vol 84 (3) ◽  
pp. 701-710 ◽  
Author(s):  
Madeleine G. Moule ◽  
Natasha Spink ◽  
Sam Willcocks ◽  
Jiali Lim ◽  
José Afonso Guerra-Assunção ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Insertion Site ◽  
Wild Type ◽  
Content Type ◽  
Growth And Survival ◽  
New Genes ◽  
Insertion Sites

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

scholarly journals New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Alba Pérez-Cantero ◽  
Loida López-Fernández ◽  
Josep Guarro ◽  
Javier Capilla
Keyword(s):  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Severe Condition ◽  
Content Type ◽  
Recommended Treatment ◽  
Highly Active ◽  
Expression Studies

ABSTRACT Invasive aspergillosis (IA) is a severe condition mainly caused by Aspergillus fumigatus, although other species of the genus, such as section Nigri members, can also be involved. Voriconazole (VRC) is the recommended treatment for IA; however, the prevalence of azole-resistant Aspergillus isolates has alarmingly increased in recent years, and the underlying resistance mechanisms in non-fumigatus species remain unclear. We have determined the in vitro susceptibility of 36 strains from section Nigri to VRC, posaconazole (POS), and itraconazole (ITC), and we have explored the role of Cyp51A and Cyp51B, both targets of azoles, in azole resistance. The three drugs were highly active; POS displayed the best in vitro activity, while ITC and VRC showed MICs above the established epidemiological cutoff values in 9 and 16% of the strains, respectively. Furthermore, expression studies of cyp51A and cyp51B in control condition and after VRC exposure were performed in 14 strains with different VRC susceptibility. We found higher transcription of cyp51A, which was upregulated upon VRC exposure, but no correlation between MICs and cyp51 transcription levels was observed. In addition, cyp51A sequence analyses revealed nonsynonymous mutations present in both, wild-type and non-wild-type strains of A. niger and A. tubingensis. Nevertheless, a few mutations were exclusively present in non-wild-type A. tubingensis strains. Altogether, our results suggest that azole resistance in section Nigri is not clearly explained by Cyp51A protein alteration or by cyp51 gene upregulation, which indicates that other mechanisms might be involved.

scholarly journals In VitroActivity of Ceftazidime-Avibactam Combination inIn VitroCheckerboard Assays

2014 ◽  
Vol 59 (2) ◽  
pp. 1138-1144 ◽  
Author(s):  
Johanna Berkhout ◽  
Maria J. Melchers ◽  
Anita C. van Mil ◽  
Wright W. Nichols ◽  
Johan W. Mouton
Keyword(s):  
Antibacterial Activity ◽  
Enterobacter Cloacae ◽  
Resistance Mechanisms ◽  
Maximum Effect ◽  
Wild Type ◽  
Dependent Effect ◽  
Content Type ◽  
Resistant Strains ◽  
Type Strains

ABSTRACTTo evaluate thein vitroeffects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55Enterobacteriaceaestrains (32Klebsiella pneumoniae, 19Escherichia coli, 1Citrobacter freundii, and 3Enterobacter cloacae) and 26 strains ofPseudomonas aeruginosa, all with known resistance mechanisms such as extended-spectrum β-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains (n= 69) were analyzed in more detail. For theEnterobacteriaceaestrains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC50and MIC90being 8 and 16 mg/liter, respectively). For the ceftazidime-resistantP. aeruginosastrains, considerable inhibition of β-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on thesein vitrodata, avibactam is a promising inhibitor of different β-lactamases, including ESBLs and carbapenemases.

scholarly journals In VitroActivity of ASP2397 against Aspergillus Isolates with or without Acquired Azole Resistance Mechanisms

2015 ◽  
Vol 60 (1) ◽  
pp. 532-536 ◽  
Author(s):  
Maiken Cavling Arendrup ◽  
Rasmus Hare Jensen ◽  
Manuel Cuenca-Estrella
Keyword(s):  
Amphotericin B ◽  
Target Gene ◽  
Antifungal Agents ◽  
Susceptibility Testing ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type

ABSTRACTASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity againstAspergillusandCandida glabrata. We compared itsin vitroactivity against wild-type and azole-resistantA. fumigatusandA. terreusisolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-typeA. fumigatusisolates, 24A. fumigatusisolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), andA. terreusisolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) againstA. fumigatusCYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistantA. fumigatusisolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active againstA. terreusCYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayedin vitroactivity againstA. fumigatusandA. terreusisolates which was independent of the presence or absence of azole target gene resistance mutations inA. fumigatus. The findings are promising at a time when azole-resistantA. fumigatusis emerging globally.

scholarly journals The Strength of Synergistic Interaction between Posaconazole and Caspofungin Depends on the Underlying Azole Resistance Mechanism of Aspergillus fumigatus

2015 ◽  
Vol 59 (3) ◽  
pp. 1738-1744 ◽  
Author(s):  
Eleftheria Mavridou ◽  
Joseph Meletiadis ◽  
Antony Rijs ◽  
Johan W. Mouton ◽  
Paul E. Verweij
Keyword(s):  
Aspergillus Fumigatus ◽  
Resistance Mechanism ◽  
Fungal Growth ◽  
Resistance Mechanisms ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Synergistic Interactions ◽  
Drug Concentrations

ABSTRACTThe majority of azole resistance mechanisms inAspergillus fumigatuscorrespond to mutations in thecyp51Agene. As azoles are less effective against infections caused by multiply azole-resistantA. fumigatusisolates, new therapeutic options are warranted for treating these infections. We therefore investigated thein vitrocombination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistantA. fumigatusisolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (ttest,P< 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P< 0.01). FIC-2 values were inversely correlated with POSA MICs (rs= −0.52,P= 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs= 0.76,P< 0.0001) and CAS (rs= 0.52,P= 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) againstA. fumigatusisolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

scholarly journals Target- and Resistance-Based Mechanistic Studies with TP-434, a Novel Fluorocycline Antibiotic

2012 ◽  
Vol 56 (5) ◽  
pp. 2559-2564 ◽  
Author(s):  
Trudy H. Grossman ◽  
Agata L. Starosta ◽  
Corey Fyfe ◽  
William O'Brien ◽  
David M. Rothstein ◽  
...  
Keyword(s):  
Tetracycline Resistance ◽  
Resistance Mechanisms ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Tetracycline Resistance Genes ◽  
Potent Inhibition ◽  
The Common ◽  
Wild Type Control

ABSTRACTTP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based andin vitroassays. InEscherichia colicells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 μg/ml) was unaffected bytet(M),tet(K), andtet(B) and increased to 0.25 and 4 μg/ml in the presence oftet(A) andtet(X), respectively. Tetracycline, in contrast, was significantly less potent (MIC ≥ 128 μg/ml) againstE. colicells when any of these resistance mechanisms were present. TP-434 showed potent inhibition inE. coli in vitrotranscription/translation (50% inhibitory concentration [IC50] = 0.29 ± 0.09 μg/ml) and [3H]tetracycline ribosome-binding competition (IC50= 0.22 ± 0.07 μM) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4- to 16-fold, compared to that of the wild-type control strain, againstPropionibacterium acnesstrains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.

scholarly journals Low Prevalence of Gram-Positive Isolates Showing Elevated Lefamulin MIC Results during the SENTRY Surveillance Program for 2015–2016 and Characterization of Resistance Mechanisms

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Rodrigo E. Mendes ◽  
Susanne Paukner ◽  
Timothy B. Doyle ◽  
Steven P. Gelone ◽  
Robert K. Flamm ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Resistance Mechanisms ◽  
Surveillance Program ◽  
Wild Type ◽  
Coagulase Negative Staphylococci ◽  
High Genetic Diversity ◽  
Content Type ◽  
Low Prevalence ◽  
Viridans Group Streptococci

ABSTRACT This study investigated the molecular mechanisms possibly associated with non-wild-type MICs for lefamulin among staphylococci and streptococci included in the lefamulin surveillance program from 2015 to 2016. A total of 2,919 Staphylococcus aureus, 276 coagulase-negative staphylococci (CoNS), 3,923 Streptococcus pneumoniae, 389 β-hemolytic, and 178 viridans group streptococci isolates were included in the surveillance studies. Eleven (0.3% of all S. aureus) S. aureus isolates with lefamulin MICs above the staphylococcal epidemiological cutoff (ECOFF) value (>0.25 μg/ml) were selected for this study. Eight (72.7%) S. aureus (lefamulin MIC, 0.5 to 4 μg/ml) isolates carried vga(A or E), one isolate (MIC, 32 μg/ml) carried lsa(E), one isolate (MIC, 16 μg/ml) had an alteration in L4, and one strain (MIC, 0.5 μg/ml) did not carry any of the investigated resistance mechanisms. A total of 14 (5.1% of all CoNS) CoNS isolates had lefamulin MICs (0.5 to >32 μg/ml) above the ECOFF. Similar to S. aureus, 8 (57.1%) CoNS (lefamulin MIC, 1 to 8 μg/ml) isolates carried vga(A or B), while 2 isolates (MIC, 4 to 32 μg/ml) carried cfr. High genetic diversity was observed among staphylococci, although 3 S. aureus isolates belonged to sequence type 398 (ST398). Among the 3 Streptococcus agalactiae and 3 viridans group streptococci (0.1% of all streptococci surveyed) isolates selected for additional characterization, all but 1 isolate carried lsa(E). This study documents a low occurrence of surveillance isolates exhibiting a non-wild-type MIC for lefamulin, and among these isolates, vga and lsa(E) prevailed in staphylococci and streptococci, respectively.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Antimicrobial Susceptibility Patterns among a Large, Nationwide Cohort of Abiotrophia and Granulicatella Clinical Isolates

2017 ◽  
Vol 55 (4) ◽  
pp. 1025-1031 ◽  
Author(s):  
Kunatum Prasidthrathsint ◽  
Mark A. Fisher
Keyword(s):  
Antimicrobial Susceptibility ◽  
Clinical Isolates ◽  
Broth Microdilution ◽  
Content Type ◽  
Geographical Regions ◽  
Susceptibility Patterns

ABSTRACTAntimicrobial susceptibility patterns from 599A. defectiva,G. adiacens, andG. elegansclinical isolates were determined by broth microdilution. We observed significant differences in susceptibility across species, particularly to penicillin and ceftriaxone, and across geographical regions.A. defectivawas the least susceptible species overall to penicillin. All isolates were susceptible to vancomycin and >90% were susceptible to levofloxacin.

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