Comparison Of Phenotypic Methods Research Articles

Direct Detection of Methicillin Resistant Staphylococci: Comparison of Phenotypic Methods with Multiplex PCR and Direct Susceptibility Testing

Bloodstream infections are augean diseases characterized by a high morbidity and mortality, related with the lag in administration of the first adequate anti-infectious agent. Clinical and epidemiological data guide the clinicians towards empirical anti-infectious treatments whose effectualness remains questionable especially in the present day milieu of multidrug-resistant organisms. Early microbiological evidence of the causative agent is hierophantic of antimicrobial stewardship. We evaluated three rapid methods for the direct identification of S. aureus from 720 positive blood cultures in comparison to Vitek 2 automated microbial identification system. Early distinction S. aureus from CoNS was attempted with Direct Tube Coagulase. Multiplex PCR assay was used to separate MRSA from MSSA by the presence of mecA gene. Direct antibiotic susceptibility (DST) from positive blood culture bottles was performed in an endeavour to reduce time and compared to the routine Vitek 2 compact antimicrobial susceptibility. For Direct tube coagulase at 4 hrs of incubation sensitivity was 79.6% while specificity was 100% however, on overnight incubation sensitivity increased to 97.9% with 100% specificity. The positive predictive value and negative predictive values were 100% and 96.9% respectively when compared to Vitek 2 identification of staphylococcal species. By DST Categorical agreement of 95% was seen for coagulase negative staphylococci microorganism-antibiotic combinations. Categorical agreement of 89.7% was seen for S. aureus microorganism-antibiotic combinations. Direct multiplex PCR testing did not misidentify any S. aureus isolate compared to Vitek 2. In 12 methicillin resistant CoNS isolates mecA gene was not detected by PCR. In total 13(3.5%) strains of staphylococci identified by DST as methicillin resistant were not identified by PCR analysis. Each of the tests has positive qualities and all may have a place in a Gram positive cocci algorithm for testing blood cultures depending on the laboratory setting, workload volume and staffing. However, rapid detection methods are a pressing priority.

The Phenotypic and Genetic Detection of the Methicillin-Resistant Staphylococcus aureus (MRSA)

Methicillin resistant strains of Staphylococcus aureus (MRSA) were identified shortly upon the introduction of methicillin into the clinical practice. The S. aureus samples were taken from patients hospitalized in General Hospital of Chania “Agios Georgios”. The strains were isolated from different pathological products, in the hospital laboratory. All isolates were tested using the cefoxitin disk diffusion, the oxacillin MIC methods and the PBP2’ latex agglutination test (bioMérieux), for the production of the Penicillin-Binding Protein 2a (PBP2a or PBP2’ protein). S. aureus ATCC 29213 was used as a negative control. We followed the detection of the mecA gene throughout the PCR method, as the standard “gold” method, in order to identify MRSA strains. Conventional methods for MRSA strains detection were compared with the PCR method. The antibiotic susceptibility testing was performed throughout the Kirby-Bauer disk diffusion method using antibiotic discs from Bioanalyse ltd. The mecA gene was found by PCR in 57.5 % of S. aureus strains, which allowed defining the isolated strains as MRSA strains. According to the oxacillin MIC values of the studied strains, 25 strains (53.2%) were identified as MRSA, 21 (44.68%) as MSSA and 1*strain (2.13%) as Borderline (BL) MRSA. The mecA gene is present in 24 of the MRSA strains with oxacillin MIC ≥ 4 being more common in strains showing the oxacillin MICs ≥ 256 (12/27). Adding the BL strains to the methicillin resistant strains, the rate of the MRSA strains increases to 26 (55.32%), the appropriate values of the MRSA strains percent, as determined by the PCR method (57.45%), which shows a concordance of 96.3% (26/27), between the results obtained by the two tests. Comparing the results obtained using the PCR method; with the oxacillin MICs and the PBP2’ latex agglutination test, concordant results were obtained for 89.36% of the strains (42/47) by oxacillin MICs and for 97.87% of the strains (46/47) by PBP2’ latex agglutination test. We conclude that the specificity of these methods is 100% for the mecA PCR method, 97.87% for the PBP2a latex method and 89.36% for the oxacillin MIC. The comparison of phenotypic methods (the PBP2a latex reaction, oxacillin MICs, the Cefoxitin disk diffusion test with the genotypic methods (the presence of the mecA gene), reveals that the PBP2a latex reaction has high sensitivity (97.87%), and can be used as an alternative method to PCR for the MRSA detection, in resource constraint settings.

Comparison of phenotypic methods for the detection of IMP producing enterobacteriaceae in queensland

Background The prevalence of carbapenemase producing enterobacteriaceae (CPE), particularly IMP producers (IMP), in Australia is increasing. Optimal methods for detection of IMP CPE are unknown. We compare six phenotypic methods to determine the most sensitive method for the detection of IMP CPE. Methods We tested 25 molecularly confirmed IMP CPE producers, including 16 Enterobacter cloacae , collected from Queensland Health hospitals. The following methods were performed: disc susceptibility testing (DST), minimum inhibitory concentrations (MICs) by E-test, broth microdilution (BMD) and Vitek 2, modified hodge test (MHT) and Carba NP test. Results Meropenem MIC range (MIC 50/90 ) was 0.25->32 (4/16), 0.25-32(2/16), 1->16(>16/>16) for BMD, E-test and Vitek 2, respectively. Seventeen percent of isolates had a Vitek 2 meropenem MIC between 1-2 |xg/mL. Using EUCAST breakpoints, all isolates tested resistant by Ertapenem E-test and all except for one isolate was resistant by ertapenem DST. CLSI breakpoints increased detection of CPE with meropenem and imipenem. Carba NP test and MHT provided positive results for all isolates. Conclusion: Our results indicate that isolates with a Vitek 2 meropenem MIC of ≥1 μg/mL should undergo further phenotypic testing. Ertapenem DST, ertapenem E-test, Carba NP test and MHT are the most sensitive methods for detection of IMP CPE.

Comparison of Phenotypic Methods and Yield Contributions of Quantitative Trait Loci for Partial Resistance to Phytophthora sojae in Soybean

ABSTRACTIn several host–pathogen systems, the detection of an individual quantitative trait locus (QTL) differed depending on the specific pathogen isolate or phenotypic assay that was used. Moreover, the contribution of a QTL toward yield is an important factor for selecting specific QTL candidates for resistance breeding. The first experiment in this study compared the QTL identified previously in a ‘Conrad’ × ‘Sloan’ F4:6 soybean [Glycine max (L.) Merr.] population challenged with Phytophthora sojae Kaufmann and Gerdemann isolate 1.S.1.1 to the QTL identified when this same population was challenged with isolates PT2004C2.S1 and OH25 using a tray test assay. In the second experiment, QTL were mapped in the same population with P. sojae isolate 1.S.1.1 using a different phenotypic assay, the layer test. In the third experiment, the impact of these QTL on soybean yield was evaluated using 20 selected recombinant inbred lines (RILs) grown in a field infested with P. sojae. Resistance QTL with smaller effects, especially those from the susceptible parent, were not consistently detected with the three isolates or the two phenotypic assays. Composite interval mapping identified a major Conrad QTL on chromosome 18 and one on 19 that were detected with all three isolates and both phenotypic assays. Recombinant inbred lines with resistance alleles at these QTL had significantly higher yields (∼1000 kg ha−1 more) than RILs with susceptible alleles. These results indicate the important role these QTL play in conferring partial resistance to P. sojae.

Comparison of phenotypic methods and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the identification of aero-tolerant Actinomyces spp. isolated from soft-tissue infections

Aero-tolerant Actinomyces spp. are an under-recognised cause of cutaneous infections, in part because identification using conventional phenotypic methods is difficult and may be inaccurate. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a promising new technique for bacterial identification, but with limited data on the identification of aero-tolerant Actinomyces spp. This study evaluated the accuracy of a phenotypic biochemical kit, MALDI-TOF MS and genotypic identification methods for the identification of this problematic group of organisms. Thirty aero-tolerant Actinomyces spp. were isolated from soft-tissue infections over a 2-year period. Species identification was performed by 16 s rRNA sequencing and genotypic results were compared with results obtained by API Coryne and MALDI-TOF MS. There was poor agreement between API Coryne and genotypic identification, with only 33% of isolates correctly identified to the species level. MALDI-TOF MS correctly identified 97% of isolates to the species level, with 33% of identifications achieved with high confidence scores. MALDI-TOF MS is a promising new tool for the identification of aero-tolerant Actinomyces spp., but improvement of the database is required in order to increase the confidence level of identification.

Comparison of phenotypic methods in predicting methicillin resistance in coagulase-negative Staphylococcus (CoNS) from animals

Phenotypic detection of methicillin resistance in coagulase-negative Staphylococcus (CoNS) of animal origin has been challenging due to the heterogeneous expression of mecA. To compare different phenotypic methods in predicting the mecA presence in CoNS, a total of 87 CoNS isolates from agricultural animals were analyzed in this study by agar dilution, disk diffusion, and broth microdilution. mecA was present in 81 CoNS isolates. Broth microdilution demonstrated the highest sensitivity of 100% in predicting the mecA presence, followed by 72.8% by agar dilution and 70.4% by disk diffusion. The results indicate that broth microdilution may be more suitable for predicting the presence of mecA in CoNS from animals than the other two methods, although staphylococcal species may also be a factor affecting the sensitivities of the methods as the top three staphylococcal species in this study were Staphylococcus lentus, Staphylococcus sciuri, and Staphylococcus xylosus (a total of 75 of 87).

Comparison of phenotypic methods and DNA hybridization for detection of methicillin-resistant Staphylococcus aureus

One hundred thirty-eight Staphylococcus aureus isolates from patients with severe staphylococcal infections were collected in 15 French hospitals. Detection of the mec gene was performed by dot blot hybridization with a specific DNA probe. Dot blot results were used to characterize the isolates as methicillin susceptible (77 isolates) or resistant (61 isolates). The isolates were screened for methicillin resistance by an agar spread method on Mueller-Hinton plates containing oxacillin (2 and 10 micrograms/ml) and were incubated at 37 degrees C, with 10(8) CFU as the inoculum. MICs of oxacillin and methicillin were determined by the agar dilution method on Mueller-Hinton plates without NaCl, by using 10(5) CFU per spot, after 24 and 48h of incubation at 30 or 37 degrees C. Moderately elevated MICs were found for 20 isolates (14.5%). The mec gene was detected in six (30%) of the isolates expressing a low level of resistance to methicillin and/or oxacillin. As determined by comparison with probe hybridization results, the spread plate method with oxacillin at 2 micrograms/ml was more sensitive (sensitivity, 100%) and specific (specificity, 100%) than agar dilution with either methicillin or oxacillin in identifying methicillin resistance or susceptibility. Determinations of methicillin and oxacillin MICs by the agar dilution method had a specificity of 99 to 100% depending on the conditions of incubation, but the sensitivity was below 85% whatever the duration or temperature of incubation.