scholarly journals Applicability and performance of EUCAST’s rapid antimicrobial susceptibility testing (RAST) on primarily sterile body fluids in blood culture bottles in laboratory routine with total lab automation

Author(s):  
Jasmin Kaur Jasuja ◽  
Stefan Zimmermann ◽  
Irene Burckhardt

AbstractOptimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST’s RAST on primarily sterile body fluids in blood culture bottles with total lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197 Staphylococcus aureus, 91 Enterococcus spp., 38 Escherichia coli, 11 Klebsiella pneumoniae and 8 Pseudomonas aeruginosa were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for P. aeruginosa and meropenem and ciprofloxacin, 1/9 (11.1%) for K. pneumoniae and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for Enterococcus spp. and vancomycin and ampicillin, respectively. Minor errors for P. aeruginosa and meropenem (1/8; 12.8%) and for E. coli and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.

Author(s):  
Gaurab Risal ◽  
Aayush Shrestha ◽  
Saroj Kunwar ◽  
Gajal Paudel ◽  
Rameshwor Dhital ◽  
...  

Background: In urinary tract infections, an important role is played by bacterial biofilms which are responsible for persistence infections together with the antimicrobial resistance. Higher resistance can be seen in biofilm forming uropathogens in comparison with free-floating bacteria. So, the present study was performed with a goal to find the prevalence of biofilm formation and also the antimicrobial resistant pattern of uropathogens.Methods: A descriptive method was conducted at Modern Technical College, Sanepa, Lalitpur in samples isolated from UTI suspected patients. The overall duration of this study was approximately 3 months. Total of 50 isolated E. coli was tested for biofilm formation and antimicrobial susceptibility testing was done by Kirby-Bauer disc diffusion method on Mueller Hinton agar as per CLSI guidelines.Results: From the 50 isolates of E. coli, 32 were biofilm producers (3 strong and 29 moderate) and 18 were weak/non-biofilm producers. Among the biofilm producers, cefotaxime was more resistant in 20 of the isolates followed by ceftriaxone in 16 and amoxyclav in 13, whereas amikacin was least resistant in 2 of the isolates.Conclusions: Among the isolated E. coli, biofilm-forming isolates showed higher antimicrobial resistance as compared to the non-biofilm producer. Thus, uropathogen should be routinely screened for biofilm formation. 


2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S731-S731
Author(s):  
Yosef Nissim ◽  
Douglas Slain ◽  
P Rocco LaSala

Abstract Background Carbapenems are broad-spectrum antibacterials that have seen increased usage for the Enterobacteriales family in recent years. While carbapenem usage has been associated with increased antibacterial resistance, there is currently a lack of data comparing the risk of reduced susceptibility selection by the two most commonly used carbapenems in the US, ertapenem (ERT) and meropenem (MER). We conducted a novel serial passage experiment with clinical isolates of Enterobacteriales to assess the impact of repeated exposure to ERT or MER on phenotypic susceptibility patterns. Methods Non-duplicate clinical Enterobacteriales isolates were selected randomly for inclusion. Antimicrobial susceptibility testing was performed by CLSI disc diffusion methods. Standardized suspensions of isolates were plated on Mueller-Hinton agar, and ERT (10mcg) and MER (10mcg) discs applied. Zones of inhibition were measured and recorded after 16-18 hours incubation. Growth from the innermost zone of inhibition around each disc was used to prepare subsequent suspensions for serial susceptibility testing. This process would be repeated daily for 10 days. Each subsequent serially-passaged isolated was tested against both ERT and MER. Daily zones of inhibition were measured and interpreted. Baseline & final susceptibilities were determined by automated methods (Vitek 2). Results Seventeen Enterobacteriaceae isolates were selected, including: Klebsiella pneumoniae (n=11), Klebsiella oxytoca (n=2), Escherichia coli (n=1), Morganella morganii (n=1), and Enterobacter cloacae (n=2). Despite a greater degree of reductions in zones of inhibition with repeated ERT exposure (vs MER), the overall 10 day trends were not found to be significant different (P=0.529). Resistance developed to ERT in six isolates compared to one MER-resistant isolate (P = 0.053). E. cloacae was the only species to show a significant change between drugs (P=0.010). Two of three isolates that developed reduced zone changes > 10mm to MER were initially exposed to ERT on an earlier plate. Conclusion This novel experiment identified the development of some nonsignificant reductions in susceptibility with ERT after serial exposure. Results from this pilot study should encourage larger well-designed studies in this area. Disclosures All Authors: No reported disclosures


Author(s):  
Kavipriya D. ◽  
Suman Susan Prakash ◽  
Sarumathi Dhandapani ◽  
Deepashree Rajshekar ◽  
Apurba Sankar Sastry

Abstract Background Timely initiation of antimicrobial therapy in patients with blood stream infection is absolutely necessary to reduce mortality and morbidity. Most clinical microbiology laboratories use conventional methods for identification and antimicrobial susceptibility testing (AST) that involve biochemical methods for identification followed by AST by disk diffusion. The aim of the current study is to assess the various errors associated with direct susceptibility testing done from blood culture broth using automated AST system-Vitek-2 compact compared with the reference method of AST done from bacterial colonies. Materials and Methods The study was conducted in a tertiary care public sector 2,200-bedded hospital in South India for a period of 6 months. The study involved positively flagged blood culture bottles that yielded single morphotype of Gram-negative organism by Gram stain. A total of 120 bacterial isolates were collected that consisted of consecutively obtained first 60 isolates of Enterobacteriaceae family (30 Escherichia coli and 30 Klebsiella pneumoniae) and consecutively obtained first 60 nonfermenters (30 Pseudomonas aeruginosa and 30 Acinetobacter baumannii). Vitek-2 AST was done from these 120 blood culture broth, following the protocol by Biomerieux, and results were obtained. Then, Vitek-2 was done from colonies (reference method) using appropriate panel for Enterobacteriaceae and nonfermenters, and results were obtained. Both the results were compared. Results Nonfermenters showed a better categorical agreement of 97.6%, as compared to Enterobacteriaceae, which showed 97%. Among Enterobacteriaceae, both E. coli and K. pneumoniae showed categorical agreement of 97% each. Conclusion The procedure of AST directly from blood culture broth represents a simple and effective technique that can reduce the turnaround time by 24 hours, which in turn benefits the clinician in appropriate utilization of antimicrobials for better patient care.


2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Konrad Gwozdzinski ◽  
Saina Azarderakhsh ◽  
Can Imirzalioglu ◽  
Linda Falgenhauer ◽  
Trinad Chakraborty

ABSTRACTThe plasmid-located colistin resistance genemcr-1confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination ofmcr-1-containingEnterobacteriaceae. Colistin susceptibility testing was performed for 50mcr-1-containingEscherichia coliandKlebsiella pneumoniaeisolates, 7 intrinsically polymyxin-resistant species,K. pneumoniaeandE. coliisolates with acquired resistance to polymyxins due tomgrBandpmrBmutations, respectively, and 32mcr-1-negative, colistin-susceptible isolates ofAcinetobacter baumannii,Citrobacter freundii,Enterobacter cloacae,E. coli,K. pneumoniae, andSalmonella entericaserovar Typhimurium. A comparison of the colistin MICs determined in CA-MH medium and those obtained in CE-MH medium was performed using both the BMD and strip-based susceptibility test formats. We validated the data using an isogenic IncX4 plasmid lackingmcr-1. Use of the CE-MH broth provides clear separation between resistant and susceptible isolates in both BMD and gradient diffusion assays; this is true for bothmcr-1-containingEnterobacteriaceaeisolates and those exhibiting either intrinsic or acquired colistin resistance. CE-MH medium is simple to prepare and overcomes current problems associated with BMD and strip-based colistin susceptibility testing, and use of the medium is easy to implement in routine diagnostic laboratories, even in resource-poor settings.


2019 ◽  
Vol 9 (2) ◽  
pp. 409-416
Author(s):  
L. V. Domotenko ◽  
I. S. Kosilova ◽  
A. P. Shepelin

At present, a rise of antimicrobial resistance requires that susceptibility of infectious agents to antimicrobial agents could be accurately evaluated as related errors may lead to selecting improper therapeutics provoking spread of drug resistance. Pathogen sensitivity to antimicrobial agents is commonly determined by a disc diffusion method. A quality of nutrient medium used in assays plays a crucial role influencing final results. In Russia, it turned out that regulatory documents such as the nationwide guidelines and clinical recommendations outlining methodology for antimicrobial susceptibility testing underlay availability in domestic market few nutrient media, including Mueller–Hinton Agar, AGV medium etc. exhibiting sometimes unsatisfactory quality. To harmonize such methodology with international requirements, theStateResearchCenterfor Applied Microbiology and Biotechnology has developed a technology and promoted manufacture of Russia-made Mueller–Hinton agar satisfying requirements of EUCAST documents, clinical guidelines, and ISO/TS 16782:2016. The main objective of this study was to compare quality of new agar product with five similar foreign media while examining 11 test strains by disc diffusion method. As a result, some of nutrient media available to the Russian market turned out to be off-standard: not all of them satisfy to the EUCAST requirements and clinical guidelines since diameter distribution for growth inhibition recommended by EUCAST for quality control does not fit into permissible range. Moreover, susceptibility of P. aeruginosa ATCC 27853 to aminoglycosides, fluoroquinolones, Meropenem, as well as S. aureus ATSS 25923 and E. faecalis ATCC 29212 to tigecycline was assessed with certain mistakes. The data obtained by us were analyzed in accordance to the new document ISO/TS 16782:2016 “Clinical laboratory testing — criterion for acceptable lots of dehydrated Mueller–Hinton agar and broth for antimicrobial susceptibility testing”, not approved yet In Russia. To determine potential reason for deviation of data from reference range, we measured concentration of bivalent metals in all nutrient media examined by atomic emission spectrometry with inductively coupled plasma. We determined new patterns affecting reliability of results on microbial antibiotic susceptibility. A need to check intralaboratory quality control of nutrient media was emphasized.  


1999 ◽  
Vol 43 (2) ◽  
pp. 283-286 ◽  
Author(s):  
D. L. Butler ◽  
C. J. Jakielaszek ◽  
L. A. Miller ◽  
J. A. Poupard

ABSTRACT Current National Committee for Clinical Laboratory Standards (NCCLS) susceptibility guidelines for quality control testing withHaemophilus influenzae do not include a β-lactamase-producing strain that could detect the deterioration of the β-lactamase inhibitor components of amoxicillin-clavulanic acid, ampicillin-sulbactam, and piperacillin-tazobactam. The objective of the study was to determine if comparable quality control results forEscherichia coli ATCC 35218, a β-lactamase-producing strain, would be produced for the three β-lactam–β-lactamase inhibitor agents with Haemophilus test medium and Mueller-Hinton medium. The criteria used in this study to determine if Haemophilus test medium was acceptable for quality control testing of E. coli ATCC 35218 was that 100% of the results obtained with an antimicrobial agent-methodology combination needed to be within the acceptable NCCLS ranges established with Mueller-Hinton medium. The MIC testing results obtained by the broth microdilution and E-test methods with amoxicillin-clavulanic acid and piperacillin-tazobactam were all within the NCCLS ranges; however, the results obtained with ampicillin-sulbactam by both methods were not within the NCCLS ranges. Acceptable results were obtained by the disk diffusion methodology with ampicillin-sulbactam and piperacillin-tazobactam but not with amoxicillin-clavulanic acid. When performing susceptibility testing with H. influenzae with the β-lactam–β-lactamase inhibitors, in addition to quality control testing with H. influenzae ATCC 49247, testing of E. coli ATCC 35218 on Haemophilus test medium is an effective way to monitor the β-lactamase inhibitors in some antimicrobial agent-methodology combinations.


Antibiotics ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 43 ◽  
Author(s):  
Rangel-Castañeda Itzia Azucena ◽  
Cruz-Lozano José Roberto ◽  
Zermeño-Ruiz Martin ◽  
Cortes-Zarate Rafael ◽  
Hernández-Hernández Leonardo ◽  
...  

Aim: This study investigated the susceptibility of Enterotoxigenic Escherichia coli to curcumin, as well as its synergistic effect with 12 antimicrobial drugs. Methods and Results: Our study shows that curcumin did not affect bacterial growth. The antimicrobial susceptibility of curcumin and antibiotic synergy were identified using disc diffusion on Mueller-Hinton agar. The strain of Enterotoxigenic Escherichia coli used was resistant to Ampicillin, Amoxicillin/Clavulanic acid, Ampicillin/Sulbactam, Ciprofloxacin, and Cefazolin. There was synergy between curcumin and the majority of antibiotics tested. Maximum synergy was observed with combinations of 330 µg/mL curcumin and Ceftazidime, followed by Cefotaxime, Amoxicillin/Clavulanic acid, Ampicillin, Aztreonam, Trimethoprim, Ciprofloxacin, Ceftriaxone, Cefazolin, Tetracycline, and Imipenem. Conclusion: Our findings indicated that curcumin might be useful as a combinatorial strategy to combat the antibiotic resistance of Enterotoxigenic Escherichia coli.


2016 ◽  
Vol 60 (3) ◽  
pp. 1725-1735 ◽  
Author(s):  
Mimi R. Precit ◽  
Daniel J. Wolter ◽  
Adam Griffith ◽  
Julia Emerson ◽  
Jane L. Burns ◽  
...  

Staphylococcus aureussmall-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST ofS. aureusSCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86S. aureusSCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin;mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.


2010 ◽  
Vol 61 (1) ◽  
pp. 67-76 ◽  
Author(s):  
A. Mavridou ◽  
E. Smeti ◽  
G. Mandilara ◽  
P. Boufa ◽  
M. Vagiona-Arvanitidou ◽  
...  

In this study ten laboratories in Greece compared the performance of reference method TTC Tergitol 7 Agar (with the additional test of β-glucuronidase production) with five alternative methods, to detect E. coli in water, in line with European Water Directive recommendations. The samples were prepared by spiking drinking water with sewage effluent following a standard protocol. Chlorinated and non-chlorinated samples were used. The statistical analysis was based on the mean relative difference of confirmed counts and was performed in line with ISO 17994. The results showed that in total, three of the alternative methods (Chromocult Coliform agar, Membrane Lauryl Sulfate agar and Trypton Bilex-glucuronidase medium) were not different from TTC Tergitol 7 agar (TTC Tergitol 7 agar vs Chromocult Coliform agar, 294 samples, mean RD% 5.55; vs MLSA, 302 samples, mean RD% 1; vs TBX, 297 samples, mean RD% −2.78). The other two alternative methods (Membrane Faecal coliform medium and Colilert 18/ Quantitray) gave significantly higher counts than TTC Tergitol 7 agar (TTC Tergitol 7 agar vs MFc, 303 samples, mean RD% 8.81; vs Colilert-18/Quantitray, 76 samples, mean RD% 18.91). In other words, the alternative methods generated performance that was as reliable as, or even better than, the reference method. This study will help laboratories in Greece overcome culture and counting problems deriving from the EU reference method for E. coli counts in water samples.


Author(s):  
Angela Aparecida Da Silva ◽  
Márcia Maria Dos Anjos ◽  
Suelen Pereira Ruiz ◽  
Lucimara Bergamo Panice ◽  
Jane Martha Graton Mikcha ◽  
...  

Neste trabalho foi avaliada a ação dos óleos essenciais de Thymus vulgaris (tomilho), Syzygium aromaticum (cravo-da-índia) e Rosmarinus officinalis (alecrim) e dos conservantes benzoato de sódio e sorbato de potássio como agentes antimicrobianos. As cepas de Staphylococcus aureus (ATCC 25923) e Escherichia coli (ATCC 25922) foram utilizadas no teste de susceptibilidade antimicrobiana usando-se a técnica de microdiluição em microplaca de 96 poços para avaliação da Concentração Inibitória Mínima (CIM) e, posteriormente, subcultivo em Mueller Hinton Agar para avaliaçãoda Concentração Bactericida Mínima (CBM). As concentrações dos óleos e conservantes sintéticos testados variaram de 15,6 a 1000μg/mL. As microdiluições utilizando inóculos bacterianos nas concentrações de 104 UFC/mL foram incubadas a 37ºC/24h. As CIM para os óleos essenciais de cravo, tomilho e alecrim foram de 550, 650 e >1000μg/mL para E. coli e 550, 800 e 1000μg/mL para S. aureus, respectivamente. No entanto, a ação bactericida dos óleos essenciais do cravo-da-índia e do tomilho foi encontrada apenas em relação a E. coli, na concentração de 550 e 850μg/mL, respectivamente. Para os dois conservantes sintéticos testados, a CIM foi >1000μg/mL, portanto não apresentaram atividade antibacteriana contra os microrganismos testados. Trabalhos futuros deverão ser realizados para verifi car a efi ciência dos antimicrobianos naturais anteriormente citados e para avaliar a possibilidade de serem utilizados na indústria de alimentos.   


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