Gradient Strip Method Research Articles

Fosfomycin-Overcoming Problematic In Vitro Susceptibility Testing and Tricky Result Interpretation: Comparison of Three Fosfomycin Susceptibility Testing Methods.

Background: Fosfomycin (FOS) is an older antimicrobial agent newly rediscovered as a possible treatment for infections with limited therapeutic options (e.g., Gram-negative bacteria with difficult-to-treat resistance, DTR), especially in intravenous form. However, for correct usage of FOS, it is necessary to have a reliable susceptibility testing method suitable for routine practice and robust interpretation criteria. Results: The results were interpreted according to 2023 interpretation criteria provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). DTR Gram-negatives were more likely to be resistant to FOS (45% in Enterobacterales and 20% in P. aeruginosa) than non-DTR (10% and 6.7%, resp.). All isolates of S. aureus were susceptible to FOS. In Gram-negatives, all agreement values were unacceptable. Etest® performed better in the DTR cohort (categorical agreement, CA, 80%) than in the non-DTR cohort (CA 45.7%). There were no very major errors (VREs) observed in P. aeruginosa. S. aureus had surprisingly low essential agreement (EA) rates (53% for MRSA and 47% for MSSA) for Etest®, but categorical agreement was 100%. Methods: A total of 130 bacterial isolates were tested and compared using the disc diffusion method (DD) and gradient strip method (Etest®) with the reference method (agar dilution, AD). The spectrum of isolates tested was as follows: 40 Enterobacterales (20 DTR vs. 20 non-DTR), 30 Pseudomonas aeruginosa (15 DTR vs. 15 non-DTR), and 60 Staphylococcus aureus (30 methicillin-susceptible, MSSA, vs. 30 methicillin-resistant, MRSA). Conclusions: Neither one of the tested methods was identified as a suitable alternative to AD. It would be beneficial to define more interpretation criteria, at least in some instances.

Antimicrobial Susceptibility of Various MRSA Clinical Isolates and the Impact of Glycopeptide MICs on Clinical and Microbiological Outcomes.

While vancomycin has remained the mainstay of the treatment for methicillin-resistant Staphylococcus aureus (MRSA) infections, there is growing evidence of the clinical impact of increased glycopeptide minimum inhibitory concentrations (MICs) in MRSA isolates. This study aimed to determine the susceptibility of various MRSA isolates to different antibiotics with antistaphylococcal activity and the impact of glycopeptide MICs on clinical and microbiological outcomes. This retrospective cohort study, conducted between 2013 and 2017, evaluated the susceptibility of MRSA strains isolated from various clinical samples to antistaphylococcal antibiotics using the gradient strip method. The clinical and laboratory features of patients infected with MRSA isolates with elevated glycopeptide MICs (>1 mg/L) and with isolates that had low glycopeptide MICs (≤1 mg/L) were compared. A total of 104 patients infected with MRSA strains were included in this study. Male sex (odds ratio [OR]=2.48, 95% confidence interval [CI]=1.01-6.10, p=0.048), two or more comorbidities (OR=2.48, 95% CI=1.03-6.50, p=0.044), history of MRSA infection (OR=4.91, 95% CI=1.70-14.28, p=0.003) and a longer hospital stay prior to MRSA infection (OR=2.32, 95% CI=1.05-7.85, p=0.040) were independent risk factors for high glycopeptide MICs. In MRSA infections with a teicoplanin MIC of >0.75mg/L, the microbiological and treatment failures were 46.2% (p=0.044) and 60.6% (p=0.042), respectively. This study showed that the critical MIC value, which suggested treatment failure as well as microbiological failure in the teicoplanin-treated MRSA infections, was >0.75 mg/L rather than >1 mg/L in our study cohort. The identification of high-risk patients;for treatment failures and mortality considering gradient strip method MIC values is crucial for the effective management of MRSA infections.

In Vitro Activity of "Old" and "New" Antimicrobials against the Klebsiella pneumoniae Complex.

The Klebsiella pneumoniae complex is a commonly isolated bacteria in human infections. These opportunistic pathogens pose a serious threat to public health due to their potential transmission to the human population. Resistance to carbapenems is a significant antimicrobial resistance mechanism, leading to limited therapeutic options. Therefore, the aim of this study was to evaluate the in vitro activity of fosfomycin, colistin, ceftazidime-avibactam, and meropenem-vaborbactam against multidrug-resistant K. pneumoniae complex strains. This study involved 160 strains of Gram-negative rods, comprising 138 K. pneumoniae and 22 K. variicola. The minimal inhibitory concentration of fosfomycin was estimated using the agar dilution method, and for colistin, the microdilution method was employed. Susceptibility to ceftazidime-avibactam and meropenem-vaborbactam was determined using the gradient strip method. All analyzed K. pneumoniae complex isolates produced extended-spectrum β-lactamases, and 60.0% exhibited carbapenemases. The majority of the analyzed strains were susceptible to fosfomycin and colistin (62.5%). Among pandrug-resistant K. pneumoniae complex isolates, the highest susceptibility was observed with colistin (43.9%). Fosfomycin demonstrated good activity against ESβLs- and VIM-positive isolates from this complex. Colistin also exhibited satisfactory in vitro activity against VIM- and KPC-positive isolates from the K. pneumoniae complex. Ceftazidime-avibactam displayed good activity against K. pneumoniae complex strains producing ESβLs, KPC, and OXA enzymes. Additionally, meropenem-vaborbactam showed satisfactory in vitro activity against ESβLs- and KPC-positive isolates from this complex.

Azithromycin susceptibility testing for salmonella enterica isolates: Comparing disk diffusion results with MIC gradient strips

Background: Enteric fever remains an imperative public health problem in developing countries. After the emergence of cephalosporin resistance in Salmonella enterica subsp. enterica serovar Typhi, azithromycin is increasingly being used for oral treatment of enteric fever. Reports of sporadic azithromycin resistance have been reported across the country, additionally, misuse of azithromycin during the COVID-19 pandemic has concerns regarding emerging azithromycin resistance. This study evaluated the reliability of the disc diffusion method as a screening test for detecting azithromycin resistance by comparing it with the minimum inhibitory concentrations (MIC) gradient strip results, in 231 typhoidal salmonellae. Material and Methods: This prospective study was conducted in the Section of microbiology of the Shaukat Khanum Memorial Cancer Hospital and Research Centre, Lahore from March 2022 to March 2023. Isolates recovered from blood cultures of patients, suffering from enteric fever were selected. Azithromycin susceptibility testing was performed both by disk diffusion and as well as gradient strips and their results were compared. Results: Among typhoidal salmonellae, a significant portion consisted of extensively drug resistant Salmonella Typhi (61.9%). Only one XDR S. Typhi was found to be resistant to azithromycin both by disk diffusion method and MIC gradient strip method, with a MIC value of 64µg/ml. The study found no discrepancy between the disk diffusion and gradient strip methods. Conclusion: The current study found no discordance between disk diffusion and gradient strip test methods for evaluating azithromycin susceptibility among typhoidal salmonellae. Keywords: Azithromycin, disk diffusion testing, enteric fever, Minimum inhibitory concentration, Salmonella Typhi

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

The Importance of Gram-stained Smears in the Diagnosis of Nocardia Infections

Nocardia species are low virulence bacteria found in nature. They can be an infectious agent, especially in patients with risk factors such as underlying immunosuppression, chronic lung disease, and malignancy. They can be easily overlooked because they are not seen frequently and has no pathognomonic symptoms. With this study, it was aimed to draw attention to the importance of microscopic examination of Gram-stained smears in the diagnosis of Nocardia infections in routine microbiology laboratories. Cases in which Nocardia spp. were detected in their clinical samples between November 2014-December 2015 in Hacettepe University Medical Faculty Hospital were included in the study. In the direct microscopic examination of Gram-stained smears of the samples arriving to the laboratory, the incubation periods of the cultures of the samples compatible with Nocardia spp. were extended. Then relevant colonies were identified by conventional microbiological methods and also by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, bioMerieux, France) automated system. Species-level identification of Nocardia isolates was performed by 16S rRNA gene sequence analysis. To demonstrate the genetic relationship between Nocardia isolates, pulsed-field gel electrophoresis (PFGE) was performed. In vitro susceptibility of the isolates against amoxicillin-clavulanate (AMC), linezolid, moxifloxacin, trimethoprim-sulfamethoxazole (TMP-SXT), amikacin, imipenem, clarithromycin, cefepime, cefotaxime, ceftriaxone, and ciprofloxacin was determined using the gradient strip method (E-test). A total of 19 Nocardia spp. strains were isolated from eight patients. Four cases exhibited repeated growth of Nocardia spp. up to a period of nine months. The most frequently isolated species was N.cyriacigeorgica, which was identified in four cases. Other species isolated from patients were N.asteroides, N.transvalensis, N.farcinicia, and N.asiatica/arthritidis. When the results obtained with DNA sequence analysis and MALDI-TOF MS were compared, 16 (84.2%) of 19 isolates were correctly identified to the genus level and 9 (47.4%) to the species level with MALDI-TOF MS, while three (15.8%) isolates could not be identified, and seven (36.8%) isolates were misidentified. According to the PFGE results, it was determined that the strains isolated from the same patient were genetically identical. All isolates were susceptible to amikacin, cefepime, cefotaxime, ceftriaxone, imipenem, linezolid, and except one isolate to TMP-SXT. Among the study isolates, the most common resistance was against ciprofloxacin (62.5%), followed by clarithromycin (37.5%). N.cyriacigeorgica was determined as the most frequently detected and the most resistant species to antibiotics in the study population. Direct microscopic examination of clinical specimens is one of the most valuable methods for the identification of Nocardia-type bacteria, which is difficult to isolate in microbiology laboratories. With this study, the importance of examining Gram-stained clinical samples was emphasized in the identification of Nocardia species, which can emerge with a wide variety of clinical forms and can be easily overlooked. In addition, antibiotic susceptibility profiles of the isolated bacteria were determinedto contribute to species-specific susceptibility profiles. Accurate identification of Nocardia species will contribute to clinical and epidemiological studies.

Acquired fluconazole resistance and genetic clustering in Diutina (Candida) catenulata from clinical samples

ObjectivesDiutina (Candida) catenulata is an ascomycetous yeast isolated from environmental sources and animals, occasionally infecting humans. The aim of this study is to shed light on the in vitro antifungal susceptibility and genetic diversity of this opportunistic yeast. MethodsForty-five D. catenulata strains isolated from various sources (including human and environmental sources) and originating from nine countries were included. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and confirmed via internal transcribed spacer ribosomal DNA barcoding. In vitro antifungal susceptibility was determined for seven systemic antifungals via the gradient strip method after 48 hours of incubation at 35°C using Etest® (Biomérieux) or Liofilchem® strips. Isolates exhibiting fluconazole minimal inhibitory concentrations (MICs) of ≥8 μg/mL were investigated for mutations in the ERG11 gene. A novel microsatellite genotyping scheme consisting of four markers was developed to assess genetic diversity. ResultsMIC ranges for amphotericin B, caspofungin, micafungin, isavuconazole, and posaconazole were 0.19–1 μg/mL, 0.094–0.5 μg/mL, 0.012–0.064 μg/mL, 0.003–0.047 μg/mL, and 0.006–0.032 μg/mL, respectively. By comparison, a broad range of MICs was noted for fluconazole (0.75 to >256 μg/mL) and voriconazole (0.012–0.38 mg/L), the higher values being observed among clinical strains. The Y132F amino acid substitution, associated with azole resistance in various Candida species (C. albicans, C. tropicalis, C. parapsilosis, and C. orthopsilosis), was the main substitution identified. Although microsatellite typing showed extensive genetic diversity, most strains with high fluconazole MICs clustered together, suggesting human-to-human transmission or a common source of contamination. DiscussionThe high rate of acquired fluconazole resistance among clinical isolates of D. catenulata is of concern. In this study, we highlight a link between the genetic diversity of D. catenulata and its antifungal resistance patterns, suggesting possible clonal transmission of resistant isolates.

Performance Evaluation of the Quantamatrix QMAC-dRAST System for Rapid Antibiotic Susceptibility Testing Directly from Blood Cultures.

Objectives: Rapid antibiotic susceptibility testing (AST) for positive blood cultures can improve patient clinical outcomes if the time to an effective antimicrobial therapy is shortened. In this study, we tested the Quantamatrix dRAST system (QMAC-dRAST), a rapid AST system based on time-lapse microscopic imagery of bacterial colony formation in agarose. Methods: Evaluation of the QMAC-dRAST was performed from 250 monobacterial blood cultures including 130 Enterobacterales, 20 non-fermentative Gram-negative bacteria, 69 staphylococci and 31 enterococci. Blood cultures were recovered from anonymous patients or from spiking experiments to enrich our study with bacterial species and resistant strains. Categorical agreement (CA), minor errors (me), major errors (ME) and very major errors (VME) were calculated in comparison to the results obtained from the BD Phoenix™ M50. Discrepancies between the Phoenix™ M50 and QMAC-dRAST results were investigated using the gradient strip method. The repeatability and reproducibility performance of the QMAC-dRAST was assessed for 16 strains, each strain being tested five times from a spiked blood culture. Results: The overall CAs for Enterobacterales, non-fermentative Gram-negative bacteria, staphylococci and enterococci were 95.1%, 91.2%, 93.4% and 94.5%, respectively. The VME percentage was below 4% for all the groups except for staphylococci, which showed a VME rate of 7%. The median time to result was 6.7 h (range: 4.7–7.9). Repeatability and reproducibility assays showed a high reliability of AST results with best and worst ratios of 98.8% and 99.6% and 95.0% and 98.3%, respectively. Conclusions: The QMAC-dRAST is a fast and reliable system to determine AST directly from monobacterial blood cultures with a major TAT reduction compared to conventional AST testing.

Susceptibility Testing of Candida auris Isolates.

Susceptibility testing of isolates of Candida auris is helpful as a guide to the selection of the most appropriate antifungal agent for treatment as different clades and strains within clades often demonstrate markedly different susceptibility profiles. Some strains are relatively susceptible to all antifungal drugs, but most demonstrate innate resistance to fluconazole, many are cross-resistant to other azoles and others demonstrate resistance to other classes of antifungal. The finding of multi-drug resistance, where an isolate is resistant to two or more classes of antifungal agent, is not uncommon, and development of resistance during a course of treatment has also been documented. This chapter describes a reference broth microdilution method for susceptibility testing and a commercially available gradient strip method.

Antibiotic resistance of Enterococcus species: 3-year data

Aim: The aim of this study is to investigate the prevalence of Enterococcus species and to evaluate susceptibilities to antimicrobial agents in a state (secondary) hospital.
 Material and Methods: A total of 1676 enterococci strains (490 E.faecium, 1146 E.faecalis, 10 E.casseliflavus/E.gallinarum and 30 other Enterococcus species) isolated from cultures obtained from January 2017 to December 2019 in XXX Hospital were included. Blood cultures were incubated in automated device (Render Biotech Co.Ltd., PRC). Other cultures were incubated with conventional methods. Grown colonies were identified by PhoenixTM 100 automated system (Becton Dickinson, USA). Identifications that need confirmation or strains identified to genus level were further evaluated with conventional techniques. Antimicrobial susceptibility testing were performed by same system, Kirby-Bauer disc diffusion and gradient strip method according to EUCAST guidelines.
 Results: 43.1%, 27.1%, 14.7% and 15.1% of enterococci were isolated from urine, blood/sterile body fluids, wound/abscess and other samples. Majority of the strains were ciprofloxacin (72.0%) and levofloxacin (74.1%) resistant, and more than 40% showed ampicillin and high-level gentamicin resistance. Glycopeptide resistance was relatively high (5.4%), especially when considering E.faecium (12.1%). There was not any tigecycline and linezolid resistance. 
 Conclusions: Antimicrobial resistance is a serious and growing public health problem affecting all countries, which is not just a topic of medicine, but multiple sectors such as commercial companies, food industry, veterinarians, etc. High percentages of resistance strongly indicate to get a local action, which should be followed by national and global one.

Antimicrobial susceptibility profiles of invasive isolates of anaerobic bacteria from a large Canadian reference laboratory: 2012–2019

Anaerobic bacteria can cause severe and life threatening infections. Susceptibility data are relatively limited on anaerobic organisms despite the clinical importance in guiding empiric treatment of infections. To determine antimicrobial susceptibility profiles of clinically significant anaerobic bacteria, isolates obtained from sterile sites submitted to Public Health Ontario Laboratory (2012–2019) were included in this study (N = 5712). Cefoxitin, clindamycin, metronidazole, meropenem, penicillin and piperacillin-tazobactam were tested using the gradient strip method with MICs interpreted based on Clinical and Laboratory Standards Institute guidelines. Bacteroides spp. (N = 958; 16.7%), Clostridium spp. (N = 798; 14.0%), Cutibacterium spp. (N =659; 11.5%) and Actinomyces spp. (N = 551; 7.0%) were the most commonly isolated genera. Bacteroides fragilis isolates were susceptible to cefoxitin (88.4%), clindamycin (68.4%), metronidazole (96.0%), meropenem (99.0%) and piperacillin-tazobactam (98.4%). Other Bacteroides spp. showed reduced susceptibility to several antimicrobials. Clostridium spp. isolates were susceptible to penicillin (69.7%), clindamycin (69.7%) and cefoxitin (76.3%); C. perfringens and C. ramosum showed distinct susceptibility profiles. Susceptibility rates among anaerobes remained relatively unchanged over 8 years with a few exceptions: C. perfringens susceptibility to clindamycin decreased from 91.3% to 60% (p = 0.03); Clostridium spp. susceptibility to penicillin similarly decreased from 82.1% to 65.9% (p = 0.03); Eggerthella spp. susceptibility to piperacillin-tazobactam decreased from 100% to 24.3% (p < 0.001); B. fragilis group susceptibility to cefoxitin decreased from 70.4% to 48.2% (p = 0.05); and Parabacteroides spp. susceptibility to piperacillin-tazobactam decreased from 100% to 25% (p = 0.01). Our findings underscore the need for ongoing surveillance and periodic monitoring of antimicrobial resistance in order to guide empiric therapy.

Antimicrobial Susceptibility and Genomic Analysis of Aliarcobacter cibarius and Aliarcobacter thereius, Two Rarely Detected Aliarcobacter Species.

Aliarcobacter cibarius and Aliarcobacter thereius are two rarely detected Aliarcobacter species. In the study, we analyzed the antimicrobial susceptibility and provide detailed insights into the genotype and phylogeny of both species using whole-genome sequencing. Thermophilic Campylobacter species are the most common bacterial foodborne pathogens causing gastroenteritis in humans worldwide. The genus Aliarcobacter is part of the Campylobacteraceae family and includes the species Aliarcobacter butzleri, Aliarcobacter cryaerophilus, Aliarcobacter skirrowii, and the rarely described Aliarcobacter cibarius, Aliarcobacter faecis, Aliarcobacter lanthieri, Aliarcobacter thereius, and Acrobarter trophiarum. Aliarcobacter are emergent enteropathogens and potential zoonotic agents. Here, we generated, analyzed, and characterized whole-genome sequences of Aliarcobacter cibarius and Aliarcobacter thereius. They were isolated from water poultry farms in Germany, cultured and identified by MALDI-TOF MS. With PCR the identity was verified. Antibiotic susceptibility testing was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, ampicillin, and cefotaxime using the gradient strip method (E-test). Whole-genome sequences were generated including those of reference strains. Complete genomes for six selected strains are reported. These provide detailed insights into the genotype. With these, we predicted in silico known AMR genes, virulence-associated genes, and plasmid replicons. Phenotypic analysis of resistance showed differences between the presence of resistance genes and the prediction of phenotypic resistance profiles. In Aliarcobacter butzleri, the nucleotide sequence of the gyrA gene (DQ464331) can show a signature mutation resulting in an amino acid change T85>I. Acrobarter cibarius and Acrobarter thereius showed the same gene as assessed by similarity annotation of the mutations 254C>G. Most of the isolates were found to be sensitive to ciprofloxacin. The ciprofloxacin-resistant Aliarcobacter thereius isolate was associated with the amino acid change T85>I. But this was not predicted with antibiotic resistance databases, before. Ultimately, a phylogenetic analysis was done to facilitate in future outbreak analysis.

Evaluation of susceptibility testing methods for Burkholderia cepacia complex: a comparison of broth microdilution, agar dilution, gradient strip and EUCAST disc diffusion

ObjectivesTo evaluate the accuracy and reproducibility of antimicrobial susceptibility testing methods in Burkholderia cepacia complex (BCC). MethodsMinocycline, ciprofloxacin, trimethoprim/sulphamethoxazole, meropenem, ceftazidime and chloramphenicol were tested against 155 BCC strains using broth microdilution at 35 ± 1°C (BMD35) in triplicate, then BMD at 30 ± 1°C (BMD30), agar dilution at 30°C and 35°C (AD30 and AD35), gradient strip (GS) and EUCAST standardized disc diffusion (DD) testing methods once. ResultsBMD35 reproducibility ranged from 70% to 84.5% for all agents. Correlations of MICs from BMD35 with BMD30 ranged from 63% to 85%, with AD35 from 32.9% to 87% and with GS methods from 36% to 83.9%. Essential agreement (EA) of MICs by GS with BMD35 ranged from 62.6% (trimethoprim-sulphamethoxazole) to 83.9% (minocycline). EA of EUCAST DD zone diameters using CLSI breakpoint criteria was between 85.8% and 97.4%, however Very Major Errors (VME) for trimethoprim/sulphamethoxazole were 31%. ConclusionsBMD at 35 ± 1°C was poorly reproducible for most agents and no method showed acceptable performance. Of particular concern were the GS results. Although this is the most commonly used method for determining MICs in laboratories, there was poor correlation with BMD35 for meropenem and trimethoprim/sulphamethoxazole. EUCAST DD correlated poorly with BMD35 MICs. This study confirms that no susceptibility method is capable of providing reproducible and accurate MICs when testing BCC.

Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Cryptic Isolates from Aspergillus Section Fumigati.

The antifungal susceptibility of Aspergillus cryptic species is poorly known. We assessed 51 isolates, belonging to seven Fumigati cryptic species, by the EUCAST reference method and the concentration gradient strip (CGS) method. Species-specific patterns were observed, with high MICs for azole drugs, except for Aspergillus hiratsukae and Aspergillus tsurutae, and high MICs for amphotericin B for Aspergillus lentulus and Aspergillus udagawae Essential and categorical agreements between EUCAST and CGS results were between 53.3 and 93.3%.

Imipenem-Relebactam Susceptibility Testing of Gram-Negative Bacilli by Agar Dilution, Disk Diffusion, and Gradient Strip Methods Compared with Broth Microdilution

This study aimed to determine whether agar dilution, research-use-only disk diffusion (Mast Group Ltd., Bootle Merseyside, UK), Etest (bioMérieux, Inc., Durham, NC), and MIC test strip (MTS) (Liofilchem, Inc., Waltham, MA) methods yield equivalent results to those of broth microdilution (BMD) for imipenem-relebactam susceptibility testing using a collection of 297 Gram-negative bacilli, including members of the order Enterobacterales and Pseudomonas aeruginosa, enriched for drug resistance. MIC and disk diameter results were interpreted using United States Food and Drug Administration breakpoints. Overall, 76.8% of the isolates tested were susceptible to imipenem-relebactam by BMD. MIC values for agar dilution, Etest, and MTS were not significantly different from that for BMD, although they tended to be 1 to 2 dilutions higher. Essential agreement was 95.6% for agar dilution, 90.6% for Etest, and 85.2% for MTS. Categorical agreement was 98.0% for agar dilution, 73.1% for disk diffusion, 96.3% for Etest, and 96.6% for MTS. In conclusion, agar dilution and Etest yielded comparable results to BMD for imipenem-relebactam.

Aliarcobacter butzleri from Water Poultry: Insights into Antimicrobial Resistance, Virulence and Heavy Metal Resistance.

Aliarcobacter butzleri is the most prevalent Aliarcobacter species and has been isolated from a wide variety of sources. This species is an emerging foodborne and zoonotic pathogen because the bacteria can be transmitted by contaminated food or water and can cause acute enteritis in humans. Currently, there is no database to identify antimicrobial/heavy metal resistance and virulence-associated genes specific for A. butzleri. The aim of this study was to investigate the antimicrobial susceptibility and resistance profile of two A. butzleri isolates from Muscovy ducks (Cairina moschata) reared on a water poultry farm in Thuringia, Germany, and to create a database to fill this capability gap. The taxonomic classification revealed that the isolates belong to the Aliarcobacter gen. nov. as A. butzleri comb. nov. The antibiotic susceptibility was determined using the gradient strip method. While one of the isolates was resistant to five antibiotics, the other isolate was resistant to only two antibiotics. The presence of antimicrobial/heavy metal resistance genes and virulence determinants was determined using two custom-made databases. The custom-made databases identified a large repertoire of potential resistance and virulence-associated genes. This study provides the first resistance and virulence determinants database for A. butzleri.

Investigation of Antimicrobial Resistance and Virulence Genes of Campylobacter Isolates from Patients in a Tertiary Hospital in Edirne, Turkey

AbstractPurpose:Campylobacter is one of the most common pathogens that cause food-borne infections worldwide. The aim of this study was to determine the antimicrobial resistance rates and the presence of multiple virulence genes in Campylobacter isolates obtained from humans. Materials and Methods: In this study, 71 Campylobacter isolates obtained from human faecal samples were used. Antimicrobial susceptibility tests were performed through the gradient strip method. The presence of virulence genes was investigated by monoplex and multiplex polymerase chain reaction. Results: The rate of resistance of the 66 Campylobacter jejuni isolates was 12.1% for erythromycin, 40.9% for tetracycline and 68.2% for ciprofloxacin. Only one of five Campylobacter coli isolates was resistant to these three antimicrobial agents. The flaB, pldA, cdtA, cadF, cdtC and ceuE genes were found in all 66 of the C. jejuni isolates. In the C. jejuni isolates, positivity rates of 92.4% for flaA, 96.7% for cdtB, 98.5% for ciaB, 90.9% for dnaJ and 96.7% for racR were observed. The flaA, flaB, ciaB, cdtA and cdtC genes were present in all C. coli isolates. Conclusions: It was detected that there is an increase in antimicrobial resistance of Campylobacter strains in our region, and most of the isolates harbour virulence genes.

Genetic Diversity of OXA-like Genes in Multidrug-Resistant Acinetobacter baumannii Strains from ICUs

This study aimed to investigate the genetic diversity of OXA-51-like, OXA-23-like, OXA-24, and OXA-58-like genes and the role of β-lactamases in carbapenem resistance among multidrug resistant Acinetobacter baumannii strains recovered from patients in intensive care units (ICUs). Non-duplicate clinical isolates of A. baumannii from ICUs that were identified as imipenem and meropenem resistant were collected. Antimicrobial susceptibilities were determined by PhoenixTM system (Becton Dickinson, USA). Minimum inhibitory concentrations (MICs) for imipenem and meropenem were determined by using gradient strip method (E-test) and interpreted according to CLSI. Presence of carbapenemase activity was determined by the modified Hodge test (MHT) and detection of metallo-β-lactamase (MBL) was performed by the double-disk synergy test (DDST) and MBL E-test. Detection of the four groups of OXA carbapenemase genes (OXA-23, OXA-24, OXA-51, and OXA-58) was carried out using a multiplex PCR assay. Sequencing of the products in both directions was performed by ABI 3130XL genetic analyzer (Life Technologies Corporation, CA, USA). The resulting DNA sequence was analyzed by the BLAST program, available at the NCBI website. Sixty-one non-duplicate, multidrug resistant clinical A. baumannii isolates were studied. MHTs were positive for all 61 A. baumannii strains, but none of them showed MBL activity. As determined through multiplex PCR, all of the 61 isolates had blaOXA-51 genes including blaOXA-64, blaOXA-66, and blaOXA-91, 50 isolates had blaOXA-23, and 11 isolates had blaOXA-58 genes. Alleles encoding OXA-24-like enzymes were not detected in any isolates. This study indicated that the major cause of carbapenem resistance in our region was OXA-type car-bapenemase encoded by blaOXA-51, blaOXA-23, and blaOXA-58 genes and as we know, this is the first report from Turkey identifying blaOXA-51-like sequences.

Antifungal Susceptibly Testing by Concentration Gradient Strip Etest Method for Fungal Isolates: A Review.

Antifungal susceptibility testing is an important tool for managing patients with invasive fungal infections, as well as for epidemiological surveillance of emerging resistance. For routine testing in clinical microbiology laboratories, ready-to-use commercial methods are more practical than homemade reference techniques. Among commercially available methods, the concentration gradient Etest strip technique is widely used. It combines an agar-based diffusion method with a dilution method that determinates a minimal inhibitory concentration (MIC) in µg/mL. Many studies have evaluated the agreement between the gradient strip method and the reference methods for both yeasts and filamentous fungi. This agreement has been variable depending on the antifungal, the species, and the incubation time. It has also been shown that the gradient strip method could be a valuable alternative for detection of emerging resistance (non-wild-type isolates) as Etest epidemiological cutoff values have been recently defined for several drug-species combinations. Furthermore, the Etest could be useful for direct antifungal susceptibility testing on blood samples and basic research studies (e.g., the evaluation of the in vitro activity of antifungal combinations). This review summarizes the available data on the performance and potential use of the gradient strip method.

Comparison of the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for routine antimicrobial disc diffusion susceptibility testing

ObjectivesThis study investigated the agreement at the categorical level between the Copan WASPLab incorporating the BioRad expert system against the SIRscan 2000 automatic for antimicrobial disc diffusion susceptibility testing. MethodsThe 338 clinical strains (67 Pseudomonas aeruginosa, 19 methicillin-resistant Staphylococcus aureus, 75 methicillin-sensitive S. aureus and 177 Enterobacterales isolates) analysed in this study were non-duplicate isolates obtained from consecutive clinical samples referred to the clinical bacteriology laboratory at Geneva University Hospitals between June and August 2019. For the WASPLab the inoculum suspension was prepared in strict accordance with the manufacturer's instruction (Copan WASP srl, Brescia, Italy) by adding 2 mL of the 0.5 McFarland primary suspension used for the SIRscan analysis into a sterile tube filled with 4 mL of sterile saline (1:3 dilution). The inoculum (2 × 30 μL loop/spreader) was spread over the entire surface of Mueller–Hinton agar plates according to the AST streaking pattern defined by Copan. The antibiotic discs were dispensed by the WASP and inoculated media were loaded on conveyors for transfer to the automatic incubators. The plates were incubated for 16 h, and several digital images were acquired. Inhibition zone diameters were automatically read by the WASPLab and were adjusted manually whenever necessary. For the SIRscan 2000 automatic, the antimicrobial disc diffusion susceptibility testing was performed according to the EUCAST guidelines. The gradient strip method was used to resolve discrepancies. ResultsThe overall categorical agreement between the compared methods reached 99.1% (797/804; 95% CI 98.2%–99.6%), 99.5% (1029/1034; 95% CI 98.9%–99.8%), and 98.8% (2798/2832; 95% CI 98.3%–99.1%) for P. aeruginosa, S. aureus and the Enterobacterales, respectively. ConclusionsWASPLab incorporating the BioRad expert system provides a fully automated solution for antimicrobial disc diffusion susceptibility testing with equal or better accuracy than other available phenotypic methods.