Comparative Evaluation of Colistin-Susceptibility Testing in Carbapenem-Resistant Klebsiella pneumoniae Using VITEK, Colistin Broth Disc Elution, and Colistin Broth Microdilution.

PURPOSE: The study aimed to compare the results of colistin-susceptibility testing performed using the automated VITEK system, colistin broth microdilution (BMD), and colistin broth disk elution (CBDE) methods. MATERIALS AND METHODS: This exploratory study was conducted in a tertiary care center in South India. Carbapenem-resistant Klebsiella pneumoniae (n = 49) isolates collected from a clinical microbiology laboratory over six months (March-September 2023) were used for the study. RESULTS: Among the 49 carbapenem-resistant Klebsiella pneumoniae isolates, 42 were found to be susceptible to carbapenem by all three methods. Seven isolates were found to be resistant to colistin using BMD and CBDE methods. Two isolates were incorrectly detected as colistin-susceptible, and one isolate was wrongly categorized as colistin-resistant using the automated VITEK system. CONCLUSION:  CBDE is a reliable and cost-effective method that can be adopted in the routine microbiology laboratory for colistin-susceptibility testing, as it does not require any specialized equipment or techniques and is 100% consistent with the gold standard BMD method. Although the automated VITEK system is used in most routine microbiological laboratories for antibiotic-susceptibility testing, it cannot be reliably used for colistin-susceptibility testing due to its high error rates (very major error rate of 28.5%; major error rate of 2.4%).

In Vitro Synergistic Effect of Colistin with Fosfomycin Against Carbapenem-Resistant Klebsiella pneumoniae.

BACKGROUND: The dwindling antibiotic reserve owing to augmented drug-resistant bacteria is a major handicap for treating physicians. Klebsiella pneumoniae, a gram-negative encapsulated member of the Enterobacteriaceae family, is one such pathogenic bacteria. Carbapenemase-producing Klebsiella pneumoniae is globally recognized as one of the most critical bacterial threats to public health due to its extremely limited treatment options. Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections pose therapeutic challenges due to simultaneous resistance to various other groups of antibiotics. In this study, we have evaluated the synergistic effect of fosfomycinagainst CRKP isolates when used in combination with colistin by applying the Checkerboard method. METHODS: A laboratory-based prospective study was conducted in the Department of Microbiology, JSS Hospital, Mysuru, for a period of one year after obtaining ethical clearance. Klebsiella pneumoniae isolates obtained from clinical samples were screened for carbapenem resistance by the VITEK-2 compact system (bioMérieux, Marcy-l'Étoile, France). The minimum inhibitory concentration (MIC) of colistin and fosfomycin was individually ascertained by broth microdilution (BMD). Finally, the synergistic activity of the fosfomycin-colistin combination was determined by the BMD-based Checkerboard method. RESULTS: Among the 50 CRKP isolates, 36 (72%) isolates showed synergism, eight (16%) isolates showed indifference and six (12%) isolates showed partial synergism, while none of them showed additivity and antagonism by the Checkerboard method. These results are found to be statistically significant (chi-square value of 116.204 and p-value of < 0.00001). CONCLUSION: This study showed a promising in-vitro synergy between the drugs fosfomycin and colistin by Checkerboard BMD testing protocol. Colistin being a reserve antibiotic, monotherapy comes with the limitations of higher chances of resistance as well as toxicity, which can be overcome by combination therapy, thereby decreasing CRKP-associated mortality rates and delivering holistic patient benefit.

Comparative evaluation of Sensititre YeastOne and CLSI M38-Ed3 reference method for determining echinocandin minimum effective concentrations against Aspergillus isolates.

The minimum inhibitory concentration (MIC) of echinocandins against Aspergillus spp. does not represent the actual inhibition threshold of echinocandins. Therefore, the recommended method to evaluate their activity is determining the minimum effective concentration (MEC) in broth microdilution, a method that is less common in clinical settings. This study aimed to assess a user-friendly commercial method, Sensititre YeastOne (SYO), to determine the effectiveness of echinocandins (caspofungin, anidulafungin and micafungin) against Aspergillus spp. Echinocandins MEC was determined against 23 isolates of Aspergillus spp. using SYO and the reference Clinical and Laboratory Standards Institute (CLSI) method. MECs were read with an inverted microscope and a reading mirror. Essential agreement (EA) between the tested methods was defined as a ±twofold dilution difference. There was a high EA (91%-100%) between the reference method and SYO in determining echinocandins MEC against Aspergillus isolates using inverted microscopy. A high EA was also observed between SYO MEC determined by inverted microscopy and a reading mirror, but different incubation times were required. SYO is a reliable, simple method for determining the MEC of echinocandins against Aspergillus isolates, preferably with an inverted microscope, and can be easily used in clinical laboratories when echinocandin susceptibility testing is required.IMPORTANCEUsing a commercial method such as Sensititre YeastOne (SYO) to determine the minimum effective concentration (MEC) of echinocandins against Aspergillus spp. has been shown to be a reliable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method. This makes it more suitable for high-volume clinical laboratories. SYO provides accurate results comparable to the standard method and could potentially improve patient care by guiding more optimal antifungal treatment choices for patients with Aspergillus infections.

Investigation of Plasmid-Mediated Colistin Resistance Genes (mcr-1-8) in Enterobacterales Isolates.

Background The escalating global rise in multidrug-resistant gram-negative bacteria presents an increasingly substantial threat to patient safety. Over the past decade, carbapenem-resistant Enterobacterales (CRE) have emerged as one of the most critical pathogens in hospital-acquired infections, notably within intensive care units. Colistin has become one of the last-resort antimicrobial agents utilized to combat infections caused by CRE. However, the use of colistin has been accompanied by a notable increase in the prevalence of colistin-resistant bacteria. This study aimed to investigate plasmid-mediated colistin resistance genes ranging from mcr-1 to mcr-8 among members of the Enterobacterales order. Materials and methods This prospective study was conducted in the microbiology laboratory of Afyonkarahisar Health Sciences University Health Research and Practice Center between May 1, 2021 and July 31, 2022. A total of 2,646 Enterobacterales isolates were obtained from all culture-positive clinical samples sent from various clinics. Of these, 79 isolates exhibiting resistance to carbapenem antibiotics were included in the study. Among the 79 isolates, the presence of mcr-1 to mcr-8 genes was investigated in 27 isolates that were shown to be resistant to colistin. The identification of bacteria at the species level and antibiotic susceptibility tests were conducted using the VITEK 2 automated system (bioMérieux, USA). Colistin resistance among Enterobacterales strains exhibiting carbapenem resistance was evaluated using the broth microdilution technique (ComASP™ Colistin, Liofilchem, Italy), in accordance with the manufacturer's instructions. Results In our in vitro investigations, the minimum inhibitory concentration (MIC) values for meropenem were determined to be >8 µg/ml, whereas for colistin, the MIC50 value was >16 µg/ml and the MIC90 value was 8 µg/ml. A total of 27 colistin-resistant strains were identified among the 79 carbapenem-resistant Enterobacterales strains analyzed. The most prevalent agent among colistin-resistant strains was Klebsiella pneumoniae (K. pneumoniae), representing 66.7% of the isolates. This was followed by Proteus mirabilis (P. mirabilis) with 29.6% and Escherichia coli (E. coli) with 3.7%. The colistin resistance rate among carbapenem-resistant strains was found to be 34.2%, with colistin MIC values in strains tested by the broth microdilution method ranging from 4 to >16 µg/ml concentrations. In polymerase chain reaction (PCR) studies, the mcr-1 gene region was successfully detected by real-time PCR in the positive control isolate. Nevertheless, none of the gene regions from mcr-1 to mcr-8 were identified in our study investigating the presence of plasmid-mediated genes using a multiplex PCR kit. Conclusion Although our study demonstrated the presence of increased colistin resistance rates in carbapenem-resistant Enterobacterales isolates, it resulted in the failure to detect genes from mcr-1 to mcr-8 by the multiplex PCR method. Therefore, it is concluded that the colistin resistance observed in Enterobacteriaceae isolates in our region is not due to the mcr genes screened, but to different resistance development mechanisms.

Development of Versatile, thermally stable, flexible, UV-resistant and antibacterial polyvinyl alcohol-Nanodiamonds composite for efficient food packaging.

This research paper reports an enhancement of thermal, optical, mechanical and antibacterial activities of the Polyvinyl alcohol-Nanodiamonds (PVA-NDs) composite required for the food packaging industry. The synthesis of composites was done by the wet processing method. The large surface area of NDs facilitated the robust interaction between the hydroxyl group and macromolecular chains of PVA to enhance the hydrogen bonding of PVA with NDs rather than PVA molecules. Thus, a reduction in PVA diffraction peak intensity was reported. NDs improved the thermal stability by preventing the out-diffusion of volatile decomposition products of PVA. The results also revealed an enhancement in tensile strength (∼60 MPa) and ductility (∼180 %). PVA-NDs composite efficiently blocked the UVC (100 %), most of the part of the UVB (∼85 % above 300 nm), and UVA (∼58 %). Furthermore, enhanced antibacterial activities were reported for PVA-NDs composite against E. coli and S. aureus. NDs accumulated around the bacterial cells prevented essential cellular functions and led to death. Hence, this composite could be a promising candidate for safe, thermally stable, strong, flexible, transparent, UV- resistant antibacterial food packaging material.

Analysis of Susceptibility or Resistance to Antimicrobial Agents.

Here were described the main three methods being used for analysis of antibiotic susceptibility or resistance of Streptococcus suis clinical isolates to antimicrobial agents: the Kirby-Bauer disk diffusion, the epsilometer test (E test), and the broth microdilution test. In each case, procedures, results, and interpretation are described, as well as their advantages or limitations when proceeds.

Skipped wells and scientific error during fosfomycin agar dilution and broth microdilution lead to inconsistent minimal inhibitory concentrations and may be cause for reevaluating testing methods for Klebsiella pneumoniae.

Despite the first-line recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are pressing barriers for optimizing its use for the treatment of non-Escherichia coli Enterobacterales UTI. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for minimal inhibitory concentration (MIC) determination is largely impractical. Using 160 clinical Klebsiella pneumoniae isolates, we sought to understand rates of skipped wells and MIC imprecision in broth microdilution (BMD) and how that compares to rates of error using AD. Though the Clinical and Laboratory Standards Institute refers to the skipped well phenomena in their recommendation against the use of BMD, there is a paucity of data on its frequency. While AD and BMD produced similar MIC50/90 values (32/256 µg/mL for AD and 64/256 µg/mL for BMD), essential agreement was poor. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells at a given concentration, as the most frequent scientific error. Growth in concentrations above the measured MIC occurred in up to 3.3% of wells and was seen within three dilutions of the MIC for BMD. Observation of single colonies either at or beyond the measured MIC for AD was also common and occurred up to 8.3% and 2.5% of the time, respectively. The frequent scientific error in both testing methods should prompt re-evaluation of AD guidelines and expansion of MIC testing methods for fosfomycin susceptibility testing, as poor agreement with another method prone to scientific error should not be the main detractor from BMD use.IMPORTANCEDespite the recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are barriers for optimizing its use. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for MIC determination is largely impractical. The use of broth microdilution (BMD) for fosfomycin testing is not recommended by the Clinical and Laboratory Standards Institute due to unsatisfactory precision and skipped wells-occurrence of no-growth in a single well before the minimal inhibitory concentration (MIC)-and trailing endpoints. We sought to understand rates of skipped wells and growth at concentrations above measured MICs in BMD and how that compares to scientific error using AD. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells for BMD and single colonies at or beyond measured MICs for AD were also common. Frequent scientific error in both methods should prompt re-evaluation of both AD and BMD for fosfomycin susceptibility testing.

Investigation of antifungal susceptibility of Aspergillus species isolated from systemic clinical specimens by different methods.

PURPOSE: Due to the potential for Aspergillus species to cause lethal infections and the rising rates of antifungal resistance, the significance of antifungal susceptibility tests has increased. We aimed to assess the sensitivities of Aspergillus species to amphotericin B (AMB), voriconazole (VOR), itraconazole (ITZ), and caspofungin (CAS) using disk diffusion (DD) and gradient diffusion (GD) methods and compare them with broth microdilution (BMD) as the reference susceptibility method. METHODS: The study involved 62 Aspergillus fumigatus, 28 Aspergillus flavus, and 16 Aspergillus terreus isolates, totaling 106 Aspergillus isolates. BMD and DD methods were performed in accordance with CLSI M38-A2 and CLSI M51-A documents, respectively. The GD method utilized nonsupplemented Mueller Hinton agar (MHA) as the medium. RESULTS: In the BMD method, the lowest minimal inhibitory concentration (MIC)90 or minimal effective concentration (MEC)90 values were observed for VOR and CAS (0.5 µg/mL and 0.06 µg/mL, respectively). AMB and ITZ MIC90 values were both 2 µg/mL. In our comparison of the GD method with the BMD method at ±2 dilution, we observed essential agreement rates of 91.6%, 99.1%, 100%, and 38.6% for AMB, VOR, ITZ, and CAS, respectively. When comparing DD and BMD methods, we found categorical agreement rates of 65.1%, 99.1%, 77.3%, and 100% for AMB, VOR, ITZ, and CAS, respectively. For GD and BMD methods, these rates were 79.2%, 99.1%, 87.8%, and 100%. CONCLUSIONS: Given the high essential and categorical agreement rates, we posit that the GD method is a viable alternative to the BMD method for AMB, ITZ and VOR but not for CAS. In addition, the use of nonsupplemented MHA in the GD method proves advantageous due to its cost-effectiveness and widespread availability compared to other growth media.

Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese.

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Evaluation of the QMAC-dRAST System Version 2.5 for Rapid Antimicrobial Susceptibility Testing of Gram-Negative Bacteria From Positive Blood Culture Broth and Subcultured Colony Isolates.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) for bloodstream infections (BSIs) facilitates the optimization of antimicrobial therapy, preventing antimicrobial resistance and improving patient outcomes. QMAC-dRAST (QuantaMatrix Inc., Korea) is a rapid AST platform based on microfluidic chip technology that performs AST directly using positive blood culture broth (PBCB). This study evaluated the performance of QMAC-dRAST for Gram-negative bacteria using PBCB and subcultured colony isolates, comparing it with that of VITEK 2 (bioMérieux, France) using broth microdilution (BMD) as the reference method. METHODS: We included 141 Gram-negative blood culture isolates from patients with BSI and 12 carbapenemase-producing clinical isolates of Enterobacterales spiked into blood culture bottles. QMAC-dRAST performance was evaluated using PBCB and colony isolates, whereas VITEK 2 and BMD were tested only on colony isolates. RESULTS: For PBCB, QMAC-dRAST achieved 92.1% categorical agreement (CA), 95.3% essential agreement (EA), with 1.8% very major errors (VMEs), 3.5% major errors (MEs), and 5.2% minor errors (mEs). With colony isolates, it exhibited 92.5% CA and 95.1% EA, with 2.0% VMEs, 3.2% MEs, and 4.8% mEs. VITEK 2 showed 94.1% CA and 96.0% EA, with 4.3% VMEs, 0.4% MEs, and 4.3% mEs. QMAC-dRAST yielded elevated error rates for specific antimicrobial agents, with high VMEs for carbapenems and aminoglycosides. The median time to result for QMAC-dRAST was 5.9 h for PBCB samples and 6.1 h for subcultured colony isolates. CONCLUSIONS: The QMAC-dRAST system demonstrated considerable strengths and comparable performance to the VITEK 2 system; however, challenges were discerned with specific antimicrobial agents, underlining a necessity for improvement.

Comparison of BD Phoenix and disk diffusion to broth microdilution for determining cefepime susceptibility among carbapenem-resistant Enterobacterales.

There are increasing reports of carbapenem-resistant Enterobacterales (CRE) that test as cefepime-susceptible (S) or susceptible-dose dependent (SDD). However, there are no data to compare the cefepime testing performance of BD Phoenix automated susceptibility system (BD Phoenix) and disk diffusion (DD) relative to reference broth microdilution (BMD) against carbapenemase-producing (CPblaKPC-CRE) and non-producing (non-CP CRE) isolates. Cefepime susceptibility results were interpreted according to CLSI M100Ed32. Essential agreement (EA), categorical agreement (CA), minor errors (miEs), major errors (MEs), and very major errors (VMEs) were calculated for BD Phoenix (NMIC-306 Gram-negative panel) and DD relative to BMD. Correlates were also analyzed by the error rate-bounded method. EA and CA for CPblaKPC-CRE isolates (n = 64) were <90% with BD Phoenix while among non-CP CRE isolates (n = 58), EA and CA were 96.6%, and 79.3%, respectively. CA was <90% with DD for both cohorts. No ME or VME was observed for either isolate cohort; however, miEs were >10% for CPblaKPC-CRE and non-CP CRE with BD Phoenix and DD tests. For error rate-bounded method, miEs were <40% for IHigh + 1 to ILow - 1 ranges for CPblaKPC-CRE and non-CP CRE with BD Phoenix. Regarding disk diffusion, miEs were unacceptable for all MIC ranges among CPblaKPC-CRE. For non-CP CRE isolates, only IHigh + 1 to ILow - 1 range was acceptable at 37.2%. Using this challenge set of genotypic-phenotypic discordant CRE, the BD Phoenix MICs and DD susceptibility results trended higher (toward SDD and resistant phenotypes) relative to reference BMD results yielding lower CA. These results were more prominent among CPblaKPC-CRE than non-CP CRE.

Carbapenem non-susceptibility overcalling by BD phoenix NMIC-500 panel.

We aimed to assess the accuracy of BD Phoenix for determining carbapenem susceptibility because we observed a decline in carbapenem susceptibility rate from the biannual cumulative data, after we transitioned to the BD Phoenix form Vitek 2 system. Between October 2021 and May 2022, we collected 82 non-duplicated Enterobacterales showing non-susceptible to at least one of the three carbapenems by BD Phoenix. We performed the broth microdilution (BMD) and disk diffusion (DD) according to the CLSI guideline. Compared to BMD, the categorical agreements for ertapenem (ERT), imipenem (IPM) and meropenem (MEPM) was 58.8%, 56.8% and 91.5% for BD Phoenix and it was 85.4%, 89.0%, and 97.6%, respectively, for DD (p value; 0.0001 for ERT and IPM, p value; 0.17 for MEPM). The major errors/minor errors for ERT, IPM, and MEPM were 14.0%/31.7%, 2.94%/40.7%, and 2.56%/6.10%, respectively for BD Phoenix, compared to 0%/14.6%, 0%/9.8%, and 0%/2.5%, for DD. While errors in the BD Phoenix showed tendency towards resistance, those in DD displayed no tendency towards either resistance or susceptibility. With DD, 21 out of the 27 isolates showing susceptible/intermediate/susceptible pattern (ERT/IPM/MEPM) and 13 out of the 16 isolates showing intermediate/susceptible/susceptible pattern (ERT/IPM/MEPM), were correctly categorized by DD. However, for 22 isolates showing resistant/susceptible/susceptible pattern (ERT/IPM/MEPM), only 13 isolates were correctly categorized by DD. In conclusion, to mitigate the risk of overcalling carbapenem non-susceptibility with BD Phoenix, it will be helpful to perform a complementary test using DD and to provide comments on the DD results to clinicians.

Novel automated antifungal susceptibility testing system for yeasts based on dual-detection algorithm of turbidimetry and colorimetry.

Introduction. The increasing prevalence and growing resistance of fungi present a significant peril to public health. There are only four classes of antifungal medicines available today, and few candidates are in clinical trials.Hypothesis/Gap Statement. Rapid and sensitive diagnostic techniques are lacking for most fungal pathogens, and those that do exist are expensive or hard to obtain.Aim. This study aimed to evaluate the feasibility of a novel automated antifungal susceptibility testing system, Fungus AST, in comparison to the broth microdilution method (BMD) recommended by the Clinical and Laboratory Standards Institute (CLSI).Methodology. A total of 101 clinical Candida spp. isolates were collected from the Zengcheng Branch of Nanfang Hospital and subjected to antifungal susceptibility testing. Antifungal susceptibility was assessed using the Fungus AST method and the BMD.Results. In this study, we introduce a novel automated antifungal susceptibility testing system, Fungus AST, which detects the turbidity and/or colour intensity of microdilution wells using a four-wavelength detection technology in real time and is designed to match the growth characteristics of strains over time. Based on our analysis, all reportable ranges of Fungus AST were suitable for clinical fungal isolates in PR China. Within ±twofold dilutions, reproducibility was 100 %. Considering the BMD as a referenced method, ten antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B, 5-flucytosine and nystatin) showed an essential agreement of >95 %. The category agreement of five antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and voriconazole) was excellent at >90 %. One Candida albicans isolate and voriconazole showed a major error (ME) (1.7 %), and no other ME or very ME agents were found.Conclusion. Given the above, it can be argued that the utilization of Fungus AST is a discretionary automated approach. More improvements are needed in Fungus AST compared to the BMD system for a wider range of clinical isolates, including different types of fungi.

Comparative evaluation of Vitek®2 and broth microdilution method for colistin susceptibility testing of Gram-negative isolates from intensive care unit in a tertiary care hospital.

INTRODUCTION: Colistin is the last resort treatment against resistant Gram-negative bacteria, necessitating reliable and rapid means for sensitivity testing of colistin. Automated systems like VITEK®2 are adopted to determine the minimum inhibitory concentration (MIC) due to easy usage. Broth microdilution (BMD) for colistin MIC was suggested by EUCAST and CLSI. OBJECTIVE: To compare and evaluate colistin MIC by BMD and VITEK®2 against Gram-negative organisms from the ICU in a tertiary care hospital. METHOD: Clinically significant organisms isolated from ICU patients were included. MIC was determined using BMD and VITEK®2. Very major error (VME), major error (ME), essential agreement (EA), categorical agreement (CA), positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity were analysed. RESULT: 533 isolates were obtained from blood (435,81.60%), respiratory samples (57,10.70%), pus and exudates (20,3.80%), urine (18,3.40%), and CSF (3,0.60%). The Enterobacterales were K. pneumoniae (185,34.70%) E. coli (73,13.70%) and E. cloacae (26,4.90%) while non-fermenters were A. baumannii (209,39.20%) and P. aeruginosa (40,7.50%). The VITEK®2 sensitivity was >99%; specificity ranged from 14.28 to 52.94%. PPV was 93.81% while NPV was 93.75%. VME ranged from 47 to 100% between isolates. ME was up to 20%. The highest VME was obtained in E. coli (100%). The total EA and CA observed were 68.5% and 99.79% respectively. CONCLUSION: Automated system VITEK®2 failed to detect the resistance in 32 (60%) isolates. The obtained VME and ME values were >3%, which is unacceptable as per the standard guidelines. EA of ≥90% wasn't obtained. Sensitivity for VITEK®2 was >99%, but had low specificity (14.28%). Hence, VITEK®2 is not reliable for colistin susceptibility testing.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of Avibacterium (Av.) paragallinarum, the causative agent of infectious coryza in chickens. For this, a total of 83 Av. paragallinarum isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15 Av. paragallinarum. The visible growth of Av. paragallinarum was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of Av. paragallinarum growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%-100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78 Av. paragallinarum were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene tet(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62 Av. paragallinarum were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes aph(6)-Id, aph(3″)-Ib, blaTEM-1B, catA2, sul2, tet(B), tet(H), and mcr-like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for Av. paragallinarum. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended. IMPORTANCE: Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Intermethod comparability analyses of gepotidacin antimicrobial susceptibility tests using a large collection of globally collected Escherichia coli and Staphylococcus saprophyticus clinical isolates.

Gepotidacin (GSK2140944) is a novel, bactericidal, first in class triazaacenaphthylene bacterial type II topoisomerase inhibitor in development for the treatment of uncomplicated urinary tract infections and gonorrhea. The performance of several antimicrobial susceptibility methods (broth microdilution, gradient diffusion, and disk diffusion) for gepotidacin were evaluated using over 5800 recent Escherichia coli and Staphylococcus saprophyticus clinical isolates. Reference broth microdilution gepotidacin MICs showed an essential agreement of 95.9 % and 98.1 % with MICs by gradient diffusion for E. coli and S. saprophyticus isolates, respectively. Gepotidacin susceptibility using disks produced by 2 manufacturers had good agreement with an R2 values of 0.95 and 99.2 % of overall zone diameters agreeing within 3 mm. A correlation with an overall R2 value of 0.72 between MICs by broth microdilution and zone diameters by disk diffusion was observed. This data should assist in the clinical development of gepotidacin and provide reliable susceptibility methods to evaluate its activity.

Assessing the colony morphotypes and antibiotic susceptibility profile of Malaysian clinical Burkholderia pseudomallei to support the use of EUCAST disk diffusion breakpoints to determine antibiotic resistance.

Burkholderia pseudomallei is intrinsically resistant to many antibiotics. This study aimed to assess bacterial colony morphotypes and the validity of using disk diffusion method (DD) to determine antibiotic resistance in Malaysian clinical B. pseudomallei isolates for ceftazidime (CAZ), meropenem (MEM), amoxicillin-clavulanate (AMC) and doxycycline (DOX). DD produced good categorical agreements exhibiting concordance of 100% with reference method, broth microdilution for CAZ and DOX, 98.6% for MEM and 97.2% for AMC. Smooth-centred colonies were most frequently observed. EUCAST DD interpretative criterion is suitable to interpret B. pseudomallei CAZ, MEM, AMC and DOX resistance. Increasing AMC MIC in B. pseudomallei is a concern.

Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

In vitro activity of ceftobiprole and dalbavancin against a collection of coagulase-negative staphylococci isolates from clinical samples with reduced susceptibility to daptomycin and/or resistant to linezolid or glycopeptides / Evaluación de la actividad in vitro de ceftobiprol y dalbavancina frente a estafilococos coagulasa negativos con sensibilidad disminuida a daptomicina y/o resistentes a linezolid o glucopéptidos aislados de muestras clínicas

Introduction:The aim was to investigate the in vitro activity of ceftobiprole and dalbavancin against a collection of coagulase-negative staphylococci (CoNS) isolates with reduced susceptibility to daptomycin or resistant to linezolid and/or glycopeptides. Methods: A total of 228 CoNS were tested using the Vitek-2 AST-626 cards (bioMérieux) and MIC of daptomycin, linezolid, vancomycin and teicoplanin were confirmed by Etest Strips (bioMérieux). Susceptibility testing for ceftobiprole and dalbavancin were performed by CLSI broth microdilution methodology. Results were interpreted according to 2021 EUCAST clinical breakpoints. Results: Ceftobiprole and dalbavancin were active against 96.0% and 93.0% of CoNS, respectively, MIC90 were 2 and 0.125mg/L. MICs of ceptobiprole were higher against S. hominis and S. haemolyticus (MIC90 4mg/L). Dalbavancin exhibited higher MICs against S. haemolyticus and CoNS with reduced susceptibility to daptomycin and resistant to teicoplanin. Conclusion: Ceftobiprole and dalbavancin demonstrated a high in vitro activity against our collection of CoNS isolates.(AU)

Introducción: El objetivo fue evaluar la actividad in vitro de dalbavancina y ceftobiprol frente a estafilococos coagulasa negativos (ECN) con sensibilidad disminuida a daptomicina y/o resistentes a linezolid o glucopéptidos. Métodos: Se testó la sensibilidad de 228 ECN con tarjetas VITEK®2 AST-626 (bioMérieux) y las CMI de daptomicina, linezolid, vancomicina y teicoplanina fueron confirmadas con tiras Etest® (bioMérieux). El ensayo de sensibilidad frente a ceftobiprol y dalbavancina se realizó mediante microdilución en caldo (metodología CLSI). Los resultados se interpretaron siguiendo los puntos de corte de EUCAST 2021. Resultados: Ceftobiprol y dalbavancina fueron activos en el 96,0 y 93% de ECN, las CMI90 fueron 2 y 0,125mg/L, respectivamente. Las CMI de ceftobiprol fueron superiores en Staphylococcus hominis y Staphylococcus haemolyticus (CMI90 4mg/L). Dalbavancina exhibió mayores CMI en S. haemolyticus y en ECN con sensibilidad disminuida a daptomicina o resistentes a teicoplanina. Conclusión: Ceftobiprol y dalbavancina han demostrado una potente actividad in vitro frente a esta colección de ECN.(AU)

Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram-negative bacilli.

Introduction: The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods - colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates. Methods: Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen's kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction. Results: Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2-84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5-98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids. Conclusion: As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.