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.

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.

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.

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.

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.

Antimicrobial Resistance Laboratory Network's multisite evaluation of the ThermoFisher Sensititre GN7F broth microdilution panel for antimicrobial susceptibility testing.

In 2017, the Centers for Disease Control and Prevention (CDC) established the Antimicrobial Resistance Laboratory Network to improve domestic detection of multidrug-resistant organisms. CDC and four laboratories evaluated a commercial broth microdilution panel. Antimicrobial susceptibility testing using the Sensititre GN7F (ThermoFisher Scientific, Lenexa, KS) was evaluated by testing 100 CDC and Food and Drug Administration AR Isolate Bank isolates [40 Enterobacterales (ENT), 30 Pseudomonas aeruginosa (PSA), and 30 Acinetobacter baumannii (ACB)]. We assessed multiple amounts of transfer volume (TV) between the inoculum and tubed 11-mL cation-adjusted Mueller-Hinton broth: 1 µL [tribe Proteeae (P-tribe) only] and 10, 30, and 50 µL, resulting in respective CFU per milliter of 1 × 104, 1 × 105, 3 × 105, and 5 × 105. Four TV combinations were analyzed: standard (STD) [1 µL (P-tribe) and 10 µL], enhanced standard (E-STD) [1 µL (P-tribe) and 30 µL], 30 µL, and 50 µL. Essential agreement (EA), categorical agreement, major error (ME), and very major error (VME) were analyzed by organism then TVs. For ENT, the average EA across laboratories was <90% for 7 of 15 ß-lactams using STD and E-STD TVs. As TVs increased, EA increased (>90%), and VMEs decreased. For PSA, EA improved as TVs increased; however, MEs also increased. For ACB, increased TVs provided slight EA improvements; all TVs yielded multiple VMEs and MEs. For ENT and ACB, Minimum inhibitory concentrations (MICs) trended downward using a 1 or 10 µL TV; there were no obvious MIC trends by TV for PSA. The public health and clinical consequences of missing resistance warrant increased TV of 30 µL for the GN7F, particularly for P-tribe, despite being considered "off-label" use.

Determination of Epidemiological Cutoff Values for Antimicrobial Resistance of Enterococcus cecorum.

Enterococcus cecorum, a commensal Gram-positive bacterium of the chicken gut, has emerged as a worldwide cause of lameness in poultry, particularly in fast-growing broilers. It is responsible for osteomyelitis, spondylitis, and femoral head necrosis, causing animal suffering, mortality, and antimicrobial use. Research on the antimicrobial resistance of E. cecorum clinical isolates in France is scarce, and epidemiological cutoff (ECOFF) values are unknown. To determine tentative ECOFF (COWT) values for E. cecorum and to investigate the antimicrobial resistance patterns of isolates from mainly French broilers, we tested the susceptibility of a collection of commensal and clinical isolates (n = 208) to 29 antimicrobials by the disc diffusion (DD) method. We also determined the MICs of 23 antimicrobials by the broth microdilution method. To detect chromosomal mutations conferring antimicrobial resistance, we investigated the genomes of 118 E. cecorum isolates obtained mainly from infectious sites and described previously in the literature. We determined the COWT values for more than 20 antimicrobials and identified two chromosomal mutations explaining fluoroquinolone resistance. The DD method appears better suited for detecting E. cecorum antimicrobial resistance. Although tetracycline and erythromycin resistances were persistent in clinical and nonclinical isolates, we found little or no resistance to medically important antimicrobials.

Contemporary Considerations for Establishing Reference Methods for Antibacterial Susceptibility Testing.

Antibacterial susceptibility testing (AST) is performed to guide therapy, perform resistance surveillance studies, and support development of new antibacterial agents. For 5 decades, broth microdilution (BMD) has served as the reference method to assess in vitro activity of antibacterial agents against which both novel agents and diagnostic tests have been measured. BMD relies on in vitro inhibition or killing of bacteria. It is associated with several limitations: it is a poor mimic of the in vivo milieu of bacterial infections, requires multiple days to perform, and is associated with subtle, difficult to control variability. In addition, new reference methods will soon be needed for novel agents whose activity cannot be evaluated by BMD (e.g., those that target virulence). Any new reference methods must be standardized, correlated with clinical efficacy and be recognized internationally by researchers, industry, and regulators. Herein, we describe current reference methods for in vitro assessment of antibacterial activity and highlight key considerations for the generation of novel reference methods.

Toward a Method for Harmonized Susceptibility Testing of Mycoplasma bovis by Broth Microdilution.

Mycoplasma bovis is a fastidious pathogen of cattle causing massive economic losses in the calf and dairy industries worldwide. Since there is no approved standard method for antimicrobial susceptibility testing (AST) of M. bovis, the Clinical and Laboratory Standards Institute has requested the development of a suitable method. Therefore, this study aimed at developing a method for harmonized broth microdilution AST of M. bovis. For this, 131 M. bovis field isolates and M. bovis strain DSM 22781T were collected and macrorestriction analysis was performed to select 15 epidemiologically unrelated M. bovis strains for method validation steps. To select a suitable broth for AST of M. bovis, growth determinations were performed using five media and growth curves were compiled. Then, susceptibility testing was performed considering the exact (precondition of five identical MICs) and essential (MIC mode, accepting a deviation of ±1 dilution step) MIC agreements to evaluate the reproducibility of MIC values using a panel of 16 antimicrobial agents. Subsequently, the remaining field isolates were tested and the suitability of quality control (QC) strains was assessed. Growth experiments showed that SP4 broth was the only one of the five media that yielded sufficient growth of M. bovis. Therefore, it was selected as the test medium for AST and homogeneous MIC values were obtained (exact and essential agreements of 36 to 100% and 92 to 100%, respectively). For all other isolates tested, easy-to-read MIC endpoints were determined with this medium. High overall MIC50 and/or MIC90 values were observed for aminoglycosides and macrolides, and some isolates showed elevated MICs of fluoroquinolones, gentamicin, and/or tiamulin. Since the MICs of four commonly used QC strains were partially not within their ranges, a 20-fold MIC testing of M. bovis DSM 22781T was performed and met the criteria for a new QC strain. For harmonized AST of M. bovis, SP4 broth seems to be suitable with an incubation time of 72 ± 2 h and further validation of M. bovis DSM 22781T as a future QC strain is recommended.

Fungistatic Effect of Phthalide Lactones on Rhodotorula mucilaginosa.

Currently, there is an increasing number of cases of fungal infections caused by opportunistic strains of the yeast Rhodotorula mucilaginosa, mainly in immunocompromised patients during hospitalization. The excessive use of antibiotics and azole compounds increases the risk of resistance to microorganisms. A new alternative to these drugs may be synthetic phthalide lactones with a structure identical to or similar to the natural ones found in celery plants, which show low toxicity and relatively high fungistatic activity. In the present study, the fungistatic activity of seven phthalide lactones was determined against R. mucilaginosa IHEM 18459. We showed that 3-n-butylidenephthalide, the most potent compound selected in the microdilution test, caused a dose-dependent decrease in dry yeast biomass. Phthalide accumulated in yeast cells and contributed to an increase in reactive oxygen species content. The synergistic effect of fluconazole resulted in a reduction in the azole concentration required for yeast inhibition. We observed changes in the color of the yeast cultures; thus, we conducted experiments to prove that the carotenoid profile was altered. The addition of lactones also triggered a decline in fatty acid methyl esters.

Reassessment of the Broth Microdilution Method for Susceptibility Testing of Helicobacter pylori.

BACKGROUND: Helicobacter pylori infection is an infectious disease and thus the eradication treatment should be guided by susceptibility testing. This study aimed to assess the applicability of broth microdilution as a routine susceptibility testing method for H. pylori. METHODS: Susceptibility profiles of clarithromycin (CLR) and levofloxacin (LEV) resistance in 76 clinical H. pylori isolates were simultaneously assessed using agar dilution and broth microdilution methods. The correlation between the minimum inhibitory concentrations (MICs) obtained by the 2 methods was assessed by means of linear regression analysis. RESULTS: The correlation between the MICs determined by broth microdilution method and agar dilution method was good for both CLR (r = 0.966) and LEV (r = 0.959). The susceptibility agreement between the 2 methods was 100% for CLR and 96.1% for LEV. Using the broth microdilution method, the false resistance was found in 3.9% (3 of 76) strains for LEV susceptibility testing. No false susceptibility was found for either CLR or LEV, and no false resistance was found for susceptibility testing of CLR. CONCLUSIONS: The broth microdilution method is suitable for routine susceptibility testing of clinical H. pylori isolates.

Evaluation of Piperacillin-Tazobactam ETEST for the Detection of OXA-1 Resistance Mechanism among Escherichia coli and Klebsiella pneumoniae.

Globally, piperacillin-tazobactam resistance among Escherichia coli and Klebsiella pneumoniae is driven by OXA-1 beta-lactamases. Expression of blaOXA-1 yields piperacillin-tazobactam MICs of 8 to 16 µg/mL, which straddle the susceptible/susceptible-dose dependent breakpoint set by the Clinical and Laboratory Standards Institute in 2022. Variability of the reference broth microdilution method (BMD) was evaluated by manufacturing BMD panels using 2 brands of piperacillin, 2 brands of tazobactam and 2 brands of cation-adjusted Mueller-Hinton broth. In addition, ETEST, which harbors an intermediate dilution of 12 µg/mL was evaluated for the ability to differentiate isolates with and without blaOXA-1. A collection of 200 E. coli and K. pneumoniae, of which 82 harbored a blaOXA-1 gene, were tested. BMD variability was on average 1.3-fold, within the accepted 2-fold variability of MICs. However, categorical agreement (CA) between BMD reads was 74.0% for all isolates and 63.4% for those with a blaOXA-1 gene and 81.3% for those without blaOXA-1 detected (P = 0.004, Pearson's Chi Square). ETEST overall CA with the BMD mode was 68.0% and essential agreement (EA) was 80.5%. For isolates with blaOXA-1, CA was 50.0% and EA was 69.5%, versus 80.5% and 88.1%, respectively, for isolates without blaOXA-1 (P < 0.0001 for both comparisons). All ETEST errors were major errors (false resistance) compared to BMD mode. However, the negative predictive value of the ETEST for the presence of blaOXA-1 was 94.1%, compared to only 74.2% negative predictive value for BMD. Clinicians and microbiologists should be aware of the challenges associated with testing piperacillin-tazobactam in regions where blaOXA-1 is prevalent.

Multilaboratory Comparison of Omadacycline MIC Test Strip to Broth Microdilution MIC against Gram-Negative, Gram-Positive, and Fastidious Bacteria.

The performance of the Liofilchem omadacycline MIC Test Strip (MTS) was evaluated in a multisite study. Three testing sites collected/tested clinical isolates and one site tested challenge isolates that totaled 175 S. aureus, 70 S. lugdunensis, 121 E. faecalis, 100 E. faecium, 578 Enterobacterales, 142 Haemophilus spp., 181 S. pneumoniae, 45 S. anginosus group, 35 S. pyogenes,and 20 S. agalactiae. MIC testing was performed by CLSI broth microdilution (BMD) and MTS. Fastidious isolates testing included BMD and MTS testing with both CLSI and EUCAST Mueller-Hinton Fastidious (MH-F). In addition, each site performed reproducibility for nonfastidious and fastidious isolates and QC by MTS and BMD. All BMD and MTS results for the QC strains were within expected ranges, with exception of one MTS HTM result for H. influenzae ATCC 49247. Among reproducibility isolates, omadacycline MTS results were within one dilution of the modal MIC for 95.2% of nonfastidious Gram-positive, 100% of Gram-negative, 99.3% and 98.5% of fastidious isolates tested on CLSI and EUCAST media, respectively. MTS results for all study isolates were within one doubling dilution of the CLSI BMD MIC for 98.9% of S. aureus, 100% of S. lugdunensis, 98.3% of E. faecalis, 100% of E. faecium, and 99.6% of Enterobacterales. Essential agreement rates for CLSI and EUCAST MH-F agar compared to CLSI BMD were 98.2% and 98.2%, for H. influenzae, 91.1% and 73.6%, for S. pneumoniae and 100% and 85-91.7% for other streptococcus species, respectively. Based on CLSI media, all categorical errors were minor errors and categorical agreement rates were >90% with exception of C. freundii, S. lugdunensis, E. faecalis, S. anginosus and S. constellatus.

Proposal of a Method for Harmonized Broth Microdilution Antimicrobial Susceptibility Testing of Avibacterium gallinarum.

Avibacterium (Av.) gallinarum is an opportunistic pathogen in poultry, which, however, has also been associated with human disease. There is currently no approved method for antimicrobial susceptibility testing of this pathogen, so this study aimed at developing a harmonized broth microdilution method for Av. gallinarum that is suitable for diagnostic laboratories. For this, the Av. gallinarum CCUG 12391T type strain and 42 field isolates were collected and their species was confirmed by using a species-specific PCR assay and biochemical reactions. To select epidemiologically unrelated isolates, ApaI macrorestriction analysis was performed. Preliminary growth experiments were conducted with six culture media, and based on the results, four media were selected to compile growth curves with four isolates. Independent repetitions of MIC determinations were then performed to evaluate the reproducibility of the values. Cation-adjusted Mueller-Hinton broth (CAMHB) was initially selected as broth medium, but did not show sufficient homogeneity of MICs. Therefore, CAMHB plus 1% chicken serum and 0.0025% NADH was selected and showed a good homogeneity of MICs after 20 h and 24 h of incubation at 35 ± 2°C. This was reflected in essential MIC agreements ranging between 96% and 100%. Testing of a larger Av. gallinarum collection (n = 43) revealed that easily readable MICs could be obtained for the type strain and all isolates. Some Av. gallinarum showed elevated MICs of enrofloxacin (n = 35), nalidixic acid (n = 35), penicillin (n = 2), tetracycline (n = 19), and/or trimethoprim-sulfamethoxazole (n = 1). By using PCR analyses, the following antimicrobial resistance genes were detected: blaTEM, dfrA14, sul2, tet(B), tet(H). The study demonstrated that the proposed medium is suitable for a harmonized broth microdilution susceptibility testing of Av. gallinarum with a recommended incubation time of 20 to 24 h.

Comparative Analysis of Bacillus cereus Group Isolates' Resistance Using Disk Diffusion and Broth Microdilution and the Correlation between Antimicrobial Resistance Phenotypes and Genotypes.

Bacillus cereus group isolates (n = 85) were screened for phenotypic resistance to 18 antibiotics using broth microdilution and CLSI M45 Bacillus spp. breakpoints. The susceptibility to 9 out of 18 antibiotics was also tested using disk diffusion method and M100 Staphylococcus spp. breakpoints when available. Overall, a high prevalence of susceptibility to clinically relevant antibiotics was identified using broth microdilution. For most tested antibiotics, a poor correlation was found between zones of inhibition and MICs. Using the broth microdilution results as a reference for comparison, we identified high error rates and low categorical agreement between results produced using disk diffusion and broth microdilution for the seven tested antibiotics with defined breakpoints. This suggests that disk diffusion should be avoided for AST of B. cereus group isolates. Further, we detected antimicrobial resistance genes with ARIBA and ABRIcate to calculate the sensitivity and specificity for predicting phenotypic resistance determined using broth microdilution based on the presence of detected antimicrobial resistance genes (ARGs). ARGs with poor sensitivity and high specificity included rph (rifampicin, 0%, 93%), mph (erythromycin, 0%, 99%), bla1 (penicillin, 29%, 100%), and blaZ (penicillin, 56%, 100%). Compared to penicillin, bla1 and blaZ had lower specificity for the prediction of ampicillin resistance. Overall, none of the ARGs had both high sensitivity and specificity, suggesting the need for further study of the mechanisms underlying phenotypic antimicrobial resistance in the B. cereus group. IMPORTANCE Bacillus cereus group includes human pathogens that can cause severe infections requiring antibiotic treatment. Screening of environmental and food isolates for antimicrobial resistance can provide insight into what antibiotics may be more effective therapeutic options based on the lower prevalence of resistance. Currently, the comparison of antimicrobial susceptibility testing results using the disk diffusion method is complicated by the fact that many previous studies have used Staphylococcus spp. breakpoints to interpret their results. In this study, we compared the results of disk diffusion interpreted using the Staphylococcus spp. breakpoints against the results of broth microdilution interpreted using Bacillus spp. breakpoints. We demonstrated that the disk diffusion method does not produce reliable results for B. cereus group isolates and should therefore be avoided. This study also provides new insight into poor associations between the presence of antimicrobial resistance genes and resistance phenotypes for the B. cereus group.

Pygidial glands of the blue ground beetle Carabus intricatus: chemical composition of the secretion and its antimicrobial activity.

Pygidial gland secretions are used as repellent defensive allomones in ground beetles. We provide the first precise data on the chemical composition and antimicrobial potency of the secretion of the blue ground beetle, as well as on the morphology of its pygidial glands. The latter structures were not previously studied chemoecologically and morphologically, and we hypothesized that their secretion may have some antimicrobial action, as is the case with certain Carabus species. Gas chromatography-mass spectrometry (GC-MS) was used to identify methacrylic and angelic acids as dominant chemicals in the secretion from individuals of three populations of the blue ground beetle in Serbia. We tested its secretion against selected strains of medically important microorganisms. The secretion exibits antimicrobial action against certain bacterial species and all tested micromycetes. The most significant antifungal effect of the secretion was against Penicillium ochrochloron, which is more sensitive to the secretion than to commercial antifungal drugs ketoconazole and bifonazole. Bifonazole achieved minimum inhibitory concentrations against Trichoderma viride at more than three times higher value than did the secretion, indicating a significant antifungal effect of the secretion against this micromycete as well. Additionally, we tested commercially available standards of two dominant chemicals in the secretion to investigate their interaction and antimicrobial role in the secretion. Finally, we describe all glandular morpho-functional units of the blue ground beetle. Our results suggest that the secretion of the blue ground beetle may serve not only defensive but also antimicrobial functions, which likely aid the survival of this beetle in the microbial-rich forest litter habitat.

Evaluation of the broth microdilution plate methodology for susceptibility testing of Mycobacterium tuberculosis in Peru.

BACKGROUND: Tuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru. METHODS: MTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods. RESULTS: MIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179). CONCLUSIONS: The BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

In vitro susceptibility of Mycoplasma iowae isolates to antimicrobial agents.

ABSTRACTMycoplasma iowae, a potential re-emerging avian pathogen mainly affecting turkeys, has been reported from many parts of the world. Poor hatchability, embryonic death, joint and skeletal abnormalities, poor ossification, runting-stunting, poor feathering and airsacculitis may be observed in infected flocks. The reduction of the severity of clinical signs and short-term control of M. iowae are performed by antibiotic treatment. However, M. iowae develops resistance more rapidly and is considered to be more resistant to antimicrobials than other avian pathogenic mycoplasmas. The aim of the present study was to determine the in vitro susceptibility of 101 M. iowae isolates and strains to ten clinically important antimicrobial agents, and to analyse and compare the susceptibility patterns of isolates of various origins and from a wide time-period. The examined reference strains showed high susceptibility to all antimicrobials except for spectinomycin. Low concentrations of tiamulin, florfenicol and oxytetracycline inhibited the growth of the clinical isolates. Nevertheless, slow tendency of increasing minimum inhibitory concentration (MIC) values was observed over time in the case of the above mentioned agents, while MIC values of enrofloxacin showed relatively rapid changes. Spiramycin, erythromycin, tilmicosin, tylosin, lincomycin and spectinomycin did not inhibit the bacterial growth in most of the cases. Isolates originating from captive game birds showed similar susceptibility profiles to isolates from industrial turkey hosts. The widely detected low susceptibility of M. iowae isolates to macrolides, lincomycin and spectinomycin, and the increase of MIC values of frequently used antimicrobials against this pathogen, emphasize the importance of targeted antibiotic therapy.RESEARCH HIGHLIGHTSAntimicrobial susceptibilities of 101 Mycoplasma iowae isolates were determined.Minimum inhibitory concentrations were determined by broth micro-dilution method.Tiamulin, oxytetracycline and florfenicol showed low MIC values.Isolates rapidly adapted to antimicrobial pressure.

Colistin resistance screening by 3 µg/ml colistin agar in Carbapenemase-producing Enterobacterales.

BACKGROUND: In low- and middle-income countries, the use of colistin in therapeutic regimens is common, to treat infections produced for Carbapenemase-producing Enterobacterales (CPE) due to limited access to the recently discovered-approved antibiotics. Furthermore, the technical limitations to perform colistin susceptibility tests make it difficult to assess the suitability of this treatment for each patient, as well as to monitor the rates of resistance. In the present study, we describe the use of agar dilution using a unique colistin concentration of 3 µg/ml to discriminate isolates with colistin resistance in CPE obtained from clinical samples. METHODS: Clinical Laboratory Standards Institute (CLSI) colistin broth microdilution method and dilution agar with a colistin concentration of 3 µg/ml were performed in 168 isolates of CPE obtained from clinical samples in Guayaquil, Ecuador. Broth microdilution was considered our gold standard using CLSI breakpoints as reference (≤2 µg/ml intermediate and ≥4 µg/ml resistant). Categorical agreement was defined as obtaining a reading within the same category with both methodologies. RESULTS: Isolates obtained from respiratory samples were the most prevalent (26.19%; n = 44). Klebsiella pneumoniae was the predominant specie (94.04%; n = 158). KPC-like carbapenemase was present in all the isolates, and interestingly, colistin resistance was not mediated by MCR-1 production. Categorical agreement between both methods resulted in 97.02%. CONCLUSION: We propose the use of dilution agar with a colistin concentration of 3 µg/ml, as a valid method for screening colistin resistance in low- and middle-income countries to monitor resistance and to perform epidemiological studies.

Azoles resistance of candida species causing vulvo-vaginitis in reproductive age women at a tertiary care setting.

Objectives: To study prevalence and resistance pattern to azoles of candida species causing vulvovaginitis in reproductive age women. Methods: Samples were collected from Hayatabad Medical Complex from November 2018 to May 2019. This cross-sectional study was conducted at the department of Microbiology, Institute of Pathology and Diagnostic Medicine, Khyber Medical University Peshawar, Pakistan. A total of 369 high vaginal swabs were collected. Candida was isolated by vaginal swabs inoculation on Sabouraud's dextrose agar (SDA). Colonies on SDA were inoculated on Candida CHROM agar to identify candida species. Wet film microscopy and Gram staining were performed. Biochemical identification was done with 20C AUX. Antifungal susceptibility testing was done by disc diffusion and broth micro-dilution methods to find the resistance pattern of azole drugs. Fluconazole, Clotrimazole, Miconazole, Voriconazole and Itraconazole were the azoles drugs used. Results: Among 43%(n=158) positive candida cases, 44%(n=85) were non pregnant women while 41%(n=73) were pregnant. The Candida species distribution of 158 isolates was as follow; Candida albicans (C. albicans) 46.2%(n=73), Candida krusei (C. krusei) 29.1%(n=46), Candida parapsilosis (C. parapsilosis) 19%(n=30) and Candida glabrata (C. glabrata) 5.7%(n=9). Overall Candida isolates were highly resistant 72%(n=113) to Fluconazole while least resistant 21.5 % (n=34) to Itraconazole. Conclusion: C. albicans is the most prevalent specie involved in Vulvovaginal candidiasis. Candida species were found to be least resistant to Itraconazole followed by Voriconazole, Miconazole, Clotrimazole and Fluconazole.