Disk Diffusion Antimicrobial Susceptibility Testing Research Articles

Evaluation of Automated Disk Diffusion Antimicrobial Susceptibility Testing Using Radian® In-Line Carousel

Antimicrobial susceptibility testing (AST) by disk diffusion provides an accurate image of bacterial growth, enabling the detection of culture purity, heterogeneous growth, and antibiotic interactions. However, this manual method is time-consuming and visual interpretation is prone to errors. To overcome these disadvantages, the Radian® In-Line Carousel (Copan, Brescia, Italy) was launched, which is a WASPLab® module dedicated to full automation of (pre)-analytical steps as well as interpretation of disk diffusion AST. However, until now, no evaluation of Radian® against manual disk diffusion has been performed. We assessed the categorical agreement (CA) between standardized disk diffusion (reference method) and Radian® using EUCAST 2021 breakpoints. We tested 135 non-duplicate strains, selected from the National EUCAST challenge panel, clinical strains, and external quality controls. The strains included Enterobacterales (n = 63), Enterococcus faecalis (n = 3), Enterococcus faecium (n = 10), Pseudomonas aeruginosa (n = 16), Staphylococcus aureus (n = 19), coagulase-negative staphylococci (n = 4), and Streptococcus spp. (n = 20). Furthermore, we explored antibiotic disk thermolability in the WASP Radian® carousel by testing 10 ATCC® strains up to 7 days. The observed CA was 95.3%, 96.3%, 93.8%, 97.3% and 98.0% for Enterobacterales, Enterococcus spp., P. aeruginosa, Staphylococcus spp. and Streptococcus spp., respectively, resulting in an acceptable overall CA for all groups. (Very) major error rates were ≤ 5% for all antibiotics. Antibiotic disk thermostability was confirmed up to 4 days in the WASP Radian® In-Line Carousel. The Radian® In-Line Carousel provides a fully automated solution for accurate disk diffusion AST, reducing workload and improving standardization and traceability. In addition, our study demonstrated the thermostability of antibiotic disks up to 4 days in the WASP Radian® In-Line Carousel.

Chemical analysis and antibacterial susceptibility of some brands of amoxicillin suspension marketed in Sagamu, Ogun, Nigeria

AbstractPoor quality medicines present a serious public health concern and have a significant impact on national clinical and economic burden. This study was carried out for comparative quality assessment of different brands of amoxicillin suspension in Sagamu community in Nigeria to determine their suitability for the therapeutic purpose. Ten brands of 125 mg/5 mL amoxicillin suspension, marketed in Sagamu community pharmacies were procured and subjected to chemical analysis using the high‐performance liquid chromatography method according to the official procedures in United State Pharmacopoeia (USP) and the microbiological analysis on different clinical isolates which include Pseudomonas aeruginosa, Escherichia coli, Streptococcus pyogene, and Staphylococcus aureus using agar disc diffusion antimicrobial susceptibility testing. The results of the high‐performance liquid chromatography revealed the percentage content of the brands A, B, C, D, E, F, G, H, I, and J to be 97.39%, 57.078%, 91.565%, 98.137%, 61.694%, 103.337%, 91.454%, 106.683%, 75.109%, and 96.895% w/v, respectively. The result of the high‐performance liquid chromatography reveals the brands (A, C, D, F, G, H, and J) had a percentage within the required range and were deemed unsuitable for therapeutic use. In the microbiological analysis, all the brands passed the antibacterial susceptibility testing except for brand (B, E, and I) which were not susceptible to S. aureus due to their zone of inhibition being less than 15 mm. Overall, this study emphasizes the importance of quality assurance in the pharmaceutical industry to ensure that only high‐quality drugs are made available to the population for optimal therapeutic benefit.

Exploiting Image Processing and Artificial Intelligence Techniques for the Determination of Antimicrobial Susceptibility

Antimicrobial susceptibility tests, achieved through the use of antibiotic-impregnated disks in a suitable laboratory environment, are conducted to determine which antibiotics are effective against the bacteria present in the body of an infected patient. The Kirby–Bauer method, a type of disk diffusion antimicrobial susceptibility test, is currently widely applied in microbiology laboratories due to its proven effectiveness. In our study, we developed an algorithm that utilizes image processing techniques to detect the inhibition zones of bacteria. A certain color depth acts as the threshold for the inhibition zone, with its radius determined according to the size of the reference object. This approach facilitates the measurement of inhibition zones and employs machine learning and deep learning to categorize antibiograms, followed by determination of whether a bacterium on the disk is sensitive or resistant to the antibiotics applied. The focus of this research is creating an automated interpretation system for antimicrobial susceptibility testing using the disk diffusion technique, thus simplifying the measurement and interpretation of inhibition zone sizes.

Molecular Analysis of the Antiseptic Resistance Genes among Multidrug-Resistant Pseudomonas aeruginosa Isolated from Burn Patients

Abstract Background: Pseudomonas aeruginosa is known to be susceptible to a variety of antibiotics and disinfectants, and this resistance has become a major problem, particularly among hospitalized patients. Objectives: The present study aims to determine the association between class 1 integrons (intI1) and genes responsible for antiseptic resistance (fabV, qacΔE1, and qacE) among multidrug-resistant (MDR) P. aeruginosa from burn patients. Materials and Methods: Sixty-four isolates from burns were identified using both standard approaches and the VITEK 2 system. Disc diffusion antimicrobial susceptibility testing was performed. Polymerase chain reaction (PCR) was utilized to identify class 1 integrons and antiseptic resistance genes using specific primers for intI1, fabV, qacE1, and qacE. Results: 64 MDR P. aeruginosa isolates were obtained from burn patients during the research period. The antibiotic susceptibility of all 64 P. aeruginosa isolates was examined. MDR resistance to at least three antibiotic classes were found in all of the isolates. The PCR results revealed that 100% of the isolates tested positive for the class 1 integron gene. According to the current findings, all isolates possessed the qacΔE1 and fabV genes. While the qacE results were being processed, 87.50% of the isolates tested positive. There was no correlation between the presence of antiseptic resistance genes and antibiotic resistance (P ˃ 0.05). Conclusion: our data suggest that P. aeruginosa isolates have profound antibiotic resistance and an antiseptic resistance gene. The integron was mostly found in all isolates, indicating that the Antiseptic resistance gene corresponds to the integron class 1.

Antimicrobial resistance, serogroups, virulence gene profiles and MLST of Escherichia coli from giant panda.

Escherichia coli is a major bacterial pathogen which causes diarrhea in the giant panda. This study investigated the biological characteristics of 100 E. coli strains isolated from fecal samples collected from 100 captive giant pandas of different age groups and sexes. A standard Kirby-Bauer disk diffusion antimicrobial susceptibility test was performed with the isolates and we then further evaluated the antibiotic resistance genes (ARGs) by high-throughput quantitative PCR. Additionally, we then analyzed O serogroups through a slide agglutination test, virulence genes and the multi-locus sequence typing (MLST) by PCR. Antimicrobial susceptibility testing demonstrated that the 100 E. coli strains were mainly resistant to ENR (68%), AM (56%), IPM (55%), AMX (54%) and CA (52%), but were susceptible to MEM and FOX. The resistance to TZP, AK, FEP, CAZ, AMS, AZM, AT and IPM was significantly related to age (p < 0.05); the resistance rate of E. coli isolated from female giant pandas to N was significantly higher than in males (p < 0.05). Forty-five different types of ARGs were found, which included a total of 2,258 ARGs, in the 100 E. coli isolates. The top 10 of detection rate of ARGs were: acrA-04, acrA-05, aacC, blaCTX-M-04, ampC-04, blaSHV-01, blaTEM, sul2, blaOXY, tetA-02. ARGs aac (6')I1, blaCTX-M-03, tetD-02, blaSHV-02 and blaOXY were significantly related to age (p < 0.05), blaSHV-02, blaNDM and ampC-04 were related to sex (p < 0.05). Twelve different O serogroups from 32 E. coli isolates were distinguished, including O4, O8, O9, O15, O18, O20, O55, O88, O112, O157, O158, and O167. The most prevalent O serotype was O20, but O28, O45, O101, O149, and O152 were not detected. Fourteen different types of virulence genes were detected in the 100 E. coli isolates, of which papA (99%) were highly detected, while hlyA, elt and estA were not detected. MLST showed that 41 STs, which had one CCs and six groups with SLVs, in the 100 E. coli strains were identified, the main type was ST37. Our results advocate the need of strict biosecurity and surveillance programs in order to prevent the spread of pathogenic bacteria in the captive giant panda population.

Investigating the Presence of Efflux Pump Genes adeI and adeJ in Clinical Antibiotic-Resistant Isolates of Acinetobacter baumannii

Background: Acinetobacter baumannii is one of the most important known causes of hospital infections worldwide that is resistant to many common antibiotics. Efflux pumps are among the main reasons behind resistance in this bacterium. Objectives: This study was conducted to investigate the presence of efflux pump genes (adeI, adeJ) in clinical antibiotic-resistant isolates of A. baumannii in Tehran hospitals. Methods: One hundred fifty clinical samples of wounds, urine, sputum, and blood were collected periodically (6 months) from Tehran hospitals. Acinetobacter baumannii was identified using common biochemical methods. After conducting biochemical tests, the final confirmation of the samples was performed by examining the blaOXA-51-like gene by molecular polymerase chain reaction (PCR) method. The disk diffusion antimicrobial susceptibility testing was performed using Mueller Hinton agar growth medium according to Clinical and Laboratory Standards Institute (CLSI) guidelines on nine antibiotics. Then the samples were investigated for the presence of adeI and adeJ genes. Results: Examining the antibiotic resistance of the isolates showed that the resistance level varied from 48.1% to 98.2%, depending on the antibiotic type. In this study, isolates showed the highest and lowest resistance to tetracycline and gentamicin, respectively. Also, positive isolates for the presence of adeI and adeJ genes showed the highest resistance to tetracycline, amikamycin, ceftazidime, and ceftriaxone. Isolates that were negative for the presence of these two genes showed the highest sensitivity to imipenem, gentamicin, and ciprofloxacin. Conclusions: In this study, the correlation of antibiotic resistance test and PCR results showed that the presence of adeI and adeJ genes in the samples significantly increased the resistance to all investigated antibiotics. Therefore, evaluating efflux pumps proves to be useful in identifying antibiotic-resistant strains and appropriate drug treatment. Of course, the role of other factors in creating resistance should not be neglected.

Assessing the quality of the anaerobic environment — a method developed to support EUCAST disk diffusion of anaerobic bacteria

The purpose of this study is to establish a method for assessing the anaerobic environment for EUCAST disk diffusion antimicrobial susceptibility testing (AST) of anaerobic bacteria on fastidious anaerobe agar with 5% mechanically defibrinated horse blood (FAA-HB). The method utilizes the association between a decrease in the metronidazole disk zone diameter and increasing oxygen levels with an aerotolerant Clostridium perfringens strain DSM 25589 (CCUG 75076 and NCTC 14679). The C. perfringens strain was tested on FAA-HB with a McFarland 1 inoculum and a metronidazole 5 μg disk. FAA-HB was incubated for 16–20 h at 35–37°C. The association between oxygen levels (0, 0.16, 1, 2, and 4% oxygen) and the metronidazole zone diameter was determined. Reproducibility at 0% oxygen was investigated as part of a European multi-centre study of disk diffusion of anaerobic bacteria. The median zone diameters (n=12) at each oxygen level were 29 mm (0%), 21 mm (0.16%), 16 mm (1%), 15 mm (2%), and 15 mm (4%). The metronidazole zone diameters at 0% oxygen from the multi-centre reproducibility-study had a median of 29 mm and a 95%-percentile range of 25–33 mm (n=236). Only one reading was below 25 mm. Based on our results, a zone diameter of ≥25 mm using a metronidazole 5 μg disk and the C. perfringens strain, tested with EUCAST recommendations, can be used to indicate that the anaerobic environment is of sufficient quality for culture and disk diffusion AST. EUCAST has included the method as part of the quality control for AST of anaerobic bacteria.

Performance evaluation of the FAST™ System and the FAST-PBC Prep™ cartridges for speeded-up positive blood culture testing.

ObjectivesAs time to appropriate antimicrobial therapy is major to reduce sepsis mortality, there is great interest in the development of tools for direct identification (ID) and antimicrobial susceptibility testing (AST) of positive blood cultures (PBC). Very recently, the FAST™ System (Qvella) has been developed to isolate and concentrate microorganisms directly from PBCs, resulting in the recovery of a Liquid Colony™ (LC) within 30 min. The LC can be used as equivalent of an overnight subcultured colony for downstream testing. We aimed to evaluate the performances of the FAST™ System and FAST-PBC Prep™ cartridges by testing the resulting LC for direct ID, AST and rapid resistance detection.Materials and methodsProspectively, FAST™ System testing was carried out on each patient’s first PBC with a monomicrobial Gram-stain result. In the second arm of the study, FAST™ System testing was carried out on blood cultures spiked with multidrug-resistant bacteria. Downstream testing using the LC included MALDI-TOF MS ID with the Bruker Biotyper® smart system, rapid resistance detection testing including the Abbott Diagnostics Clearview™ PBP2a SA Culture Colony Test (PBP2a) and the Bio-Rad βLACTA™ Test (βLT). AST was performed using the Becton Dickinson Phoenix™ System or by Bio-Rad disk diffusion using filter paper disk following EUCAST 2020 breakpoint criteria.ResultsFAST™ System testing was completed on 198 prospective PBCs and 80 spiked blood cultures. After exclusion of polymicrobial blood cultures, performance evaluation compared with standard of care results was carried out on 266 PBCs. Concordant, erroneous and no ID results included 238/266 (89.5%), 1/266 (0.4%), 27/266 (10.2%) PBCs, respectively. Sensitivity and specificity for PBP2a were 100% (10/10) and 75% (15/20), respectively. Sensitivity and specificity for βLT were 95.8% (23/24) and 100% (42/42), respectively. Categorical agreement for all 160 tested strains was 98% (2299/2346) with 1.2% (8/657) very major errors and 0.7% (10/1347) major errors.ConclusionFAST™ System testing is a reliable approach for direct downstream testing of PBCs including MALDI-TOF MS ID, BD Phoenix™ and Bio-Rad disk diffusion AST as well as rapid resistance testing assays. Next steps include optimal integration of the FAST™ System in the PBC workflow with a view toward clinical studies.

Evaluation of ten brands of pre-poured Mueller-Hinton agar plates for EUCAST disc diffusion testing

ObjectivesMueller-Hinton agar (MHA) is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing (AST). We have previously investigated the quality of dehydrated MHA from several manufacturers. In this study, we evaluated the performance of ten commercial brands of pre-poured MHA plates. MethodsAST was performed according to EUCAST methodology and results analyzed against targets and ranges in EUCAST quality control (QC) tables. MHA plates from different brands were tested in triplicate against four non-fastidious QC strains. The agar depth and pH were measured for all products. ResultsThe best performance was observed for MHA from Becton Dickinson (BBL MHA II), bioMérieux (MHE agar) and Hardy Diagnostics, for which >97% of zone diameters were within QC ranges and >60% on target ±1 mm. The poorest performance was seen for plates from HiMedia (MHA and MHA no. 2), where 20% and 18% of readings were outside the QC ranges, respectively. The differences in pH and agar depth of the products were small and mostly within EUCAST specifications. DiscussionThe accuracy and reproducibility of disc diffusion AST depends on standardised procedures and high-quality discs and media. The performance among ten brands of pre-poured MHA plates differed significantly. The results indicate a poorer performance for pre-poured commercial plates as compared to in-house prepared plates from dehydrated powder of corresponding brands in our previous study. Manufacturers and clinical laboratories have a shared responsibility for the quality of AST. EUCAST provides QC criteria to be used both by manufacturers and laboratories.

Risk factors associated with the occurrence of multiresistant Staphylococcus spp. isolated from bovine subclinical mastitis in northern Brazil

Staphylococcus bacteria are often associated with subclinical bovine mastitis. This study aimed to identify multiresistant Staphylococcus spp. associated with subclinical mastitis and the associated risk factors. Twenty-three dairy farms with a history of decrease in milk production, located in the lower Acre region, Brazil, were selected. An epidemiological questionnaire was provided in all farms. All animals were examined using the California Mastitis Test (CMT) and their milk samples were collected for bacterial culture. After isolation and identification, the disk diffusion antimicrobial susceptibility test was performed against nine classes of antimicrobials. Of the 339 cows examined using the CMT, 108 had mastitis. A total of 229 milk samples were collected from individual teats. MALDI-TOF MS found isolates belonging to eight species of Staphylococcus, in 101 of these samples. S. chromogenes (58.4%) demonstrated strongest resistance to the nine classes of antimicrobial active principles. Nineteen isolates with multidrug resistance phenotypic profile were identified. This phenotypic expression indicates wide circulation of resistant genes in this species. The presence of multidrug resistance in Staphylococcus spp. in this study was correlated with lack of water for cleaning the corral, which is a preventive factor, minimizing the transmission and persistence of pathogens in the farms.

High Prevalence of Antimicrobial Resistance and Integron Gene Cassettes in Multi-Drug-Resistant Klebsiella pneumoniae Isolates From Captive Giant Pandas (Ailuropoda melanoleuca).

Multi-drug-resistant Klebsiella pneumoniae (MDR K. pneumonia) is increasingly being reported with corresponding increase in morbidity and mortality all over the world. However, limited information is available concerning MDR K. pneumonia in giant pandas. The objective of this study was to grasp the drug resistance profile of MDR K. pneumonia isolated from giant pandas. A total of 182 K. pneumoniae isolates were collected from fresh feces of 94 captive giant pandas of different ages and sex and separated by season. We performed a standard disk diffusion antimicrobial susceptibility test with the isolates and further evaluated the antibiotic resistance genes (ARGs) of multi-drug-resistant strains by high-throughput quantitative PCR. In addition, we then analyzed mobile genetic elements (MGEs), integron gene cassettes, and the multi-locus sequence typing of multi-drug-resistant strains by PCR. Antimicrobial susceptibility testing results demonstrated that a total of 30 (16.5%) K. pneumoniae isolates showed multiple drug resistances. The thirty MDR K. pneumonia isolates were mainly resistant to amoxicillin (100.0%), doxycycline (86.7%), chloramphenicol (60.0%), compound trimethoprim (60.0%) and trimethoprim (56.7%). Fifty different types of antibiotic resistance genes were found, which included a total of 671 antibiotic resistance genes, in the 30 multi-drug-resistant isolates. The top ten resistance genes were: vanTC-02, aacC, blaCTX-M-04, blaSHV-01, blaSHV-02, ampC-04, blaOXY, tetD, blaTEM and tetA-02. Thirteen mobile genetic elements were detected, of which IS26 (96.67%) and intI1 (96.67%) had the highest frequency. The thirty MDR K. pneumonia isolates were negative for the traA, traF, tnsA, IS1133, ISpa7, ISkpn6, intI2 and intI3 genes. Moreover, a further investigation of integrons revealed that two types of specific gene cassettes (dfrA12 + orfF + aadA2 and dfrA12 + orfF) were identified in class 1 integrons. Multi-locus sequence typing results showed that 22 STs in the thirty MDR K. pneumonia isolates were identified, the main type was ST37 (5/30). Our results illustrate that effective surveillance and strict biosecurity strategies should be taken to prevent the spread of multi-drug-resistant bacteria, and monitor the emergence of mobile genetic elements and integrons.

Multi-Drug Resistant (MDR) and carbapenemase co-producing Gram-negative bacilli in northwest of Iran

Carbapenemase enzymes confer clinically significant resistance to Gram-negative bacteria, only a few antibiotics with adverse side effects and/or unfavorable in vivo efficacy remains. This study sought to determine the prevalence of carbapenemase producing gram-negative organisms and also investigate the antibacterial susceptibility profile of carbapenem-resistant isolates. In this study, over a two-year period (2017–2019) a total of 140 imipenem and/or meropenem resistant gram negative bacilli were collected from various specimens sent to microbiology laboratory of university hospital. Disk diffusion antimicrobial susceptibility test was used to evaluate MDR isolates; also multiplex polymerase chain reaction and sequencing were performed for confirmation of carbapenemase production. In disk diffusion method, all isolates were resistant to cefazolin. High rate of resistance was observed against imipenem (98.6%), followed by cefotaxime (97.1%), trimethoprim-sulfamethoxazole (91.4%). Colistin was found as the most effective agent with only 5.7% of resistance, followed by amikacin. Based on PCR results and subsequent sequencing, 46.4% of the isolates were genetically recognized to be carrying at least one carbapenemase gene. Encoded carbapenemases were OXA-48-like (18.6%), VIM-2 (15%), IMP-1 (9.3%), KPC (5.7%), NDM-1 (3.6%), and AIM-1 (2.1%). All isolates were negative for SIM, BIC, SPM, GIM and DIM enzymes. Five isolates were co-harbored blaVIM-2 and blaOXA-48. Moreover, co-harbored carbapenemase genes were observed in six isolates as 6 two-genes-harbored profiles. Our results indicate a relatively high incidence of carbapenemase genes. In addition, the emergence of isolates harboring multiple carbapenemases is of great concern, since these isolates will result in a broad-spectrum antibiotic resistance. Molecular characterization of carbapenemase producers can be helpful tool to offer an effective treatment policy. The worrisome high rate of antibiotic resistance highlights the necessity of active surveillance to antibiotic prescription and\\or self-medication by patients.

Antimicrobial Resistance Patterns of Escherichia coli Isolated from Sheep and Beef Farms in England and Wales: A Comparison of Disk Diffusion Interpretation Methods.

Little data exist on the levels of antimicrobial resistance from bacteria isolated from British sheep and beef cattle. The aim of this study was to investigate antimicrobial resistance patterns on sheep and beef farms in England and Wales using multiple interpretation methods. Fecal samples (n = 350) from sheep and beef cattle were collected from 35 farms. Disk diffusion antimicrobial susceptibility testing against ten antimicrobials was carried out for 1115 (699 sheep, 416 beef) β-glucuronidase-positive Escherichia coli isolates. Susceptibility was interpreted using clinical breakpoints, which determine clinically resistant bacteria, and epidemiological and livestock-specific cut-off values, which determine microbiological-resistant bacteria (non-wild type). Using livestock-specific cut-off values, a high frequency of wild type for all ten antimicrobials was observed in isolates from sheep (90%) and beef cattle (85%). Cluster analysis was performed to identify patterns in antimicrobial resistance. Interpretation of susceptibility using livestock-specific cut-off values showed a cluster of isolates that were non-wild type to cefotaxime and amoxicillin/clavulanic acid, whereas clinical breakpoints did not. A multilevel logistic regression model determined that tetracycline use on the farm and soil copper concentration were significantly associated with tetracycline non-wild type isolates. The results suggest that using human clinical breakpoints could lead to both the under-reporting and over-reporting of antimicrobial resistance in sheep and beef cattle.

Interpreting Burkholderia pseudomallei disc diffusion susceptibility test results by the EUCAST method

Interpreting Burkholderia pseudomallei disc diffusion susceptibility test results by the EUCAST method

Lignin from oil palm empty fruit bunches: Characterization, biological activities and application in green synthesis of silver nanoparticles

Lignin was extracted from oil palm empty fruit bunches under four different conditions. The lignin samples were characterized and employed in the green synthesis of silver nanoparticles. Two-dimensional HSQC NMR analysis showed that lignins extracted under more aggressive conditions (3.5% acid, 60 min) exhibited less signals and thus, presented a more degraded chemical structure. Additionally, those lignins obtained under harsh conditions (3.5% acid, 60 min) exhibited higher antioxidant capacity than those obtained under mild conditions (1.5% acid, 20 min). Formation of lignin-mediated silver nanoparticles was confirmed by color change during their synthesis. The surface plasmon resonance peaks (423–427 nm) in UV–visible spectra also confirmed the synthesis of AgNPs. AgNPs showed spherical shape, polycrystalline nature and average size between 18 and 20 nm. AgNPs, in suspension, presented a negative Zeta potential profile. Lignin was assumed to contribute in the antioxidant capacity exhibited by AgNPs. All AgNPs presented no significant differences on the disk diffusion antimicrobial susceptibility test against E. coli. The minimum inhibitory concentration of HAL3-L AgNPs (62.5 μg·mL−1) was better than other physicochemically produced AgNPs (100 μg·mL−1).

Antimicrobial Resistance among Community-Acquired Uropathogens in Mashhad, Iran.

Background Antimicrobial resistance among community-acquired uropathogens is an emerging concern over the past decades that warrants a continuing reevaluation of the appropriateness of recommended empiric antimicrobial regimens for treatment of urinary tract infections (UTIs). Aims To describe the microbial spectrum and resistance profile of community-acquired uropathogens and predictors of isolation of resistant strains. Methods Between October 2017 and June 2019, individuals who visited the outpatient clinics for diagnosis of UTIs or screening of asymptomatic bacteriuria were included in the study if they were tested for urine culture in one of the three main medical diagnostic laboratories of Mashhad, Iran. The standard disk diffusion antimicrobial susceptibility testing was used, with the Clinical and Laboratory Standards Institute (CLSI) threshold cutoffs for susceptibility of isolated uropathogens. Results Three hundred thirty cases were included with a median age of 47 years. Two hundred seventy-six (83.6%) were female. The most common isolated uropathogens were Escherichia coli in 201 (60.9%) cases and Klebsiella species in 46 (13.9%) cases. E. coli isolates showed the highest rates of susceptibility to nitrofurantoin (89.3%), cefixime (75%), and gentamicin (72.4%). Exposure to antibiotics in the past 3 months was a predictor of resistance to ciprofloxacin (OR: 2.8, 95% CI: 1.33–6.28), and older age was a predictor of resistance to TMP-SMX (OR: 2.1, 95% CI: 1.07–3.97) among E. coli isolates. Conclusion. E. coli and Klebsiella species accounted for about two-thirds of community-acquired uropathogens. In regard to the high susceptibility rates, nitrofurantoin was identified as the first-choice agent for empiric treatment of community-acquired cystitis, while cefixime and gentamicin might be the second-choice alternatives. Ciprofloxacin and TMP-SMX, on the other hand, cannot be considered appropriate agents for empiric therapy of community-acquired UTIs, particularly in those who had exposure to antibiotics in the past 3 months and the elderly.

High prevalence of multi-drug resistances and diversity of mobile genetic elements in Escherichia coli isolates from captive giant pandas.

The purpose of this study was to characterize the antimicrobial resistance produced by mobile genetic elements and integron gene cassettes in Escherichia coli isolated from the feces of captive giant pandas. We performed a standard disk diffusion antimicrobial susceptibility test with 84 E. coli isolates and further evaluated the mobile genetic elements and integron gene cassettes. The antimicrobial susceptibility test demonstrated that 43.37% (36/84) of the isolates showed multiple drug resistances. The E. coli isolates mainly showed resistance to aztreonam (86.90%, 73/84) and amoxicillin/clavulanic acid (80.95%, 68/84). The most frequently observed resistance patterns were ampicillin/amoxicillin-clavulanic acid (13.10%, n=11), and doxycycline/amoxicillin-clavulanic acid (4.76%, n=4). Further analyses detected 11 mobile genetic elements, of which merA (54/84, 64.30%) had the highest frequency. All isolates were negative for intI3, traA, tnpU, traF, tnp513, tnsA, ISkpn7, ISpa7, ISkpn6, and ISCR1. We further analyzed antimicrobial resistance-related integrons among 30 E. coli isolates (the 27 intI1-positive isolates and the 3 intI2-positive isolates); six gene cassette profiles (dfrA17+aadA5, aadA2, dfrA12+aadA2, dfrA1+aadA1, dfrA1, and aadA1) were identified in the 27 intI1-positive isolates, but not in the three intI2-positive ones. Our study sheds light on the prevalence of multiple drug resistances and the diversity of mobile genetic elements in E. coli isolates, and highlights the necessity to monitor antibiotic resistance in more E. coli strains from captive giant pandas.

Increased Azithromycin Susceptibility of Multidrug-Resistant Gram-Negative Bacteria on RPMI-1640 Agar Assessed by Disk Diffusion Testing.

Increasing antibiotic resistances and a lack of new antibiotics render the treatment of Gram-negative bacterial infections increasingly difficult. Therefore, additional approaches are being investigated. Macrolides are not routinely used against Gram-negative bacteria due to lack of evidence of in vitro effectiveness. However, it has been shown that Pseudomonas spp. are susceptible to macrolides in liquid RPMI-1640 and clinical data suggest improvement in patients’ outcomes. So far, these findings have been hardly applicable to the clinical setting due to lack of routine low-complexity antimicrobial susceptibility testing (AST) for macrolides. We therefore optimized and compared broth microdilution and disk diffusion AST. Multidrug-resistant Gram-negative bacteria (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa) were tested for azithromycin susceptibility by disk diffusion and broth microdilution in Mueller–Hinton and RPMI-1640 media. Azithromycin susceptibility of Enterobacteriaceae and a subgroup of P. aeruginosa increased significantly on RPMI-1640 agar compared to Mueller–Hinton agar. Further, a significant correlation (Kendall, τ, p) of zone diameters and minimal inhibitory concentrations (MICs) was found on RPMI-1640 agar for E. coli (−0.4279, 0.0051), E. cloacae (−0.3783, 0.0237) and P. aeruginosa (−0.6477, <0.0001). Performing routine disk diffusion AST on RPMI-1640 agar may lead to the identification of additional therapeutic possibilities for multidrug-resistant bacterial infections in the routine clinical diagnostic setting.

EUCAST evaluation of 21 brands of Mueller–Hinton dehydrated media for disc diffusion testing

ObjectivesMueller–Hinton (MH) agar is recommended by EUCAST and CLSI for disc diffusion antimicrobial susceptibility testing. Using EUCAST methodology, we evaluated the performance of 21 internationally available brands of dehydrated MH agar from 17 manufacturers. MethodsMH plates were prepared in-house and evaluated against four quality control (QC) strains tested in triplicate, using EUCAST disc diffusion methodology. This resulted in 30 disc–QC strain combinations and 90 readings per MH brand. All brands were tested blindly and in parallel. Results were evaluated against targets and ranges in the EUCAST QC tables. The agar depth, pH and concentration of five cations were measured for all brands. ResultsSix brands of MH agar (Bio-Rad, Biolife, Oxoid, Sigma MH 2, BD BBL MH II and CRITERION) demonstrated excellent performance, with ≥99% of zone diameter readings within QC ranges and ≥70% on target ±1 mm. The poorest performance was seen for Biolab and Merck MH, with 10% (9/90) and 23% (21/90) of readings outside the QC ranges, respectively. Of all readings, 4.9% (93/1890) were out of range, mainly related to trimethoprim sulfamethoxazole (n = 25), aminoglycosides (n = 25) and fluoroquinolones (n = 15). The cation content differed considerably between the agars, and for four brands pH values were outside the acceptable range 7.2–7.4. DiscussionsThis study evaluated the performance and content of 21 brands of MH dehydrated media. Six brands showed excellent performance with all investigated antimicrobial classes. Others exhibited problems with one or more classes of agents. This could partly be explained by differences in concentration of specific chemical components and pH.

Breakpoint beware: reliance on historical breakpoints for Enterobacteriaceae leads to discrepancies in interpretation of susceptibility testing for carbapenems and cephalosporins and gaps in detection of carbapenem-resistant organisms.

Carbapenem-resistant Enterobacteriaceae (CRE) are an important public health and infection prevention threat. CRE are typically detected via phenotypic antimicrobial susceptibility testing (AST), for which interpretive standards were modified in recent years. Our objective was to measure the impact of breakpoint changes on AST interpretation for CRE. Zone sizes from disk diffusion AST for Enterobacteriaceae isolates recovered from clinical cultures over a 1-year period (n = 10,183) and CRE from clinical and environmental sources from the USA and Pakistan (n = 342) were evaluated. Results were interpreted according to historical (CLSI M100-S19) and current (CLSI M100-S29) breakpoints. Interpretive errors were calculated according to the FDA definitions. Using current breakpoints as the reference standard, 56 (17%) very major (false susceptibility) errors occurred for cefepime and 13 (45%) very major errors for meropenem interpretation using historical breakpoints in clinical isolates of Enterobacteriaceae, corresponding to 12 carbapenemase-producing CRE that would have been missed during the 1-year period. For confirmed blaKPC CP-CRE clinical and environmental isolates (n = 149), the very major error rate for historic breakpoints was 8%, 30%, 63%, and 0% for cefepime, meropenem, imipenem, and ertapenem, respectively. For blaKPC isolates, the use of historical breakpoints would have led to 42 (28%) reports of false susceptibility to meropenem. Failure to adopt updated AST breakpoints may lead to reports of false susceptibility for antimicrobials commonly used to treat Gram-negative infections and preclude recognition of CRE. Such errors could negatively impact patient care and hamper infection control and public health efforts.