Quantitative Susceptibility Testing Research Articles

Quantitative drug susceptibility testing for Mycobacterium tuberculosis using unassembled sequencing data and machine learning.

There remains a clinical need for better approaches to rapid drug susceptibility testing in view of the increasing burden of multidrug resistant tuberculosis. Binary susceptibility phenotypes only capture changes in minimum inhibitory concentration when these cross the critical concentration, even though other changes may be clinically relevant. We developed a machine learning system to predict minimum inhibitory concentration from unassembled whole-genome sequencing data for 13 anti-tuberculosis drugs. We trained, validated and tested the system on 10,859 isolates from the CRyPTIC dataset. Essential agreement rates (predicted MIC within one doubling dilution of observed MIC) were above 92% for first-line drugs, 91% for fluoroquinolones and aminoglycosides, and 90% for new and repurposed drugs, albeit with a significant drop in performance for the very few phenotypically resistant isolates in the latter group. To further validate the model in the absence of external MIC datasets, we predicted MIC and converted values to binary for an external set of 15,239 isolates with binary phenotypes, and compare their performance against a previously validated mutation catalogue, the expected performance of existing molecular assays, and World Health Organization Target Product Profiles. The sensitivity of the model on the external dataset was greater than 90% for all drugs except ethionamide, clofazimine and linezolid. Specificity was greater than 95% for all drugs except ethambutol, ethionamide, bedaquiline, delamanid and clofazimine. The proposed system can provide quantitative susceptibility phenotyping to help guide antimicrobial therapy, although further data collection and validation are required before machine learning can be used clinically for all drugs.

Nationwide genome surveillance of carbapenem-resistant Pseudomonas aeruginosa in Japan.

Japan is a country with an approximate 10% prevalence rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA). Currently, a comprehensive overview of the genotype and phenotype patterns of CRPA in Japan is lacking. Herein, we conducted genome sequencing and quantitative antimicrobial susceptibility testing for 382 meropenem-resistant CRPA isolates that were collected from 78 hospitals across Japan from 2019 to 2020. CRPA exhibited susceptibility rates of 52.9%, 26.4%, and 88.0% against piperacillin-tazobactam, ciprofloxacin, and amikacin, respectively, whereas 27.7% of CRPA isolates was classified as difficult-to-treat resistance P. aeruginosa. Of the 148 sequence types detected, ST274 (9.7%) was predominant, followed by ST235 (7.6%). The proportion of urine isolates in ST235 was higher than that in other STs (P = 0.0056, χ2 test). Only 4.1% of CRPA isolates carried the carbapenemase genes: blaGES (2) and blaIMP (13). One ST235 isolate carried the novel blaIMP variant blaIMP-98 in the chromosome. Regarding chromosomal mutations, 87.1% of CRPA isolates possessed inactivating or other resistance mutations in oprD, and 28.8% showed mutations in the regulatory genes (mexR, nalC, and nalD) for the MexAB-OprM efflux pump. Additionally, 4.7% of CRPA isolates carried a resistance mutation in the PBP3-encoding gene ftsI. The findings from this study and other surveillance studies collectively demonstrate that CRPA exhibits marked genetic diversity and that its multidrug resistance in Japan is less prevailed than in other regions. This study contributes a valuable data set that addresses a gap in genotype/phenotype information regarding CRPA in the Asia-Pacific region, where the epidemiological background markedly differs between regions.

National genomic surveillance integrating standardized quantitative susceptibility testing clarifies antimicrobial resistance in Enterobacterales

Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the blaCTX-M extended-spectrum β-lactamase genes, blaCTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC β-lactamase genes were blaCMY-2 and blaDHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of blaCMY-2 and blaDHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

Minimum inhibitory concentrations and sequencing data have to be analysed in more detail to set provisional epidemiological cut-off values forMycobacterium tuberculosiscomplex

There is a clear need for a high-quality broth microdilution (BMD) assay for quantitative antimicrobial susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC) [1]. The BMD plate developed by the CRyPTIC Consortium, therefore, represents an important advance [2]. However, the analysis of the resulting data has several limitations. Provisional epidemiological cut-off values for M. tuberculosis complex need to be set carefully <http://bit.ly/3XjJ8px> C.U. Köser is a visiting scientist at the Department of Genetics, University of Cambridge, and research associate at Wolfson College, University of Cambridge. Feedback from G. Kahlmeter, T. Schön and J. Turnidge was gratefully received. We thank L. Sonnenkalb for clarifications regarding her work.

Next-Generation Sequencing of Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated from Patients Hospitalized in the University Hospital Facilities.

Carbapenem-resistant (CR) Klebsiella pneumoniae represents an urgent worldwide threat. We focused on CR K. pneumoniae in selected facilities of the University Hospital Bratislava (UHB) to investigate sequence types (STs), clonal relatedness, and antimicrobial resistance. Suspected carbapenem-nonsusceptible K. pneumoniae strains were obtained from hospitalized patients. Further examination included carbapenemase confirmation, cgMLST, and quantitative susceptibility testing. Of the total 41 CR K. pneumoniae strains, 26 (63.4%) were determined as ST11 in hospital No. 1; of these ST11, 22 (84.6%) were found in the first internal clinic. Six (14.6%) ST258 and three (7.3%) ST584 occurred in hospital No. 2; the most, i.e., four (66.7%), ST258 were detected in the geriatric clinic. In hospital No. 3, we found two (4.8%) ST584 and one (2.4%) ST258. Of the ST11 set, 24 (92.3%) produced NDM-1. Furthermore, seven (87.5) ST258 and five (100%) ST584 strains generated KPC-2. Antimicrobial resistance was as follows: ertapenem 97.6%, meropenem 63.4%, tigecycline 7.3%, eravacycline 7.3%, and colistin 2.5%. We revealed a presumably epidemiological association of ST11 with transmission, particularly in the first internal clinic of hospital No. 1, while ST258 and ST584 were related to interhospital dissemination between medical facilities No. 2 and No. 3. It is essential to prevent the circulation of these pathogens within and between healthcare facilities.

In-vitro evaluation of antimicrobial and antioxidant activity of Embelica officinalis extract on different strains on gram positive and negative bacteria

The present study was carried out to evaluate the in vitro antimicrobial and antioxidant activity of Emblica officinalis extract powder. The antimicrobial activity was assessed against gram positive and gram negative bacteria namely E.coli, Pseudomonas fluoresces, Bacillus subtilis, S.aureus, by agar well diffusion method. Quantitative antimicrobial susceptibility testing method, i.e., determination of minimum inhibitory concentration (MIC) by serial broth dilution was carried out on the different bacterial strains. The diameter of the zone of inhibition (ZOI) for the strains tested against was recorded and compared with the standard (Gentamicin, in case of bacteria). The results of the present study also revealed that Amla extract powder can scavenge free radicals or reactive oxygen species under in vitro conditions. The elimination of hydrogen peroxide by Amla powder may be attributed to phenolic compounds. The total phenolic content in the Emblica officinalis extract powder measured by the Folin Ciocalteau reagent in terms of gallic acid equivalents (GAE) was found to be 390.8mg/g. The possible reason for antibacterial activity of Amla extract powder might be the presence of tannins in the fruits.

Whole-Genome Sequencing for Resistance Level Prediction in Multidrug-Resistant Tuberculosis.

ABSTRACTDefining the precise relationship between resistance mutations and quantitative phenotypic drug susceptibility testing will increase the value of whole-genome sequencing (WGS) for predicting tuberculosis drug resistance. However, a large number of WGS data sets currently lack corresponding quantitative phenotypic data—the MICs. Using MYCOTBI plates, we determined the MICs to nine antituberculosis drugs for 154 clinical multidrug-resistant tuberculosis isolates from the Shenzhen Center for Chronic Disease Control in Shenzhen, China. Comparing MICs with predicted drug-resistance profiles inferred by WGS showed that WGS could predict the levels of resistance to isoniazid, rifampicin, streptomycin, fluoroquinolones, and aminoglycosides. We also found some mutations that may not be associated with drug resistance, such as EmbB D328G, mutations in the gid gene, and C−12T in the eis promoter. However, some strains carrying the same mutations showed different levels of resistance to the corresponding drugs. The MICs of different strains with the RpsL K88R, fabG1 C−15T mutations and some with mutations in embB and rpoB, had MICs to the corresponding drugs that varied by 8-fold or more. This variation is unexplained but could be influenced by the bacterial genetic background. Additionally, we found that 32.3% of rifampicin-resistant isolates were rifabutin-susceptible, particularly those with rpoB mutations H445D, H445L, H445S, D435V, D435F, L452P, S441Q, and S441V. Studying the influence of bacterial genetic background on the MIC and the relationship between rifampicin-resistant mutations and rifabutin resistance levels should improve the ability of WGS to guide the selection of medical treatment regimens.IMPORTANCE Whole-genome sequencing (WGS) has excellent potential in drug-resistance prediction. The MICs are essential indications of adding a particular antituberculosis drug dosage or changing the entire treatment regimen. However, the relationship between many known drug-resistant mutations and MICs is unclear, especially for rarer ones. The results showed that WGS could predict resistance levels to isoniazid, rifampicin, streptomycin, fluoroquinolones, and aminoglycosides. However, some mutations may not be associated with drug resistance, and some others may confer various MICs to strains carrying them. Also, 32.3% of rifampicin (RIF)-resistant strains were classified as sensitive to rifabutin (RFB), and some mutations in the rpoB gene may be associated with this phenotype. Our data on the MIC distribution of strains with some rarer mutations add to the accumulated data on the resistance level associated with such mutations to help guide further research and draw meaningful conclusions.

1312. Evaluation of a Multiplexed PCR Pneumonia Panel in a Tertiary Care Medical Center

Abstract Background Syndromic PCR testing for lower respiratory pathogens may give rapid, actionable results to aid in management decisions for suspected pneumonia cases. We sought to evaluate the performance of a multiplexed PCR pneumonia panel compared to routine microbiologic work-up in a tertiary care patient population. Methods Sputum and bronchoalveolar lavage (BAL) samples from Keck Medical Center (Los Angeles, CA) inpatients submitted for clinical microbiology work-up Dec 2019-Jun 2020 were tested by a multiplexed PCR panel (FilmArray Pneumonia Panel, BioFire Diagnostics). We compared panel results for typical bacterial pathogens to those of quantitative culture and susceptibility testing. We retrospectively determined the incidence of non-panel respiratory pathogens as detected by standard of care tests in this patient cohort. Results 68 of 180 samples yielded 80 positive bacterial PCR results: 34 were detected by both PCR panel and culture and 46 by PCR panel only, yielding a sensitivity of 100% (34/34) for pathogens detected and specificity of 73.1% (114/156) among negative cultures (normal flora or no growth). Concordant results had PCR Bin values ≥10^5 copies/mL whereas all 18 targets detected at 10^4 copies/mL were culture-negative. Among resistance gene targets, the panel detected 12 MRSA specimens, of which MRSA grew in only 4 cultures; E. coli and CTX-M in 1 specimen from which grew normal flora; and multiple gram-negative organisms and KPC in 1 specimen from which culture isolated carbapenem-resistant P. aeruginosa. Quantitation from positive BAL cultures (n=25) correlated weakly with PCR Bin values (R-squared=0.17). Non-PCR panel pathogens were detected in 22 of 180 (12.2%) specimens through routine methods (16 molds, 3 AFB, and 3 non-fermenter gram-negative bacteria). Conclusion The pneumonia panel had excellent sensitivity for its target bacterial pathogens, but results were often positive in negative cultures. This could be due to antecedent antibiotic therapy, differences in reporting threshold versus culture, or inability of PCR to discern results from normal flora. Non-panel pathogens were detected in a significant proportion in our population. The pneumonia panel should be implemented and interpreted carefully with consideration of antimicrobial stewardship. Disclosures All Authors: No reported disclosures

Identification of bacterial antibiotic resistance genes in next-generation sequencing data (review of literature).

The spread of antibiotic-resistant human bacterial pathogens is a serious threat to modern medicine. Antibiotic susceptibility testing is essential for treatment regimens optimization and preventing dissemination of antibiotic resistance. Therefore, development of antibiotic susceptibility testing methods is a priority challenge of laboratory medicine. The aim of this review is to analyze the capabilities of the bioinformatics tools for bacterial whole genome sequence data processing. The PubMed database, Russian scientific electronic library eLIBRARY, information networks of World health organization and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) were used during the analysis. In this review, the platforms for whole genome sequencing, which are suitable for detection of bacterial genetic resistance determinants, are described. The classic step of genetic resistance determinants searching is an alignment between the query nucleotide/protein sequence and the subject (database) nucleotide/protein sequence, which is performed using the nucleotide and protein sequence databases. The most commonly used databases are Resfinder, CARD, Bacterial Antimicrobial Resistance Reference Gene Database. The results of the resistance determinants searching in genome assemblies is more correct in comparison to results of the searching in contigs. The new resistance genes searching bioinformatics tools, such as neural networks and machine learning, are discussed in the review. After critical appraisal of the current antibiotic resistance databases we designed a protocol for predicting antibiotic resistance using whole genome sequence data. The designed protocol can be used as a basis of the algorithm for qualitative and quantitative antimicrobial susceptibility testing based on whole genome sequence data.

A sensitive EZMTT method provides microscale, quantitative and high-throughput evaluation of drug efficacy in the treatment of Mycobacterium tuberculosis infectious diseases

Drug resistance has become a serious public health problem in mycobacterial infectious diseases. Here, we investigated a water soluble tetrazolium salt (EZMTT)-based detection method to provide an easy, safe and quantitative antimycobacterial susceptibility test (AMST) method, especially for targeting early detection of loss of drug susceptibility in mycobacteria. After a single addition of the EZMTT detection reagent at the inoculation of mycobacteria culture, the AMST was continuously analyzed in a sealed 96-well plate (100 μl), or a sealed tube to ensure biosafety. Using Mycobacterium tuberculosis H37Ra as the model strain, the EZMTT assay was developed with high reproducibility (Z factor of 0.64) for facile measurements of growth and drug susceptibility. In the comparative AMST study, the 7-day EZMTT method identified not only the same set of drug resistance as the other two methods (the 30-day traditional Löwenstein Jensen solid medium assay and the 10–14 day 8 ml Mycobacteria Growth Indicator Tube liquid method), but also additional strains with loss of drug susceptibility. In conclusion, we demonstrated that the EZMTT-based AMST assay in a sealed microtiter plate has great potential for routine use in medical diagnosis and drug screening to battle the unmet medical need in the treatment of multi- and extensive-drug resistant mycobacteria.

Administration of nitrofurantoin in dogs with lower urinary tract infections: 14 cases (2013-2019).

The purpose of this study was to describe the clinical use of nitrofurantoin in client-owned dogs with lower urinary tract infections. The primary aim was to describe the patient population, dosage, treatment duration, outcome and side effects. Medical records in an institution were retrospectively reviewed from July 2013 to January 2019. Nitrofurantoin was prescribed in this clinical population of 14 client-owned dogs for lower urinary tract infections. Recurrent urinary tract infection was the clinical diagnosis in all dogs. Each dog's urinary tract infection was associated with a nitrofurantoin-susceptible, multidrug-resistant uropathogen. The median dosage and duration of nitrofurantoin treatment was 4.3mg/kg by mouth every 8hours for 14 days. Twelve of the 14 dogs had successful outcomes including bacteriologic cure (n=9), clinical cure (2) and resolution of target bacteria (1). Treatment failures (n=2) were associated with uropathogens developing progressive nitrofurantoin resistance. In some dogs with recurrent lower urinary tract infections, nitrofurantoin may be an effective antibiotic for treatment of nitrofurantoin-susceptible uropathogens. Treatment failures were associated with progressive uropathogen resistance. Urine bacterial culture and quantitative susceptibility testing are essential to initiating and monitoring treatment due to the multidrug-resistant isolates and, in some cases, persistent bacteriuria in the face of treatment.

Agreement of Quantitative and Qualitative Antimicrobial Susceptibility Testing Methodologies: The Case of Enrofloxacin and Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the causal agent of colibacillosis, one of the most common bacterial infections in the poultry sector. Antimicrobial susceptibility testing (AST) is essential for rational and prudent antimicrobial therapy. Subsequently, uniformity in test results from the various testing methodologies used in diagnostic laboratories is pivotal. The aim of this study was therefore to evaluate the agreement between different AST methods in determining fluoroquinolone resistance in APEC. Twenty APEC isolates were selected and subjected to four different susceptibility tests: the quantitative microbroth dilution, agar dilution and gradient strip tests, and the qualitative disk diffusion method. The experiments were performed in triplicate. Categorical agreement, essential agreement and different errors were assessed. Moreover, agreement was also evaluated by calculating intraclass correlation coefficients (ICCs) for the quantitative tests and determining the Pearson correlation coefficients for the agreement between the disk diffusion method and the quantitative tests. Categorical agreement and essential agreement when compared with the microbroth technique ranged from 85–95% and 85–100%, respectively. No very major errors (false susceptible) and only one major error (false resistant) and minor errors (results involving an intermediary category) were detected. The calculated ICC values of the three quantitative tests fluctuated around 0.970 (range 0.940–0.988). There was a high negative correlation between the disk diffusion method and the other tests (correlation coefficients ranging from −0.979 to −0.940), indicating a clear inverse relationship between the minimum inhibitory concentration value and the zone diameter of growth inhibition. In conclusion, the overall agreement between the four different testing methodologies was very high. These results confirm the reliability of the disk diffusion and gradient strip test methods as substantiated alternatives, next to the gold standard agar and microbroth dilution, for fluoroquinolone susceptibility testing of APEC isolates.

Assessment of asymptomatic bacteriuria and sterile pyuria among antenatal attendants in hospitals in northern Ghana

BackgroundAsymptomatic bacteriuria (ASB) and sterile pyuria (SP) are complexities of UTI whose prevalence are not known in the northern sector of Ghana. Our aim was to determine the occurrence of sterile pyuria and asymptomatic bacteriuria among pregnant women accessing antenatal care at a secondary and tertiary care hospitals in Tamale, northern Ghana.MethodsA cross sectional study was conducted by screening 530 pregnant women with no signs of acute urinary tract infection attending antenatal clinic for a period of 6 months. Midstream urine was collected for microscopy, quantitative urine culture and antibiotic susceptibility testing. Data analysis was carried out using the Statistical Package for Social Sciences version 20.ResultsAsymptomatic bacteriuria was respectively 20 and 35.5% at Tamale Central and Tamale Teaching Hospital out of the 390 and 90 women screened. Sterile pyuria was found among 66% of the 50 women presenting at Tamale Central Hospital. More than 64% of isolates recovered from ASB patients were S. aureus and coagulase negative Staph. (CoNS). Escherichia coli was the dominant species among members of the enterobacteriaceae isolated. Highest susceptibility was recorded against gentamicin and amikacin while most resistance was to Ampicillin, cotrimoxazole, chloramphenicol and nitrofurantoin. Resistance to imipenem and vancomycin were 28.8 and 52%, with strains showing multiple drug resistance of between 81 and 92%.ConclusionThe prevalence of asymptomatic bacteriuria is appreciably higher (20–35.5%) than documented rates in the southern sector of the country. The presence of sterile pyuria which may be an indication of asymptomatic renal impairment and most often overlooked in antenatal management is 66%. Empirical treatment of UTIs at the Tamale Central and Teaching Hospital without confirmation of susceptibility may result in treatment failure. It is necessary to screen and treat pregnant women for ASB and SP due to the complications associated with these conditions.

Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis

Whole-genome sequencing allows rapid detection of drug-resistant Mycobacterium tuberculosis isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinical M. tuberculosis isolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

Dynamic Sessile-Droplet Habitats for Controllable Cultivation of Bacterial Biofilm.

Bacterial biofilms play essential roles in biogeochemical cycling, degradation of environmental pollutants, infection diseases, and maintenance of host health. The lack of quantitative methods for growing and characterizing biofilms remains a major challenge in understanding biofilm development. In this study, a dynamic sessile-droplet habitat is introduced, a simple method which cultivates biofilms on micropatterns with diameters of tens to hundreds of micrometers in a microfluidic channel. Nanoliter plugs are utilized, spaced by immiscible carrier oil to initiate and support the growth of an array of biofilms, anchored on and spatially confined to the micropatterns arranged on the bottom surface of the microchannel, while planktonic or dispersal cells are flushed away by shear force of aqueous plugs. The performance of the aforementioned method of cultivating biofilms is demonstrated by Pseudomonas aeruginosa PAO1 and its derived mutants, and quantitative antimicrobial susceptibility testing of PAO1 biofilms. This method could significantly eliminate corner effects, avoid microchannel clogging, and constrain the growth of biofilms for long-term observations. The controllable sessile droplet-based biofilm cultivation presented in this study should shed light on more quantitative and long-term studies of biofilms, and open new avenues for investigation of biofilm attachment, growth, expansion, and eradication.

Polymyxin B Etest® compared with gold-standard broth microdilution in carbapenem-resistant Enterobacteriaceae exhibiting a wide range of polymyxin B MICs

ObjectivesPolymyxins have been revitalized to combat carbapenem-resistant Enterobacteriaceae (CRE). However, evaluating the activity of these agents by traditional broth dilution methods is not practical for busy clinical laboratories. We compared polymyxin B activity using two quantitative susceptibility testing methods, Etest® and broth microdilution (BMD), against CRE isolates from patients at an academic medical centre. MethodsPolymyxin B activity against 70 CRE clinical isolates was determined by Etest® according to the manufacturer and by BMD according to CLSI guidelines. Pseudomonas aeruginosa ATCC® 27853 and Escherichia coli NCTC 13846 served as quality control strains. The EUCAST colistin susceptibility breakpoint of Enterobacteriaceae (≤2 mg/L) was used. Essential agreement was isolates with an MIC within 1 log2 dilution over total isolates. Categorical agreement was number of isolates in the same susceptibility category (susceptible or resistant) over total isolates. Major and very major error rates were calculated using number of susceptible and number of resistant isolates, respectively, as the denominator. McNemar's test was used for determining a difference in susceptibility between methods. ResultsThe CRE isolates were primarily Klebsiella spp. (49%) and Enterobacter spp. (36%). Polymyxin B susceptibility was significantly higher by Etest® compared with BMD (97% versus 77%; p 0.0001). Categorical agreement was 80%, but essential agreement was low (10%). False non-susceptibility was never observed by Etest® (BMD reference), but the very major errors were high (88%). ConclusionsEtest® reporting of false susceptibility may result in inappropriate antibiotic use and treatment failure clinically. We do not recommend using Etest® for polymyxin B susceptibility testing for routine patient care.

Effects of Diagnostic Work-Up on Medical Decision-Making for Canine Urinary Tract Infection: An Observational Study in Danish Small Animal Practices.

BackgroundClinical signs of urinary tract disease in dogs often lead to prescription of antibiotics. Appropriate diagnostic work‐up could optimize treatment and reduce the risk of inappropriate use of antibiotics.Hypothesis/ObjectivesTo describe and evaluate the impact of diagnostic work‐up on decision to treat (DTT) and choice of antibiotic treatment (COT) for dogs presenting with clinical signs of urinary tract disease.AnimalsOne hundred and fifty‐one dogs presenting to 52 Danish veterinary practices.MethodsProspective, observational study. Clinical signs, diagnostic work‐up, and prescriptions were recorded. Urine samples were submitted to a reference laboratory for quantitative bacterial culture (QBC) and susceptibility testing. The laboratory results were used as reference for assessing the appropriateness of DTT and COT.ResultsIn the majority of dogs, veterinarians performed dipstick (99%), microscopic examination of urine (80%) and bacterial culture (56%). Fifty‐one percent of dogs had urinary tract infection (UTI) based on reference QBC. Appropriate DTT was made for 62% of the dogs, while 36% were over‐prescribed and 2% under‐prescribed. Inappropriate use of second‐line agents was found in 57% of the UTI cases. Performing microscopy—but not culture—significantly impacted DTT (P = 0.039) while no difference was seen in COT (P = 0.67). The accuracy of in‐house microscopy and culture were 64.5 and 77%, respectively. Conclusions and Clinical Importance: Over‐prescription of antibiotics was common among dogs with suspected UTI, regardless of the diagnostic work‐up performed. Test inaccuracy under practice conditions and incoherence between diagnostic test results and decision‐making both explained inappropriate and unnecessary use of antibiotics.

Microorganisms Associated with Prostatitis Using Indwelling Urinary Catheters in Okigwe, Imo State, Nigeria

Indwelling urinary catheters are standard medical devices utilized in both hospital and nursing home settings to relieve urinary retention and urinary incontinence in a prostatitis patient. The microorganisms associated with prostatitis using urinary catheters was carried out in Okigwe, Imo State using culture technique. 200 patients were examined for prostate specific antigens (PSA) using quantitative and qualitative tests and antibiotic susceptibility tests were also done. Out of 200 patients tested for PSA, 119 (59.5%) and 129 (64.5%) were positive for quantitative and qualitative respectively. 85 patients were catheterized with 80 (94.15%) having bacterial isolates while 75 (65.5%) of the 115 uncatheterized patients having bacterial growth. The organisms isolated from catheterized and uncatheterized patients were Escherichia coli 55 (3.5%), Klebsiella spp 12 (7.8%), Staphylococcus aureus 42 (27.0%), Streptococcus 20 (12.9%), Protus spp 13 (8.4%) and Pseudomanas 13 (8.4%). Higher bacterial loads were observed in the catheterized patients urine than in the uncatheterized. Streptomycin, Ceftriaxone and Augumentine were the drugs of choice in the sensitivity tests while high antimicrobial resistant rates were observed with Ampiclox, Septrin and Chlorophenicol. Generally, high prevalence rate of PSA and bacterial pathogens were reported in patients of high age (50 and above years). This calls for proper medical checks for men of 50 years and above. This check will prevent the development of prostatitis which could lead to fertility problems because of difficulty in ejaculation in prostatitis patients.

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis.

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis.

Revisiting the mutant prevention concentration to guide dosing in childhood tuberculosis.

The mutant prevention concentration (MPC) is a well-known concept in the chemotherapy of many bacterial infections, but is seldom considered in relation to tuberculosis (TB) treatment, as the required concentrations are generally viewed as unachievable without undue toxicity. Early studies revealed single mutations conferring high MICs of first- and second-line anti-TB agents; however, the growing application of genomics and quantitative drug susceptibility testing in TB suggests a wide range of MICs often determined by specific mutations and strain type. In paediatric TB, pharmacokinetic studies indicate that despite increasing dose recommendations, a proportion of children still do not achieve adult-derived targets. When considering the next stage in anti-TB drug dosing and the introduction of novel therapies for children, we suggest consideration of MPC and its incorporation into pharmacokinetic studies to more accurately determine appropriate concentration targets in children, to restrict the growth of resistant mutants and better manage drug-resistant TB.