Microbiological Breakpoints Research Articles

Trimeric Tobramycin/Nebramine Synergizes β-Lactam Antibiotics against Pseudomonas aeruginosa.

β-Lactam antibiotics remain one of the most effective therapeutics to treat infections caused by Gram-negative bacteria (GNB). However, since ancient times, bacteria have developed multiple resistance mechanisms toward this class of antibiotics including overexpression of β-lactamases, suppression of porins, outer membrane impermeability, overexpression of efflux pumps, and target modifications. To cope with these challenges and to extend the lifetime of existing β-lactam antibiotics, β-lactamase inhibitors are combined with β-lactam antibiotics to prevent antibiotic inactivation by β-lactamases. The combination therapy of an outer membrane permeabilizer with β-lactam antibiotics is an alternative approach to overcoming bacterial resistance of β-lactams in GNB. This approach is of particular interest for pathogens with highly impermeable outer membranes like Pseudomonas aeruginosa. Previous studies have shown that outer membrane permeabilizers can be designed by linking tobramycin and nebramine units together in the form of dimers or chimeras. In this study, we developed trimeric tobramycin and nebramine-based outer membrane permeabilizers presented on a central 1,3,5-triazine framework. The resultant trimers are capable of potentiating outer membrane-impermeable antibiotics but also β-lactams and β-lactam/β-lactamase inhibitor combinations against resistant P. aeruginosa isolates. Furthermore, the microbiological susceptibility breakpoints of ceftazidime, aztreonam, and imipenem were reached by a triple combination consisting of an outer-membrane permeabilizer/β-lactam/β-lactamase inhibitor in β-lactam-resistant P. aeruginosa isolates. Overall, our results indicate that trimeric tobramycins/nebramines can rescue clinically approved β-lactams and β-lactam/β-lactamase inhibitor combinations from resistance.

Comparative analysis of clinical breakpoints, normalized resistance interpretation and epidemiological cut-offs in interpreting antimicrobial resistance of Escherichia coli isolates originating from poultry in different farm types in Tanzania.

Existing breakpoint guidelines are not optimal for interpreting antimicrobial resistance (AMR) data from animal studies and low-income countries, and therefore their utility for analysing such data is limited. There is a need to integrate diverse data sets, such as those from low-income populations and animals, to improve data interpretation. There is very limited research on the relative merits of clinical breakpoints, epidemiological cut-offs (ECOFFs) and normalized resistance interpretation (NRI) breakpoints in interpreting microbiological data, particularly in animal studies and studies from low-income countries. The aim of this study was to compare antimicrobial resistance in Escherichia coli isolates using ECOFFs, CLSI and NRI breakpoints. A total of 59 non-repetitive poultry isolates were selected for investigation based on lactose fermentation on MacConkey agar and subsequent identification and confirmation as E. coli using chromogenic agar and uidA PCR. Kirby Bauer disc diffusion was used for susceptibility testing. For each antimicrobial agent, inhibition zone diameters were measured, and ECOFFs, CLSI and NRI bespoke breakpoints were used for resistance interpretation. According to the interpretation of all breakpoints except ECOFFs, tetracycline resistance was significantly higher (TET) (67.8 -69.5 %), than those for ciprofloxacin (CIPRO) (18.6 -32.2 %), imipenem (IMI) (3.4 -35 %) and ceftazidime (CEF) (1.7 -45.8 %). Prevalence estimates of AMR using CLSI and NRI bespoke breakpoints did not differ for CEF (1.7 % CB and 1.7 % COWT), IMI (3.4 % CB and 4.0 % COWT) and TET (67.8 % CB and 69.5 % COWT). However, with ECOFFs, AMR estimates for CEF, IMI and CIP were significantly higher (45.8, 35.6 and 64.4 %, respectively; P<0.05). Across all the three breakpoints, resistance to ciprofloxacin varied significantly (32.2 % CB, 64.4 % ECOFFs and 18.6 % COWT, P<0.05). AMR interpretation is influenced by the breakpoint used, necessitating further standardization, especially for microbiological breakpoints, in order to harmonize outputs. The AMR ECOFF estimates in the present study were significantly higher compared to CLSI and NRI.

Characterization and Transferability of erm and tet Antibiotic Resistance Genes in Lactobacillus spp. Isolated from Traditional Fermented Milk.

Lactobacillus is a widely used bacteria and consumed through various fermented foods and beverages. Strains have been shown to carry resistance genes and mobile genetic elements with their ability to transfer the resistance to sensitive pathogenic strains. To study this, 4 cultures of Lactobacillus were isolated from traditional fermented milk. The isolates were able to grow up to 4% (w/v) NaCl concentration and 45°C temperature, and showed > 97% 16S rRNA gene similarities with Lactobacillus fermentum. All the isolates were phenotypically screened for the presence of antibiotic resistance. Minimum inhibitory concentration (MIC) as microbiological breakpoints were observed against a varied class of antibiotics. Isolates AKO 94.6, DVM 95.7, and NIFTEM 95.8 were explicitly resistant to ampicillin, ciprofloxacin and vancomycin with MIC well beyond the maximum range of 256µg/ml in the E-strip test. While isolate SKL1 was sensitive to ampicillin and showed MIC at 0.25µg/ml but resistant to streptomycin and trimethoprim (MIC > 256µg/ml). Molecular characterization showed the presence of tet(M) gene in three isolates SKL1, DVM 95.7, and NIFTEM 95.8 which was chromosomally associated resistance determinants while erm(B) resistance gene was detected in isolates DVM 95.7 and NIFTEM 95.8 only which was a plasmid associated gene and could be transferrable conjugally. Gene for Tn916 family (xis) was also observed in isolates DVM 95.7 and NIFTEM 95.8. Transferability of antibiotic resistance to pathogenic recipient strains was examined in isolates DVM 95.7 and NIFTEM 95.8 in different food matrices. The highest conjugation frequency with ~ 10-1 was obtained in alfalfa seed sprouts. This study reports the presence of acquired gene resistance in Lactobacillus species and dissemination to susceptible strains of bacteria in different food matrices. 16S rRNA gene sequences of isolates were uploaded to the NCBI GenBank database to retrieve the accession number.

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis

ObjectivesThe first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. MethodsDuring 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. ResultsFollowing the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015–0.06) mg/L and 0.12 (0.06–0.25) mg/L for isoniazid, 0.25 mg/L (0.25–0.5) and 0.5 mg/L (0.12–0.5) for levofloxacin, and 0.5 mg/L (0.5–1.0) and 0.5 mg/L (0.5–1.0) for amikacin. ConclusionsBoth SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.

Antimicrobial resistance of commensal Escherichia coli from food-producing animals in Russia.

Background and Aim:Commensal Escherichia coli is an important indicator of antimicrobial resistance (AMR) in animals and food of animal origin. Therefore, it was recommended by the World Health Organization and OIE for inclusion in resistance surveillance programs. At the same time, the data on E. coli isolates from animals in Russia are scarce. The aim of this work was to determine the current prevalence of resistance and genetic markers of non-pathogenic commensal E. coli collected from major food-producing animals (poultry, pigs, and cows) in different regions of Russia and to compare these data with data from other countries to prioritize antimicrobials for limiting their use according to the National Action PlanMaterials and Methods:Samples (n=306) were collected from biomaterial of chicken, turkey, cows, and pigs raised on 11 farms in the European part of Russia, Siberia, and North Caucasus. Isolates (n=306) of E. coli were tested for resistance to 11 antimicrobials from ten classes using the broth microdilution method. MICs were interpreted against EUCAST microbiological and clinical breakpoints. For data analysis and statistical processing, the AMRcloud online platform was used. The data are presented in comparison with other countries.Results:In Russia, higher levels of microbiological and clinical resistance of E. coli to critically important antimicrobials, including colistin, cefotaxime, and ciprofloxacin, were found compared to other countries, especially in poultry: About 30% of isolates from chicken were resistant to colistin, 8% to cefotaxime, and 88% to ciprofloxacin according to EUCAST ECOFFs. Only 10% of isolates from cows were resistant to cefotaxime. About 47% of isolates of E. coli from chicken had a moderate relative resistance for ampicillin and 56% for tetracycline. For most antimicrobials, isolates from cows demonstrated a lower resistance than isolates from poultry and pigs. All tested isolates from chicken, turkey, and pigs showed a simultaneous microbiological resistance to at least three classes of antimicrobials. No pan-resistant isolates were found.Conclusion:High levels of AMR of commensal E. coli from poultry, especially for critically important drugs, are a matter of concern and should be taken into account when choosing antimicrobials to be restricted for use in animal husbandry according to the National Action Plan.

1556. Assessment of Translational In Vitro and Animal Pharmacokinetic–Pharmacodynamic Data Used to Support Drug Development of Recent Tetracycline Derivatives

BackgroundNonclinical (animal and in vitro) models are commonly used during the development of antibacterial drugs. Pharmacokinetic (PK) and pharmacodynamic (PD) data obtained from these nonclinical models are used to generate a PK-PD target, which can then be bridged to humans in a probability of PK-PD target attainment (PTA) analysis to support selection of the dose regimen for phase 3 trials and in vitro susceptibility testing criteria (breakpoints) to guide clinical usage.MethodsTwo recently approved tetracycline antibacterial drugs, eravacycline (ERV) and omadacycline (OMD), were evaluated. PK-PD data from nonclinical models and clinical microbiological response were collected from each of the respective clinical pharmacology reviews and assessments published by FDA and EMA, respectively. The highest MICs (minimum inhibitory concentrations) reflecting 80% success in the ability of the drug to inhibit growth in the target bacteria were identified from clinical and nonclinical data and termed the MIC cutoff. The nonclinical MIC cutoff was obtained from the PTA analysis using the PK-PD targets from animal studies. The clinical MIC cutoff was obtained from microbiological response (microbiological intent-to-treat population) data from clinical trial experience. The ratios of the clinical and nonclinical MIC cutoffs were calculated and used to evaluate potential discrepancies between the animal model prediction and clinical trial experience.ResultsThe drug development programs for ERV and OMD included murine infection models and in vitro models to characterize PK-PD. The clinical to nonclinical MIC cutoff ratios ranged from 4 to 32. Higher values of the MIC cutoff signify that the drug can treat larger proportions of the bacterial population; therefore, high clinical to nonclinical MIC cutoff ratios signify that the drugs had more activity in reducing the bacterial population in clinical than in nonclinical studies.ConclusionThus, the nonclinical models for ERV and OMD under-predicted microbiological response and breakpoints. While nonclinical models are generally useful, more characterization of translational factors may be needed to allow nonclinical models to be more predictive of clinical trial outcomes.Disclosures All authors: No reported disclosures.

Assessment of antibiotic susceptibility in Lactobacillus isolates from chickens

BackgroundThe aim of this study was to determine the susceptibility of 88 Lactobacillus isolates derived from chickens to antibiotic substances and to detect drug-resistance genes.ResultsThe minimal inhibitory concentration of 13 antimicrobial substances was determined by the broth microdilution method, and resistance genes were detected by PCR. We recorded a high prevalence of resistance to tiamulin (90% resistant isolates), tetracyclines (74%) and lincomycin (70%), and a moderately high frequency of resistance to enrofloxacin (48%), macrolides (42%), aminoglycosides (12.5–31%), ampicillin (26%) and chloramphenicol (23%). Multi-drug resistance was observed in 79.5% of isolates. The presence of resistance genes was generally correlated with phenotypic resistance, but some molecular determinants were also recorded in susceptible isolates. Among tetracycline resistance genes, the most frequently identified was tetW (45% isolates), followed by tetM (26%) and tetL (24%). The ermB, ermC and lnuA genes, associated with resistance to macrolides and lincosamides, were observed in 39, 12 and 39% of isolates, respectively. Among genes determining resistance to aminoglycoside antibiotics, we identified ant(6)-Ia (10% of isolates), aac(6′)-Ie-aph(2′)-Ia (8%), aph(2″)-Ic (6%) and aadE (4.5%). The cat gene was present in 32 isolates, including 8 of 20 found to be resistant to chloramphenicol. Two genes encoding efflux pumps were identified—the acrA gene was present in all isolates tested, and 10 of 79 lactobacilli determined to be phenotypically resistant to tiamulin contained the lsaE gene. We were unable to explain the resistance mechanism of Lactobacillus isolates to ampicillin, but showed that it did not involve the production of β-lactamases.ConclusionsOur findings indicate that intestinal lactobacilli should be considered a reservoir of resistance genes and that antibiotics must be used prudently in poultry production. The data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorization of susceptible and resistant strains within the genus Lactobacillus.

Prolonged or Continuous Infusion of IV Antibiotics as Initial Treatment Strategy.

Prolonged or Continuous Infusion of IV Antibiotics as Initial Treatment Strategy.

Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese

The aim of this study was to determine the antibiotic susceptibility of 93 Lactobacillus strains isolated from domestic geese raised on Polish farms. The minimal inhibitory concentration (MIC) of 13 antimicrobial substances was determined by the broth microdilution method.All strains were sensitive to the cell wall inhibitors ampicillin and amoxicillin (MIC ≤ 8 μg/ml). Resistance to inhibitors of protein synthesis and to fluoroquinolone inhibitors of replication was found in 44.1% and 60.2% of isolates, respectively; 26.9% strains were resistant to neomycin (MIC ≥ 64 μg/ml), 23.6% to tetracycline (MIC ≥ 32 μg/ml), 15% to lincomycin (MIC ≥ 64 μg/ml), 18.3% to doxycycline (MIC ≥ 32 μg/ml), 9.7% to tylosin (MIC ≥ 32 μg/ml), 56% to flumequine (MIC ≥ 256 μg/ml) and 22.6% to enrofloxacin (MIC ≥ 64 μg/ml).Bimodal distribution of MICs indicative of acquired resistance and unimodal distribution of the high MIC values indicative of intrinsic resistance were correlated with Lactobacillus species. Eleven (11.8%) strains displayed multiple resistance for at least three classes of antibiotics.Data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorisation of susceptible and resistant strains of Lactobacillus genus and help to assess the hazards associated with the occurrence of drug resistance among natural intestinal microflora.

Functional properties of Lactobacillus strains isolated from dairy products

Twenty-four acid- and bile-tolerant lactobacilli isolates from dairy products were identified and further in vitro characterized for the presence of functional traits potentially useful for probiotic applications, which included desirable and undesirable traits, such as biofilm formation, ability to inhibit intestinal pathogens, antibiotic susceptibility, and enzyme activity. The majority of examined strains were susceptible to certain antimicrobial agents (streptomycin, gentamicin, clindamycin, erythromycin, tetracycline, quinupristin-dalfopristin), except for three strains of Lactobacillus rhamnosus with minimal inhibitory concentration levels for streptomycin higher than the microbiological breakpoints (≥32μg/mL), which are considered as resistant. Undesirable traits such as α-chymotrypsin or N-acetyl-β-glucosaminidase activities were not detected, but low β-glucuronidase, and moderate and high β-glucosidase activities were recorded in nine strains, which were eliminated from further examination together with three isolates showing unsuitable antibiotic resistance. Of the remaining 12 isolates, 4 (Lactobacillus fermentum 202, Lactobacillus gallinarum 7001, L. rhamnosus 183, and Lactobacillus plantarum L2-1) manifested an outstanding potential to inhibit selected intestinal pathogens in an agar spot test, including Escherichia coli and Salmonella spp., and simultaneously demonstrated strong biofilm-forming capacity. In conclusion, the results of our in vitro experiments showed that the above four strains had a potential probiotic value and met the criteria to be identified as a possible probiotic microorganism, with the necessity of verification through well-designed in vivo experimental, clinical, and technological studies before the strains can be used as probiotics or as starter probiotic cultures.

Susceptibility of bifidobacteria of animal origin to selected antimicrobial agents.

Strains of the genus Bifidobacterium are frequently used as probiotics, for which the absence of acquired antimicrobial resistance has become an important safety criterion. This clarifies the need for antibiotic susceptibility data for bifidobacteria. Based on a recently published standard for antimicrobial susceptibility testing of bifidobacteria with broth microdilution method, the range of susceptibility to selected antibiotics in 117 animal bifidobacterial strains was examined. Narrow unimodal MIC distributions either situated at the low-end (chloramphenicol, linezolid, and quinupristin/dalfopristin) or high-end (kanamycin, neomycin) concentration range could be detected. In contrast, the MIC distribution of trimethoprim was multimodal. Data derived from this study can be used as a basis for reviewing or verifying present microbiological breakpoints suggested by regulatory agencies to assess the safety of these micro-organisms intended for the use in probiotics.

Characterization and Transfer of Antibiotic Resistance in Lactic Acid Bacteria from Fermented Food Products

The study provides phenotypic and molecular analyses of the antibiotic resistance in lactic acid bacteria (LAB) from fermented foods in Xi'an, China. LAB strains (n = 84) belonging to 16 species of Lactobacillus (n = 73), and Streptococcus thermophilus (n = 11) were isolated and identified by sequencing their 16S rRNA gene. All strains were susceptible to ampicillin, bacitracin, and cefsulodin, and intrinsically resistant to nalidixic acid, kanamycin, and vancomycin (except L. bulgaricus, L. acidophilus, and S. thermophilus, which were susceptible to vancomycin). Some strains had acquired resistance for penicillin (n = 2), erythromycin (n = 9), clindamycin (n = 5), and tetracycline (n = 14), while resistance to gentamycin, ciprofloxacin, streptomycin, and chloramphenicol was species dependent. Minimum inhibitory concentrations presented in this study will help to review microbiological breakpoints for some of the species of Lactobacillus. The erm(B) gene was detected from two strains of each of L. fermentum and L. vaginalis, and one strain of each of L. plantarum, L. salivarius, L. acidophilus, L. animalis, and S. thermophilus. The tet genes were identified from 12 strains of lactobacilli from traditional foods. This is the first time, the authors identified tet(S) gene from L. brevis and L. kefiri. The erm(B) gene from L. fermentum NWL24 and L. salivarius NWL33, and tet(M) gene from L. plantarum NWL22 and L. brevis NWL59 were successfully transferred to Enterococcus faecalis 181 by filter mating. It was concluded that acquired antibiotic resistance is well dispersed in fermented food products in Xi'an, China and its transferability to other genera should be monitored closely.

Antibiotic susceptibility of members of the Lactobacillus acidophilus group using broth microdilution and molecular identification of their resistance determinants

The range of antibiotic susceptibility to 13 antibiotics in 101 strains of the Lactobacillus acidophilus group was examined using the lactic acid bacteria susceptibility test medium (LSM) and broth microdilution. Additionally, microarray analysis and PCR were applied to identify resistance genes responsible for the displayed resistant phenotypes in a selection of strains. In general, narrow as well as broad unimodal and bimodal MIC distributions were observed for the Lactobacillus acidophilus group and the tested antimicrobial agents. Atypically resistant strains could be determined by visual inspection of the obtained MIC ranges for ampicillin, chloramphenicol, clindamycin, erythromycin, quinupristin/dalfopristin, streptomycin and tetracycline. For most of these atypically resistant strains underlying resistance determinants were found. To our knowledge erm(A) was detected in lactobacilli for the first time within this study. Data derived from this study can be used as a basis for reviewing present microbiological breakpoints for categorization of susceptible and resistant strains within the Lactobacillus acidophilus group to assess the safety of microorganisms intended for use in food and feed applications.

Susceptibility to tetracycline and erythromycin of Lactobacillus paracasei strains isolated from traditional Italian fermented foods

The aim of this study was to evaluate the susceptibility of 197 isolates of Lactobacillus paracasei, isolated from Italian fermented products coming from different geographical areas, to tetracycline and erythromycin, two antimicrobials widely used in clinical and animal therapy. Isolation media were supplemented with antibiotics according to the microbiological breakpoints (BPs) defined by European Food Safety Authority (EFSA). Isolates were identified at the species level and were typed by rep-PCR using the (GTG) 5 primer. A total of 121 genotypically different strains were detected and their phenotypic antimicrobial resistance to tetracycline and erythromycin was determined as the minimum inhibitory concentration (MIC) using the broth microdilution method. The presence of the genes ermB, ermC and tetL, tetM, tetS, tetW, in the phenotypically resistant isolates was investigated by PCR. Tetracycline induction of tetM expression on representative resistant strains, grown in medium either lacking or containing the antibiotic, was also analyzed by RT-PCR. Among the 121 tested strains, 77.7% were susceptible to tetracycline (MIC ≤ 4 µg/ml; BP = 4 µg/ml) and 94.2% to erythromycin (MIC ≤ 1 µg/ml; BP = 1 µg/ml). Overall, 27 strains appeared to be resistant to tetracycline (16 ≤ MIC ≤ 128 µg/ml ) (Tet R), and 7 of them were also resistant to erythromycin (MIC ≥ 1024 µg/ml) (Erm R). The tetM and ermB genes were the most frequently detected in the Tet R and/or Erm R strains. The tetM expression was induced by antibiotic addition to the growth medium. Our study confirmed that L. paracasei is quite sensitive to tetracycline and erythromycin, but the high level of resistance of Erm R strains suggested that acquired resistance took place. Further investigations are required to analyze whether the genes identified in L. paracasei isolates might be horizontally transferred to other species. Since “commensal” bacteria, which L. paracasei belongs to, may play an active role in the spreading of antibiotic resistance, a series of measures inspired from a principle of precaution should be taken before they are used as commercial starters or probiotic cultures in food products, complemented by a more prudent use of antibiotics in agriculture, veterinary, and human medicine.

Prevalence and Mechanism of Resistance against Macrolides, Lincosamides, and Streptogramins amongEnterococcus faeciumIsolates from Food-Producing Animals and Hospital Patients in Belgium

The prevalence of acquired resistance to streptogramins, macrolides, and lincosamides and the genetic background of this resistance was investigated in Enterococcus faecium strains isolated from food-producing animals and hospital patients 4-5 years after the ban of streptogramins as growth promoters. The minimum inhibitory concentrations (MICs) of quinupristin/dalfopristin (Q/D), virginiamycin M1 (virgM1), erythromycin (ery), tylosin (tyl), and lincomycin (lin) were determined by the agar dilution method for E. faecium isolates derived from pigs (80), broilers (45), and hospitalized patients (103). Resistance or susceptibility was interpreted using a microbiological criterion and breakpoints recommended by the Clinical Laboratory Standards Institute (CLSI), if available. The isolates were also screened by PCR for erm(B), lnu(A), lnu(B), mef(A/E), vat(D), vat(E), vga(A), vga(B), and vgb(A) genes. Acquired resistance to Q/D, virgM1, ery, tyl, and lin was detected in 34%, 96%, 46%, 46%, and 69% of the porcine strains, respectively. For broiler strains this was 15% (Q/D), 98% (virgM1), 69% (ery), 71% (tyl), and 89% (lin) and for human strains 23% (Q/D), 65% (virgM1), 54% (ery), 52% (tyl), and 60% (lin). Strains showing cross-resistance against macrolides and lincosamides almost always carried the erm(B) gene. This gene was present in 64% of the Q/D-resistant isolates. Only in two human and three broiler Q/D- and virgM1-resistant isolates, a combination of the erm(B) and vat(D) or vat(E) genes was found. The genetic background of resistance could not be determined in the other Q/D- or virgM1-resistant strains. This study demonstrates that streptogramin resistance is frequently present in strains from hospitalized patients and food-producing animals, but the genetic basis hitherto mostly remains obscure.

Comparison of methods for the determination of antimicrobial resistance in Campylobacter spp. human and the food chain isolates

With a microdilution method, using the commercial diagnostic test Sensititre Susceptibility Plates for Campylobacter MIC (Trek Diagnostic Systems, Cleveland, OH, USA), disk diffusion and agar dilution method, resistance to six antimicrobial agents were examined in a reference strain &lt;i&gt;Campylobacter jejuni&lt;/i&gt; ATCC 33560 and 73 thermo-tolerant isolates of &lt;i&gt;Campylobacter&lt;/i&gt; spp. For the microdilution method and all tested antimicrobial agents, our determined values of microbiological breakpoints of resistant strains were suggested as the minimum inhibitory concentration (MIC&lt;sub&gt;R&lt;/sub&gt;) for ciprofloxacin &amp;ge; 0.5, erythromycin &amp;ge; 4, gentamicin &amp;ge; 4, nalidixic acid &amp;ge; 32 and tetracycline &amp;ge; 4 &amp;mu;g/ml. On the basis of our study results, strains resistant to clindamycin were MIC&lt;sub&gt;R&lt;/sub&gt; &amp;ge; 2 &amp;mu;g/ml for the dilution methods and a zone diameter R ≤ 16 mm for the disk diffusion method. Comparison of the results of the resistance examination, a microdilution method and disk diffusion method with the reference agar dilution method, showed that all compared methods yielded identical results with the exception of the resistance determination in erythromycin and nalidixic acid. The errors were mostly the result of the interpretation criteria for MIC&lt;sub&gt;R&lt;/sub&gt; of agar dilution method and different conditions of cultivation used. However, the compared methods, provide results comparable with the reference method having greater convenience of measurement.

Antibiotic Susceptibility Profiles of Lactobacillus reuteri and Lactobacillus fermentum

Lactobacillus reuteri and Lactobacillus fermentum, which are commonly used as food processing aids and probiotics, can potentially act as reservoirs of antibiotic resistance genes. Acquired resistance genes may be transferred via the food chain or in the gastrointestinal tract to pathogenic bacteria. Knowledge of the distributions of antibiotic MICs for a species is needed when using a phenotypic method to assess the presence of acquired resistance genes. In the present study, 56 L. reuteri and 56 L. fermentum strains that differed by source and spatial and temporal origin were assessed for antibiotic susceptibility using an Etest kit and a broth microdilution protocol. L. fermentum strains displayed a uniform distribution of MICs for all six antibiotics tested. L. reuteri strains had a bimodal distribution of MICs or a distribution with MICs above the test range for 7 of the 14 antibiotics tested. Genetic relatedness was observed among L. reuteri strains with high MICs for both ampicillin and tetracycline and among strains with high MICs for both erythromycin and clindamycin. Results obtained with the Etest and the broth microdilution method corresponded well with each other. Thus, further research may make it possible to define microbiological breakpoints for distinguishing between strains with and without acquired resistance genes.

Call for the international adoption of microbiological breakpoints for fluoroquinolones and Streptococcus pneumoniae

The use of current Clinical and Laboratory Standards Institute levofloxacin breakpoints for assessing fluoroquinolone resistance in Streptococcus pneumoniae is inadequate for detecting isolates possessing first-step parC mutations. Consequently, the risk for development of fluoroquinolone resistance is greatly underestimated. Adopting microbiological breakpoints for fluoroquinolones and S. pneumoniae, where parC mutations are rare in susceptible isolates, more accurately describes the emergence of resistance and may help to prevent a number of future fluoroquinolone treatment failures. Additionally, we propose that the use of a second fluoroquinolone marker, such as ciprofloxacin, offers the best prediction for detecting an isolate possessing a first-step parC mutation.

The Integrated Use of Pharmacokinetic and Pharmacodynamic Models for the Definition of Breakpoints

For fluoroquinolones AUC/MIC ratios are known to correlate with clinical outcomes for patients suffering from respiratory tract infections (RTI) and complicated skin and skin structure infections (cSSSI). This paper describes the results of a population PK/PD analysis followed by Monte Carlo simulations to estimate clinical outcome and the microbiological breakpoints for a 400 mg once-daily moxifloxacin (MFX) treatment schedule. Based on PK data from 416 subjects, a non-compartmental population PK model was developed first to describe the expected exposure (AUC) distribution in humans. Height and gender were the main population covariates with moderate influence on PK variability. Albumin, bilirubin, and creatinine clearance (as derived from serum creatinine according to Cockroft and Gault) had a mild effect on AUC. Residual unexplained variability of AUC was low (13.1%). To describe the PD function the MIC distribution pattern of more than 3,000 isolates of S. pneumoniae as the representative pathogen for RTI (MIC90, range: 0.125; 0.006-4 mg/l) was built into the population PK/PD model for RTI, while 126 isolates of methicillin-susceptible Staphylococcus aureus strains (MIC90, range: 0.125; 0.03-4 mg/l) were the basis for the PD function in cSSSI. Simulations for 20,000 (RTI) and 4,000 (cSSSI) subjects were performed to evaluate the AUC/MIC characteristics for moxifloxacin for these two diseases. Overall, a target hit rate was THR = 99% for RTI, while it amounted to THR = 97.5% for cSSSI when applying a threshold of AUIC > 30 [h] as the PK/PD surrogate parameter which is predictive for a positive clinical outcome. A target hit rate of THR = 93.6 % (RTI) and 97.3% (cSSSI), respectively, was predicted when assuming that an AUIC of > 125 [h] is indicative of clinical success (as shown for ciprofloxacin and severe RTIs due to gram-negative infections). In clinical trials with patients receiving 400 mg moxifloxacin once daily for the treatment of community-acquired pneumonia (CAP) success rate was approximately 93.5%. From the simulations performed for RTI an analysis of the overall likelihood of therapeutic failure broken down according to MICs suggests that the risk of a negative clinical outcome at a MIC = 1 mg/l is approximately 0.25% (for MIC = 2 mg/l: predicted likelihood approximately 0.5%) assuming that a cutoff of AUIC = 30 [h] is applicable. Likewise, for cSSSI the probability to fail is predicted as 1.6% at a MIC = 2 mg/l (no strains with MICs between 0.5 and 1 mg/l available from the clinical isolates). These findings are in line with the breakpoint definition of the former National Committee for Clinical Laboratory Standards (NCCLS) for MFX (=1 mg/L to differentiate between susceptible and intermediately susceptible microorganisms; = 2 mg/l to separate intermediate from resistant pathogens). The results of the investigation indicate that the noncompartmental PK/PD model for MFX is suitable to predict clinical outcomes in CAP and cSSSI caused by gram-positive aerobe pathogens. They confirmed that a 400 mg once-daily dosing regimen is suitable to treat these diseases successfully. They are in agreement with the microbiological breakpoints determined by independent methods by the Clinical and Laboratory Standards Institute (CLSI) (former NCCLS).

The susceptibility to growth-promoting antibiotics of Enterococcus faecium isolates from pigs and chickens in Europe

To establish the susceptibility of Enterococcus faecium isolates from pigs and chickens to antimicrobial growth promoters that either were or had been in use in the European Union. Samples were taken at abattoirs in two successive years (mid-1998-mid-1999, year 1; mid-1999-mid-2000, year 2) from chickens (France, The Netherlands, Sweden, UK) and pigs (Denmark, The Netherlands, Spain, Sweden). E. faecium was isolated from faecal samples at national laboratories and sent to a central laboratory where MICs of avilamycin, avoparcin, bacitracin, flavophospholipol, spiramycin, tylosin and virginiamycin were determined. Microbiological breakpoints were allocated on the basis of MIC distributions, and comparison was made between host species, country of origin and year of sample. In total, 2567 isolates were obtained from chickens and 1742 from pigs. In all countries, resistance to avoparcin (banned in 1997) was uncommon, but resistance to bacitracin and flavophospholipol was common (and was probably largely intrinsic). The prevalence of resistance was similar in chicken and pig isolates, with the exception of avilamycin, to which resistance was commoner among chicken isolates. The removal of four compounds as growth promoters (bacitracin, spiramycin, tylosin, virginiamycin) between years 1 and 2 appeared to result in a significant decrease in resistance to three of them-spiramycin, tylosin and virginiamycin, with no change in resistance to bacitracin, but an increase in resistance to avilamycin (not discontinued). Associated resistance was shown between some of the compounds. Resistance prevalence declined rapidly following removal of growth promoters in pigs and chickens, suggesting that in the absence of selective pressure, a susceptible population began to replace phenotypically resistant strains. Associated resistance between different compounds, where seen, could have resulted from either shared resistance mechanisms or from carriage of resistance genes on the same plasmid. Multiresistance to streptogramins, macrolides and glycopeptides was rare.