Microbioassay of Antimicrobial Agents

A previously described agar-diffusion technique for microbioassay of antimicrobial agents has been modified to increase sensitivity of the technique and to extend the range of antimicrobial agents to which it is applicable. This microtechnique requires only 0.02 ml of an unknown test sample for assay, and is capable of measuring minute concentrations of antibiotics in buffer, serum, and urine. In some cases, up to a 20-fold increase in sensitivity is gained relative to other published standardized methods and the error of this method is less than +/-5%. Buffer standard curves have been established for this technique, concurrently with serum standard curves, yielding information on antimicrobial serum-binding and demonstrating linearity of the data points compared to the estimated regression line for the microconcentration ranges covered by this technique. This microassay technique is particularly well suited for pediatric research and for other investigations where sample volumes are small and quantitative accuracy is desired. Dilution of clinical samples to attain concentrations falling with the range of this assay makes the technique readily adaptable and suitable for general clinical pharmacological studies. The microassay technique has been standardized in buffer solutions and in normal human serum pools for the following antimicrobials: ampicillin, methicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, cephaloglycin, cephalexin, cephaloridine, cephalothin, erythromycin, rifamycin amino methyl piperazine, kanamycin, neomycin, streptomycin, colistin, polymyxin B, doxycycline, minocycline, oxytetracycline, tetracycline, and chloramphenicol.     

Field trial evaluating the influence of prophylactic and therapeutic antimicrobial administration on antimicrobial resistance of fecal Escherichia coli in dairy calves

The objective of this study was to describe the influence of in-feed and therapeutic antimicrobials on resistance in commensal fecal Escherichia coli isolated from preweaned calves. Four groups of 30, day-old calf-ranch calves were enrolled and raised until 4 weeks of age. Groups 1 to 3 were raised without antimicrobials in the feed. Group 1 was isolated from the other groups and received no antimicrobial therapy. Group 2 was housed on the calf ranch and did not receive antimicrobial therapy, whereas groups 3 and 4 could be treated with antimicrobials. Group 4 was fed neomycin and tetracycline HCl in the milk replacer. Fecal samples were collected from calves on days 1, 14, and 28. Three E. coli isolates per sample were evaluated for susceptibility to 12 antimicrobials. Cluster analysis was used to group isolates having similar susceptibility patterns. Cumulative logistic regression was used to evaluate factors associated with increasing levels of multiple antimicrobial resistance. In-feed antimicrobials were associated with higher levels of multiple antimicrobial resistance in fecal E. coli.f In calves not receiving in-feed antimicrobials, older calves had higher levels of resistance compared to day-old calves. Individual antimicrobial therapy increased resistance in these calves but appeared to be transient. There was no environmental influence on resistance in E. coli populations among study groups.     

Field Trial Evaluating the Influence of Prophylactic and Therapeutic Antimicrobial Administration on Antimicrobial Resistance of Fecal Escherichia coli in Dairy Calves

The objective of this study was to describe the influence of in-feed and therapeutic antimicrobials on resistance in commensal fecal Escherichia coli isolated from preweaned calves. Four groups of 30, day-old calf-ranch calves were enrolled and raised until 4 weeks of age. Groups 1 to 3 were raised without antimicrobials in the feed. Group 1 was isolated from the other groups and received no antimicrobial therapy. Group 2 was housed on the calf ranch and did not receive antimicrobial therapy, whereas groups 3 and 4 could be treated with antimicrobials. Group 4 was fed neomycin and tetracycline HCl in the milk replacer. Fecal samples were collected from calves on days 1, 14, and 28. Three E. coli isolates per sample were evaluated for susceptibility to 12 antimicrobials. Cluster analysis was used to group isolates having similar susceptibility patterns. Cumulative logistic regression was used to evaluate factors associated with increasing levels of multiple antimicrobial resistance. In-feed antimicrobials were associated with higher levels of multiple antimicrobial resistance in fecal E. coli.f In calves not receiving in-feed antimicrobials, older calves had higher levels of resistance compared to day-old calves. Individual antimicrobial therapy increased resistance in these calves but appeared to be transient. There was no environmental influence on resistance in E. coli populations among study groups.     

Antimicrobial resistance patterns of Campylobacter from feedlot cattle*

AIMS: This study examined 448 Campylobacter strains isolated in 1999 and 2000 from US feedlot cattle for resistance to 12 antimicrobials. METHODS AND RESULTS: Isolates were tested for antimicrobial susceptibility using the E-test method. Approximately 60% (n = 267) were resistant to one or more antimicrobials, and 19.6% (n = 88) were resistant to two or more antimicrobials. Of the Campylobacter jejuni isolates, 49.1% (n = 187) were resistant to tetracycline, 10.2% (n = 39) were resistant to nalidixic acid, 8.4% were resistant to trimethoprim/sulfamethoxazole, and 1.8% (n = 7) were resistant to ciprofloxacin. Resistance to any of the other eight antimicrobials was 1.3% or less, but 14.4% (n = 55) were resistant to two or more antimicrobials. In the Campylobacter coli group, 65.7% (n = 44) were resistant to tetracycline, 52.2% (n = 35) were resistant to trimethoprim/sulfamethoxazole, 22.4% (n = 15) were resistant to nalidixic acid, and 9.0% (n = 6) were resistant to ciprofloxacin. Resistance to any of the remaining eight antimicrobials was 3.0% or less, although 49.3% (n = 33) were resistant to two or more antimicrobials. CONCLUSIONS: Although antimicrobials are widely used in US feedlot cattle production, our results demonstrate generally low levels of resistance to a broad range of commonly used antimicrobials relative to other recent studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Resistance data on Campylobacter isolated from this major US livestock commodity is lacking. This overview enhances current knowledge and provides a basis for further studies.     

A real-time PCR antibiogram for drug-resistant sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t)<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.     

Expression of a novel mycobacterial phosphodiesterase successfully lowers cAMP levels resulting in reduced tolerance to cell wall–targeting antimicrobials

cAMP and antimicrobial susceptibility in mycobacteriaAntimicrobial tolerance, the ability to survive exposure to antimicrobials via transient nonspecific means, promotes the development of antimicrobial resistance (AMR). The study of the molecular mechanisms that result in antimicrobial tolerance is therefore essential for the understanding of AMR. In gram-negative bacteria, the second messenger molecule 3′’,5′’-cAMP has been previously shown to be involved in AMR. In mycobacteria, however, the role of cAMP in antimicrobial tolerance has been difficult to probe due to its particular complexity. In order to address this difficulty, here, through unbiased biochemical approaches consisting in the fractionation of clear protein lysate from a mycobacterial strain deleted for the known cAMP phosphodiesterase (Rv0805c) combined with mass spectrometry techniques, we identified a novel cyclic nucleotide-degrading phosphodiesterase enzyme (Rv1339) and developed a system to significantly decrease intracellular cAMP levels through plasmid expression of Rv1339 using the constitutive expression system, pVV16. In Mycobacterium smegmatis mc²155, we demonstrate that recombinant expression of Rv1339 reduced cAMP levels threefold and resulted in altered gene expression, impaired bioenergetics, and a disruption in peptidoglycan biosynthesis leading to decreased tolerance to antimicrobials that target cell wall synthesis such as ethambutol, D-cycloserine, and vancomycin. This work increases our understanding of the role of cAMP in mycobacterial antimicrobial tolerance, and our observations suggest that nucleotide signaling may represent a new target for the development of antimicrobial therapies.     

The US national antimicrobial resistance monitoring system.

The use of antimicrobial agents in food animals can select for resistant bacterial pathogens that may be transmitted to humans via the commercial meat supply. In the USA, the FDA's Center for Veterinary Medicine regulatory duties require a determination that antimicrobial drugs are safe and effective for use in food animals. In addition, a qualitative assessment of risks to human health from antimicrobial resistance requires development. This risk assessment process is supported by data generated by the FDA's National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria. NARMS data on antimicrobial susceptibility among Salmonella, Campylobacter, Escherichia coli and Enterococcus is collected. Research activities defining the genetic bases of resistance helps to understand the potential public health risks posed by the spread of antimicrobial resistance from food animal antimicrobial use. These activities help insure that antimicrobials are used judiciously to promote human and animal health.     

江苏部分地区食源性和人源沙门氏菌的多重耐药性研究

从江苏省部分地区收集了117个沙门氏菌分离株,其中食物源和人源菌株分别有81株和36株。16种抗生素敏感性试验表明,有111个分离株对2种或2种以上的抗生素有耐药性,人源沙门氏菌分离株的抗生素耐药率比食物源的高,单一抗生素以链霉素耐药率(92.3%,108/117)最高。对5种或5种以上抗生素耐药的分离株有59株(50.4%),其中对特定六种抗生素:氨苄青霉素、氯霉素、链霉素、磺胺、四环素和卡那霉素耐药(ACSSuTK,R型)的菌株有12株。设计18对耐药基因和I类整合子保守区的引物,对36株有不同来源和耐药特征的多重耐药菌株进行耐药基因和I类整合子的检测,PCR扩增结果与抗生素敏感性表型一致。有30株细菌携带有I类整合子,大小为0.3、0.6、1.0、1.2和1.6kb,其中1.6kb(aadA5-dfr17)大小的整合子在25株细菌中分布(24/36)。接合试验表明,氨苄青霉素、氯霉素、链霉素、甲氧苄氨嘧啶和四环素的耐药特性是由接合性质粒携带。结果显示,耐药基因多数由I类整合子和质粒携带,可以通过接合试验发生转移,可移动的DNA成分可能在耐药特性的转移和分布中起到重要作用。     

Stress responses as determinants of antimicrobial resistance in Gram-negative bacteria

Bacteria encounter a myriad of potentially growth-compromising conditions in nature and in hosts of pathogenic bacteria. These 'stresses' typically elicit protective and/or adaptive responses that serve to enhance bacterial survivability. Because they impact upon many of the same cellular components and processes that are targeted by antimicrobials, adaptive stress responses can influence antimicrobial susceptibility. In targeting and interfering with key cellular processes, antimicrobials themselves are 'stressors' to which protective stress responses have also evolved. Cellular responses to nutrient limitation (nutrient stress), oxidative and nitrosative stress, cell envelope damage (envelope stress), antimicrobial exposure and other growth-compromising stresses, have all been linked to the development of antimicrobial resistance in Gram-negative bacteria - resulting from the stimulation of protective changes to cell physiology, activation of resistance mechanisms, promotion of resistant lifestyles (biofilms), and induction of resistance mutations.     

Characterization of antimicrobial resistant Escherichia coli isolated from processed bison carcasses

AIM: To determine the phenotypic and genotypic antimicrobial susceptibility profiles of Escherichia coli from bison carcasses. METHODS AND MATERIALS: The antimicrobial resistance of 138 E. coli isolates recovered from processed bison carcasses was determined by using the National Antimicrobial Resistance Monitoring System panels, polymerase chain reaction assays, plasmid analysis and conjugation studies. RESULTS: Resistance to 14 of the 16 antimicrobials was observed. Twenty-three (16.7%) isolates displayed resistance to at least one antimicrobial agent. The most prevalent resistances were to tetracycline (13.0%), sulfamethoxazole (7.9%) and streptomycin (5.8%). No resistance was observed to amikacin and ciprofloxacin. Further analysis of 23 antimicrobial-resistant E. coli isolates showed the presence of resistance genes corresponding to their phenotypic profiles. Results of conjugation studies carried out showed most isolates tested were able to transfer their resistance to recipients. CONCLUSION: This study indicated that multidrug-resistant E. coli isolates are present in bison. However, the resistance rate is lower than that reported in other meat species. SIGNIFICANCE AND IMPACT OF THE STUDY: The beneficial effects of antimicrobial-free feeding practice in bison may be promoting a reduction in the prevalence of antimicrobial resistance in commensal flora of bison.     

The antimicrobial susceptibility of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> isolates using three different methods and their genetic relatedness

Background  

Stenotrophomonas maltophilia is inherently resistant to many antimicrobials. So far, antimicrobial susceptibility tests for S. maltophilia have not been fully standardized. The purpose of the study was to compare the susceptibility of S. maltophilia isolates against seven different antimicrobials using three different methods and to investigate their genetic relatedness.     

Resveratrol Antagonizes Antimicrobial Lethality and Stimulates Recovery of Bacterial Mutants

Reactive oxygen species (ROS; superoxide, peroxide, and hydroxyl radical) are thought to contribute to the rapid bactericidal activity of diverse antimicrobial agents. The possibility has been raised that consumption of antioxidants in food may interfere with the lethal action of antimicrobials. Whether nutritional supplements containing antioxidant activity are also likely to interfere with antimicrobial lethality is unknown. To examine this possibility, resveratrol, a popular antioxidant dietary supplement, was added to cultures of Escherichia coli and Staphylococcus aureus that were then treated with antimicrobial and assayed for bacterial survival and the recovery of mutants resistant to an unrelated antimicrobial, rifampicin. Resveratrol, at concentrations likely to be present during human consumption, caused a 2- to 3-fold reduction in killing during a 2-hr treatment with moxifloxacin or kanamycin. At higher, but still subinhibitory concentrations, resveratrol reduced antimicrobial lethality by more than 3 orders of magnitude. Resveratrol also reduced the increase in reactive oxygen species (ROS) characteristic of treatment with quinolone (oxolinic acid). These data support the general idea that the lethal activity of some antimicrobials involves ROS. Surprisingly, subinhibitory concentrations of resveratrol promoted (2- to 6-fold) the recovery of rifampicin-resistant mutants arising from the action of ciprofloxacin, kanamycin, or daptomycin. This result is consistent with resveratrol reducing ROS to sublethal levels that are still mutagenic, while the absence of resveratrol allows ROS levels to high enough to kill mutagenized cells. Suppression of antimicrobial lethality and promotion of mutant recovery by resveratrol suggests that the antioxidant may contribute to the emergence of resistance to several antimicrobials, especially if new derivatives and/or formulations of resveratrol markedly increase bioavailability.     

Bayesian estimation of multivariate normal mixtures with covariate‐dependent mixing weights,with an application in antimicrobial resistance monitoring

Bacteria with a reduced susceptibility against antimicrobials pose a major threat to public health. Therefore, large programs have been set up to collect minimum inhibition concentration (MIC) values. These values can be used to monitor the distribution of the nonsusceptible isolates in the general population. Data are collected within several countries and over a number of years. In addition, the sampled bacterial isolates were not tested for susceptibility against one antimicrobial, but rather against an entire range of substances. Interest is therefore in the analysis of the joint distribution of MIC data on two or more antimicrobials, while accounting for a possible effect of covariates. In this regard, we present a Bayesian semiparametric density estimation routine, based on multivariate Gaussian mixtures. The mixing weights are allowed to depend on certain covariates, thereby allowing the user to detect certain changes over, for example, time. The new approach was applied to data collected in Europe in 2010, 2012, and 2013. We investigated the susceptibility of Escherichia coli isolates against ampicillin and trimethoprim, where we found that there seems to be a significant increase in the proportion of nonsusceptible isolates. In addition, a simulation study was carried out, showing the promising behavior of the proposed method in the field of antimicrobial resistance.     

Microtechnique for Most-Probable-Number Analysis

A microtechnique based on the most-probable-number (MPN) method has been developed for the enumeration of the ammonium-oxidizing population in soil samples. An MPN table for a research design ([8 by 12] i.e., 12 dilutions, 8 replicates per dilution) is presented. A correlation of 0.68 was found between MPNs determined by the microtechnique and the standard tube technique. Higher MPNs were obtained with the microtechnique with increased accuracy in endpoint determinations being a possible cause. Considerable savings of time, space, equipment, and reagents are observed using this method. The microtechnique described may be adapted to other microbial populations using various types of media and endpoint determinations.     

Antimicrobial resistance of Pseudomonas aeruginosa biofilms

Resistance to antimicrobial agents is the most important feature of biofilm infections. As a result, infections caused by bacterial biofilms are persistent and very difficult to eradicate. Although several mechanisms have been postulated to explain reduced susceptibility to antimicrobials in bacterial biofilms, it is becoming evident that biofilm resistance is multifactorial. The contribution of each of the different mechanisms involved in biofilm resistance is now beginning to emerge.     

Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections

Strains of Salmonella spp. with resistance to antimicrobial drugs are now widespread in both developed and developing countries. In developed countries it is now increasingly accepted that for the most part such strains are zoonotic in origin and acquire their resistance in the food-animal host before onward transmission to humans through the food chain. Of particular importance since the early 1990s has been a multiresistant strain of Salmonella typhimurium definitive phage type (DT) 104, displaying resistance to up to six commonly used antimicrobials, with about 15% of isolates also exhibiting decreased susceptibility to ciprofloxacin. Mutations in the gyrA gene in such isolates have been characterised by a PCR LightCycler-based gyrA mutation assay, and at least four different mutations have been identified. Multiple resistance (to four or more antimicrobials) is also common in the poultry-associated pathogens Salmonella virchow and Salmonella hadar, with an increasing number of strains of these serotypes exhibiting decreased susceptibility to ciprofloxacin. Multiple resistance is also being found in other serotypes in several other European countries, and has been associated with treatment failures. For Salmonella typhi, multiple drug resistance is now the norm in strains originating in the Indian subcontinent and south-east Asia. Such multiresistant strains have been responsible for several epidemics and some of these have been associated with contaminated water supplies. Furthermore, an increasing number of multiresistant strains of S. typhi are now exhibiting decreased susceptibility to ciprofloxacin, with concomitant treatment failures. In developed countries antimicrobial resistance in zoonotic salmonellas has been attributed to the injudicious use of antimicrobials in food-producing animals. It is hoped that the application of Codes of Practice for the use of such agents, which have been prepared by the pharmaceutical industry in response to widespread international concern about the development of drug resistance in bacterial pathogens, will now result in a widespread reduction in the incidence of drug-resistant salmonellas in food production animals and humans on an international scale.     

Prevalence and antimicrobial resistance of Campylobacter in US dairy cattle

AIMS: To obtain an overview of the prevalence and antimicrobial resistance of Campylobacter in faeces of US dairy cows in 2002. METHODS AND RESULTS: Faeces from 1435 cows, representing 96 dairy operations in 21 US states, were collected for the culture of Campylobacter. A total of 735 Campylobacter strains were isolated (51.2% positive samples) with 94 operations positive (97.9%) for Campylobacter. From this collection, 532 isolates (473 Campylobacter jejuni and 59 Campylobacter coli) were randomly selected for susceptibility testing to eight antimicrobials: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. The C. jejuni isolates exhibited resistance to tetracycline (47.4%), nalidixic acid (4.0%) and ciprofloxacin (2.5%), while the C. coli strains exhibited some resistance to all antimicrobials except chloramphenicol and ciprofloxacin. Only 3.6% of the C. jejuni isolates were resistant to two or more antimicrobials but 20.3% of the C. coli strains were multiresistant. CONCLUSIONS: On most operations, at least one cow was positive for Campylobacter and more than half of the cows sampled were shedding Campylobacter. The C. coli isolates had significantly higher levels of resistance to macrolides and to tetracycline compared with the C. jejuni strains, but were susceptible to ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a high prevalence of Campylobacter on US dairy operations; however, US dairy cattle have not been recognized as a major source of human infection compared with poultry. Campylobacter coli appears to develop antimicrobial resistance more readily than C. jejuni from the same environment.     

Distribution and characterization of ampicillin- and tetracycline-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>from feedlot cattle fed subtherapeutic antimicrobials

Background  

Feedlot cattle in North America are routinely fed subtherapeutic levels of antimicrobials to prevent disease and improve the efficiency of growth. This practice has been shown to promote antimicrobial resistance (AMR) in subpopulations of intestinal microflora including Escherichia coli. To date, studies of AMR in feedlot production settings have rarely employed selective isolation, therefore yielding too few AMR isolates to enable characterization of the emergence and nature of AMR in E. coli as an indicator bacterium. E. coli isolates (n = 531) were recovered from 140 cattle that were housed (10 animals/pen) in 14 pens and received no dietary antimicrobials (control - 5 pens, CON), or were intermittently administered subtherapeutic levels of chlortetracycline (5 pens-T), chlortetracycline + sulfamethazine (4 pens-TS), or virginiamycin (5 pens-V) for two separate periods over a 9-month feeding period. Phenotype and genotype of the isolates were determined by susceptibility testing and pulsed field gel electrophoresis and distribution of characterized isolates among housed cattle reported. It was hypothesized that the feeding of subtherapeutic antibiotics would increase the isolation of distinct genotypes of AMR E. coli from cattle.     

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibiliy of Campylobacter isolates of food and human clinical origin

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis.Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3–26.6% for tetracycline and 33.3–36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.     

Investigation of integrons/cassettes in antimicrobial-resistant Escherichia coli isolated from food animals in China

In this study,326 Escherichia coli isolates from food animals collected during the last four decades in China were characterized using antimicrobial susceptibility testing and screening for integrons/cassettes.Minimum inhibitory concentration(MIC) testing indicated that the antimicrobial resistance of E.coli has increased since the 1970s.The findings of this study present a warning to veterinary practitioners about the excessive use of antimicrobials,and suggest the necessity for surveillance and control of antimicrobial resistance in veterinary clinical medicine in China.