Concentrations Of Cephalosporins Research Articles

Susceptibility of HEK293 and RPTEC Cell Lines to Nephrotoxic Effects of Cefuroxime and Cefepime: A Comparative Study

Researchers need to identify the nephrotoxic properties of medicinal products both during preclinical development and when exploring options to optimise pharmacotherapy. The main challenge is to find an experimental model for assessing drug-induced nephrotoxicity that reflects in vivo conditions as closely as possible.The aim of the study was to compare the susceptibility of HEK293 and RPTEC cell lines used as experimental models for assessing the nephrotoxicity of cefuroxime and cefepime.Materials and methods. The study investigated HEK293 and RPTEC cell lines cultured on plates with 0.4 µm pore membrane inserts. The cell lines were incubated for 3 days with cefuroxime and cefepime (cephalosporins excreted primarily by the kidneys). The medicinal products were added to the basal part of the well at concentrations of 50 and 150 µg/mL (cefuroxime) or 30 and 120 µg/mL (cefepime) twice a day. After incubating the cells with cefuroxime and cefepime for 24, 48, and 72 hours, the authors determined the expression levels of the SLC22A6 and SLC22A8 genes encoding organic anion transporters by a reverse transcription polymerase chain reaction. The authors considered caspase 3 and caspase 7 activation indicative of the nephrotoxic effect of cephalosporins; they evaluated this indicator by a fluorometric assay after 24, 48, and 72 hours of incubation.Results. According to the study, the expression of the SLC22A6 and SLC22A8 genes decreased with cephalosporin transport in both cell lines. The decrease occurred in the RPTEC cell line earlier than in the HEK293 cell line. The authors observed caspase 3 and caspase 7 activation only in the RPTEC cell line after incubation with cefuroxime and cefepime at low concentrations (50 and 30 µg/mL, respectively) for 72 hours and at high concentrations (150 and 120 µg/mL, respectively) for 24 hours.Conclusions. The RPTEC cell line exhibits higher susceptibility to cefuroxime and cefepime toxic effects than the HEK293 cell line due to higher transporter gene expression. Higher cephalosporin concentrations accelerate caspase 3 and caspase 7 activation in the RPTEC cell line. The experimental model based on the RPTEC cell line is a promising tool for the analysis of the nephrotoxic properties of a wide range of medicinal products.

Antibiotics in the Environment: Prescribing Risks to Non-Target Organisms

Background: The cephalosporins class is among the most widely used group of antimicrobials worldwide. Antibiotics, together with other drugs and personal care products, make up a group of emerging contaminants. The effects of exposure to this group of chemical contaminants on non-target organisms are not well understood, as they are still poorly studied. Therefore, this study evaluated the phytotoxicity of five cephalosporins in Lactuca sativa. Methods: Lettuce seeds were exposed to different concentrations of antibiotics (25 to 500 mg/L) for 5 days in the dark. After this period, the germination percentage and the wet and dry weights were recorded. Results: The highest tested concentration (500 mg/L) inhibited the germination of lettuce seeds (p < 0.05); there was a decrease in dry weight when exposed to a first-generation cephalosporin (p < 0.05). Additionally, there was a significantly negative influence (p < 0.05) on the fresh weight, especially in the group that evaluated the exposure of seeds to 25 mg/L of Cefepime. Conclusions: We emphasize that there is no record of environmental concentrations of cephalosporins in soil, and therefore, we can indicate that it is possible to have environmental damage resulting from the inappropriate and constant disposal of cephalosporins in the environment.

Corrosion inhibition of carbon steel by three kinds of expired cephalosporins in 0.1 M H2SO4

It is difficult to avoid corrosion to carbon steel in acidic solutions, thus, in order to inhibit the corrosion of carbon steel during the acid pickling process, inhibitors must be added to the acid pickling solutions. In this work, the corrosion inhibition of carbon steel by three kinds of expired cephalosporins in 0.1 M H2SO4 was investigated by electrochemical, weight loss and scanning electron microscopy (SEM) methods. For the carbon steel electrode, the electrochemical results revealed that the current density was decreased, while, the charge-transfer resistance was increased in the presence of cephalosporins in 0.1 M H2SO4 corrosive solution. The weight loss results confirmed that the addition of cephalosporins decreased the corrosion rate of carbon steel. Further, the inhibition efficiency was increased with the increase in the concentration of cephalosporins. The SEM analysis of carbon steel surface was performed to confirm the effect of cephalosporins on the corrosion inhibition. The presented research demonstrated the efficient use of expired drugs for effective corrosion inhibition which could be applied to the industries at the large scale.

Characteristics of third-generation cephalosporin-resistant Salmonella from retail chicken meat produced by integrated broiler operations

Integrated broiler operations, which control and operate vertically through all phases of the chicken industry, have applied biosecurity and sanitation practices, housing technologies, feeding regimens, and antibiotic applications in different ways to improve food safety. The objective of this study was to compare the prevalence and antimicrobial resistance profiles of Salmonella isolates recovered from 6 different integrated broiler operations and to analyze the characteristics of extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC β-lactamase (pAmpC)-producing Salmonella isolates. Among 336 chicken meat samples, 57 were observed to be positive for Salmonella. However, the prevalence varied from 6.8% to 45.8% in chicken meat, indicating variations in Salmonella occurrence among the operations. Salmonella Albany was the dominant serovar, followed by Salmonella Virchow. In the antimicrobial resistance test, nalidixic acid-resistant isolates were the most prevalent (73.7%), followed by isolates resistant to ampicillin (49.1%) and tetracycline (42.1%). Among 14 third-generation cephalosporin-resistant isolates, 9 (64.3%) ESBL/pAmpC-producing isolates were only obtained from 2 operations: blaCTX-M-15 (n = 7) and blaCTX-M-79 (n = 1) for ESBL genes and blaCMY-2 (n = 1) for pAmpC. All ESBL/pAmpC-positive isolates exhibited high minimum inhibitory concentrations (≥128 μg/mL) of most cephalosporins and showed multidrug resistance. The transfer of ESBL/pAmpC genes was confirmed in transconjugants, which had the same genes and similar resistance patterns as those of the donor. Our findings suggest that Salmonella with resistance to third-generation cephalosporins can now be found in association with integrated broiler operations, providing data to support the development of monitoring and prevention programs for the development and spread of antimicrobial resistance in integrated broiler operations.

Infections Caused by Carbapenemase-Producing Klebsiella pneumoniae: Microbiological Characteristics and Risk Factors.

The spread of carbapenemase-producing Klebsiella pneumoniae (CPKP) worldwide is a serious problem. This retrospective, matched case–control, parallel study in a tertiary teaching hospital analyzed the microbiological and clinical characteristics of CPKP infection, focusing on the risk factors for carbapenem resistance and patient mortality. The hospital department with the highest incidence of CPKP infections was the intensive care unit. All CPKP strains examined were positive for blakpc-2, and 84.8% of CPKP were ST11. Hypervirulent phenotype was identified in 22.7% of the patients with CPKP, with these strains displaying a high incidence of positivity for entB, ybtS, and iutA. Multivariate conditional logistic regression analysis demonstrated that Pitt bacteremia score >4, prior stomach tube, continuous renal replacement therapy (CRRT), and previous carbapenem exposure were associated with CPKP infection. Higher albumin concentration and use of cephalosporins after diagnosis were strong prognostic factors for crude 28-day mortality. Further, high APACHE II score, CRRT, use of carbapenems after diagnosis, and bacteremia were risk factors for crude in-hospital mortality. CPKP isolates showed clonal spread and were resistant to most antibiotics, resulting in higher financial burden. Critical illness was associated with increased mortality.

Construction of a Quantitative Structure Activity Relationship (QSAR) Model to Predict the Absorption of Cephalosporins in Zebrafish for Toxicity Study

Cephalosporins are beta-lactam antibiotics that are widely used in China. Five generations of cephalosporins have been introduced in clinical practice to date; moreover, some new candidates are also undergoing clinical evaluations. To improve the success rates of new drug development, we need to have a comprehensive understanding about the relationship between the structure of cephalosporins and the toxicity that it induces at an early stage. In the cephalosporins toxicity study using zebrafish, the drug absorption is a key point. In this study, we determined the absorption of cephalosporins in zebrafish during toxicity test. The internal concentrations of 19 cephalosporins in zebrafish were determined using a developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Furthermore, a quantitative structure-activity relationship (QSAR) model was established by multilinear regression; moreover, it was used to predict the absorption of cephalosporins in zebrafish. During leave-one-out cross-validation, a satisfactory performance was obtained with a predictive ability (q2) of 0.839. The prediction ability of the model was further confirmed when the predictive ability (q2) was 0.859 in external prediction. The best QSAR model, which was based on five molecular descriptors, exhibited a promising predictive performance and robustness. In experiments involving drug toxicity, the developed QSAR model was used to estimate internal concentrations of cephalosporins. Thus, the toxicity results were correlated with the internal concentration of the drug within the larvae. The developed model served as a new powerful tool in zebrafish toxicity tests.

Effect of Benzalkonium Chloride Adaptation on Sensitivity to Antimicrobial Agents and Tolerance to Environmental Stresses in Listeria monocytogenes.

Listeria monocytogenes is an important food-borne pathogen that can persist in food processing environments and thus contaminate food products. Benzalkonium chloride (BC) is a common disinfectant widely used in food industry. Selective pressure associated with exposure to BC may result in adaptation to this agent in L. monocytogenes. In this study, the effect of BC adaptation on susceptibility to antimicrobial agents and tolerance to environmental stresses, as well as the role of efflux pumps in BC adaptation were investigated in Listeria monocytogenes. Exposure of L. monocytogenes to progressively increasing concentrations of BC led to adaptation not only to BC but also to several other antimicrobial agents with different modes of action, including cefotaxime, cephalothin, ciprofloxacin, and ethidium bromide (EtBr), indicating that the disinfectant BC has the ability to select for antibiotic resistance. Reserpine, an efflux pump inhibitor, reduced minimum inhibitory concentrations (MICs) of cephalosporins, ciprofloxacin, and EtBr in BC adapted strains, indicating that efflux pumps are involved in cross-adaptation to these antimicrobial agents. Our results showed that expression levels of the efflux pump MdrL in the BC adapted strains increased significantly relative to the corresponding wild-type strains (P < 0.05), with the highest increase in one BC adapted strain named HL06BCA. Moreover, the knockout mutant HL06BCAΔmdrL showed impaired growth compared to that of HL06BCA when exposed to 2 μg/ml of BC. It suggests that efflux pump MdrL is associated with BC adaptation in L. monocytogenes. However, we did not find mdrL to be associated with cross-adaptation to cephalosporins, ciprofloxacin, and EtBr in HL06BCA. Additionally, increased sensitivity to acid, alkali, osmotic, ethanol, and oxidative stresses was observed in most strains after repeated exposure to BC. These results suggest rotation of different disinfectant is helpful to maintain high effectiveness of BC toward L. monocytogenes and ethanol and hydrogen peroxide are at least the appropriate candidates.

Evaluation of antibiotic resistant lactose fermentative opportunistic pathogenic Enterobacteriaceae bacteria and blaTEM-2 gene in cephalosporin wastewater and its discharge receiving river

This study investigated the concentration of cephalosporin, the resistant levels of lactose fermentative opportunistic pathogenic Enterobacteriaceae bacteria (LFOPEB) against seven antibiotics and one cephalosporin-resistant gene in cephalosporin wastewater (CPWW) treatment plant and its discharge receiving river. Although large numbers of bacteria have been removed during the CPWW treatment process, the antibiotic resistant rates of the isolates to β-lactam antibiotics significantly increased (p = 0.032) after treatment, while the percentage of resistant LFOPEB to non-β-lactam antibiotics did not change dramatically. Furthermore, the discharge of the effluent of CPWW treatment plant (CPWWeff) led to an obvious increase in the percentages of β-lactam antibiotic-resistant LFOPEB and relative abundance of the blaTEM-2 gene in the downstream receiving river (RWdown) in comparison with those in the upstream receiving river (RWup). The antibiotic resistant phenotypes of isolates in the influent of CPWW treatment plant (CPWWin), CPWWeff and RWdown appeared to be seriously affected by the cephalosporin residues, which suggested that main antibiotic resistance phenotypes in antibiotic contaminated water were closely associated with its antibiotic composition. Therefore, CPWW treatment process has been proved to result in selective growth of ARB and proliferation of ARG. Besides, CPWWeff was also proved to be an important supplier of ARB and ARG to the receiving river.

Validated LC-MS/MS method for simultaneous analysis of 21 cephalosporins in zebrafish for a drug toxicity study

Zebrafish model was used to perform this drug toxicity study. In this study, we monitored the absorption of drugs in zebrafish. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed in this experiment. The LC-MS/MS method was used for the simultaneous quantification of 21 cephalosporins in zebrafish. To prepare samples, rapid protein precipitation was performed with 1% formic acid in methanol. All the analytes were separated on a C18 column. These analytes were analyzed by performing multiple-reaction monitoring (MRM) scans in positive electrospray ionization mode. The ranges for limits of detection (LOD) and quantification (LOQ) were as follows: 0.01–10 ng/mL (S/N ≥ 3) and 0.05–25 ng/mL. Intra-and inter-day accuracy of all 21 cephalosporins were within the range of 80%–120%. Intra-and inter-day precision were within the range of 0.4%–11.2%. All the 21 analytes were analyzed within 10 min. In drug toxicity studies, the present method can be used to determine the internal concentrations of cephalosporins in zebrafish following the administration of drugs.

Evaluation of the relationship between the minimum inhibitory concentration of ceftiofur and third-generation cephalosporins in Escherichia coli isolates from food-producing animals

To enable future comparison of the antimicrobial susceptibility data between bacteria obtained from animals and humans, it is necessary to compare the relationships between minimum inhibitory concentrations (MICs) of veterinary and human medicine. We evaluated the relationship between the MIC of ceftiofur (CTF) and the MICs of other third-generation cephalosporins (TGCs): cefotaxime (CTX), cefpodoxime (CPDX), and ceftazidime (CAZ), determined by the broth microdilution method using 118 cefazolin-resistant Escherichia coli isolates from food-producing animals. Using the Clinical and Laboratory Standards Institute criteria, very major classification errors were observed only in CAZ (17.8%, 21 of 118); major and minor errors were observed in all TGCs (CTX: 0.8% [1 of 118] and 9.3% [11 of 118]; CPDX: 9.3% [11 of 118] and 6.8% [8 of 118]; CAZ: 2.5% [3 of 118] and 9.3% [11 of 118], respectively). The Spearman correlation coefficients between the MICs of CTF and CTX, CPDX, and CAZ were 0.765, 0.731, and 0.306, respectively. The sensitivity and specificity values were 100.0% and 81.8% for CTX, 99.0% and 27.3% for CPDX, and 76.0% and 86.4% for CAZ compared with CTF. The C-statistic was 0.978 for CTF and CTX, 0.953 for CPDX, and 0.798 for CAZ. For the TGCs evaluated in our study, testing for CTX susceptibility results showed the highest correlation with the results given when testing for CTF susceptibility.

Hfq mutation confers increased cephalosporin resistance in Klebsiella pneumoniae

Klebsiella pneumoniae (K. pneumoniae), is an opportunistic pathogen raising significant public health concerns owing to its multi-drug resistance. Hfq, one of the main RNA-binding proteins, is a key post-transcriptional regulator. This protein is closely related to virulence and resistance in various pathogenic bacteria. Although the role of hfq in K pneumoniae virulence has been explored, its influence on resistance remains largely unknown. The aim of this study was to investigate the role of hfq in the resistance of K. pneumoniae to cephalosporins. An hfq mutant was constructed, and its resistance to cephalosporins was investigated. The hfq mutant exhibited over 16-fold higher cephalosporin resistance than that exhibited by the wild type. Time-kill curve analysis showed that the hfq mutant could survive under higher concentrations of cephalosporins than the wild-type strain could. Quantitative RT-PCR showed that expression levels for 8 out of the 9 penicillin-binding proteins, which are the targets of cephalosporins, were downregulated in the hfq mutant. Taken together, contrary to its role in many other bacteria, hfq is involved in a negative regulation of K. pneumoniae resistance to cephalosporins by downregulating the expression of penicillin-binding proteins.

Antimicrobial Resistance Mechanisms and Genetic Diversity of Multidrug-Resistant Acinetobacter baumannii Isolated from a Teaching Hospital in Malaysia.

Multidrug-resistant (MDR) Acinetobacter baumannii has increasingly emerged as an important nosocomial pathogen. The aim of this study was to determine the resistance profiles and genetic diversity in A. baumannii clinical isolates in a tertiary medical center in Malaysia. The minimum inhibitory concentrations of carbapenems (imipenem and meropenem), cephalosporins (ceftazidime and cefepime), and ciprofloxacin were determined by E-test. PCR and sequencing were carried out for the detection of antibiotic resistance genes and mutations. Clonal relatedness among A. baumannii isolates was determined by REP-PCR. Sequence-based typing of OXA-51 and multilocus sequence typing were performed. One hundred twenty-five of 162 (77.2%) A. baumannii isolates had MDR phenotype. From the 162 A. baumannii isolates, 20 strain types were identified and majority of A. baumannii isolates (66%, n = 107) were classified as strain type 1 and were positive for ISAba1-blaOXA-23 and ISAba1-blaADC and had mutations in both gyrA and parC genes at positions, 83 and 80, resulting in serine-to-leucine conversion. REP-PCR analysis showed 129 REP types that generated 31 clones with a 90% similarity cutoff value. OXA-66 variant of the blaOXA-51-like genes was predominantly detected among our A. baumannii clinical isolates belonging to ST195 (found in six clones: 1, 8, 9, 19, 27, and 30) and ST208 (found in clone 21). The study helps us in understanding the genetic diversity of A. baumannii isolates in our setting and confirms that international clone II is the most widely distributed clone in Universiti Kebangsaan Malaysia Medical Centre, Malaysia.

Susceptibility to cephalosporins of bacteria causing intramammary infections in dairy cows with a high somatic cell count in Germany

The objective of this cross-sectional study was to determine the minimal inhibitory concentrations of cephalosporins of the first (cefalonium and cefapirin) and fourth generation (cefquinome) against bacteria isolated from intramammary infections in dairy cows with elevated somatic cell counts in Germany. Additionally, possible regional differences of the minimal inhibitory concentrations within Germany should be evaluated. In total, 6936 quarter milk samples from cows with a somatic cell count >200,000cells/ml were taken in 43 herds.The concentrations of the first generation cephalosporins inhibiting at least 90% of the isolates of a pathogen (MIC90) were ≥64μg/ml against Gram-negative bacteria and enterococci whereas the respective MIC90 against the other Gram-positive bacteria were ≤4μg/ml. The MIC90 of cefquinome were ≥16μg/ml against Gram-negative bacteria, bacilli and enterococci, and ≤2μg/ml against the other Gram-positive bacteria.Only the minimal inhibitory concentrations against coagulase-negative staphylococci differed significantly between regions in parametric survival models with shared frailties for the herds. However, the minimal inhibitory concentrations of cefquinome against staphylococci were higher than the minimal inhibitory concentrations of the tested cephalosporins of the first generation. Therefore, cefquinome should not be the first choice to treat staphylococcal mastitis in dairy cows.

Modified Deacetylcephalosporin C Synthase for the Biotransformation of Semisynthetic Cephalosporins.

Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher kcat/Km values. The efficacy and industrial scalability of one of the selected variants, CefFGOS, were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS. 7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directed-evolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization.

Gonococcal infections in Austria: a long-term observation of prevalence and resistance profiles from 1999 to 2014.

The increase in minimum inhibitory concentrations (MICs) of cephalosporins for Neisseria gonorrhoeae has given rise to concerns regarding potentially untreatable gonococcal infections. The goal was to ascertain the prevalence of gonorrhea in a Viennese patient group and determine resistance patterns. Another objective was to evaluate resistance profiles and MIC values of gonococcal isolates in an Austria-wide surveillance project. From 1999 to 2014, 350,000 individuals were tested for gonococci at the Viennese Outpatient Clinic. In addition, from 2010 to 2014, the MICs of recommended antibiotics was determined in 3,584 gonococcal isolates, initially by agar dilution and breakpoint determination, and, from 2012 onwards, by Etest®. During the observation period, the prevalence of gonorrhea increased eightfold, with a significantly greater number of quinolone, penicillin, and tetracycline- resistant strains. In gonococcal strains isolated from across Austria, there was an increase in cefixime and ceftriaxone MICs toward breakpoints. Twenty-one isolates showed cefixime resistance, and while there was an increase in azithromycin resistance from 0.9 % (2013) to 3.2 % (2014), no resistance to ceftriaxone was observed. Currently, there is no imminent risk of untreatable gonorrhea in Austria. However, continuing the use of gonococcal cultures as a diagnostic tool for establishing resistance profiles is essential in order to monitor trends in the development of Neisseria (N.) gonorrhoeae resistance.

Voltammetric determination of cefotaxime using potassium peroxomonosulfate

The presented article is dedicated to the development of new procedure for quantitative voltammetric determination of cefotaxime powder for injection solution preparation in the form of corresponding S-oxide in weak acidic medium using potassium hydrogenperoxomonosulfate (KHSO5) as an analytical reagent. Voltammogramms of cefotaxime S-oxide solutions for different concentrations of cephalosporins were scanned. There are two peaks on the voltammetric curve of the cefotaxime S-oxide solution: at -0.65 V (that corresponds to potassium peroxomonosulfate) and -1.3 V (peak height was rising proportionally to cefotaxime concentrations increasing) that has been chosen as analytical. The calibration curve method can be easily applied. Linearity has been studied over a drug concentration range from 1·10-4 to 1·10-3 mol L-1. The correlation coefficient r=0.999. Precision and accuracy have been studied by analyzing five replicates of the sample solutions at three concentrations levels. The calculated relative standard deviations were below 1.75 %, d ≤ -1.1 % indicating excellent precision of the proposed procedure. The Limit of Detection (LOD) and the Limit of Quantification (LOQ) were calculated (LOD=1.2·10-5 mol L-1 and LOQ= 4·10-5 mol L-1). The proposed voltammetric method is sensitive enough, accurate, precise, replicable and linear to enable determination of lower amounts of drug. These advantages encourage the application of the method in routine quality control of cefotaxime in industrial laboratories.

Opposite effects of cefoperazone and ceftazidime on S‑ribosylhomocysteine lyase/autoinducer-2 quorum sensing and biofilm formation by an Escherichia coli clinical isolate.

To investigate the effects of subminimum inhibitory concentrations of cephalosporins on bacterial biofilm formation, the biofilm production of 52 Escherichia (E.) coli strains was examined following treatment with cephalosporin compounds at 1/4 minimum inhibitory concentrations (MICs). Ceftazidime (CAZ) inhibited biofilm formation in seven isolates, while cefoperazone (CFP) enhanced biofilm formation in 18 isolates. Biofilm formation of E. coli E42 was inhibited by CAZ and induced by CFP. Therefore, using reverse transcription-polymerase chain reaction, the expression of the biofilm-modulating genes of this isolate was investigated. To monitor the production of the autoinducer of quorum sensing in E. coli, autoinducer-2 (AI-2) production was detected by measuring the bioluminescence response of Vibrio harveyi BB170. Antisense oligonucleotides (AS-ODNs) targeting S-ribosylhomocysteine lyase (luxS) inhibited the expression of the luxS gene in E. coli. CAZ at 1/4 MIC reduced luxS mRNA levels and the production of AI-2, whereas CFP at 1/4 MIC had the opposite effect. AS-ODNs targeting luxS significantly decreased the aforementioned inhibitory effects of CAZ and the induction effects of CFP on E. coli biofilm formation. Therefore, biofilm formation by the E. coli clinical isolate E42 was evoked by CFP but attenuated by CAZ at sub-MICs, via a luxS/AI-2-based quorum sensing system.

Treatment of Extended-Spectrum Beta-Lactamase Enterobacteriaceae With Cefepime: The Dose Matters, Too

To the Editor—We read Lee et al's article in the 15 February 2013 issue of Clinical Infectious Diseases with great interest [1]. In this retrospective cohort, Lee et al showed that 17 propensity-score-matched patients had increased mortality when treated with cefepime, as compared to carbapenems, as definitive therapy for extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae. This finding was only relevant to organisms with minimum inhibitory concentrations (MICs) in the 2–8 µg/mL range. Based on their results, the authors conclude that cefepime therapy may only be appropriate to treat ESBL-producing organisms when the MIC is ≤1 µg/mL. Although we agree that there are limited data supporting the clinical use of cefepime for ESBL-producing organisms, we cannot entirely dismiss the efficacy of cefepime for pathogens with elevated but susceptible MICs based on these results. With the recent change in the Clinical and Laboratory Standards Institute recommendations to simply report MICs instead of performing confirmatory phenotypic testing for ESBL production, the assumption is that the organism's β-lactamase gene is irrelevant as long as pharmacodynamic targets remain attainable with the most commonly utilized dose of the drug [2]. Indeed, Andes and Craig have shown that maintaining a free cephalosporin concentration above the MIC for 70% of the dosing interval is associated with optimal microbial killing in both non-ESBL- and ESBL-producing organisms in the neutropenic mouse model, irrespective of genotype [3]. As Lee et al point out, several authors have described the difficulty in achieving meaningful pharmacodynamic probability of target attainment (PTA) for cefepime with organisms in the higher (2–8 µg/mL) MIC ranges. As shown in the Monte Carlo simulation performed by Roos et al, a dose of 2 g every 12 hours resulted in insufficient PTA for pathogen MICs of 4–8 µg/mL in the critically ill population, whereas 2 g every 8 hours was able to achieve meaningful PTA [4]. These data are in concordance with the findings of Bhat et al, who showed that a group of bacteremic patients with organism MICs of 8 µg/mL treated with cefepime 1–2 g every 12 hours had higher mortality compared to those with lower MICs [5]. As the present study only included 11 patients (5 of whom had died at 30 days) with MICs in the 2–8 µg/mL range [1], it is difficult to conclude that cefepime is ineffective, especially without specification of the MIC distribution and dosing regimens utilized. For instance, were all 5 failures treated with 1 g every 8 hours for organisms with an MIC of 8 µg/mL? Given the recent increase in prevalence of carbapenemase-producing organisms, it is essential to minimize the use of carbapenems whenever possible. There are data suggesting that traditional doses of cefepime (1 g every 8 hours or 2 g every 12 hours) may be inadequate to treat infections due to Enterobacteriaceae with elevated cefepime MICs (≥4 µg/mL), including ESBL-producing organisms; however, we are not aware of any trials evaluating aggressive cefepime doses (2 g every 8 hours, 1 g every 6 hours, or extended infusion strategies) to optimize the chances of achieving PTA and treat infections due to Enterobacteriaceae with higher cefepime MICs. Although carbapenems remain the drug of choice for ESBL-producing organisms, further clinical research evaluating cefepime dosing with pharmacodynamic optimization should be performed.

Partitioning of cephalosporins in acetone/NaCl two-phase system

Partitioning of several kinds of semi-synthetic cephalosporins in acetone/NaCl two-phase system was investigated. The system is composed of hydrophilic solvent, inorganic salt and water. The phase diagram of acetone/NaCl system was made by titration method. Cephalosporin concentration was determined by HPLC. Experimental results showed that cephalosporin concentration and temperature have a little effect on the partition of cephalosporins. NaCl concentration and acetone concentration affect the partition to a certain extent. When these concentrations increase, cephalosporins move from top phase (acetone phase) to bottom phase (salt phase). The effect of pH on the partition is great. At low pH, cephalosporins stay mainly in top phase. When pH increases to 3.8, most of cephalosporins transfer to bottom phase. It is practicable to extract cephalosporins by adjusting pH of the two-phase system.

Strategies to overcome extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in shigellae

Oral cephalosporins and mecillinam are used to treat Shigella infections, but are compromised by extended-spectrum β-lactamases (ESBLs) and plasmid AmpC β-lactamases. Potential solutions include combining an oral or intravenous cephalosporin with a β-lactamase inhibitor (BLI) or using an oral penem. These strategies were examined using Escherichia coli transconjugants and clinical isolates with ESBLs or AmpC, as a proxy for shigellae. The Clinical and Laboratory Standards Institute agar dilution method was used with inocula of 104 and 106 colony-forming units/spot. ESBLs conferred resistance to the cephalosporins and mecillinam, at least at high inoculum, although: (i) ceftibuten was significantly compromised only by SHV and CTX-M-15 ESBLs, but not by TEM or CTX-M-9 and -14; (ii) cefdinir was little affected by TEM-type ESBLs, and mecillinam was little affected by CTX-M-9 group enzymes. The BLI clavulanic acid reduced the minimum inhibitory concentrations (MICs) of cephalosporins and mecillinam to ≤2mg/L for ESBL-producers, even at high inocula; sulbactam in particular and tazobactam were less effective, especially against SHV types. Strains with AmpC were resistant to all cephalosporins±inhibitors, but mecillinam remained active (MIC=1mg/L) against a strain with AmpC alone, whereas strains with TEM-1+AmpC were susceptible to mecillinam+clavulanic acid at ≤2mg/L. Faropenem was active against all ESBL- and AmpC-producers at 4mg/L, with little inoculum effect or inhibitor potentiation. In conclusion, cephalosporin+clavulanic acid combinations overcame ESBLs, with ceftibuten+clavulanic acid being particularly promising. Mecillinam+clavulanic acid and faropenem overcame both ESBLs and AmpC enzymes. Clinical utility will depend also on a drug's ability to reach intracellular shigellae in the intestinal epithelium and this deserves exploration for clavulanic acid and faropenem.