Concentrations Of Cefotaxime Research Articles

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.

Extended spectrum β-lactamase-producing Escherichia coli forms filaments as an initial response to cefotaxime treatment.

Backgroundβ-lactams target the peptidoglycan layer in the bacterial cell wall and most β-lactam antibiotics cause filamentation in susceptible Gram-negative bacteria at low concentrations. The objective was to determine the initial morphological response of cephalosporin resistant CTX-M-1-producing E. coli to cefotaxime and to determine whether the response depended on the growth phase of the bacterium and the concentration of antibiotic.ResultsTwo antibiotic resistant strains carrying blaCTX-M-1 on the chromosome and on an IncI1 plasmid and three sensitive strains were used in this study. The resistant strains displayed elongated cells when exposed to cefotaxime at sub-inhibitory as well as therapeutic concentrations (1 to 512 mg/L of cefotaxime) in both lag and early exponential phase, suggesting that the elongation was an initial response mechanism to the antibiotic. Normal sized cells were the dominant cell type in exponential and stationary growth phase. No elongated cells were seen in cultures without cefotaxime. In cultures with high concentrations of cefotaxime (128–512 mg/L), no growth other than initial filamentation was observed, but spheroplats appeared after 14–17 hours in cultures of the resistant strains. Filaments were also observed in sensitive control strains with sub-inhibitory concentrations of cefotaxime.ConclusionsWe showed that E. coli resistant to β-lactams by an extended-spectrum β-lactamase, blaCTX-M-1, produced filaments when exposed to cefotaxime. The filament formation was restricted to early growth phases and the time the cells grew as filaments was antibiotic concentration dependent. This indicates that antibiotic resistant E. coli undergo the same morphological changes as sensitive bacteria in the presence of β-lactam antibiotic. It was showed that the filament formation was an initial response to the antibiotics.

In vitro prediction of the evolution of GES-1 β-lactamase hydrolytic activity.

Resistance to β-lactams is constantly increasing due to the emergence of totally new enzymes but also to the evolution of preexisting β-lactamases. GES-1 is a clinically relevant extended-spectrum β-lactamase (ESBL) that hydrolyzes penicillins and broad-spectrum cephalosporins but spares monobactams and carbapenems. However, several GES-1 variants (i.e., GES-2 and GES-5) previously identified among clinical isolates display an extended spectrum of activity toward carbapenems. To study the evolution potential of the GES-1 β-lactamase, this enzyme was submitted to in vitro-directed evolution, with selection on increasing concentrations of the cephalosporin cefotaxime, the monobactam aztreonam, or the carbapenem imipenem. The highest resistance levels were conferred by a combination of up to four substitutions. The A6T-E104K-G243A variant selected on cefotaxime and the A6T-E104K-T237A-G243A variant selected on aztreonam conferred high resistance to cefotaxime, ceftazidime, and aztreonam. Conversely, the A6T-G170S variant selected on imipenem conferred high resistance to imipenem and cefoxitin. Of note, the A6T substitution involved in higher MICs for all β-lactams is located in the leader peptide of the GES enzyme and therefore is not present in the mature protein. Acquired cross-resistance was not observed, since selection with cefotaxime or aztreonam did not select for resistance to imipenem, and vice versa. Here, we demonstrate that the β-lactamase GES-1 exhibits peculiar properties, with a significant potential to gain activity against broad-spectrum cephalosporins, monobactams, and carbapenems.

Ultrastructural Changes in Clinical and Microbiota Isolates of Klebsiella pneumoniae Carriers of Genes bla SHV, bla TEM, bla CTX-M, or bla KPC When Subject to β-Lactam Antibiotics.

The aim of this study was to characterize the ultrastructural effects caused by β-lactam antibiotics in Klebsiella pneumoniae isolates. Three K. pneumoniae clinical isolates were selected for the study with resistance profiles for third-generation cephalosporins, aztreonam, and/or imipenem and with different resistance genes for extended-spectrum β-lactamases (ESBL) or Klebsiella pneumoniae carbapenemase (KPC). Two K. pneumoniae isolates obtained from the microbiota, which were both resistant to amoxicillin and ampicillin, were also analyzed. In accordance with the susceptibility profile, the clinical isolates were subjected to subminimum inhibitory concentrations (sub-MICs) of cefotaxime, ceftazidime, aztreonam, and imipenem and the isolates from the microbiota to ampicillin and amoxicillin, for analysis by means of scanning and transmission electron microscopy. The K. pneumoniae isolates showed different morphological and ultrastructural changes after subjection to β-lactams tested at different concentrations, such as cell filamentation, loss of cytoplasmic material, and deformation of dividing septa. Our results demonstrate that K. pneumoniae isolates harboring different genes that encode for β-lactamases show cell alterations when subjected to different β-lactam antibiotics, thus suggesting that they possess residual activity in vitro, despite the phenotypic resistance presented in the isolates analyzed.

Effect of Ticarcillin, Cefotaxime and Carbenicillin doses on Efficacy of Tissue Culture Parameters Associated with Durum wheat Transformation

Agrobacterium-mediated genetic trans-formation is one of the most efficient and widely used method for plant transformation. In this study three types of β-lactam antibiotics (ticarcillin, cefotaxime and carbenicillin) individu-ally and in different concentrations and combi-nations, were used to suppress post co-cultivation growth of Agrobacterium tumefa-ciens (strain EHA105, harboring binary vector PBI 121) during plant transformation. High concen-tration of antibiotics used for eliminating Agro-bacterium has negative effect on callus growth, plant regeneration and embryo germination. Therefore the optimal concentration of antibi-oticthat efficiently prevents growth of Agrobac-terium without impairing the above parameters needs to be identified. Different concentrations of ticarcillin, cefotaxime, and carbenicillin (50–250 mg/l) individually and in combination (25–100 mg/l) were added to callusing, regeneration and germination media. Individual antibiotics at 200 mg/l and in combination at 50 mg/l and above were effective for control of agrobacte-rium. In the medium containing combination of ticarcillin, cefotaxime and carbenicillin (50 mg/l each), relative callus weight gain (21.73 mg), percentage plant regeneration (80%) and percentage germination rate (66.12%) was high-er than media containing otherwise effective doses of single antibiotic (200 and 250 mg/l) or combinations of three antibiotics at the rate of 75 mg/l and 100 mg/l of each.

Effect of ambroxol on the concentration of cefotaxime in the bronchoalveolar lavage fluid of rats with pulmonary fibrosis

This study aimed to investigate the effect of ambroxol on the concentration of cefotaxime in the bronchioalveolar lavage fluid of rats with bleomycin-induced pulmonary fibrosis. A total of 54 Wistar male rats were randomly divided into three groups, namely the normal control group, model group and ambroxol group. On experimental day 0, the rats were intratracheally instilled with bleomycin (5 mg/kg body weight) or sterile saline. The rats in the ambroxol group were then treated with ambroxol (35 mg/kg/day) intraperitoneally. On days 7, 14, 28 after instillation, six rats from each group were sacrificed, bronchial alveolar fluids were recovered and the lungs were collected for histopathological examination following the injection of cefotaxime (600 mg/kg) intravenously. The concentration of cefotaxime in the bronchial alveolar fluids was assayed by a liquid chromatography-mass spectrometry method. On day 7, the concentration of cefotaxime in the bronchial alveolar fluid of the ambroxol group was lower than that of the model group. On day 14, the concentration of cefotaxime in the bronchial alveolar fluids of the ambroxol group was higher than that of the model group, and the difference between these groups was significant statistically (P<0.001). On day 28, the concentration of cefotaxime in the bronchial alveolar fluids of the ambroxol group decreased sharply, and was lower than that of the model group (P=0.126). These results indicate that ambroxol increased the concentration of cefotaxime in the bronchial alveolar fluids at the primary fibrosis stage.

Preventing infective complications following leech therapy: elimination of symbiotic Aeromonas spp. from the intestine of Hirudo verbana using antibiotic feeding.

Hirudotherapy is often used successfully in modern medicine, especially in plastic and reconstructive surgery. However, Aeromonas infections are the most common complications of post-operative leech application. Hence, prophylactic antibiotic administration is recommended before and during leech therapy. It has been confirmed that patient safety and achieving the desired therapeutic effect depend mainly on the microbiologic purity of the animals used. The aims of this study were to find a safe and practical way to eradicate symbiotic Aeromonas spp. occuring in the intestine of Hirudo verbana. Leeches were fed artificially with 1.5 mL of sterile defibrinated sheep blood supplemented with ciprofloxacin (CIP) or cefotaxime (CTX), at bacteriostatic concentrations of 0.2 mcg/mL or 1.5 mcg/mL, and bactericidal concentrations of 20 mcg/mL or 50 mcg/mL, respectively. Bacteria were isolated from the leech intestines before and after feeding at different time intervals: 1, 7, 14, 21, and 28 d. Biochemical identification of bacterial isolates from water samples and intestines of H. verbana using the API-NE20 test showed that A. veronii biovar sobria was predominant. Bacteria belonging to the genus Aeromonas were detected in all control leeches. The results showed that optimum eradication of bacteria from leech intestines was obtained using 20 mcg/mL of CIP and 50 mcg/mL of CTX, which decreased the number of Aeromonas spp. to undetectable levels for two weeks after feeding in all treated leeches. A statistically significant reduction in the number of bacterial colonies (p<0.0001) was observed in leeches treated with bacteriostatic concentrations of CIP or CTX; no bacterial growth was found on the plates after only seven days of feeding with antibiotics. All water samples in which the leeches were kept before treatment were contaminated with Aeromonas spp., whereas these samples were negative after antibiotic feeding of animals. All leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated or cefotaxime-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy.

First case of pregnant women bacteraemia and probable early-onset neonatal infection due to Aerococcus urinae.

First case of pregnant women bacteraemia and probable early-onset neonatal infection due to Aerococcus urinae.

CTX-M-1 β-lactamase expression in Escherichia coli is dependent on cefotaxime concentration, growth phase and gene location

Knowledge about the regulatory mechanisms of CTX-M β-lactamase-encoding genes in Escherichia coli is limited. The objectives of this study were to determine the growth response of CTX-M-1-producing E. coli exposed to cefotaxime and to investigate how blaCTX-M-1 expression at mRNA and protein levels is influenced by cefotaxime concentration, growth phase and gene location (chromosome versus plasmid). Two isogenic E. coli strains, MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, containing blaCTX-M-1 on the chromosome and on a wild-type IncI1 plasmid, respectively, were constructed and the MIC of cefotaxime was determined. Growth of the two strains was studied in the presence of increasing concentrations of cefotaxime ranging from 0 to 512 mg/L. The levels of mRNA and protein in different growth phases and at different cefotaxime concentrations were studied by qPCR and selected-reaction-monitoring MS, respectively. The MICs of cefotaxime were 168 and 252 mg/L for MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, respectively. Both strains displayed a prolonged lag phase when exposed to cefotaxime. The mRNA of blaCTX-M-1 and CTX-M-1 protein levels increased in the presence of high cefotaxime concentrations and varied with growth phase. Higher mRNA expression levels were detected for MG1655/CTX-M-1 compared with MG1655/IncI1/CTX-M-1, but a higher protein level was found for MG1655/IncI1/CTX-M-1 compared with MG1655/CTX-M-1, the latter corresponding well with the higher MIC for this strain. blaCTX-M-1 mRNA expression and CTX-M-1 protein levels were dependent on cefotaxime concentration, growth phase and gene location. These results provide insight into the expression of cephalosporin resistance in CTX-M-1-producing E. coli, improving our understanding of the relationship between antimicrobial therapy and the expression of resistance mechanisms.

Optimizing antibacterial treatment in acute pancreatitis (experimental and clinical research)

Aim. To examine the effectiveness of interstitial electrophoresis in antibacterial treatment in animals with experimental pancreatitis and in patients with acute pancreatitis. Methods. Cefotaxime concentration was studied by highly effective chromatography at different routes of administration in 40 rats of both gender with body weight of 150-250 g at the department of surgery of Kazan State Medical Academy and department of pharmacology of Kazan State Medical University. The clinical part of the research compared treatment results in group of patients (n=27) in whom interstitial electroforesis was performed together with antibacterial treatment with group of patients (n=26) who were treated conventionally. Results. Antibiotic introduction using galvanizing in experimental pancreatitis is more effective compared with traditional antibiotic introduction 24 hours after acute pancreatitis modulation. In the clinical part of the research in the main group complications were registered in 14.8% of cases, localized forms of infection (3 effusions, 1 retroperitoneal abscess) were the most common. In control group complications were seen in 34.6% of cases, generalized infections were the most common type (omental and retroperitoneal abscess - in 15.4% of cases, retroperitoneal pyogenic cellulitis - in 19.2% of cases). So, in the basic group less invasive procedures were more common compared with laparotomy, marsupialization, lumbotomy being more common in control group. Mortality rate was 11.1% in the main group and 15.4 in control group (р 0.05). Conclusion. Using interstitial electrophoresis in antibacterial treatment decreases the frequency of purulent complications, with localized forms of the infection being more prevalent, and allows to decrease the mortality rate in patients with acute destructive pancreatitis.

Prediction of major antibiotic resistance in Escherichia coli and Klebsiella pneumoniae in Singapore, USA and China using a limited set of gene targets

Antibiotic resistance in Gram-negative bacteria, especially Enterobacteriaceae, can be conferred by a large number of different acquired resistance genes, although it appears that relatively few dominate. A previous survey of Escherichia coli and Klebsiella pneumoniae isolates from Sydney, Australia, revealed that a limited set of genes could reliably predict resistance to third-generation cephalosporins (3GCs) and aminoglycosides. Here we tested E. coli and K. pneumoniae isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration of ≥2μg/mL from China and Singapore, with significantly higher resistance rates than Australia, as well as the USA. Few targets were needed to predict non-susceptibility to 3GCs (95/95; 100%) and gentamicin (47/51; 92%). The gene types detected here are consistent with previous surveys in similar countries with similar resistance rates, where the majority of 3GC resistance can be explained by blaCTX-M genes. This study identified a limited set of genes capable of predicting resistance to 3GC and aminoglycoside antibiotics and implies a restriction in the global resistance gene pool that can be exploited for diagnostic purposes.

Proteomic changes in extended-spectrum beta-lactamase-producing Escherichia coli strain under cefotaxime selection

Proteomics can be used to study the metabolic pathways and mechanisms involved in antimicrobial resistance. The aim of this comparative proteomic study was to establish the overall changes in the proteome of a naturally occurring ESBL-producing E. coli strain (C5478) stressed with its minimal inhibitory concentration (2 μg/mL) of cefotaxime, compared to the proteome of the same strain without antimicrobial stress, by using 2-D gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparative proteomic analysis revealed that the abundance of numerous protein species changed in the strain stressed by CTX compared to the non-stressed wild-type strain. A total of 188 spots were excised from the 2-DE gel of the wild-type strain, 112 of which were successfully identified by MALDI-TOF MS, representing 110 different proteins. Concerning the 2-DE gel of the CTX-stressed bacteria, 171 spots were excised and 156 were identified, representing 143 different proteins. The proteins identified in both strains were categorized according to their biological functions. These proteins were involved in metabolism, protein synthesis, cell division, stress responses, and antimicrobial resistance, among others. These findings will be helpful to further understand not only the antimicrobial resistance mechanisms, but also the role of wild animals as reservoirs in spreading antimicrobial-resistant bacteria into the environment.

Spectrophotometric determination of cefotaxime by using sodium 1,2-naphthoquinone-4-sulfonate

A novel method is established to determine cefotaxime by using sodium 1,2-naphthoquinone-4-sulfonate (NQS) as a chromogenic reagent. A russety product can be formed owing to the nucleophilic substitution reaction between cefotaxime and NQS in 0.1 M solution of NaOH. The maximal absorption wavelength of the product is 489 nm. Absorbance is linear with the concentration of cefotaxime in the range of 3.8–114.6 mg/L, and the regression equation is A = 0.04481 + 0.00567c (mg/L), with a correlation coefficient of 0.9992. The detection limit and the RSD are 3 mg/L and 1.2%, respectively. The average recovery is in the range of 98.9–101.2%. The results indicate that the method could be applied to the determination of cefotaxime in injection.

Polymeric hydrogels with memory effect for immobilization of binary drug combinations

Hydrogels with memory effect (imprinted hydrogels) with respect to a combination of cefotaxime and diclofenac were synthesized from 2-hydroxyethyl methacrylate and functional monomers (acrylic acid and N,N-dimethylacrylamide). The hydrogels obtained show increased ability to sorb cefotaxime and especially diclofenac. Prolonged release of cefotaxime was revealed, becoming more pronounced with an increase in the cefotaxime concentration in the solution from which it is sorbed with the hydrogel. The effect of selective sorption of diclofenac with imprinted hydrogels from the diclofenac-cefotaxime drug combination was discovered.

Relationship between the distribution of cefepime minimum inhibitory concentrations and detection of extended-spectrum β-lactamase production among clinically important Enterobacteriaceae isolates obtained from patients in intensive care units in Taiwan: Results from the Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART) in 2007

The data on susceptibility of important cephalosporins against four Enterobacteriaceae members producing potential extended-spectrum β-lactamase (ESBL) collected from Taiwanese intensive care units are lacking. Minimum inhibitory concentrations (MICs) of cefotaxime, ceftazidime, and cefepime were determined using agar dilution method, against Escherichia coli (n = 344), Klebsiella pneumoniae (n = 359), Enterobacter cloacae (n = 103), and Proteus mirabilis (n = 78). Susceptibilities of these isolates to three cephalosporins were assessed according to MIC breakpoints recommended by the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013. The double-disk synergy test using disks containing cefepime (30 μg) with or without clavulanate (10 μg) was applied to confirm production of ESBL for isolates with cephalosporin MIC ≥ 2 μg/mL. A total of 175 isolates were verified as ESBL producers. The rates of cefepime susceptibility among the ESBL-producing isolates, according to CLSI (EUCAST) criteria, were 56.7% (22.4%) for E. coli, 61.3% (12.0%) for K. pneumoniae, 57.9% (31.6%) for E. cloacae, and 71.4% (7.1%) for P. mirabilis. Using different cefepime MIC breakpoints (MICs ≥ 16 μg/mL recommended by CLSI criteria and ≥ 2 μg/mL by EUCAST criteria) to define nonsusceptibility, we found that both criteria were poorer at predicting ESBL producers among K. pneumoniae and E. cloacae than among the other two species. In addition, we also found that the cefepime MIC level of 1.0 μg/mL best distinguished non-ESBL- from ESBL-producing K. pneumoniae and E. cloacae. To detect ESBLs, CLSI should revise the cefepime MIC breakpoint against Enterobacteriaceae.

Oral DAV131, a Charcoal-Based Adsorbent, Inhibits Intestinal Colonization by β-Lactam-Resistant Klebsiella pneumoniae in Cefotaxime-Treated Mice

Antibiotics excreted into the intestinal tract, such as broad-spectrum cephalosporins, disrupt the indigenous microflora, affect colonization resistance (CR), and promote intestinal colonization by resistant bacteria. We tested whether oral DAV131, a charcoal-based adsorbent, would prevent colonization by a cefotaxime (CTX)-resistant Klebsiella pneumoniae strain (PUG-2) in CTX-treated mice. Mice received CTX, saline, CTX and DAV131, or saline and DAV131 for 3 days before oral challenge with 10(6) CFU of PUG-2. The fecal CTX concentrations and counts of PUG-2 were assayed. Fecal CTX disappeared when DAV131 was given concomitantly with CTX (P < 0.05), and the area under the curve of PUG-2 fecal density was significantly reduced (P < 0.01). In conclusion, reducing intestinal antibiotic exposure with DAV131 may reduce colonization by resistant strains during treatment compared to treatment with CTX only. This might open new possibilities for decreasing the impact of antibiotics on the intestinal microbiota during treatments.

Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae

Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study, 152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration (MIC) of ≥2μg/mL were collected from seven major hospitals in Sydney (Australia) in 2008–2009. Nearly all of those with a MIC in excess of European Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or more representatives of only seven gene types capable of explaining this phenotype, and this included 96% of those with a MIC≥2μg/mL to any one of these drugs. Similarly, 97% of associated gentamicin-non-susceptibility (MIC≥8μg/mL) could be explained by three gene types. In a country like Australia, with a background prevalence of resistance to third-generation cephalosporins of 5–10%, this equates to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic application. This is an important proof-of-principle that should be tested in other geographic locations.

Electrooxidation and determination of cefotaxime on Au nanoparticles/poly (L-arginine) modified carbon paste electrode

A simple and sensitive electrochemical sensor based on Au nanoparticles/poly (L-arginine) modified carbon paste electrode (AuNPs/Parg/CPE) was constructed and utilized to determine cefotaxime. Scanning electron microscope (SEM) image showed that the L-arginine (L-arg) had been electropolymerized on the CPE and the immobilized Au nanoparticles (AuNPs) were spherical in shape. Fourier transform infrared spectra (FTIR) indicated that the poly (L-arginine) (Parg) film was successfully modified on CPE. Electrochemical impedance spectroscopy (EIS) and cyclic voltammogram (CV) illustrated the Parg and AuNPs efficiently decreased the charge transfer resistance value of electrode and improved the electron transfer kinetic between analytes and electrode. The electrooxidation of cefotaxime on the modified electrode was performed by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The results showed the response current of cefotaxime at AuNPs/Parg/CPE increased 17times than that of bare CPE. The calibration curve was linear over the cefotaxime concentration range of 0.01–100.0μM with a detection limit (S/N=3) of 2.3nM. This proposed method has been applied to determine cefotaxime in pharmaceutical formulations and human serum samples, and the results were satisfactory.

Susceptibility patterns to extended-spectrum cephalosporins among Enterobacteriaceae harbouring extended-spectrum β-lactamases using the updated Clinical and Laboratory Standards Institute interpretive criteria

We examined the effect of applying the updated 2010 Clinical and Laboratory Standards Institute (CLSI) susceptibility breakpoints for extended-spectrum cephalosporins (ESCs) to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. In total, 202 ESBL-producing, plasmidic AmpC- and carbapenemase-negative isolates derived from separate patients were collected from three Greek hospitals during 2007–2011, including 150 Escherichia coli, 43 Klebsiella pneumoniae and 9 Enterobacter cloacae clinical isolates. ESBLs were detected using the ESBL CLSI confirmatory test and PCR assays. Sequencing analysis showed that 91 (45.0%) of the ESBL-producers carried the blaCTX-M-3 gene, 66 (32.7%) carried the blaCTX-M-15 gene and the remaining 45 (22.3%) carried the blaSHV-5 gene. Minimum inhibitory concentrations for cefotaxime, ceftazidime and cefepime were determined by the agar dilution method. Based on the new CLSI breakpoints, 13 (6.4%) of the ESBL-producers were susceptible to cefotaxime, 90 (44.6%) to ceftazidime and 112 (55.4%) to cefepime; as many as 145 (71.8%) were susceptible to at least one ESC. Among the 150 E. coli, 12 (8.0%), 87 (58.0%) and 79 (52.7%) were susceptible to cefotaxime, ceftazidime and cefepime, respectively, whilst among the 43 K. pneumoniae, 1 (2.3%), 3 (7.0%) and 25 (58.1%) were susceptible to the above ESCs, respectively. None of the nine E. cloacae were susceptible to cefotaxime and ceftazidime, but all except one were susceptible to cefepime. By implementation of the new 2010 CLSI breakpoints, a considerable proportion of ESBL-possessing Enterobacteriaceae would be reported as susceptible, mostly to ceftazidime and cefepime, leading to possible infection control and therapeutic implications.

A novel flow cytometric assay for rapid detection of extended-spectrum beta-lactamases

The rapid detection of extended-spectrum beta-lactamases (ESBLs) is a challenge for most clinical microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control. The aim of our study was to develop a rapid detection assay of ESBL producers based upon flow cytometric analysis. Antimicrobial susceptibility testing followed by molecular characterization of blaTEM, blaSHV or blaCTX-M genes was performed on clinical isolates (41 ESBL positive and 20 ESBL negative) and isolates expressing well-characterized beta-lactamases, including ESBLs (n = 13), plasmid AmpCs (n = 3), oxacillinases (n = 5) and carbapenemases (n = 3). Additionally, two ATCC strains recommended by CLSI for susceptibility testing were used as controls. The flow cytometry analysis protocol involved an incubation of bacterial cells with different concentrations of ceftazidime (1, 2 and 4 mg/L) and cefotaxime (4, 8 and 16 mg/L) for 1 and 2 hours, in the presence and absence of clavulanic acid; subsequently, cells were stained with the fluorescent dye Bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], a lipophilic anion able to diffuse across depolarized membranes. Additionally, CFU counts were performed. Susceptible isolates displayed increased fluorescence after 1 hour of incubation; conversely, the increase of the depolarized population was only observed after incubation with clavulanic acid associated with ceftazidime or cefotaxime in ESBL producers. An excellent correlation was obtained between the number of non-depolarized bacteria quantified by flow cytometry and by conventional CFU assays. A novel, accurate and fast flow cytometric assay is available to detect the presence of ESBLs.