First detection of extended-spectrum cephalosporin- and fluoroquinolone-resistant Escherichia coli in Australian food-producing animals

This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n = 324) from Australian food-producing animals [cattle (n = 169), porcine (n = 114), poultry (n = 32) and sheep (n = 9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0–88.6%), ampicillin (9.4–71.1%), trimethoprim/sulfamethoxazole (11.1–67.5%) and streptomycin (21.9–69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.

The permeability parameter of the outer membrane of pseudomonas aeruginosa Varies with the concentration of a test substrate, cephalosporin C

The permeability parameter (C) for the movement of cephalosporin C across the outer membrane of Pseudomonas aeruginosa was measured using the widely accepted method of Zimmermann & Rosselet. In one experiment, the value of C varied continuously from 4·2 to 10·8 cm3 min-1 (mg dry wt cells)-1 over a range of concentrations of the test substrate, cephalosporin C, from 50 to 5 μm. Dependence of C on the concentration of test substrate was still observed when the effect of a possible electric potential difference across the outer membrane was corrected for. In quantitative studies of β-lactam permeation the dependence of C on the concentration of β-lactam should be taken into account.

Effect of the addition of invert sugar on the production of cephalosporin C in a fed-batch bioreactor

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modelling and optimization of the cephalosporin C production bioprocess in a fed-batch bioreactor with invert sugar as substrate

Cephalosporin C production process optimization was studied based on four experiments carried out in an agitated and aerated tank fermenter operated as a fed-batch reactor. The microorganism Cephalosporium acremonium ATCC 48272 (C-10) was cultivated in a synthetic medium containing glucose as major carbon and energy source. The additional medium contained a hydrolyzed sucrose solution as the main carbon and energy source and it was added after the glucose depletion. By manipulating the supplementary feed rate, it was possible to increase antibiotic production. A mathematical model to represent the fed-batch production process was developed. It was observed that the model was applicable under different operation conditions, showing that optimization studies can be made based on this model. (C) 1999 Elsevier B.V. Ltd. All rights reserved.

The dead core model applied to beads with immobilized cells in a fed-batch cephalosporin C production bioprocess

Viable cells immobilized in inert supports are currently studied for a wide range of bioprocesses. The intrinsic advantages of such systems over suspended cultures incite new research, including studies on fundamental aspects as well as on the industrial viability of these non-conventional processes. In aerobic culture of filamentous fungi, scale-up is hindered by oxygen mass transfer limitation through the support material and bioprocess kinetics must be studied together with mass transfer limitation. In this work, experimental and simulated data of cephalosporin C production were compared. Concentrations in the bulk fermentation medium and cellular mass profiles inside the bioparticles are focused. Immobilized cells were used in a tower bioreactor, operated in fed-batch mode. To describe the radial variation of oxygen concentration within the pellet, a dead core model was used. Despite the extremely low sugar concentrations, bioreaction rates in the pellets were limited by the dissolved oxygen concentration. Cell growth occurs only in the outer layers, a result also confirmed by scanning electron microscopy. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor

Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess. Other alternatives for the production of beta -lactam antibiotics, such as a cephalosporin C fed-batch p recess in an aerated stirred-tank bioreactor with free cells of Cepha-losporium acremonium or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch profess. In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium. In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations, For this study's model, modifications were introduced to take into account the influence of supplementary medium flow rate. The balance equations considered the effect of oxygen limitation inside the bioparticles. In the Monod-type rate equations, eel concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate. The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval. The simulation results showed that the proposed model fit well with the experimental data,and based on the experimental data and the mathematical model an optimal mass flow rate to maximize the bioprocess productivity could be proposed.

Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor

The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina (<44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity. (C) 2004 Wiley Periodicals, Inc.

Studies on the respiration rate of free and immobilized cells of Cephalosporium acremonium in cephalosporin C production

Bioprocesses using filamentous fungi immobilized in inert supports present many advantages when compared to conventional free cell processes. However, assessment of the real advantages of the unconventional process demands a rigorous study of the limitations to diffusional mass transfer of the reagents, especially concerning oxygen. In this work, a comparative study was carried out on the cephalosporin C production process in defined medium containing glucose and sucrose as main carbon and energy sources, by free and immobilized cells of Cephalosporium acremonium ATCC 48272 in calcium alginate gel beads containing alumina. The effective diffusivity of oxygen through the gel beads and the effectiveness factors related to the respiration rate of the microorganism were determined experimentally. By applying Monod kinetics, the respiration kinetics parameters were experimentally determined in independent experiments in a complete production medium. The effectiveness factor experimental values presented good agreement with the theoretical values of the approximated zero-order effectiveness factor, considering the dead core model. Furthermore, experimental results obtained with immobilized cells in a 1.7-L tower bioreactor were compared with those obtained in 5-L conventional fermenter with free cells. It could be concluded that it is possible to attain rather high production rates working with relatively large diameter gel beads (ca. 2.5 mm) and sucrose consumption-based productivity was remarkably higher with immobilized cells, i.e., 0.33 gCPC/kg sucrose/h against 0.24 gCPC/kg sucrose/h in the aerated stirred tank bioreactor process. (C) 1999 John Wiley & Sons, Inc.

Metabolic engineering of beta-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporinrelated products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. cluysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via beta-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of beta-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of beta-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis. (C) 2012 Elsevier Inc. All rights reserved.

First countrywide survey of third-generation cephalosporin-resistant Escherichia coli from broilers, swine, and cattle in Switzerland

The herd prevalence of third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) was determined for broilers (25.0% [95% confidence interval (CI) 17.6-33.7%]), pigs (3.3% [(95% CI 0.4-11.5%]), and cattle (3.9% [95% CI 0.5-13.5%]), using a sampling strategy that was representative of the livestock population slaughtered in Switzerland between October 2010 and April 2011. The 3GC-R-Ec isolates were characterized by the measurement of the MICs of various antibiotics, microarray analyses, analytical isoelectric focusing, polymerase chain reaction and DNA sequencing for bla genes, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. CMY-2 (n = 12), CTX-M-1 (n = 11), SHV-12 (n = 5), TEM-52 (n = 3), CTX-M-15 (n = 2), and CTX-M-3 (n = 1) producers were found. The majority of CMY-2 producers fell into 1 PFGE cluster, which predominantly contained ST61, whereas the CTX-M types were carried by heterogeneous clones of E. coli, as shown by the numerous PFGE profiles and STs that were found. This is the first national Swiss study that focuses on the spread of 3GC-R Enterobacteriaceae among slaughtered animals.

Extended-spectrum cephalosporin-resistant Escherichia coli in community, specialized outpatient clinic and hospital settings in Switzerland

OBJECTIVES Resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli can be due to the production of ESBLs, plasmid-mediated AmpCs (pAmpCs) or chromosomal AmpCs (cAmpCs). Information regarding type and prevalence of β-lactamases, clonal relations and plasmids associated with the bla genes for ESC-R E. coli (ESC-R-Ec) detected in Switzerland is lacking. Moreover, data focusing on patients referred to the specialized outpatient clinics (SOCs) are needed. METHODS We analysed 611 unique E. coli isolated during September-December 2011. ESC-R-Ec were studied with microarrays, PCR/DNA sequencing for blaESBLs, blapAmpCs, promoter region of blacAmpC, IS elements, plasmid incompatibility group, and also implementing transformation, aIEF, rep-PCR and MLST. RESULTS The highest resistance rates were observed in the SOCs, whereas those in the hospital and community were lower (e.g. quinolone resistance of 22.6%, 17.2% and 9.0%, respectively; P = 0.003 for SOCs versus community). The prevalence of ESC-R-Ec in the three settings was 5.3% (n = 11), 7.8% (n = 22) and 5.7% (n = 7), respectively. Thirty isolates produced CTX-M ESBLs (14 were CTX-M-15), 5 produced CMY-2 pAmpC and 5 hyper-expressed cAmpCs due to promoter mutations. Fourteen isolates were of sequence type 131 (ST131; 10 with CTX-M-15). blaCTX-M and blaCMY-2 were associated with an intact or truncated ISEcp1 and were mainly carried by IncF, IncFII and IncI1plasmids. CONCLUSIONS ST131 producing CTX-M-15 is the predominant clone. The prevalence of ESC-R-Ec (overall 6.5%) is low, but an unusual relatively high frequency of AmpC producers (25%) was noted. The presence of ESC-R-Ec in the SOCs and their potential ability to be exchanged between hospital and community should be taken into serious consideration.

Extended-spectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health?

Escherichia coli, Salmonella spp. and Acinetobacter spp. are important human pathogens. Serious infections due to these organisms are usually treated with extended-spectrum cephalosporins (ESCs). However, in the past two decades we have faced a rapid increasing of infections and colonization caused by ESC-resistant (ESC-R) isolates due to production of extended-spectrum-β-lactamases (ESBLs), plasmid-mediated AmpCs (pAmpCs) and/or carbapenemase enzymes. This situation limits drastically our therapeutic armamentarium and puts under peril the human health. Animals are considered as potential reservoirs of multidrug-resistant (MDR) Gram-negative organisms. The massive and indiscriminate use of antibiotics in veterinary medicine has contributed to the selection of ESC-R E. coli, ESC-R Salmonella spp. and, to less extent, MDR Acinetobacter spp. among animals, food, and environment. This complex scenario is responsible for the expansion of these MDR organisms which may have life-threatening clinical significance. Nowadays, the prevalence of food-producing animals carrying ESC-R E. coli and ESC-R Salmonella (especially those producing CTX-M-type ESBLs and the CMY-2 pAmpC) has reached worryingly high values. More recently, the appearance of carbapenem-resistant isolates (i.e., VIM-1-producing Enterobacteriaceae and NDM-1 or OXA-23-producing Acinetobacter spp.) in livestock has even drawn greater concerns. In this review, we describe the aspects related to the spread of the above MDR organisms among pigs, cattle, and poultry, focusing on epidemiology, molecular mechanisms of resistance, impact of antibiotic use, and strategies to contain the overall problem. The link and the impact of ESC-R organisms of livestock origin for the human scenario are also discussed.

Temporal trends of extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae isolates in in- and outpatients in Switzerland, 2004 to 2011

Increasing trends for invasive infections with extended-spectrum cephalosporin-resistant (ESC-R) Enterobacteriaceae have been described in many countries worldwide. However, data on the rates of ESC-R isolates in non-invasive infections and in the outpatient setting are scarce. We used a laboratory-based nationwide surveillance system to compare temporal trends of ESC-R rates in Escherichia coli and Klebsiella pneumoniae for in- and outpatients in Switzerland. Our data showed a significant increase in ESC-R rates from 1% to 5.8% in E. coli (p<0.001) and from 1.1% to 4.4% in K. pneumoniae (p=0.002) during an eight-year period (2004–2011). For E. coli, the increase was significantly higher in inpatients (from 1.2% to 6.6%), in patients residing in eastern Switzerland (from 1.0% to 6.2%), in patients older than 45 years (from 1.2% to 6.7%), and in male patients (from 1.2% to 8.1%). While the increase in inpatients was linear (p<0.001) for E. coli, the increase of ESC R K. pneumoniae isolates was the result of multiple outbreaks in several institutions. Notably, an increasing proportion of ESC-R E. coli was co-resistant to both trimethoprim-sulfamethoxazole and quinolones (42% in 2004 to 49.1% in 2011, p=0.009), further limiting the available oral therapeutic options.

Occurrence and Genetic Characteristics of Third-Generation Cephalosporin-Resistant Escherichia coli in Swiss Retail Meat

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the blaCTX-M-1 (79.4%), blaCMY-2 (17.6%), blaCMY-4 (1.5%), and blaSHV-12 (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The blaCMY-2 and blaCMY-4 genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.