Staphylococcus aureus surface attachment selectively influences tolerance against charged antibiotics.

The threat of infection during implant placement surgery remains a considerable burden for millions of patients worldwide. To combat this threat, clinicians employ a range of anti-infective strategies and practices. One of the most common interventions is the use of prophylactic antibiotic treatment during implant placement surgery. However, these practices can be detrimental by promoting the resilience of biofilm-forming bacteria and enabling them to persist throughout treatment and re-emerge later, causing a life-threatening infection. Thus, it is of the utmost importance to elucidate the events occurring during the initial stages of bacterial surface attachment and determine whether any biological processes may be targeted to improve surgical outcomes. Using gene expression analysis, we identified a cellular mechanism of S. aureus which modifies its cell surface charge following attachment to a medical grade titanium surface. We determined the upregulation of two systems involved in the d-alanylation of teichoic acids and the lysylation of phosphatidylglycerol. We supported these molecular findings by utilizing synchrotron-sourced attenuated total reflection Fourier-transform infrared microspectroscopy to analyze the biomolecular properties of the S. aureus cell surface following attachment. As a direct consequence, S. aureus quickly becomes substantially more tolerant to the positively charged vancomycin, but not the negatively charged cefazolin. The present study can assist clinicians in rationally selecting the most potent antibiotic in prophylaxis treatments. Furthermore, it highlights a cellular process that could potentially be targeted by novel technologies and strategies to improve the outcome of antibiotic prophylaxis during implant placement surgery. STATEMENT OF SIGNIFICANCE: The antibiotic tolerance of bacteria in biofilm is a well-established phenomenon. However, the physiological adaptations employed by Staphylococcus aureus to increase its antibiotic tolerance during the early stages of surface attachment are poorly understood. Using multiple techniques, including gene expression analysis and synchrotron-sourced Fourier-transform infrared microspectroscopy, we generated insights into the physiological response of S. aureus following attachment to a medical grade titanium surface. We showed that this phenotypic transition enables S. aureus to better tolerate the positively charged vancomycin, but not the negatively charged cefazolin. These findings shed light on the antibiotic tolerance mechanisms employed by S. aureus to survive prophylactically administered antibiotics and can help clinicians to protect patients from infections.

Prevalence of Efflux Pump and Porin-Related Antimicrobial Resistance in Clinical Klebsiella pneumoniae in Baghdad, Iraq.

Klebsiella pneumoniae is an opportunistic bacterium that causes many infections, including septicemia, pneumonia, urinary tract infection, and liver abscesses. There are many mechanisms for antibiotic resistance and K. pneumonia is considered a multidrug-resistant pathogen. This study aimed to find the correlation between the susceptibility of K. pneumonia to certain antibiotics with the porin-related resistance and pumps mechanisms. In total, two genes that are responsible for porin formation were considered in the current study OmpK-35gene and OmpK-36 gene, in addition to other four genes (CfiaS, CfiaL, MFS, and MdtK genes) related to an efflux pump mechanism of antibiotic resistance. The bacterial resistance was investigated towards five cephalosporins (Cefazolin, Cefoxitin, Ceftazidime, Ceftriaxone, and Cefepime) and two carbapenems (imipenem and ertapenem). Clinical samples, including blood, swabs, and urine, consisting of 20 specimens for each group, were collected from patients who attended three hospitals in Baghdad. The VITEK-2 system and genetic tests (polymerase chain reaction and sequencing) of bacterial isolates were applied to confirm the diagnosis of K. pneumoniae and detect the antibiotic sensitivity profile. The results showed that 51 (85%) and 15 (25%) of the total 60 isolates had positive results for OmpK-35 and Omp-K36 genes, respectively. The MFS and MdtK genes were observed (70-88.3%) in cephalosporin-resistant isolates of K. pneumoniae. There were no significant variations of bacterial resistance genes of antibiotics within the specimen groups. It was concluded that the bacterial resistance of the selected antibiotics was elevated markedly with the loss of the OmpK-36 gene with a high expression of MFS and MdtK genes and a slight minimal occurrence in the new generation of carbapenems. The best antimicrobial agent was ertapenem with a percentage of 0% of resistance in all bacterial isolates.

Overproduction of Chromosomal ampC ß-Lactamase Gene Maintains Resistance to Cefazolin in Escherichia coli Isolates.

Cefazolin, an active in vitro agent against Escherichia coli, is used to treat urinary and biliary tract infections. Cefazolin is used widely as an antibiotic, and the increase in the emergence of cefazolin-resistant E. coli in many countries is a major concern. We investigated the changes in the susceptibility of E. coli clinical isolates to cefazolin following exposure. A total of 88.9% (16/18 strains) of the strains acquired resistance to cefazolin. All strains with an MIC to cefazolin of 2 µg/mL became resistant. The expression of chromosomal ampC (c-ampC) increased up to 209.1-fold in the resistant strains. Moreover, 11 of the 16 E. coli strains (68.8%) that acquired cefazolin resistance maintained the resistant phenotype after subculture in cefazolin-free medium. Therefore, the acquisition and maintenance of cefazolin resistance in E. coli strains were associated with the overexpression of c-ampC. Mutations in the c-ampC attenuator regions are likely to be maintained and are one of the key factors contributing to the increase in the number of cefazolin-resistant E. coli worldwide. IMPORTANCE This study is the first to demonstrate that mutations in the chromosomal-ampC attenuator region are responsible for the emergence of cefazolin resistance in Escherichia coli strains. The resistance was maintained even after culturing E. coli without cefazolin. This study highlights one of the key factors contributing to the increase in the number of cefazolin-resistant E. coli strains, which can pose a considerable challenge for treating common infections, such as urinary tract infections.

Exploring the Role of L10 Loop in New Delhi Metallo-ß-lactamase (NDM-1): Kinetic and Dynamic Studies.

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

Pharmacokinetics of Total and Unbound Cefazolin during Veno-Arterial Extracorporeal Membrane Oxygenation: A Case Report.

Extra-corporeal membrane oxygenation (ECMO) therapy could affect effective drug concentrations via adsorption onto the oxygenator or associated circuit. We describe a case of a 25-year-old female who required a veno-arterial ECMO therapy for refractory cardiac arrest due to massive pulmonary embolism. She had mild renal dysfunction as a result of the cardiac arrest. A total of 2 g of intravenous cefazolin 8-hourly was administered. Pre- and post-oxygenator blood samples were collected at 0, 1, 4, and 8 h post antibiotic administration. Samples were analyzed for total and unbound cefazolin concentrations. Protein binding was ∼60%. Clearance was reduced due to impaired renal function. The pharmacokinetics of cefazolin appear to not be affected by ECMO therapy and dosing adjustment may not be required.

Perioperative use of cefazolin ameliorates postoperative cognitive dysfunction but induces gut inflammation in mice.

BACKGROUND: Emerging evidence indicates that long-time use of multiple antibiotics can induce cognitive dysfunction via gut dysbiosis. Cefazolin is often used for 3 to 5 days to prevent perioperative infection. This study is to detect the impact of perioperative use of cefazolin on inflammatory responses and postoperative cognition. METHODS: The anti-inflammatory effect of cefazolin was determined in mouse C8-B4 microglial cells treated with lipopolysaccharide (LPS). Interleukin (IL)-6 and IL-1ß at 6 and 24 h after LPS treatment were detected. Six- to 8-week-old CD-1 mice were subjected to laparotomy. Cefazolin at 300 mg/kg was injected intraperitoneally 1 h before surgery and then once per day for 5 days after surgery. Their learning and memory were assessed by Barnes maze and fear conditioning tests which started 1 week after the surgery. The brain and colon were harvested 24 h and 6 days after surgery to determine inflammatory cytokines. The colon and its luminal contents were harvested 6 and 19 days after surgery for the determination of bacteria flora. Cefazolin concentrations in the serum and brain were measured 0.5, 1, and 2 h after cefazolin injection. RESULTS: IL-6 and IL-1ß levels were decreased by 250 µg/ml cefazolin in the LPS-stimulated C8-B4 cells. Laparotomy increased the time for mice to identify the target hole in the Barnes maze on day 1 and day 8 after training sessions and reduced context-related freezing behavior in fear conditioning test. Cefazolin attenuated these surgical effects but reduced context-related freezing behavior in mice without surgery. IL-6 in the hippocampus and cerebral cortex, IL-1ß in the cerebral cortex, and IL-6 and IL-1ß in the serum and colon were increased 24 h after laparotomy. Cefazolin attenuated these effects. Cefazolin treatment for 5 days in mice without surgery induced colon dysbiosis and increased IL-6 and IL-1ß in the colon and IL-1ß in the cerebral cortex. Colon dysbiosis disappeared in mice treated with cefazolin alone but persisted in mice with surgery and cefazolin 19 days after surgery. High cefazolin concentrations in the serum but not in the brain were detected after cefazolin injection. CONCLUSIONS: These results suggest that cefazolin has a direct anti-inflammatory effect and can attenuate surgery-induced postoperative memory and learning impairment in mice. Cefazolin alone may induce cognitive dysfunction possibly by transient gut dysbiosis in mice without surgery.

Real-time activity monitoring of New Delhi metallo-ß-lactamase-1 in living bacterial cells by UV-Vis spectroscopy.

We report an UV-Vis method for monitoring the hydrolysis of the ß-lactam antibiotics inside living bacterial cells. Cell-based studies demonstrated that the hydrolysis of cefazolin was inhibited by three known NDM-1 inhibitors. This approach can be applied to the monitoring of reactions in a complex biological system, for instance in medical testing.

Antibiotic stability related to temperature variations in elastomeric pumps used for outpatient parenteral antimicrobial therapy (OPAT).

Background: Elastomeric pumps can be used for the continuous administration of antimicrobials in the outpatient setting. A potentially limiting factor in their use is the stability of antimicrobials. Objectives: To investigate under real-life conditions the temperature variations of antibiotic solutions contained in elastomeric pumps, and to examine under such conditions the stability of five antibiotics. Methods: Healthy volunteers carried the elastomeric pumps in carry pouches during their daily activities. A thermologger measured the temperatures every 15 min over 24 h. Antibiotic concentrations were measured by HPLC coupled to tandem MS. Results: During daytime, the temperature of solutions in the pumps increased steadily, warming to >30°C. During the night, when the pumps were kept attached to the waist, the temperatures reached up to 33°C. The use of white carry pouches avoided excessive temperature increases. Over seven experiments, cefazolin, cefepime, piperacillin and tazobactam were found to be stable over 24 h. Flucloxacillin showed a mean decrease in concentration of 11% ( P = 0.001). Conclusions: Real-life situations can cause significant temperature rises in elastomeric pumps, thereby potentially increasing the risk of antibiotic degradation. Patients should be instructed to avoid situations causing excessive temperature increases. Despite these temperature variations, cefazolin, cefepime, piperacillin and tazobactam were found to be stable over 24 h. A moderate degradation was noticed for flucloxacillin, albeit most probably not to an extent that might impair anti-infective efficacy.

Drug release behavior of electrospun twisted yarns as implantable medical devices.

In this study, twisted drug-loaded poly(L-lactide) (PLLA) and hybrid poly(L-lactide)/poly(vinyl alcohol) (PLLA/PVA) yarns were produced using an electrospinning technique based on two oppositely charged nozzles. Cefazolin, an antibiotic drug was incorporated in the yarn fibers by addition to the PLLA electrospinning solution. Morphological studies showed that independent of the twist rate, uniform and smooth fibers were formed. The diameter of the electrospun fibers in the yarns decreased at higher twist rates but produced yarns with larger diameters. At increasing twist rates the crystallinity of the fibers in the yarns increased. In the presence of cefazolin the fiber diameter, yarn diameter and crystallinity were always lower than in the non-drug loaded yarns. In addition the yarn mechanical properties revealed a slightly lower strength, modulus and elongation at break upon drug loading. The effect of the twist rate on the cefazolin in vitro release behavior from both PLLA and hybrid yarns revealed similar profiles for both types of drug-loaded yarns. However, the total amount of drug released from the hybrid PLLA/PVA yarns was significantly higher. The release kinetics over a period of 30 d were fitted to different mathematical models. Cefazolin release from electrospun PLLA yarns was governed by a diffusion mechanism and could best be fitted by Peppas and Higuchi models. The models that were found best to describe the drug release mechanism from the hybrid PLLA/PVA yarns were a first-order model and the Higuchi model.

Quantification of cefazolin in serum and adipose tissue by ultra high performance liquid chromatography-Tandem mass spectrometry (UHPLC-MS/MS): application to a pilot study of obese women undergoing cesarean delivery.

Higher doses of cefazolin are required in obese patients for preoperative antibiotic prophylaxis, owing to its low lipophilicity. An ultra high performance liquid chromatography-tandem mass spectrometry method was developed to quantify cefazolin in serum and adipose tissue from 6 obese patients undergoing cesarean delivery, and using stable-isotope labeled cefazolin as an internal standard. The method has a 2µg/g lower limit of quantitation. The concentration in adipose tissue was 3.4±1.6µg/mL, which is less than half of the reported minimum inhibitory concentration of 8µg/mL for cefazolin. Serum cefazolin concentrations were more than 30-fold higher than in adipose tissue.

Skeletal muscle and plasma concentrations of cefazolin during complex paediatric spinal surgery.

BACKGROUND: Surgical site infections (SSIs) can have devastating consequences for children who undergo spinal instrumentation. Prospective evaluations of prophylactic cefazolin in this population are limited. The purpose of this study was to describe the pharmacokinetics and skeletal muscle disposition of prophylactic cefazolin in a paediatric population undergoing complex spinal surgery. METHODS: This prospective pharmacokinetic study included 17 children with adolescent idiopathic scoliosis undergoing posterior spinal fusion, with a median age of 13.8 [interquartile range (IQR) 13.4-15.4] yr and a median weight of 60.6 (IQR 50.8-66.0) kg. A dosing strategy consistent with published guidelines was used. Serial plasma and skeletal muscle microdialysis samples were obtained during the operative procedure and unbound cefazolin concentrations measured. Non-compartmental pharmacokinetic analyses were performed. The amount of time that the concentration of unbound cefazolin exceeded the minimal inhibitory concentration for bacterial growth for selected SSI pathogens was calculated. RESULTS: Skeletal muscle concentrations peaked at a median of 37.6 (IQR 26.8-40.0) µg ml(-1) within 30-60 min after the first cefazolin 30 mg kg(-1) dose. For patients who received a second 30 mg kg(-1) dose, the peak concentrations reached a median of 40.5 (IQR 30.8-45.7) µg ml(-1) within 30-60 min. The target cefazolin concentrations for SSI prophylaxis for meticillin-sensitive Staphylococcus aureus (MSSA) and Gram-negative pathogens were exceeded in skeletal muscle 98.9 and 58.3% of the intraoperative time, respectively. CONCLUSIONS: For children with adolescent idiopathic scoliosis undergoing posterior spinal fusion, the cefazolin dosing strategy used in this study resulted in skeletal muscle concentrations that were likely not to be effective for intraoperative SSI prophylaxis against Gram-negative pathogens.

[Recombinant cephalosporin-acid synthesase: optimisation of expression in E.coli cells, immobilisation and application for biocatalytic cefazolin synthesis].

Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and "leaderless" CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (МETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.

Cefazolin concentration in surgically created wounds treated with negative pressure wound therapy compared to surgically created wounds treated with nonadherent wound dressings.

OBJECTIVE: To compare cefazolin concentrations in biopsied tissue samples collected from surgically created wounds treated with negative pressure wound therapy to those collected from surgically created wounds treated with nonadherent dressings. STUDY DESIGN: Prospective, controlled, experimental study. ANIMALS: Adult female spayed Beagles (n = 12). METHODS: Full thickness cutaneous wounds were created on each antebrachium (n = 24). Immediately after surgery, cefazolin (22 mg/kg intravenously [IV]) was administered to each dog and continued every 8 hours during the study. The right wound was randomly assigned to group I or group II whereas the wound on the contralateral antebrachium was assigned to the other group. Group I wounds were treated with negative pressure wound therapy (NPWT) and group II wounds were treated with nonadherent dressings for 3 days. Dressings were changed and tissue biopsies obtained from wound beds at 24 hours intervals for both groups. Cefazolin wound tissue and plasma concentrations were measured by liquid chromatography mass spectrometry (LC-MS/MS). Blood samples for measuring plasma cefazolin concentrations were collected before biopsy sampling. At the time of surgery and at each subsequent bandage change, wound beds were swabbed and submitted for aerobic and anaerobic culture. RESULTS: After initiating cefazolin treatment, wound tissue antibiotic concentrations between treatment groups were not significantly different at any sampling time. Similarly, after initiating cefazolin treatment, plasma cefazolin concentrations were not significantly different at any sampling time for individual dogs. CONCLUSIONS: Using a canine experimental model, NPWT treatment of surgically created wounds does not statistically impact cefazolin tissue concentrations when compared with conventional nonadherent bandage therapy.

Plasma and tissue cefazolin concentrations in obese patients undergoing cesarean delivery and receiving differing pre-operative doses of drug.

BACKGROUND: Patients undergoing cesarean delivery typically receive a 1-g to 2-g dose of cefazolin as pre-operative antibacterial prophylaxis. This traditional dosage may not provide an adequate tissue concentration of cefazolin in obese patients during the peri-operative period. This study compared the tissue concentrations of prophylactic cefazolin administered as a either a 2-g or a 4-g dose prior to cesarean delivery in obese patients. METHODS: Twenty obese patients (first trimester body mass index [BMI] >35) who underwent cesarean delivery completed this randomized study. Eleven patients received 2 g of cefazolin, and nine received 4 g. Blood and subcutaneous tissues were collected at the times of the incision and closure. Myometrial biopsies were collected at uterine closure. A cefazolin concentration threshold of 4 mcg/g for tissue samples was used as a surrogate adequate concentration. Plasma and tissue cefazolin concentrations were compared for the two doses. RESULTS: Mean plasma, umbilical cord, and myometrial cefazolin concentrations were significantly higher in the 4-g treatment group (p<0.05). Subcutaneous incision site tissue obtained at time of incision creation also was significantly higher in the 4-g group than in the 2-g group (40.11±24.10 mcg/g vs. 18.36±6.68 mcg/g; p=0.0005). Subcutaneous tissue concentrations at closure were significantly different in the two dosage groups (34.89±17.42 mcg/g vs. 21.73±16.02 mcg/g; p=0.044). All tissue samples were above the target of 4 mcg/g. Body morphometry did not correlate with the variability in cefazolin tissue concentration. No surgical site infections, endometritis, or other adverse effects were observed. CONCLUSIONS: Administering a prophylactic dose of 4 g of cefazolin produced blood and tissue cefazolin concentrations that were significantly higher than concentrations obtained from a 2-g dose in patients with BMIs between 35 and 60 kg/m(2) undergoing cesarean delivery. It is unclear if the larger cefazolin dose produces a more protective anti-infective effect than that obtained with the more traditional 2-g dose for cesarean delivery in obese patients.

Cefazolin plasma protein binding and its covariates in neonates.

Cefazolin (CFZ) is highly and saturably bound to human serum albumin (HSA) in adults. We aim to describe CFZ protein binding and its covariates in neonates. In neonates to whom intravenous CFZ (50 mg/kg) was administered prior to a surgical procedure, total and unbound CFZ plasma concentrations (mg/l) were determined at 0.5, 2, 4 and 8 h after CFZ administration. Linear and multiple regression analyses were used to document covariates of unbound CFZ fraction. The Wilcoxon signed-rank test was used for the paired analysis of unbound CFZ fractions. In 40 patients with a median weight of 2,767 (range 830-4,200) g and a postmenstrual age (PMA) of 39 (25-45) weeks, 131 samples were collected. The median unbound CFZ fraction was 0.39 (0.10-0.73). Linear regression of unbound CFZ fraction versus unbound CFZ plasma concentration (R (2) = 0.39) had a slope significantly different from zero (p < 0.001). In a multiple regression analysis, albuminaemia, total CFZ concentration, indirect bilirubinaemia and PMA resulted in an R (2) value of 0.496. The median unbound CFZ fraction at the peak concentration (0.46, range 0.28-0.69) was significantly higher compared to the trough level (0.36, range 0.17-0.73) (p < 0.001). The between- and within-patient saturability of CFZ plasma protein binding were documented in neonates. The median unbound CFZ fraction in neonates is higher than in adults and depends partly on albuminaemia, total CFZ concentration, indirect bilirubinaemia and PMA. Integration of CFZ protein binding in future pharmacokinetic/pharmacodynamic research is warranted in order to optimise neonatal CFZ dosing. We recommend protein binding assessment in the neonatal pharmacokinetic evaluation of highly protein-bound or clinically relevant drugs.

Are bone and serum cefazolin concentrations adequate for antimicrobial prophylaxis?

BACKGROUND: Antimicrobial prophylaxis is considered beneficial for preventing surgical-site infections in clean orthopaedic surgery. However, whether tissue concentrations of cefazolin achieve the minimum inhibitory concentration for the targeted contaminants have yet to be clarified. QUESTIONS/PURPOSES: We asked whether 2 g of cefazolin would enable effective serum and bone concentrations relative to the current minimum inhibitory concentration for cefazolin-resistant coagulase-negative Staphylococci and methicillin-sensitive Staphylococcus aureus. PATIENTS AND METHODS: We enrolled 43 patients (THA, n = 16; TKA, n = 27) scheduled for primary THAs and primary TKAs. Subjects were given 2 g of cefazolin intravenously before incision. One blood sample and two bone samples were collected from each subject before tourniquet deflation before any additional dose. All samples were assayed at the same laboratory. Minimum inhibitory concentration values were defined based on nationwide surveys. RESULTS: Mean (± standard deviation) serum concentration was 170.3 ± 51.3 µg/mL (range, 99.3-370.3 µg/mL). Mean bone concentration was 32.3 ± 15.2 µg/g (range, 11.4-70.0 µg/g) in THA, and 16.0 ± 10.4 µg/g (range, 6.3-46.3 µg/g) in TKA. All serum and bone concentrations exceeded the minimum inhibitory concentration for methicillin-sensitive S. aureus, but some serum levels were marginal and no bone levels exceeded the minimum inhibitory concentration for cefazolin-resistant coagulase-negative Staphylococcus. CONCLUSIONS: Our data suggest intravenous administration of 2 g of cefazolin achieves the minimum inhibitory concentration for methicillin-sensitive S. aureus in serum and bone, but not the minimum inhibitory concentration for cefazolin-resistant coagulase-negative Staphylococcus in bone, resulting in a potential risk of deep surgical site infections in THAs and TKAs.

Hydrolysis of cefazolin by enzymes produced by Pseudomonas aeruginosa after exposure to ceftazidime in vitro.

BACKGROUND/AIM: Sometimes resistance of Pseudomonas aeruginosa (Ps. aeruginosa) is developed during antibiotic treatment, in spite of the initial susceptibility in vitro. The aim of this study was to use an in vitro model for the study of the development of resistant strains of Ps. aeruginosa after a short exposure to ceftazidime, and to study the hydrolysing capacity of beta-lactamases produced by the resistant strains. METHODS: Among 563 clinical strains of Ps. aeruginosa, 37 multisensitive strains were collected for the study. After being identified, strains with simultaneous sensitivity to 5 expanded spectrum cephalosporins were chosen. For each strain, the minimal inhibitory concentration (MIC) of the 5 expanded spectrum cephalosporins was determined, and the production of extended spectrum beta-lactamases (ESBL) was excluded by the double-disc synergy diffusion test. Strains non producing ESBL were cultivated in concentrations of ceftazidime equal to MICx2 and MICx4. After 24 hours of culture, the development of resistant strains was estimated and the cephalosporinase activity of the produced beta-lactamases was determined by their ability to hydrolyse cefazolin. Hydrolysis of cefazolin was studied by measuring the change of its absorbance on 272 nm using a Shimadzu 160A spectrophotometer. The hydrolyzing capacity of the enzymes was expressed as the percentage of the antibiotic, which was hydrolysed in 10 sec. RESULTS: A total of 60% and 50% of strains developed resistant strains after exposure to ceftazidime in concentration MICx2 and MICx4, respectively. The hydrolyzing capacity of the original strains was 15-36% while the hydrolyzing capacity of the resistant strains was 10-73%. Totally 64% of the resistant strains expressed higher hydrolyzing capacity than the original strains. CONCLUSION: Regardless of the susceptibility test results, Ps. aeruginosa presented a high tendency to develop resistant strains after a short exposure to ceftazidime in vitro. In most cases the resistant strains expressed higher cephalosporinase activity than the original strains, suggesting derepression of chromosomal beta-lactamases. Our model offers a simple, inexpensive and rapid method for detecting resistance of Ps. aeruginosa developed due to derepression of beta-lactamases, and for discriminating resistant strains with derepressed beta-lactamases from strains that developed other mechanisms of resistance.

Cefazolin plasma protein binding saturability during pregnancy.

This article aims to document cefazolin (CFZ) plasma binding and its covariates during pregnancy and compare these observations with previously reported observations in nonpregnant adults. Maternal CFZ plasma samples were collected during in utero surgery. The unbound CFZ fraction was reported by median and range. Correlation (Spearman) and multiple regression were used to identify covariates (total CFZ concentration, albuminemia, gestational age) of the unbound CFZ fraction. Observations during pregnancy were compared with observations in nonpregnant adults (unpaired t test, multiple regression). Plasma (N = 130) samples were collected during 30 interventions. The median unbound CFZ fraction was 0.25 (range 0.14-0.41). Correlations between the unbound CFZ fraction and total CFZ plasma concentration (0.46), time after administration (-0.38), albuminemia (-0.39) and gestational age (-0.19) were statistically significant. The median unbound CFZ fraction was higher during pregnancy when compared to observations in nonpregnant adults (0.25 vs. 0.19, P < 0.001). In a multiple-regression model, total plasma CFZ concentration and albuminemia were covariates of the unbound CFZ fraction (r(2) = 0.4). The concept of saturability of CFZ plasma protein binding has been confirmed during pregnancy, but the free CFZ fraction is higher, likely explained by the lower albuminemia during pregnancy.

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli.

Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic beta-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a K(m) of about 5 microM with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.