Effect of the Association and Evaluation of the Induction to Adaptation of the (+)-α-pinene with Commercial Antimicrobials against Strains of Escherichia coli.

BACKGROUND: The increasing and inappropriate use of antibiotics has increased the number of multidrug-resistant microorganisms to these drugs, causing the emergence of infections that are difficult to control and manage by health professionals. As an alternative to combat these pathogens, some monoterpenes have harmful effects on the bacterial cell membrane, showing themselves as an alternative in combating microorganisms. Therefore, the positive enantiomer α -pinene becomes an alternative to fight bacteria, since it was able to inhibit the growth of the species Escherichia coli ATCC 25922, demonstrating the possibility of its use as an isolated antimicrobial or associated with other drugs. AIMS: The aim of this study is to evaluate the sensitivity profile of E. coli ATCC 25922 strain against clinical antimicrobials associated with (+) -α-pinene and how it behaves after successive exposures to subinhibitory concentrations of the phytochemicals. METHODS: The minimum inhibitory concentration (MIC) was determined using the microdilution method. The study of the modulating effect of (+) -α-pinene on the activity of antibiotics for clinical use in strains of E. coli and the analysis of the strain's adaptation to the monoterpene were tested using the adapted disk-diffusion method. RESULTS: The results demonstrate that the association of monoterpene with the antimicrobials ceftazidime, amoxicillin, cefepime, cefoxitin and amikacin is positive since it leads to the potentiation of the antibiotic effect of these compounds. It was observed that the monoterpene was able to induce crossresistance only for antimicrobials: cefuroxime, ceftazidime, cefepime and chloramphenicol. CONCLUSION: It is necessary to obtain more concrete data for the safe use of these combinations, paying attention to the existence of some type of existing toxicity reaction related to the herbal medicine and to understand the resistance mechanisms acquired by the microorganism.

Role of age and sex in determining antibiotic resistance in febrile urinary tract infections.

OBJECTIVES: To identify the age- and sex-specific antimicrobial susceptibility patterns of Gram-negative bacteria (GNB) in outpatient febrile urinary tract infections (UTIs) in Korea. METHODS: A total 2262 consecutive samples collected from patients aged 1-101 years with febrile UTIs, during the period January 2012 to December 2014, were analyzed in this multicentre, retrospective cohort study. RESULTS: The sensitivities to cefotaxime and cefoxitin were over 85% for females but under 75% for males. Sex played an important role in the susceptibility of GNB to cefotaxime (p<0.001) and cefoxitin (p<0.001). The sensitivity to ciprofloxacin (age >20 years) was under 75% in both sexes, and was not influenced by sex (p=0.204). Age distributions of the incidences of resistance to cefotaxime, cefoxitin, and ciprofloxacin (age >20 years) were similar to the age distribution of the incidence of GNB, which indicates that the resistance patterns to these drugs were not affected by age (Kolmogorov-Smirnov test, female/male: p=0.927/p=0.509, p=0.193/p=0.911, and p=0.077/p=0.999, respectively). CONCLUSIONS: Age is not a considerable factor in determining the antibiotic resistance in febrile UTIs. Ciprofloxacin should be withheld from both sexes until culture results indicate its use. Second- or third-generation cephalosporins such as cefoxitin and cefotaxime can be used empirically only in females.

Failure of the Amikacin, Cefoxitin, and Clarithromycin Combination Regimen for Treating Pulmonary Mycobacterium abscessus Infection.

In a hollow-fiber model, we mimicked the drug exposures achieved in the lungs of humans treated with standard amikacin, clarithromycin, and cefoxitin combination therapy for Mycobacterium abscessus infection. At optimal dosing, a kill rate of -0.09 (95% confidence interval, -0.04 to 0.03) log10 CFU per ml/day was achieved over the first 14 days, after which there was regrowth due to acquired drug resistance. Thus, the standard regimen quickly failed. A new regimen is needed.

In vivo effect of flucloxacillin in experimental endocarditis caused by mecC-positive staphylococcus aureus showing temperature-dependent susceptibility in vitro.

Methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene (mecC-MRSA) exhibited at 37°C MICs of oxacillin close to those of methicillin-susceptible S. aureus (MSSA). We investigated whether at this temperature, mecC-MRSA strains respond to flucloxacillin treatment like MSSA strains, using a rat model of endocarditis. Flucloxacillin (human-like kinetics of 2 g intravenously every 6 h) cured 80 to 100% of aortic vegetations infected with five different mecC-MRSA strains. These results suggest that mecC-MRSA infections may successfully respond to treatment with ß-lactams.

Pharmacological study of cefoxitin as an alternative antibiotic therapy to carbapenems in treatment of urinary tract infections due to extended-spectrum-ß-lactamase-producing Escherichia coli.

Cefoxitin could be an alternative to carbapenems in extended-spectrum-beta-lactamase-producing Escherichia coli (ESBL-EC) infections. However, pharmacological and clinical data regarding cefoxitin are limited. Using a recent pharmacological model and the MICs of ESBL-EC collected from pyelonephritis, we determined the probabilities to reach four pharmacological targets: free cefoxitin concentrations above the MIC during 50% and 100% of the administration interval (T>MIC = 50% and T>MIC = 100%, respectively) and free cefoxitin concentrations above 4× MIC during 50% and 100% of the administration interval (T>4MIC = 50% and T>4MIC = 100%, respectively). Cefoxitin could be used to treat ESBL-EC pyelonephritis, but administration modalities should be optimized according to MICs in order to reach pharmacological targets.

Antibiotic resistant Staphylococcus aureus in hospital wastewaters and sewage treatment plants with special reference to methicillin-resistant Staphylococcus aureus (MRSA).

AIMS: To investigate the presence of methicillin-resistant Staphylococcus aureus (MRSA) in untreated hospital wastewaters (UHWW), their transmission into the receiving sewage treatment plant (STP) and survival through the STP treatment. METHODS AND RESULTS: Over eight consecutive weeks of sampling, we isolated 224 Staph. aureus strains from UHWW-1, UHWW-2 and its receiving STP inlet (SI) and post-treatment outlet (SO). These strains were typed using the PhP typing method and RAPD-PCR and tested for their antibiotic resistance patterns. Resistance to cefoxitin and the presence of mecA gene identified MRSA isolates. In all, 11 common (C) and 156 single (S) PhP-RAPD types were found among isolates, with two multidrug resistant (MDR) C-types found in H2, SI and SO. These C-type strains also showed resistance to cefoxitin and vancomycin. The mean number of antibiotics to which the strains from UHWW were resistant (5.14 ± 2) was significantly higher than the STP isolates (2.9 ± 1.9) (P < 0.0001). Among the 131 (68%) MRSA strains, 24 were also vancomycin resistant. MDR strains (including MRSA) were more prevalent in hospital wastewaters than in the STP. CONCLUSION: This study provides evidence of the survival of MRSA strains in UHWWs and their transit to the STP and then through to the final treated effluent and chlorination stage. SIGNIFICANCE AND IMPACT OF THE STUDY: This preliminary study identifies the need to further investigate the load of MRSA in hospitals' wastewaters and possible their survival in STPs. From a public health point of view, this potential route of hospital MRSA dissemination is of great importance.

Cefoxitin as an alternative to carbapenems in a murine model of urinary tract infection due to Escherichia coli harboring CTX-M-15-type extended-spectrum ß-lactamase.

We investigated the efficiency of the cephamycin cefoxitin as an alternative to carbapenems for the treatment of urinary tract infections (UTIs) due to Escherichia coli producing CTX-M-type extended-spectrum ß-lactamases. The susceptible, UTI-inducing E. coli CFT073-RR strain and its transconjugant CFT073-RR Tc (pbla(CTX-M-15)), harboring a bla(CTX-M-15) carrying-plasmid, were used for all experiments. MICs of cefoxitin (FOX), ceftriaxone (CRO), imipenem (IMP), and ertapenem (ETP) for CFT073-RR and CFT073-RR Tc (pbla(CTX-M-15)) were 4 and 4, 0.125 and 512, 0.5 and 0.5, and 0.016 and 0.032 µg/ml, respectively. Bactericidal activity was similarly achieved in vitro against the two strains after 3 h of exposure to concentrations of FOX, IMI, and ETP that were 2 times the MIC, whereas CRO was not bactericidal against CFT073-RR Tc (pbla(CTX-M-15)). The frequencies of spontaneous mutants of the 2 strains were not higher for FOX than for IMP or ETP. In the murine model of UTIs, mice infected for 5 days were treated over 24 h. Therapeutic regimens in mice (200 mg/kg of body weight every 3 h or 4 h for FOX, 70 mg/kg every 6 h for CRO, 100 mg/kg every 2 h for IMP, and 100 mg/kg every 4 h for ETP) were chosen in order to reproduce the percentage of time that free-drug concentrations above the MIC are obtained in humans with standard regimens. All antibiotic regimens produced a significant reduction in bacterial counts (greater than 2 log(10) CFU) in kidneys and bladders for both strains (P < 0.001) without selecting resistant mutants in vivo, but the reduction obtained with CRO against CFT073-RR Tc (pbla(CTX-M-15)) in kidneys was significantly lower than that obtained with FOX. In conclusion, FOX appears to be an effective therapeutic alternative to carbapenems for the treatment of UTIs due to CTX-M-producing E. coli.

Effect of antibiotic prescribing in primary care on meticillin-resistant Staphylococcus aureus carriage in community-resident adults: a controlled observational study.

The objectives of this study were to investigate the relationship between primary care antibiotics prescribed within 2 months and 12 months and the carriage of meticillin-resistant Staphylococcus aureus (MRSA) in nasal flora from a large representative sample of community-resident adults. S. aureus isolates were obtained from nasal samples submitted by UK resident adults aged ≥ 16 years registered with 12 general practices in the former Avon and Gloucestershire health authority areas. Individual-level antibiotic exposure data during the 12 months prior to providing the samples were collected from the primary care electronic records. MRSA status was determined by measuring resistance to cefoxitin. In total, 6937 adults were invited to take part, of whom 5917 returned a nasal sample. S. aureus was identified in 946 samples and a total of 761 participants consented to primary care record review and had complete data for the analyses. There was no evidence of an association between any antibiotic in the previous 2 months and MRSA isolation, with an adjusted odds ratio (aOR) of 1.33 [95% confidence interval (CI) 0.12-15; P=0.8]. There was a suggestion of an association between any antibiotic use in the previous 12 months and MRSA, with an aOR of 2.45 (95% CI 0.95-6.3; P=0.06). In conclusion, there is a suggestion that antibiotics prescribed within 12 months is associated with the carriage of MRSA, but not within 2 months, although the 2-month analysis had fewer data subjects and was therefore underpowered to detect this association. A larger study would be able to clarify these associations further.

Efficacy of amoxycillin-clavulanate in an experimental model of murine pneumonia caused by AmpC-non-hyperproducing clinical isolates of Escherichia coli resistant to cefoxitin.

The algorithms included in most automated systems used for antimicrobial susceptibility testing (e.g., Vitek 2) consider that Escherichia coli isolates resistant to cefoxitin are AmpC-hyperproducers and, consequently, resistant also to amoxycillin-clavulanate. However, a recent study revealed that 30% of E. coli clinical isolates resistant to cefoxitin remained susceptible in vitro to amoxycillin-clavulanate. The aim of the present study was to evaluate the in-vivo efficacy of amoxycillin-clavulanate in the treatment of an experimental model of pneumonia, using two clonally related isolates (with identical repetitive extragenic palindromic sequence (REP)-PCR patterns) of AmpC-non-hyperproducing and OmpF-lacking E. coli (Ec985 and Ec571) that were resistant to cefoxitin and susceptible to cefotaxime and amoxycillin-clavulanate. MICs were determined using a microdilution technique, and in-vitro bactericidal activity was tested using time-kill assays. The in-vivo efficacy of amoxycillin, amoxycillin-clavulanate and cefotaxime against both isolates was tested in a murine pneumonia model using immunocompetent C57BL/6 mice. Ec571 (a TEM-1/2 producer) was resistant to amoxycillin, whereas Ec985 (a TEM-1/2 non-producer) was susceptible. Amoxycillin, amoxycillin-clavulanate and cefotaxime were bactericidal for Ec985, and amoxycillin-clavulanate and cefotaxime were bactericidal for Ec571 at different concentrations and time-points, as determined using time-kill assays. Treatment with amoxycillin, amoxycillin-clavulanate and cefotaxime reduced the bacterial lung concentration of Ec985 compared with non-treated controls (p <0.05), whereas amoxycillin-clavulanate and cefotaxime showed efficacy against Ec571 when compared with the control and amoxycillin groups (p <0.05). Regardless of the exact underlying mechanism(s) of resistance, amoxycillin-clavulanate was effective in the experimental murine model in the treatment of pneumonia caused by AmpC-non-hyperproducing strains of E. coli resistant to cefoxitin.

Detection of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in effluents and sludge of a hospital sewage treatment plant.

AIMS: To detect ESBL (extended-spectrum beta-lactamase)-producing Klebsiella pneumoniae present in the effluents and sludge of a hospital sewage treatment plant, evaluating the treatment plant's potential to remove these micro-organisms. METHODS AND RESULTS: Twenty samples (crude sewage, UASB reactor effluent, filtered effluent and sludge) were collected in the period from May to December 2006, in order to analyse antimicrobial susceptibility and to check ESBL production, the disc-diffusion and the combined disc methods were used. Total and faecal coliform concentrations were also determined. ESBL-producing K. pneumoniae were detected in all samples analysed, representing 46 x 5% of the total strains isolated. Among the non-ESBL-producing strains, 26% were multiresistant and one strain resistant to eight of the nine antimicrobials tested was detected in the treated effluent. CONCLUSIONS: The hospital wastewater treatment plant did not show a satisfactory efficacy in removing pathogenic micro-organisms, allowing for the dissemination of multiresistant bacteria into the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The inefficacy of hospital wastewater treatment plants can result in routes of dissemination of multiresistant bacteria and their genes of resistance into the environment, thus contaminating water resources, and having serious negative impact on public health.

Evaluation of a cefoxitin 30 microg disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus.

OBJECTIVES: To evaluate the performance of a cefoxitin 30 microg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36 degrees C, for detection of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: A total of 457 S. aureus, including 190 MRSA of several defined PFGE types and a number of low-level resistant isolates, were tested with a cefoxitin 30 microg disc on Iso-Sensitest agar, using a semi-confluent inoculum and overnight incubation at 35-36 degrees C. This method was compared with the standard SRGA (Swedish Reference Group for Antibiotics) method (oxacillin 1 microg disc on Iso-Sensitest agar supplemented with 5% defibrinated horse blood, confluent growth and 24 h incubation in ambient air at 30 degrees C). RESULTS: The cefoxitin method was excellent, with a sensitivity of 100% and a specificity of 99% using an interpretative zone diameter of S > or = 29 mm and R < 29 mm. Its performance was much better than the SRGA method, which with this collection of difficult strains had a sensitivity of only 78% using the current breakpoint of S > or = 12 mm. CONCLUSION: We suggest that the cefoxitin method should replace that currently recommended by the SRGA for the detection of MRSA, and that it would fit well into BSAC methodology.

A luminescent Escherichia coli biosensor for the high throughput detection of beta-lactams.

A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of beta-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for beta-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various beta-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams. Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-beta-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of beta-lactam action.

Use of cephalosporins for prophylaxis and therapy of polymicrobial infection in mice.

Cefoxitin, cefotetan, and cefmetazole were compared in 10-day therapy of intra-abdominal and subcutaneous infections caused by three organisms: Bacteroides fragilis and Bacteroides thetaiotaomicron combined with either Escherichia coli or Staphylococcus aureus. Intra-abdominal infection was caused by B. fragilis plus B. thetaiotaomicron plus E. coli. Therapy was initiated immediately before inoculation or was delayed for 8 h. Mortality was 14 of 30 (47%) for saline-treated mice, and all survivors developed abscesses. Immediate therapy reduced mortality and the percentage of mice with abscesses (in survivors), respectively, to 17 and 20% with cefoxitin, 0 and 13% with cefotetan, and 0 and 17% with cefmetazole, and the numbers of all bacteria were reduced by all the cephalosporins. Delayed therapy reduced mortality and abscess formation, respectively, to 20 and 8% of mice with cefoxitin, 10 and 93% with cefotetan, and 7 and 96% with cefmetazole. B. thetaiotaomicron survived in all abscesses treated with cefotetan and cefmetazole. Subcutaneous abscesses were caused by each organism alone or in combinations of one aerobe (S. aureus or E. coli) and one or two Bacteroides species. Early therapy reduced the numbers of all bacteria independent of their in vitro susceptibility. All agents reduced the number of each Bacteroides species with either E. coli or S. aureus. However, when therapy was delayed, cefotetan and cefmetazole were less effective than cefoxitin against B. thetaiotaomicron. Cefotetan was the most active agent against E. coli, and cefmetazole was the most effective against S. aureus. These data illustrate the efficacy of all tested cephalosporins in the prophylaxis of polymicrobial infections.

Anti-tumor necrosis factor antibody reduces mortality in the presence of antibiotic-induced tumor necrosis factor release.

The systemic tumor necrosis factor (TNF) response has been extensively studied during infection. In addition, antibiotics that cause cell-wall lysis have been associated with endotoxinemia and, therefore, could trigger TNF release. We studied the effects of pretreatment with cefoxitin and/or anti-TNF antibody on mortality and early (90 minutes) and delayed (6 hours) serum TNF levels in a murine model of mixed Escherichia coli/Bacteroides fragilis peritonitis. At low and intermediate inocula levels, cefoxitin, but not anti-TNF antibody, prevented death, and low serum TNF levels were noted in all groups. At the highest inoculum level, mortality was uniform in control, cefoxitin, and anti-TNF antibody groups, and a significant elevation in serum TNF levels was seen only at the 6-hour point in animals receiving cefoxitin. The addition of anti-TNF antibody to cefoxitin at this inoculum level abrogated the 6-hour rise in serum TNF levels and reduced mortality to 40%. These results emphasize that the cytokine response in disease is dependent on both the nature of the insult and other forms of therapeutic interventions.

Therapeutic interchange of ampicillin-sulbactam for cefoxitin.

A therapeutic interchange program based on microbial patterns within an institution is described. A change in anaerobic susceptibility patterns, increased prevalence of enterococcal infections, and cost factors provided the rationale for the therapeutic interchange of ampicillin-sulbactam for cefoxitin. Ampicillin-sulbactam was recommended for prophylaxis in intraabdominal or gynecological surgery as well as for treatment for gynecological infections. Cefoxitin was restricted to penicillin-allergic patients and women who were pregnant or breast-feeding. The transition from cefoxitin to ampicillin-sulbactam proceeded smoothly as a result of preliminary education of pharmacists and physicians. Pharmacists participated in continuing-education programs and received concise guidelines for the interchange and follow-up instructions; physicians learned of the program from the drug newsletter published by the pharmacy department. Three months after the program began, only one physician was resistant to the interchange. After the program began, 11 antimicrobials, including cefoxitin, were used less frequently and ampicillin-sulbactam use increased. No adverse clinical consequences from the interchange were detected. A therapeutic interchange program based on institution-specific microbial patterns and educational efforts by the pharmacy department produced a change in physician prescribing.

Evaluation of cephalosporins/cephamycins with antianaerobic activity by integrating microbiologic and pharmacokinetic properties.

When evaluating antimicrobial agents, in vitro microbiologic activity and pharmacokinetics are important factors, but these data are usually not assessed simultaneously. The purpose of the study was to compare cefoxitin, cefotetan, ceftizoxime, cefotaxime (CT), desacetylcefotaxime (DACT), and CT/DACT (1:1 ratio) by integrating their microbiologic activity against clinical isolates of Bacteroides fragilis with their pharmacokinetic properties. Minimal inhibitory concentrations (MIC) were determined by the agar dilution method. Steady-state serum concentration--time profiles were simulated for 2-gm doses in a 70-kg patient using ADAPT software and pharmacokinetic data from published studies. Serum protein binding (%) of each agent was also obtained from published studies and used to calculate the unbound serum concentration--time profiles. As estimates of pharmacodynamic activity, time below the MIC (T less than MIC) and percentage of the dosing interval below the MIC (% INT less than MIC) were calculated for individual isolates using total and unbound serum concentrations. Data analysis included MIC50, MIC90, range, breakpoint susceptibility, and analysis of variance for T less than MIC and % INT less than MIC (Scheffé post-hoc test, P less than 0.05). The MIC90 of cefotetan was at least a twofold dilution lower than the other agents. However, using unbound (pharmacologically active) serum concentrations, T less than MIC and % INT less than MIC for ceftizoxime (at a simulated eight-hour dosing interval) were significantly smaller than with the other antibiotic regimens. Integration of in vitro and pharmacokinetic data may provide additional information to assist in the evaluation of antimicrobials. For B fragilis from our institution, the pharmacodynamic profile of unbound ceftizoxime is superior to the other antianaerobic cephalosporins/cephamycins tested.

Comparison of the inoculum effect of cefoxitin and other cephalosporins and of beta-lactamase inhibitors and their penicillin-derived components on the Bacteroides fragilis group.

We compared the inoculum effects for 109 recent clinical isolates of the Bacteroides fragilis group of cefoxitin, cefotetan, ceftizoxime, ceftriaxone, and three beta-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) and their penicillin-derived components. Bactericidal activity was assayed and morphologic changes were monitored for selected strains exhibiting a large inoculum effect. Ceftizoxime demonstrated the largest inoculum effect, followed by cefotetan and ceftriaxone. The large inoculum effect of ceftizoxime and ceftriaxone was correlated with filamentous transformation at the high inoculum (10(8) CFU/ml) and lack of bactericidal activity suggesting drug destruction or inactivation. Cefotetan was bactericidal for B. fragilis isolates but not for other members of the B. fragilis group. Cefoxitin showed the least inoculum effect and was consistently bactericidal at high (10(8) CFU/ml), standard (10(6) CFU/ml), and low (10(4) CFU/ml) inocula, followed by ampicillin-sulbactam. Piperacillin-tazobactam and ticarcillin-clavulanic acid showed an intermediate inoculum effect. The degree of inoculum effect observed generally correlated with bactericidal activity at all inocula.

Cefoxitin in the treatment of aerobic and anaerobic infections. Prospective correlation of in vitro susceptibility methods with clinical outcome.

Anaerobic isolates from patients with intra-abdominal infection were tested in vitro by eight methods for susceptibility to cefoxitin. Broth disk elution correlated poorly with clinical outcome. The clinical breakpoint for anaerobic susceptibility to cefoxitin was less than or equal to 32 micrograms/ml.