LC-9018 treatment enhances survival in gram-negative burn wound sepsis

Mice pretreated with the LC-9018 showed enhanced survival compared to controls when burned and infected with P. aeruginosa and K. pneumoniae strains. Burned skin quantitative bacterial counts in LC-9018 treated and controls were similar but kidney and liver counts of treated mice were significantly lower. Increased peritoneal cell counts in LC-9018 treated groups suggested enhanced survival and lower organ counts resulted from increased microbial clearance. Survival in LC-9018 pretreated P. aeruginosa infected burned mice was reduced in neutropenic or macrophage blockaded mice which supported this hypothesis. Burned mice treated with LC-9018 at times postburn but pre-P. aeruginosa infection showed significantly enhanced survival. Thus LC-9018 treatment might be useful for increasing survival from burn wound sepsis and further investigation is warranted.     

Activity of combinations of ceftazidime, imipenem and pefloxacin against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa

The chequerboard technique was used to look for synergistic combinations of ceftazidime, imipenem and pefloxacin. The synergistic combinations were used in vivo in mice experimentally infected with Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. In vitro ceftazidime/imipenem, ceftazidime/pefloxacin and pefloxacin/imipenem combinations showed synergistic effects against Staphylococcus aureus and S. typhimurium and additive effects against P. aeruginosa. Only the ceftazidime/pefloxacin combination was synergistic against E. coli while the ceftazidime/imipenem and pefloxacin/imipenem combinations resulted in additive effects. In vivo, combination of ceftazidime/imipenem against E. coli infection and the pefloxacin/imipenem combination against S. typhimurium infection were protective.     

The metabolic activation of 2-aminofluorine, 4-aminobiphenyl, and benzidine by cytochrome P-450-107S1 of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen of the human urinary bladder. Similar to rat liver S9, the cell-free extract from P. aeruginosa caused significant increase of histidine reversion numbers with the Salmonella typhimurium tester strain TA98 in the Ames Salmonella mutagenicity assay in the presence of either 2-aminofluorene, 4-aminobiphenyl, or benzidine procarcinogens. The presence of cytochrome P-450 protein in the cell-free extract was demonstrated by the carbon monoxide difference spectrum. We employed gene knockout technology to inactivate one of the three known putative cytochrome P-450 genes of P. aeruginosa, namely CYP107S1, which we postulated to be the most likely to induce activation. The ampicillin resistant gene from PUC19 DNA confers carbenicillin resistance to P. aeruginosa. We inserted a synthetic ampicillin gene flanked by 40 base-pairs of the 5′ and 3′ untranslated region of the CYP gene by electroporating the synthetic gene into electrocompetent P. aeruginosa cells. CYP107S1 knockout strains were selected on 1000 μg/ml carbenicillin plates. A single cloned carbenicillin resistant colony was isolated and used to determine its mutagenic capacity using Ames Salmonella mutagenicity assay. The results showed that Salmonella TA98 tester strain returned the number of revertants to its baselines level indicating the lack of metabolic activation of procarcinogens in the P. aeruginosa CYP107S1 knockout cell-free extract. In addition, the characteristic cytochrome P-450 peak determined by the carbon monoxide difference spectrum was completely absent in the cell-free extract from this CYP107S1 knockout strain bacterium. Homologous recombination of the synthetic ampicillin gene on the CYP 107S1 P-450 locus was confirmed by PCR on purified genomic DNA extracted from the knockout bacterium. The metabolic activation of tested procarcinogens is, therefore, carried out by CYP107S1 in P. aeruginosa.     

High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin

This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3′)-VI. The enzyme from P. aeruginosa PA40 was purified to >70% homogeneity. The K_m of amikacin for this enzyme was 1.4 μM, the V_max/K_m ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3′)-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3′)-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed.     

Influence of adjunct azithromycin on the mortality of experimental Pseudomonas aeruginosa sepsis

The aim of this study was to determine the in vivo influence of azithromycin subinhibitory concentrations on mortality in a peritonitis-sepsis model. One hour after an intraperitoneal injection of Pseudomonas aeruginosa, mice were randomized to receive: ceftazidime, 500 mg/kg SC q4h×two doses alone; azithromycin, 20 mg/kg SC×one dose alone; ceftazidime plus azithromycin×one dose; ceftazidime plus azithromycin×two doses (1 and 24 h); ceftazidime plus prophylactic azithromycin (three doses at −48, −24, 1 h); or no treatment (control). A significant decrease in the rate of mortality was observed in animals treated with all ceftazidime plus azithromycin groups when compared with those receiving ceftazidime alone. These data indicate a potential role for adjunctive azithromycin therapy in P. aeruginosa infection.     

Role of outer membrane protein OprD and penicillin-binding proteins in resistance of Pseudomonas aeruginosa to imipenem and meropenem

The main mechanism of imipenem resistance in Pseudomonas aeruginosa is via downregulation of the gene for OprD porin. In a previous study, it was shown that the level of resistance did not parallel with the degree of downregulation of the porin gene, thus arguing for the existence of other resistance mechanisms. Penicillin-binding protein (PBP) 2 and PBP3 are involved in carbapenem resistance in Escherichia coli. The genes for PBPs were sequenced in three imipenem-resistant clinical strains and these strains were conjugated with two susceptible P. aeruginosa PA0 strains, selecting for auxotrophic markers. In all the clinical and resistant isolates there was no obvious elevation of AmpC cephalosporinase. The active sites of PBP1b (ponB), PBP2 (pbpA), PBP3 (pbpB) and PBP6 (dacC) had no mutations in any of the examined strains. Production of oprD mRNA was significantly lower in clinical strains and transconjugants after selection for the proB marker (PA4565 at 5113 kb). The clinical strains had alterations in OprD that were not found in transconjugants. Our findings suggest that PBPs do not play a role in imipenem resistance in the clinical strains examined here, but that a regulatory gene for oprD contributing to carbapenem resistance is located close to the proB gene.     

Antimicrobial activity of imipenem against isolates from complicated urinary tract infections

Antimicrobial activity of imipenem was measured using 4725 strains isolated from patients with complicated urinary tract infections (CUTIs) between 1988 and 2000. Imipenem was inactive against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, Enterococcus faecium and some non-fermenting Gram-negative rods. Resistant strains (MIC>16 mg/l) were observed in Staphylococcus haemolyticus (22%), Enterococcus faecalis (4%), Enterococcus avium (8%), Serratia marcescens (5%) and Pseudomonas aeruginosa (7%). Although the prevalence of imipenem-resistant strains of S. aureus, S. epidermidis and P. aeruginosa was sporadically high in some years, no steady increase was seen over the period. Resistant strains were rare in other major uropathogenic species. These results suggest that imipenem is still one of the most reliable antimicrobial drugs.     

A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets

This study, Vétoquinol S.A. epidemiosurveillance, was conducted from 1994 to 2001 in order to determine the susceptibility (by MIC determination) to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals). Strains from infected pets originated from six European countries. Isolates were collected from urinary infections (Escherichia coli), respiratory infections (Pasteurella multocida), dermatological infections (Staphylococcus intermedius, Pseudomonas aeruginosa) and otitis (S. intermedius, P. aeruginosa). The MIC distribution for each species was the same both before and after the launch of marbofloxacin in 1995. In E. coli, a resistant population was present before the use of marbofloxacin; this resistance was induced by co- or cross-resistance to other antibiotics used previously. Over this period, there was no significant evolution of MIC₉₀ for any bacterial species studied and no development of resistance was observed. Marbofloxacin was the most active antibiotic against P. multocida isolates and had the lowest MIC. No difference in MIC distribution was seen between the S. intermedius (unimodal distribution) isolated from dermatological infections and those from otitis. This was also true for P. aeruginosa. The use of marbofloxacin was not found to have induced a significant increase or spread of resistant bacteria.     

Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China

, , , and . Partial structural determination of hepatotoxic peptides from Microcystis aeruginosa (cyanobacterium) collected in ponds of central China. Toxicon 26, 1213–1217, 1988.—Waterbloom samples of the colonial cyanobacterium Microcystis aeruginosa, collected in fish ponds at the Hydrobiological Institute, Wuhan, People's Republic of China, were hepatotoxic to mice. Lyophilized cells had an ₅₀ (i.p. mouse; 40 mg/kg) and signs of poisoning similar to that reported for other cyanobacterial hepatotoxic peptides. Two toxins, with an ₅₀ (i.p. mouse) of 40 and 150 μg/kg, were isolated using gel filtration and high performance liquid chromatography. The amino acid composition and mol.wt (994) of the 40 μg/kg toxin was the same as that for microcystin-LR, while the 150 μg/kg toxin had an amino acid composition and mol.wt (1048) different from any of the reported cyanobacteria heptapeptide toxins reported to date.     

Toxicity to medaka fish embryo development of okadaic acid and crude extracts of Prorocentrum dinoflagellates

Chronic and subchronic toxicity following exposure to the DSP (Diarrhetic shellfish poisoning) toxin okadaic acid (OA) is receiving increasing attention as a public human health biohazard. However information on ecological impacts induced by proliferation of the OA producing dinoflagellate Prorocentrum is scarce. In order to analyse the toxicity of these substances, in vivo experiments were conducted on medaka fish (Oryzias latipes) embryos used as an experimental model. The study was focused on two strains of benthic Prorocentrum species, P. arenarium and P. emarginatum, naturally found in the Indian Ocean. Sample extracts (crude extracts, CE) were obtained from algal cultures and their toxic potential was explored. Their OA (and derivatives) content was evaluated by two methods: one based on chemical analysis using HPLC-MS, the other based on screening the inhibiting effect on protein phosphatase PP2A. P. arenarium extracts inhibit PP2A and the active toxin was confirmed as being OA by HPLC-MS. In contrast, P. emarginatum showed negative results regardless of the method used. The development of medaka fish embryos kept in medium containing pure OA or Prorocentrum CE was examined. Survival rates were reduced up to 100% depending on the concentrations used of both OA and CE of P. arenarium, while no effect was observed with CE of P. emarginatum. Anatomopathological studies of surviving embryos indicate that OA treatment resulted in significant increases in liver and digestive tract areas compared to controls. P. arenarium treated surviving embryos exhibited significant quantitative increases of global body and vitellus areas. Together, our results indicate that the toxic effects to medaka embryos development of pure OA and P. arenarium extracts containing OA are distinguishable. The differences may indicate the presence of additional toxic substance(s) (or molecules able to modulate OA impact) in the P. arenarium CE that probably are not present in P. emarginatum.     

Pseudomonas aeruginosa biofilms are more susceptible to ciprofloxacin than to tobramycin

The objective of this study was to determine and compare the biofilm elimination concentrations (BEC: the concentration which reduced the viability of biofilm organisms by at least 99.9%) of ciprofloxacin and tobramycin for Pseudomonas aeruginosa, a common cause of nosocomial biomaterial-related infections. Bacterial biofilms were produced in the modified. Robbins device using continuous culture flow at 60 ml/h for 40–44 h, and the sessile organisms were then exposed to either ciprofloxacin or tobramycin at a range of concentrations for 12 or 36h. The BEC of ciprofloxacin was 5 μg/ml for the 12 and 36 h treatments, a value 10 × greater than the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In contrast, the BEC of tobramycin was > 100 μ/ml after 12h and 75 ug/ml following 36 h of drug exposure, that is 75–100 × the MIC and MBC. The results demonstrated that the BEC is a more suitable indicator of the antibiotic susceptibility of P. aeruginosa biofilms than the MIC and MBC. Ciprofloxacin was significantly more effective than tobramycin in the treatment of P. aeruginosa adherent to biomaterials. With respect to clinical application, if the intention of antibiotic use is to eradicate bacteria adherent to devices, only biofilm-active agents should be used.     

亚最低杀菌浓度环丙沙星体外诱导铜绿假单胞菌标准质控菌株耐药研究

目的:研究亚最低杀菌浓度(minimum bactericidal concentration,Sub-MBC)环丙沙星体外诱导对铜绿假单胞菌MIC值影响,为防止临床不合理使用抗菌药物导致细菌耐药性,提供新思路。方法:以铜绿假单胞菌质控菌株ATCC27853为研究对象,以环丙沙星为诱导耐药抗菌药物,用微量稀释法首先测定其MBC值,然后用1/2MBC值环丙沙星浓度进行体外诱导培养,每天观察其MBC值变化,及时调整诱导浓度为变化后MBC值的1/2,直到诱导菌株MBC值升高到原始MBC值的64倍时,停止诱导,并记录诱导天数;诱导出的耐药菌株传代3 d,再采用微量稀释法对其MBC进行测定,并通过全自动微生物药敏仪对其进行耐药性鉴定。结果:铜绿假单胞菌标准质控菌株的环丙沙星MBC值为0.5μg·ml^-1;采用0.25μg·ml^-1环丙沙星浓度体外诱导7 dMBC值明显升高,诱导30 d升高至原始菌株MBC的64倍;诱导出的铜绿假单胞菌针对环丙沙星的耐药菌株,经3 d传代后,微量稀释法测定其MBC仍为原始菌株的64倍,全自动药敏分析仪鉴定为环丙沙星耐药菌。结论:亚最低杀菌浓度环丙沙星可体外诱导铜绿假单胞菌标准质控菌株出现耐药,且耐药性随亚最低杀菌浓度诱导时间延长而增加,诱导出的铜绿假单胞菌耐药菌株的耐药性能稳定传代,提示低剂量使用抗菌药物可导致细菌产生耐药性。     

The in vivo assessment of safety and gastrointestinal survival of an orally administered novel probiotic, Propionibacterium jensenii 702, in a male Wistar rat model

This study aimed to evaluate in vivo gastrointestinal survival and safety of orally administered probiotic bacterium, Propionibacterium jensenii 702, using a male Wistar rat model. A high dose of 10¹⁰ cfu/rat/day of P. jensenii 702 was fed to each rat for 81 days. The repeated dose toxicity and translocation of P. jensenii 702 into rat tissues were evaluated, along with the rat faecal β-glucuronidase activities and dairy propionibacteria counts. Results showed that P. jensenii 702 had no adverse effect on general health status, body weight gain, visceral organs and faecal β-glucuronidase activities. No viable cells of P. jensenii 702 were recovered from blood and tissue samples (mesenteric lymph nodes, liver and spleen) of rats, and no treatment-associated illness or death was observed. Faecal dairy propionibacteria counts reached 10⁸ cfu/g after 36 days treatment and remained between 10⁸–10⁹ cfu/g till the end of 81 days treatment. The results indicate that P. jensenii 702 was able to survive the in vivo gastrointestinal tract transit of rats, with no adverse affects on the animals. However, further human clinical trials are required before strain P. jensenii 702 could be incorporated into food for human consumption as probiotics.     

Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era?

The evolving problem of antimicrobial resistance in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae has led to the emergence of clinical isolates susceptible to only one class of antimicrobial agents and eventually to pandrug-resistant (PDR) isolates, i.e. resistant to all available antibiotics. We reviewed the available evidence from laboratory and clinical studies that reported on polymyxin-resistant and/or PDR P. aeruginosa, A. baumannii or K. pneumoniae clinical isolates. Eleven laboratory studies reported on isolates with resistance to polymyxins, three of which (including two surveillance studies) also included data regarding PDR isolates. In addition, two clinical studies (from Central and Southern Europe) reported on the clinical characteristics and outcomes of patients infected with PDR isolates. These data suggest that polymyxin-resistant or PDR P. aeruginosa, A. baumannii and K. pneumoniae clinical isolates are currently relatively rare. However, they have important global public health implications because of the therapeutic problems they pose. The fears for the dawn of a post-antibiotic era appear to be justified, at least for these three Gram-negative bacteria. We must increase our efforts to preserve the activity of available antibiotics, or at least expand as much as possible the period of their use, whilst intense research efforts should be focused on the development and introduction into clinical practice of new antimicrobial agents.     

Aerosol treatment with cefoperazone or gentamicin protects granulocytopenic mice from acute Pseudomonas aeruginosa pneumonia

The usefulness of the aerosol route for delivery of either cefoperazone (CEF) or gentamicin (GEN) for the treatment of acute Pseudomonas aeruginosa pneumonia was assessed in mice rendered granulocytopenic by treatment with 200 mg/kg cyclophosphamide. Aerosol delivery of CEF (60 μg/ml final concentration in lung homogenate) was significantly more effective (90% survival) than the single intraperitoneal (i.p.) inoculation of 640 mg/kg CEF (0% survival) (P < 0.001). Similarly, aerosol delivery of gentamicin (11 μg/ml final concentration in lung homogenate) was significantly more effective (100% survival) than single or multiple inoculation of a total i.p. dose of 16 mg/kg (0% survival) (P < 0.001). Pulmonary half-life of both antibiotics after aerosol administration was higher than that obtained after i.p. inoculation.     

Role of efflux mechanisms on fluoroquinolone resistance in Streptococcus pneumoniae and Pseudomonas aeruginosa

Prokaryotic efflux mechanisms can effectively increase the intrinsic resistance of bacteria by actively transporting antibiotics out of cells, thus reducing the effective concentration of these agents. The fluoroquinolones, similar to most other antimicrobial classes, are susceptible to efflux mechanisms, particularly in Gram-negative organisms, such as Pseudomonas aeruginosa. Resistant P. aeruginosa clones isolated after fluoroquinolone therapy frequently over express at least one of the multiple efflux pump mechanisms found in this organism. Gram-positive bacteria, such as Streptococcus pneumoniae, also possess efflux mechanisms, though their effect on fluoroquinolone resistance seems to be more limited and selective. In the future, efflux pump inhibitors may offer effective adjunctive therapy to antibiotics for the treatment of difficult infections by efflux mutants. In the meantime, appropriate antibiotic selection and optimal dosing strategies should aim to eradicate the causative pathogen before a resistant efflux mutant can emerge.     

Influence of the cell wall on ciprofloxacin susceptibility in selected wild-type Gram-negative and Gram-positive bacteria

The susceptibility of several wild-type bacteria to ciprofloxacin and accumulation of the drug in these bacteria were evaluated. Species studied included Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Bacillus cereus. Ciprofloxacin susceptibility was measured for each strain using two different methods: the minimal inhibitory concentration and the bactericidal index. Significant differences were observed between the results derived from these two methods. Whereas the minimal inhibitory concentration was low in all strains tested, ciprofloxacin’s bactericidal activity, as indicated by the bactericidal index, varied with the species studied. To determine whether this finding was due to variations in cell envelope permeability to ciprofloxacin (i.e. to combined cell uptake and efflux), we studied ciprofloxacin accumulation using spectrofluorometry. In Gram-negative bacteria, differences in permeability can lead to altered susceptibility to antibiotics. In fact, the combination of slow uptake and efficient efflux seems to be crucial to the characteristic poor susceptibility of P. aeruginosa to ciprofloxacin. However, the low level of activity of ciprofloxacin against S. aureus and two Bacillus species may have resulted from the drug’s interaction with its target enzymes (i.e. topoisomerase IV in S. aureus and DNA gyrase in Bacillus spp.) rather than diminished permeability.     

Selenium and viral virulence

A mouse model of coxsackievirus-induced myocarditis is being used to investigate nutritional determinants of viral virulence. This approach was suggested by research carried out in China which showed that mice fed diets composed of low selenium ingredients from a Keshan disease area suffered more extensive heart damage when infected with a coxsackie B4 virus than infected mice fed the same diet but supplemented with selenium by esophageal intubation. Selenium deficiency in our mice increased the virulence of an already virulent strain of coxsackievirus B3 (CVB3/20) and also allowed conversion of a non-virulent strain (CVB3/0) to virulence. Such conversion of CVB3/0 was accompanied by a change in the viral genome to more closely match that of the virulent virus, CVB3/20. As far as the authors are aware, this is the first report of host nutrition influencing the genetic make-up of an invading pathogen. Nutritionists may need to consider this mechanism of increased viral virulence in order to gain a better understanding of diet/infection relationships.     

Contemporary in vitro spectrum of activity summary for antimicrobial agents tested against 18569 strains non-fermentative Gram-negative bacilli isolated in the SENTRY Antimicrobial Surveillance Program (1997-2001)

The frequency of occurrence and antimicrobial susceptibility patterns of 18 569 non-fermentative Gram-negative bacilli consecutively collected as part of the SENTRY Antimicrobial Surveillance Program were summarized. The isolates were tested by the broth microdilution method in three coordinator laboratories using common reagents and reference methodologies. The most frequently isolated pathogen was Pseudomonas aeruginosa (11 968 isolates; 64.5%) followed by Acinetobacter spp. (3468 isolates; 18.7%) and Stenotrophomonas maltophilia (1488 isolates; 8.0%). The lowest resistance rates for P. aeruginosa documented were for amikacin (8%), meropenem (10%) and cefepime (10%), and all fluoroquinolones tested showed similar resistance rates (22–24%). The most active compounds against Acinetobacter spp. were the carbapenems, imipenem (11% resistance) and meropenem (12% resistance) followed by cefepime (31% resistance) and gatifloxacin (32% resistance). Very few compounds showed reasonable in vitro activity against S. maltophilia, with the most active antimicrobial agents being trimethoprim/sulphamethoxazole, gatifloxacin and levofloxacin (5–6% resistance). Resistance surveillance among these organisms remains necessary to guide empirical antimicrobial therapy, especially for these less frequently isolated and difficult to test pathogens.