Tetsufumi Koga

Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin.

Interactions between bacterial biofilms formed by Pseudomonas aeruginosa and clarithromycin, a macrolide having no anti-P. aeruginosa activity, were investigated. P. aeruginosa incubated for 10 days on membrane filters formed biofilms on the surfaces of the filters. The biofilms were characterized by dense colonizations of bacteria and thick membranous structures that covered the colonies. Treatment of the biofilms with a relatively low concentration of clarithromycin for 5 days resulted in an eradication of the membranous structures. Quantitative analysis of alginate and hexose was done to evaluate the quantity of polysaccharides in or on the biofilms. Treatment of the biofilms with clarithromycin decreased the quantity of alginate and hexose and therefore perhaps the quantity of polysaccharides as well. Eradication of the membranous structures of biofilms, or the decrease in the quantity of polysaccharides, resulted in an increase in the rate of penetration of antibiotics through bacterial biofilms. In vivo therapeutic effects of ofloxacin in the rat infection model, in which the biofilm mode of growth of P. aeruginosa is characteristic, were enhanced by oral coadministration of clarithromycin. It is suggested that clarithromycin eradicated glycocalyx produced by P. aeruginosa, or suppressed the production of glycocalyx, by unknown mechanisms and thereby enhanced the therapeutic efficacies of other antimicrobial agents against infections caused by P. aeruginosa. Images

Interaction between clarithromycin and biofilms formed by Staphylococcus epidermidis.

Interactions between clarithromycin and biofilms formed by Staphylococcus epidermidis were investigated by using a clarithromycin-resistant strain. Treatment of the colonization with a relatively low concentration of clarithromycin resulted in the eradication of slime-like structure and a decrease in the quantity of hexose. Another result was increased penetration of antibiotics through the biofilm of S. epidermidis. Images

In Vitro and In Vivo Antibacterial Activities of CS-023 (RO4908463), a Novel Parenteral Carbapenem

ABSTRACT CS-023 (RO4908463, formerly R-115685) is a novel 1β-methylcarbapenem with 5-substituted pyrrolidin-3-ylthio groups, including an amidine moiety at the C-2 position. Its antibacterial activity was tested against 1,214 clinical isolates of 32 species and was compared with those of imipenem, meropenem, ceftazidime, ceftriaxone, ampicillin, amikacin, and levofloxacin. CS-023 exhibited a broad spectrum of activity against gram-positive and -negative aerobes and anaerobes, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, penicillin-resistant Streptococcus pneumoniae (PRSP), β-lactamase-negative ampicillin-resistant Haemophilus influenzae, and Pseudomonas aeruginosa. CS-023 showed the most potent activity among the compounds tested against P. aeruginosa and MRSA, with MICs at which 90% of isolates tested were inhibited of 4 μg/ml and 8 μg/ml, respectively. CS-023 was stable against hydrolysis by the β-lactamases from Enterobacter cloacae and Proteus vulgaris. CS-023 also showed potent activity against extended-spectrum β-lactamase-producing Escherichia coli. The in vivo efficacy of CS-023 was evaluated with a murine systemic infection model induced by 13 strains of gram-positive and -negative pathogens and a lung infection model induced by 2 strains of PRSP (serotypes 6 and 19). Against the systemic infections with PRSP, MRSA, and P. aeruginosa and the lung infections, the efficacy of CS-023 was comparable to those of imipenem/cilastatin and vancomycin (tested against lung infections only) and superior to those of meropenem, ceftriaxone, and ceftazidime (tested against P. aeruginosa infections only). These results suggest that CS-023 has potential for the treatment of nosocomial bacterial infections by gram-positive and -negative pathogens, including MRSA and P. aeruginosa.

[43] In vitro and in Vivo models of bacterial biofilms

Publisher Summary This chapter focuses on several methods that have been developed in the laboratory to form bacterial biofilm models in vitro and in vivo , as well as several methods developed to investigate the nature of biofilm bacteria. Ideal properties sought in an in vitro bacterial biofilm model are uniform film formation, uniform reproducibility, and the ability to be investigated quantitatively. However, the ability to mimic a highly complicated in vivo condition is desirable in an in vitro model. There have been relatively few reports on in vivo models of biofilm growth. One of the most difficult challenges to overcome is to devise a durable infection model. Generally, it takes several days for infected bacteria to form biofilms in an infected tissue or organ, and it takes several days longer to investigate the therapeutic effect of a drug after the biofilm mode of growth of the infected bacteria has been established. Ideally, the infection should continue in a stable condition for more than 7–10 days. Respiratory tract infections, urinary tract infections, infective endocarditis, foreign body infections, and so forth are typical infectious diseases in which the biofilm mode of bacterial growth is sometimes characteristic. Therefore, these diseases should be taken into account in making a useful in vivo experimental biofilm infection model.

Potent In Vitro Activity of Tomopenem (CS-023) against Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

ABSTRACT Tomopenem (formerly CS-023) is a novel 1β-methylcarbapenem with broad-spectrum coverage of gram-positive and gram-negative pathogens. Its antibacterial activity against European clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was compared with those of imipenem and meropenem. The MICs of tomopenem against MRSA and P. aeruginosa at which 90% of the isolates tested were inhibited were 8 and 4 μg/ml, respectively, and were equal to or more than fourfold lower than those of imipenem and meropenem. The antibacterial activity of tomopenem against MRSA was correlated with a higher affinity for the penicillin-binding protein (PBP) 2a. Its activity against laboratory mutants of P. aeruginosa with (i) overproduction of chromosomally coded AmpC β-lactamase; (ii) overproduction of the multidrug efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN; (iii) deficiency in OprD; and (iv) various combinations of AmpC overproduction, MexAB-OprM overproduction, and OprD deficiency were tested. The increases in the MIC of tomopenem against each single mutant compared with that against its parent strain were within a fourfold range. Tomopenem exhibited antibacterial activity against all mutants, with an observed MIC range of 0.5 to 8 μg/ml. These results suggest that the antibacterial activity of tomopenem against the clinical isolates of MRSA and P. aeruginosa should be ascribed to its high affinity for PBP 2a and its activity against the mutants of P. aeruginosa, respectively.

In vivo pharmacodynamic activity of tomopenem (formerly CS-023) against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in a murine thigh infection model.

ABSTRACT Tomopenem (formerly CS-023) is a novel carbapenem with broad-spectrum activities against diverse hospital pathogens, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). We examined the in vivo pharmacodynamic characteristics of tomopenem against P. aeruginosa and MRSA by using a neutropenic murine thigh infection model with P. aeruginosa 12467 (MIC, 1 μg/ml) and MRSA 12372 (MIC, 2 μg/ml). The mice had 106 to 107 CFU/thigh of each strain 2 h after inoculation and were treated for 24 h with a fractionated administration of tomopenem given at intervals of 3, 6, 12, and 24 h. The serum protein binding of tomopenem was 17.4%. The efficacy of tomopenem in both infection models was enhanced by frequent dosing, which indicates that the efficacy is driven by the time above MIC (TMIC). In a sigmoid model, the cumulative percentages of the 24-h period that the concentrations of free, unbound fractions of the drug exceeded the MIC under steady-state pharmacokinetic conditions (f%TMICs) were best correlated with efficacy when R2 was 0.79 and 0.86 against P. aeruginosa and MRSA, respectively. Other pharmacokinetic and pharmacodynamic (PK-PD) indexes for the free, unbound fractions, the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) and the maximum concentration of the drug in serum divided by the MIC (Cmax/MIC), showed poor correlation with efficacy when R2 was ≤0.42. The f%TMIC values required for a static effect, 1-log kill, and 2-log kill against P. aeruginosa were 29, 39, and 51, respectively, which were similar to those for meropenem, for which the values were 24, 33, and 45, respectively. Against MRSA, the values for tomopenem were 27, 35, and 47. In conclusion, the pharmacodynamic characteristics of tomopenem were similar to those of meropenem against P. aeruginosa, and there was no difference between the target values for P. aeruginosa and MRSA required for efficacy in this study.

Affinity of Tomopenem (CS-023) for Penicillin-Binding Proteins in Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa

ABSTRACT Tomopenem (formerly CS-023), a novel 1β-methylcarbapenem, exhibited high affinity for penicillin-binding protein (PBP) 2 in Staphylococcus aureus, PBP 2 in Escherichia coli, and PBPs 2 and 3 in Pseudomonas aeruginosa, which are considered major lethal targets. Morphologically, tomopenem induced spherical forms in E. coli and short filamentation with bulges in P. aeruginosa, which correlated with the drugs PBP profiles. The potential of resistance of these bacteria to tomopenem was comparable to that to imipenem.

Experimental Helicobacter pylori gastric infection in miniature pigs.

An experimental Helicobacter pylori infection in miniature pigs was developed and investigated. Eighteen miniature pigs were inoculated with an H. pylori strain that has high virulence in mice at c. 5 x 10(10) cfu. H. pylori infection in miniature pigs was achieved by the administration of agar 1% in brucella broth with fetal bovine serum 10% just before inoculation. The bacterial colonisation and distribution were analysed by mapping of viable cell counts in the stomach in pigs of three different ages. The mapping assay was achieved on post-infection day 3 for the 5-day-old and 2-week-old pigs, and between days 41 and 43 for 3-month-old pigs. The highest cell counts were observed in 5-day-old pigs, which averaged 4.9 x 10(6) cfu/g of mucosa (n = 4). The bacteria were colonised mainly in the cardiac and fundus gland region in the 5-day-old and 2-week-old pigs, whereas the colonisation sites did not depend on the region in the 3-month-old pigs. Biopsy assay of the antral mucosa of a 3-month-old pig after H. pylori infection showed that this infection persisted for >22 months. Serum antibody against H. pylori was detected in the infected pigs but not in the uninfected animal. Immunostaining demonstrated the presence of bacteria on the epithelial surface of the infected pigs. A microscopic finding common to all the infected pigs, focal gastritis with infiltration of lymphocytes detected on the lesser curvature of the stomach, resembled the microscopic appearance in H. pylori-infected human patients. These results suggest that miniature pigs might be a suitable model for studying H. pylori infection.

The effect of colonisation by Helicobacter pylori in Praomys (Mastomys) natalensis on the incidence of carcinoids

An animal model of experimental gastric Helicobacter pylori infection has been developed in the Z strain of Praomys (Mastomys) natalensis; this animal has been reported to develop gastric carcinoids and adenocarcinoma spontaneously. In the present study, male and female Mastomys were killed at 1, 2, 4, 8 and 16 weeks after H. pylori inoculation. Colonisation of H. pylori was maintained in the stomachs of all animals for up to 16 weeks. H. pylori were mainly found in the antrum. Lymphoid infiltration appeared in the antral lamina propria and submucosa in all male and female animals from 4 to 16 weeks after inoculation. On microscopic examination after immunostaining for H. pylori, the organisms were detected in the antral mucus layer of the gastric epithelium. Serum immunoglobulin G specific for H. pylori could be detected 2 weeks after inoculation in female and 4 weeks after inoculation in male Mastomys, and persisted throughout the 16-week study period. At 18 months after inoculation, H. pylori positive rates for male and female Mastomys were 15 of 21 and 7 of 27, respectively. Carcinoids developed in 27 of 100 inoculated and in 49 of 100 uninoculated male, and in 5 of 100 inoculated and in 21 of 100 uninoculated female animals at 18 months after inoculation. Adenocarcinoma developed in 1 of 100 male Mastomys in both the inoculated and uninoculated groups, but in none of the female animals in either the inoculated or uninoculated groups. These results indicate that antrum-predominant colonisation by H. pylori caused the decrease in incidence of carcinoid formation in Mastomys.

Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P3-sparing S1P1 agonists

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.