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Macrolides are a well-known family of oral antibiotics whose antibacterial spectrum of activity covers most relevant bacterial species responsible for respiratory infectious disease. In recent years, it has been reported that macrolides have not only bactericidal activity but also direct immunomodulating activity in mammals. In this study, we observed new physiological activity of macrolides and examined whether various macrolides induce apoptosis in human leukemia cell lines. We investigated the effects of 13 different macrolides on the viability of Jurkat and HL-60 cells. Among all the macrolides used in this study, rokitamycin, a semisynthetic macrolide with a 16-member ring, effectively induced cell death. Rokitamycin induced DNA fragmentation and caspase activation, resembling the progression of apoptosis. Moreover, rokitamycin reduced the mitochondrial transmembrane potential and released cytochrome c from mitochondria to the cytosol, suggesting that mitochondrial perturbation is involved in rokitamycin-induced apoptosis. These results suggest that rokitamycin possesses not only bactericidal activity but also pro-apoptotic activity in human leukemia cells. Keywords:: macrolide, rokitamycin, apoptosis, mitochondria, caspase

Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.

The present study demonstrates the in vitro effectiveness of the macrolide rokitamycin and the phenothiazine compound chlorpromazine against Acanthamoeba castellanii . Growth curve evaluations revealed that both drugs inhibit trophozoite growth in dose- and time-dependent ways. The effects of both drugs when they were used at the MICs at which 100% of isolates are inhibited were amoebistatic, but at higher doses they were amoebicidal as well as cysticidal. Experiments showed that when rokitamycin was associated with chlorpromazine or amphotericin B, rokitamycin enhanced their activities. Furthermore, low doses of rokitamycin and chlorpromazine, alone or in combination, blocked the cytopathic effect of A. castellanii against WKD cells derived from the human cornea. These results may have important significance in the development of new anti- Acanthamoeba compounds.

Streptococcus pyogenes isolates with the same pulsed-field patterns were recovered from the throat cultures of a child with tonsillopharyngitis before and after treatment with miocamycin, a 16-membered macrolide. The initial isolate was macrolide-susceptible, but the isolates after the treatment were resistant to 14 and 15-membered macrolides and had two amino acid (65WR66) deletions in ribosomal protein L4.

In 1998 we conducted a multicentre study in Spain on the susceptibility of Streptococcus pyogenes isolates to different 14-, 15- and 16-membered macrolides and clindamycin, in which the number of strains examined was proportional to the number of inhabitants in each geographical area. The aim of the present work was to re-examine the antimicrobial susceptibility of S. pyogenes in 2001, using the same methodology and centres as in 1998, to determine the different susceptibility phenotypes to macrolides-lincosamides, and to compare the results from the 2 years by statistical tests. A total of 529 unique isolates of S. pyogenes, collected in 21 laboratories, were studied. Throat swabs provided 417 isolates (78.8%), and the remaining 112 were from other sources. Four hundred and thirty-five (82.2%) were isolated from children and 94 (17.8%) from adults. One hundred and fifty-seven (29.7%) of the isolates were resistant to erythromycin and azithromycin, whereas resistance to miocamycin, a 16-membered macrolide, was 1.5%. The prevalence of resistance to clindamycin was 1.3%. The majority (98.7%) of the 157 erythromycin-resistant strains presented the M phenotype. When we compared the results obtained in 1998 and 2001, we observed a statistically significant increase in resistance to erythromycin and azithromycin (P = 0.02, chi(2) test), but not to clindamycin or miocamycin (P = 0.47, chi(2) test with Yates' correction). The significant increase in the prevalence of resistance to some macrolides of S. pyogenes in Spain underscores the need for continuous surveillance of antimicrobial resistance in this species.

This study aimed to evaluate the bactericidal rates of levofloxacin, gatifloxacin, penicillin, meropenem and rokitamycin against seven isolates of Bacillus anthracis clinically isolated between 1960 and 1970. After determination of MIC and MBC, time-kill experiments were carried out. Antimicrobial activity was evaluated at concentrations equal to 1 x, 2 x, 4 x and 8 x MIC after 0, 3, 6, 12 and 24 h of incubation with the drugs. Bactericidal activity was defined as a decrease in bacterial count of at least 3 log10. All the isolates were susceptible to all the antibiotics, by considering the antistaphylococcal breakpoints. Levofloxacin was bactericidal at 1 x MIC after 24 h and at 4 x MIC after 12 h, and gatifloxacin was bactericidal at 2 x MIC after 24 h and at 8 x MIC after 12 h. Meropenem, rokitamycin and penicillin also showed bactericidal activity at concentrations of 4 x and 8 x MIC, respectively, but only after 24 h incubation; after the same time, meropenem and rokitamycin showed a more marked killing than penicillin at 2 x MIC.

This in-vitro study was designed to identify the enzyme(s) involved in the major metabolic pathway of rokitamycin, i.e. the formation of leucomycin A7, and to assess possible interactions of the drug with rat liver microsomes. Formation of leucomycin A7 was NADPH-independent and was not appreciably inhibited by anti-rat NADPH cytochrome P-450 reductase serum or cimetidine, a nonspecific inhibitor of cytochrome P-450 isoforms. Eadie-Hofstee plots for the formation of leucomycin A7 were indicative of apparently monophasic behaviour for six rat liver microsomes tested. The mean (± s.d.) kinetic parameters, Km, Vmax and Vmax/Km, for the formation of leucomycin A7 from rokitamycin were 47 ± 13 μm, 390 ± 56 nmol min−1 (mg protein)−1 and 8.6 ± 1.6 mL min−1 (mg protein)−1, respectively. Three esterase inhibitors (100 μM), bis-nitrophenylphosphate, physostigmine and metrifonate inhibited the formation of leucomycin A7 by more than 60%. Metabolism of rokitamycin was inhibited by terfenadine, but not by mequitazine, whereas chlorpheniramine and theophylline activated the formation of leucomycin A7. Rokitamycin, leucomycin A7, leucomycin V, erythromycin and clarithromycin were weak inhibitors of CYP3A-catalysed 3-hydroxylation of quinine with mean IC50 values ranging from 71 to > 100 μM. It is concluded that in rat liver microsomes the formation of leucomycin A7 from rokitamycin is catalysed mainly by an esterase (possibly cholinesterase, EC3.1.1.8), but not by cytochrome P-450 enzyme(s). Although in this in-vitro animal study CYP3A activity was barely inhibited by rokitamycin, the possibility cannot be totally discounted in man when rokitamycin is co-administered with drugs metabolized by CYP3A.

The purpose of this study was to evaluate possible interaction of nifedipine with erythromycin or rokitamycin in the intestinal mucosa. Male beagle dogs were orally administered nifedipine (10 mg), with or without oral pre-medication with erythromycin (300 mg), and 300 mg erythromycin or rokitamycin twice a day for 3 days. The experiments were of randomized cross-over design with a two-week wash-out period between dosing regimens. Erythromycin pre-medication for 3 days resulted in a significant increase in the area under the serum nifedipine concentration-time curve (AUC), whereas the curve for one nifedipine metabolite (M-2) decreased significantly. When the effects of erythromycin on the metabolism of nifedipine were studied using dog liver microsomes it was found that erythromycin significantly inhibited formation of M-2 but not of the metabolite M-1. These results indicate that formation of M-2 from M-1 in the liver might be reduced by erythromycin premedication. To avoid possible metabolism in the gut, the dogs were then administered 8 mg nifedipine into the peritoneal cavity, with or without multiple dose pre-treatment with erythromycin for 3 days. After intraperitoneal administration of nifedipine, the maximum concentration (Cmax) of nifedipine increased significantly. After pre-administration of erythromycin the relative bioavailability of nifedipine after oral administration was increased compared with injection into the peritoneal cavity. In-vitro study using rat intestinal microsomes and the in-vivo rat intestinal loop technique also showed that pre-administration of erythromycin inhibits nifedipine metabolism in the small intestine.

The effects of erythromycin, clarithromycin and rokitamycin on the metabolism of nifedipine were studied in vitro and in situ. Erythromycin, clarithromycin or rokitamycin added to nifedipine did not inhibit the formation of metabolite, M-1, of nifedipine, whereas pretreatment with erythromycin or clarithromycin significantly (p < 0.05) inhibited its formation. Only erythromycin significantly (p < 0.05) inhibited the formation of M-2 from M-1. These observations agreed with the results obtained using an in situ rat intestinal loop technique. As assessed by the concentrations of nifedipine and M-2 in jugular and portal vein blood, the inhibition of nifedipine metabolism by erythromycin was greater after multiple doses than after a single dose. Moreover, our results suggest that rokitamycin is a less potent inhibitor of nifedipine metabolism.

In an open, randomized, crossover trial, the clinical efficacy and effect on total serum immunoglobulin E (IgE) levels of a macrolide—miocamycin—and of a beta-lactam—amoxicillin/clavulanate—were compared in patients with infectious exacerbations of bronchial asthma. Of the 42 patients admitted, 33 were evaluated for efficacy and tolerability of the treatments and 16 for the effect on serum IgE levels. Both treatments were effective in treating infectious exacerbations. Mild side effects (gastralgia) occurred in 19% of the miocamycin-treated patients, compared with 32% (generally, worsening of the asthma attacks) in the amoxicillin/clavulanate-treated patients. Total IgE serum levels were not affected by miocamycin, whereas they increased after treatment with amoxicillin/clavulanate (statistically significant difference). It would thus appear reasonable to prefer macrolides to beta-lactams in the treatment of asthmatic patients (at least as far as the tested drugs are concerned).

The efficacies of macrolide antibiotics, rokitamycin, leukomycin, josamycin and erythromycin were investigated against 63 strains of M. pneumoniae included FH strain which were isolated from clinical specimens obtained in 1986-1987, 1991, 1992-1993. Values of MICs and MBCs of these antibiotics were evaluated by two methods, broth dilution method and M. pneumoniae L cell infection system. In broth dilution method, MIC50 and MBC50 of rokitamycin were 0.0063-0.025 microgram/ml and 0.025-0.2 microgram/ml respectively. The ratios of MBC50/MIC50 were 1-32. MIC50 of leukomycin, josamycin and erythromycin were 0.0063-0.05 micrograms/ml, 0.0025-0.05 microgram/ml, and 0.0125-0.05 microgram/ml respectively. MBC50 of these drugs were 0.0125-50 micrograms/ml, 25-50 micrograms/ml, and 25-50 micrograms/ml respectively. The ratios of MIC50/MBC50 were 2-800, 500-2000, 500-4000. MIC50 of rokitamycin and leukomycin to the isolates during period from 1992-1993 were higher as compare to those of isolates during 1986 to 1991. On the other hand, rokitamycin markedly reduced the numbers of CFU in the growth phase when added at the concentrations of 16 and 4 times the MIC, but the other macrolides were reduced slightly CFU at the concentrations of 4 times, the MIC. In L cell infectious system, MIC50 and MBC50 of rokitamycin were 0.005-0.0125 microgram/ml and 0.05-0.1 microgram/ml respectively. The ratios of MBC50/MIC50 were 1-2 Ratios of leukomycin, josamycin, and erythromycin were 0.05-0.1 microgram/ml, 0.05-0.2 microgram/ml and 0.050-0.1 microgram/ml respectively. The MBC50 of these drugs were 0.2-0.4 micrograms/ml, 0.8-1.6 microgram/ml, 0.8-1.6 microgram/ml. Ratios of MBC50/MIC50 were 2-8, 4-32, 8-32.(ABSTRACT TRUNCATED AT 250 WORDS)

Amorphous 16-membered macrolide compounds were prepared by spray drying, and the physicochemical stability of the amorphous states has been investigated. Josamycin, rokitamycin, midecamycin, 3-O-acetyl-4"-O-isovaleryltylosin, miocamycin and 4"-O-(4-methoxyphenyl)acetyltylosin were used as macrolide compounds. From X-ray powder diffractometry and differential scanning calorimetric (DSC) measurements, all the spray dried compounds were recognized to be in a glassy state. Miocamycin and 4"-O-(4-methoxyphenyl)acetyltylosin glasses showed crystallization and fusion peaks on DSC thermograms, and were crystallized after 6 months of storage at 313 K and 75% relative humidity. Other compounds showed only a glass transition peak on each DSC thermogram, and the glassy state was maintained stably against humidity. It was found that the physicochemical stability of the glassy state of miocamycin was closely related to the inlet temperature of the spray drying process.

Pharmacokinetic interaction between ponsinomycin-nicoumalone was studied in six subjects who received an 8 mg oral dose of racemic nicoumalone alone and 4 days into an oral regimen of ponsinomycin 800 mg twice daily. The concentrations of R(+) and S(-)-nicoumalone in plasma were measured using a stereospecific h.p.l.c. assay. The disposition characteristics of nicoumalone enantiomers in the control phase of this study were similar to those reported previously with the exception of the data for one subject whose oral clearance for S(-)-nicoumalone was seven times lower than those in the other subjects. A statistically significant effect of ponsinomycin on the kinetics of R(+) and S(-)-nicoumalone was not demonstrated.

Tonsillitis is the most common disease in the otorhinolaryngeal location. For most patients, tonsillitis is attributed to a group A streptococcal infection if the throat culture is positive for that organism or to a viral infection if the throat culture is negative. However, recent studies have shown that Chlamydia trachomatis can produce tonsillar infection. In this study, we evaluated the efficacy of Rokitamycin, a 16-membered ring macrolide antibiotic agent, in the treatment of tonsillitis associated with C. trachomatis. In 26 of 28 (92.9%) patients from whom C. trachomatis was isolated, the organism was eradicated by antimicrobial treatment with Rokitamycin of five days to three weeks' duration. In 25 of the 26 patients, they were totally free of tonsillar symptoms.

To study the antimicrobial susceptibility and prevalence of the different phenotypes and genotypes of macrolide resistance in group A streptococci isolated in Spain in 2004, and to compare the results with those obtained in 1998 and 2001 using the same methodology and centres.A total of 530 unique isolates of Streptococcus pyogenes collected in 21 laboratories from 16 geographic areas (regions) in Spain were used. Antimicrobial susceptibility testing was performed using the agar dilution method. Discs containing erythromycin or clindamycin were used to recognize the phenotypes of macrolide-lincosamide-streptogramin (MLS) resistance. Genes encoding macrolide-lincosamide resistance were detected by PCR.Resistance to erythromycin was 21.7% [95% confidence interval (CI) 16.5-26.3]. The resistance to azithromycin was 21.5%, whereas the resistance to miocamycin and to clindamycin was 6.6% (95% CI 3.0-8.9). Thirty-one (5.8%) of the isolates were resistant to telithromycin. Of the 115 erythromycin-resistant isolates, 67.8% had the M phenotype, representing 14.7% of all the isolates tested. Thirty-five isolates (30.5% of the erythromycin-resistant strains and 6.6% of all the isolates) had the MLS(B) constitutive phenotype. There was a high prevalence of resistance to telithromycin (88.6%) among the 35 strains with the MLS(B) constitutive phenotype. When we compared these results with those from previous studies (1998 and 2001), we found a significant increase in the MLS(B) constitutive phenotype (P0.001), and a significant decrease in the M phenotype (P0.005) was noted.The significant increase in the prevalence of resistance to clindamycin and miocamycin, and the prevalence of resistance to telithromycin reached in a short period of time from the introduction of its use, underscore the need for continuous surveillance of antimicrobial resistance in S. pyogenes in Spain.

Erythromycin-resistant Streptococcus pneumoniae isolates from young carriers were tested for their antimicrobial susceptibility; additionally, inducibility of macrolide and clindamycin resistance was investigated in pneumococci carrying erm (A), erm (B), or mef (A). Of 125 strains tested, 101 (81%) were multidrug resistant. Different levels of induction were observed with erythromycin, miocamycin, and clindamycin in erm (B) strains; however, in erm (A) strains only erythromycin was an inducer. Induction did not affect macrolide MICs in mef (A) strains.

The effects of erythromycin (a 14-membered ring macrolide) and rokitamycin (a 16-membered ring macrolide) on the viability of the Streptococcus pyogenes M phenotype were studied by means of flow cytometry and fluorescence microscopy by using a combination of two fluorochromes (syto 9 and propidium iodide) that stains live bacteria green and dead bacteria red. In order to apply the flow cytometry, a bacterial sonication procedure was expressly set up to separate single cells from the long, intralaced S. pyogenes chains of up to 30 to 40 cells that have previously prevented the application of flow cytometry to this type of bacteria. The association of flow cytometry using an appropriate sonication procedure, together with a combination of fluorescent probes, offered the possibility of very quickly investigating the different microbiological effects of rokitamycin at 2 μg/ml, which was active on the S. pyogenes M phenotype, and of erythromycin at doses of up to 32 μg/ml, which was not.

The Group B streptococcus (Streptococcus agalactiae) is a pathogen of increasing importance in human disease. We therefore studied the susceptibility of clinical isolates of S. agalactiae to penicillin G, erythromycin, azithromycin and clindamycin using National Committee for Clinical Laboratory Standards methodology, and we also determined the phenotypes of macrolide-lincosamide susceptibility and the resistance genes implicated in a group of selected isolates of the different phenotypes. We used 221 isolates collected between 1997 and 1999 in two Health Authority Areas in Móstoles and Granada, Spain. The minimal concentration for 90% inhibition (MIC90) for penicillin G was 0.12 mg/L and all the isolates tested were susceptible. One hundred and eighty-five (83.7%) were susceptible to erythromycin and azithromycin and 191 (86.4%) were susceptible to miocamycin and clindamycin. Twenty-three isolates (10.4%) had a constitutive MLSB phenotype, seven (3.2%) an inducible phenotype, and six (2.7%) an M phenotype. All except one of the MLSB phenotype isolates tested (n = 23) carried erm genes; in two strains with the mef (A) gene, all the M phenotype (n = 6) isolates tested carried mef genes, while erm and mef (A) genes were absent in all the macrolide-lincosamide-susceptible (n = 12) isolates tested. In our environment, resistance to macrolide and lincosamide in S. agalactiae was present in 10-16% of the isolates. The majority of resistant strains had the MLSB phenotype.

The aim of this study was to use atomic force microscopy (AFM), an innovative type of micro- scopy, to investigate the different behaviours of erythromycin A (a 14-membered ring) and rokitamycin (a 16-membered ring) in disrupting the morphology of Streptococcus pyogenes with the M phenotype. AFM scanning and sensing of the topography of a sample makes it possible to obtain simultaneous high-resolution digital measurements of the x, y and z coordinates at any point on the bacteria surface. The images obtained before and 2, 4 and 6 h after incubation with erythromycin A (32 mg/L) and rokitamycin (2 mg/L) clearly show that not even high concentrations of erythromycin A interfere with the M phenotype of S. pyogenes, whereas rokitamycin has a progressive action that leads to the formation of abnormally large cells, the loosening of chain structure and the formation of clusters.

The in vitro activities of the new agents linezolid, quinupristin-dalfopristin, moxifloxacin, and trovafloxacin were determined and compared with those of penicillin, clindamycin, and four macrolides against 53 erythromycin-resistant Streptococcus pneumoniae , 117 S. pyogenes (64 erythromycin-susceptible and 53 -resistant), and 101 S. agalactiae (53 erythromycin-susceptible and 48 -resistant) isolates. Differentiation of macrolide resistance phenotypes was performed by the double-disk method. The genetic basis for macrolide resistance in 52 strains was also determined. The M phenotype was found in 84.9, 6.3, and 1.9% of S. pyogenes , S. agalactiae , and S. pneumoniae isolates, respectively. These strains were susceptible to miocamycin and clindamycin. Strains with the inducible phenotype accounted for 27.1% of S. agalactiae isolates and 9.4% each of S. pyogenes and S. pneumoniae isolates. All erythromycin-resistant isolates were also resistant to the 14- and 15-membered macrolides tested. Strains with all three phenotypes were susceptible to ≤2 μg of linezolid per ml. Quinupristin-dalfopristin exhibited good in vitro activity against all strains, irrespective of their resistance to erythromycin (MICs at which 90% of the isolates tested were inhibited [MIC 90 s], 0.2 to 1 μg/ml). Against the erythromycin-resistant S. pyogenes and S. agalactiae strains, moxifloxacin and trovafloxacin were the most active agents (MIC 90 s, 0.1 μg/ml). The new antimicrobials evaluated may be alternative agents to treat infections caused by macrolide-resistant as well as macrolide-susceptible streptococci.

The plasma pharmacokinetics for a single intravenous dose (10 mg/kg body weight) of miocamycin (a 16-membered macrolide drug) was investigated in Holando Argentino cattle (n = 5). Blood drug concentrations were determined by a microbiological method and data were best-fitted to a two-compartment open model. The pharmacokinetic profile consisted of a short distribution phase (t1/2 alpha = 7.41 +/- 0.53 min), followed by an extended terminal elimination phase (t1/2 beta = 2.49 +/- 0.23 h). The volume of distribution at steady-state was large (2.13 +/- 0.17 l/kg), suggesting extensive tissue distribution, the clearance value was 0.60 +/- 0.03 l/h.

In Spain, the frequency of resistance of group A streptococci (GAS) to erythromycin was very low, increasing from 0 to 0.7% in 1989 and 1990 (1, 7) to approximately 3% in 1991 to 1994 (2). The purpose of the present study was to determine the susceptibilities of recent isolates of GAS to macrolides and, in the case of resistant isolates, to ascertain their patterns of erythromycin resistance. We also studied the in vitro activities of penicillin G and clindamycin. A total of 222 isolates of GAS were collected from April to August 1996 from outpatient samples in Madrid, Spain. A total of 203 isolates were throat swab samples and 19 were from other sources. Identification was done by biochemical tests according to standard criteria (8). Penicillin G, erythromycin, azithromycin, miocamycin, and clindamycin were tested. The MICs were determined by the agar dilution method with Mueller-Hinton agar supplemented with 5% defibrinated horse blood. The plates were incubated overnight at 35°C in a 5% carbon dioxide atmosphere. The procedures for susceptibility testing were those recommended by the National Committee for Clinical Laboratory Standards (6). The MIC breakpoint for miocamycin resistance was >4 μg/ml, as defined by the Comite de l’Antibiogramme de la Societe Francaise de Microbiologie (10). Erythromycin resistance was classified according to the results of disk diffusion, with a double-disk test with erythromycin and clindamycin disks, as previously described (9), to test for inducible resistance. The MIC ranges and the MICs at which 50 and 90% of isolates were inhibited (MIC50s and MIC90s, respectively) for the 222 isolates of Streptococcus pyogenes are given in Table ​Table1.1. Penicillin remained exquisitely active, with an MIC90 of 0.03 μg/ml. TABLE 1 In vitro susceptibilities of 222 recent GAS strains to five antimicrobial agents Thirty-nine (17.6%) of the isolates tested were resistant to erythromycin (MIC breakpoint, ≥1 μg/ml). In contrast, only 0.5% of the S. pyogenes isolates were resistant to clindamycin (MIC90, ≤0.12 μg/ml). The resistance to both 14- and 15-membered macrolides tested was 17.6%, whereas the resistance to miocamycin, a 16-membered macrolide, was 0.5%. Thirty-seven (95%) of 39 erythromycin-resistant GAS were susceptible to clindamycin (MICs, ≤0.12 μg/ml) and miocamycin (MICs, 0.5 to 1 μg/ml). One isolate was resistant to erythromycin (MIC, >64 μg/ml), azithromycin (MIC, >16 μg/ml), miocamycin (MIC, >64 μg/ml), and clindamycin (MIC, >64 μg/ml), which indicates cross-resistance to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSB phenotype). Another erythromycin-resistant strain was susceptible to clindamycin but showed an inducible type of erythromycin resistance. All erythromycin-resistant isolates were also resistant to the 15-membered macrolide azithromycin. However, the 16-membered macrolide miocamycin showed good activity against these isolates. The MICs for 37 of 39 erythromycin-resistant isolates were moderate (8 to 32 μg/ml), but the MICs of clindamycin were the same as those for the erythromycin-susceptible isolates (≤0.12 μg/ml); this pattern has been described with the M phenotype (11) and “novel resistance” (9). With this phenotype, susceptibility to clindamycin and streptogramin B antibiotics but resistance to 14- and 15-membered macrolides is seen (11). The mechanism of erythromycin resistance involves active efflux. The rate of isolation of strains with this novel phenotype among the erythromycin-resistant GAS of our work is even higher than that recently reported in the United States (75%) (11), although the incidence of erythromycin-resistant GAS is rare in this country (3). However, 50% of erythromycin-resistant GAS isolated in Japan (5) and in Taiwan (4) were also resistant to clindamycin and tetracycline. In conclusion, our report documents the emergence of high rates of resistance to erythromycin in S. pyogenes in Spain. Strains with the M phenotype account for the majority of those isolates, which in a sample of this size represents a significant finding. Clindamycin remained active against these strains and might be useful for treatment of patients with clinical failure after phenoxymethyl penicillin treatment and for penicillin-allergic patients. We suggest that clinical laboratories review their local susceptibility data. If erythromycin resistance rates are high, we recommend that susceptibilities to both macrolides and clindamycin in S. pyogenes isolates be routinely tested.

A survey of antimicrobial resistance in Streptococcus pyogenes, performed within the framework of a national surveillance program, has revealed a dramatic increase in resistance of S. pyogenes to erythromycin in most areas of Italy. In virtually all the centers that provided data for 3 consecutive years, the incidence of erythromycin-resistant strains increased twofold to 20-fold from 1993 to 1995 and was greater than 30% in five of the 14 centers participating in the study. The clonality of erythromycin-resistant isolates was studied in 15 strains isolated from different patients at the Institute of Microbiology of Verona University (Verona). The features of the Verona isolates and the substantially different rates of erythromycin and clindamycin resistance observed in most centers suggest that the spread of different resistance genes in multiple clones might be occurring throughout the country.

The activity of rokitamycin, a new macrolide with a 16-member ring, was tested against Borrelia burgdorferi in vitro. The antibiotic had a lower MIC at which 50% of the isolates are inhibited than erythromycin, the parent 14-member macrolide, but the same MIC at which 50% of the isolates are inhibited as the other recent 14- and 15-member macrolides, like clarithromycin and azithromycin. The MBC was equal to the MIC at which 50% of the isolates are inhibited, so rokitamycin can be considered bactericidal against B. burgdorferi. The sensitivity of the Borrelia strains tested was not correlated with the particular species Burgdorferi sensu stricto, B. garinii, and B. afzelii or with the number of subcultures of the isolates.

A survey of five Nocardia spp. with respect to susceptibility towards three macrolides (erythromycin, rokitamycin, and midecamycin) showed that the Nocardia spp. have different susceptibility profiles. Most of the resistance was due to the inactivation of the macrolides by phosphorylation, glycosylation, reduction, deacylation, or a combination thereof.

The inhibitory effects of azithromycin (AZM), a new 15-membered macrolide antibiotic, on the production of exotoxin A, total protease, elastase, and phospholipase C by Pseudomonas aeruginosa were determined, and the virulence-suppressing effects of AZM were compared with those of erythromycin (EM), roxithromycin (RXM), and rokitamycin (RKM). The effect of exposure of P. aeruginosa PA103 or B16 in cultures to sub-MICs of these macrolide antibiotics on the production of exoenzymes was determined. AZM suppressed the in vitro production of extracellular and intracellular exotoxin A by P. aeruginosa PA103 more than did EM, even at a concentration of only 2 micrograms/ml. At concentrations of between 4 and 32 micrograms/ml, AZM also inhibited total protease, elastase, and phospholipase C production by P. aeruginosa B16 more than did EM, RXM, and RKM. AZM was effective in suppressing exotoxin A and total protease production through 24 h of incubation in the presence of drug at sub-MICs, but it had no significant effect on either the growth of P. aeruginosa or its total protein production. Moreover, at a concentration of 4 micrograms/ml, AZM suppressed exoenzyme production by other strains of P. aeruginosa more than did EM. These findings indicate that AZM, EM, RXM, and RKM each has an inhibitory effect on exoenzyme production separate from the antimicrobial effect and that, of these macrolides, AZM has the strongest virulence-suppressing effect.

The aim of this study was to compare the clinical and bacteriologic effectiveness of miocamycin (Miocamin, Merck) as compared to benzathine penicillin G in the treatment of streptococcal pharyngitis. One hundred forty nine patients (aged 2 to 15 years) with culture proven Group A streptococcal pharyngitis were randomly assigned to receive miocamycin (15 mg/kg/day bid per os) or one injection of 600,000 or 1,200,000 units of benzathine penicillin G. The clinical response was similar in both groups, in terms of fever duration (16 +/- 14 hours with miocamycin vs 13 +/- 13 hours with penicillin) and normalization of appetite (87.7% of children with miocamycin vs 95.8% of children with penicillin after three days). Bacteriologic eradication of streptococcus was achieved in 66% of children treated with penicillin and 32% of those treated with miocamycin (p0.001). We conclude that a single benzathine penicillin is more effective eradicating streptococcus pyogenes than miocamycin in children with streptococcal pharyngitis.

The effects of rokitamycin, a macrolide antibiotic, on the binding of bilirubin to albumin were examined in vitro using blood plasma from young rats with hyperbilirubinemia, bilirubin-supplemented serum separated from human cord blood, and the human serum from a newborn infant with icterus gravis neonatorum. Rokitamycin at concentrations from 1 to 500 micrograms/ml (the entire range over which experiments could be conducted) had little or no effect on the concentration of unbound bilirubin in any of the incubation mixtures used. This result suggests that rokitamycin has no effect on the binding of bilirubin to albumin.

In a previous paper we reported that rokitamycin (RKM) which killed some types of RKM-susceptible staphylococci bound cohesively to ribosomes obtained from such bacteria whereas other macrolides such as erythromycin and josamycin, which are generally known to be bacteriostatic, bound to these ribosomes only reversibly. From this observation, we speculated that such cohesive binding of RKM to certain ribosomes probably resulted in cell killing (ENDOU, K. et al., FEMS Microbiology Letters, 72: 93-96, 1990). However, this speculation was based only on circumstantial evidence and we did not show directly that reversible binding of RKM to ribosomes from other strains would bring about bacteriostasis only. A clinically isolated strain, Staphylococcus aureus S704, was found to be susceptible to RKM, mycinamicin and tylosin as well as lincosamide and streptogramin type B antibiotics but not to other macrolides (erythromycin, josamycin, rosamicin, etc.). RKM showed bacteriostatic, but not bactericidal activity, on the strain. Determinant(s) responsible for the bacteriostatic phenotype was transferred into strain NCTC8325 using bacteriophage 80L2; the obtained transductant was referred to as strain 8325MMT7. The drug bound reversibly, not cohesively, to the ribosomes from both strains S704 and 8325MMT7, confirming our earlier hypothesis that rokitamycin can cause bacteriostasis or cell death depending upon whether it binds reversibly or cohesively to the ribosomes of a given strain.

We determined the minimum inhibitory concentrations (MICs) of rokitamycin (TMS-19-Q, RKM), a macrolide antimicrobial agent, against strains of various bacterial species isolated from enteritis patients, and compared them with those of josamycin (JM), erythromycin (EM) and ofloxacin (OFLX). MIC90 of RKM against 147 strains of Campylobacter jejuni, and each 25 strains of Shigella spp., Salmonella spp. and diarrheagenic Escherichia coli were 1.56, 200, 800 and 200 micrograms/ml, respectively. There was only one RKM resistant (MIC greater than 100 micrograms/ml) C. jejuni strain, while most of the strains of the other species were resistant to RKM. MIC values of the other drugs were all similar to those of RKM. MIC90 of OFLX against 147 strains of C. jejuni was 0.78 micrograms/ml, lower than other drugs.

YM133, the 4"-O-(4-methoxyphenyl)acetyltylosin, is a new macrolide. The in vitro activity of YM133 was compared with those of erythromycin, josamycin, and rokitamycin by an agar dilution method. YM133 inhibited 90% of the tested isolates of Streptococcus pneumoniae, Legionella spp., and anaerobic bacteria at less than or equal to 1.56 micrograms/ml. The drug inhibited 90% of erythromycin-resistant staphylococci and Streptococcus pyogenes at less than or equal to 50 micrograms/ml. YM133 showed activity against erythromycin-, josamycin-, and rokitamycin-resistant (MIC greater than or equal to 100 micrograms/ml) strains of staphylococci, streptococci, Bacteroides spp., and Clostridium spp. Enterococci were less susceptible to other YM133-like macrolides. Unlike other macrolides, YM133 showed killing activity, and the MBC/MIC ratios of YM133 for several strains were 1:32, whereas those of erythromycin were 4:1,024. In a time-kill curve study, the reduction of viable cells started within 2 h after the addition of YM133.

Evaluation of the in vitro activity of rokitamycin (RKM) against Chlamydia trachomatis in cycloheximide treated HeLa 229 cells and McCoy cells by comparing with five drugs including doxycycline (DOXY), minocycline (MINO), ofloxacin (OFLX), ampicillin (ABPC) and erythromycin (EM) with regard to assaying minimal inhibitory concentrations (MICs), minimal lethal concentrations (MLCs), and by yield reduction assays: 1) direct treatment of Chlamydial organisms with various concentrations of antibiotics before inoculation, 2) pre-treatment of host cell (HeLa 229) with the antibiotics before they are infected and 3) treatment of already infected cultures (48 hrs after infection) with antibiotics. The yield of Chlamydia was determined by both assaying the infectivity of Chlamydia and/or Chlamydiazyme value (from Abott Co Ltd USA). It was found that similar MIC was obtained among the drugs tested (except EM) in both HeLa 229 cell and McCoy cell assay system. The MLC of RKM (0.3 micrograms/ml) was the same as that of OFLX and was significantly lower than that of other drugs tested. When Chlamydial organisms and the host cells were treated with various concentrations (25-0.1 micrograms/ml) of the drug, the infectivity and the growth of Chlamydia was noteworthily decreased with RKM treatment. Infectivity of Chlamydia in an already infected cultures also decreased with RKM treatment within 24 hours when comparing the value of the control. In other drugs treatment, 96 hours or more hours were required for obtaining the same infectivity as RKM.(ABSTRACT TRUNCATED AT 250 WORDS)

The susceptibilities of 100 clinical isolates of Campylobacter jejuni to rokitamycin, a new macrolide, and eight other oral antibiotics were tested by using a broth microdilution method. Rokitamycin demonstrated antibacterial activity that was higher than those of fosfomycin and the tetracyclines and comparable to those of erythromycin and clindamycin, whereas it was slightly less active than the quinolones.

Miokamycin (MOM), a new macrolide antibiotic, was tested for mutagenicity in the Ames Salmonella test and the dominant lethal assay. MOM was not mutagenic against S. typhimurium strains TA1535-1538, TA100 and TA98 both with and without activation by S-9 mix. No increases in the frequencies of induced dominant lethal effects were found in MOM treated mice. MOM did not have mutagenic activities on either system.

9, 3"-Di-O-acetyl midecamycin (Mom) showed in vitro antibacterial activity against clinical isolates of Staphylococcus aureus and was effective against some of the strains resistant to erythromycin (Em) and josamycin (Jm). The distribution pattern of the level of drug-sensitivity of the isolates to Mom was similar to that of Jm but different from that of Em. Mom- or Jm-resistant strains gradually or rapidly developed among S. aureus strains, that were sensitive or resistant to other macrolide antibiotics. There was no significant difference between Mom and Jm as regards the rate of development of resistant strains. These mutants were always resistant to both Mom and Jm. Mom, like Jm, was effective against Em-inducible strains of S. aureus. The in vivo study demonstrated that Mom was more potent than Jm, similar in its potency to Em against systemic staphylococcal infections, and that it was effective against the infection due to an Em-resistant clinical isolate of S. aureus. Mom was more effective than either Jm or Em against a staphylococcal kidney infection in mice.

Synergistic effects of peptide and macrolide antibiotics against Pseudomonas aeruginosa were investigated in vitro and in vivo. Synergistic effects were evaluated by estimating the number of viable bacteria at varying intervals in the logarithmic growth phase. These bacteria were treated concurrently with polymyxin B (PL) at the final concentration of 1.56 U/ml and with 9,3"-di-O-acetylmidecamycin (MOM) at varying concentrations. Synergistic effect was observed when PL was used with MOM at 3.13 and 12.5 microgram/ml respectively. When MOM at 50 microgram/ml was used with PL, the viable bacterial count was reduced to below 1/300 of the control to which PL alone had been added. Thus, the synergistic effect was remarkable. Similar results were obtained when colistin methanesulfonate (CL) was used instead of PL. Subsequently, attempts were made to determine if this action could also be found in in vivo experiments using mice. PL or CL was injected intramuscularly and midecamycin (MDM) or MOM was administered once or repeatedly by the oral route. Simultaneously, Pseudomonas aeruginosa strain IFO 3455 was inoculated intraperitoneally to mice. In the case of treatment once or repeatedly using both PL and MDM or MOM, the survival rate of infected mice increased significantly compared to single treatment by PL alone. Thus, the synergistic effects were demonstrated in four experiments. (The significance levels for the experiments were P = 0.070, 0.015, 0.042 and 0.024). Similar results were obtained when strain No. 5 was used to infect mice (P = 0.0096, 0.0027). When CL and MOM were given to mice once prior to infection with strain No. 5, synergistic effects were obtained as well (P = 0.010, 0.034).

A radioimmunoassay system has been developed for the measurement of two major metabolites of 9,3"-diacetylmidecamycin, Mb-6 and Mb-12. A radioimmunoassay for Mb-6 was performed by using anti-Mb-6 serum and a [125I]tyramined Mb-6 derivative as a radiolabeled antigen. The labeled antigen was prepared by the chloramine T method. The antiserum was obtained from a rabbit immunized with Mb-6 conjugated to bovine serum albumin. The obtained antiserum was cross-reactive with two other metabolites of 9,3"-diacetylmidecamycin, Mb-2 and Mb-12, in addition to Mb-6. This Mb-6 radioimmunoassay system could detect Mb-6 concentrations as low as 100 pg/ml of serum. The coefficients of variation were 4.5% (intra-assay) and 5.1% (inter-assay). A radioimmunoassay for Mb-12, using anti-midecamycin serum and a [125I]tyramined-Mb-12 derivative, has also been developed. The antiserum was cross-reactive only with Mb-12 among the 9,3"-diacetylmidecamycin metabolites. This Mb-12 radioimmunoassay system could detect Mb-12 concentrations as low as 2 ng/ml. The intra- and inter-assay variances were 5.9 and 5.8%, respectively.

Fifty strains of Mycoplasma pneumoniae in L cells were tested for susceptibility to macrolide antibiotics. Rokitamycin, a new macrolide antibiotic, was most active against these organisms, with an MIC for 90% of strains of 0.007 microgram/ml. The MICs of erythromycin, josamycin, and kitasamycin for 90% of strains were 0.03, 0.03, and greater than or equal to 0.06 microgram/ml, respectively. Based on these results, rokitamycin is a promising antibiotic for the treatment of mycoplasmal infections, and further clinical investigations are needed.

The accumulation of macrolide antibiotics in Bacteroides fragilis ATCC 25285 was increased in the order erythromycin, josamycin, and rokitamycin, depending on hydrophobicity. The half-times of efflux were also prolonged in the same order. Furthermore, MICs of the antibiotics were correlated with the extent of hydrophobicity. These findings suggest that the macrolide antibiotics are accumulated in B. fragilis by means of their hydrophobic properties, and the efficient accumulation of the drugs may explain the susceptibility of this gram-negative bacterium to macrolides.

Dry syrup of Rokitamycin (RKM.DS), a new macrolide antibiotic with 16-membered ring developed by Toyo Jozo Co., Ltd., was used on cases of upper deep cervical lymphadenitis (UDCL) and of otitis media with exudatives (OME) likely caused by chlamydia infections, and its clinical effects were investigated. 1) In four cases of UDCL (C. psittaci infections), daily doses of 20.0-28.6 mg/kg were administered for 10-42 consecutive days and good efficacies were observed with two cases of excellent results and two cases of good results. 2) In one case of OME (C. trachomatis infection) a daily dose of 30.0 mg/kg was administered for 15 consecutive days and good effectiveness was observed. 3) No side effect was observed in any of the cases. From the above results, it became clear that RKM.DS could effectively used as a therapeutic agent for UDCL and OME in children.

The in vitro activities of several 14-, 15- and 16-membered macrolides were compared with that of erythromycin. In general, 14-membered macrolides such as erythromycin, clarithromycin, and flurithromycin were more active against streptococci and Bordetella pertussis than was the 15-membered macrolide azithromycin, which was more active than 16-membered macrolides such as miocamycin and rokitamycin. Clarithromycin was the most active compound against Streptococcus pyogenes, pneumococci, Listeria monocytogenes, and Corynebacterium species. Legionella pneumophila was most susceptible to miocamycin, clarithromycin, and rokitamycin. Branhamella catarrhalis, Neisseria gonorrhoeae, and Haemophilus influenzae were most susceptible to azithromycin. Azithromycin and dirithromycin were the most active compounds against Campylobacter jejuni. MICs of 16-membered macrolides for strains expressing inducible-type resistance to erythromycin were less than or equal to 1 microgram/ml, whereas none of the compounds had activity against strains expressing constitutive-type resistance. The MICs of roxithromycin, miocamycin, rokitamycin, and josamycin increased in the presence of human serum, whereas MICs of the other compounds either were unchanged or decreased.

The effects of rokitamycin (RKM), a macrolide antibiotic, on young rats with hyperbilirubinemia were investigated. RKM at a dose of 1, 000 mg/kg was orally administered to 14-day-old rats with hereditary, non-hemolytic hyperbilirubinemia (homozygous Gunn rats, total plasma bilirubin concentration : about 7 mg/dl). Animals given 10 ml/kg of 0.5% carboxymethyl cellulose (CMC) were used as control. Plasma total bilirubin concentration, plasma unbound bilirubin concentration and derebellar bilirubin level did not significantly change during 1, 3, 6 and 24 hours after administration of RKM or CMC. There was no significant difference in plasma total bilirubin concentration, plasma unbound bilirubin concentration and cerebllar bilirubin level between RKM-treated and control animals at 1, 3, 6 and 24 hours after the administration. No localized yellow discoloration of the brain tissue (non-cerebellar parts) was noted at 1, 3, 6 and 24 hours after administration of either RKM or CMC.