In Vitro Susceptibilities of Neisseria gonorrhoeae to Fatty Acids and Monoglycerides

The susceptibility of Neisseria gonorrhoeae to several medium-chain fatty acids and their 1-monoglycerides was tested at a short inactivation time of 1 min. The results indicate that monocaprin, a monoglyceride of capric acid (10 carbon atoms, no double bonds), causes the fastest and most effective killing of all strains of N. gonorrhoeae tested.     

In Vitro Activity of Rosaramicin Against Chlamydia trachomatis

In vitro, rosaramicin was highly active against Chlamydia trachomatis when added to the culture system 1 or 48 h after inoculation with the chlamydia.     

In Vitro Susceptibility of 30 Strains of Chlamydia trachomatis to Rosamicin

A total of 13 of 30 clinical isolates of Chlamydia trachomatis were susceptible in vitro to 0.01 μg of rosamicin per ml. Only two of these strains were susceptible to tetracycline or erythromycin at this level. The results suggest that rosamicin may be useful for the treatment of chlamydial urethritis.     

In Vitro Activities of BMS-284756 against Chlamydia trachomatis and Recent Clinical Isolates of Chlamydia pneumoniae

The in vitro activities of BMS-284756 (a novel des-fluoroquinolone), levofloxacin, moxifloxacin, and clarithromycin were tested against 5 strains of Chlamydia trachomatis and 20 isolates of Chlamydia pneumoniae. The MIC at which 90% of the isolates were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by BMS-284756 for all isolates of C. pneumoniae and C. trachomatis was 0.015 microg/ml (range, 0.015 to 0.03 microg/ml). BMS-284756 was the most active quinolone tested.     

In Vitro Activity of Nemonoxacin,a Novel Nonfluorinated Quinolone Antibiotic,against Chlamydia trachomatis and Chlamydia pneumoniae

The in vitro activities of nemonoxacin, levofloxacin, azithromycin, and doxycycline were tested against 10 isolates each of Chlamydia trachomatis and Chlamydia pneumoniae. The MICs at which 90% of the isolates of both C. trachomatis and C. pneumoniae were inhibited (MIC₉₀s) were 0.06 μg/ml (range, 0.03 to 0.13 μg/ml). The minimal bactericidal concentrations at which 90% of the isolates were killed by nemonoxacin (MBC₉₀s) were 0.06 μg/ml for C. trachomatis (range, 0.03 to 0.125 μg/ml) and 0.25 for C. pneumoniae (range, 0.015 to 0.5 μg/ml).     

In Vitro Activity of AZD0914, a Novel DNA Gyrase Inhibitor,against Chlamydia trachomatis and Chlamydia pneumoniae

The in vitro activities of AZD0914, levofloxacin, azithromycin, and doxycycline against 10 isolates each of Chlamydia trachomatis and Chlamydia pneumoniae were tested. For AZD0914, the MIC₉₀s for C. trachomatis and C. pneumoniae were 0.25 μg/ml (range, 0.06 to 0.5 μg/ml) and 1 μg/ml (range, 0.25 to 1 μg/ml), respectively, and the minimal bactericidal concentrations at which 90% of the isolates were killed (MBC₉₀s) were 0.5 μg/ml for C. trachomatis (range, 0.125 to 1 μg/ml) and 2 μg/ml for C. pneumoniae (range, 0.5 to 2 μg/ml).     

In Vitro Activities of Thirteen β-Lactam Antibiotics Against Chlamydia trachomatis

The in vitro activity (minimal inhibitory concentration and minimal lethal concentration) of 13 β-lactam antibiotics against two laboratory strains of Chlamydia trachomatis was compared. No useful activity could be detected.     

In Vitro Activity of CEM-101, a New Fluoroketolide Antibiotic,against Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae

The in vitro activities of CEM-101, telithromycin, azithromycin, clarithromycin, and doxycycline against 10 isolates each of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae were tested. The MIC at which 90% of the isolates of both C. trachomatis and C. pneumoniae were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by CEM-101 were 0.25 μg/ml (ranges, 0.125 to 0.5 μg/ml for C. trachomatis and 0.25 to 1.0 μg/ml for C. pneumoniae).The ketolides are a subclass of macrolides, which were designed specifically to overcome macrolide-resistant respiratory pathogens. Ketolides lack the cladinose sugar, which is replaced with a 3-ketone group. Ketolides bind to a secondary region on domain II of the 23S rRNA subunit (4). The ketolides are acid stable and have activity against a broad range of respiratory pathogens, including multiresistant pneumococci, Haemophilus influenzae, Legionella species, Mycoplasma pneumoniae, and Chlamydia (Chlamydophila) pneumoniae (4, 6, 8, 12). Currently only one ketolide, telithromycin, has approval from the Food and Drug Administration (FDA). However, after reports of rare but serious cases of hepatotoxicity and reports of visual disturbances and loss of consciousness that were associated with telithromycin, the FDA restricted its use in February 2007 (3, 10). Cethromycin, a ketolide in clinical development, has been studied for the treatment of respiratory infections, and a new drug application (NDA) for community-acquired bacterial pneumonia (CABP) is under review by FDA (4). We compared the in vitro activity of CEM-101, a new fluoroketolide antibiotic, to those of telithromycin, azithromycin, clarithromycin, and doxycycline against 10 isolates each of Chlamydia trachomatis and C. pneumoniae.Isolates of C. trachomatis included standard isolates from the ATCC (E-BOUR, F-IC-CAL3, C-HAR32, J-UW-36, L2434, D-UW-57kx, and B-HAR-36) and clinical isolates N18 (cervical), N19 (cervical), and 7015 (infant eye). Isolates of C. pneumoniae tested included a reference strain (TW 183), 9 isolates from children and adults with pneumonia from the United States (AR39, T2023, T2043, W6805, CWL 029, and CM-1), an isolate from a child with pneumonia from Japan (J-21), and 2 isolates from bronchoalveolar lavage specimens from patients with human immunodeficiency virus infection and pneumonia from the United States (BAL15 and BAL16).CEM-101, telithromycin, azithromycin, clarithromycin, and doxycycline were provided as powders, solubilized according to the manufacturers'' instructions, and frozen in 1-ml aliquots of 2,048 μg/ml. Drug suspensions were made fresh each time the assay was run. Susceptibility testing of C. pneumoniae was performed with cycloheximide-treated HEp-2 cells grown in 96-well microtiter plates (7, 8). Each well was inoculated with 0.1 ml of the test strain diluted to yield 10³ to 10⁴ inclusion-forming units per ml; the plates were centrifuged at 1,700 × g for 1 h and incubated at 35°C for 1 h. Wells were then aspirated and overlaid with medium containing 1 μg/ml of cycloheximide and serial 2-fold dilutions of the test drug. After incubation at 35°C for 72 h, cultures were fixed and stained for inclusions with fluorescein-conjugated antibody to the chlamydial lipopolysaccharide genus-specific antigen (Pathfinder; Bio-Rad, Redmond, WA). The MIC was the lowest antibiotic concentration at which no inclusions were seen. The minimal bactericidal concentration (MBC) was determined by aspirating the antibiotic-containing medium, washing wells twice with phosphate-buffered saline, and adding antibiotic-free medium. The infected cells were frozen at −70°C, thawed, passed onto new cells, incubated for 72 h, and then fixed and stained as described above. The MBC was the lowest antibiotic concentration that resulted in no inclusions after passage. All tests were run in duplicate.The results for C. trachomatis are shown in Table ​Table1.1. The in vitro activity of CEM-101 against C. trachomatis was similar to that of azithromycin but less than those of telithromycin, clarithromycin, and doxycycline. The MIC₉₀ and MBC which was bactericidal against 90% of the isolates (MBC₉₀) of CEM-101 were 0.25 μg/ml. The MIC₉₀s for telithromycin, azithromycin, clarithromycin, and doxycycline were 0.06, 0.125, 0.125, and 0.06 μg/ml, respectively.

TABLE 1.

Activities of CEM-101 and other antibiotics against 10 isolates of C. trachomatis

Sequencing of Gyrase and Topoisomerase IV Quinolone-Resistance-Determining Regions of Chlamydia trachomatis and Characterization of Quinolone-Resistant Mutants Obtained In Vitro

The L2 reference strain of Chlamydia trachomatis was exposed to subinhibitory concentrations of ofloxacin (0.5 μg/ml) and sparfloxacin (0.015 μg/ml) to select fluoroquinolone-resistant mutants. In this study, two resistant strains were isolated after four rounds of selection. The C. trachomatis mutants presented with high-level resistance to various fluoroquinolones, particularly to sparfloxacin, for which a 1,000-fold increase in the MICs for the mutant strains compared to the MIC for the susceptible strain was found. The MICs of unrelated antibiotics (doxycycline and erythromycin) for the mutant strains were identical to those for the reference strain. The gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes of the susceptible and resistant strains of C. trachomatis were partially sequenced. A point mutation was found in the gyrA quinolone-resistance-determining region (QRDR) of both resistant strains, leading to a Ser83→Ile substitution (Escherichia coli numbering) in the corresponding protein. The gyrB, parC, and parE QRDRs of the resistant strains were identical to those of the reference strain. These results suggest that in C. trachomatis, DNA gyrase is the primary target of ofloxacin and sparfloxacin.     

In vitro activity of dirithromycin against Chlamydia trachomatis.

Dirithromycin is a new macrolide antibiotic with an active metabolite, erythromycylamine. We evaluated the in vitro activities of both drugs against 16 isolates of Chlamydia trachomatis and compared them with that of doxycycline. In vitro susceptibility testing was performed with McCoy cell monolayers. The MIC was defined as the lowest concentration of antibiotic without inclusions. The MBC was defined as the lowest concentration of antibiotic yielding no inclusions after passage onto 24-h-old antibiotic-free McCoy cell monolayers. Dirithromycin and erythromycylamine appeared to be equally effective against these 16 strains of C. trachomatis (MIC for 90% of strains tested, 1 mg/ml; MBC for 90% of strains tested, 2 micrograms/ml). Both were less active than doxycycline (MIC for 90% of strains tested, 0.06 micrograms/ml; MBC for 90% of strains tested, 0.12 micrograms/ml). The combination of dirithromycin and erythromycylamine appeared to be additive.     

In vitro antichlamydial activity of garenoxacin against Chlamydia trachomatis

Garenoxacin showed the most potent chlamydial activity against Chlamydia trachomatis D/UW-3/Cx among three tested quinolones and azithromycin. The DNA gyrase genes, gyrA and gyrB, of C. trachomatis D/UW-3/Cx were cloned and the GyrA and GyrB subunits of DNA gyrase protein were separately expressed as histidine-tagged proteins in Escherichia coli. The mean 50% inhibitory concentration (IC(50)) of garenoxacin against the supercoiling activity of C. trachomatis D/UW-3/Cx gyrase was 2.9 ± 0.4 μg/ml, which was the most potent inhibitory activity against DNA gyrase among the quinolones tested in this study. At an extracellular concentration of 0.5 μg/ml, the cellular-to-extracellular concentration ratio of garenoxacin was 15.3 ± 1.3, equivalent to that of moxifloxacin and greater than that of levofloxacin. In a time-kill experiment, after exposure to garenoxacin at a concentration of 0.5 μg/ml at 0-6, 5-11, and 24-30 h after infection, the percentages of recoverable chlamydial inclusion-forming units were 11.1 ± 3.3, 0.6 ± 0.1, and 2.6 ± 0.5%, respectively. On transmission electron microscopy observation, after exposure to garenoxacin at 24-30 h after infection, some C. trachomatis elementary bodies remained in the inclusion body; however, the reticulate bodies were completely disrupted. In conclusion, garenoxacin is expected to be a useful quinolone in the treatment of infectious diseases caused by C. trachomatis.     

In Vitro Activities of Several Antimicrobial Agents against Recently Isolated and Genotyped Chlamydia trachomatis Urogenital Serovars D through K

A systematic evaluation of the susceptibility of all Chlamydia trachomatis urogenital serovars (D through K) to levofloxacin, erythromycin, doxycycline, clarithromycin, and azithromycin was performed. All C. trachomatis serovars had comparable susceptibilities with respect to the various antimicrobials tested, thus confirming the homogeneous data so far obtained regarding the susceptibility of C. trachomatis to antimicrobial agents.Infections caused by Chlamydia trachomatis serovars D through K are among the most prevalent causes of urogenital diseases worldwide (4). Although chlamydial infections are often asymptomatic, they can cause long-term sequelae, including pelvic inflammatory disease, chronic pelvic pain, and tubal factor infertility (5).In the past, doxycycline has been regarded as the first-choice antibiotic therapy for C. trachomatis genital infections (10) but is subject to problems of compliance (1). Subsequently, a variety of broad-spectrum antibiotics, including fluoroquinolones and macrolides, have been recommended for the treatment of pelvic inflammatory disease associated with C. trachomatis (3, 7).The 2006 Centers for Disease Control and Prevention (CDC) sexually transmitted disease (STD) treatment guidelines (2) indicated doxycycline and azithromycin as recommended agents against chlamydial urogenital infection and erythromycin, levofloxacin, and ofloxacin as alternative drugs.So far, few studies have evaluated the susceptibilities of the various C. trachomatis serovars to antimicrobial agents (8). This study was undertaken to evaluate the in vitro activity of several antimicrobial agents known to be active against C. trachomatis in order to test the sensitivity of all C. trachomatis serovars (D through K) associated with nondisseminating sexually transmitted disease. The antimicrobial drugs used were levofloxacin, erythromycin, doxycycline, clarithromycin, and azithromycin.A total of 50 C. trachomatis strains were tested. Of these, 5 were reference strains (Ic Cal-8 serovar D, Bour serovar E, 392-F serovar G, 580 serovar H, and UW-36 serovar G) and 45 were strains isolated from urethral swabs obtained from male patients with nongonococcal urethritis or from women with cervicitis, isolated in our laboratory at S. Orsola University Hospital, Bologna, Italy, over the years 2005 and 2006. About 65% of the patients were from Italy, 24% from East Europe, 4% from Africa, 3% from America, 2% from West Europe, and 2% from Asia.C. trachomatis serovars were identified by using amplification and sequencing of the major outer membrane protein gene (ompA). DNA of all 45 C. trachomatis isolates and 5 reference strains, grown in LLC-MK2 cells, was extracted for molecular analysis employing a commercially available kit (tissue kit; Qiagen, Düsseldorf, Germany) and used as a template for amplification of a 1,050-bp chlamydial ompA gene fragment. The reaction was performed according to the method of Lan et al. (6). The amplicons were purified using a Wizard purification kit (Promega, WI), and each PCR product was sequenced twice in each direction (Bio-Fab Research, Rome, Italy). The nucleotide sequences were compared with the same regions of the C. trachomatis reference serovars in the GenBank database using the BLAST at the National Center for the Biotechnology Information (www.ncbi.nlm.nih.gov/).From the analyses of the sequencing data, 8 C. trachomatis serovars, deduced from genotypes, were found in the following order of prevalence among the 45 C. trachomatis strains isolated: 9 serovar D, 8 serovar G, 7 serovar E, 7 serovar F, 4 serovar I, 4 serovar J, 4 serovar K, and 2 serovar H strains. The molecular typing of the five reference strains (two C. trachomatis type D, one type E, one type J, and one type H) confirmed the previous serotyping.The antimicrobial drugs levofloxacin (GlaxoSmithKline, Verona, Italy), doxycycline, erythromycin, azithromycin (Sigma, Milan, Italy), and clarithromycin (Abbott, Latina, Italy) were provided as powders and solubilized according to the instructions of manufacturers. Antimicrobial susceptibility testing was performed with LLC-MK2 cells grown in 24-well plates with Eagle''s minimum essential medium. Each of the 24-well plates was inoculated with 5 × 10³ inclusion-forming units (IFU) per milliliter. After centrifugation at 1,700 × g for 1 h, the medium was removed and replaced with medium containing different concentrations of antimicrobial drugs (3). After incubation at 35°C for 48 h, infected monolayers were washed with phosphate-buffered saline (PBS), fixed with methanol, and stained for inclusion with a fluorescein-conjugated monoclonal antibody specific for the chlamydial lipopolysaccharide genus-specific antigen (Meridian Diagnostics, Inc., Cincinnati, OH). The MIC was defined as the lowest concentration that reduced the number of inclusions more than 90%, compared with the level for drug-free controls. The minimal bactericidal concentration (MBC) was measured by aspirating the antibiotic-containing medium, washing the monolayer twice with PBS, and incubating it in growth antibiotic-free medium for 48 h at 35°C (3). Cells were fixed and chlamydial inclusions were stained as described above (3). The MBC was the lowest concentration of the drug reducing more than 90% demonstrable inclusions after monolayers were reincubated in antimicrobial-free medium. All tests were run in triplicate.The MICs and MBCs of the antimicrobial agents are reported in Table ​Table1.1. The MICs of levofloxacin against C. trachomatis serovars ranged between 0.25 and 0.5 μg/ml, whereas the MBCs ranged between 0.25 μg/ml and 1.0 μg/ml. The MICs of erythromycin ranged between 0.5 μg/ml and 1.0 μg/ml, whereas the MBCs ranged between 1.0 μg/ml and 2.0 μg/ml. Doxycycline showed MIC values ranging between 0.03 μg/ml and 0.06 μg/ml, whereas the MBCs ranged between 0.06 μg/ml and 0.125 μg/ml. The MICs of clarithromycin ranged between 0.015 μg/ml and 0.06 μg/ml, whereas the MBCs ranged between 0.03 μg/ml and 0.125 μg/ml. Finally, azithromycin showed MIC values ranging between 0.25 μg/ml and 0.5 μg/ml, whereas the MBCs ranged between 0.5 μg/ml and 1.0 μg/ml. All agents except levofloxacin were bactericidal by MBC at 1 to 2 times the MIC. No differences have been detected in tests run in triplicate.

TABLE 1.

In vitro MICs and MBCs of five antimicrobial drugs against 50 C. trachomatis strains

In vitro synergy of clindamycin and aminoglycosides against Chlamydia trachomatis.

The importance of Chlamydia trachomatis as an etiologic agent in the development of pelvic inflammatory disease (PID) is well documented. Although there are numerous antimicrobial agents that are effective against C. trachomatis, one of the most frequent combinations that is used to treat PID is clindamycin and gentamicin. The efficacy of clindamycin as the sole treatment for chlamydial infections has been questioned. In fact, the Centers for Disease Control (Atlanta, Ga.) has recommended the use of doxycycline following clindamycin and gentamicin treatment of PID confirmed or suspected to be caused by C. trachomatis. This study was designed to determine whether there is any synergistic in vitro activity between clindamycin and gentamicin or tobramycin on inhibition of C. trachomatis replication. In this experiment, the MIC of clindamycin decreased two- to threefold when an aminoglycoside was added. This occurred even though aminoglycosides by themselves had essentially no effect against C. trachomatis. The mechanism of this interaction is uncertain.     

Inactivation of Herpesvirus hominis Types 1 and 2 by Silver Nitrate In Vitro and In Vivo

Herpes simplex virus (HSV) types 1 and 2 (two strains each) were inactivated at different rates in vitro by 40 muM AgNO(3). The inactivation of HSV type 1 strains was virtually complete in 10 to 15 min, whereas almost half of the infectivity of HSV type 2 strains survived this exposure. One strain of type 1 inoculated into rabbit eyes was almost completely inactivated by 1% AgNO(3) solution dropped into the eye 20 min later, so that there was markedly reduced viral replication and less corneal herpetic disease. One strain of HSV type 2 in the rabbit eye was not effectively inactivated by 1% AgNO(3). From these results, it seems likely that AgNO(3) instillation into the eyes of a newborn who has passed through a birth canal infected with HSV might prevent eye infection with HSV type 1 but not with type 2. The greater resistance of HSV type 2 strains to chemical inactivation in vitro and in vivo may be of medical concern.     

In vitro activity of azithromycin (CP-62,993) against Chlamydia trachomatis and Chlamydia pneumoniae.

The in vitro susceptibilities of 49 strains of Chlamydia trachomatis and 3 strains of Chlamydia pneumoniae to azithromycin and tetracycline or doxycycline were determined. The MIC of azithromycin ranged from < or = 0.06 to 1.0 micrograms/ml, the MIC of tetracycline ranged from 0.03 to 0.12 micrograms/ml, and the MIC of doxycycline ranged from 0.015 to 0.06 micrograms/ml against C. trachomatis. The MIC ranges for C. pneumoniae were 0.12 to 0.25 micrograms/ml for azithromycin and 0.06 to 0.12 micrograms/ml for tetracycline. All minimal chlamydicidal concentrations were either equal to the MIC or one or two dilutions higher. No strains resistant to these antibiotics were detected. In vitro activity shows that azithromycin is highly active against C. trachomatis and C. pneumoniae.     

In vitro inhibitory effects of hinokitiol on proliferation of Chlamydia trachomatis

The inhibitory effects of hinokitiol (beta-thujaplicin) on Chlamydia trachomatis D/UW-3/Cx were shown by MIC, minimum lethal concentration (MLC), and preinoculation minimal microbicidal concentration assays using HeLa 229 cells. The MIC and the MLC were both 32 microg/ml. Further evaluation of hinokitiol as a topical agent against C. trachomatis is warranted.     

In vitro activity of beta-lactam drugs and sulbactam against Chlamydia trachomatis.

We tested the in vitro activity of ampicillin, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, and sulbactam against 18 recent clinical isolates of Chlamydia trachomatis and two ATCC strains. Ampicillin (MIC50, 256 micrograms/ml) and sulbactam (MIC50, 128 micrograms/ml) demonstrated some activity against C. trachomatis, but cefoperazone had little to no activity. At 2-3 dilutions below the MIC, C. trachomatis treated with ampicillin or sulbactam, but not cefoperazone, formed small inclusions that remained small on passage onto antibiotic-free McCoy cells. It appears that ampicillin and sulbactam suppress rather than kill C. trachomatis.     

四种国产大环内酯类抗菌药物体外抗沙眼衣原体和肺炎衣原体敏感性研究

目的 研究国产必特螺旋霉素、乙酰螺旋霉素、红霉素和阿奇霉素等4种大环内酯类药物对于沙眼衣原体和肺炎衣原体体外药物敏感性试验,评估其抗衣原体作用,以指导临床用药.方法 细胞培养和免疫荧光包涵体染色技术测定4种国产大环内酯类抗菌药物对于沙眼衣原体和肺炎衣原体体外MIC.结果 对于沙眼血清型B,必特螺旋霉素、红霉素和阿奇霉素体外MIC为0.5μg/ml,乙酰螺旋霉素为4μg/ml.对于沙眼血清型D,必特螺旋霉素与阿奇霉素体外MIC均为0.25μg/ml,红霉素0.5μg/ml,乙酰螺旋霉素2μg/ml.对于肺炎衣原体,红霉素体外MIC≤0.016μg/ml,阿奇霉素和必特螺旋霉素均为0.032μg/ml,乙酰螺旋霉素0.5μg/ml.结论 国产必特螺旋霉素、红霉素和阿奇霉素体外抗沙眼衣原体(血清型B和D)和肺炎衣原体作用可靠,但乙酰螺旋霉素则较差.     

婴幼儿沙眼衣原体肺炎的临床特征及基因型分布

目的 了解湖南郴州地区3个月以内的婴幼儿沙眼衣原体(Chlamydozoa trachomatis,CT)肺炎临床特征、基因型分布及致病力差异,为临床的诊治提供理论依据.方法 采集719例肺炎患儿支气管肺泡灌洗(bronchoalveolar lavage fluid,BALF)标本,用实时荧光定量PCR法检测CT,再...