In Vitro Activity of Thimerosal against Ocular Pathogenic Fungi

The in vitro activity of thimerosal versus those of amphotericin B and natamycin was assessed against 244 ocular fungal isolates. The activity of thimerosal against Fusarium spp., Aspergillus spp., and Alternaria alternata was 256 times, 512 times, and 128 times, respectively, greater than that of natamycin and 64 times, 32 times, and 32 times, respectively, greater than that of amphotericin B. Thimerosal''s antifungal activity was significantly superior to those of amphotericin B and natamycin against ocular pathogenic fungi in vitro.The problem of keratomycosis in developing countries like China is more acute because of its higher incidence and the unavailability of effective antifungals (18, 28, 30). To date, only fluconazole and natamycin are commercially available for ocular use in China. Fluconazole has high bioavailability against Candida spp., but Fusarium spp. and Aspergillus spp. are resistant to it (3, 27, 29). Fusarium spp. and Aspergillus spp. are more commonly associated with keratomycosis, while Candida spp. are rarely implicated as etiological agents of keratomycosis in China (23, 26). Natamycin is the only topical ophthalmic antifungal compound approved in the United States (14). Natamycin is poorly soluble in water. After topical application, natamycin penetrates the cornea and conjunctiva poorly and effective drug levels are not achieved in either the cornea or the aqueous humor (15); it is therefore useful only in the treatment of superficial keratomycosis. Due to the relative unavailability of effective antifungals, keratomycosis fails to resolve in many of the patients who receive antifungal treatment; some patients experience vision loss and eventually corneal perforation, ultimately require penetrating keratoplasty, or even enucleation or evisceration (20, 28). Therefore, it is very important and urgent to explore broad-spectrum antifungals to effectively suppress a wide variety of ocular fungal pathogens and to develop new antifungal eye drops to combat this vision-threatening infection.Thimerosal is a preservative commonly used in ophthalmic solutions, otic drops, topical medicine, and vaccines because of its bactericidal property. However, the efficacy of thimerosal against ocular pathogenic fungi has not been evaluated so far. The present study was performed to determine the antifungal activity of thimerosal versus those of amphotericin B and natamycin against ocular pathogenic fungi in vitro. To our knowledge, this is the first study to determine the antifungal activity of thimerosal against main ocular pathogenic fungi. Results obtained in this study may contribute to the development of new antifungal eye drops.Two hundred forty-four strains of fungi isolated from patients with keratomycosis from the Henan Eye Institute in Zhengzhou, China, were investigated. These isolates were identified based on morphology by standard methods (22, 23, 25). They included 136 Fusarium isolates, 98 Aspergillus isolates, and 10 Alternaria alternata isolates. Candida parapsilosis ATCC 22019 was used as quality control for each test.Thimerosal (Yili Pharmaceutical Co. Ltd., Beijing, China), amphotericin B (Bristol-Myers Squibb, Princeton, NJ), and natamycin (Yinxiang Biotechnology Co. Ltd., Zhejing, China) were studied. They were all dissolved in 100% dimethyl sulfoxide. The stock solutions were prepared at concentrations of 400 μg/ml for thimerosal and 1,600 μg/ml for amphotericin B and natamycin. Drug dilutions were made in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid. Final concentrations ranged from 0.0078 to 4 μg/ml for thimerosal and from 0.0313 to 16 μg/ml for amphotericin B and natamycin.A broth microdilution method was performed by following the Clinical and Laboratory Standards Institute M38-A2 document (13). The final inoculum was 0.4 × 10⁴ to 5 × 10⁴ CFU/ml. Following incubation at 35°C for 48 h, the MIC was determined as the lowest concentration of amphotericin B, natamycin, or thimerosal that prevented any discernible growth.The MIC range and mode, the MIC for 50% of the strains tested (MIC₅₀), and the MIC₉₀ were provided for the isolates with the SPSS statistical package (version 13.0). For calculation, any high off-scale MIC was converted to the next higher concentration.The in vitro activities of thimerosal, amphotericin B, and natamycin against the isolates are summarized in Tables ​Tables11 and ​and2.2. When comparing the MIC₉₀s of thimerosal with those of natamycin and amphotericin B, the activity of thimerosal against Fusarium spp. is 256 times greater than that of natamycin and 64 times greater than that of amphotericin B, the activity of thimerosal against Aspergillus spp. is 512 times greater than that of natamycin and 32 times greater than that of amphotericin B, and the activity of thimerosal against Alternaria alternata is 128 times greater than that of natamycin and 32 times greater than that of amphotericin B. Therefore, thimerosal''s effect was significantly superior to those of amphotericin B and natamycin against main ocular pathogenic fungi in vitro.

TABLE 1.

In vitro susceptibilities of ocular Fusarium isolates to thimerosal, amphotericin B, and natamycin

In Vitro Activity of Netilmicin, Gentamicin, and Amikacin

The in vitro activity of netilmicin (Sch 20569), a new semisynthetic derivative of gentamicin, was compared with that of gentamicin and amikacin. One hundred and ninety-two clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus were tested using both agar and broth dilution techniques. Netilmicin was comparable to gentamicin, with the following exceptions: (i) for Serratia marcescens and P. aeruginosa, gentamicin was more active than netilmicin; (ii) all strains of Escherichia coli, Klebsiella, Enterobacter, Proteus mirabilis, and Citrobacter freundii, which were resistant to gentamicin, were susceptible to netilmicin; (iii) some strains of S. marcescens, indole-positive Proteus, and Providencia, which were resistant to gentamicin, were susceptible to netilmicin. Netilmicin was more active than amikacin for all Enterobacteriaceae and S. aureus and equal to amikacin in activity against gentamicin-susceptible strains of P. aeruginosa. All strains of P. aeruginosa, resistant to gentamicin, were also resistant to netilmicin but were susceptible to amikacin. Minimal inhibitory concentrations (MICs) obtained with broth and agar showed no significant differences except for P. mirabilis, where broth MICs were twofold greater than agar MICs, and for P. aeruginosa, where agar MICs were twofold higher than broth MICs. The minimal bactericidal concentration (MBC) was either identical to or within one twofold dilution of the MIC for the strains tested. A 100-fold increase in inoculum size produced less increase in MIC and MBC with netilmicin than with gentamicin or amikacin.     

In Vitro Activity of Thienamycin

The in vitro activity of thienamycin was tested against 135 aerobic and anaerobic bacteria. The compound was highly active against resistant gram-negative bacilli and penicillin-resistant Straphylococcus aureus. The antianaerobic spectrum of the drug seemed to be comparable to that of metronidazole.     

In Vitro Activity of Netilmicin

The activity of netilmicin against a variety of bacteria was similar to that of gentamicin, sisomicin, and tobramycin, but it was less active than these three drugs against Pseudomonas aeruginosa. Synergy with penicillin G against enterococci was demonstrated.     

In Vitro Activity of Ceftazidime-Avibactam Combination in In Vitro Checkerboard Assays

To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae, 19 Escherichia coli, 1 Citrobacter freundii, and 3 Enterobacter cloacae) and 26 strains of Pseudomonas aeruginosa, all with known resistance mechanisms such as extended-spectrum β-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains (n = 69) were analyzed in more detail. For the Enterobacteriaceae strains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC₅₀ and MIC₉₀ being 8 and 16 mg/liter, respectively). For the ceftazidime-resistant P. aeruginosa strains, considerable inhibition of β-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on these in vitro data, avibactam is a promising inhibitor of different β-lactamases, including ESBLs and carbapenemases.     

Antibacterial Activity of Nitropyrroles, Nitrothiophenes, and Aminothiophenes In Vitro

The antibacterial activities of nitropyrroles, nitrothiophenes, and aminothiophenes were studied. Replacement of the nitro group with an amino group enhanced the activity of the thiophene compounds.     

In Vitro Activity of Tobramycin and Gentamicin

The in vitro antimicrobial activity of tobramycin and gentamicin was compared against 362 bacterial isolates. The minimal inhibitory concentration (MIC) of tobramycin was fourfold less than the MIC of gentamicin against most of 119 Pseudomonas organisms. Gentamicin and tobramycin had similar in vitro activity against Enterobacteriaceae and Staphylococcus aureus. Proteus rettgeri were commonly resistant to both tobramycin and gentamicin. The 10-mug tobramycin disc separated resistant (MIC >/=5 mug/ml) and susceptible (MIC <5 mug/ml) organisms in 359 of 362 tested. In disc diffusion testing, the tobramycin and gentamicin zone diameters were found to vary significantly with concentrations of magnesium ions in the media employed. The MIC of tobramycin varied with the size of the inoculum, and tobramycin was most effective at a neutral pH.     

In Vitro Activity of LY127935

LY127935 is a unique new beta-lactam antibiotic. Its activity against 536 clinical isolates was studied by using microdilution methods of susceptibility testing and compared with the activities of cefamandole, cefoxitin, and cephalothin. The lowest concentrations required to inhibit at least 90% of strains tested (MIC(90)s) of LY127935 for Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae ranged from 2 to 8 mug/ml. The MIC(90)s for other staphylococci and streptococci were higher. The MIC(90)s for Enterobacteriaceae and Pseudomonas species ranged from 0.12 to 8 mug/ml and 8 to >32 mug/ml, respectively. The MIC(90)s for anaerobes ranged from 2 to >32 mug/ml. As determined by MIC(90)s, LY127935 was consistently the least active antibiotic against facultatively anaerobic gram-positive cocci and the most active against aerobic and facultatively anaerobic gram-negative bacilli. Its position with respect to activity against anaerobes varied from being the most active against Bacteroides fragilis and Clostridium perfringens to the least active against anaerobic cocci. In a population of multidrug-resistant isolates, concentrations of 8 mug or less of LY127935 per ml inhibited 82% of Enterobacteriaceae; concentrations of 32 mug or less per ml inhibited 100% of Enterobacteriaceae and 40% of P. aeruginosa. Increasing the inoculum size by 100-fold did not increase the minimal inhibitory concentrations of LY127935 or cefoxitin but did increase minimal inhibitory concentrations of cefamandole and cephalothin for some Enterobacteriaceae. All four drugs were bactericidal; minimal bactericidal concentrations were the same or one concentration higher than minimal inhibitory concentrations for 91 to 96% of strains tested. The broad spectrum and marked in vitro activity of LY127935 make it a promising new antibiotic.     

Antibacterial Activity of Cinoxacin In Vitro

Cinoxacin is a new synthetic compound similar chemically and in antimicrobial activity to oxolonic acid and nalidixic acid. It is most effective against Escherichia coli and Proteus mirabilis, but at concentrations expected in the urine it is inhibitory for all species of Enterobacteriaceae. Relative to nalidixic acid, cinoxacin has slightly greater inhibitory and bactericidal activity, less inoculum effect probably due to less heterogeneity in the susceptibility of bacterial cells, and less inhibition by high concentrations of serum protein. Both drugs are more active in an acid than an alkaline medium. Glucose can specifically antagonize the inhibitory effect against P. mirabilis. In urine the bactericidal rate and effect are decreased. Resistance to cinoxacin can be developed quickly by serial transfers in vitro. Some nonresistant organisms remained viable in bactericidal drug concentrations. The in vivo importance of the favorable features of cinoxacin must be determined by clinical trials.     

In Vitro Antibacterial Activity of Spectinomycin

The in vitro inhibitory and bactericidal activities of spectinomycin hydrochloride were tested against a variety of bacteria. The antibiotic was inhibitory at 31.2 mug/ml to most strains of Escherichia coli, Klebsiella, Enterobacter, and Staphylococcus epidermidis. Concentrations of antibiotic exhibiting bactericidal activity exceeded the inhibitory concentration by at least fourfold. Regression graphs were plotted for results obtained with 30-, 100-, 200-, and 300-mug spectinomycin discs; tentative interpretative standards are proposed.     

Comparison of In Vitro Activity of Cephalexin, Cephradine, and Cefaclor

Inhibitory activity of cephalexin, cephradine, and cefaclor was compared by the WHO-ICS agar dilution technique. Cefaclor was substantially more active against staphylococci, streptococci, gonococci, meningococci, Haemophilus, Escherichia coli, Klebsiella pneumoniae, Citrobacter diversus, Proteus mirabilis, salmonellae, and shigellae than was cephalexin, which in turn was more active than cephradine. Cefaclor appeared to be less resistant to staphylococcal penicillinase than did the other two agents. None of these cephalosporins was active against Enterobacter, Serratia, indole-positive Proteeae, Pseudomonas, or Bacteroides fragilis.     

Comparative Activity In Vitro of Ticarcillin, BL-P1654, and Carbenicillin

The activity of ticarcillin, BL-P1654, and carbenicillin was compared in vitro using a microtiter tube dilution test in Mueller-Hinton broth against 50 recent clinical isolates each of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella species, Enterobacter species, Proteus species, and Pseudomonas aeruginosa. Bactericidal end points were determined using a modified Steers replicator. Ticarcillin was generally two to four times more active against all organisms tested except S. epidermidis against which BL-P1654 was most active. Median minimum inhibitory concentrations in micrograms per milliliter were for S. aureus: ticarcillin (6.2), carbenicillin (12.5), BL-P1654 (25); for S. epidermidis: BL-P1654 (1.6), ticarcillin (3.2), carbenicillin (3.2); for E. coli: ticarcillin (3.2), BL-P1654 (6.2), carbenicillin (6.2); for Klebsiella sp.: >100 for all three drugs; for Enterobacter sp.: ticarcillin (3.2), carbenicillin (6.2), BL-P1654 (12.5); for Proteus sp.: ticarcillin (1.6), carbenicillin (1.6), BL-P1654 (3.2); for P. aeruginosa: ticarcillin (31), BL-P1654 (62), carbenicillin (125). Bactericidal end points were dependent on both the drug and the species but were in general no more than twofold more than the minimum inhibitory concentration with the exception of BL-P1654 against P. aeruginosa. BL-P1654 was bactericidal for only 60% of the strains tested at a concentration of 500 μg/ml.     

5溴汉防己甲素逆转多药耐药的体内体外研究

本研究旨在探讨5一溴汉防己甲素（BrTet）和汉防己甲素（Tet）对K562／A02细胞多药耐药性的逆转作用。采用MTT法检测阿霉素（ADM）联合应用BrTet、TeL,对K562／A02细胞和K562细胞增殖的影响;采用流式细胞术（FCM）检测细胞内ADM浓度和P糖蛋白（P—gp）的表达;采用RT—PCR测定细胞mdrl基因mRNA表达水平。建立裸鼠皮下移植瘤模型,比较BrTet、Tet在体内的逆转耐药作用。结果发现,BrTet在0．25、0．5以及1μmol／L浓度条件下对K562／A02细胞多药耐药性的逆转作用呈剂量依赖性。流式细胞术检测提示,BrTet显著增加K562／A02细胞内ADM浓度,并呈剂量依赖性,同时抑制P—gp的表达,下调mdrl mRNA的表达。在荷瘤鼠模型中,BrTet明显增加ADM对K562／A02移植瘤的抗肿瘤作用,从单用ADM的5．8％上升至26．1％,而在K562移植瘤中没有显著差异。结论：BrTet在体内体外试验中均显示出显著的逆转MDR作用,其活性可能与抑制P—gp的过度表达和增加抗肿瘤药物的积聚有关。     

In Vitro Activity of Cinoxacin, an Organic Acid Antibacterial

The activity of cinoxacin, a synthetic organic acid antimicrobial agent, was studied and found to have in vitro activity similar to that previously reported for nalidixic acid.     

In Vitro Antimycobacterial Activity of 5-Chloropyrazinamide

5-Chloropyrazinamide and 5-chloropyrazinoic acid were evaluated for in vitro activity against Mycobacterium tuberculosis, Mycobacterium bovis, and several nontuberculous mycobacteria by a broth dilution method. 5-Chloropyrazinamide was more active than pyrazinamide against all organisms tested. It is likely that this agent has a different mechanism of action than pyrazinamide.     

In Vitro Activity of Penicillins Against Anaerobes

The in vitro susceptibility of 162 anaerobic isolates from clinical material were tested to pencillin G, BL-P1654, and carbenicillin. Penicillin G and BL-P1654 showed good activity against Bacteroides fragilis, but only 60% of strains were susceptible to carbenicillin at achievable blood levels (128 μg/ml).     

Comparative In Vitro Activity of Sch 20656, Netilmicin, Gentamicin, and Tobramycin

Sch 20656 and netilmicin (Sch 20569), two new semisynthetic aminoglycosides, were as active as gentamicin and tobramycin against Enterobacteriaceae. Against Pseudomonas aeruginosa, Sch 20656 was the least active, whereas netilmicin was active against many highly gentamicin-resistant isolates.     

Cefuroxime, a New Cephalosporin Antibiotic: Activity In Vitro

Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to beta-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinase-producing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-beta-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many beta-lactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range.     

In Vitro and In Vivo Antibacterial Activity of T-1220, a New Semisynthetic Penicillin

T-1220, sodium 6-[d-(-)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)- α-phenylacetamido] penicillanate, is a new semisynthetic penicillin derivative that possesses a broad spectrum of in vitro antibacterial activity against gram-positive and gram-negative bacteria. T-1220 is more effective than carbenicillin (CB-PC) against Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus species, and Serratia marcescens. Addition of human serum to culture media did not significantly alter the antibacterial activity of T-1220. Greater bactericidal activity toward various strains of gram-negative bacteria was demonstrated with T-1220 than with CB-PC. T-1220, like penicillin G, was hydrolyzed by penicillinase, but was sable to a type IV penicillinase produced by P. aeruginosa strains. In vivo antibacterial activities of T-1220, ampicillin (AB-PC), and CB-PC were compared, using systemic infections of mice with P. aeruginosa, K. pneumoniae, and Escherichia coli. The 50% effective doses (milligrams per kilogram) of T-1220 were consistently lower than those of AB-PC and CB-PC.     

In Vitro Antimicrobial Activity of MSI-78, a Magainin Analog

MSI-78 is a cationic peptide with broad-spectrum antimicrobial activity and is being developed as a topical agent. We compared the in vitro activity of MSI-78 with those of ofloxacin and other antibiotics against fresh clinical isolates. Based on MIC distribution statistics, strains for which the MSI-78 MIC was ≤64 μg/ml were assumed to be susceptible for purposes of this report. Of 411 aerobic isolates tested, 91% were susceptible to MSI-78, compared to 91% for ofloxacin and 92% for ciprofloxacin. Only enterococci consistently required ≥64 μg of MSI-78/ml for inhibition. MSI-78 demonstrated bactericidal activity equivalent to that of ofloxacin. Of 61 anaerobes, 97% were susceptible to MSI-78. Of 10 isolates of Candida albicans, 3 were inhibited by MSI-78 at 24 h. Further studies of this compound appear to be warranted.