Inhibition of the reproduction of a herpes simplex I virus carrying mutations in the thymidine kinase and DNA polymerase genes

Sensitivity of Herpes Simplex Virus type I (HSV-I) mutants carrying genetic defect in the DNA polymerase and thymidine kinase genes to the action of some drugs was studied. TK- mutant of HSV-I was resistant to Ara-T and ACG and sensitive to PAA, Ara-A as well as to ribavirin and ADEA. PAAr mutant of HSV-I was resistant to PAA, Ara-A, ACG and sensitive to Ara-T, ribavirin and ADEA. A double mutant of HSV-I-TK-, PAAr was resistant to all drugs, except for ribavirin and ADEA. To inhibit reproduction of HSV with genetic defect, it is important using drugs of independent mode of action on the function of defective viral gene.     

Susceptibilities of phosphonoacetic acid and acyclovir resistant varicella-zoster virus mutants to 9-beta-arabinofuranosyladenine and 1-beta-arabinofuranosylcytosine

The DNA polymerase activity, and susceptibilities to 9-beta-D-arabinofuranosyladenine(ara-A) and 1-beta-arabinofuranosylcytosine(ara-C) of a phosphonoacetic acid resistant mutant (PAA-R) of varicella-zoster virus (VZV) selected in the presence of PAA were examined. The DNA polymerase activity of PAA-R was inhibited less than that of the parent strain by PAA in vitro. PAA-R was resistant to acyclovir and also to both ara-A and ara-C. The susceptibilities to ara-A and ara-C of four acyclovir resistant mutants selected in the presence of acyclovir, and also resistant to PAA, were examined. Two variants were resistant, one was slightly resistant, and one was sensitive to both drugs. These cross-resistances and susceptibilities of VZV variants to PAA, ACV, ara-A and ara-C should be considered in chemotherapy of VZV infections.     

Acyclovir-resistant mutants of herpes simplex virus type 1 express altered DNA polymerase or reduced acyclovir phosphorylating activities.

The biochemical properties of four acyclovir-resistant mutants are described. Two of these mutants, PAAr5 and BWr, specified nucleotidyl transferase (DNA polymerase) activities which were less sensitive to inhibition by acyclovir triphosphate than their wild-type counterparts. Another mutant, IUdRr, exhibited reduced ability to phosphorylate acyclovir. The fourth mutant, ACGr4, both induced an altered DNA polymerase and failed to phosphorylate appreciable amounts of acyclovir. BWr, a new acyclovir-resistant mutant derived from the Patton strain of herpes simplex virus type 1, induced a DNA polymerase resistant to inhibition by acyclovir triphosphate, but, unlike the polymerases induced by PAAr5 and ACGr4, still sensitive to phosphonoacetic acid. Resistance of BWr to acyclovir mapped close to the PAAr locus and was separable from mutations in the herpes simplex virus thymidine kinase gene by recombination analysis.     

Effect of β-Lactamase Location in Escherichia coli on Penicillin Synergy

Resistance to ampicillin in Escherichia coli is due generally to the presence of a beta-lactamase (penicillinase). Resistant strains have been found to fall into two groups: those with high-level resistance (1,000 mug/ml or greater) and those with low-level resistance (8 to 250 mug/ml). Most of the high-level resistant organisms posses beta-lactamases whose synthesis is episomally mediated. These strains release penicillinase from the cell when they are subjected to osmotic shock. Low-level resistant strains do not release the enzyme with osmotic shock. High-level resistant strains are not susceptible to the synergistic action of a penicillinase-resistant penicillin with ampicillin. Seventy eight per cent of low-level resistant strains are susceptible to the synergistic action of ampicillin and oxacillin. The two types of beta-lactamases are similar in regard to most properties; both enzymes are subject to competitive inhibition by penicillinase-resistant penicillins. The difference in location in the cell might explain why only some strains of E. coli are susceptible to the synergistic action of penicillin combinations.     

Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus.

Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.     

Antiviral activity of recombinant interferon-alpha-2b in combination with certain antioxidant

In vitro activity of interferon-alpha-2b in combination with various antioxidants against the influenza virus and Herpes simplex was studied. The standard strains and a clinical strain of Herpes simplex isolated from a patient with resistance to acyclovir were used. The in vitro studie showed that antioxidants, such as alpho-tocoferol acetate (vitamin E), Unithiol and ascorbic acid had a significant antiinfluenzae and antiherpetic action on the influenza virus A/H5N1 and Herpes simplex variants. They protected up to 100% of the cell monolayer from the virus cytopathic effect. The taurin solutions had no antiviral activity irrespective of the infection dose. Combinations of interferon-alpha-2b with alpha-tocopherol acetate (vitamin E), Unithiol or ascorbic acid showed a significant synergistic effect: the antiviral activity of interferon increased several times. The antiinfluenza activity of interferon-a-2b in the presence of various concentrations of taurin did not change.     

Selection and characterization of mutant S49 T-lymphoma cell lines resistant to phosphonoformic acid: evidence for inhibition of ribonucleotide reductase

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.     

粘菌素对耐碳青霉烯类抗生素鲍曼不动杆菌的抗菌作用

感染耐碳青霉烯的鲍曼不动杆菌(CR-Ab)常与高发病率和死亡率相关联,而可供选择的治疗方案有限,大多基于与粘菌素联用。长期用药导致CR-Ab对粘菌素也产生一定抗性。为了评估含有或不含有粘菌素的不同抗菌组合对从CR-Ab感染患者收集的CR-Ab临床分离株的体外抗菌活性,本研究从本院就诊的患者中收集CR-Ab菌株,通过常量肉汤稀释法(MBD)测定最低抑菌浓度(MICs),通过定性(棋盘法)和定量(即杀菌测试)方法评估各组药物协同活性。结果发现所有菌株均是碳青霉烯类抗性的,且其中两株菌对粘菌素有抗性。棋盘法结果表明含粘菌素的组合在不同处理时间下具有完全协同作用,粘菌素+万古霉素和粘菌素+利福平表现出最高的协同增效作用;不含粘菌素的组合则在35.7%的菌株中观察到完全协同作用。杀菌测试表明粘菌素+美罗培南、粘菌素+替加环素和美罗培南+替加环素组合对粘菌素敏感和低粘菌素抗性的菌株具有杀菌和协同作用,而只有粘菌素+万古霉素和粘菌素+利福平组合表现出持久的杀菌活性。     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

In vitro assessment of the antibacterial effects of the combinations of fosfomycin,colistin, trimethoprim and nitrofurantoin against multi-drug–resistant Escherichia coli

MDR UPEC has become a global health challenge. Our study investigates the pairwise interactions among FOS, COL, NIT and TRI against 29 UPEC strains using the checkerboard method. The synergistic combinations are further evaluated for their bactericidal effects against the most resistant strain (MRS) using the time-kill method. The results showed that 100% of these strains were resistant to TRI and NIT, whereas 75·86% of them were susceptible to FOS and COL. Among all tested strains, only seven strains were highly resistant to all used antibiotics. Remarkably, FOS/COL, COL/NIT and COL/TRI combinations represent the most effective synergistic (fractional inhibitory concentration index <1) combinations against the seven strains at MICs lower than the susceptible breakpoint ranges, followed by FOS/NIT and FOS/TRI, which achieved synergistic interactions against 1/7 and 2/7 of these strains. Importantly, the bactericidal effects (reduction ≥3·0 log₁₀ CFU per ml) were only observed with FOS/COL, COL/NIT and COL/TRI combinations against MRS after 24 h of post-treatment. Our data suggested that FOS/COL, COL/NIT and COL/TRI combinations could be a promising option against MDR UPEC infections. Additionally, FOS/NIT and FOS/TRI probably represent a good option for MDR UPEC with lower MICs.     

Isolation of drug resistant mutants of varicella-zoster virus: cross resistance of acyclovir resistant mutants with phosphonoacetic acid and bromodeoxyuridine

Mutants of Varicella-Zoster Virus (VZV) which are resistant to phosphonoacetic acid (PAA), bromodeoxyuridine (BuDR), and acyclovir (ACV) were obtained by serial passages of VZV with increasing concentrations of these drugs. A PAA-resistant mutant and a BuDR-resistant mutant were found also to be resistant to ACV. Five of 8 ACV-resistant mutants acquired resistance to PAA, but none acquired resistance to BuDR. The BuDR-resistant mutant did not induce viral thymidine kinase (TK) activity, but all the ACV-resistant mutants selected in ACV showed viral TK activity which was suppressed with anti-VZV serum and had almost the same electrophoretic mobility as that of the parent strain on polyacrylamide gel electrophoresis in non-denaturing conditions. However, in competitive TK assay with ACV, 2 of 8 ACV-resistant mutants showed no change of phosphorylation of radioactive thymidine, while the other 6 showed decreased phosphorylation of radioactive thymidine. It was suggested that TK induced by the former 2 ACV-resistant mutants had lost affinity to ACV, and so the mutants could grow in the presence of ACV. Thus of the 8 ACV-resistant mutants selected in ACV, 2 were sensitive to PAA with altered TK activity, 5 were resistant to PAA with unaltered TK activity, and 1 was sensitive to PAA with unaltered TK activity, and may have altered DNA polymerase activity to ACV, retaining sensitivity to PAA. These results suggest that resistance of VZV to ACV results from alterations in the virus-specified TK or DNA polymerase, as demonstrated in HSV resistant to ACV.     

Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro.

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.     

Synergistic action of combinations of penicillin and perhydroacridine derivatives

The effect of combinations of perhydroacridine derivatives such as 10-nitroso-trans-anti-cys-perhydroacridine (MT-2) and 10-isopropylamino-trans-anti-cys-perhydroacridine (MT-6) with benzylpenicillin on growth of two penicillinase-producing strains of Staphylococcus aureus was studied. The combination components were added to the culture simultaneously or at different periods i.e. the second component was added after preliminary exposure of the bacterial cells to the first component for 1-6 hours at 37 degrees C. It was shown that duration and efficacy of the combination synergistic action were directly proportional to the time of the component addition. The highest synergistic action of the combinations was observed when both the components were simultaneously added to the staphylococcal culture. The combinations were less efficient when the bacterial cells were preliminarily incubated with the perhydroacridines. Addition of the perhydroacridine derivatives after the strain contact with the penicillin resulted in elimination of the combination synergistic action. Thin-layer chromatography did not reveal complexing between the penicillin and perhydroacridines.     

Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro

In this study the in vitro activities of seven antibiotics (ciprofloxacin, ceftazidime, tetracycline, trimethoprim, sulfamethoxazole, polymyxin B and piperacillin) and six phytochemicals (protocatechuic acid, gallic acid, ellagic acid, rutin, berberine and myricetin) against five P. aeruginosa isolates, alone and in combination are evaluated. All the phytochemicals under investigation demonstrate potential inhibitory activity against P. aeruginosa. The combinations of sulfamethoxazole plus protocatechuic acid, sulfamethoxazole plus ellagic acid, sulfamethoxazole plus gallic acid and tetracycline plus gallic acid show synergistic mode of interaction. However, the combinations of sulfamethoxazole plus myricetin shows synergism for three strains (PA01, DB5218 and DR3062). The synergistic combinations are further evaluated for their bactericidal activity against P. aeruginosa ATCC strain using time-kill method. Sub-inhibitory dose responses of antibiotics and phytochemicals individually and in combination are presented along with their interaction network to suggest on the mechanism of action and potential targets for the phytochemicals under investigation. The identified synergistic combinations can be of potent therapeutic value against P. aeruginosa infections. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals).     

Effects of Antifungal Agents Alone and in Combination Against Candida glabrata Strains Susceptible or Resistant to Fluconazole

The rise of Candida spp. resistant to classic triazole antifungal agents has led to a search for new therapeutic options. Here, we evaluated combinations of antifungals in a checkerboard assay against two groups of Candida glabrata strains: one containing fluconazole-susceptible clinical isolates (FS) and another containing fluconazole-resistant laboratory derivative (FR). The most synergistic combination observed was amphotericin B + flucytosine (synergistic for 61.77 % of FS strains and 76.47 % of FR strains). The most antagonistic combination observed was ketoconazole + flucytosine (FS 61.77 % and FR 55.88 %). Surprisingly, most combinations evidenced indifferent interactions, and the best synergism appeared when amphotericin B and flucytosine were combined against both groups of isolates.     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

Haemocyte changes in resistant and susceptible strains of D. melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi

Two strains of Drosophila melanogaster (resistant and susceptible) were parasitized by a virulent or avirulent strain of the parasitoid wasp Leptopilina boulardi. The success of encapsulation depends on both the genetic status of the host strain and the genetic status of the parasitoid strain: the immune cellular reaction (capsule) is observed only with the resistant strain-avirulent strain combination. The total numbers of host haemocytes increased in all 4 combinations, suggesting that an immune reaction was triggered in all hosts. Resistant host larvae infected with the virulent or avirulent strains of parasitoid wasp had slightly more haemocytes per mm(3) than did susceptible host larvae at the beginning of the reaction (less than 15 h post-parasitization). This difference disappeared later. Only the virulent parasitoid strain caused the production of a high percentage of altered lamellocytes (from a discoid shape to a bipolar shape), half the total number of lamellocytes are altered. This suggests that the alteration of lamellocyte shape alone is not sufficient to explain the lack of capsule formation seen in resistant hosts parasitized by the virulent strain. Lastly, there were very few altered lamellocytes in resistant or susceptible hosts parasitized by the avirulent parasitoid strain, two combinations in which no capsule was formed. As is now established for Drosophila-parasitoid interactions, virus-like particles contained in the long gland of the female wasp affect the morphology of the lamellocytes. The results presented here are further proof of the action (direct or indirect) of virus like particles of the virulent strain on lamellocytes.     

Flocculent phenotypes in wine yeasts

The floc-forming ability of highly flocculent wine yeasts isolated from musts and wines was tested for susceptibility to heat and proteinase treatments. Four phenotypes were discriminated by treatments with pronase, proteinase K, trypsin and chymotrypsin. The most common phenotype was irreversibly lost only upon treatment with pronase, whereas the floc-forming ability was resistant to the action of proteinase K, trypsin and chymotrypsin. Another flocculent phenotype, represented by only one strain 6789, was resistant to the action of all proteolytic enzymes. The effect of high temperature on floc-forming ability in the presence or absence of Ca²⁺ions resulted in all the possible combinations and did not aid further general discrimination of flocculent phenotypes in Saccharomyces cerevisiae strains from wine.     

Sensitivity of arabinosyladenine-resistant mutants of herpes simplex virus to other antiviral drugs and mapping of drug hypersensitivity mutations to the DNA polymerase locus.

Seven herpes simplex virus mutants which have been previously shown to be resistant to arabinosyladenine were examined for their sensitivities to four types of antiviral drugs. These drugs were a pyrophosphate analog, four nucleoside analogs altered in their sugar moieties, two nucleoside analogs altered in their base moieties, and one altered in both. The seven mutants exhibited five distinct phenotypes based on their sensitivities to the drugs relative to wild-type strain KOS. All mutants exhibited resistance to acyclovir and arabinosylthymine, as well as marginal resistance to iododeoxyuridine, whereas all but one exhibited resistance to phosphonoformic acid. The mutants exhibited either sensitivity or hypersensitivity to other drugs tested--2'-nor-deoxyguanosine, 5-methyl-2'-fluoroarauracil, 5-iodo-2'-fluoroarauracil, and bromovinyldeoxyuridine--some of which differed only slightly from drugs to which the mutants were resistant. These results suggest ways to detect and treat arabinosyladenine-resistant isolates in the clinic. Antiviral hypersensitivity was a common phenotype. Mutations conferring hypersensitivity to 2'-nor-deoxyguanosine in mutant PAAr5 and to bromovinyldeoxyridine in mutant tsD9 were mapped to nonoverlapping regions of 1.1 and 0.8 kilobase pairs, respectively, within the herpes simplex virus DNA polymerase locus. Thus, viral DNA polymerase mediates sensitivity to these two drugs. However, we could not confirm reports of mutations in the DNA polymerase locus conferring resistance to these two drugs. All of the mutants exhibited altered sensitivity to two or more types of drugs, suggesting that single mutations affect recognition of the base, sugar, and triphosphate moieties of nucleoside triphosphates by viral polymerase.     

Sensitivity of Brucella melitensis to various antibiotics, drug preparations and their combinations

In vitro activity of 13 antibiotics and drug preparations as well as some of their combinations was studied with respect to Brucella melitensis. It was shown that the strains of B. melitensis were sensitive to sisomicin, amikacin, gentamicin, cefotaxime, rifaprim +, nitroxoline and trimethoprim. Variations in the doxycycline sensitivity depending on the strain were observed. The strains of B. melitensis were resistant to cephalexin, phosphomycin, chinoxydine, dioxydine and oxolinic acid. Combinations of sisomicin and trimethoprim at a ratio of 1:10 and sisomicin and nitroxoline at a ratio of 1:2 had a summation effect.