Correlation between antibiotic sensitivity testing by conventional and conductivity measurements

A high correlation in the susceptibilities of 44 strains isolated from clinical material to a variety of antimicrobial agents was found between the minimum inhibitory concentration and conductivity measurements. Some discrepancy was found for vancomycin and teicoplanin when tested against 21 strains of staphylococci. Similarly, discrepancies were seen with azlocillin, chloramphenicol and gentamicin when tested against strains of aerobic and facultatively anaerobic Gram-negative bacilli. In the majority of cases in which discrepancies were noted, this reflected differences in relative, and not absolute, susceptibility. With five strains of Pseudomonas, however, there was a poor correlation with three of four antibiotics studied. The advantage of this technique is that sensitivities are available within 4-6 h.     

Comparative Study of the Efficacy of Seven Paper-Reagent Strips and Conventional Biochemical Tests in Identifying Gram-negative Organisms

Gram-nagative organisms were tested with commercially available reagentimpregnated strips (PATHO-TEC). Of the 291 strains, all were tested by using seven paper tests and their conventional counterparts. Excellent correlation was obtained with the oxidase, phenylalanine-deaminase, and Voges-Proskauer tests. Indole tests made on liquid medium cultures also gave complete correlation, but some false-negative results with indole-positive Proteus strains were obtained when growth from solid medium was tested by the strip method. Paper strip urease tests were positive within 2 hr with all Klebsiella and some Serratia, Herellea, and Citrobacter strains as well as with Proteus strains. Approximately 15% of citrate strip test results differed from those of the conventional tests, and reproducibility was poor on retest. The lysine decarboxylase strip test showed a number of discrepancies and posed problems of interpretation and readability. Paper reagent strip methods are simple and convenient and merit further development to increase the specificity of those which depend on pH change up to that achieved with the Voges-Proskauer, oxidase, phenylalanine, and indole methods.     

Comparison of Agar Dilution and Broth Microdilution Methods of Anaerobic Antimicrobial Susceptibility Testing using Several Veterinary Antibiotics against Clostridium perfringens Strains Originating from Porcine and Avian Sources

A broth microdilution and a reference agar dilution method was used to determine minimal inhibitory concentrations (MICs) of five veterinary antimicrobials when tested against 96 animal-derived and six American Type Culture Collection (ATCC) strains of Clostridium perfringens. These antimicrobials [bacitracin methylenedisalicylate (bacitracin-MD), tylosin, virginiamycin, erthromycin and tetracycline] are approved for use in animal feed at different levels for growth enhancement, control, and treatment of a variety of enteric diseases. For bacitracin-MD, MICs were higher using the broth microdilution method when compared to the agar dilution method. The two methods had the lowest agreement when using bacitracin-MD compared to the method agreements of other antimicrobials tested (only 34.3% of the C. perfringens tested within ±1 doubling dilution). Tylosin MICs were lower by the broth microdilution method but had better agreement between methods with 75.5% of the C. perfringens tested within ±1 doubling dilution. Good correlation between methods was found for virginiamycin, tetracycline, and erythromycin with 85.3, 76.5, 81.4% of the C. perfringens tested within ±1 doubling dilution, respectively. Differences in susceptibility to individual antimicrobials were found among the avian and porcine strains by both methods. For avian strains, bacitracin-MD, tylosin, and erthromycin MIC₉₀values had differences of at least four doubling dilutions between methods. There were biases toward higher bacitracin-MD and lower tylosin and erythromycin MIC₉₀values using the broth microdilution method. MIC₉₀values against porcine and ATCC strains were more comparable between methods for all five antimicrobials than those generated against avian strains but the biases were still present. Most animal-derived strains were inhibited by approved livestock feed levels of the antimicrobials. Caution should be used when evaluating the potential effectiveness of feed-based antimicrobials against C. perfringens when results are generated using an in vitro test that may not be in agreement with the reference agar dilution method.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Effect of Mesua ferrea Linn. flower extract on Salmonella

Based on its traditional uses in folk medicine, the whole flower extract of Mesua ferrea Linn. was tested for its in vitro antimicrobial efficacy against five different strains of Salmonella spp. All the strains were found to be highly sensitive to the extract, MIC of the extract against each organism being 50 microg/ml. The extract was tested in vitro for its mode of antibacterial activity against S. Typhimurium NCTC 74 and it was found to be bactericidal in action. In vivo studies of this extract offered significant protection to Swiss albino mice at doses approximately 2 and 4 mg/mouse when challenged with 50 median lethal dose of S. Typhimurium NCTC 74. Further, the extract caused statistically significant reduction in viable count of the strain in liver, spleen and heart blood of challenged mice.     

Characteristics of myxobacteria isolated from the surface of freshwater fish

A study was made of 32 nonpathogenic myxobacterial isolates obtained from a variety of fish taken in the Pacific Northwest. Morphological, cultural, biochemical, and serological studies were carried out on these strains. All were found to be members of the genus Cytophaga. Two myxobacterial strains pathogenic to fish were also included in this study for comparative purposes. These pathogenic organisms were found to be culturally and physiologically similar to some of the nonpathogenic strains. Antiserum against the pathogenic species, however, showed no cross agglutination when tested against the other myxobacterial isolates. As a result, serological procedures appear promising as a rapid means for distinguishing pathogenic myxobacteria from one another and from saprophytic myxobacteria commonly found on fish.     

Antimicrobial potentiality of a phenothiazine group of antipsychotic drug-prochlorperazine

The antipsychotic drug, prochlorperazine (Pcp), was tested for its antimicrobial efficacy against 103 strains belonging to both gram positive and gram negative bacteria. The drug was found to possess maximum activity against Staphylococcus aureus, Vibrio cholerae and Shigella spp. Pcp was moderately active against E. coli but most of the strains belonging to Bacillus spp, Klebsiella spp, Salmonella spp and Lactobacillus spp were found to be resistant to this drug. The drug was tested for its mode of antibacterial activity against Shigella dysenteriae 1 and it was found to be bacteriostatic in action. In in vivo studies, Pcp offered significant protection to Swiss albino mice at concentrations of 0.75 micro g/g (P < 0.01) and 1.5 microg/g (P < 0.001) body weight when challenged with 50 median lethal dose of Salmonella typhimurium NCTC 74. Thus the result depicts that prochlorperazine may emerge as a strong antimicrobial drug to replace the conventional antibiotics and to overcome the problem of drug resistance.     

Pathogenic significance of Acinetobacter calcoaceticus: analysis of experimental infection in mice

The phenomenon that mixed infection with certain species of bacteria and Acinetobacter calcoaceticus is more virulent than single infection was analyzed experimentally. In mixed infections with A. calcoaceticus paired with either Escherichia coli, Serratia marcescens, or Pseudomonas aeruginosa, the virulence of the latter three organisms was markedly increased over that of single infections only by slime-producing strains of A. calcoaceticus. Of the 100 strains of A. calcoaceticus tested, 14 had slime-producing ability. There was scarcely any difference in the chemical components of the slimes of the two strains tested, but the components of the slime of P. aeruginosa were different from those of these strains. The slime of these two strains exhibited lethal activity in mice, but no correlation was found between the amount of slime produced and the virulence. The slime enhanced the virulence of E. coli, S. marcescens, and P. aeruginosa when it was inoculated along with their viable cells. Furthermore, the slime exhibited potent cell-impairing activity against mouse neutrophils both in vitro and in vivo. This activity was considered to be mainly responsible for the enhancement of virulence in mixed infections.     

In vitro activity of cefamandole, cefoxitin, cefuroxime, and carbenicillin, alone and in combination with aminoglycosides against Serratia marcescens.

Synergistic antibiotic studies were undertaken to compare the effectiveness of two new beta-lactamase resistant cephalosporins, cefamandole, and carbenicillin, with four aminoglycosides against clinical strains of Serratia marcescens. The strains demonstrated various combinations of resistance and/or susceptibility to the antibiotics tested. Tobramycin was the most effective aminoglycoside when used in combination with beta-lactam antibiotics. Carbenicillin and cefamandole demonstrated similar activity with aminoglycosides in synergy experiments. Tobramycin-carbenicillin was found to be the superior pairs as indicated by the total number of strains inhibited. This combination was the only one effective against certain high drug resistant strains and the strain resistant to all four aminoglycosides. Carbenicillin or cefamandole with tobramycin exhibited comparable activity against multiple drug resistant organisms. However, mutants significantly more resistant to cefamandole developed during susceptibility testing. The findings of this study have clinical relevance for treating infections by this formidable pathogen.     

Fimbriae and haemagglutinating properties in Edwardsiella and Levinea.

Using the haemagglutinating procedure, the presence of fimbriae was revealed in all 34 Edwardsiella strains tested and in 3 Levinea strains out of 17 examined. The great majority of fimbriate Edwardsiella strains caused haemagglutination of MR type; only two strains of Edwardsiella and all fimbriate strains of Levinea brought about haemagglutination of MS type. All the strains exhibited a similar spectrum of haemagglutinating activity when they were tested against red cells of various animal species. Under the electron microscope MR fimbriae of Edwardsiella were found to be thicker than MS fimbriae (6.5 nm and 6.0 nm respectively). MS fimbriae of Levinea had 6.5 nm in diameter.     

A biphasic approach to the determination of the phenotypic and genotypic diversity of some anaerobic,cellulolytic, thermophilic,rod-shaped bacteria

A biphasic approach, involving a numerical phenetic and a phylogenetic study, was used to determine diversity among some anaerobic, cellulolytic, thermophilic, rod-shaped bacteria. Ninety two characters were determined for 51 strains in the numerical taxonomy study, and partial 16S rDNA sequences from 16 isolates were compared. Both the phenetic and phylogenetic data indicate diversity within this group of organisms, and reveal the lack of similarity between sporogenous and asporogenous isolates. The results of the phylogenetic study demonstrate the lack of relationship of the majority of the strains studied to previously studied thermophilic bacteria. In general, good correlation exists between the two data sets, but discrepancies arise when strains with a high level of similarity are examined. The need for caution in the interpretation of data obtained from such a biphasic approach is discussed.     

The Activity of Pipemidic Acid and Piromidic Acid against Escherichia coli In Static Culture and in Conditions Simulating the Treatment of Bacterial Cystitis

The activity of the new pyridopyrimidine compounds, pipemidic and piromidic acids, against Escherichia coli was compared with that of nalidixic acid in vitro . In a static turbidimetric system nalidixic and pipemidic acids were found to be more active than piromidic acid when tested against sensitive E. coli strains, but pipemidic acid was the most active of the three compounds against nalidixic acid-resistant clinical isolates. When tested in an in vitro model designed to mimic the conditions in which bacteria and drug interact in the treatment of bacterial cystitis, all three drugs were found to be able to suppress bacterial growth for long periods, but a high, sustained drug level was necessary in order to prevent the emergence of resistant variants.     

Bactericidal activity of human sera with a physiologic or genetic defect of the complement system

The serum of patient suffering genetically conditioned C2 defect showed a weak bactericidal activity against Pseudomonas aeruginosa strains. Out of 23 tested strains one was susceptible comparing with 16 ones sensitive to normal human serum. The normal cord serum exerted a bactericidal effect on 8 strains. All sera were found to be active against Salmonella strains tested.     

Interaction with homologous erythrocytes of rat T cells which act as aggressors in the mixed lymphocyte reaction.

Substantial percentages of T-enriched spleen lymphocytes or thymocytes of inbred rats were found to form rosettes with the RBC of homologous strains. When an excess of RBC was used, essentially all of the rosette-forming subpopulation of lymphocytes was removed when the rosettes were separated by centrifugation. After depletion of the lymphocytes, reactive with RBC of one strain, most of the lymphocytes reactive with RBC of other strains could be recovered in the supernatant. A very large percentage of lymphocytes of the BN strain formed rosettes when a mixture of the RBC of five other strains was tested; the percentage was, however, somewhat lower than that predicted on the basis of complete additivity. Rosettes dissociated when warmed to 37 degrees C. The lymphocytes recovered were unable to form rosettes again. In nearly all instances, the subpopulation that formed rosettes with RBC of a given strain included essentially all of the lymphocytes that acted as aggressors against peripheral leukocytes or mytomycin C-treated thymocytes of that strain; the lymphocytes in the supernatant always retained activity as aggressors against the leukocytes of one or more other strains and retained their responsiveness to PHA. Lymphocytes recovered from rosettes, by warming to 37 degrees C, were highly reactive as aggressors in the MLR against the strain providing the RBC. Varying degrees of reactivity were noted against leukocytes of other strains.     

Bacteriocinogenic properties of Staphylococci isolated from the oral cavity

Two hundred ninety two staphylococcal strains were isolated out of 130 saliva samples taken from children and adults, among which 116 were coagulase-positive and 176 coagulase negative. Bacteriocinogenic activity against Staphylococcus aureus strain Oxford 209P was found in 13 (4.5%) of the strains only. On the other hand, when a set of 15 sensitive staphylococcal strains selected by cross checking was used for the study 260 (89.0%) strains were found to be bacteriocinogenic. It was found that a higher percentage of coagulase-positive staphylococcal strains is sensitive to staphylococcins than of coagulase-negative strains. However, mean zone of inhibition is smaller in the case of former than of the latter strains. It was shown, that in the case of active strains a positive correlation exists between a percentage of coagulase-positive and negative strains inhibited by them and also between a percentage of all inhibited strains and a mean diameter if the growth inhibition zone. Simultaneous occurrence in saliva of two or more staphylococcal strains was found in 106 persons examined. In 93.4% of those cases coexisting strains did not show antagonistic properties: in remaining 6.6% despite of the number of simultaneously existing strains in oral cavity only one strain showed antagonistic properties against the remaining strains.     

Identification of environmental Serratia plymuthica strains with the new combo panels type 1S

Automated systems are required when numerous samples need to be processed, offering both high through put and test of a multiple simultaneously. This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Neg Panels Type 1S with conventional biochemical methods for identifying ten environmental Serratia plymuthica strains. High correlation between both methods were observed for all the 21 tests evaluated, and the MicroScan system was found capable of correctly identifying all S. plymuthica strains tested. In all tests, the percentage of correlation was 100%, except in raffinose test (91%).     

Analysis of the two-peptide bacteriocins lactococcin G and enterocin 1071 by site-directed mutagenesis

The two peptides (Lcn-alpha and Lcn-beta) of the two-peptide bacteriocin lactococcin G (Lcn) were changed by stepwise site-directed mutagenesis into the corresponding peptides (Ent-alpha and Ent-beta) of the two-peptide bacteriocin enterocin 1071 (Ent), and the potencies and specificities of the various hybrid constructs were determined. Both Lcn and, to a lesser extent, Ent were active against all the tested lactococcal strains, but only Ent was active against the tested enterococcal strains. The two bacteriocins thus differed in their relative potencies to various target cells, despite their sequence similarities. The hybrid combination Lcn-alpha+Ent-beta had low potency against all strains tested, indicating that these two peptides do not interact optimally. The reciprocal hybrid combination (i.e., Ent-alpha+Lcn-beta), in contrast, was highly potent, indicating that these two peptides may form a functional antimicrobial unit. In fact, this hybrid combination (Ent-alpha+Lcn-beta) was more potent against lactococcal strains than wild-type Ent was (i.e., Ent-alpha+Ent-beta), but it was inactive against enterococcal strains (in contrast to Ent but similar to Lcn). The observation that Ent-alpha is more active against lactococci in combination with Lcn-beta and more active against enterococci in combination with Ent-beta suggests that the beta peptide is an important determinant of target cell specificity. Especially the N-terminal residues of the beta peptide seem to be important for specificity, since Ent-alpha combined with an Ent-beta variant with Ent-to-Lcn mutations at positions 1 to 4, 7, 9, and 10 was >150-fold less active against enterococcal strains but one to four times more active against lactococcal strains than Ent-alpha+Ent-beta. Moreover, Ent-to-Lcn single-residue mutations in the region spanning residues 1 to 7 in Ent-beta had a more detrimental effect on the activity against enterococci than on that against lactococcal strains. Of the single-residue mutations made in the N-terminal region of the alpha peptide, the Ent-to-Lcn mutations N8Q and P12R in Ent-alpha influenced specificity, as follows: the N8Q mutation had no effect on activity against tested enterococcal strains but increased the activity 2- to 4-fold against the tested lactococcal strains, and the P12R mutation reduced the activity >150-fold and only approximately 2-fold against enterococcal and lactococcal strains, respectively. Changing residues in the C-terminal half/part of the Lcn peptides (residues 20 to 39 and 25 to 35 in Lcn-alpha and Lcn-beta, respectively) to those found in the corresponding Ent peptides did not have a marked effect on the activity, but there was an approximately 10-fold or greater reduction in the activity upon also introducing Lcn-to-Ent mutations in the mid-region (residues 8 to 19 and 9 to 24 in Lcn-alpha and Lcn-beta, respectively). Interestingly, the Lcn-to-Ent F19L+G20A mutation in an Lcn-Ent-beta hybrid peptide was more detrimental when the altered peptide was combined with Lcn-alpha (>10-fold reduction) than when it was combined with Ent-alpha ( approximately 2-fold reduction), suggesting that residues 19 and 20 (which are part of a GXXXG motif) in the beta peptide may be involved in a specific interaction with the cognate alpha peptide. It is also noteworthy that the K2P and A7P mutations in Lcn-beta reduced the activity only approximately 2-fold, suggesting that the first seven residues in the beta peptides do not form an alpha-helix.     

Chitinolytic activity of Bacillus Cohn.--phytopathogenic fungus antagonist

Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, beta-1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r x,y = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.     

Innate defense against influenza A virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.