Survey of modifying enzymes and plasmids in amikacin-resistant Serratia marcescens

Forty amikacin-resistant strains of Serratia marcescens isolated from four different hospitals (A, B, C, and D) were examined for modifying enzymes and plasmids. Twenty-one of the isolates produced acetyltransferase that modified amikacin. Eighteen of the 21 acetyltransferase-bearing isolates were from different inpatients in hospital A and the other three were from hospital C. Amikacin resistance was mediated by conjugative plasmid of 24 megadaltons in 15 of the 18 acetyltransferase-bearing isolates of hospital A and by nonconjugative plasmids, derivatives of the 24-megadalton plasmids, in the remaining three isolates of the same hospital. The 24-megadalton plasmid determined aminoglycoside acetyltransferase (6') IV. This plasmid-borne enzyme conferred amikacin resistance on S. marcescens but not on Escherichia coli K12. The frequency of transfer of the 24-megadalton plasmid from the S. marcescens isolate to E. coli K12 by conjugation was approximately 10(-7) (transconjugants/donors) and was 0.1% of that between E. coli strains. In acetyltransferase-bearing isolates from hospital C, the enzyme was mediated by a nonconjugative plasmid in one case and could not be associated with a plasmid in the remaining two cases. Neither enzymes nor plasmids could be associated with amikacin resistance of the isolates of the other two hospitals.     

Factors affecting the pressure resistance of some Campylobacter species

AIMS: To compare pressure resistance between strains of Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter fetus, and to investigate the effect of suspending medium on pressure resistance of sensitive and more resistant strains. METHODS AND RESULTS: Six strains of C. jejuni and four each of C. coli, C. lari and C. fetus were pressure treated for 10 min at 200 and 300 MPa. Individual strains varied widely in pressure resistance but there were no significant differences between the species C. jejuni, C. coli and C. lari. Campylobacter fetus was significantly more pressure sensitive than the other three species. The pressure resistance of C. jejuni cultures reached a maximum at 16-18 h on entry into stationary phase then declined to a minimum at 75 h before increasing once more. Milk was more baroprotective than water, broth or chicken slurry but did not prevent inactivation even of a resistant strain at 400 MPa. CONCLUSIONS: Pressure resistance varies considerably between species of Campylobacter and among strains within a species, and survival after a pressure challenge will be markedly influenced by culture age and food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the strain variation in pressure resistance and protective effects of food, Campylobacter sp. do not present a particular problem for pressure processing.     

Determination of metabolic resistance mechanisms in pyrethroid‐resistant and fipronil‐tolerant brown dog ticks

Rhipicephalus sanguineus (Latreille) (Ixodida: Ixodidae) is a three‐host dog tick found worldwide that is able to complete its' entire lifecycle indoors. Options for the management of R. sanguineus are limited and its' control relies largely on only a few acaricidal active ingredients. Previous studies have confirmed permethrin resistance and fipronil tolerance in R. sanguineus populations, commonly conferred by metabolic detoxification or target site mutations. Herein, five strains of permethrin‐resistant and three strains of fipronil‐tolerant ticks were evaluated for metabolic resistance using synergists to block metabolic enzymes. Synergist studies were completed with triphenyl phosphate (TPP) for esterase inhibition, piperonyl butoxide (PBO) for cytochrome P450 inhibition, and diethyl maleate (DEM) for glutathione‐S‐transferase inhibition. Additionally, increased esterase activity was confirmed using gel electrophoresis. The most important metabolic detoxification mechanism in permethrin‐resistant ticks was increased esterase activity, followed by increased cytochrome P450 activity. The inhibition of metabolic enzymes did not have a marked impact on fipronil‐tolerant tick strains.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.     

Resistance of aphis gossypii (Homoptera: Aphididae) to fenvalerate and imidacloprid and activities of detoxification enzymes on cotton and cucumber

Resistance of two strains of cotton aphid, Aphis gossypii Glover, to fenvalerate and imidacloprid were determined on cotton (Gossypium hirsutum L.) and cucumber (Cucumis sativa L.) after resistance selection of one strain to fenvalerate for 16 consecutive generations, and of a second strain to imidacloprid for 12 consecutive generations on cotton in greenhouses. Dose-response and activities of detoxication enzymes of the fenvalerate-resistant strain (R-fenvalerate), the imidacloprid-resistant strain (R-imidacloprid), and a susceptible strain (S) were determined. After 16 consecutive generations of selection, resistance of A. gossypii to fenvalerate increased >29,000-fold and to imidacloprid 8.1-fold. On cucumber. resistance of the R-fenvalerate strain to fenvalerate increased 700-fold and to imidacloprid 3.6-fold. However, the most significant finding in this study was that the R-imidacloprid strain exhibited cross-resistance to fenvalerate, with a resistance ratio of 108.9-fold on cotton and 3:3.5-fold on cucumber, whereas the R-fenvalerate strain did not show significant cross-resistance to imidacloprid on either plant species. Both resistant strains of A. gossypii were more resistant to fenvalerate on cotton than on cucumber, whereas their susceptibility to imidacloprid on otton and cucumber were not significantly different. The response of the S strain to fenvalerate and imidacloprid were similar on cotton and Cucumber. Activities of acetylcholinesterase (AChE) and alpha-naphthylacetate (alpha-NA) esterases of A. gossypii were significantly different among the three strains, with the R-fenvalerate strains having the highest, followed by the R-imidacloprid strain, and the S strain the lowest. The activities of the AChE and alpha-NA esterases for all three strains were also significantly higher on cotton than on cucumber. The resistance mechanism and resistance management strategies for the R-fenvalerate and R-imidacloprid strains of A. gossypii to fenvalerate and imidacloprid on cotton and cucumber are discussed.     

Overproduction of peroxide-scavenging enzymes in Escherichia coli suppresses spontaneous mutagenesis and sensitivity to redox-cycling agents in oxyR-mutants.

Mutations that suppressed the H2O2 sensitivity of Escherichia coli oxyR- strains caused elevated levels of one three enzymes that destroy organic and hydrogen peroxides: catalase-hydroperoxidase I (the katG gene product), catalase-hydroperoxidase II (controlled by katEF) or alkyl hydroperoxide reductase (specified by the ahp genes). The continuous high-level expression of any one of these enzymes also conferred resistance in an oxyR deletion mutant against other compounds such as N-ethylmaleimide and the superoxide-generator menadione. Overproduction of alkyl hydroperoxide reductase, but not of the catalases, gave resistance to the organic oxidant cumene hydroperoxide. The E. coli delta oxyR strains also exhibited a strongly elevated frequency of spontaneous mutagenesis, as reported for such mutants in Salmonella typhimurium. This mutagenesis was greatly diminished by the individual overexpression of these scavenging enzymes. All of these phenotypes--enzyme overproduction, resistance to oxidants and suppression of spontaneous mutagenesis--remained linked upon transduction of the mutant katG or ahp genes. Peroxides thus appear to mediate the toxicity of a variety of redox agents, and are produced in sufficient quantity during normal metabolism to cause a substantial increase in 'spontaneous' mutations in cells that lack adequate antioxidant defenses.     

Pyrethroid resistance in Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) and implications for its management in Australia

Abstract  The study was conducted to characterise the underlying resistance mechanisms responsible for high levels of pyrethroid resistance in Frankliniella occidentalis (Pergande) in Australia. Seven commercially available pyrethroids (acrinathrin, alpha-cypermethrin, bifenthrin, deltamethrin, esfenvalerate, permethrin and tau-fluvalinate) were evaluated against seven F. occidentalis strains collected from ornamentals, fruit and vegetables in three states of Australia. A Potter spray tower was used to test for pyrethroid resistance and all field strains were found to be resistant, with resistance ratios ranging from 15-fold deltamethrin to 1300-fold tau-fluvalinate. The two most resistant strains were further tested for detoxification enzymes that could be involved in resistance. Three synergists, piperonyl butoxide (PBO), diethyl maleate (DEM) and profenofos, which, respectively, inhibit the enzymes cytochrome P-450 monooxygenases, glutathione S -transferases and esterases, were used. The synergism data indicate that multiple mechanisms may be involved in pyrethroid resistance in Australian populations of F. occidentalis . Among the three synergists, PBO considerably reduced pyrethroid resistance in the selected strains compared with DEM and profenofos. The practical implication for PBO use to suppress pyrethroid resistance in F. occidentalis is elaborated.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability I. Dihydrofolate reductase,thymidylate synthethase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems

Three strains of Listeria monocytogenes (NCTC 11994, a poultry isolate and the Scott A strain) were exposed to a range of pressures (300, 350, 375, 400 and 450 MPa) in 10 mmol l⁻¹ phosphate-buffered saline (PBS) at pH 7·0 for up to 30 min at ambient temperature. Generally, increasing the magnitude and duration of compression resulted in increasing levels of inactivation, although the inactivation kinetics varied depending on the strain and pressure applied. The three strains also exhibited a wide variation in their resistance to high pressure. The resistance of the three strains to high pressure (375 MPa) was also assessed in a series of model food systems containing one of each of the three main food constituents: protein (1, 2, 5 and 8% w/v bovine serum albumin in PBS), carbohydrate (1, 2, 5 and 10% w/v glucose in PBS) and lipid (olive oil (30% v/v) in PBS emulsion). Overall, increasing the concentrations of bovine serum albumin (BSA) and glucose in the suspending medium resulted in decreasing levels of inactivation of all three strains; however, the minimum concentration of BSA and glucose required to increase survival to a level greater than that observed in PBS alone varied depending on the strain and on the duration of the treatment. The survival of all three strains was greater in the olive oil/PBS emulsion than in PBS alone at all treatment times.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability : II. Permeability changes to amethopterin and other folates in the drug-resistant mutant

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

The potential for enhanced fungicide resistance in Beauveria bassiana through strain discovery and artificial selection

Our objectives were to determine the (1) natural variation in fungicide resistance among Beauveria bassiana strains, (2) potential to increase fungicide resistance in B. bassiana through artificial selection, and (3) stability of virulence in selected B. bassiana strains. Fungicides included dodine, fenbuconazole, and triphenyltin hydroxide, which are commonly used in pecan and other horticultural crops. Comparison of seven B. bassiana strains indicated some are substantially more resistant to fungicides than others; a commercial strain (GHA) was less resistant than all wild strains isolated from pecan orchards. Artificial selection resulted in enhanced fungicide resistance in the GHA strain but not in a mixed wild strain. Removal of selection pressure for three passages did not reduce the enhanced fungicide resistance. Sub-culturing with exposure to fungicides did not affect the GHA strain's virulence to pecan weevil, Curculio caryae, larvae, whereas fungicide exposure increased virulence in a mixed wild population of B. bassiana.     

Effect of synergists on the oral and topical toxicity of azamethiphos to organophosphate-resistant houseflies (Diptera: Muscidae).

Dermal and oral toxicities of azamethiphos were determined in two organophosphate-resistant and one susceptible strain of houseflies, Musca domestica L. The 594vb strain was 1,967-fold more resistant to azamethiphos when compared with the susceptible Chemical Specialties Manufacturers Association (CSMA) strain by dermal application. When the compound was administered orally to the 594vb strain, we observed only a 15-fold resistance. In contrast, the Yachiyo strain, which show 1,500-fold resistance to diazinon and which has known multiple mechanisms for organophosphate resistance, showed only 6-fold resistance to azamethiphos by topical application and 4-fold resistance by oral administration. Azamethiphos administered dermally and orally was equally toxic to the CSMA and Yachiyo strains. However, when azamethiphos was administered to the 594vb strain, the insecticide was 71 times more toxic orally than by the dermal route. This result indicated involvement of a cuticular penetration factor in the resistance mechanism. The effect on azamethiphos toxicity of piperonyl butoxide (PB), an inhibitor of the monooxygenases, and tributylphosphorotrithioate (DEF), an esterase inhibitor, was investigated in the three strains. Pretreatment of the flies with PB, DEF, or PB+DEF before topical application of azamethiphos resulted in a significant decrease in LD50s in all the strains. The degree of synergism, however, varied depending upon the strains and the synergists. Similar results were obtained when azamethiphos was administered orally following pretreatment of the flies with PB+DEF. We attribute the high level of azamethiphos resistance in the 594vb strain partly to increased degradation by oxidative and hydrolytic enzymes. The hydrolytic enzymes are more important, but other factors including reduced cuticular penetration and insensitive acetylcholinesterase may be involved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of the aminoglycosidestreptothricin resistance gene cluster ant(6)-sat4-aph(3 ')-III in commensal viridans group streptococci.

High-level aminoglycoside resistance was assessed in 190 commensal erythromycin-resistant alpha-hemolytic streptococcal strains. Of these, seven were also aminoglycoside-resistant: one Streptococcus mitis strain was resistant to high levels of kanamycin and carried the aph(3 ')-III gene, four S. mitis strains were resistant to high levels of streptomycin and lacked aminoglycoside-modifying enzymes, and two S. oralis strains that were resistant to high levels of kanamycin and streptomycin harbored both the aph(3 ')-III and the ant(6) genes. The two S. oralis strains also carried the ant(6)-sat4- aph(3 ' ')-III aminoglycoside-streptothricin resistance gene cluster, but it was not contained in a Tn5405-like structure. The presence of this resistance gene cluster in commensal streptococci suggests an exchange of resistance genes between these bacteria and enterococci or staphylococci.     

Trimethoprim resistance in enterococci: microbiological and biochemical aspects

Synergy was found between sulphonamide and trimethoprim in ratios 1:1 and 20:1 against both trimethoprim-sensitive enterococci (17 strains) and trimethoprim-resistant enterococci (23 strains). Many of these strains were resistant to kanamycin, tetracycline, streptomycin and/or erythromycin. Resistance to kanamycin, but not to trimethoprim, was clearly associated with the presence of a plasmid of molecular weight 35-45 Md. Elimination of this plasmid in three out of four highly trimethoprim resistant strains brought about loss of resistance to both kanamycin and trimethoprim. Furthermore, it was possible to transfer trimethoprim resistance from three of five highly resistant strains, but not from three strains with low-grade resistance. It is concluded that resistance to trimethoprim in enterococci can be encoded on a plasmid, and that the gene responsible may be on a transposon. No significant differences were found between the specific activities of dihydrofolate reductase from trimethoprim-sensitive and -resistant strains. The enzyme from resistant strains was several orders of magnitude less susceptible to inhibition by trimethoprim than was the enzyme from sensitive strains.     

In Vitro Induced Anaerobic Resistance to Metronidazole In Trichomonas Vaginalis

ABSTRACT. Resitance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 μ/ml) for 12 to 21 months. the resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 μ/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 μ/ml at 48-h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 μ/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 μ/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 μ/ml metronidazole. the strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [¹⁴C]-metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.     

Effects of Regulatory Mutations upon Methionine Biosynthesis in Saccharomyces cerevisiae: Loci eth2-eth3-eth10

The effects of mutations occurring at three independent loci, eth2, eth3, and eth10, were studied on the basis of several criteria: level of resistance towards two methionine analogues (ethionine and selenomethionine), pool sizes of free methionine and S-adenosyl methionine (SAM) under different growth conditions, and susceptibility towards methionine-mediated repression and SAM-mediated repression of some enzymes involved in methionine biosynthesis (met group I enzymes). It was shown that: (i) the level of resistance towards both methionine analogues roughly correlates with the amount of methionine accumulated in the pool; (ii) the repressibility of met group I enzymes by exogenous methionine is either abolished or greatly lowered, depending upon the mutation studied; (iii) the repressibility of the same enzymes by exogenous SAM remains, in at least three mutants studied, close to that observed in a wild-type strain; (iv) the accumulation of SAM does not occur in the most extreme mutants either from endogenously overproduced or from exogenously supplied methionine: (v) the two methionine-activating enzymes, methionyl-transfer ribonucleic acid (tRNA) synthetase and methionine adenosyl transferase, do not seem modified in any of the mutants presented here; and (vi) the amount of tRNA^met and its level of charging are alike in all strains. Thus, the three recessive mutations presented here affect methionine-mediated repression, both at the level of overall methionine biosynthesis which results in its accumulation in the pool, and at the level of the synthesis of met group I enzymes. The implications of these findings are discussed.     

Development and stability of insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad

Three populations of the leafminer, Liriomyza trifolii (Burgess), were collected from commercial ornamental production greenhouses in the United States and tested for susceptibility to three commercial insecticides. A leaf dip bioassay of leaves containing young (1-2-d-old) larvae was used. Based on larval mortality and compared with a susceptible laboratory reference colony, the three strains varied in spectrum and level of resistance to the insecticides. CA-1, collected from Gerbera daisy, was moderately resistant to cyromazine (18.1-fold) and abamectin (22.0-fold), but highly resistant to spinosad (> 188-fold). CA-2, collected from chrysanthemums, was not resistant to abamectin, had a low level of resistance to cyromazine (8.2-fold), but was extremely resistant to spinosad (1,192-fold). GA-1, collected from chrysanthemums, had very low levels of resistance to cyromazine (5.4-fold) and spinosad (1.9-fold) but was moderately resistant to abamectin (30.6-fold). When reared in the absence of insecticide selection pressure, all three strains reverted to approximately the level of the reference strain. The CA-1 strain reverted in nine generations to cyromazine; however, the lowest levels of abamectin and spinosad resistance reverted to was 3.1-fold at F8 and 3.2 at the F10, respectively. The CA-2 strain reverted in five generations to both cyromazine and spinosad. GA-1 reverted in five generations to abamectin. Based on the results, resistance to these three insecticides was unstable. Additionally, there was no cross-resistance among these three insecticides.     

Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes

Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites.     

Comparison of the catalytic and immunological properties of beta-glucosidases from three strains of Saccharomyces lactis

beta-Glucosidase from Saccharomyces lactis strains Y-123 (B(h)), Y-14 (B(m)), and Y-1057A (B(1)) was partially purified. The pH optima, Michaelis constants, and activation energies were determined for the hydrolysis of p-nitro-phenyl-beta-d-glucoside by each of the enzymes. Differences among these constants were not enough to account for the low specific activity of beta-glucosidase in strains Y-14 and Y-1057A. Enzyme-inhibitor constants were measured for a series of alkyl and aryl glucosides. In general, the three enzymes are arylglucosidases. Tris(hydroxymethyl)-aminomethane inhibited all three enzymes in an uncompetitive fashion. The inhibition was antagonized by Mg(++). An antiserum was prepared to the highly purified (200-fold) beta-glucosidase from strain Y-123. The nature and degree of cross-reaction between the three beta-glucosidases was investigated by double diffusion in agar and neutralization tests. Spur formation in the immunodiffusion tests and similar equivalence points in the neutralization tests indicated a strong degree of cross-reaction between the three enzymes. The ratio of enzyme activity to antigenicity was used to compare the relative molecular activity of beta-glucosidase in the three strains. Each strain produced the same amount of beta-glucosidase per milligram of cell protein. The results are consistent either with a lower turnover number for the beta-glucosidase in strains Y-14 and Y-1057A or with the production of beta-glucosidase with a "normal" turnover number and enough cross-reacting material to effectively reduce the specific activity to the observed levels.     

Does cypermethrin affect enzyme activity,respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

Insecticides cause a range of sub‐lethal effects on targeted insects, which are frequently detrimental to them. However, targeted insects are able to cope with insecticides within sub‐lethal ranges, which vary with their susceptibility. Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) to sub‐lethal exposure to the pyrethoid insecticide cypermethrin. We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure. Lower respiration rate and lower activity were also expected in insecticide‐resistant insects since these traits are also likely to favor survivorship under insecticide exposure. Curiously though, cypermethrin did not affect activity of digestive and energy metabolism enzymes, and even reduced the activity of some enzymes (particularly for cellulase and cysteine‐proteinase activity in this case). There was strain variation in response, which may be (partially) related to insecticide resistance in some strains. Sub‐lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects, which is likely to impair their fitness. However, such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced). Walking activity varies with strain and may minimize insecticide exposure, which should be a concern, particularly if associated with (physiological) insecticide resistance.