Decolorization and biodegradation of Reactive Blue 13 by Proteus mirabilis LAG.

The decolorization and biodegradation of Reactive Blue 13 (RB13), a sulphonated reactive azo dye, was achieved under static anoxic condition with a bacterial strain identified as Proteus mirabilis LAG, which was isolated from a municipal dump site soil near Lagos, Nigeria. This strain decolorized RB13 (100mg/l) within 5h. The formation of aromatic amine prior to mineralization was supported by Fourier transform infrared spectrometry (FTIR), which revealed the disappearance of certain peaks, particularly those of the aromatic C-H bending at 600-800 cm(-1). Gas chromatography-mass spectrophotometry (GCMS) analysis of the dye metabolite showed the presence of sodium-2(2-formyl-2-hydroxyvinyl) benzoate, with a tropylium cation as its base peak, this suggested the breakage of naphthalene rings in RB13. The detection of azoreductase and laccase activities suggested the enzymatic reduction of azo bonds prior to mineralization. In addition, phytotoxicity studies indicated the detoxification of RB13 to non-toxic degradation products by this strain of P. mirabilis LAG.

Substrate profile variation and drug resistance patterns of â - lactamase producing Shigella species isolated from diarrhoeal patients in Lagos, Nigeria.

The number and trend of antibiotic resistance by Shigella species recovered from food and diarrhoeal stools are on the increase in Nigeria and has resulted in a high frequency of hospitalisation. Increased cost of disease management, and higher mortality in children. This study exposes 51 â-lactamase producing Shigella isolates from Lagos to some newly introduced drugs in the country. The drugs include â-lactam - â-actamase inhibitor antibiotics. â-lctam substrate hydrolysis and inhibitory effects of clavulanate were also investigated in-vitro. Results obtained revealed that all the isolates showed high level resistance to tetracycline, ampicillin, streptomycin, co-trimoxazole and amoxicillin with an MIC range of 128 - 1024 microg/ml. The isolates were susceptible to piperacillin, tobramycin, aztreonam and ofloxacin (0.03 - 8 microg/ ml). 18.2 - 40.9% of S. flexneri and S. dysenteriae showed low level resistance to cefuroxime and cefotaxime (MIC = 4 - 16 microg/ ml). Among the â-lactam - â- lctamase inhibitors tested, only piperacillin-tazobactam showed 100% resistance. Hydrolysis of â -lctam substrate was found to be species dependent in decreasing order of S. flexneri, S. dysenteriae, S. sonnei and S.boydii. An IC50 range of 0.8-2.4 mM was also observed in these isolates. Our data indicate that the incidence of multidrug resistance is high among â-lactamase producing Shigella isolates in Lagos, Nigeria. While the third generation cephalosporins should be used with cautions, some of the newly introduced drugs have the prospects of being used in the future control and management of shigellosis in the country.

Patterns and properties of haemagglutinins expressed by Shigella serogroups in Lagos, Nigeria.

Forty-five strains of Shigella were screened for haemagglutinin production and broad-spectrum haemagglutination reaction. Mannose-sensitive haemagglutinin (MSHA) was found in 22 strains [Shigella flexneri (7), S. dysenteriae (7), S. sonnei (3), and S. boydii (5)]. Eighteen strains harboured mannose-resistant haemagglutinin (MRHA), and 8 strains were observed to be non-haemagglutinating to guinea pig erythrocyte. With the exception of human erythrocytes (O, A, B, and AB), the observed MSHA and MRHA also agglutinated the erythrocytes of rabbit, sheep, rat, chicken, and horse, suggesting a broad-spectrum haemagglutinating property. Haemagglutinins of S. flexneri and S. dysenteriae elicited a relatively stronger haemagglutinating activity with agglutinability to chicken and rabbit erythrocytes enhanced by trypsinization. Haemagglutination reaction with guinea pig erythrocyte was generally inhibited by sialic acid, while simple sugars, such as D-glucose, D-galactose, N-acetylgalactosamine, N-acetylglucosamine, and D-rhamnose, elicited no inhibitory effect. The results of the study revealed broad-spectrum haemagglutinin expression by circulating Shigella strains in Nigeria.

Effects of Ocimum gratissimum L essential oil at subinhibitory concentrations on virulent and multidrug-resistant Shigella strains from Lagos, Nigeria.

Ocimum gratissimum leaf extracts have been extensively demonstrated to be effective against the various aetiologic agents of diarrhoea, including Shigellae. However, the mechanism of the shigellocidal action of this plant remains to be understood. This study investigated the effects of O. gratissimum essential oil (EO) at subinhibitory concentrations of 0.75 and 1.0 microg/ml on virulence and multidrug-resistant strains of 22 Shigella isolates from Nigeria. Compared with untreated Shigella strains, O. gratissimum EO caused significant decreases (p<0.01) in extracellular protease activity, o-lipopolysaccharide rhamnose content and incidence of invasiveness mediated as keratoconjunctivitis in guinea pig. The disparity in extracellular protease activity and o-lipopolysacharide rhamnose between the two treatment groups was also found to be significant (p<0.05), suggesting greater anti-virulent effects of O. gratissimum oil at 1.0 microg/ml. Antibiotic susceptibility testing revealed that the EO of O. gratissimum reduced the MICs of antibiotics to which Shigellae showed resistance by 9.8-53.1% and fluoroquinolones by 18.2-45.5%. The results of this study strongly suggest inhibition of extracellular protease and expression of O-LPS rhamnose in Shigellae by O. gratissimum EO. The future use of O. gratissimum- antibiotic combinations as a therapeutic measure against shigellosis is discussed.

Evidence for chromosomal determination of fungicidal activity in strains of Lactobacillus brevis and Lactobacillus fermentum isolated from fermented foods.

The genetic basis of the fungicidal activity of strains of Lactobacillus brevis and L. fermentum isolated from indigenous fermented foods was determined. A 5.5-kb plasmid was isolated from L. brevis while L. Fermentum was found to harbor no plasmid. Plasmid curing indicated no correlation between the plasmid and the fungicidal activity of the Lactobacillus species. The fungicidal activity of the isolated organisms can be supposed to be mediated by the chromosome. No antibiotic resistance genetic markers were detected on the plasmid and hence it was classified as cryptic.

Epidemiology of shigellosis in Lagos, Nigeria: trends in antimicrobial resistance.

Antimicrobial susceptibility of Shigella spp. and Escherichia coli, isolated from diarrhoeal patients in Lagos, was studied from March 1999 to February 2000. Four hundred fifty-nine isolates were identified as shigellae (62) and E. coli (397). Shigella flexneri, S. dysenteriae, S. boydii, and S. sonnei accounted, respectively, for 51.6%, 17.7%, 17.7%, and 13% of the total number of shigellae isolated. Eleven cases of shigellosis occurred in the age group of 0-9 years, 22 cases in the age group of 10-19 years, and 29 cases in the age group of > or = 20 years. Of the 397 E. coli isolates, 11 were enteropathogenic E. coli (EPEC), and 7 of these strains were isolated with shigellae from stools of patients aged 0-9 year(s) (71.4%) and 10-19 years (28.6%). Over 70% of the Shigella isolates were resistant to two or more drugs, including ampicillin and tetracycline. Twenty-one distinct multidrug resistance patterns were observed in these isolates. During 1990-2000, resistance to ampicillin increased from 70% to 90%, co-trimoxazole from 77% to 85%, chloramphenicol from 71% to 77%, streptomycin from 71% to 79%, and nalidixic acid from 0% to 11.3%. Resistance to tetracycline decreased from 89% to 79% but with MIC50 and MIC90 values outside the susceptible range. While resistance to ciprofloxacin and ofloxacin remained nil with MIC50 and MIC90 values of 0.008 and 0.0016 microgram/mL respectively. The results of this study revealed the endemicity of shigellosis with S. flexneri as the predominant serogroup in Lagos. Children and young adults were at a higher risk of severe shigellosis. The results also suggest that ampicillin, tetracycline, co-trimoxazole, and streptomycin should not be used as the first-line drugs in the treatment of shigellosis. Nalidixic acid should still be selectively used for treatment, while ciprofloxacin and ofloxacin can be ideal alternatives.

Shigellocidal properties of three Nigerian medicinal plants: Ocimum gratissimum, Terminalia avicennoides, and Momordica balsamina.

The prevalence of multidrug-resistant shigellae is an important concern in the treatment of shigellosis in many developing countries, and other therapies, including herbal agents, may provide an important alternative to antimicrobial agents. In this study, three Nigerian medicinal plants: Ocimum gratissimum, Terminalia avicennoides, and Momordica balsamina were investigated for their activities against multidrug-resistant Shigella species isolated from patients with bacilliary dysentery in Lagos. Decoctions of O. gratissimum and concoctions of O. gratissimum and T. avicennoides at crude concentration of 3,000 micrograms/mL markedly inhibited the growth of all isolates tested. Zones of inhibition indicating susceptibilities of the organisms varied from 18.3 to 21.5 mm for Shigella dysenteriae, 15.3 to 16.3 mm for S. flexneri, 18.8 to 19.3 mm for S. sonnei, and 16.5 mm for S. boydii. Except S. flexneri, minimum inhibitory concentration and minimum bactericidal concentration revealed a higher shigellocidal property of O. gratissimum/T. avicennoides concoction than other extracts in S. dysenteriae (300-515.6 vs 337.5-1,312.5 micrograms/mL), S. sonnei (309.4-543.8 vs 403.1-1,312.5 micrograms/mL), and S. boydii (243.8-337.5 vs 253-1,312.5 micrograms/mL). O. gratissimum showed a greater shigellocidal effect against the S. flexneri isolates, while extracts of M. balsamina possessed low shigellocidal potential. The results suggest that aqueous extracts of O. gratissimum and T. avicennoides as decoctions and concoctions could be useful in the treatment of shigellosis and should be clinically evaluated specially in Nigerian region.

PCR-based identification of Bacillus thuringiensis isolated from soil samples in Nigeria.

Six isolates of Bacillus thuringiensis isolated from soil samples confirmed to be toxic to mosquito larvae were differentiated using a PCR-Based technique. Three of these isolates initially identified using a serological technique were further differentiated with the PCR amplification of the delta-endotoxin target sequences. Using the total DNA of isolates as template, at least four isolates yielded amplicons one or all the crystal protein genes, cryI a, b, c, or II with sizes ranging from 238-1070 bp. None of these isolates yielded an amplicon for any of Cry IV A, B and D tested. Of the four isolates identified by PCR technique one isolate remained unidentified by serology.

Characterization of a filtrable 3'-nucleotidase of Leishmania donovani.

A plasma membrane 3'-nucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) having a apparent subunit molecular mass of 38 kDa but filtrable through a Centriprep-10 microconcentrator (Amicon) membrane was purified from Leishmania donovani promastigotes. The enzyme activity has an optimum at pH around 7.5. EDTA strongly inhibited the enzyme activity which was fully restored only by Co2+, from various metal ions added. Citrate ions, Zn2+ or dithiothreitol were also strongly inhibitory. The enzyme is apparently located on the inner side of the parasite plasma membrane. The substrate specificity and kinetics of the filtrable enzyme are similar to those of the non-filtrable outer-surface-membrane-bound 3'-nucleotidase reported by Gbenle and Dwyer (Gbenle, G.O. and Dwyer, D.M. (1992) Biochem. J. 285, 41-46). Therefore, it is suggested that both enzymes are implicated in the supply of nucleosides to the parasite.

Purification and properties of 3'-nucleotidase of Leishmania donovani.

A surface membrane 3'-nucleotidase from Leishmania donovani promastigotes has been purified to SDS/PAGE homogeneity. The enzyme has apparent subunit molecular mass of 38 kDa, pI 5.8 and a broad pH optimum, 5.5-7.5. EDTA partially inhibited the enzyme activity, which was fully restored by Co2+; Mg2+, Ca2+ or Mn2+ had no effect on the activity. ZnCl2 or dithiothreitol at 1 mM was inhibitory at pH 7.5, but was without effect at pH 5.5, whereas at both pH values 5 mM of either compound inhibited the enzyme. The substrate-specificity of the purified enzyme is restricted to ribonucleoside 3'-phosphates. 3'-AMP and 3'-IMP are the best substrates, whereas ADP, ATP, 2'-deoxyadenosine 3'-phosphate and 5'-AMP are competitive inhibitors of the enzyme. The enzyme showed low latency in intact-cell preparations. The kinetic properties and the surface membrane localization of the enzyme suggest its implication in the formation of nucleosides from 3'-nucleotides of the parasite's host.

Trypanosoma brucei: calcium-dependent endoribonuclease is associated with inhibitor protein.

T. brucei cytoplasmic calcium-dependent alkaline ribonuclease activity from DEAE-cellulose fractionation was separated into endoribonuclease and exoribonuclease activities by hydroxyapatite chromatography. T. brucei cytoplasmic extract markedly decreased the endoribonuclease activity, but slightly potentiated the activities of the exoribonuclease and bovine ribonuclease A. While the endoribonuclease was activated by trypsin, the exoribonuclease and bovine ribonuclease A were partially inactivated by trypsin. The endoribonuclease was activated by p-chloromercuribenzoate or N-ethylmaleimide; the exoribonuclease was not affected by these sulfhydryl group reagents. Free ribonuclease was separated from the latent endoribonuclease by 1 M NaCl-Sephadex G-100 gel filtration. The results demonstrate that T. brucei cytoplasm contains a latent endoribonuclease consisting of ribonuclease and inhibitor protein.

Study of plasmid screening amongst pathogenic bacteria isolated in Nigeria.

Clinical isolates of Neisseria gonorrhoeae, Campylobacter jejuni, Escherichia coli, Shigella dysenteriae, Shigella boydii, Yersinia spp. and Salmonella spp. were screened for the presence of plasmids. Approximately 80% of these strains harboured plasmids ranging in molecular weight from 1.0 to 45 x 10(6) daltons.

Localization of 3'-nucleotidase and calcium-dependent endoribonuclease in the plasma-membrane of Trypanosoma brucei.

We have characterized a 3'-nucleotidase activity of T. brucei. The enzyme has a pH optimum of 8.7, is inactivated by chelating agents and stimulated by divalent cations. It is inhibited by Zn2+, Mn2+, pyrophosphate and the trypanocidal drug suramin for which it has a Ki of 3 microM. From cell fractionation experiments it is concluded that the enzyme is located in the plasma membrane. Alkaline 3'-endoribonuclease is also located in the plasma membrane of T. brucei and this activity shares a great number of properties with the 3'-nucleotidase activity, including its sensitivity to suramin. The possibility that both 3'-nucleotidase and endonuclease activities are catalyzed by the same enzyme cannot be excluded.

Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates.

A method for detecting structure in marginally stable forms of a protein is described. The principle is to measure amide proton exchange rates in the absence and presence of varying concentrations of a denaturant. Unfolding of structure by the denaturant is reflected by an acceleration of amide proton exchange rates, after correction for the effects of the denaturant on the intrinsic rate of exchange. This exchange-rate test for structure makes no assumptions about the rate of exchange in the unfolded state. The effects of 0-8 M urea and 0-6 M guanidinium chloride (GdmCl) on acid- and base-catalyzed exchange from model compounds have been calibrated. GdmCl does not appear to be well-suited for use in the exchange-rate test; model compound studies show that the effects of GdmCl on intrinsic exchange rates are complicated. In contrast, the effects of urea are a more uniform function of denaturant concentration. Urea increases acid-catalyzed, and decreases base-catalyzed, rates in model compounds. The exchange-rate test is used here to study structure formation in the S-protein (residues 21-124 of ribonuclease A). In conditions where an equilibrium folding intermediate of S-protein (I3) is known to be populated (pH 1.7, 0 degree C), the exchange-rate test for structure is positive. At higher temperatures (greater than 32 degrees C) I3 is unfolded, but circular dichroism data suggest that residual structure remains [Labhardt, A. M. (1982) J. Mol. Biol. 157, 357-371].(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous isolation of cytoplasmic endoribonuclease and exoribonuclease of Trypanosoma brucei.

An endoribonuclease and an exoribonuclease have been isolated simultaneously from the cytoplasm of Trypanosoma brucei by hydroxyapatite column chromatography. The endoribonuclease produced oligonucleotides from poly(adenylic acid) with 5'-phosphate and 3'-OH termini. The exoribonuclease produced only ribonucleoside 5'-phosphates from poly(adenylic acid). The relative rates of degradation of synthetic homopolynucleotides by the endoribonuclease under standard conditions were in the order poly(adenylic acid) greater than poly(uridylic acid) poly(cytidylic acid); for the exoribonuclease the order was poly(adenylic acid) poly(uridylic acid) greater than poly(cytidylic acid). Natural transfer and ribosomal RNAs were also degraded by both enzymes, while DNA was resistant to them. The optimal pH of activity for each enzyme was 7.5-8.0. Both ribonucleases require Ca2+ for maximum enzymatic activity.

Calcium-dependent endoribonuclease of Trypanosoma brucei has a base-preferential affinity to adenylate.

The substrate specificity of a calcium-dependent endoribonuclease of Trypanosoma brucei cytoplasm has been further determined. The actions of the enzyme on transfer RNA, ribosomal RNA and various synthetic polyribonucleotides indicate that the enzyme degrades double-stranded as well as single-stranded RNAs; while it preferentially hydrolyses polyribonucleotides having adenylic acid residues, and has a pronounced preference for poly (adenylic acid). Its apparent Michaelis constant (Km) values using different substrates also suggest a base-preferential affinity of the enzyme to adenylate. The relative activity of the ribonuclease against homopolyribonucleotides is poly(A) greater than poly(U) greater than poly(C); while poly(G) is completely resistant to the activity. Poly(A) segments on poly(A)-rich RNA are selectively hydrolyzed by the endoribonuclease. A possible implication of this enzyme in the post-transcriptional modification and turnover of mRNA molecules is suggested.

A calcium dependent endoribonuclease from Trypanosoma congolense cytoplasm.

A major endoribonuclease whose activity depends on the presence of calcium ions was isolated from the cytoplasm of Trypanosoma congolense. Like the Ca2+-dependent endoribonuclease of T. brucei cytoplasm, this enzyme degraded poly(A) preferentially but also degraded to a lesser extent poly(U), tRNA and rRNA to give 5'-phosphoribosyl oligonucleotides. Neither enzyme hydrolyzes poly(G) or DNA. Both ribonucleases are inhibited by polyanions and heavy metal ions, and are heat-labile; N-ethylmaleimide has no effect on either activity. However, unlike the Ca2+-RNase of T. brucei cytoplasm, this endoribonuclease is also stimulated by Mg2+, acts maximally at around pH 8.9, and is virtually inactivate towards poly(C). It is inhibited by iodoacetamide but unaffected by spermine. Its molecular weight is about 42 000 as determined by gel filtration.

Studies on the catalytic properties of the calcium-dependent endoribonuclease of Trypanosoma brucei cytoplasm.

A calcium-dependent endoribonuclease isolated from Trypanosoma brucei cytoplasm has been further characterized. The products of exhaustive endonuclease digestion of poly(A), which the enzyme cleaved preferentially, were small oligonucleotides terminated by a 5'-phosphoryl group. Its apparent Km value was 0.8 mM using poly (A), and 3.3 mM using transfer RNA. The base specificity of the endoribonuclease was further verified by using synthetic heteropolyribonucleotides as substrates. The enzymatic reaction was inhibited by polyamines, polyanions, iodoacetic acid and the heavy metals, but only very slightly by N-ethylmaleimide and iodoacetamide. A possible implication of the enzyme in the turnover of messenger RNA is discussed.