Prevalence and antibiotic resistance profiles of Gram negative bacilli associated with urinary tract infections (UTIs) in Karachi, Pakistan.

The aim of this investigation was to determine the prevalence and antibiotic resistance profiles of Gram negative bacilli (GNB) responsible for urinary tract infections (UTIs). Urine specimens were cultured on Cysteine Lactose Electrolyte Deficient Agar (CLED) medium and pathogenic GNB were identified by conventional biochemical methods and automated profile index (API) system and further subjected to antibiotic sensitivity testing by disk diffusion method. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were encountered as most frequent GNB in sequence. Among them E. coli (71%) was the most prevalent GNB. About 77% E. coli isolates of indoor patients and 59% of outdoor patients were found resistant to Cefotaxime. Kleb. pneumoniae were 100% resistant to Ampicillin. Higher resistance in Ps. aeruginosa was noticed in isolates of indoor patients i.e. Ciprofloxacin (76%), Cefoperazone-sulbactam (60%), Ceftazidime (59%), Piperacillin-tazobactam (53%), Imipenem (49%) and Amikacin (39%) in contrast to that of outdoor patients. Slightly lower resistance in Acinetobacter baumannii against Ampicillin (86%), Nitrofurantoin (81%) and Fosfomycin (12%) was witnessed in indoor patients' urine specimens compared to outdoor patients' urine. Polymyxin B, Imipenem, Fosfomycin, Piperacillin-tazobactam, Cefoperazone-sulbactam, Amikacin and Nitrofurantoin were most effective in GNB induced UTIs. This study revealed elevated resistance profiles in GNB against Ampicillin, Amoxicillin-clavulanate, Cefotaxime, Aztreonam, Ciprofloxacin, Nalidixic acid and Trimethoprim/sulfamethoxazole. Emergence of antibiotic resistant GNB was due to the frequent use and misuse of antibiotics in our region.

Industrially important hydrolytic enzyme diversity explored in stove ash bacterial isolates.

Extreme environments merit special attention and significance because of the possible existence of thermophilic microorganisms in such ecological niches. Keeping this in mind indigenous stove ash samples were explored for extremophilic bacteria in term of their biodiversity. Accordingly, this study reports 37 bacterial isolates from the local wood run oven (Tandoor) ash samples. All the isolated strains belong to genus Bacillus on the bases of morpho-cultural and biochemical considerations. The average temperature tolerance profile was >45°C thereby, indicating towards the thermophilic nature of the isolated strains. The Bacillus isolates were screened for 10 different hydrolytic enzymes (cellulase, xylanase, amylase, pectinase, caseinase, keratinase, lipase, esterase, dextranase and ß-galactosidase) by plate screening method using the medium incorporated with specific substrate(s). It was found that keratinase was produced by all the isolates while, 36 (97.2%) isolates showed caseinase and esterase production. Amylase was produced by 35(94.6%) isolates and 34 (91.8%) isolates were able to degrade Tween-80 and xylan as substrate for lipase and xylanase respectively. The enzyme, ß-galactosidase was produced by 31 (89.1%) of the isolates. Cellulase and dextranase were produced by 26 (70.2%) and 22 (59.4%) isolates respectively. None of the isolates could (under the existing conditions) produce pectin-hydrolyzing enzyme. According to the Tukey's post hoc test, significant difference was found between the mean enzyme index of all the (screened) enzymes. Thus, the isolated bacterial strains with diverse hydrolytic potential may be of great value and relevance for the existing (national) industrial setups.

Evolutionary root defines the structural basis of high and low level vancomycin resistance in enterococci.

D-alanyl-D-lactate (Dlac) and D-alanyl-D-serine (Dser) ligases respectively mediates high and low level vancomycin resistance among enterococci. To date, the evolutionary relationship of both ligases is largely unaddressed. Also poorly understood are the molecular differences in the magnitude of vancomycin resistance. To address the mention, we constructed the phylogenetic tree of all vancomycin resistance conferring ligases with the wild type ligases (Dala). Multiple sequence alignment and tertiary structures of the structurally unresolved proteins were constructed by homology modeling. Phylogenetic tree revealed that both Dlac and Dser are profoundly different from Dala as a result of continuous selection pressure. Separate clustering of Dlac and Dser also highlighted the structural basis of molecule in maintaining different level of resistance as exhibited by the bacteria. This notion was further augmented as the functionally key region, omega loop (ω-loop), was found relatively more structured in only Dlac. Moreover, the critically active residue, His-243/244, was also noticed to be restricted in Dlac and found replaced by non polar residues in Dser. The present study not only provides protein structural explanation of the different intensities of vancomycin resistance among enterococci, but also presents yet another example for the scope of evolutionary science in biomedicine.

Isolation-drug resistance profile and molecular characterization of indigenous typical and atypical mycobacteria.

One hundred and fifty mycobacterial isolates from different pathological Labs. of Karachi were collected and screened as acid fast. On the bases of phenotypic and biochemical results, it was found that, 58.66% isolates were typical mycobacteria while 41.33% belonged to atypical mycobacteria. The individual percentages of different mycobacterial species include: M. xenopi 35%, M. thermoresistible 19 %, M. terrae complex 6 %, M. marinum 6 %, M. fortuitum 6 %, M. kansasii 25 % and M. tuberculosis 58.66 %. The sensitivity of mycobacterial isolates was determined against 5 first line, 3 second line and 1 third line anti-tuberculosis drugs. The highest number of the isolates (typical and atypical mycobacteria) offered resistance against isoniazid and streptomycin. Clarithromycin was found to be the drug of choice as regards the drug sensitivity in case of atypical mycobacterial isolates. A total of 40 isolates were subjected to PCR based identification and differentiation of 16S rRNA gene(s). Accordingly, 37.5% isolates were identified as typical mycobacteria while 25% were identified as atypical mycobacteria. These findings carry significance because a detailed research based identification (PCR and Multiplex PCR based) regarding indigenous mycobacteria has been reported for the first time in Pakistan. However, both the approaches (conventional and molecular methods) have experimental importance while identifying these organisms.

Bacteriocin production by indigenous marine catfish associated Vibrio spp.

Fifty strains of genus Vibrio were isolated (identified) from healthy and diseased marine catfish(es). The isolates were screened for bacteriocin (vibriocin) production. About 32% isolates were found bacteriocin producers. The best producer was identified as Vibrio anguillarum AVP10. The maximum production of vibriocin AVP10 was manifested at 29 degrees C at pH 7, after 18-20 h of incubation. Vibriocin activity was enhanced in the presence of citrate-phosphate buffer. The vibriocin AVP10 withstands autoclaving temperature and showed activity even after prolonged chloroform treatment. Proteolytic enzymes inhibited its activity, while lipolytic enzyme had no effect. It was found bioactive only against intrageneric bacterial strains. Mode of action of vibriocin AVP10 varies with the indicator (sensitive) culture used i.e. bactericidal effects was exerted against V. anguillarum AVS9 while bacteriostatic effect was shown against entero-toxigenic E. coli.

Comparative study of two bacteriocins produced by representative indigenous soil bacteria.

The aim of this research work was to identify and characterized the bacteriocins produced by soil-associated bacteria. Bacillocin from Bacillus brevis Bb and pyocin from Pseudomonas aeruginosa Pa were found bioactive only against gram-positive bacteria tested. Maximum production of both the bacteriocins was observed at 32 degrees C in BHI medium. Production of both the bacteriocins started in the early exponential growth phase while the maximum production was observed during the stationary phase. Bacillocin Bb remained stable during 1-9 pH while pyocin Pa remained stable at pH 1-11. Both of the bacterocins were found resistant to high temperature (100 degrees C for 30 min), detergents (1% solutions of EDTA, Tween 20, Tween 80 and SDS) and organic solvents (1% solutions of Ethanol, Butanol, Methanol, Propanol, Chloroform, and Acetone). Activity of both was completely lost after proteinase K treatment suggesting their protein nature. Titre of bacillocin Bb was estimated to be 5280 AU/mL while the titre for pyocin Pa was calculated as 640 AU/mL. Both of the bacteriocins showed bacteriolytic mode of action against the indicator Bacillus strain BC31 and were found <10 KDa in their molecular mass.

Significance of prostate specific antigen in prostate cancer patients and in non cancerous prostatic disease patients.

OBJECTIVE: To evaluate the significance of prostate specific antigent (PSA) and scan in prostate cancer patients and in non cancerous prostatic disease patients. METHODS: The study was carried out in Radioimmunoassay (RIA) lab, KIRAN Hospital, Karachi during 2002 to 2006. A total of 149 serum samples were collected in which 93 samples were biopsy positive prostate cancer patients referred to KIRAN hospital for treatment. The other 56 samples were collected from patients having other prostatic diseases and advised PSA tests by physicians and urologists from 'The Lab' Karachi. The PSA (total) was measured by immunoradiometric assay (IRMA) in which two monoclonal antibodies against two different epitopes of PSA molecules were used. The results were correlated with bone scan and age of the patients. RESULTS: A total of 149 patient samples were analyzed in which 93 were from biopsy positive prostate cancer patients and 56 from patients having other prostatic diseases. Out of 93 prostate cancer patients sample, 74 (79.6%) had elevated PSA level (>4 ng/ml) and 19 (20.4%) had PSA within normal range (<4 ng/ml). Among 74 elevated PSA level cases, 48 (64.8%) had a positive bone scan and 26 (35.2%) had negative bone scan. Minimum age recorded was 40 years (Mean age 66.4 +/- 9.1 years). In fifty six (56) serum samples which were collected from 'The Lab' and having other prostatic diseases, 49 (87.5%) had PSA within normal range (< or =4 ng/ml). CONCLUSION: Prostate specific antigen has a significant role for the diagnosis of prostate cancer (and has a significant correlation with bone scan). Immunoradiometric assay has a sensitivity of 79.5% and specificity of 87.5% for prostate cancer.

Enterococci vs coliforms as a possible fecal contamination indicator: baseline data for Karachi.

Fecal contamination of drinking water is the major cause of water borne illnesses. For long time coliforms are exploited as fecal contamination indicator. However, recent studies indicate low survival rate of coliforms in stress conditions, hence it's use as indicator of fecal pollution is being abandoned in many parts of the developed world. Implementation of such strategy demands availability of local data in the cities like Karachi. The present study provides a comparison between coliforms and enterococcal load and its variation in sewage samples collected (June, August and November, 2006) from eighteen towns of Karachi. All the diluted samples were selective media to obtain colony-forming units (CFU) mainly for coliforms and enterococci. The bacteria isolated were identified on the basis of conventional microbiological methods. Observations thus obtained were subjected to rigorous statistical analysis. The total load of enterococci was found in range of 1.27-8.47 X 10(7) as compared to coliforms (3.03-13.9 X 10(7)). However, segregation of data reveals greater inter town variability in CFU/ml both in coliforms and enterococci as suggested by their cumulative standard deviation +/-1.5 X 107. Furthermore, CFU/ml of both coliforms and enterococci also varies to variable scale when collected at different time intervals and at intra town level. Conclusively, the studies suggest high survival rate and lower variability of Enterococci compared to escherichia hence indicating its potential advantage to be used as fecal contamination indicator.

Coliphage hsa as a model for antiviral studies/spectrum by some indigenous bacteriocin like inhibitory substances (BLIS).

Coliphage HSA was isolated from a raw sewage sample (collected from a local sewage treatment plant). The phage was analyzed by spot and tube lysis followed by plaque assay. Phage titre (plaque forming units i.e. PFU) was found to be 4.2 x 103 PFU/mL. Further purification of the phage was achieved by acid-precipitation method. Genomic identification of the coliphage HSA (done by fluorescent staining using acridine orange) revealed it to be a dsDNA bacterial virus. Staphylococcin188, Enterocins AAR-71, AAR-74, and Erwiniocin NA4 were screened for their antiphage activity by plaque assay. Accordingly, all the bacteriocin preparations possess demonstrable antiphage activity witnessed as a reduction in PFU after treatment. In the case of Staphylococcin 188, the number dropped up to 40 PFU/mL, Enterocin AAR-71 and Erwiniocin NA4 treatment reduced it to a zero PFU level, while Enterocin AAR-74 could reduce PFU to 50 (after addition of a constant volume, 500uL, of each of the crude bacteriocin preparations). Transmission Electron Microscopy studies revealed the phage to have an icosahedral head with a long tail and tail fibers.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189.

Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.

Isolation and biochemical characterization of mutacin VSM43 isolated from human oral Streptococcus mutans VSM43.

Bacteriocins produced by mutans streptococci are known as mutacins. Streptococcus mutans VSM43 isolated from human clinical oral cavity was screened for the production of mutacin. It can inhibit the growth of other mutans streptococci, many other gram-positives and some gram-negative bacteria. Average size of the inhibitory zone ranged between 12-20mm. The inhibitory activity could not be related to organic acids, bacteriophages and hydrogen peroxide. Mutacin VSM43 was protease sensitive, remained active within a pH range of 2-8, and lost activity after heating at 100 degrees C for 30 min. Mutacin VSM43 was dialyzable through dialysis membrane (pore size 12,000 Da). A titre of 1280 arbitrary/activity units per ml (AU/mL) was shown against Staphylococcus aureus AB211. Lacuna frequency percentage (LF%) against Streptococcus mutans VSMD and Staphylococcus aureus AB211 was 37% and 49% respectively. We are convinced mutacin VSM43 may be a parallel candidate for use against dental caries.

Antimicrobial spectrum, production and mode of action of staphylococcin 188 produced by Staphylococcus aureus 188.

Staphylococcus aureus 188 has been shown to produce bacteriocin-like inhibitory substance known as staphylococcin 188. It has a broad-activity spectrum against Micrococcus luteus, Streptococcus pneumoniae, Streptococcus faecalis, Streptococcus viridans, Corynebacterium diphtheriae and several staphylococcus species. The arbitrary unit of staphylococcin 188 against Micrococcus luteus was 1280 AU/mL. Its production with simultaneous measurement of activity was monitored and was found to produce maximum amount of staphylococcin after 7 hours of incubation. Mode of action of the staphylococcin 188 on the sensitive cells was bactericidal rather than bacterioloytic.

Urinary tract infections associated with multidrug resistant enteric bacilli: characterization and genetical studies.

Urinary tract infections (UTIs) are among the most commonly prevalent infections in clinical practice. Escherichia coli is the causative agent in about 85% of community-acquired UTIs, followed by Klebsiella that accounts for 6 to 17% of such infections. Present study is based on the isolation-identification and antibiotic resistance pattern of about 60 indigenous bacterial isolates from UTI patients. Prevalence rates were consistent with those from major recent studies reported in the literature, i.e. 73% isolates were identified as E. coli, 16% as K. pneumoniae and 11% as Proteus sp. Bases of identification included morpho-cultural and biochemical characteristics. To assess the breadth of multidrug resistance among these isolates, culture medium incorporation method was employed using ampicillin, fosfomycin, chloramphenicol, tetracycline, and three aminoglycosides (kanamycin, gentamicin, and streptomycin). Of these isolates, 30% offered multidrug resistance to three or more agents. Among multidrug resistant isolates, 100% were resistant to ampicillin, 47% to streptomycin, 41% to chloramphenicol, gentamicin and tetracycline, 35% offered resistance to kanamycin while only 6% showed resistance to fosfomycin. After curing treatment with acridine orange, some of the isolates lost their resistance, thereby indicating the extrachromosomal location of the resistance determinants. Plasmid DNA (bearing multidrug resistant genes) was isolated from the uncured cells, and was stably transformed into the competent cured recipient cells.

Biophysicochemical characterization of Pyocin SA189 produced by Pseudomonas aeruginosa SA189

Abstract Pseudomonas aeruginosa, in spite of being a ubiquitous organism (as it is found in soil, water, and humans), is also an opportunistic pathogen. In order to maintain its diversity in the community, it produces various toxic proteins, known as, bacteriocins. In the present study, pyocin SA189, which is a bacteriocin produced by P. aeruginosa SA189 (isolated from a clinical sample) was characterized. P. aeruginosa SA189, as identified by the conventional and 16S rRNA gene amplification, produced pyocin SA189 of molecular weight of 66 k Da. The pyocin showed antimicrobial activity against several clinically relevant Gram-positive and Gram-negative bacteria and was substantially stable for wide ranges of temperature and pH. Furthermore, the pyocin also retained its biological activity upon treatment with metal ions, organic solvents, and various proteolytic and lipolytic enzymes. The data from the growth kinetics indicated that the maximum bacteriocin production occurred in the late log phase. Overall, our results signify the potential of pyocin SA189 as a bio-control agent.