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Mirani Z.A.,Microbiological Analytical Center | Aziz M.,Bahauddin Zakariya University | Khan S.I.,Microbiological Analytical Center
Journal of Antibiotics | Year: 2015

The present study was conducted to investigate the significance of small colony variants (SCVs) in biofilm life cycle of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). All of these MRSA and MSSA isolates were recovered from different food commodities. Molecular typing showed that 21 MRSA isolates carry SCCmecA type IV and belong to agr type II. Out of 15 MSSA isolates, 7 were found to carry agr type II, 5 agr type I and 2 agr type III. All of the MRSA isolates studied adopted biofilm mode of growth after exposure to sublethal doses of oxacillin. MSSA isolates, on the other hand, were biofilm producers by nature, that is, without exposure to any stress. The biomass of the biofilm reaches its maximum thickness after 48h of incubation at 351C. It was noticed that biofilm population consists of wild type and SCVs. Moreover, the number of SCVs increases with the age of biofilm. The SCVs of MRSA were unable to readopt biofilm mode of growth independently, irrespective of the presence or absence of oxacillin. The SCVs of MSSA, on the other hand, quickly revert to normal life just after a single subculture and show biofilm formation without any stress. Molecular studies showed a parallel reduction in the expression of the genes icaA, sigβ and sarA, and also in the extracellular matrix production in SCVs of MRSA. This might be due to oxacillin as it seems to be a stress factor responsible for induction of biofilm formation in MRSA isolates. Contrary to the wild type, SCVs are metabolically inactive and do not respond to oxacillin, which is only active against the growing cells. Therefore, stress-responsive genes, that is, sigβ and sarA, are not induced. Conversely, MSSA isolates are natural biofilm producers without induction through any known factors. © 2015 Japan Antibiotics Research Association.


PubMed | University of Karachi, Hallym University, Taj Fisheries Pvt Ltd, Microbiological Analytical Center and 2 more.
Type: | Journal: The open medicinal chemistry journal | Year: 2016

In this article we describe development and validation of stability indicating, accurate, specific, precise and simple Ion-pairing RP-HPLC method for simultaneous determination of paracetamol and cetirizine HCl along with preservatives i.e. propylparaben, and methylparaben in pharmaceutical dosage forms of oral solution and in serum. Acetonitrile: Buffer: Sulfuric Acid (45:55:0.3 v/v/v) was the mobile phase at flow rate 1.0 mL min(-1) using a Hibar() Lichrosorb() C18 column and monitored at wavelength of 230nm. The averages of absolute and relative recoveries were found to be 99.3%, 99.5%, 99.8% and 98.7% with correlation coefficient of 0.9977, 0.9998, 0.9984, and 0.9997 for cetirizine HCl, paracetamol, methylparaben and Propylparaben respectively. The limit of quantification and limit of detection were in range of 0.3 to 2.7 ng mL(-1) and 0.1 to 0.8 ng mL(-1) respectively. Under stress conditions of acidic, basic, oxidative, and thermal degradation, maximum degradation was observed in basic and oxidative stress where a significant impact was observed while all drugs were found almost stable in the other conditions. The developed method was validated in accordance with ICH and AOAC guidelines. The proposed method was successfully applied to quantify amount of paracetamol, cetirizine HCl and two most common microbial preservatives in bulk, dosage form and physiological fluid.


PubMed | Dow University of Health Sciences, Bahauddin Zakariya University and Microbiological Analytical Center
Type: Journal Article | Journal: The Journal of antibiotics | Year: 2016

Present study is based on 20 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from different food items. These isolates were identified on the basis of colony morphology, Gram staining and growth on different selective and differential media. Studies on 16S RNA and positive reactions on DNase agar and Prolex Latex Agglutination system confirm it as Staphylococcus aureus. Oxacillin susceptibility testing and PCR with mecA gene-specific primer results showed that these isolates are MRSA-carrying mecA gene that belongs to SCCmecA type IV and also harbor agr type II. Phenotypic study revealed that these isolates adopt biofilm mode of growth after exposure to subinhibitory doses of oxacillin. The biofilm and cell surface hydrophobicity have a strong correlation. It was noticed that affinity to hexadecane (apolar-solvent) of planktonic cells was low, suggesting its hydrophilic character. However, as the cells are exposed to oxacillin, they adopt biofilm mode of life and the affinity to apolar solvent increases, indicating a hydrophobic character. In biofilm consortia, the cells with more hydrophobic surfaces show incomplete septation and produce multicellular aggregates. This is due to reduced expression of atl gene. This was confirmed by real-time PCR studies. Moreover, the planktonic or wild-type phenotypes of these isolates were more tolerant to antibacterial effect of the fatty acids used; that is, cis-2-decanoic acid and cis-9-octadectanoic acid. These fatty acids were more effective against biofilms. After exposure to these fatty acids, established biofilms were dispersed and surviving cells were unable to readopt biofilm mode of life. The planktonic or wild-type phenotypes produce fatty acid-modifying enzyme (FAME) to inactivate the bactericidal activity of fatty acids by esterification to cholesterol. The biofilm indwellers are metabolically inactive and unable to produce FAME; hence, they are vulnerable to antibiofilm effect of cis-2-decanoic acid and cis-9-octadectanoic acid.


Li Z.,Ocean University of China | Lu Z.,Ocean University of China | Khan M.N.,Ocean University of China | Khan M.N.,Microbiological Analytical Center | And 2 more authors.
Food and Chemical Toxicology | Year: 2014

The present study aimed to investigate the relationship between protein carbonylation and changes of the IgE reactivity of turbot parvalbumin (PV) following electron beam (EB) irradiation. The concentration of protein carbonyls, specific IgE binding, and IgE binding inhibition between irradiated and oxidized PV were assessed. Irradiation resulted in a 3-fold enhancement in the protein carbonyl content. In purified PV irradiated with a 10-kGy dose, specific IgE binding was reduced by 91.2±6.2%. When raw PV was treated with reactive oxygen species (ROS), the protein carbonyl content increased 17.6-fold, with the specific IgE binding being reduced by 87.9±6.5% at an ROS concentration of 10nmol/mL. The IgE binding inhibition between irradiated and oxidized PV was investigated using an inhibition ELISA. Results showed that oxidized PV can inhibit the binding between irradiated PV and specific IgE with an IC50 of 8.2-58ng according to different doses of irradiation. These findings suggest that EB irradiation reduces specific IgE binding, probably by the induction of protein carbonylation. © 2014 Elsevier Ltd.


Rong R.,Ocean University of China | Lin H.,Ocean University of China | Wang J.,Ocean University of China | Khan M.N.,Ocean University of China | And 2 more authors.
Aquaculture | Year: 2014

Vibrio parahaemolyticus is closely associated with oysters in China, which are normally consumed raw or lightly cooked. Bacteriophages are a safe bio-controlling agent, and have been recognized in aquaculture for their pathogen reduction properties. This study investigated the potential application of the bacteriophage VPp1 during depuration to reduce V. parahaemolyticus in oysters at different multiplicity of infection (MOI) and temperature levels. Oysters were infected with 105, 106, and 107 colony-forming units (CFU)/mL of V. parahaemolyticus in the seawater and each infected group was treated with three different MOI values: 10, 1, and 0.1. Infected oysters were depurated in non-recirculating seawater at 22°C, 20°C, 16°C, and 12°C for 36h. Results revealed that temperatures <20°C were safe for oyster rearing. Depuration at 16°C with 0.1 MOI was the best condition for reducing V. parahaemolyticus in oysters, which decreased by 2.35-2.76logCFU/g within 36h. This study provides the basis for the use of bacteriophages in depuration techniques to eliminate V. parahaemolyticus in oysters. © 2013 Elsevier B.V.


Mirani Z.A.,Microbiological Analytical Center | Jamil N.,University of Karachi
Journal of the College of Physicians and Surgeons Pakistan | Year: 2013

Objective: To study the genomic organization of vancomycin resistance in a local isolate of vancomycin resistant Staphylococcus aureus (VRSA). Study Design: Experimental study. Place and Duration of Study: Department of Microbiology, University of Karachi, January 2008 through December 2010. Methodology: A vancomycin-resistant Staphylococcus aureus (VRSA-CP2) isolate (MIC 16 μg/ml) was isolated from a local hospital of Karachi. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene. The vancomycin MIC was re-confirmed by E-test. For the genetic determination of vancomycin resistance, in-vitro amplification of vanA cassette was performed by using plasmid DNA of CP2, CP2's transformant as template on MWG Thermo-Cycler. Amplified products of vanR, vanS, vanH, vanA, vanY, orf2, orf1D, orf2E, orf-Rev and IS element genes were subjected to Sanger's electrophoresis based sequence determination using specific primers. The Basic Local Alignment Search Tool (BLAST) algorithm was used to identify sequences in GenBank with similarities to the vanA cassette genes. Results: The vancomycin-resistant isolate CP2 was found to be resistant to oxacillin, chloramphenicol, erythromycin, rifampicin, gentamicin, tetracycline and ciprofloxacin, as well. The isolate CP2 revealed four bands: one of large molecular size ~56.4 kb and three of small size ~6.5 kb, ~6.1 kb and ~1.5 kb by agarose gel electrophoresis indicating the presence of 3 plasmids. The plasmid DNA of isolate CP2 was analyzed by PCR for the presence of the van cassettes with each of the vanA, vanB and vanC specific primers. It carried vanA cassette, which comprises of vanR, vanS, vanH, vanA, vanY, and orf2. The vanA cassette of isolate CP2 also carried an insertion element (IS). However, it did not show the PCR product for orf1. Vancomycin resistance was successfully transferred from the donor CP2 to a vancomycin-sensitive recipient S. aureus. The MIC of vancomycin for the transformant was 16 μg/ml, similar to the parent isolate CP2. Nucleotide sequencing of the PCR product showed similarity with van genes of enterococci and other VRSA reported from different parts of the world. Conclusion: Sequence of vanA cassette of CP2 showed partial homology with vancomycin resistant enterococci, VRSA vanA cassette element recorded in gene bank NCBI.


Han F.,Ocean University of China | Li M.,Ocean University of China | Lin H.,Ocean University of China | Wang J.,Ocean University of China | And 3 more authors.
Journal of Industrial Microbiology and Biotechnology | Year: 2014

Shewanella putrefaciens has been identified as a specific spoilage organism commonly found in chilled fresh fish, which contributes to the spoilage of fish products. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish. Endolysins, which are lytic enzymes produced by bacteriophages, have been considered an alternative to control bacterial growth, and have been useful in various applications, including food preservation. We report here, for the first time, the complete genome sequence of a novel phage Spp001, which lyses S. putrefaciens Sp225. The Spp001 genome comprises a 54,789-bp DNA molecule with 67 open reading frames and an average total G + C content of 49.42%. In silico analysis revealed that the Spp001 open reading frames encode various putative functional proteins, including an endolysin (ORF 62); however, no sequence for genes encoding the holin polypeptides, which work in concert with endolysins, was identified. To examine further the lytic activity of Spp001, we analyzed the lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S. putrefaciens, using diffusion and turbidimetric assays. The results show that the partially purified extract contained endolysin, as indicated by a high hydrolytic activity towards bacterial peptidoglycan decrease in the OD590 value by 0.160 in 15 min. The results will allow further investigation of the purification of natural Spp001 endolysin, the extension of Spp001 host range, and the applications of the phage-encoded enzymes. © Society for Industrial Microbiology and Biotechnology 2014.


Peng Y.,Ocean University of China | Jin Y.,Ocean University of China | Lin H.,Ocean University of China | Wang J.,Ocean University of China | And 2 more authors.
Journal of Microbiological Methods | Year: 2014

For rapid and quantitative detection of Vibrio parahaemolyticus, a method combining the specific lysis of bacteriophages with a bacterial luciferase-flavin mononucleotide:nicotinamide adenine dinucleotide oxidoreductase bioluminescent system in vitro was developed. A V. parahaemolyticus detection system was established by optimizing three main influencing factors: bacteriophage titer, volume ratio of the bacteriophage to its host bacterium, and lysis time. A standard curve between the number of bacteria and the luminescence intensity of the coupled enzyme system was studied and revealed a good linear relationship. More than 107colony-forming units (cfu)·ml-1 bacteria in pure culture and >108cfu·ml-1 bacteria in oyster samples were readily detected without pre-enrichment. Furthermore, >100cfu·ml-1 bacteria in oyster samples were readily detected after 4h of enrichment culture. Because of its rapid detection, high specificity, and simplicity in operation, this method is an effective tool for detecting living bacteria in food and environmental samples. © 2014 Elsevier B.V.


Mirani Z.A.,Microbiological Analytical Center | Mirani Z.A.,University of Karachi | Jamil N.,University of Karachi
Journal of Infection and Chemotherapy | Year: 2013

This study was designed to analyze the effect of vancomycin on the cytoplasmic membrane fatty acid (FA) composition of vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-intermediate resistant S. aureus (VISA), and vancomycin-susceptible S. aureus. One low-level vancomycin-resistant isolate (LLR-VRSA) termed CP2, along with two vancomycin intermediate-resistant S. aureus isolates (VISA-CP1) and Mu50 (ATCC 700699), were studied. The LLR-VRSA isolate CP2, recovered from the blood sample of a postoperative cardiac patient, exhibited vanA type vancomycin resistance [minimum inhibitory concentration (MIC) 16 μg/ml], and the vanA cassette was located on a plasmid. CP1, isolated from the pus sample of the same patient, exhibited vancomycin intermediate resistance (MIC 8 μg/ml) in the absence of the vanA, vanB, or vanC gene. As susceptible controls, we used PSA (vancomycin MIC 2 μg/ml), which was isolated from the pus sample of a neonate, and S. aureus (ATCC 29213). Membrane FA analysis was carried out using gas chromatography coupled with mass spectrometry. For this purpose, CP1, CP2, Mu50, and the susceptible control isolates were grown in the presence and absence of vancomycin. Comparative analysis showed an increase in the relative proportion of unsaturated FAs during growth under vancomycin stress. The isolate CP2 (LLR-VRSA) exhibited a higher MIC to vancomycin than the other isolates used in present study (16 μg/ml) and under vancomycin stress conditions, quantitatively, it showed a high rate of conversion of saturated to unsaturated membrane FAs than CP1, Mu50 (VISA isolate) and the susceptible control PSA. The rate of saturated-to-unsaturated FA conversion increased as the concentration of vancomycin in the growth media was increased. Therefore, it is concluded that S. aureus tend to modify their membrane lipid chemistry from saturated to unsaturated in order to survive in a vancomycin stress environment. © 2012 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases.


Liu H.,Ocean University of China | Lin H.,Ocean University of China | Mu Q.,Shandong Haizhibao Ocean Science and Technology Co. | Lu X.,Ocean University of China | And 3 more authors.
Innovative Food Science and Emerging Technologies | Year: 2014

The NADH luminescence assay is a rapid, sensitive and easy-to-perform bacterial detection method. However, the detection limit is approximately 107 CFU/mL, which is inadequate for many applications. The purpose of this study is to amplify luminescence assay signals by converting NAD(P)+ to NAD(P)H to provide a more sensitive method for the detection of bacteria. Under optimal conditions, the luminescence intensity correlated well with the bacterial count (R2 = 0.98) and the detection limit was 1.05 × 105 CFU/mL The sensitivity of this novel bioluminescence enzymatic cycling method is nearly 102 times higher than previous bioluminescence methods. Thus, this improved method can be used to rapidly determine the total viable count with higher sensitivity. Industrial relevance This improved method can be used to rapidly determine the total viable count with higher sensitivity in food. This study lays the foundation for the future development of a fast detector using bacterial bioluminescence. © 2014 Elsevier Ltd.

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