Mohamed Nawaz

A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria.

A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments.

Identification and Characterization of Class 1 Integron Resistance Gene Cassettes among Salmonella Strains Isolated from Imported Seafood

ABSTRACT A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.

Molecular Characterization of Fluoroquinolone-Resistant Aeromonas spp. Isolated from Imported Shrimp

ABSTRACT Sixty-three nalidixic acid-resistant Aeromonas sp. isolates were obtained from imported shrimp. Phylogenetic analysis of gyrB sequences indicated that 18 were A. enteropelogenes, 26 were A. caviae, and 19 were A. sobria. Double missense mutations in the quinolone resistance-determining region (QRDR) of gyrA at codon 83 (Ser→Val/Ile) and codon 92 (Leu→Met) coupled with a point mutation of parC at codon 80 (Ser→Ile/Phe) conferred high levels of quinolone resistance in the isolates. A majority of A. enteropelogenes and A. caviae strains harbored toxin genes, whereas only a few A. sobria strains harbored these genes. The fluoroquinolone-resistant Aeromonas spp. exhibited higher cytotoxicity than fluoroquinolone-sensitive, virulent Aeromonas spp. to rat epithelial cells.

Plasmid-Mediated Quinolone Resistance in Pseudomonas putida Isolates from Imported Shrimp

ABSTRACT Fourteen quinolone-resistant Pseudomonas putida isolates were recovered from imported frozen shrimp sold in the United States. Two isolates harbored plasmids with qnrA and qnrB genes. PCR and DNA sequencing of quinolone resistance-determining regions identified novel substitutions in GyrA (His139→Glu and Thr128→Ala) and GyrB (Thr442→Asn, Gly470→Ala, and Ile487→Pro) and previously reported substitutions in GyrB (Asp489→Glu) and ParC (Thr105→Pro).

Draft Genome Sequence of Multidrug-Resistant Enterococcus faecium Clinical Isolate VRE3, with a Sequence Type 16 Pattern and Novel Structural Arrangement of Tn1546

ABSTRACT Multidrug-resistant Enterococcus faecium has emerged as a nosocomial pathogen that may infect the body at various sites, including the gastrointestinal tract, and has serious implications in human health and disease. Here, we present the draft genome sequence of clinical strain VRE3, which exhibited a sequence type 16 (ST16) pattern and carried truncated Tn1546, a mobile genetic element encoding a high level of vancomycin resistance.

Direct In-Gel Hybridization of DNA With Digoxigenin-Labeled Probes

In-gel hybridization with digoxigenin (DIG)-labeled probes has been shown to detect complementary DNA sequences in dried agarose gels. Gels dried at room temperature or at 55 degrees C in an oven do not show detectable changes in the sensitivity of detection. However, gels dried under vacuum seem to lose the sensitivity by approx 8- to 10-fold. In-gel hybridization after blotting high molecular weight T7 DNA (40 kbp) onto nylon membranes has been demonstrated to transfer the DNA to the membrane inefficiently. In-gel hybridization of DIG-labeled probes with the complementary DNA sequences has been determined to detect as little as 0.05 ng of 40-kbp T7 DNA and single copies of the erythromycin resistance marker gene ermA. Nonradioactive in-gel hybridization provides better quantitation of nucleic acids than filter hybridization in Southern and Northern blot analysis.

Draft Genome Sequences of Two Methicillin-Resistant Clinical Staphylococcus aureus Isolates

ABSTRACT Here, we report the draft genome sequences of two methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates, hospital-associated perirectal isolate 32S (ST 239) from a colitis tracheostomy patient and community-associated MRSA isolate 42S (ST 772) from a hepatic-splenomegaly patient in Rawalpindi, Pakistan.

Genomic Sequence of a Clinical Vancomycin-Resistant Reference Strain, Enterococcus faecalis ATCC 51299

ABSTRACT In this paper, we present a draft genome sequence of a quality control reference strain, Enterococcus faecalis ATCC 51299 (multilocus sequencing type [MLST] ST6), which is sensitive to teicoplanin but resistant to vancomycin. It is used in an agar screening test for streptomycin, gentamicin, and vancomycin resistance and the resistance marker vanB.