Mohamed S. Nawaz

Biochemical and Molecular Characterization of Tetracycline-Resistant Aeromonas veronii Isolates from Catfish

ABSTRACT Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.

Degradation of organic cyanides by pseudomonas aeruginosa

A bacterium capable of utilizing acetonitrile (methyl cyanide) as the sole source of carbon and nitrogen was isolated from soil and identified asPseudomonas aeruginosa. This bacterium could also utilize and oxidize numerous lower-mol-wt nitrile compounds and their corresponding amides as growth substrates. A metabolite of acetonitrile in the culture medium was determined to be ammonia. The accumulation of ammonia in the culture medium was proportional to the concentration of the substrate and the inoculum. Cell extracts of the bacterium contained activities corresponding to nitrile aminohydrolase (E C 3.5.5.1) and amidase (E C 3.5.1.4), which regulate the degradation of acetonitrile. Both enzymes were inducible and hydrolyzed a wide range of substrates, and it was determined that the specific activity of amidase was far greater than the activity of nitrile aminohydrolase.

Molecular characterization of tetracycline-resistant genes and integrons from avirulent strains of Escherichia coli isolated from catfish.

A study was undertaken to investigate the occurrence of tetracycline-resistant genes and to characterize the integrons present in Escherichia coli isolated from catfish. Sixty-three tetracycline-resistant E. coli strains were isolated from the intestinal contents of 407 farm-raised catfish. All strains were resistant to multiple antibiotics. A polymerase chain reaction (PCR) assay detected tetA in the DNA of 15 of 63 (25.0%) isolates by amplifying a PCR amplicon measuring 957 bp. Oligonucleotide primers targeting a 436-bp region of tetB successfully amplified a PCR amplicon from 47 of 63 (77.0%) isolates, indicating that tetB was predominant. Oligonucleotide primers specific for tetC amplified a 589-bp PCR amplicon from 3 of 63 (5%) isolates. Eleven (17.0%) of the isolates contained both tetA and tetB genes. Class I integrons amplified from the genomic DNA of 14 of 63 (22.0%) isolates measured 1.6 and 1.8 kb. Sequence analysis of the 1.6 kb integrons indicated the presence of three different gene cassettes: a dfrA12, conferring resistance to trimethoprim; an open reading frame, orfF, a hypothetical protein of unknown function; and aadA2, conferring resistance to aminoglycosides. Sequence analysis of the 1.8-kb integron indicated the presence of dfrA17 and aadA5. PCR assays for the detection of the six predominant virulence genes failed to amplify any genes from the genomic DNA. Pulsed-field gel electrophoresis using XbaI identified 16 distinct macro restriction patterns among the 63 isolates. The dendrogram analysis indicated that the DNA from 4 of 16 isolates had a similarity index of 90.0%. Our results indicate that the use of oxytetracycline and Romet 30 (sulfadimethoxine and ormetoprim) in farm-raised catfish may select for multiple antibiotic-resistant E. coli that could serve as a reservoir of tetracycline, trimethoprim, and aminoglycoside resistance genes.

Isolation and characterization of acetonitrile utilizing bacteria.

SummaryBacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (14C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.

Degradation of Aliphatic Amide Mixture by Immobilized and Nonimmobilized Cells of Pseudomonas sp.

A strain of Pseudomonas sp. capable of utilizing acrylamide as the sole source of carbon and nitrogen was tested for its ability to degrade an aliphatic amide mixture containing acrylamide, propionamide, butyramide, and methacrylamide. Batch cultures of Pseudomonas sp. degraded butyramide and propionamide at the rate of 115 μmol/h and at the rate of 127 μmol/h to butyric and propionic acids and ammonia in 48 h. Acrylamide and methacrylamide were degraded to acrylic acid and methacrylic acid in 72 h at the rate of 97 and 78 μmol/h, respectively. Faster rates of degradation could be obtained when the bacteria was immobilized

Detection of Type III Secretion System Virulence and Mutations in gyrA and parC Genes Among Quinolone-Resistant Strains of Pseudomonas aeruginosa Isolated from Imported Shrimp

Twenty Pseudomonas aeruginosa isolates were recovered from imported frozen raw shrimp sold in the United States. Isolates were tested for antimicrobial susceptibility to quinolones and analyzed for mutations in quinolone resistance-determining regions, presence of type III secretion system genes, and genetic relatedness using pulsed-field gel electrophoresis. All isolates were resistant to nalidixic acid. Polymerase chain reaction assays detected exoS, exoT, exoU, and exoY among isolates. Eight unique pulsed-field gel electrophoresis clusters were generated. Mutations were found in gyrA at codon 83 (Ile to Thr) and in parC at codon 87 (Leu to Ser). Together, these findings reveal that imported shrimp may harbor virulent and quinolone-resistant strains of P. aeruginosa.