Genetic characterization of clinical acanthamoeba isolates from Japan using nuclear and mitochondrial small subunit ribosomal RNA.

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

Characterization of integrons and antimicrobial resistance genes in clinical isolates of Gram-negative bacteria from Palestinian hospitals.

Sixty Gram-negative bacterial isolates were collected from Palestinian hospitals in 2006. Thirty-two (53.3%) isolates showed multidrug resistance phenotypes. PCR and DNA sequencing were used to characterize integrons and antimicrobial resistance genes. PCR screening showed that 19 (31.7%) and five (8.3%) isolates were positive for class 1 and class 2 integrons, respectively. DNA-sequencing results for the captured antimicrobial resistance gene cassettes within class 1 integrons identified the following genes: dihydrofolate reductases, dfrA1, dfrA5, dfrA7, dfrA12, dfrA17 and dfrA25; aminoglycoside adenyltransferases, aadA1, aadA2, aadA5, aadA12 and aadB; aminoglycoside acetyltransferase, aac(6')-Ib; and chloramphenicol resistance gene, cmlA1. ESBL were identified in 25 (41.7%) isolates. The identified ESBL were bla(CTX-M-15), bla(CTX-M-56), bla(OXA-1), bla(SHV-1), bla(SHV-12), bla(SHV-32) and bla(TEM-1) genes. Moreover, we characterized the plasmid-mediated quinolone resistance genes, aac(6')-Ib-cr and qnrB2, which were detected in seven (11.7%) and two (3.3%) isolates, respectively. In this study various types of antibiotic resistance genes have been identified in Gram-negative bacteria from Palestinian hospitals, many of which are reported in the Middle East area for the first time.

Multiple-subgenotype infections of Giardia intestinalis detected in Palestinian clinical cases using a subcloning approach.

To evaluate the geographic distribution of Giardia intestinalis genotypes in Nablus, West Bank, Palestine, a genotyping study was performed using clinical fecal samples. Microscopic examination confirmed that 8 of 69 (11.6%) samples were G. intestinalis positive, and subsequent genotyping analyses targeting the small-subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase (GDH) genes revealed the G. intestinalis genotypes within the 8 samples. Of these 8 samples, 6 were clustered with assemblage A-II and the remaining 2 samples were clustered with assemblage B by 18S rRNA gene analysis; however, direct sequencing of the GDH gene segments from the latter 2 samples showed a mixed infection profile. To assess those samples, we employed a subcloning approach and successfully isolated 6 independent assemblage B subgenotypes. These partial GDH gene sequences (393 bp) had 15 single-nucleotide polymorphisms, all of which were synonymous transition substitutions at the third nucleotide position of codons. From the results, we concluded that the highly polymorphic gene loci such as GDH gene locus might provide us an opportunity to obtain a detailed molecular data even from the samples with multiple-subgenotype mixed infections. Therefore, subcloning approach is recommended in genotyping studies, especially in those conducted in giardiasis-endemic areas, where the repeated and cumulative infections could be commonly expected.

Proteus mirabilis clinical isolate harbouring a new variant of Salmonella genomic island 1 containing the multiple antibiotic resistance region.

OBJECTIVES: A clinical isolate of Proteus mirabilis strain 18306, which displayed the multidrug resistance phenotype of Salmonella genomic island 1 (SGI1), was examined for the presence of this island including its multiple antibiotic resistance genomic region. METHODS: P. mirabilis 18306 was isolated in March 2006 from a patient in Palestine with diabetic foot infection. Antibiotic susceptibility tests and various molecular techniques, including PCR, cloning and DNA sequencing were used for detection and characterization of SGI1 in P. mirabilis 18306. RESULTS: P. mirabilis 18306 showed the typical multidrug resistance phenotype of SGI1 as it was resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline, in addition to trimethoprim and nalidixic acid. Molecular characterization showed that P. mirabilis 18306 harboured a structure similar to SGI1, except that the aadA2 gene, which confers resistance to streptomycin and spectinomycin, of standard SGI1 had been replaced with dfrA15, which confers resistance to trimethoprim. Furthermore, the nucleotide sequence of the extrachromosomal circular form of SGI1 in P. mirabilis was found to be identical to that of Salmonella Typhimurium DT104. However, PCR results showed that P. mirabilis 18306 was negative for the left and right junctions which represent the integration sites of SGI1 into Salmonella enterica chromosome. Hence, this new variant of SGI1 may be integrated at a different site into the chromosome of P. mirabilis 18306. Tn1826-derived class 2 integron, which carries only two gene cassettes, sat2 and aadA1, was also identified in this strain. CONCLUSIONS: In this study, we identified a new variant SGI1 containing the multiple resistance genomic region in a multidrug-resistant strain of P. mirabilis. This is the first report for SGI1 in a genus other than Salmonella.