Emergence of CTX-M-3, TEM-1 and a new plasmid-mediated MOX-4 AmpC in a multiresistant Aeromonas caviae isolate from a patient with pneumonia

Aeromonas species rarely cause pulmonary infection. We report, for what is believed to be the first time, a case of severe pneumonia in a cancer patient caused by Aeromonas caviae. Detailed microbiological investigation revealed that this isolate carried three β-lactamase-encoding genes (encoding MOX-4, CTX-M-3 and TEM-1) conferring resistance to all β-lactams but imipenem. The β-lactamase with a pI of 9.0 was transferred by conjugation and associated with a 7.3 kb plasmid, as demonstrated by Southern blot hybridization. Analysis of the nucleotide and amino acid sequences showed a new ampC gene that was closely related to those encoding the MOX-1, MOX-2 and MOX-3 β-lactamases. This new plasmid-mediated AmpC β-lactamase from China was named MOX-4. This is believed to be the first report of MOX-4, CTX-M-3 and TEM-1 β-lactamases in a multiresistant A. caviae.

Characterization of plasmid-mediated quinolone resistance by the qnrS gene in Escherichia coli isolated from healthy chickens and pigs

The prevalence of <I>qnr</I> and <I>qepA</I> genes in 660 <I>Escherichia coli</I> isolates was investigated in healthy animals from 30 pig farms and 30 chicken farms in Taiwan from January 2005 to February 2006 by the polymerase chain reaction. The <I>qnrS</I> gene, but not <I>qnrA, qnrB, </I> and <I>qepA</I> were detected in 12/360 pig isolates (3.33%) and in 6/300 chicken isolates (2%). Southern blot hybridization analysis indicated that <I>qnrS</I> was located on plasmids ranging in size from 50–165 kb. Eleven of the 18 <I>qnrS</I> positive isolates which showed a high ciprofloxacin resistance phenotype (minimum inhibitory concentration ≥ 8 mg/l) also had amino acid sequence variations in chromosomal quinolone resistance-determining regions of <I>gyrA</I> and <I>parC</I>. Only two <I>qnrS</I>-positive isolates carried the <I>aac(6’)-Ib-cr</I>variant that mediates FQ acetylation. For the high percentage resistance of cephalosporins, the<I> bla</I>_CTX-M gene was also examined in <I>qnrS</I>-positive isolates. The <I>bla</I>_CTX-M gene was detected in fifteen isolates (15/18, 83.3%) of which 12 isolates were <I>bla</I>_CTX-M-1 and three isolates were <I>bl</I>_CTX-M-15. This study demonstrated a close linkage between the <I>qnrS</I> gene and <I>bla</I>_CTX-M-1, suggesting CTX-M and Qnr-based mechanisms might be co-emerging in <I>E. coli</I> strains isolated from healthy chickens and pigs under selective pressure of quinolone and cephalosporine administration.

Genetic Transformation of Garlic (Allium sativum L.) by Particle Bombardment

Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic calluses, derived from garlic cultivar `GT96-1', were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L-1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated.

Tolerance to Citrus mosaic virus in Transgenic Trifoliate Orange Lines Harboring Capsid Polyprotein Gene

Trifoliate orange plants (Poncirus trifoliata) were transformed with a binary vector containing the capsid polyprotein (pCP) gene of Citrus mosaic virus (CiMV) via Agrobacterium tumefaciens LBA4404. Transformation was performed on the epicotyl segments obtained from a young seedling that was grown in the dark. Southern blot hybridization analysis showed that the transgene was stable in the transgenic lines after regeneration and propagation by grafting. Transgenic lines were screened for tolerance to CiMV by mechanical inoculation. Infection was monitored 30, 60, 90, and 120 days after inoculation by reverse transcription-polymerase chain reaction. The transgenic line 24 had the lowest infection rate (7.1%) at 60 days after inoculation, in contrast to that of nontransgenic plants (65.1%).The response of other lines to inoculation ranged from susceptibility to moderate tolerance.

Plasmid-Borne Genes Code for an Angular Dioxygenase Involved in Dibenzofuran Degradation by Terrabacter sp. Strain YK3

ABSTRACT The genes responsible for angular dioxygenation of dibenzofuran in actinomycetes were cloned by using a degenerate set of PCR primers designed by using conserved sequences of the dioxygenase alpha subunit genes. One sequence of alpha subunit genes was commonly amplified from four dibenzofuran-utilizing actinomycetes: Terrabacter sp. strains YK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18. A 5.2-kb PstI fragment encoding the alpha and beta subunits of the terminal dioxygenase, ferredoxin, and ferredoxin reductase (designated dfdA1 to dfdA4, respectively) was cloned from the large circular plasmid pYK3 isolated from Terrabacter sp. strain YK3. We confirmed that transcription of the dfdA1 gene was induced by dibenzofuran in Terrabacter sp. strain YK3. Southern blot hybridization analysis revealed that this type of dioxygenase gene is distributed among diverse dibenzofuran-utilizing actinomycetes. However, genes homologous to dfdA1 were not detected in dibenzofuran utilization-deficient mutants of Terrabacter, Rhodococcus, and Microbacterium species. When the dfdA1 to dfdA4 genes were introduced into a non-dibenzofuran-degrading mutant of Rhodococcus sp. strain YK2, strain YK2-RD2, which had spontaneously lost the gene homologous to dfdA1, the ability to degrade dibenzofuran was restored. Analysis of the breakdown products indicated that DfdA has angular dioxygenase activity. This dfdA transformant degraded several aromatic compounds, indicating that the novel angular dioxygenase possesses broad substrate specificity.

Identification of a Locus Involved in Systemic Dissemination of Yersinia enterocolitica

ABSTRACT A putative LysR-type transcriptional activator, Hre20, was identified previously in an in vivo expression technology screen designed to identify factors which are expressed early during infection by Yersinia enterocolitica (G. M. Young and V. L. Miller, Mol. Microbiol. 25:319–328, 1997). An insertion in hre20, now designated rscR, resulted in increased splenic dissemination of bacteria during infection in a BALB/c mouse model. A nonpolar mutation was generated inrscR, and examination of this strain in the BALB/c mouse model demonstrated that the mutation in rscR was responsible for the increased dissemination to the spleen that was seen in the original experiments. RscR is homologous to the LysR family of transcriptional regulators; thus, a screen was undertaken to identify genes regulated by RscR. A strain containing an insertion in the chromosomal rscR gene and carrying rscR on a plasmid under the control of the inducible araBAD promoter was mutagenized with an mTn5Km-2 transposon containing a promoterless lacZY. Eighteen insertions were identified which appeared to respond to levels of RscR, and these were classified into four allelic groups based on Southern blot hybridization analysis. Representative members were sequenced from three allelic groups. Sequencing revealed insertions in an ORF with no known homologues, a homologue of OmpF of Serratia marcescens, and a locus (designated rscBAC) with similarity to thehmwABC locus of Haemophilus influenzae. ThehmwABC locus promotes adherence of H. influenzae to host cells (S. J. Barenkamp and J. W. St. Geme III, Infect. Immun. 62:3320–3328, 1994; J. W. St. Geme III, S. Falkow, and S. J. Barenkamp, Proc. Natl. Acad. Sci. USA 90:2875–2879, 1993). A strain containing a deletion mutant of rscA, the hmwA homologue, exhibits increased splenic dissemination of bacteria during infection in a BALB/c mouse model, similar to the rscR mutant. This suggests that the phenotype of an rscR mutant is due to the loss of RscA.

Resistance to Bean pod mottle virus in Transgenic Soybean Lines Expressing the Capsid Polyprotein

Transgenic fertile soybean plants were generated from somatic embryos of soybean (Glycine max) cv. Jack transformed via particle bombardment with the capsid polyprotein (pCP) gene of Bean pod mottle virus(BPMV). The plant transformation vector (pHIG/BPMV-pCP) utilized in these experiments contained the BPMV-pCP coding sequence, an intron-containing GUS gene, and the hygromycin phosphotransferase gene. Southern blot hybridization analysis showed that 19 transgenic soybean plants selected for resistance to hygromycin contained the genes for GUS and BPMV-pCP. The progeny of five of these transgenic soybean plants (plants 137, 139, 157, 183, and 186) were characterized in detail. An additional transgenic plant (plant 200) contained the intron-GUS and hygromycin resistance genes, but lacked the BPMV-pCP gene and was used as a negative control. Southern blot hybridization analysis of the five transgenic plants showed the presence of three copies of the T-DNA in a similar banding pattern suggesting that they were derived from a single transformation event. Western and northern blot analyses showed that the expression levels of BPMV-pCP and pCP transcript were high in these five pCP plants. Infectivity assays with detached leaves demonstrated that all five pCP plants exhibited resistance to virus infection because they accumulated lower levels of BPMV compared with plant 200 and nontransformed controls. Unlike the T2 progeny of line 183-1 that segregated with respect to the pCP gene and, consequently, to BPMV resistance, the T2 progeny of the homozygous line 183-2 showed little or no symptoms in response to rub-inoculation with virions of a severe strain of BPMV. Although BPMV accumulation was evident in leaves on which viruliferous beetles were allowed a 72-h inoculation access period, the upper noninoculated leaves of the T2 progeny of line 183-2 plants were symptomless and accumulated little or no virus. Because the progeny of this homozygous transgenic line exhibited systemic resistance, they could potentially be useful in generating commercial cultivars resistant to BPMV.

Characterization of Insertions of IS476and Two Newly Identified Insertion Sequences, IS1478 and IS1479, in Xanthomonas campestris pv. campestris

ABSTRACT Thirty-two plasmid insertion mutants were independently isolated from two strains of Xanthomonas campestris pv. campestris in Taiwan. Of the 32 mutants, 14 (44%), 8 (25%), and 4 (12%) mutants resulted from separate insertions of an IS3 family member, IS476, and two new insertion sequences (IS), IS1478 and IS1479. While IS1478does not have significant sequence homology with any IS elements in the EMBL/GenBank/DDBJ database, IS1479 demonstrated 73% sequence homology with IS1051 in X. campestrispv. dieffenbachiae, 62% homology with IS52 inPseudomonas syringae pv. glycinea, and 60% homology with IS5 in Escherichia coli. Based on the predicted transposase sequences as well as the terminal nucleotide sequences, IS1478 by itself constitutes a new subfamily of the widespread IS5 family, whereas IS1479, along with IS1051, IS52, and IS5, belongs to the IS5 subfamily of the IS5 family. All but one of the IS476 insertions had duplications of 4 bp at the target sites without sequence preference and were randomly distributed. An IS476 insertion carried a duplication of 952 bp at the target site. A model for generating these long direct repeats is proposed. Insertions of IS1478 and IS1479, on the other hand, were not random, and IS1478 and IS1479 each showed conservation of PyPuNTTA and PyTAPu sequences (Py is a pyrimidine, Pu is a purine, and N is any nucleotide) for duplications at the target sites. The results of Southern blot hybridization analysis indicated that multiple copies of IS476, IS1478, and IS1479 are present in the genomes of all seven X. campestris pv. campestris strains tested and several X. campestrispathovars.