Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

Analysis of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant A. baumannii (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 A. baumannii isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive A. baumannii (CSAB)) were collected. Carbapenemase genes (bla KPC, bla VIM, bla IMP, bla NDM, and bla OXA-23-like) and biofilm-formation-related virulence genes (abal, bfms, bap, and cusE) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a bla OXA-23-like-negative isolate. All 219 CRAB isolates were negative for bla KPC, bla VIM, bla IMP, and bla NDM, while bla OXA-23-like was detected in 218 isolates. The detection rates for abal, bfms, bap, and cusE in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array aacA4-catB8-aadA1 with relatively strong PcH2 promoter was detected in class 1 integrons. The bla OXA-23-like-negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying bla OXA-72, bla OXA-259, and bla ADC-26. In conclusion, bla OXA-23-like was the main reason for CRAB's resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the bla OXA-72, bla OXA-259, and bla ADC-26 was reported.

Molecular Characterization of Class 1 Integrons and Carbapenem-Resistant Genes in Enterobacter cloacae Complex Isolates.

Enterobacter cloacae complex (ECC) widely exists in the hospital environment and is one of the important conditional pathogens of hospital-acquired infection. To investigate the distribution of integrons and carbapenem-resistant genes in clinical ECC, 70 isolates of ECC from non-sputum specimens were collected. Class 1 and class 2 integron integrase gene intI1 and intI2, as well as common carbapenem-resistant genes, blaKPC, blaVIM, blaIMP, blaNDM, blaGES, and blaOXA-23, were screened. Gene cassette arrays and common promoters of class 1 integron together with subtypes of carbapenem-resistant genes were determined by sequencing. Resistant rates to commonly used antimicrobial agents between class 1 integron-positive and integron-negative ECC isolates were analyzed. The whole-genome of blaNDM-7 harboring Enterobacter hormaechei was sequenced and the sequence around blaNDM-7 was analyzed. Twenty isolates were positive for intI1. Nineteen different antimicrobial-resistant gene cassettes and 11 different gene cassette arrays, including aadA22-lnuF, were detected in this study. Common promoters of class 1 integron PcH1, PcW, PcW-P2, and PcH2 were detected in 12, 4, 3, and 1 isolates, respectively. The rates of antimicrobial resistance of intI1-positive isolates were higher than those of intI1-negative isolates to clinical commonly used antimicrobial agents. Carbapenem-resistant genes blaKPC-2, blaNDM-1, blaNDM-2, and blaNDM-7 were detected in 2, 1, 1, and 1 isolates, respectively. blaNDM-7 was located between bleMBL and IS5. To the best of our knowledge, this study reported for the first time of blaNDM-7 in ECC isolate in China.

Molecular characterization of class 1 integrons in carbapenem-resistant Enterobacterales isolates.

OBJECTIVE: Carbapenem-resistant Enterobacterales (CRE) infections result in higher treatment costs and mortality rates. Integrons play important roles in emergence and spread of antibiotic resistant genes. To get a better understand on the effects of integron on CRE resistance, distribution of common carbapenemase genes and class 1 integron in clinical CRE isolates were investigated. METHOD: Carbapenemase genes, including blaKPC, blaVIM, blaIMP, blaNDM, blaGES, blaVEB and blaOXA-23, were screened in 161 CRE isolates and subtypes of these genes were confirmed through sequence analysis. Class 1 integron was screened and common promoter and gene cassette arrays were determined by sequencing. The resistant rates to clinical commonly used antibiotics between integron positive and integron negative CRE isolates were compared. RESULTS: Of 161 CRE isolates, the most prevalent carbapenemase gene was blaKPC-2, which was detected in 139 isolates, including 99 Klebsiella pneumoniae. Class 1 integron was detected in 78 isolates. Twenty different gene cassettes, including two carbapenemase genes blaVEB-1 and blaIMP-4, and nine different gene cassette arrays, including blaVEB-1-aadB-arr-2-cmlA5-blaOXA-10-aadA1, aadB-catB8-blaOXA-10-aadA1-dfrA1-aacA4 and blaIMP-4-qacG-aacA4-catB3, were detected. Five types of common promoters were identified. Relative weak promoter PcH1 was the dominant type. Resistant rates of CRE isolates containing class 1 integrons to ceftazidime, amikacin, trimethoprim/sulfamethoxazole and gentamicin were higher than those without class 1 integrons (P < 0.05). CONCLUSION: Class 1 integrons play important roles in the emergence and spread of CRE resistance. To the best of our knowledge, this is the first report of aadB-catB8-blaOXA-10-aadA1-dfrA1-aacA4 and blaIMP-4-qacG-aacA4-catB3 in the same Providencia rettgeri isolate and blaVEB-1-aadB-arr-2-cmlA5-blaOXA-10-aadA1 in P. rettgeri.

Influence of class 2 integron integrase concentration on gene cassette insertion and excision in vivo.

Integron can capture and express antimicrobial resistance gene cassettes and plays important roles in horizontal gene transfer. The establishment of a complete in vitro reaction system will help to reveal integron integrase mediated site-specific recombination process and regulation mechanism. As an enzymatic reaction, the concentration of integrase is assumed to have a great influence on the reaction rate. To determine the influence of different concentrations of integrase on the reaction rate and to find the best range of enzyme concentration were essential to optimizing the in vitro reaction system. In this study, plasmids with gradient transcription levels of class 2 integron integrase gene intI2 under different promoters were constructed. Among plasmids pI2W16, pINTI2N, pI2W, and pI2NW, intI2 transcription levels ranged from about 0.61-fold to 49.65-fold of that in pINTI2N. And the frequencies of gene cassette sat2 integration and excision catalyzed by IntI2 were positively correlated with the transcription levels of intI2 within this range. Western blotting results indicated high expression of IntI2 partly existed in the form of an inclusion body. When compared with Pc of class 1 integron, the spacer sequence of PintI2 can increase the strength of PcW but decrease the strength of PcS. In conclusion, the frequencies of gene cassette integration and excision were positively correlated with the concentration of IntI2. intI2 driving by PcW with PintI2 spacer sequence can obtain the optimum IntI2 concentration required to achieve the maximum recombination efficiency in vivo in this study.

Two-dimensional PCR for detecting class 1, 2 and 3 integrons.

Integrons can capture and express foreign gene cassettes through site-specific recombination and are important genetic elements in spreading antibiotic resistance genes among bacteria. We have developed a two-dimensional PCR technology (2D-PCR) based on the base quenching probe technology in detecting three major integrons at the same time. The minimum detection limits were evaluated by detecting three plasmids each harboring different types of integron with different concentrations. The specificity of this method was evaluated by screening and typing three major types of integrons in 105 clinical Proteus isolates, and the results were compared with those of traditional PCR. Results indicated that the melting temperature (Tm) difference of the three genes was about 10 °C and was very easy to be distinguished. The minimum detection limits of intI1, intI2 and intI3 were all below 102 copies/µl. The detection results of clinical isolates were consistent with those of traditional PCR. This developed rapid, economic and high-throughput 2D-PCR based method can detect three main classes of integron at the same reaction, and can be applied to clinical isolates in large-scale integron screening and typing.

Comparison of Class 2 Integron Integrase Activities.

Integrons play important roles in the dissemination of antimicrobial resistant genes among bacteria. Class 2 integrons usually has an internal stop codon, TAA, in integrase genes (intI2), leading to a truncated integrase, IntI2*. However, a few class 2 integrons with a natural full-length integrase have been reported. In this study, the sequences of natural full-length intI2 were extracted from INTEGRALL database and analyzed. A total of 236 sequences of intI2 were retrieved from INTEGRALL database, only seven of which were natural full-length intI2 genes and could be divided into five types according to their coding amino acid sequence. Quantitative real-time PCR was used to detect gene cassette sat2 integration and excision efficiency catalyzed by different natural full-length IntI2s. The results showed that all five IntI2s could catalyze attI2 × attCsat2 integration and attCdfrA1/sat2 × attCsat2/aadA1 excision in Escherichia coli. Integration and excision frequency catalyzed by IntI2A176 was highest and was about twofold as high as those catalyzed by IntI2S175_A176. The secondary structure of the IntI2 was predicted by online software. Polymorphisms of these five IntI2s were limited within residues 172, 174, 175, 176 and 256, and these residues were all far away from the predicted DNA binding regions or catalyzed sites. Influence of amino acid sequence polymorphisms of these natural full-length IntI2s on their catalyzed activities is limited.

HIF-1α forms regulatory loop with YAP to coordinate hypoxia-induced adriamycin resistance in acute myeloid leukemia cells.

Despite the improvement in acute myeloid leukemia (AML) treatments, most patients had a poor prognosis and suffered from chemoresistance and disease relapse. Therefore, there is an urgent need for elucidation of mechanism(s) underlying drug resistance in AML. In the present study, we found that AML cells showed less susceptibility to adriamycin (ADR) in the presence of hypoxia, while inhibition of hypoxia-inducible factor 1α (HIF-1α) by CdCl2 can make AML cells re-susceptibile to ADR even under hypoxia. Moreover, HIF-1α is overexpressed and plays an important role in ADR-resistance maintenance in resistant AML cells. We further found hypoxia or induction of HIF-1α can significantly upregulate yes-associated protein (YAP) expression in AML cells, and resistant cells express a high level of YAP. Finally, we found that YAP may not only enhance HIF-1α stability but also promote HIF-1α's activity on the target gene pyruvate kinase M2. In conclusion, our data indicate that HIF-1α or YAP may represent a therapeutic target for overcoming resistance toward adriamycin-based chemotherapy in AML.

Characterization of Beta-Lactamases in Bloodstream-Infection Escherichia coli: Dissemination of blaADC - 162 and blaCMY- 2 Among Bacteria via an IncF Plasmid.

OBJECTIVES: To describe the molecular characteristics of beta-lactamases in bloodstream-infection Escherichia coli isolated from elderly patients, and to determine the genotypic patterns of bla CMY - 2 and bla ADC - 162. METHODS: A total of 50 bloodstream-infection E. coli isolates were obtained from patients aged > 50 years at Shanghai Sixth People's Hospital South Campus during 2015-2018. The isolates were subjected to beta-lactamase detection using phenotypic and molecular methods. Beta-lactamase genes were verified by sequencing and the phylogenetic relationships of the isolates were analyzed by multilocus sequence typing (MLST). The transferability of plasmids carrying bla CMY- 2 and bla ADC- 162 genes was verified by conjugation experiments and plasmid replicon typing. RESULTS: Eight beta-lactamase subtypes were detected in 50 isolates of bloodstream-infection E. coli. bla TEM- 1 (21/50) was the most common beta-lactamase gene, followed by bla CTX-M- 14 (8/50), bla OXA- 27 (5/50), bla CTX-M- 27 (3/50), bla CTX-M- 65 (1/50), bla ADC- 162 (1/50), and bla CMY- 2 (1/50). Of these, bla ADC- 162 (ST95-A), and bla CMY- 2 (ST95-B2) have not previously been reported in bloodstream-infection E. coli. In 21 isolates, beta-lactamase genes were located on conjugative plasmids belonging to incompatibility groups FrepB (n = 7), FIA (n = 1), FIC (n = 2), K (n = 8), N (n = 1), and I (n = 1), and bla CTX-M was associated with the common elements ISEcp1, IS903, and IS26, but with special sequences (region V, region Y, and region W) for ISEcp1 in 14 isolates. CONCLUSION: To the best of our knowledge, this study provides the first molecular characterization of beta-lactamase genes in E. coli isolated from the bloodstream in elderly patients. Beta-lactamase genes were detected at a relatively high frequency in elderly patients with bloodstream E. coli infections. Plasmid replicon analysis showed that horizontal dissemination of beta-lactamase genes was mainly mediated by IncK and IncF plasmids, which could encode multidrug resistance genes. The study also provides the first report of ISAba1-bla ADC - 162-tnpA and ISEcp1-bla CTX-M- 14-IS903-bla CMY- 2-blc-sugE in E. coli, and demonstrates IncF plasmid-mediated bla ADC - 162 and bla CMY- 2 gene dissemination among bacteria.

Polymorphisms of Gene Cassette Promoters of the Class 1 Integron in Clinical Proteus Isolates.

OBJECTIVE: To describe the polymorphisms of gene cassette promoters of the class 1 integron in clinical Proteus isolates and their relationship with antibiotic resistance. METHODS: Polymorphisms of the gene cassette promoter in 153 strains of Proteus were analyzed by PCR and nucleotide sequencing. Variable regions of atypical class 1 integrons were detected by inverse PCR and nucleotide sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyze the phylogenetic relationships of class 1 integron-positive clinical Proteus isolates. Representative beta-lactamase genes (bla), including bla TEM,bla SHV,bla CTX-M-1,bla CTX-M-2,bla CTX-M-8,bla CTX-M-9,bla CTX-M-25 and bla OXA-1, and plasmid-mediated quinolone resistance (PMQR) genes including qnrA, qnrB, qnrC, qnrD, qnrS, oqxA, oqxB, qepA, and aac(6')-Ib were also screened using PCR and sequence analysis. RESULTS: Fifteen different gene cassette arrays and 20 different gene cassettes were detected in integron-positive strains. Of them, aadB-aadA2 (37/96) was the most common gene cassette array. Two of these gene cassette arrays (estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3) have not previously been reported. Three different Pc-P2 variants (PcS, PcWTGN-10, PcH1) were detected among the 96 Proteus strains, with PcH1 being the most common (49/96). Strains carrying the promoters PcS or PcWTGN-10 were more resistant to sulfamethoxazole, gentamicin and tobramycin than those carrying PcH1. Strains with weak promoter (PcH1) harbored significantly more intra- and extra-integron antibiotic resistance genes than isolates with strong promoter (PcWTGN-10). Further, among 153 isolates, representative beta-lactamase genes were detected in 70 isolates (bla TEM-1, 54; bla OXA-1, 40; bla CTX-M-3, 12; bla CTX-M-14, 12; bla CTX-M-65, 5; bla CTX-M-15, 2) and representative PMQR genes were detected in 87 isolates (qnrA, 6; qnrB, 3; qnrC, 5; qnrD, 46; qnrS, 5; oqxA, 7; aac(6')-Ib, 13; aac(6')-Ib-cr, 32). CONCLUSION: To the best of our knowledge, this study provides the first evidence for polymorphisms of the class 1 integron variable promoter in clinical Proteus isolates, which generally contain relatively strong promoters. Resistance genotypes showed a higher coincidence rate with the drug-resistant phenotype in strong-promoter-containing strains, resulting in an ability to confer strong resistance to antibiotics among host bacteria and a relatively limited ability to capture gene cassettes. Moreover, strains with relatively weak integron promoters can "afford" a heavier "extra-integron antibiotic resistance gene load". Furthermore, the gene cassettes estX, psp and the gene cassette arrays estX-psp-aadA2-cmlA1, estX-psp-aadA2-cmlA1-aadA1a-qacI-tnpA-sul3 have been confirmed for the first time in clinical Proteus isolates. Beta-lactamase genes and PMQR were investigated, and bla TEM-1 and bla OXA-1 were the most common, with qnrD and aac (6')-Ib-cr also being dominant.

PdII -Catalyzed Oxidative Tandem aza-Wacker/Heck Cyclization for the Construction of Fused 5,6-Bicyclic N,O-Heterocycles.

A PdII -catalyzed oxidative tandem cyclization was developed for the construction of fused 5,6-bicyclic N, O-heterocycles. This reaction was enabled by the combined use of a 3-methylpyridine ligand and pentafluorobenzoic acid additive. A range of heterocyclic products with different substituents could be prepared in moderate to good yields via this methodology. Several transformations, including a scaled-up preparation of product 2 a, were also carried out showing the good applicability of our methodology.

Characterization of aminoglycoside resistance and virulence genes among Enterococcus spp. isolated from a hospital in China.

This study investigated the aminoglycoside resistance phenotypes and genotypes, as well as the prevalence of virulence genes, in Enterococcus species isolated from clinical patients in China. A total of 160 enterococcal isolates from various clinical samples collected from September 2013 to July 2014 were identified to the species level using the VITEK-2 COMPACT system. The antimicrobial susceptibilities of the identified Enterococcus strains were determined by the Kirby-Bauer (K-B) disc diffusion method. PCR-based assays were used to detect the aminoglycoside resistance and virulence genes in all enterococcal isolates. Of 160 Enterococcus isolates, 105 were identified as E. faecium, 35 as E. faecalis, and 20 isolates were classified as "other" Enterococcus species. High-level aminoglycoside resistance (HLAR) for gentamicin, streptomycin, and both antibiotics was identified in 58.8, 50, and 34.4% of strains, respectively. The most common virulence gene (50.6% of isolates) was efaA, followed by asa1 (28.8%). The most prevalent aminoglycoside resistance genes were aac(6')-Ie-aph(2''), aph(2')-Id, aph(3')-IIIa, and ant(6')-Ia, present in 49.4%, 1.3%, 48.8% and 31.3% of strains, respectively. Overall, E. faecium and E. faecalis were most frequently associated with hospital-acquired enterococcal infections in Zhejiang Province. All aminoglycoside resistance genes, except aph(2'')-Id, were significantly more prevalent in HLAR strains than amongst high level aminoglycoside susceptible (HLAS) strains, while there was no significant difference between HLAR and HLAS strains in regard to the prevalence of virulence genes, apart from esp, therefore, measures should be taken to manage infections caused by multi-drug resistant Enterococcus species.

A novel functional class 2 integron in clinical Proteus mirabilis isolates.

OBJECTIVES: To describe a novel functional class 2 integron that was found in clinical Proteus mirabilis isolates. METHODS: Class 1 and 2 integrons were screened by PCR in 153 clinical Proteus isolates. The variable regions of class 1 and 2 integrons were determined by restriction analysis and sequencing. The mutations of internal stop codons in class 2 integrons and their common promoters were also determined by sequencing. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to analyse the phylogenetic relations of class 2 integron-positive P. mirabilis isolates. RESULTS: Class 1 integrons were detected in 96 (63%) of 153 Proteus isolates: eight different gene cassette arrays were detected, including dfrA32-ereA1-aadA2, which was detected for the first time in P. mirabilis. Class 2 integrons were detected in 101 (66%) of 153 Proteus isolates: four different gene cassette arrays were detected, including dfrA1-catB2-sat2-aadA1, which was detected for the first time in a class 2 integron. A novel functional class 2 integron was detected in 38 P. mirabilis isolates with a common promoter (-35 TTTAAT|16 bp|-10 TAAAGT). The variable region of this functional class 2 integron contained dfrA14 and three novel open reading frames with unknown functions. Very similar ERIC-PCR fingerprinting patterns were detected in these 38 P. mirabilis isolates and were different from other class 2 integron-positive isolates. CONCLUSIONS: A novel functional class 2 integron was found for the first time in P. mirabilis. These functional class 2 integron-harbouring P. mirabilis isolates were likely to be clonally spread in our hospital.

Diversity of gene cassette promoter variants of class 1 integrons in uropathogenic Escherichia coli.

Class 1 integrons play important roles in the emergence and horizontal transfer of antibiotic resistance genes among bacteria. The gene cassette promoter variants Pc or Pc-P2 of class 1 integrons not only drive the transcription of downstream gene cassettes, they also correlate with the excision and integration efficiency of the capture exogenous gene cassettes. In this study, the diversity of Pc or Pc-P2 variants of class 1 integrons and their association with antibiotic resistance phenotypes were analyzed in 132 uropathogenic Escherichia coli strains. Class 1 integrons were detected in 95 (72 %) strains. Sixteen different gene cassettes, 11 different gene cassette arrays and six different Pc or Pc-P2 variants were detected. The most prevalent gene cassettes were those that conferred resistance to trimethoprim, aminoglycosides, and chloramphenicol. The most prevalent promoter was PcH1, a relatively weak promoter. Certain gene cassette arrays or gene cassettes were mainly associated with the same Pc or Pc-P2 in different strains. Strains harboring class 1 integrons with relatively strong promoters had higher resistance rates to, or higher MIC(50) for, amikacin, chloramphenicol and tobramycin than those with relatively weak promoters. To the best of our knowledge, this is the first report of the diversity of class 1 integron Pc or Pc-P2 variants in uropathogenic E. coli strains.

Expression and purification of SenX3 from the Mycobacterium tuberculosis strain H37Rv in Escherichia coli.

Infection with the bacterium Mycobacterium tuberculosis (MTB) causes tuberculosis, a pulmonary infection that may be fatal if left untreated. Misuse or mismanagement of tuberculosis drugs may lead to drug-resistant pathogen forms that are difficult to treat and contribute to a global health problem. The MTB SenX3/RegX3 signal transduction system allows bacteria to externally sense the environment and mediate an appropriate internal response; SenX3 is also associated with MTB virulence, suggesting that this protein may provide a potential therapeutic target. To investigate the role of SenX3 and MTB drug resistance, SenX3 was cloned, expressed and purified in Escherichia coli. SenX3 was cloned from the genome of the MTB strain H37Rv by polymerase chain reaction and an internal NcoI restriction site was destroyed by site-directed mutagenesis to allow cloning into the pET-28b prokaryotic expression vector. SenX3 expression from the resulting pET­28b-mSenX3 plasmid was induced with isopropyl ß-D-thiogalactoside and the protein was purified using Ni-NTA agarose affinity chromatography. A pure protein of the expected size was identified. The examination of purified SenX3 protein is considered to enable the in­depth investigation of SenX3-mediated drug resistance.

Transcription of integron-harboured gene cassette impacts integration efficiency in class 1 integron.

Class 1 integrons play important roles in the dissemination of antibiotic resistance genes among bacteria. Generally, class 1 integron consists of an integrase gene (intI1), a recombination site (attI1) and a promoter (Pc) that drives the transcription of the downstreamed gene cassettes. Occasionally, there is a second promoter P2 downstream of the Pc promoter. Several Pc variants and Pc-P2 combinations have been defined and they display different transcription strengths, but the influence of the transcription of integron-harboured gene cassette on the integration efficiency has never been comprehensively studied. In this study, the integration frequencies of gene cassettes into the attI1 sites that downstream of four different Pc variants as well as their combinations with P2 promoter were measured. The results showed that there was an inverse correlation between the strength of Pc promoter and the integration efficiency and, with the same Pc promoter, the integration efficiency was significantly decreased when a P2 promoter preceded the attI1 site. Our findings indicate there is a relationship between the transcription of integron-harboured gene cassette and the integration of exogenous gene cassettes. The interrelationship between these two relatively independent processes may throw a light on our understanding the regulation system of class 1 integron.

The pheV phenylalanine tRNA gene Klebsiella pneumoniae clinical isolates is an integration hotspot for possible niche-adaptation genomic islands.

Horizontally acquired genomic islands may allow bacteria to conquer and colonize previously uncharted niches. Four Klebsiella pneumoniae tRNA gene insertion hotspots (arg6, asn34, met56, and pheV) in 101 clinical isolates derived from blood, sputum, wound, bile or urine specimens were screened by long-range PCR for the presence or absence of integrated islands. The pheV phenylalanine tRNA gene was the most frequently occupied site and harbored at least three entirely distinct types of islands: (1) KpGI-1, a 3.7 kb island coding for four proteins, three of which showed high similarity to two hypothetical proteins and a Gcn5-related N-acetyltransferase in Salmonella enterica, (2) KpGI-2, a 6.4 kb island coding for five proteins including a truncated phage-like integrase, two helicase-related proteins, and a homolog of the functionally elusive Fic protein, and (3) KpGI-3, a 12.6 kb island which carried seven fimbriae-related genes, first identified in MGH78578. Consistent with the niche-adaptation hypothesis, KpGI-1-like islands which coded for the putative acetyltransferase were significantly over-represented in sputum isolates as compared to urine (P < 0.001), blood (P < 0.05) or bile (P < 0.05) derived isolates. Despite the unique nature of KpGI-2, likely homologs of orf5_KpGI-2 that coded for Fic were also found at undefined locations in six other clinical isolates, though none possessed the other KpGI-2 genes. We propose that the pheV-associated islands described in this study may contribute to fine tuning and adaptation of K. pneumoniae strains toward preferred infection and/or colonization pathways.

A novel and rapid method for determining integration frequency catalyzed by integron integrase intI1.

We developed a faster and more convenient method to determine integration frequency mediated by integron integrase intI1. This method based on real-time fluorescent quantitative PCR. By using this method, we revealed that the integration frequency of aadA2 gene cassette was 1.87x10(-4) when integrase intI1 was present, and the background frequency was less than 5.23x10(-8) without integrase intI1.