Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection

The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intll (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intll genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intll gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.

Loop-mediated isothermal amplification assays for screening of bacterial integrons

BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

Antimicrobial Resistance Patterns and Integron Carriage of Escherichia coli Isolates Causing Community-Acquired Infections in Turkey

We aimed to observe antimicrobial resistance patterns and integron carriage of Escherichia coli isolates causing community-acquired infections. Two hundred sixty-eight E. coli strains were obtained from outpatients with various infections at different polyclinics at the 82nd Year of State Hospital in Rize, Turkey. Susceptibility to antimicrobials was tested using a disk diffusion method. The presence of integrons was examined using PCR with specific primers. Positive PCR results were confirmed by sequencing. A broth mating method was used for conjugation assays. Extragenic palindromic-PCR was performed using the oligonucleotide primer BOXA1R. Resistance frequency for ampicillin, trimethoprim/sulfamethoxazole, and tetracycline was determined as 50.6%, 33.5%, and 36.8% respectively. No strains were resistant to amikacin. Seventy isolates were positive for the intI1 gene, of which 49 carried gene cassettes. Eleven isolates were positive for the intI2 gene, eight of which carried gene cassettes. Seven gene cassettes (dfrA1, dfrA5, dfrA7, dfrA17, aadA1, aadA5, and sat2) were predominantly harbored in integrons. We detected conjugative plasmids harboring integrons in two E. coli strains. Four strain clusters were yielded by BOX-PCR fingerprints showing that they were clonally related. No apparent relationship occurred among class 1 and 2 integron-carrying strains. We conclude that integrons are widespread in genetically variable E. coli strains and will continue to mediate dissemination of resistance genes in the community.

Molecular Epidemiology of Integron-Associated Antimicrobial Gene Cassettes in the Clinical Isolates of Acinetobacter baumannii from Northern Taiwan

BACKGROUND: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. METHODS: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. RESULTS: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR) results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. CONCLUSIONS: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.

Serovariedades de Salmonella enterica subespecie enterica en porcinos de faena y su resistencia a los antimicrobianos / Serovars of Salmonella enterica subspecies enterica and its antimicrobial resistance in slaughterhouse pigs

Se realizó un estudio para determinar la prevalencia de Salmonella y sus serovariedades en cerdos de faena, para evaluar sus perfiles de resistencia a los antimicrobianos y para conocer la presencia de integrones de clase 1 como posibles reservorios de resistencia. A partir de un total de 386 muestras de porcinos provenientes de cuatro frigoríficos de las provincias de Buenos Aires y de Santa Fe (Argentina), se identificaron 93 (24,1%) cepas de Salmonella enterica subespecie enterica, 52 (55,9%) de contenido cecal y 41 (44,1%) de nódulo linfático ileocecal. Se hallaron 13 serovariedades de S. enterica, las más prevalentes fueron S. Schwarzengrund, S. Heidelberg, S. subespecie I 6,8:e,h:-, S. Derby y S. Bredeney. Se probaron 15 antimicrobianos por el método de dilución en agar: amikacina, gentamicina, ciprofloxacina, cefalotina, cefotaxima, enrofloxacina, fosfomicina, polimixina-B, tetraciclina, cloranfenicol, estreptomicina, trimetoprima-sulfametoxazol, ampicilina, nitrofurantoína y ácido nalidíxico. Según se estableció mediante la determinación de la CIM, el 73% de las cepas de S. enterica subespecie enterica fueron sensibles a todos los antimicrobianos probados. Se observó resistencia a tetraciclina en 24 (25,8%) de las 93 cepas, a cloranfenicol en 22 (23,7%), a estreptomicina en 22 (23,7%) a trimetoprima-sulfametoxazol en 20 (21,5%), a ampicilina en 18 (19,4%), a nitrofurantoína en 3 (3,2%) y a ácido nalidíxico en 3 (3,2%). Algunos aislamientos de S. Typhimurium, S. Heildelberg, S. Derby y S. Orion presentaron multirresistencia y portaban el gen de la integrasa clase 1. Los mayores porcentajes de resistencia correspondieron a los antimicrobianos habitualmente utilizados en veterinaria y en las explotaciones porcinas.

A study was carried out in order to determine the prevalence of Salmonella and its serovars among porcine slaughterhouses, to evaluate the antimicrobial resistance profiles and to know the presence of class 1 integrons as possible reservoir of resistance. From a total of 386 samples from four porcine slaughterhouses of Buenos Aires and Santa Fe Provinces (Argentina), 93 (24,1%) Salmonella enterica subspecies enterica strains were identified, 52 (55,9%) from cecal contents and 41 (44,1%) from ileocecal lymph nodes. Thirteen serovars of S. enterica were found, the most prevalent were: S. Schwarzengrund, S. Heidelberg, S. subspecie I 6,8:e,h:-, S. Derby and S. Bredeney. Fifteen antimicrobials by the agar dilution method were tested: amikacin, gentamicin, ciprofloxacin, cephalotin, cefotaxime, enrofloxacin, fosfomycin, polimixin-B, tetracycline, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, ampicillin, nitrofurantoin, and nalidixic acid. According to the CIM determination, 73% Salmonella enterica subspecies enterica strains were sensible to all the antimicrobials tested. Antimicrobial resistance was observed to tetracycline in 24 (25,8%) of 93 strains, to chloramphenicol in 22 (23,7%), to streptomycin in 22 (23,7%), to trimethoprim-sulfamethoxazole in 20 (21,5%), to ampicillin in 18 (19,4%), to nitrofurantoin in 3 (3,2%) and to nalidixic acid in 3 (3,2%). Some isolates of S. Typhimurium, S. Heidelberg, S. Derby, S. Orion showed multidrug resistance and carried the class 1 integrase gene. The highest percentage of resistance corresponded to the antimicrobials currently used in veterinary and porcine farms.

Optimization of Streptomyces bacteriophage phiC31 integrase system to prevent post integrative gene silencing in pulmonary type II cells

phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phiC31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1alpha (EF1alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1alpha promoter when combined with phiC31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with phiC31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

Prevalence of extended-spectrum beta-lactamase and class 1 integron integrase gene intl1 in Escherichia coli from Thai patients and healthy adults.

Among 120 Escherichia coli isolates from Thai patients, 37 and 9 isolates were extended-spectrum beta-lactamase (ESBL) and suspected ESBL producers respectively while 5 E. coli isolates from 120 Thai healthy adults were suspected ESBL producers. Integrase (intl1) gene was detected in 99% of the clinical and 87% of the non-clinical isolates. Among 37 ESBL producers, percent recovery of bla(TEM), bla(CTX-M), bla(SHV) and bla(VEB) was 78%, 78%, 8% and 8%, respectively. Twenty-five isolates of ESBL producers carried both bla(TEM) and bla(CTX-M), 2 isolates carried 3 genes (bla(TEM), blac(CTX-M), and bla(SHV)) and 3 showed no detectable bla gene. Among the 14 suspected ESBL producers, intl1 and bla(TEM) were detected in 13 isolates. ESBL producers from clinical samples were resistant to most of the tested antimicrobial agents compared to non-ESBL producers and isolates from healthy adults with about half of the latter susceptible to all tested antimicrobial agents. Only one clinical isolate was resistant to imipenem. Susceptibility to trimethoprim/sulfamethoxazole among the clinical isolates in ESBL producer group (27%) and non-producer group (33%) were comparable, whereas the percent susceptibility of the non-clinical isolates was about twice that of the clinical isolates.

Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Development of thymic lymphomas in mice disrupted of Brca2 allele in the thymus

Germ-line mutations in BRCA2 predispose to early-onset cancer. Homozygous mutant mouse, which has Brca2 truncated in exon 11 exhibit paradoxic occurrence of growth retardation and development of thymic lymphomas. However, due to its large embryonic lethality, cohort studies on the thymic lymphomas were not feasible. With the aid of Cre-loxP system, we demonstrate here that thymus-specific disruption of Brca2 allele without crossing it to p53-mutant background leads to the development of thymic lymphomas. Varying from 16 weeks to 66 weeks after birth, 25% of mice disrupted of Brca2 in the thymus died of thymic lymphomas, whereas previous report did not observe lymphomagenesis using similar Cre-loxP system. Future analysis of thymic lymphomas from these mice presented here will provide information on the cooperative mutations that are required for the BRCA2-associated pathogenesis of cancer.

Cre-loxP recombination vectors for promoter studies

For promoter studies the cloning, subcloning and transfer to different plasmid vectors usually requires use of restriction enzymes and ligation reactions. One obstacle is the nucleotide polymorphisms of eukaryotic genomic DNA, which has the consequence that a sequence often differs from published sequences. Therefore sequencing, rigorous restriction enzyme analysis or introduction of suitable sites has to be performed prior to cloning and subcloning. In addition, conventional methods using restriction enzymes, insert purifications and ligations is expensive and labour demanding. We have developed a fast, efficient and inexpensive Cre recombinase-loxP based method, which allows cloning of promoter regions and subcloning of these into a variety of vectors in a restriction enzyme independent manner. We here demonstrate that expression of a number of reporter genes and a therapeutic gene from both a viral and 2 mammalian promoters cloned by this recombinase method have activities comparable to conventionally cloned plasmids.

Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP

OBJECTIVE: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed andconfirmed by PCR and digesti on analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR.CONCLUSION: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.