Irp2 Gene Research Articles

Detection Of Escherichia Coli Virulence Genes ( Pap , Afa , Iha Ipr2 And Hly ) Isolated From Patients With Urinary Tract Infections In Babylon Province, Iraq

The current study included a collection of 110 urine samples of both sexes of different ages for patients with urinary tract infections, who consulted Al-Hilla Teaching Hospital and the Children and Maternity Hospital in Al-Hilla city, through the period from May 2020 to December 2020, to investigate some virulence factors associated with Escherichia coli bacterium. The results of the biochemical tests revealed that 74 (67.3%) isolates were belonging to E. coli that was isolated from the urine samples. The results also indicated that the females were more infected 47 (63.5 %) with urinary tract infections than males 27 (36.4 %). Some virulence factor genes have also been investigated using the polymerase chain reaction (PCR) technique, which are (pap, afa, iha, ipr2 and hly genes). The PCR results for E.coli genes revealed that, 11 isolates contain pap gene (7 ( 14.8 %) and 4 ( 14.8 %)), while 9 isolates possesses afa gene ( 6 ( 12.7 %) and 3 ( 11.1 %)), whereas 5 isolates have gene iha (3 ( 6.3 %) and 2 ( 7.4 % )) , In addition to 30 isolates have irp2 gene (21 ( 44.6 % ) and 9 ( 33.3 % )) and 27 isolates contain hly gene ( 19 ( 40.4 %) and 8 ( 29.6 %)). These percentages are for females and males respectively. Conclusions: E.coli is the bacterium that dominant the UTIs. Females are more likely to develop UTIs than males. The irp2 gene responsible for taking iron is the most genetically studied genome that causes UTIs, followed by the gene hly responsible for producing α-hemolysin enzyme second among the gene of the virulence factors under study.

Evaluation of the Virulence Gene Irp2 in Iraqi Patients of Urinary Tract Infections and Other Community-Acquired Illnesses

A total of sixty-two isolates were tested to assess the presence of the irp2 gene in different isolates of Klebsiella pneumoniae. The isolates discussed in this study were obtained from patients who had acquired illnesses either within the hospital premises or in the surrounding vicinity. These isolates were sourced from three hospitals located in Baghdad, namely Al-Imam Ali, Al-Zaafaranya, and Ibin-Albady hospitals. One interesting thing about Klebsiella pneumoniae is that it makes siderophores, especially yersiniabactin. This is because of a gene that controls this trait. The application of DNA sequencing methodologies has facilitated the identification of the irp2 gene in 44% of Klebsiella pneumoniae. According to amino acid sequences and differentiation of nucleotide, the current work reports findings on the identification of the K. pneumoniae irp2 gene isolates collected from patients in Iraq. This event represents the initial recorded occurrence of such detection. The presence of this gene is considered an unconventional human pathogen. The aim is to explore the correlation between genetic analysis and the diagnosis of genetic variation by examining isolates documented in the global GenBank database (LC791754.1, LC791755.1, LC791756.1, LC791757.1, LC791758.1, LC791759.1, LC791760.1). Additionally, it seeks to provide insights into the magnitude of genetic variation observed within these isolates.

Molecular Prevalence and Geographical Variations of Carbapenem-Resistant Klebsiella pneumoniae ST15 Isolates in a Tertiary Hospital in Ningbo, China.

BACKGROUND In China, the most prevalent type of CRKP is ST11, but the high-risk clone ST15 has grown in popularity in recent years, posing a serious public health risk. Therefore, we investigated the molecular prevalence characteristics of ST15 CRKP detected in a tertiary hospital in Ningbo to understand the current potential regional risk of ST15 CRKP outbreak. MATERIAL AND METHODS We collected and evaluated 18 non-duplicated CRKP strains of ST15 type for antibiotic resistance. Their integrons, virulence genes, and resistance genes were identified using polymerase chain reaction (PCR), and their homology was determined using MALDI-TOF MS. RESULTS The predominant serotype of 18 ST15 CRKP strains was K5. ST15 CRKP exhibited the lowest antimicrobial resistance to Cefoperazone/sulbactam (11.1%), followed by trimethoprim/sulfamethoxazole (22.2%). Resistance gene testing revealed that 14 out of 18 ST15 CRKP strains (77.8%) carried Klebsiella pneumoniae carbapenemase 2 (KPC-2), whereas all ST15 CRKP integrons were of the intI1 type. Furthermore, virulence gene testing revealed that all 18 ST15 CRKP strains carried ybtS, kfu, irp-1, and fyuA genes, followed by the irp-2 gene (17 strains) and entB (16 strains). The homology analysis report showed that 2 clusters had closer affinity, which was mainly concentrated in classes C and D. CONCLUSIONS The ST15 CRKP antibiotic resistance rates demonstrate clear geographical differences in Ningbo. Additionally, some strains carried highly virulent genes, indicating a possible evolution towards carbapenem-resistant highly virulent strains. To reduce the spread of ST15 CRKP, we must rationalize the clinical use of antibiotics and strengthen resistance monitoring to control nosocomial infections.

Investigation of Genes Encoding Siderophores in Escherichia coli Isolated from Cystitis Patients

Pathogenic bacteria represented a challenge in acquiring iron as it is typically bound to heme or by proteins like transferrin or lactoferrin in the body, to overcome this challenge, uropathogenic Escherichia.coli uses various strategies to capture iron, with siderophores being one of these strategies. Therefore, in this study aimed to identify the specific gene types (iucA, iroN and irp2) responsible for siderophore production in uropathogenic E.coli in the presence of FeSO4. And potential implications for understanding the role of siderophores in cystitis. A total of 300 urine samples were collected from patients with signs and symptoms of UTIs. The boiling method was used for DNA extraction, followed by the analysis of the presence of 3 siderophore genes using PCR. FeSO4 preparation performed using serial dilution method to prepare solution known concentration (10-1, 10-2, 10-3, 10-4, and 10-5), Iron sulfate was prepared and tested on EMB medium, the bacteria were cultured by spreading method and impregnated fitter paper, and bacterial growth was measured using a colony counter instrument. A total of 45 isolates of E.coli are recovered from (300) patients suffering from UTIs. Based on the results, it was found that 26 out of the 45 bacterial isolates contained the irp2 gene, while 5 isolates contained the iroN gene. While the third gene, iucA, was not present in any of the bacterial isolates. Additionally, the data showed in the presence of FeSO4 at concentrations of 10-4 showed a more prominent effect in increasing bacterial growth rate, however, cultures supplemented with iron at concentrations of 10-3 and 10-5 showed moderate growth and low growth at concentration 10-2, while a concentration of 10-1 had an inhibitory effect on growth of the E.coli. A set of 45 urine sample of E.coli isolated from outpatient in Al- Najaf center, it was found that the irp2 gene is one of the most prevalent genes in bacterial isolates, indicating its importance as a virulence factor in UTIs caused by E.coli. I found this study as well, the concentration of FeSo4 plays a significant role in its impact on the growth rate and gene expression of E.coli.

The emergence of multi-drug resistant and virulence gene carrying Escherichia coli strains in the dairy environment: a rising threat to the environment, animal, and public health.

Escherichia coli is a common inhabitant of the intestinal microbiota and is responsible for udder infection in dairy cattle and gastro-urinary tract infections in humans. We isolated E. coli strains from a dairy farm environment in Xinjiang, China, and investigated their epidemiological characteristics, phenotypic and genotypic resistance to antimicrobials, virulence-associated genes, and phylogenetic relationship. A total of 209 samples were collected from different sources (feces, slurry, water, milk, soil) and cultured on differential and selective agar media (MAC and EMB). The presumptive identification was done by the VITEK2 system and confirmed by 16S rRNA gene amplification by PCR. Antimicrobial susceptibility testing was done by micro-dilution assay, and genomic characterization was done by simple and multiplex polymerase chain reaction (PCR). A total of 338 E. coli strains were identified from 141/209 (67.5%) of the samples. Most of the E. coli strains were resistant to sulfamethoxazole/trimethoprim (62.43%), followed by cefotaxime (44.08%), ampicillin (33.73%), ciprofloxacin (31.36%), tetracycline (28.99%), and a lesser extent to florfenicol (7.99%), gentamicin (4.44%), amikacin (1.77%), and fosfomycin (1.18%). All of the strains were susceptible to meropenem, tigecycline, and colistin sulfate. Among the resistant strains, 44.4% were identified as multi-drug resistant (MDR) showing resistance to at least one antibiotic from ≥3 classes of antibiotics. Eighteen out of 20 antibiotic-resistance genes (ARGs) were detected with sul2 (67.3%), blaTEM (56.3%), gyrA (73.6%), tet(B) (70.4%), aph(3)-I (85.7%), floR (44.4%), and fosA3 (100%, 1/1) being the predominant genes among different classes of antibiotics. Among the virulence-associated genes (VAGs), ompA was the most prevalent (86.69%) followed by ibeB (85.0%), traT (84.91%), ompT (73.96%), fyuA (23.1%), iroN (23.1%), and irp2 gene (21.9%). Most of the E. coli strains were classified under phylogenetic group B1 (75.45%), followed by A (18.34%), C (2.96%), D (1.18%), E (1.18%), and F (0.30%). The present study identified MDR E. coli strains carrying widely distributed ARGs and VAGs from the dairy environment. The findings suggested that the dairy farm environment may serve as a source of mastitis-causing pathogens in animals and horizontal transfer of antibiotic resistance and virulence genes carrying bacterial strains to humans via contaminated milk and meat, surface water and agricultural crops.

Molecular typification of Escherichia coli from community-acquired urinary tract infections in Mexico

One hundred and five uropathogenic Escherichia coli (UPEC) strains from patients with community-acquired urinary tract infections were characterized according to phylogenetic group, virulence factors, serogroup, antibiotic resistance, and genotype. The pathogenic phylogenetic groups (B2, D, and F) were found in 71.4% of the tested strains. Among them, the main uropathogenic serogroups were O8, O25, and O75, in which 97.1% of the strains had a multidrug-resistant profile. Sixteen virulence genes were analysed using a combination of polymerase chain reaction (PCR) assays, with the fimH, irp-2, iutA, aer, iucC, PAI, sat, iroN, usp, and cnf1 genes being mainly found in pathogenic phylogroups. The E. coli O25b-ST131 clone was identified in 32% of the strains assigned to the pathogenic phylogroup B2. These findings demonstrate that virulence genes encoding adhesin components, iron-acquisition systems, toxins, and pathogenicity-associated islands were highly prevalent among the pathogenic phylogroup of UPEC strains.

Pandemic Clones of CTX-M-15 Producing Klebsiella pneumoniae ST15, ST147, and ST307 in Companion Parrots.

Psittacine birds are commonly kept as companion birds and the maintenance of these birds in captivity may represent a zoonotic risk and contribute to the propagation of multidrug-resistant and β-lactamase extended-spectrum (ESBLs)-producing pathogens. This study aimed to identify and characterize strains of the Klebsiella pneumoniae complex isolated from diseased psittacine birds, determining virulence and resistance profiles. K. pneumoniae strains were isolated from 16 birds (16/46). All strains carried more than three virulence genes, with a high frequency of fimH and kpn (93.75%), uge (87.52%), and irp-2 (81.25%) genes. The antimicrobial susceptibility revealed that 3/16 strains were ESBL producers. Genomic analysis revealed that CTX-M-15-positive strains belonged to sequence types (STs) ST15, ST147, and ST307, characterized as international clones associated with outbreaks of healthcare-associated infections (HAIs) worldwide.

Virulence, Antimicrobial Resistance and Biofilm Production of Escherichia coli Isolates from Healthy Broiler Chickens in Western Algeria

The aim of this study was to assess the virulence, antimicrobial resistance and biofilm production of Escherichia coli strains isolated from healthy broiler chickens in Western Algeria. E. coli strains (n = 18) were identified by matrix-assisted laser desorption–ionization time-of-flight mass spectrometry. Susceptibility to 10 antibiotics was determined by standard methods. Virulence and extended-spectrum β-lactamase (ESBL) genes were detected by PCR. The biofilm production was evaluated by microplate assay. All the isolates were negative for the major virulence/toxin genes tested (rfbE, fliC, eaeA, stx1), except one was stx2-positive. However, all were resistant to at least three antibiotics. Ten strains were ESBL-positive. Seven carried the β-lactamase blaTEM gene only and two co-harbored blaTEM and blaCTX-M−1 genes. One carried the blaSHV gene. Among the seven strains harboring blaTEM only, six had putative enteroaggregative genes. Two contained irp2, two contained both irp2 and astA, one contained astA and another contained aggR, astA and irp2 genes. All isolates carrying ESBL genes were non-biofilm producers, except one weak producer. The ESBL-negative isolates were moderate biofilm producers and, among them, two harbored astA, two irp2, and one aggR, astA and irp2 genes. This study highlights the spread of antimicrobial-resistant E. coli strains from healthy broiler chickens in Western Algeria.

Antibiotic Resistance and Virulence Genes of Escherichia coli Isolated from Patients with Urinary Tract Infections After Kidney Transplantation from Deceased Donors.

ObjectiveThis study aimed to determine the prevalence of antibiotic resistance and virulence genes of Escherichia coli strains among patients with urinary tract infections (UTIs) after kidney transplantation from deceased donors.MethodsBetween January 2014 and June 2018, 64 patients who received kidney transplants from deceased donors at our institution developed a UTI due to E. coli. Polymerase chain reaction was used to detect virulence genes in E. coli strains. The Kirby–Bauer method was used to evaluate the antibiotic susceptibility pattern of the isolates.ResultsAmong the study cohort, 46 (71.9%) UTIs were community-acquired (CA), and 18 (28.1%) were hospital-acquired (HA). The percentages of isolated E. coli strains that showed antibiotic resistance were as follows: 92.2% to ampicillin, 76.6% to cefalotin, 81.3% to carbenicillin, 29.7% to ciprofloxacin, 62.5% to cotrimoxazole, 35.9% to gentamicin, 34.4% to levofloxacin, 28.1% to norfloxacin, 68.8% to pefloxacin, 57.8% to trimethoprim/sulfamethoxazole, and 20.3% to amikacin. HA E. coli showed higher resistance to ciprofloxacin, cotrimoxazole, trimethoprim/sulfamethoxazole and amikacin, compared with CA E. coli (P<0.05). The most prevalent virulence genes among the E. coli strains were fim (64.1%), followed by irp2 (56.3%), iroN (46.9%), pap GII (45.3%), sfa (31.3%), pap (25%), iuc (23.4%), pap GI (15.6%), pap GIII (14.1%), hly (9.4%), and cnf (4.7%). The irp2 and iroN genes were found more frequently in the HA E. coli than in the CA E. coli (P<0.05).ConclusionThe E. coli strains, especially HA E. coli, isolated from UTI patients after kidney transplantation from deceased donors showed resistance to multiple antibiotics and harbored numerous virulence genes. These findings provide insight for genetic characterizations and epidemiological studies of E. coli strains causing UTIs in patients after kidney transplantation from deceased donors.

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

To construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

Metabolic Conversion and Removal of Manganese Ferrite Nanoparticles in RAW264.7 Cells and Induced Alteration of Metal Transporter Gene Expression.

BackgroundManganese Ferrite Nanoparticles (Mn-IONPs) are widely used in biomedical field and their cytotoxicity has been initially explored, but the mechanism remains obscure. The nano-bio interactions are believed to be crucial for cytotoxicity mechanism, while little data have been acquired.MethodsMn-IONPs were synthesized by thermal decomposition of acetylacetonate precursor. After physicochemical characterization, we analyzed the metabolic conversion and removal of Mn-IONPs in RAW264.7 cells by Prussian blue staining, TEM, HRTEM and elemental quantitative analysis, followed by gene expression evaluation using quantitative RT-PCR.ResultsMn-IONPs were successfully synthesized. Both the uptake and cytotoxicity of Mn-IONPs on RAW264.7 cells were time- and dose-dependent. After internalized, Mn-IONPs were passed to daughter cells with passages on. Meanwhile, Mn-IONPs were exocytosed and digested to metal ions and further excreted out, resulted in the labeling rate and ions contents decreased gradually. As ion influx related genes, the expressions of ZIP14, IRP2, FtH and DMT1 were suppressed within 24 hours but overexpressed to a plateau at the 48th hour in a dose-dependent manner. At the 72nd hour, ZIP14 and DMT1 mRNA levels decreased toward normal, while IRP2 and FtH kept up-regulated. As efflux related genes, FPN, SLC30A10 and Hamp2 genes were up-regulated within 24–72 hours; SPCA1 was suppressed at the 24th and 72nd hour, while overexpressed at the 48th hour. All the efflux related genes’ mRNA had a dose-dependent increasing manner at the corresponding time points.ConclusionMn-IONPs showed time- and dose-dependent cytotoxicity and cell labeling rate in RAW264.7 cells. Accompanying with the intracellular catabolic breakdown and exocytosis of Mn-IONPs, RAW264.7 cells also secreted and re-uptook manganese and iron ions to maintain intracellular homeostasis in the succeeding passages. And the metabolic conversion of Mn-IONPs in RAW264.7 cells can affect the expression of ZIP14, DMT1, FPN, SLC30A10, IRP2, FtH, Hamp2 and SPCA1 genes.

Antimicrobial resistance and virulence genes in the Escherichia coli isolates obtained from ostrich.

Escherichia coli (E.coli) is a normal intestinal microflora of birds, including ostrich. However, some strains are pathogenic to ostrich. There is a lack of knowledge concerning the differences between commensal and pathogenic E.coli isolates of ostrich. This research aimed to characterize the antimicrobial susceptibility profile and virulence gene content of presumptive commensal and pathogenic E.coli isolates of ostrich. A total of 129 bacterial isolates (commensal strains no. = 45; pathogenic strains no. = 84) were obtained from ostriches. The resistance profile of these isolates was investigated by the disk diffusion method and PCR. Also, the strains were screened for virulence-associated genes of avian pathogenic E.coli (APEC). The study showed that the highest and the lowest antimicrobial resistance were against oxytetracycline and gentamicin, respectively. Oxytetracycline, florfenicol, and streptomycin resistance rate in pathogenic isolates were higher than commensal ones (p < 0.05). Also, tetA, blaTEM, and aac(3)-IV resistance genes were more prevalent in pathogenic than commensal isolates (p < 0.05). More than half of the isolates had no virulence-associated genes. The multiplex PCR results showed that irp2 gene was more prevalent in pathogenic than commensal E.coli (p < 0.05). Nevertheless this was not the case with the other genes. Our results indicated a low frequency of antimicrobial resistance and virulence genes in E.coli isolates of ostriches. Antimicrobial susceptibility profile and virulence gene content of E.coli isolates of ostriches differ between presumptive commensal and pathogenic strains. However, more analyses are needed to discriminate these isolates.

A survey for phylogenetic relationship; presence of virulence genes and antibiotic resistance patterns of avian pathogenic and uropathogenic Escherichia coli isolated from poultry and humans in Yazd, Iran

IntroductionExtra-intestinal infections in humans and poultry are caused by Extra-intestinal Pathogenic Escherichia coli strains (ExPEC) such as Avian Pathogenic E. coli (APEC) and Uropathogenic E. coli (UPEC). This study aimed to compare the presence of four pathogenic genes and antimicrobial resistance patterns of APEC and UPEC isolates, as well as investigating the phylogenetic relationship between these isolates by the Repetitive element sequence-based PCR (rep-PCR) method. Materials and methods112 E. coli isolates were collected from poultry specimens in farms and 88 E. coli isolates from human urinary tract infection in the patients of the hospital in Yazd city during March 2015 to July 2016. Susceptibility testing was carried out using the disk diffusion method. PCR was performed to investigate the presence of fimH, iucC, sitA, and irp2 genes. Data were analyzed using SPSS software (version 16) and Chi-square test. Phylogenetic analysis was performed and analyzed by gel-comparison software for the rep-PCR method. ResultsThe frequency of fimH, iucC, sitA, and irp2 genes was 67.8, 78.5, 84.8 and 89.2% for APEC and 96.5, 79.5, 86.3 and 75% for UPEC, respectively. Poultry strains exhibited multi-drug resistance to 16 antibiotics with the highest resistance to nalidixic acid and erythromycin, and all human isolates showed multi-drug resistance. The phylogenetic relationship was not found between human and poultry isolates by the rep-PCR method. ConclusionHigh antibiotic resistance was observed in isolates related to humans and poultry. Due to the presence of fimH, iucC, sitA, and irp2 genes in APEC and UPEC isolates, these genes are suggested to be effective in extra-intestinal E. coli infections. The phylogenetic relationship between the two pathotypes was not found by rep-PCR. Therefore, the hypothesis of transferring APEC to humans requires further investigation.

A virulence gene typing scheme for Photobacterium damselae subsp. piscicida, the causative agent of fish photobacteriosis, reveals a high prevalence of plasmid-encoded virulence factors and of type III secretion system genes

Fish photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) constitutes one of the major diseases affecting marine aquaculture worldwide. Although several virulence factors have been described in this pathogen, its pathobiology is still poorly understood. Two virulence factors, the exotoxin AIP56 and the siderophore piscibactin, are encoded by plasmids. Notably, presence of genes of a type III secretion system (T3SS) has been recently reported for the first time in this subspecies. However, the prevalence of these virulence genes in the populations of Pdp remains unstudied. Here, we have designed a PCR-based typing scheme for the detection of plasmid (pPHDP10)-borne aip56 gene encoding AIP56 exotoxin, plasmid (pPHDP70)-borne irp2 and frpA genes encoding piscibactin biosynthesis and transport proteins respectively, and for detection of the T3SS genes vscD and vcrD. The analysis of 103 isolates between 1963 and 2015, from different host species and geographical locations, has revealed that aip56 is highly prevalent, with only 6 isolates negative for aip56. The virulence plasmid pPHDP70 was present in 89 European and 2 Japanese isolates, refuting previous observations of a linkage of this plasmid exclusively to European strains. Most notably, 74 isolates tested positive for the T3SS genes, and their presence in photobacteriosis outbreaks could be traced back to 1980. Remarkably, we noticed that spontaneous rifampicin-resistant Pdp colonies underwent the loss of the T3SS genes vscD and vcrD, concomitantly with the loss of additional T3SS gene markers and of the putative plasmid-related genes parAB and traC, suggesting that the Pdp T3SS system is encoded within an unstable plasmid. This study unveils an unsuspected high prevalence of a putative plasmid-borne T3SS in this pathogen, and provides evidence that photobacteriosis outbreaks can be caused by distinct Pdp genotypes.

Phylogenetic typing and virulence gene profile of pathogenic and commensal avian Escherichia coli in Iran: a notable finding

The aim of this study was to compare phylogenic groups and virulence genes profiles of Escherichia coli isolated from poultry suffering from Colibacillosis with E.coli in the intestinal flora of healthy poultry. For this purpose, var, tsh, iucd, papc, irp2, and iss virulence genes of E. coli were investigated by multiplex PCR in 70 isolates (35 obtained from healthy samples and the other 35 isolated were attained form those with Colibacillosis) in Ardabil province, northwest of Iran. The phylogenic groups were also determined through use of PCR multiplex. Among them, 32 isolates (91%) of samples taken from poultry with Colibacillosis and 27 isolates (77%) from healthy samples contained papc gene. The results for iucd, var, and irp2 genes were 17 healthy and 20 diseased isolates, 23 healthy and 28 diseased isolates, and 32 healthy and 29 diseased isolates, respectively. In the case of tsh gene, only one isolate from healthy samples included the mentioned gene; while iss gene was observed in none of the isolates. In terms of phylogeny groups, except one, all the isolates belonged to the phylogenic group D. Results of present research revealed that the frequency of virulence genes and the diversity of phylogeny groups in isolates from healthy and diseased samples are not significantly different in the studied area.

Effect of Helicobacter pylori, black tea and sodium bicarbonate on iron metabolism and MDCK cell survival

Background: Iron deficiency anemia is the most common nutritional disorder in the world. Diet and Helicobacter pylori infection are among the main causes of this disorder. Objective: In this study, the effect of black tea extract and sodium bicarbonate with Helicobacter pylori on the genes involved in iron absorption and storage, as well as cell proliferation, were studied. Methods: Simultaneous cultivation of MDCK and Helicobacter pylori cell lines was performed at concentrations of 10, 20, 40 and 80 μg/ml of tea extract and 30, 40, 60 and 100 mM sodium bicarbonate at 24 and 48 hours. The effect of treatment on cell survival was investigated by trypan blue staining and expression of MYC, TFRC, FTH1, IRP2, IRP1, and NDRG1 genes by real-time PCR and analyzed by ANOVA and independent T-test. Findings: There was no significant change in the expression of the genes involved in iron metabolism under the influence of tea, sodium bicarbonate and Helicobacter pylori treatment in MDCK cell line. Upregulation MYC gene expression was observed in the presence of Helicobacter pylori after 24 hours treatment with tea extract, and sodium bicarbonate, and in the absence of Helicobacter pylori upregulation with tea extract after 48 hours (P<0.05). Also upregulation NDRG1 gene expression was seen after tea extract treatment of cells with or without Helicobacter pylori in both 24 and 48 h (P<0.05). Conclusion: Sodium bicarbonate and tea each one alone didn’t not influence iron status. This study suggests that reduction of tea intake could be served as a risk prevention strategy.

Iron Overload Plays an Important Role in ER Stress-Induced Insulin Resistance in Human Skeletal Muscle Cells

Aim: In this study, we investigated the relationship and molecular mechanisms between iron-overload and ER-stress-induced insulin resistance in human skeletal muscle cells. Methods: Intracellular iron was measured using calcein AM. Iron metabolism-related genes were analyzed by immunoblotting and PCR. The study was conducted using iron supplementation, which was achieved by palmitate, FeSO4 and FeCl3 administration, and iron reduction, which occurred by DFO, DS and Bapta AM administration, and knockdown of tfR1 or IRP2 genes, to investigate the effects of iron metabolism on insulin sensitivity in HSMMs. Intracellular calcium was detected using Fluo-3/AM staining. TfR1 internalization regulated by calcium was detected by GFP-labelled transferrin. Results: Endoplasmic reticulum stress by tunicamycin, thapsigargin, or palmitate evoked insulin resistance and simultaneously increased intracellular iron. Iron chelator, DFO, dramatically prevented ER-stress inducer-induced insulin resistance and iron donor impaired insulin sensitivity in vitro and in vivo through activation of JNK. Among several iron metabolism-related genes, tfR1 plays a predominant role for maintaining iron homeostasis in HSMMs. Treatment of ER-stress inducer positively regulated translocation of tfR1 by intracellular calcium, but protein levels of tfR1 did not change. Iron reduction, by adding iron chelator, calcium chelator, or knockdown of tfR1 or IRP2, dramatically prevented ER-stress-induced insulin resistance through the reduction of iron-overload. Conclusions/Interpretation: The current study shows ER-stress inducer evoked insulin resistance through iron overload. Reduction of intracellular iron, by iron chelator, significantly prevents insulin resistance. Therefore, attempts to block iron overload might be a strategy for preventing insulin resistance and diabetes. Disclosure K. Lee: None. R. Cui: None. D. Kim: None. S. Choi: None. W. Lee: None. Y. Kang: None. T. Kim: None. H. Moon: None. J. Jeon: None. S. Han: None. H. Kim: None.

Antibiotic resistance pattern and virulence genes content in avian pathogenic Escherichia coli (APEC) from broiler chickens in Chitwan, Nepal

BackgroundAvian pathogenic Escherichia coli (APEC) are causative agent of extraintestinal infections, collectively known as colibacillosis, which results significant losses in poultry industries. The extraintestinal survival of E. coli is facilitated by numerous virulence factors which are coded by virulence genes. This study was conducted to find out the pattern of antibiotic resistance and virulence genes content in the APEC strains isolated from broiler chickens at National Avian Disease Investigation Laboratory and Veterinary Teaching Hospital, Rampur, Chitwan, Nepal.ResultsA total of 50 E. coli strains were isolated from 50 colibacillosis suspected broiler chickens. Out of 50 isolates of E. coli, 47 (94%) showed resistant to three or more antimicrobials. The highest levels (22%) of multidrug-resistant E. coli were observed for five different types of antimicrobials. Antibiogram profiles of 50 E. coli strains showed the maximum resistance to ampicillin (98%), followed by co-trimoxazole (90%), and doxycycline (62%). The highest intermediate resistance was shown by colistin (50%) and the highest sensitivity was against amikacin (84%), followed by nitrofurantoin (55%). Based on the genetic criteria, 45 (90%) E. coli isolates were considered as pathogenic (APEC) which contained more than five virulence genes. Out of total APEC genes detected, we found the combination of iss, iucD, hlyF, ompT, iroN, and iutA genes were mostly associated with the APEC and additionally, to some lesser extent irp2, papC, Cva/cvi, and tsh genes showed the critical role for virulent traits of APEC strains.ConclusionIn this study, high prevalent of antimicrobial resistant pattern was found with avian pathogenic E. coli strains isolated from broiler chickens. To our knowledge, this is the first molecular analysis which confirmed the prevalence of APEC strains in poultry sector in Nepal. These finding suggest the need of surveillance and intervention system to control misuse of antibiotics and APEC outbreak in the poultry farm.

Divergence of affinities, serotypes and virulence factor between CTX-M Escherichia coli and non-CTX-M producers

This study was undertaken to discern the differences of the multi-locus sequence typing (MLST), O serogroups, and virulence factors among 34 CTX-M-1 Escherichia coli, 49 CTX-M-9 strains and 23 non-CTX-M isolates from chickens in Henan province, China. The MLST scheme yielded 34 sequence types, in which ST155 and ST359 were frequent (17% and 15%, respectively) and associated with zoonotic disease. The irp-2 (20% versus 2%, P = 0.0001), traT (85.3% versus 56.5%, P = 0.019), and sfaS (70.6% versus 0, P = 0.021) were significantly more prevalent in CTX-M-1 E. coli than in non-CTX-M producers. Also, CTX-M-9 isolates carried more irp-2 (17% versus 2%, P = 0.023), iroN (71.4% versus 39.1%, P = 0.019), and iss (79.6% versus 39.1%, P = 0.002) genes. In conclusion, although the 106 isolates encompassed a great genetic diversity, the CTX-M isolates harbored more virulence factor genes than non-CTX-M producers.

The irp2 Genes in High Pathogenicity Islands are Involved in the ROS Generation and Increases Antioxidase Levels in the PMNs Caused by Porcine Pathogenic Escherichia coli

Polymorphonuclear leukocytes (PMNs) release large quantities of reactive oxygen species (ROS) to kill pathogens. Escherichia coli (E. coli) strains containing a Yersinia high-pathogenicity island (HPI) display increased virulence that is attributable to increased iron scavenging activity, which enhances bacterial growth and limits the availability of iron for use by innate immune cells. ROS generation requires the catalysis of iron. The irp2 gene has been confirmed to be the main gene involved in the synthesis of HPI. In the present study, the effect of pathogenic HPI-positive Yunnandominant (O152) E. coli strains on the respiratory oxidative stress response from PMNs in Yunnan Saba pigs were not explored. The results showed that E. coli containing the HPI can reduce ROS generation by competitively consuming iron in the surrounding environment, while inducing high levels of antioxidases in PMNs. We discovered a novel mechanism by which the HPI protects E.coli from ROS and enhances its virulence in PMNs. These results increase our understanding of the interaction between pathogenic E. coli and host PMNs in Saba pigs.