B2 Isolates Research Articles

Morphological Identification and Antimicrobial Activity Test to Water Quality Using Bandotan (Ageratum conyzoides) Leaves

Water cleaning technology using coagulation has already been carried out use a plants in the coagulation process has never been done before, especially using plants that have potential to be a source of natural coagulants is Bandotan (Ageratum conyzoides). Plants that have the potential to be a source of natural coagulants are plants that contain a lot of protein. Each type of protein has an isoelectric point at a different pH like isoelectric point, the protein will have a neutral charge. . The calculation results of the number of colonies in samples were B1, B2, and B3 with dilutions of 266. 105, 9. 107 and 104. 109 CFU/mL. The results of Gram staining identification showed that the B1, B2 and B3 isolates had the shape are basil, basil and coccus (round) with Gram positive. The bacteria thrive in different habitats, so bacteria from the sample have been very cloudy to pH of 6.0 and 8.0 because neutralophiles properties. From the B3 isolate sample have high clear zone for Gram positive (S. aureus) of 13 mm more than Gram negative ( E. coli) of 11 mm. So that, the bandotan is able to inhibit the growth of pathogenic bacteria in the water

Isolation, Screening and Identification of Biosurfactant Producing Strain Nocardiopsis dassonvillei var B2 From Oil Contaminated Soil.

Petroleum and other oil manufacturing industries contribute to environmental pollution by releasing hazardous hydrocarbons. Biosurfactants offer a sustainable solution for mitigating oil pollution through emulsification processes, safeguarding agricultural soils, aquatic ecosystems, and human health. This study focuses on isolating, screening, and identifying actinomycetes producing biosurfactant from oil-polluted soil in the naval dockyard of Visakhapatnam. Biosurfactant production was successfully achieved utilizing Kim's medium, which was supplemented with olive oil serving as the carbon source. The evaluation involved preliminary identification tests, including oil displacement, Parafilm-M, and lipase activity assays, using sodium lauryl sulfate as the standard reference. Surface tension and emulsification index measurements were conducted, and the chemical composition of glycolipids and phospholipids was elucidated using phenol-sulfuric acid and phosphate assays. Glycolipids, specifically identified as rhamnolipids, were confirmed via cetyltrimethylammonium bromide (CTAB) testing and quantitatively analyzed using the orcinol method. The cell-free broth exhibited antagonistic activity against Gram-positive and negative bacilli.16S rRNA sequencing-based phylogenetic analysis was carried out by the NCIM, Pune, with the gene sequence being deposited in GenBank. Further characterization of isolate B2 included scanning electron microscopy (SEM) analysis, as well as physiological and biochemical assays. This study highlights the ability of Nocardiopsis dassonvillei var. B2, isolated from oil-polluted soil, to produce biosurfactants, specifically glycolipids identified as rhamnolipids. Our findings represent the first reported instance of biosurfactant production from isolate B2 originating from the naval dockyard in Visakhapatnam, Andhra Pradesh, India.

Longitudinal study of avian pathogenic Escherichia coli (APEC) serogroups associated with disease in Georgia poultry using molecular serology and virulence gene analysis

ABSTRACT Avian pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality, and production loss to the poultry industry worldwide. Here, we characterized 569 E. coli isolates from avian-diagnosed colibacillosis cases from the state of Georgia, USA. In total, 339 isolates were assigned into 32 serogroups with the majority classifying as O78, O2, O25, O8, O1, O86, O18, and O15. Serogroup O25 was found to link with broilers, while broiler breeders were more often associated with serogroup O1 and pet/ hobby birds with serogroup O8. In addition, some serogroups (O1) were more prevalent in the summer and fall. Analysis for virulence-associated genes (VAGs) found 23.20% of isolates did not harbour any genes linked with the APEC pathotype, while ColV plasmid-associated genes (iroN, ompT, hlyF, iss, and aerJ) were frequently detected among most isolates (with 80–96% prevalence) and some of these genes were linked with serogroup. Phylogenetic analysis, classified isolates into phylogenetic groups B2 (34%), F (19%), A (15%), and G (9%). The phylogenetic group B2 isolates also harboured the highest number of VAGs. This study highlights that the current APEC-causing disease in birds in the State of Georgia has identified several emerging serogroups possessing several VAGs that could potentially lead to challenges in colibacillosis control. RESEARCH HIGHLIGHTS Several emerging APEC serogroups were observed in Georgia poultry populations. An association between APEC serogroups and bird type was observed. The prevalence of different APEC serogroups was influenced by season. A multiplex PCR assay targeting common serogroups of APEC in Georgia poultry was developed.

Genomic characterization of third-generation cephalosporin-resistant Escherichia coli strains isolated from diseased dogs and cats: Report from Japanese Veterinary Antimicrobial Resistance Monitoring

This study investigates the genomic characteristics of canine and feline cefotaxime (CTX, a third-generation cephalosporin)-resistant Escherichia coli using the JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring System, a nationwide monitoring.In this study, whole-genome sequencing (WGS) was performed on 51 canine and 45 feline CTX-resistant E. coli isolates, with certain isolates subjected to pulsed-field gel electrophoresis with S1 nuclease for plasmid–chromosome separation.The most common blaCTX-M genes were blaCTX-M-27 (dogs: 11/51 [21.6 %]; cat: 10/45 [22.2 %]), followed by blaCTX-M-14 (dogs: 10/51 [19.6 %]; cats: 10/45 [22.2 %]), and blaCTX-M-15 (dogs: 9/51 [17.6 %]; cats: 5/45 [11.1 %]). Besides β-lactamase genes, all isolates harbored mdf(A), a multidrug efflux pump, with resistance genes for aminoglycosides, sulfonamides, trimethoprims, macrolides and tetracyclines. None of the isolates had carbapenemase genes, such as blaOXA-48, blaNDM, and blaIMP, whereas most of the isolates showed double mutations in gyrA and parC, which affected quinolone resistance. For the isolates separately analyzed for plasmid and chromosomal DNA via WGS, the majority of CTX-M genes were present on the plasmids. Some plasmids also harbored the same combination of resistance genes and plasmid replicon type, although they differed from isolates derived from different areas of Japan. The predominant plasmids were blaCTX-M-27,aadA5, aph(6)-Id, aph(3”)-Ib, sul1, sul2, tet(A), dfrA17, and mph(A) on IncF. The predominant combination of ST131, O25:H4, and B2 isolates comprised the largest cluster in the minimum spanning tree and the ST131 E. coli harboring blaCTX-M-27 from human in Japan was closely related to these isolates.The results indicated that CTX-resistant canine and feline E. coli harbored multiple plasmids carrying the same combination of resistance genes and emphasizes the need to prevent the spread. Data AvailabilityAll raw short-read sequence data have been deposited in the DNA Data Bank of Japan. (DRR Run No, DRR335726–335821).

HOST RANGE AND CROSS INFECTIVITY STUDIES OF CORYNESPORA CASSIICOLA

Corynespora infecting cotton has wide host range and due to its importance in blackgram earlier in Andhra Pradesh an investigation was carried to test the ability of the pathogen, Corynespora cassiicola isolated from cotton could cross infect blackgram crop and vice versa. Isolates were collected from the major cotton and blackgram (B-1 to B-6) growing areas viz., Guntur, Prakasam and Krishna districts. Experimental design was completely randomized in a factorial arrangement of 12x3x2 (12 isolates, 3 areas and 2 hosts) and replicated thrice. Cross inoculation experiments demonstrated variation in the level of latent period, infectivity and per cent disease index (PDI) among C. cassiicola isolates. The incubation period was 4 to 7 days in cotton for cotton isolates with highest PDI (32.94) in C-5 and lowest in C-2 isolates (20.77 PDI). It was 3 and 6 days in blackgram with blackgram isolates; the highest PDI (38.05) was recorded in B-1 and lowest in B-6 (16.06). However, C6 (8.28) and B5 (11.21) displayed the highest virulenceindex, respectively. Cross infectivity studies registered the highest PDI with C3 isolate (24.82) and the lowest in C5 isolate (12.48) in blackgram; whereas PDI was highest with B2 isolate (15.90) and B6 isolate (5.59) in cotton. In terms of virulence, C1 (4.50) and B4 (2.44) exhibited the highest virulence index, respectively.

Genotypic characteristics of Uropathogenic Escherichia coli isolated from complicated urinary tract infection (cUTI) and asymptomatic bacteriuria-a relational analysis.

Uropathogenic Escherichia coli (UPEC) is the predominant agent causing various categories of complicated urinary tract infections (cUTI). Although existing data reveals that UPEC harboured numerous virulence determinants to aid its survival in the urinary tract, the reason behind the occurrence of differences in the clinical severity of uninary tract infections (UTI) demonstrated by the UPEC infection is poorly understood. Therefore, the present study aims to determine the distribution of virulence determinants and antimicrobial resistance among different phylogroups of UPEC isolated from various clinical categories of cUTI and asymptomatic bacteriuria (ASB) E. coli isolates. The study will also attempt a relational analysis of the genotypic characteristics of cUTI UPEC and ASB E. coli isolates. A total of 141 UPEC isolates from cUTI and 160 ASB E. coli isolates were obtained from Universiti Malaya Medical Centre (UMMC). Phylogrouping and the occurrence of virulence genes were investigated using polymerase chain reaction (PCR). Antimicrobial susceptibility of the isolates to different classes of antibiotics was determined using the Kirby Bauer Disc Diffusion method. The cUTI isolates were distributed differentially among both Extraintestinal Pathogenic E. coli (ExPEC) and non-ExPEC phylogroups. Phylogroup B2 isolates were observed to possess the highest average aggregative virulence score (7.17), a probable representation of the capability to cause severe disease. Approximately 50% of the cUTI isolates tested in this study were multidrug resistant against common antibiotics used to treat UTI. Analysis of the occurrence of virulence genes among different cUTI categories demonstrated that UPEC isolates of pyelonephritis and urosepsis were highly virulent and had the highest average aggregative virulence scores of 7.80 and 6.89 respectively, compared to other clinical categories. Relational analysis of the occurrence of phylogroups and virulence determinants of UPEC and ASB E. coli isolates showed that 46.1% of UPEC and 34.3% of ASB E. coli from both categories were distributed in phylogroup B2 and had the highest average aggregative virulence score of 7.17 and 5.37, respectively. The data suggest that UPEC isolates which carry virulence genes from all four virulence genes groups studied (adhesions, iron uptake systems, toxins and capsule synthesis) and isolates from phylogroup B2 specifically could predispose to severe UTI involving the upper urinary tract. Therefore, specific analysis of the genotypic characteristics of UPEC could be further explored by incorporating the combination of virulence genes as a prognostic marker for predicting disease severity, in an attempt to propose a more evidence driven treatment decision-making for all UTI patients. This will go a long way in enhancing favourable therapeutic outcomes and reducing the antimicrobial resistance burden among UTI patients.

Clinical isolates of hepatitis B virus genotype C have higher in vitro transmission efficiency than genotype B isolates.

Serum samples were collected from 54 hepatitis B e antigen (HBeAg)-positiveChinese patients infected with hepatitis B virus (HBV)subgenotype B2 or C2. They were compared for transmission efficiency using same volume of samples or infectivity using same genome copy number. Adding polyethylene glycol (PEG)during inoculation did not increase infectivity of fresh samples but markedly increased infectivity following prolonged sample storage. Differentiated HepaRG cells infected without PEG produced more hepatitis B surface antigen (HBsAg)and higher HBsAg/HBeAgratio than sodium taurocholate cotransporting polypeptide (NTCP)-reconstitutedHepG2 cells infected with PEG. They better supported replication of core promoter mutant in contrast to wild-type (WT)virus by HepG2/NTCP cells. Overall, subgenotype C2 samples had higher viral load than B2 samples, and in general produced more HBeAg, HBsAg, and replicative DNA following same-volume inoculation. Precore mutant was more prevalent in subgenotype B2 and had reduced transmission efficiency. When same genome copy number of viral particles was inoculated, viral signals were not necessarily higher for three WT C2 isolates than four WT B2 isolates. Using viral particles generated from cloned HBV genome, three WT C2 isolates showed slightly reduced infectivity than three B2 isolates. In conclusion, subgenotype C2 serum samples had higher transmission efficiency than B2 isolates in association with higher viral load and lower prevalence of precore mutant, but not necessarily higher infectivity. PEG-independent infection by HBV viremic serum samples is probably attributed to a labile host factor.

Mobile colistin resistance (MCR), extended-spectrum beta-lactamase (ESBL) and multidrug resistance monitoring in Escherichia coli (commensal and pathogenic) in pig farming: need of harmonized guidelines and clinical breakpoints.

Current data on antimicrobial resistance in pig production is essential for the follow-up strategic programs to eventually preserve the effectiveness of last-resort antibiotics for humans. Here, we characterized 106 Escherichia coli recovered in routine diagnosis (2020-2022) from fecal sample pigs, belonging to 74 Spanish industrial farms, affected by diarrhea. The analysis of virulence-gene targets associated with pathotypes of E. coli, determined 64 as pathogenic and 42 as commensal. Antimicrobial susceptibility testing (AST) performed by minimal inhibitory concentration (MIC) assay, was interpreted by applying breakpoints/cut-off values from the different standards EUCAST/TECOFF 2022, CLSI VET ED5:2020, and CASFM VET2020. Comparisons taking EUCAST as reference exhibited moderate to high correlation except for enrofloxacin, neomycin, and florfenicol. Of note, is the lack of clinical breakpoints for antibiotics of common use in veterinary medicine such as cefquinome, marbofloxacin, or florfenicol. AST results determined multidrug resistance (MDR) to ≥3 antimicrobial categories for 78.3% of the collection, without significant differences in commensal vs pathogenic isolates. Plasmid-mediated mobile colistin resistance gene (mcr) was present in 11.3% of 106 isolates, all of them pathogenic. This means a significant decrease compared to our previous data. Furthermore, 21.7% of the 106 E. coli were ESBL-producers, without differences between commensal and pathogenic isolates, and mcr/ESBL genes co-occurred in 3 isolates. Phylogenetic characterization showed a similar population structure (A, B1, C, D, and E), in both commensal and pathogenic E. coli, but with significant differences for B1, C, and E (38.1 vs 20.3%; 19 vs 1.6%; and 7.1 vs 25%, respectively). Additionally, we identified one B2 isolate of clone O4:H5-B2-ST12 (CH13-223), positive for the uropathogenic (UPEC) status, and in silico predicted as human pathogen. We suggest that a diagnosis workflow based on AST, detection of mcr and ESBL genes, and phylogenetic characterization, would be a useful monitoring tool under a "One-Health" perspective.

Colibactin possessing E. coli isolates in association with colorectal cancer and their genetic diversity among Pakistani population

Colorectal cancer (CRC) is the third most prevalent cause of tumorigenesis and several pathogenic bacteria have been correlated with aggressive cases of cancer i.e., genotoxin (colibactin) producing Escherichia coli (E. coli). This study was designed to investigate the genetic diversity of clb+clb+ E. coli strains and their association with CRC. Pathogenic E. coli isolates from colorectal biopsies were characterized based on phylotypes, antibiotic resistance pattern, and (Enterobacterial Repetitive Intergenic Consensus Sequence-based Polymerase Chain Reaction) ERIC-PCR. Furthermore, isolates were screened for the presence of the Pks (polyketide synthase) Island specifically targeting colibactin genes A and Q. The selective clb+clb+ isolates were subjected to cytotoxicity assay using Human embryonic kidney (HEK) cell lines. We revealed that 43.47% of the cancer-associated E. coli isolates were from phylogroup B2 comparatively more pathogenic than rest while in the case of healthy controls no isolate was found from B2. Moreover, 90% were found positive for colibactin and pks (polyketide synthase) island, while none of the healthy controls were found positive for colibactin genes. All healthy and cancer-associated isolates were tested against 15 antibiotic agents, we observed that cancer-associated isolates showed a wide range of resistance from 96% against Nalidixic acid to 48% against Doxycycline. Moreover, E. coli isolates were further genotyped using ERIC-PCR, and selected clb+clb+ E. coli isolates were subjected to cytotoxicity assay. We recorded the significant cytotoxic activity of clb+clb+ E. coli phylogroup B2 isolates that might have contributed towards the progression of CRC or dysbiosis of healthy gut microbiota protecting against CRC pathogenesis. Our results revealed a significant p<0.023 association of dietary habits and hygiene p<0.001with CRC. This is the first study to report the prevalence of E. coli phylogroups and the role of colibactin most virulent phylogroup B2 among Pakistani individuals from low socioeconomic setup.

Possible step-up in prevalence for Escherichia coli ST131 from fecal to clinical isolates: inferred virulence potential comparative studies within phylogenetic group B2

BackgroundEscherichia coli sequence type (ST)131 is an important urinary tract pathogen, and is responsible for considerable healthcare-associated problems and costs worldwide. A better understanding of the factors that contribute to its rapid worldwide spread may help in arresting its continual spread. We studied a large collection of fecal and urinary E. coli ST131 and E. coli non-ST131 phylogenetic group B2 isolates, from women, men and children, in regional NSW, Australia.ResultsWe found out that there was a step up in ST131 prevalence (and possibly in virulence) from fecal to clinical (urinary) isolates in general, and specifically among ciprofloxacin resistant isolates, in the 3 host groups. Furthermore, our results revealed that the inferred virulence potential of the ST131 isolates (as measured by VF gene scores) was much higher than that of non-ST131 phylogenetic group B2 isolates, and this was much more pronounced amongst the urinary isolates. This finding suggests presence of possible E. coli phylogenetic B2 subgroups with varying levels of virulence, with ST131 being much more virulent compared to others. A strong association between ST131 and fluoroquinolone (FQ) resistance was also demonstrated, suggesting that FQ use is related to ST131 emergence and spread. Specifically, about 77% of ST131 isolates from women and men, and 47% from children, were extended spectrum β- lactamase (ESBL) producers. Moreover, FQ resistant ST131 ESBL isolates on average harbored more VF genes than all other isolates.ConclusionsThe strong association between ST131 prevalence and FQ resistance amongst the studied isolates suggests that FQ use is related to ST131 emergence and spread. Furthermore, our results demonstrate that FQ resistance and a plurality of VF genes can exist together in ST131, something that has traditionally been regarded as being inversely related. This may partly contribute to the emergence and worldwide spread of ST131.

Association between Escherichia coli with NotI-restriction resistance and urinary tract infections.

Escherichia coli is the most common cause of urinary tract infections (UTIs). It is widely accepted that uropathogenic E. coli (UPEC) mainly emerge from the distal gut microbiota. Identification of bacterial characteristics that are able to differentiate UPEC from fecal commensal strains will facilitate the development of novel strategies to detect and monitor the spread of UPEC. Fifty fecal commensal, 83 UTI-associated and 40 biliary tract infection (BTI)-associated E. coli isolates were analyzed. The NotI restriction patterns of chromosomal DNA in the isolates were determined by pulse-field gel electrophoresis. The phylogenetic types and the presence of 9 known virulence genes of each isolate were determined by PCR analyses. Additionally, the susceptibilities of the isolates to antibiotics were revealed. Then the associations of NotI resistance with UTI-associated isolates, phylotypes, and antibiotic resistance were assessed. NotI resistance was correlated with UTI-associated isolates, compared to the fecal isolates. Consistently, NotI-resistant isolates harbored a greater number of virulence factors and mainly belonged to phylotype B2. Additionally NotI resistance was correlated with chloramphenicol resistance among the bacteria. Among the fecal, UTI-associated and BTI-associated groups, the distribution of NotI-resistant group B2 isolates was correlated with UTI-associated bacteria. NotI resistance alone is a potential marker for distinguishing fecal strains and UPEC, while the combination of NotI resistance and B2 phylogeny is a candidate marker to differentiate UPEC from fecal and other extraintestinal pathogenic E. coli. Additionally, NotI resistance may be valuable for assessing the potential of chloramphenicol resistance of E. coli.

Extended Spectrum β-Lactamase Activity and Cephalosporin Resistance in Escherichia coli from U.S. Mid-Atlantic Surface and Reclaimed Water.

Phylogenetic distribution and extended spectrum β-lactamase (ESBL) activity of Escherichia coli recovered from surface and reclaimed water in the mid-Atlantic U.S. were evaluated. Among 488 isolates, phylogroups B1 and A were the most and least prevalent, respectively. Water type, but not season, affected phylogroup distribution. The likelihood of detecting group A isolates was higher in reclaimed than pond (P < 0.01), freshwater river (P < 0.01) or brackish river (P < 0.05) water. Homogeneity in group distribution was lowest in pond water, where group B1 comprised 50% of isolates. Only 16 (3.3%) isolates exhibited phenotypic resistance to one or more cephalosporins tested and only four had ESBL activity, representing groups B1, B2 isolates, and D. Phylogroup was a factor in antimicrobial resistance (P < 0.05), with group A (8.7%) and D (1.6%) exhibiting the highest and lowest rates. Resistance to cefoxitin was the most prevalent. Multi- versus single drug resistance was affected by phylogroup (P < 0.05) and more likely in groups D and B1 than A which carried resistance to cefoxitin only. The most detected β-lactam resistance genes were blaCMY-2 and blaTEM. Water type was a factor for blaCTX-M gene detection (P < 0.05). Phenotypic resistance to cefotaxime, ceftriaxone, cefuroxime and ceftazidime, and genetic determinants for ESBL-mediated resistance were found predominantly in B2 and D isolates from rivers and reclaimed water. Overall, ESBL activity and cephalosporin resistance in reclaimed and surface water isolates were low. Integrating data on ESBL activity and β-lactam resistance among E. coli populations can inform decisions on safety of irrigation water sources and One Health. IMPORTANCE Extended spectrum β-lactamase (ESBL) producing bacteria, that are resistant to a broad range of antimicrobial agents, are spreading in the environment but data remain scarce. ESBL-producing Escherichia coli infections in the community are on the rise. This work was conducted to assess presence of ESBL-producing E. coli in water that could be used for irrigation of fresh produce. The study provides the most extensive evaluation of ESBL-producing E. coli in surface and reclaimed water in the mid-Atlantic United States. The prevalence of ESBL producers was low and phenotypic resistance to cephalosporins (types of β-lactam antibiotics) was affected by season but not water type. Data on antimicrobial resistance among E. coli populations in water can inform decisions on safety of irrigation water sources and One Health.

Phylogenetic Groups/B2 Subgroup Distributions, Serogrouping and Identification of Virulence Factors in Extended-Spectrum Cephalosporin-Resistant Escherichia coli Strains Isolated from the Stool of Healthy Children Under 10 Years Old

Background: Segregation of Escherichia coli (E. coli) into the phylogenetic groups was observed in the experiments so that group B2 contained the enteropathogenic E. coli (EPEC) strains and extraintestinal pathogenic E. coli (ExPEC). Objectives: This study aimed to identify B2 phylogenetic groups in the extended-spectrum Cephalosporins resistant E. coli isolated from the stool of healthy children under 10 years old. Methods: One hundred E. coli resistant to broad-spectrum Cephalosporins were collected from the feces of healthy children under 10. Subsequently, we grouped phylogenetic via PCR based on the genes yjaA, chuA, arpA, as well, as TspE4.C2. Then, according to Clermont et al.’s study, we used two individual multiplex PCRs for identifying B2 sub-groups (I-X subgroups). Serogroup typing with the 12 O-antigen was analyzed via PCR, and finally, 10 virulence genes (cnf1, papG, ibeA, malX, usp, cdt, eae, bfp, and afa-Dr) were identified with PCR. Results: The age range of the healthy children was between 1 and 10 years. The B2 and unknown phylogroups were the most common strains in this study. The most common B2 subgroups were I (2%), IX (1%), V (8%), IV, V, VII (1%), IX, V (3%), IX, V, III, I (1%), IX, V, III, VII, I (%1), V, I (6%), V, III, I (3%), and V, III, VII (1%), with each subgroup carrying distinctive sets of ExPEC virulence markers. The results also showed that 29% of E. coli in the healthy children had malX and 23% had papGII. It was also found that 32% of the strains isolated from the healthy children had antigens O2 and 36% were unknown. Conclusions: In this study, 27% of the strains belonged to B2 phylogroup and 6% to B1 phylogroup. Moreover, serogroups O2, O16, and O25 were predominant and belonged to B2 phylogroup. Moreover, malX, papGII, usp, papGIII, aggR, and eae virulence genes also had the highest to lowest supply among the tested strains, respectively. Moreover, B2 isolates were shown to have further virulence-related genes in comparison to the non B2 isolates.

Application of calcium oxide as an efficient phase separation agent in temperature-induced counter-current homogenous liquid-liquid extraction of aflatoxins from dried fruit chips followed by high-performance liquid chromatography-tandem mass spectrometry determination.

A temperature-induced counter-current homogenous liquid-liquid extraction procedure performed in a burette has been proposed for the isolation of aflatoxins B1, B2, G1, and G2 from various fruit chip samples. In this method, a homogenous solution of deionized water and cyclohexylamine is added to the solid sample and the resulted mixture is vortexed. In the following, the liquid phase is taken and passed through the burette filled with a mixture of calcium oxide (as a phase separation agent) and sand (to avoid clumping the calcium oxide). By doing so, the temperature of the solution is increased by hydration of calcium oxide and consequently, the homogenous state is broken and the aflatoxins are migrated into the resulted tiny droplets of cyclohexylamine. This phase is collected on the top of the solution owing to its low density with respect to an aqueous solution. Numerous parameters which can affect the efficiency of the suggested approach were evaluated and under the best situations, great repeatability, low limits of determination and quantification, and high extraction recoveries were acquired. In the end, the suggested approach was employed for the quantification of the selected aflatoxins in various fruit chips samples marketed in Tabriz City, Iran.

Persistence of Antibiotic-Resistant Escherichia coli Strains Belonging to the B2 Phylogroup in Municipal Wastewater under Aerobic Conditions.

Escherichia coli is classified into four major phylogenetic groups (A, B1, B2, and D) that are associated with antibiotic resistance genes. Although antibiotic-resistant E. coli is commonly detected in municipal wastewater, little is known about the relationship between the phylogenetic groups and antibiotic-resistant E. coli in wastewater. In this study, the survival of E. coli in wastewater and the changes to the relationships between each phylogroup and the antibiotic-resistant profiles of E. coli isolates from wastewater were investigated under aerobic conditions for 14 days. The isolates were classified into the phylogroups A, B1, B2, and D or others by multiplex PCR. In addition, the susceptibility of the isolates to 11 antibiotics was assessed with the minimum inhibitory concentration (MIC) assay. While E. coli counts decreased in the wastewater with time under aerobic conditions, the prevalence of phylogroup B2 had increased to 73% on day 14. Furthermore, the MIC assay revealed that the abundance of antibiotic-resistant E. coli also increased on day 14. After batch-mixing the experiments under aerobic conditions, the surviving antibiotic-resistant E. coli included mainly multidrug-resistant and beta-lactamase-producing isolates belonging to phylogroup B2. These results suggest that the phylogroup B2 isolates that have acquired antibiotic resistance had a high survivability in the treated wastewater.

Prevalence of common carbapenemase genes and multidrug resistance among uropathogenic Escherichia coli phylogroup B2 isolates from outpatients in Wasit Province/ Iraq.

Carbapenems are the last resort antimicrobials for the treatment of extended spectrum β-lactamases (ESBLs) producing Enterobacteriaceae. Emergence of carbapenems resistant group B2 uropathogenic E. coli (UPEC) is a major concern because of their high virulence. Prevalence of these enzymes and multidrug resistance (MDR) among B2 UPEC isolates from Iraqi outpatients with acute urinary tract infection (UTI) was evaluated in this research. Urine cultures were performed and the isolates were identified biochemically. Escherichia coli isolates were tested for phylogroup reference by quadraplex PCR, then B2 isolates were detected for antimicrobial resistance by disc diffusion test and carbapenemase genes by PCR. Escherichia coli was the most prevalent among Gram-negative isolates (66.6%) and B2 was the most detected phylogroup among E. coli isolates (33.9%). Most of B2 isolates showed high resistance rates to tested antimicrobials, especially β-lactams with MDR revealed in 100% of them. Whereas, low resistance rates were noted against carbapenems, aminoglycosides and nitrofurantoin. Carbapenemase genes were detected in 76.3% of B2 isolates. Of which, blaOXA-48 was the most frequent (57.8%), followed by blaPER (47.3%), blaKPC (15.7%), blaVEB and blaVIM (10.5%, for each). Whereas, blaGES and blaIMP genes were not found. Coproduction of these genes occurred among 17 isolates. The combination of blaOXA-48 and blaPER was the most frequent (41.1%). All carbapenemase producing isolates were MDR. These results revealed high prevalence of carbapenemase genes and MDR among B2 UPEC recovered in this study. In the study area. it is strongly advised to use aminoglycosides and nitrofurantoin for empirical treatment of UPEC.

A 21-Year Survey of Escherichia coli from Bloodstream Infections (BSI) in a Tertiary Hospital Reveals How Community-Hospital Dynamics of B2 Phylogroup Clones Influence Local BSI Rates.

ABSTRACTThis is a longitudinal study comprising 649 Escherichia coli isolates representing all 7,165 E. coli bloodstream infection (BSI) episodes recorded in a hospital (1996 to 2016). Strain analysis included clonal identification (phylogenetic groups/subgroups, STc131 subclades, pulsed-field gel electrophoresis [PFGE], and whole-genome sequencing [WGS]), antibiotic susceptibility (13 antibiotics), and virulence-associated genes (VAGs; 29 genes). The incidence of E. coli BSI increased from 1996 to 2016 (5.5 to 10.8 BSI episodes/1,000 hospitalizations, average 7 to 8/1,000). B2 isolates predominate (53%), with subgroups B2-I (STc131), B2-II, B2-IX, and B2-VI representing 25%, 25%, 14%, and 9%, respectively. Intertwined waves of community-acquired (CA) plus health care-associated and community-onset health care-associated (HCA) and hospital-acquired (HA) episodes of both B2 and non-B2 phylogroups occurred. A remarkable increase was observed only for B2-I-STc131 (C1/C2 subclades), with oscillations for other B2 subgroups and phylogroups throughout the years. Epidemic and persistent clones (comprising isolates with highly similar/identical PFGE types and genomes differing in 6 to 173 single nucleotide polymorphisms [SNPs]) of B2-I (STc131), B2-II (STc73), B2-III (STc127), B2-IX (STc95), and B2-VI (STc12) were recovered from different patients, most at hospital admission, for long periods (2 to 17 years), and extended-spectrum beta-lactamase (ESBL) producers or resistance to ciprofloxacin in B2 isolates was almost restricted to B2-I (STc131) subclade C. STc131 contributed to increasing the B2 rates but only transiently altered the E. coli population structure. The increase of E. coli BSI was determined by waves of CA+HCA BSI episodes that predate the waves of HA BSI. Besides the risk of hospital transmission that led to temporal increases in BSI, this study suggests that E. coli populations/clones from community-based healthy individuals may occasionally have an epidemic structure and provide a source of transmissible strains influencing the HA BSI incidence.IMPORTANCE Sepsis is the third leading cause of mortality in Western countries and one of the Global Health Threats recognized by the WHO since 2017. Despite Escherichia coli constituting the most common cause of bloodstream infections (BSI), its epidemiology is not fully understood, in part due to the scarcity of local and longitudinal studies. Our work analyzes the long-term dynamics of E. coli causing bacteremia in a single institution and reveals waves of different clonal lineages that emerge periodically and successfully spread afterward in both the community and hospitals. Because the origin of E. coli bloodstream infections is the gut, the microbiota of healthy individuals might occasionally have an epidemic structure, providing a source of E. coli strains to influence the incidence of hospital BSI. The study complements previous fractionated observations focusing on specific E. coli lineages or antibiotic-resistant isolates in the last decades and helps to understand the epidemiology of E. coli BSI and the dynamics of pandemic clones.

ZnO, TiO2 and Ag nanoparticles impact against some species of pathogenic bacteria and yeast.

The economic approaches for manufacturing the nanoparticles with physical and chemical effects and limited resistance to antibiotics have been progressed recently due to the rise of microbial resistance to antibiotics. This research aimed to study the antimicrobial efficacy of silver nanoparticles Ag, ZnO, and Tio2 nanoparticles against Salmonella typhimurium and Brucella abortus and Candida albicans. Two isolates of Salmonella and two isolates of Brucella abortus were isolated from food spastically meat and blood specimens, respectively. Candida albicans were isolated from the patient's mouth with oral candidiasis (oral thrush) and confirmed diagnosis by API 20C test. The antimicrobial susceptibility of Salmonella typhimurium and B. abortus isolates were performed against nine different antibiotics. Silver nanoparticles consisting of AgNPs size (90) nm, ZnO NPs size (20, 50) nm as well as TiO2 NPs size (10, 50) nm, were used. UV-Visible spectrophotometer was used to characterize silver nanoparticles. The highest resistance of Candida albicans was seen for fluconazole, Clotrimazole and Itraconazole. The results of the Minimum Inhibitory Concentration (MIC) of nanoparticles against Salmonella typhimurium showed the average MIC of Tio2-10nm and Tio2-50nm were 5000 and 2500 μg\ml for S1 and S2 isolates, respectively. The isolated Brucella abortus (B1 and B2) showed sensitivity to NPs with different MIC. The average MIC for Ag-90nm was 5000 and 2500 µg/ml for B1 and B2 isolates, respectively. The findings suggest NP solution has fungicidal and bactericidal impacts on the tested microorganisms so they can be suitable for multiple applications of the biomedical field such as developing new antimicrobial agents.

​Control Activity and Antibiotic Gene Detection of Endophytic Bacteria in Suppressing Cocoa Black Pod Disease (Phytophthora palmivora Butl.)

Background: Black pod rot disease of cocoa caused by (Phytophthora palmivora Butl.) is one of the major diseases on cocoa plantations worldwide. Many attempts have been made to prevent or reduce the infection of pathogens, but they have not provided optimum results. This study aims to detect antibiotic genes in endophytic bacteria that can suppress cocoa black pod disease. Methods: Eight endophytic bacteria were isolated from healthy cocoa pods and twigs that showed potentials in suppressing P. palmivora growth in vitro were used in suppressing of black pod rot disease in vivo tests. Antibiotic biosynthesis-related genes from eight endophytic bacterial isolates were confirmed by using PCR method, which includes phenazine-1-carboxylic (PCA), pyrrolnitrin (PRN), phenazine-1-carboxamide acid (PCN), pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DPAG/Phl). Result: The endophytic bacteria, 4RSI, 5BR B3 and 2RW B2 isolates showed the highest disease suppressing index to black pod rot disease in vivo, i.e., 70.27%, 70.08% and 56.64%. The isolates 5BR B3 and 2RW B2 DNA yielded PCR product by using PCA primers (1400 bp), PRN primers (700 bp) and DAPG primers (1600 bp), while the 5RSI isolate yield PCR product using PRN primers only. Endophytic bacterial isolates 5BRB3 and 2RW B2 provided partial disease suppression to pod rot disease by inhibit pathogen growth and antibiotic compounds production.

Affinity-Guided Isolation and Identification of Procyanidin B2 from Mangosteen (Garcinia mangostana L.) Rinds and its In Vitro LPS Binding and Neutralization Activities.

Garcinia mangostana L. (mangosteen) is a tropical fruit that has been used for medicinal purposes in Southeast Asia for centuries. With an interest in its applications to treat infection, we sought to investigate the bioactive constituents of mangosteen and identified the phenolic compound procyanidin B2 from the mangosteen pericarp by examining lipopolysaccharide (LPS) binding capacity. The LPS binding and neutralization activities of procyanidin B2 were determined by a combination of biophysical and in silico techniques. The affinity of procyanidin B2 to LPS was 1.61 × 10-5M. Procyanidin B2 significantly neutralized LPS and selectively inhibited the LPS-induced release of tumor necrosis factor (TNF)-α from RAW264.7 cells in a dose-dependent manner. Binding thermodynamics revealed favorable hydrogen bonding and hydrophobic interactions between procyanidin B2 and LPS. Molecular simulations suggested that hydrogen bonding and hydrophobic interactions were involved in the binding process. These findings have, for the first time, shed light on the anti-inflammatory properties of procyanidin B2 through LPS binding and neutralization and provided a promising lead for the development of antiendotoxin agents.