Phylogenic Groups Research Articles

2846. Microbiological Characteristics of the Patients with Acute Pyelonephritis who experienced Urinary Tract Infections within prior to 1 year

Abstract Background Approximately 15% of patients with acute pyelonephritis (APN) experience another urinary tract infection (UTI) within 1 year in South Korea. The purpose of this study is to examine the microbiological characteristics of patients with APN who experienced UTIs within prior to 1 year. Methods A retrospective cohort study was performed in a tertiary-care hospital in South Korea from July 2019 to December 2021. All female patients who were diagnosed with community acquired APN at admission were recruited. Patients who had prolonged hospitalization period due to medical problems that were not associated with APN treatment were excluded. We regarded recurrent group as the cases which have history of UTI within 1 year. Types of causative organism, major antibiotic resistance of E.coli isolated from patients' urine or blood, and the genetic characteristics of E.coli were compared between recurrent group and non-recurrent group. Results A total of 285 APN patients were finally included in this study, of which 41 (14.4%) were belong to recurrent group. E.coli was the most common causative pathogens in both groups, but the proportions of some pathogens, such as Klebsiella pneumonia (17.1 vs. 4.7%, P=0.007) and Pseudomonas aeruginosa (5.7 vs. 0.5%, P=0.014) were higher in the recurrent group than the non-recurrent group. There were no significant differences between the two groups in the proportions of antibiotic resistances to E.coli, such as amikacin (0 vs. 1.2%, P=0.597), cefotaxime (25.0 vs. 22.5%, P=0.788), and fluoroquinolone (20.8 vs. 28.9%, P=0.409), etc. The most prevalent β-lactamase and PMQR determinant were CTX-M-1G (44.4 vs 60.0%, P=0.308) and aac(6’)-lb-cr (50.0 vs 36.2%, P=0.303), respectively, and the predominant clone was ST131 (22.2 vs 17.8%, P=0.744) in both groups. However, there were no significant differences in the proportions of phylogenic groups, resistance genes, and virulence factors between the two groups. Conclusion E.coli was the most common causative pathogens in both groups, but the proportion of pathogens except E.coli was higher in the recurrent group than the non-recurrent group. There were no significant differences between the two groups in the proportions of antibiotic resistances to E.coli, phylogenic groups, resistance genes, and virulence factors. Disclosures All Authors: No reported disclosures

Microsatellite-based diversity analysis and the development of core-set germplasm in Pakistani barley lines.

Illustrating the population structure and genetic diversity in selected germplasm resources (after three year multi locations trials) plays a key role which directly utilize the selection of lines in a population for accumulative trait breeding in crops. In order to further understand, the structure of population and genetic variability, we explored 100 selected lines, cultivated for three consecutive years (2016-2019) in swat, University of Malakand, Khyber Pakhtunkhwa Pakistan and Provinces of China (Chongqing and Beijing) with 33 mapped SSR markers. The integrated population structure analysis in a core of hundred germplasm with Pakistani origin with three approved commercial barley cultivars have strong stratification that allowed their division into four major subpopulations (i.e. PI, PII, PIII and PIV) and an admixture subpopulation, with 52, 9, 15 and 27 germplasm respectively. A total of 133 alleles were identified with mean value of 0.80 Polymorphic information content. The number of alleles detected by the system varied from two alleles amplified to as six with an average of 4.03 per SSR marker pair. The gene diversity ranged from 0.56 to 0.98 with an average of 0.82 in selected germplasm resources. Based on the SSR data, the 100 selected germplasm with three cultivars were classified into four main phylogenetic Linages (LI, LII, LIII and LIV) which corresponded to the phylogenic grouping in genotypes. We assembled a core set of 20 barley genotypes (~1/5 of original population size) to sustain sufficient mapping of SSR marker with Phenotype, in which we proposed four SSR markers, Bmac0040, Bmac0134, Bmag0125 and Bmag0211 for malt gene and marker (Bmac0399) for tolerance to salinity gene, which will be applicable for marker assisted breeding in barley gene resources.

Genomic characterisation of multi-drug resistant Escherichia coli and Klebsiella pneumoniae co-harbouring mcr-1 and mcr-3 genes on a single plasmid from paediatric clinical cases

Emergence of plasmid-born mobile colistin resistance (mcr) gene, a growing concern in healthcare. Therefore, this study aims to genomically characterize multidrug-resistant Escherichia coli and Klebsiella pneumoniae co-harboring the mcr-1 and mcr-3 genes in young children. E. coli (n=3) and K. pneumoniae (n=2) were collected from abdominal secretion and blood, respectively. The isolates were screened using tryptone soya broth (TSB) with 4µl/ml polymyxin-B. Growing bacteria were identified using the VITEK-2 system, MALDI-TOF and 16s RNA sequencing, followed by antibiotic susceptibility testing, as well as metallo-β-lactamase (MBL) and extended spectrum β-lactamase (ESBL) production was detected. Afterwards, strains were subjected to molecular screening targeting mcr variants, and ESBLs/MBL encoding genes. Conjugation, PFGE, southern hybridisation, MLST, and Phylogenic group detection were performed. Plasmid-genome sequencing and bioinformatic analysis were performed. E. coli named EC-19-322, 323, and 331 and K. pneumoniae isolates named KP-19-225 and 226 harboured both mcr-1 and mcr-3 genes. These strains were also found to be resistant to more than three classes of antibiotics. The conjugation experiment revealed the presence of mcr-1 and mcr-3 on the single plasmid and the transmission frequency was 10-2 to 10-3. Both strains were found to be able to produce ESBLs and MBL. E. coli EC-19-322 and 323 were identified as an ST131(O25a:H41), SP-19-331as an ST1577 (O16:H30) and K. pneumoniae as an ST231 (K2). All E. coli strains belonged to phylogenetic group B2, and the results of PFGE supported the MLST findings. This study, reported the co-occurrence of mcr-1 and mcr-3 genes on single plasmid in pathogenic ESBLs/MBL-producing E. coli and K. pneumoniae isolated from young children.

Rab11 rescues muscle degeneration and synaptic morphology in the park13/+ Parkinson model of Drosophila melanogaster

Mutation in parkin and pink1 is associated with Parkinson’s disease (PD), the most common movement disorder characterized by muscular dysfunction. In a previous study, we observed that Rab11, a member of the small Ras GTPase family, regulates the mitophagy pathway mediated by Parkin and Pink1 in the larval brain of the Drosophila PD model. Here, we describe that the expression and interaction of Rab11 in the PD model of Drosophila is highly conserved across different phylogenic groups. The loss of function in these two proteins, i.e., Parkin and Pink1, leads to mitochondrial aggregation. Rab11 loss of function results in muscle degeneration, movement disorder and synaptic morphological defects. We report that overexpression of Rab11 in park13 heterozygous mutant improves muscle and synaptic organization by reducing mitochondrial aggregations and improving cytoskeleton structural organization. We also show the functional relationship between Rab11 and Brp, apre-synaptic scaffolding protein, required for synaptic neurotransmission. Using park13 heterozygous mutant and pink1RNAi lines, we showed reduced expression of Brp and consequently, there were synaptic dysfunctions including impaired synaptic transmission, decreased bouton size, increase in the bouton numbers, and the length of axonal innervations at the larval neuromuscular junction (NMJ). These synaptic alterations were rescued with the over-expression of Rab11 in the park13 heterozygous mutants. In conclusion, this work emphasizes the importance of Rab11 in rescuing muscle degeneration, movement dysfunction and synaptic morphology by preserving mitochondrial function in the PD model of Drosophila.

Phylogenetic analysis and antibiotic resistance of Escherichia coli isolated from wild and domestic animals at an agricultural land interface area of Salaphra wildlife sanctuary, Thailand.

Domestic and wild animals are important reservoirs for antibiotic-resistant bacteria. This study aimed to isolate Escherichia coli from feces of domestic and wild animals at an agricultural land interface area of Salaphra Wildlife Sanctuary, Thailand, and study the phylogenic characteristics and antibiotic resistance in these isolates. In this cross-sectional, descriptive study, we randomly collected ground feces from free-ranging wild animals (deer and elephants) and domestic animals (cattle and goats). All fecal samples were inoculated onto MacConkey agar plates, and lactose-fermenting colonies were identified as E. coli. Antibiotic susceptibility of the E. coli isolates was determined using the disc diffusion method. Polymerase chain reaction assays were used to detect antibiotic resistance and virulence genes. We obtained 362 E. coli isolates from the collected fecal samples. The E. coli isolates were categorized into four phylogenetic groups according to the virulence genes (chuA, vjaA, and TspE4C2). Phylogenetic Group D was predominant in the deer (41.67%) and elephants (63.29%), whereas phylogenetic Group B1 was predominant in the cattle (62.31%), and phylogenetic Groups A (36.36%) and B2 (33.33%) were predominant in the goats. Antibiotic susceptibility testing revealed that most antibiotic-resistant E. coli were isolated from domestic goats (96.96%). Among the 362 E. coli isolates, 38 (10.5%) were resistant to at least one antibiotic, 21 (5.8%) were resistant to two antibiotics, and 6 (1.66%) were resistant to three or more antibiotics. Ampicillin (AMP) was the most common antibiotic (48.48%) to which the E. coli were resistant, followed by tetracycline (TET) (45.45%) and trimethoprim-sulfamethoxazole (3.03%). One isolate from an elephant was resistant to five antibiotics: AMP, amoxicillin, sulfisoxazole, TET, and ciprofloxacin. Determination of antibiotic resistance genes confirmed that E. coli isolates carried antibiotic resistance genes associated with phenotypic resistance to antibiotics. Most antibiotic-resistant E. coli belonged to phylogenic Groups A and B1, and most non-resistant E. coli belonged to phylogenic Groups B2 and D. Monitoring E. coli isolates from wild and domestic animals showed that all four phylogenic groups of E. coli have developed antibiotic resistance and are potential sources of multidrug resistance. High levels of antibiotic resistance have been linked to domestic animals. Our results support strengthening surveillance to monitor the emergence and effects of antibiotic-resistant microorganisms in animals.

Azithromycin resistance genes in Escherichia coli isolated from wastewater: Characterization and modeling-based evaluation of factors affecting the prevalence

Azithromycin is becoming an effective alternative medicine against antibiotic resistant Escherichia coli (E. coli), and in parallel, is frequently detecting in wastewater and fresh water media. The current study aimed to evaluate the presence of azithromycin resistance genes (AzRGs) in E. coli isolated from urban wastewater for which there is a serious lack of information. For this, the samples were collected from four vital points of an urban wastewater treatment plant (WWTP), analyzed for physicochemicfial parameters, and identified for the azithromycin resistant isolates. The isolates were then subjected to 13 other extensively used antibiotics (from seven different classes), including amikacin, aztreonam, ceftazidime, ciprofloxacin, cefepime, ceftriaxone, cefotaxime, doxycycline, gentamicin, imipenem, norfloxacin, tetracycline, and trimethoprim/sulfamethoxazole; detected for AzRGs, phylogenic group, and molecular typing. The results clearly showed that: a) except for amikacin in all samples and gentamicin in effluent samples, E. coli isolates were resistant to all other the antibiotics studied in the mean respective values of 53.8%, 90.4%, 50%, 40.4%, 86.5%, 86.5%, 63.5%, 13.5%, 73.1%, 46.2%, 65.4%, 69.2%; b) for 75% of them (i.e. except for amikacin, gentamicin, and trimethoprim/sulfamethoxazole), the antibiotic resistance prevalence in effluent samples were significantly higher than that of sludge (p-value = 0.011), aeration (p-value = 0.023), and even influent (p-value = 0.038) samples, suggesting that WWTP facilities acquiring the resistance gene; c) The high values of multiple antibiotic resistance (MAR) index in almost all of the isolates (0.21–0.91) were categorized them in high-risk group, though the values were not found to be influenced by WWTP. Surprisingly, from 10 AzRGs, only two genes of mph(A) and erm(B) were detected in 100% and 67.3% of the isolates, respectively. The prevalence of erm(B) was significantly influenced by MAR index (p-value = 0.004), which MAR in turn, influenced by physicochemical parameters of wastewater in the relative effectiveness of COD (36%) > EC (29%) > TS (16%) > TDS (9%) > temperature (6%) > pH (4%), as modeled using artificial neural network (ANN) approach (R2 = 0.85 and MSE = 0.006302). The phylogenetic group A (59.6%) and clusters of III and V with 23 and 18 isolates, respectively, were dominant among the isolates which both showed a high correlation with MAR indices and therefore erm(B) prevalence. These results were interesting as highlight the effect of WWTP on acquiring antibiotic resistance and AzRGs prevalence in E. coli. Therefore, defining new strategies for relieving the challenge is of great importance.

Identification of Candidate Genes Regulating Drought Tolerance in Pearl Millet.

Pearl millet is an important crop of the arid and semi-arid ecologies to sustain food and fodder production. The greater tolerance to drought stress attracts us to examine its cellular and molecular mechanisms via functional genomics approaches to augment the grain yield. Here, we studied the drought response of 48 inbreds representing four different maturity groups at the flowering stage. A set of 74 drought-responsive genes were separated into five major phylogenic groups belonging to eight functional groups, namely ABA signaling, hormone signaling, ion and osmotic homeostasis, TF-mediated regulation, molecular adaptation, signal transduction, physiological adaptation, detoxification, which were comprehensively studied. Among the conserved motifs of the drought-responsive genes, the protein kinases and MYB domain proteins were the most conserved ones. Comparative in-silico analysis of the drought genes across millet crops showed foxtail millet had most orthologs with pearl millet. Of 698 haplotypes identified across millet crops, MyC2 and Myb4 had maximum haplotypes. The protein–protein interaction network identified ABI2, P5CS, CDPK, DREB, MYB, and CYP707A3 as major hub genes. The expression assay showed the presence of common as well as unique drought-responsive genes across maturity groups. Drought tolerant genotypes in respective maturity groups were identified from the expression pattern of genes. Among several gene families, ABA signaling, TFs, and signaling proteins were the prospective contributors to drought tolerance across maturity groups. The functionally validated genes could be used as promising candidates in backcross breeding, genomic selection, and gene-editing schemes in pearl millet and other millet crops to increase the yield in drought-prone arid and semi-arid ecologies.

Community Fecal Carriage and Molecular Epidemiology of Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Escherichia coli from Healthy Children in the Central South China.

BackgroundFecal carriage of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) and carbapenemase-producing E. coli (CP-EC) is well reported among hospitalized adults and children. However, there are few studies on the carriage prevalence and ESBL-EC and CP-EC genotypes among healthy children in China.Patients and MethodsStool samples were collected from 330 students in 2021 from three randomly selected primary schools in Changsha, China. ESBL-EC and CP-EC were screened using CHROMagarTM chromogenic plates. ESBL and carbapenemase production was confirmed using the double-disc synergy test and a modified carbapenem inactivation method, respectively. Antimicrobial susceptibility was tested using the broth microdilution method. Resistance determinants, virulence factors, and phylogenetic groups were determined by PCR and sequencing. Multi-locus sequence typing (MLST) was performed (seven housekeeping genes were amplified and sequenced) on the phylogenic group B2 E. coli to detect high-risk clonal strains such as ST131 E. coli. Then, ST131 E. coli were characterized based on ST131 clades, O-type, and fimH alleles.ResultsIn total, 118 (35.8%) ESBL-EC and 3 (0.9%) CP-EC were isolated. blaCTX-M was the most common genotype (27.1%), identified in all ESBL-EC, except one, which carried blaSHV-12. One isolate with mcr-1 was found amongst ESBL-EC, whereas all three CP-EC carried blaNDM-1. The predominant sequence type (ST) clones in group B2 were ST131 and ST1193. The prevalence of ST131 E. coli was 9.9%, displaying serotypes O16 and O25b, fimH alleles 30, 41, and 89, and ST131 clades A and C1-M27.ConclusionIn this study, high carriage rate of ESBL-EC was found among healthy children, and the dominant ESBL was CTX-M-14. In addition, high-risk clones (ST131 and ST1193) were also detected. This emphasizes the importance of monitoring ESBL-EC in community settings.

Genetic diversity and multidrug resistance of phylogenic groups B2 and D in InPEC and ExPEC isolated from chickens in Central China

BackgroundAvian colibacillosis is an infectious bacterial disease caused by avian pathogenic Escherichia coli (APEC). APEC causes a wide variety of intestinal and extraintestinal infections, including InPEC and ExPEC, which result in enormous losses in the poultry industry. In this study, we investigated the prevalence of InPEC and ExPEC in Central China, and the isolates were characterized using molecular approaches and tested for virulence factors and antibiotic resistance.ResultsA total of 200 chicken-derived E. coli isolates were collected for study from 2019 and 2020. The prevalence of B2 and D phylogenic groups in the 200 chicken-derived E. coli was verified by triplex PCR, which accounted for 50.53% (48/95) and 9.52% (10/105) in ExPEC and InPEC, respectively. Additionally, multilocus sequence typing method was used to examine the genetic diversity of these E. coli isolates, which showed that the dominant STs of ExPEC included ST117 (n = 10, 20.83%), ST297 (n = 5, 10.42%), ST93 (n = 4, 8.33%), ST1426 (n = 4, 8.33%) and ST10 (n = 3, 6.25%), while the dominant ST of InPEC was ST117 (n = 2, 20%). Furthermore, antimicrobial susceptibility tests of 16 antibiotics for those strains were conducted. The result showed that more than 60% of the ExPEC and InPEC were resistant to streptomycin and nalidixic acid. Among these streptomycin resistant isolates (n = 49), 99.76% harbored aminoglycoside resistance gene strA, and 63.27% harbored strB. Among these nalidixic acid resistant isolates (n = 38), 94.74% harbored a S83L mutation in gyrA, and 44.74% harbored a D87N mutation in gyrA. Moreover, the prevalence of multidrug-resistant (MDR) in the isolates of ExPEC and InPEC was 31.25% (15/48) and 20% (2/10), respectively. Alarmingly, 8.33% (4/48) of the ExPEC and 20% (2/10) of the InPEC were extensively drug-resistant (XDR). Finally, the presence of 13 virulence-associated genes was checked in these isolates, which over 95% of the ExPEC and InPEC strains harbored irp2, feoB, fimH, ompT, ompA. 10.42% of the ExPEC and 10% of the InPEC were positive for kpsM. Only ExPEC isolates carried ibeA gene, and the rate was 4.17%. All tested strains were negative to LT and cnf genes. The carrying rate of iss and iutA were significantly different between the InPEC and ExPEC isolates (P < 0.01).ConclusionsTo the best of our knowledge, this is the first report on the highly pathogenic groups of InPEC and ExPEC in Central China. We find that 50.53% (48/95) of the ExPEC belong to the D/B2 phylogenic group. The emergence of XDR and MDR strains and potential virulence genes may indicate the complicated treatment of the infections caused by APEC. This study will improve our understanding of the prevalence and pathogenicity of APEC.

Genetic diversity and transmission patterns of Burkholderia pseudomallei on Hainan island, China, revealed by a population genomics analysis.

Burkholderia pseudomallei is a Gram-negative soil-dwelling bacillus that causes melioidosis, a frequently fatal infectious disease, in tropical and subtropical regions. Previous studies have identified the overall genetic and evolutionary characteristics of B. pseudomallei on a global scale, including its origin and transmission routes. However, beyond its known hyperendemicity foci in northern Australia and Southeast Asia, the distribution and genetic characteristics of B. pseudomallei in most tropical regions remain poorly understood, including in southern China. Here, we sequenced the genomes of 122 B. pseudomallei strains collected from Hainan, an island in southern China, in 2002–2018, to investigate the population structure, relationships with global strains, local epidemiology, and virulence and antimicrobial-resistance factors. A phylogenetic analysis and hierarchical clustering divided the Hainan strains into nine phylogenic groups (PGs), 80 % of which were concentrated within five major groups (group 1: corresponding to minor sequence types [STs], 12.3 %; group 3: ST46 and ST50, 31.1 %; group 9: ST58, 13.1 %; group 11: ST55, 8.2 %; group 15: mainly ST658, 15.6%). A phylogenetic analysis that included global strains suggested that B. pseudomallei in Hainan originated from Southeast Asian countries, transmitted in multiple historical importation events. We also identified several mutual transmission events between Hainan and Southeast Asian countries in recent years, including three importation events from Thailand and Singapore to Hainan and three exportation events from Hainan to Singapore, Malaysia, and Taiwan island. A statistical analysis of the temporal distribution showed that the Hainan strains of groups 3, 9, and 15 have dominated the disease epidemic locally in the last 5 years. The spatial distribution of the Hainan strains demonstrated that some PGs are distributed in different cities on Hainan island, and by combining phylogenic and geographic distribution information, we detected 21 between-city transmission events, indicating its frequent local transmission. The detection of virulence factor genes showed that 56 % of the Hainan strains in group 1 encode a B. pseudomallei -specific adherence factor, boaB, confirming the specific pathogenic characteristics of the Hainan strains in group 1. An analysis of the antimicrobial-resistance potential of B. pseudomallei showed that various kinds of alterations were identified in clinically relevant antibiotic resistance factors, such as AmrR, PenA and PBP3, etc. Our results clarify the population structure, local epidemiology, and pathogenic characteristics of B. pseudomallei in Hainan, providing further insight into its regional and global transmission networks and improving our knowledge of its global phylogeography.

Circulation and characterisation of Equid herpesvirus‐3 strains in France between 2010 and 2021

Background: Equine coital exanthema (ECE) is an acute viral infection of horses caused by equid herpesvirus-3 (EHV-3). The presence of the virus and its economic impact is well described in different countries such as Argentina or Japan. However, even though it is included in the HBLB code of practice (UK) and in the list of diseases monitored by the RESPE (FR), only a few studies have been performed in Europe. Objectives: To determine the circulation of EHV-3 in France and measure the efficacy of new antiviral compounds against EHV-3. Study design: Retrospective field study and in vitro experiments. Methods: 71 field samples from suspected ECE cases, and collected between 2010 and 2021, were analysed by qPCR (DNA polymerase gene) and phylogenetic analysis (gG gene) was performed on 10 positive samples. Antiviral compounds (aciclovir, ganciclovir, valganciclovir, decitabine, idoxuridine, pritelivir, cidofovir, aphidicolin, vidarabine) previously described with antiviral properties against EHV-1, were evaluated using Real-Time Cell Analysis (RTCA) on equine dermal cells with the reference strain EHV-3 VR3522, cytopathic effect monitoring (microscopy) and viral load quantification (qPCR). Results: 16/71 (22%) suspected ECE cases were confirmed positive by PCR with 0 to 3 cases per year. French strains were clustered into two of the three observed phylogenic groups, but there was no correlation between the phylogeny and the time of collection or location. Aphidicolin was the most effective compound with an EC50 value of 2.43±0.02 μM (RTCA) and 1.63±0.48 μM (qPCR). Pritelivir and cidofovir were less potent molecules against EHV-3 (EC50 >50 μM). Main limitation: Antiviral compounds were tested against the reference strain only. Conclusions: This study demonstrates for the first time the presence of EHV-3 over a twelve-year period in France. In vitro studies show the antiviral efficacy of aphidicolin.

Proteolytic Processing, Maturation, and Unique Synteny of Streptomyces Hemagglutinin, SHA

Blood-type B specific hemagglutinin (L-rham/D-gal-specific lectin) secreted by Streptomyces sp. 27S5 (SHA) was purified over 40 years ago. Despite the loss of the original strain and the lack of genomic information, the amino acid sequence of its SHA was recently determined based on peptides matching to a hypothetical protein of S. lavendulae, differing only by a single amino acid, A108E (J Biol Chem 293:368, 2018). Recombinant SHA (rSHA A108E) exhibited binding to L-rham on Lactobacillus casei (Shirota) and blood type B-specificity, supporting the notion that rSHA A108E is equivalent to the authentic SHA. To address questions how SHA was biosynthesized and secreted by the lost strain as well as what biological roles SHA may have, we needed to identify SHA-secreting S. strains that are identical to or at least comparable to the lost strain. We took advantage of the IMC's collection of over 40,000 actinomycete strains. Out of 5,000 strains with available 16S rRNA gene signatures, 67 strains shared significant 16S rRNA sequence homology with S. lavendulae, and were examined. We found that 17 S. lavendulae-related strains secreted SHA homologues in cultures. Seven SHA homologues were purified to homogeneity by gum arabic affinity chromatography. All homologues were secreted as pro-SHA proteins having larger masses than archived SHA. Proteolytic processing of the pro-SHAs occurred during and after purification, which indicated that associated proteases apparently converted pro-SHAs into mature SHAs with molecular masses similar to that of the archived SHA. Pro-SHAs did not exhibit hemagglutination (HA) activity, but bound to gum arabic gels, suggesting that pro-SHAs have one active glycan binding site. The processed SHA proteins appear to possess specific HA activities equivalent to that of the archived SHA. Comparison of strain-specific 16S rRNA and SHA sequences revealed phylogenetic inconsistencies, which cannot be explained by the deletion and horizontal transmission repeated in this phylogenic group alone. Genome analysis of 1,234 Streptomyces strains from the database resulted in the discovery of 18 strains encoding SHA genes. Of those, 15 strains were newly identified and 16 strains revealed a unique syntenic region which encodes the SHA gene, supporting limited propagation of SHA in the course of evolution. In short, we succeeded in purifying SHA homologues from seven strains. The lost strain Streptomyces sp. 27S5, however, was exceptional, because it expressed and secreted a processed form of SHA protein at levels that were at least 4 times higher than those of the two best-SHA homologue-producing strains. Apart from structural and functional studies on homologous SHA proteins, this study led to an intriguing discovery, i.e., the identification of SHA gene in syntenic regions. It is plausible that SHA would play a role in the unknown function achieved by the ensemble of accompanying genes in synteny.

1667. Change in characteristics of community-onset ciprofloxacin-resistant E. coli isolates causing community-acquired acute pyelonephritis in South Korea

BackgroundThe aim of this study was to examine the change in characteristics of community-onset ciprofloxacin-resistant (CIP-R) E. coli isolates causing community-acquired acute pyelonephritis (CA-APN) in South Korea between 2010-2011 and 2017-2018.Methods E. coli samples isolated from the blood or urine were collected from patients with CA-APN aged 19 years and more who were admitted to 8 Korean hospitals from September 2017 to August 2018, prospectively. One isolate was collected from each patient. Phylogenetic typing, multilocus sequence typing (MLST), and molecular characterization of β-lactamase resistance and plasmid-mediated quinolone resistance (PMQR) determinants were performed. The data were compared with those from the previous study with same design in 2010-2011.ResultsA total of 346 and 300 isolates were collected during 2017-2018 and 2010-2011, respectively. Among them, 76 (22.0%) and 77 (25.7%) were CIP-R isolates. Significantly higher antimicrobial resistance against ampicillin (75.7% vs. 100%, P < 0.001) and cefotaxime (23.9% vs. 77.9%, P < 0.001) were observed for isolates in 2017-2018 compared to those in 2010-2011. The proportion of phylogenic group B2 had increased significantly (44.7% vs. 79.2%, P < 0.001). As for MLST, the proportion of ST131 (27.6% vs. 66.2%, P < 0.001) had increased while that of ST393 (18.4% vs. 3.9%, P =0.004) had decreased significantly. Higher proportion of CIP-R E. coli isolates in 2017-2018 had extended-spectrum β-lactamase (ESBL)/plasmid-mediated AmpC β-lactamase (PABL) (23.7% vs. 79.2%, P < 0.001) and PMQR determinant (11.8% vs. 40.8%, P < 0.001) compared to those in 2010-2011.Phlogenetic tree Analyzed by SplitsTree ConclusionAmong uropathogenic CIP-R E. coli isolates in South Korea, ST131 predominance had become more prominent and the proportion of containing ESBL/PABL and/or PMQR determinants had increased.Disclosures All Authors: No reported disclosures

Virulence genotypes and phylogenetic distribution of the community-onset Escherichia coli

Background: The concept that not all E. coli are equally able to cause UTI, and that the uropathogenic E. coli (UPEC) strains possess distinctive traits that confer enhanced virulence potential compared to other source E. coli, is supported by abundant epidemiologic and experimental evidence. Virulence factors (VFs) are the specific genes encoding a diverse array of specialized properties that are postulated to contribute to virulence. The aim of this study was to find the prevalence difference of virulence genes and phylogenetic lineages between E. coli from community-onset urinary tract infection (CO-UPEC) and hepatobiliary infection (CO-HBEC) compared to rectal commensal E. coli (RCEC) in a healthy population. Methods and materials: We conducted a case-control study of patients with positive UPEC & HBEC between 2006 and 2008 at a teaching hospital in South Korea. Community-onset (CO) was the isolated from the patients from whom the specimen was obtained within 2 calendar days of hospitalization or at outpatient department. We also collected the rectal commensal E. coli (RCEC) of healthy people. All isolates underwent PCR-based microarray for phylotyping and detection of 11 VFs, including adhesins (prf, sfa, drb, prsGj96, papGad), toxins (hly, cnf1), siderophores (iroN, aer), capsule (kpsMT), and other (ompT). The virulence score was defined as the number of virulence genes identified. Results: A total of 548 CO-UPEC and 140 HBEC with 486 CREC isolates were collected. Compared with CREC, CO-UPEC showed higher VFs (3.92 ± 2.22 vs. 2.28 ± 1.90, p < 0.001) and CO-HBEC averaged fewer VFs (1.56 ± 1.64 vs. 2.28 ± 1.90, p < 0.001). Overall distribution of phylogenic group were B2(52.5%) and D(33%) in CO-UPEC, A(37.9%) and D(31.4%) in CO-HBEC and A(38.3%) and B2(29%) in RCEC. Conclusion: In conclusion, we observed the differences of VFs between clones from community- onset UPECs and HBECs compared to RCEC, and this result explain that UPECs would be more virulent than HBECs which could be less virulent than RCECs considering only VFs distribution. In phylogenetic analysis, each group of E. coli showed different phylogenic distribution, which imply group B E. coli might be more specifically associated with uropathogenecity.

Fox rabies outbreaks in the republic of Buryatia: Connections with neighbouring areas of Russia, Mongolia and China.

The Republic of Buryatia (RB) is located southeast of Lake Baikal and shares a long border with Mongolia. This region of Russia was rabies-free from 1982 to 2010. The first outbreak of fox rabies in RB was identified in 2011, about 30km from the Russian-Mongolian border. We assessed the possible pathways to further spread the 'steppe' phylogenetic lineage of the rabies virus near the northeastern limits of its known distribution. All rabies cases were located 30-210km north of the Russia-Mongolia border, with a distance of up to 320km from each other. Rabies has spread to the north across steppe landscapes and river valleys, with foxes being the main natural hosts of the infection. All RABV isolates from RB belong to the 'steppe' phylogenetic lineage, and three major phylogenic groups could be separated. Group 1 contains sequences from RB, Mongolia, China (Inner Mongolia), and bordering regions of Russia. Group 2 is formed by isolates from other regions of Eastern Siberia, which have no borders with RB and foreign countries. Group 3 contains samples from Western Siberia with endemic fox rabies since the 1950s. The most probable cause of fox rabies epizooty in the RB was multiple drift of the RABV across the Russian-Mongolian border. Our data show that after 2010, fox rabies affected new areas in Central Asia and extended to the north and northeast. Affected areas are similar to the Mongolian-type steppes in their zoogeographical aspect. Closely related genetic lineages of RABV are circulating in RB, Mongolia and the nearest areas of China. International cooperation is necessary to prevent the spread of rabies in the bordering territories of these countries.

Detection of Extended-Spectrum β-Lactamases (ESBL) Producing Enterobacteriaceae from Fish Trapped in the Lagoon Area of Bizerte, Tunisia.

Extended-spectrum β-lactamase and their molecular mechanism in Enterobacteriaceae were analyzed in 126 fish samples of 9 various wild species, living in the lagoon of Bizerte in Tunisia. Fifty-nine (59) Gram-negative strains were isolated and identified as Escherichia coli (n = 24), Klebsiella pneumonia (n = 21), Citrobacter freundii (n = 8), and Shigella boydii (n = 6). Forty-seven ESBL producers were identified using the synergic test. β-Lactamase genes detected were blaCTX-M-1 (E. coli/15; K. pneumonia/8; C. freundii/1; Sh. boydii/1), blaCTX-M-1+ blaOXA-1 (E. coli/4; K. pneumonia/3), blaCTX-M-1+ blaTEM-1-a (K. pneumonia/2), blaCTX-M-15+ blaTEM-1-a (K. pneumonia/1; Sh. boydii/1), blaCTX-M-15+ blaOXA-1 (K. pneumonia/1), blaCTX-M-15 (E. coli/3; K. pneumonia/1; Sh. boydii/3), and blaCTX-M-9 (C. freundii/3). Most strains (84.7%) showed a multiresistant phenotype. qnrA and qnrB genes were identified in six E. coli and in ten E. coli+one K. pneumonia isolates, respectively. The resistance to tetracycline and sulfonamide was conferred by the tet and sul genes. Characterization of phylogenic groups in E. coli isolates revealed phylogroups D (n = 20 strains), B2 (n = 2), and A (n = 2). The studied virulence factor showed prevalence of fimA genes in 9 E. coli isolates (37.5%). Similarly, no strain revealed the three other virulence factors tested (eae, aer, and cnf1). Our findings confirmed that the lagoons of Bizerte may be a reservoir of multidrug resistance/ESBL-producing Enterobacteriaceae. This could lead to indisputable impacts on human and animal health, through the food chain.

Occurrence of ESBL-producing Escherichia coli in soils subjected to livestock grazing in Azores archipelago: an environment-health pollution issue?

Antibiotics are successful drugs used in human and animal therapy; however, they must be considered as environmental pollutants. This study aims to isolate and characterize the extended-spectrum β-lactamase (ESBL) producing Escherichia coli soil from Azores Archipelago subjected to livestock agricultural practices. Twenty-four soil samples were collected from three different pasture systems with different number of cattle heads, and from a control site. Antibiotic susceptibility method was performed by Kirby-Bauer disk diffusion method against 16 antibiotics, and the presence of genes encoding lactamases, antimicrobial resistance genes, virulence factors, and phylogenetic groups was determined by polymerase chain reaction (PCR). Nine ESBLs were recovered from the three grazing sites, and all isolates presented the beta-lactamase genes blaCTX-M-3 and blaSHV. E. coli isolates were resistance to tetracycline and streptomycin and harbored the tetB, strA, and strB genes. One isolate also showed resistance to sulfonamides, and the genes sul1 and sul2 were detected. The isolates were grouped into the following phylogenic groups: B1 (n = 6), D (n = 2), and A (n = 1). The presence of antibiotics and resistance genes in soils may be the source to the development of antimicrobial resistance, which may have negative consequences in human and animal health.

Prevalence and characteristics of extended-spectrum β-lactamases-producing Escherichia coli from broiler chickens at different day-age

Commensal Escherichia coli from the poultries have been considered as reservoirs of extended-spectrum β-lactamases (ESBL)-encoding genes. Between May 2018 and March 2019, a total of 340 E. coli isolates were obtained from apparently healthy broiler chickens from 20 to 40 D old, distributed in 17 small-scale commercial farms. Finally, 45 isolates (8 from 20-day-old broiler chickens, 14 from 30-day-old ones, and 23 from 40-day-old ones) were identified as ESBL producers, which were further investigated to shed light on the virulence gene profiles, phylogenetic groups, and multilocus sequence types and to detect the ESBL plasmid-mediated quinolone resistance determinant (PMQR) genes as well as the mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC. Molecular analysis showed that phylogenic group A and B1 accounted for 66.7% of the ESBL producers. The overall occurrence of virulence genes ranged from 5.1% (cva) to 86.7% (papC). Twenty (44.4%) ESBL producers were considered as biofilm producers with moderate or heavy biofilm formation. The most predominant specific CTX-M subtype was blaCTX-M-14 (n = 19), followed by blaCTX-M-9 (n = 17), blaCTX-M-55 (n = 9), blaCTX-M-15 (n = 6), blaCTX-M-1 (n = 5), and blaCTX-M-65 (n = 4). Additionally, PMQR genes were identified in 86.7% of ESBL producers, qnrS (n = 21) was the most dominant PMQR gene, followed by the aac(6′)-Ib-cr (n = 15), qnrB (n = 12), and qnrA (n = 9), and all of them co-expressed with β-lactamase genes. All PMQR-positive isolates harbored simultaneously at least 1 mutation in the QRDR of gyrA and parC. Forty-five ESBL producers were assigned to 33 sequence types, and the most frequent sequence types (STs) was ST10 (n = 5) and followed by ST95 (n = 3). Additionally, ST302, ST88, ST410, ST187, and ST23 were represented by 2 ESBL producers, respectively, and the remaining ones exhibited diverse ST. Moreover, the prevalence of ESBL producers, the biofilm-forming ability, and the occurrence of the QRDR mutations among the E. coli isolates were characterized by gradually increased with advancing age of broiler chickens.

Exploring the metabolomic diversity of plant species across spatial (leaf and stem) components and phylogenic groups

BackgroundPlants have been used as an important source of indispensable bioactive compounds in various cosmetics, foods, and medicines. However, the subsequent functional annotation of these compounds seems arduous because of the largely uncharacterized, vast metabolic repertoire of plant species with known biological phenotypes. Hence, a rapid multi-parallel screening and characterization approach is needed for plant functional metabolites.ResultsFifty-one species representing three plant families, namely Asteraceae, Fabaceae, and Rosaceae, were subjected to metabolite profiling using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and ultrahigh-performance liquid chromatography quadrupole orbitrap ion trap tandem mass spectrometry (UHPLC-Q-orbitrap-MS/MS) as well as multivariate analyses. Partial least squares discriminant analysis (PLS-DA) of the metabolite profiling datasets indicated a distinct clustered pattern for 51 species depending on plant parts (leaves and stems) and relative phylogeny. Examination of their relative metabolite contents showed that the extracts from Fabaceae plants were abundant in amino acids, fatty acids, and genistein compounds. However, the extracts from Rosaceae had higher levels of catechin and ellagic acid derivatives, whereas those from Asteraceae were higher in kaempferol derivatives and organic acids. Regardless of the different families, aromatic amino acids, branch chain amino acids, chlorogenic acid, flavonoids, and phenylpropanoids related to the shikimate pathway were abundant in leaves. Alternatively, certain amino acids (proline, lysine, and arginine) as well as fatty acids levels were higher in stem extracts. Further, we investigated the associated phenotypes, i.e., antioxidant activities, affected by the observed spatial (leaves and stem) and intra-family metabolomic disparity in the plant extracts. Pearson’s correlation analysis indicated that ellagic acid, mannitol, catechin, epicatechin, and quercetin derivatives were positively correlated with antioxidant phenotypes, whereas eriodictyol was positively correlated with tyrosinase inhibition activity.ConclusionsThis work suggests that metabolite profiling, including multi-parallel approaches and integrated bioassays, may help the expeditious characterization of plant-derived metabolites while simultaneously unraveling their chemodiversity.

Computational analysis of drug like candidates against Neuraminidase of Human Influenza A virus subtypes

Abstract Influenza virus is an enveloped virus having segmented genome, coded by eight (negative-sense) RNA segments. The RNA segments are packed in a nucleocapsid protein. Neuraminidase (NA), an exosialidase, is the major antigen as well as vital virulence factor of the virus and is coded by the 6th segment of RNA. Eleven subtypes of NA are characterized for influenza A and the N1–N9 are divided into two phylogenic groups. NA is an active target for development of potent antivirals or Neuraminidase inhibitors (NAIs), which are reported to interact with multiple conserved residues in the active site of NA resulting in inhibition of virion release and prevention of viral spread to nearby cells. The current computational study focuses on docking of eleven different drug-like molecules including the known NAIs such as oseltamivir and zanamivir against crystal structures of NA of human Influenza A virus subtypes of both phylogenic groups. The docking results of this study indicated that zanamivir was the most active drug candidate against various NA subtypes followed by laninamivir. The study provides insights on interactions as well as cross-reactivity between lead compounds and NA proteins of various influenza subtypes. Observations on the efficacy of these NAIs assessed by the computational study have to be validated through in vitro and in vivo studies for successfully translating them as potential therapeutic agents to treat infection by various subtypes of influenza.