Tra Gene Research Articles

Early embryonic transcriptomes of Zeugodacus tau provide insight into sex determination and differentiation genes.

Zeugodacus tau (Walker) is an invasive pest. The sterile insect technique is an environment-friendly method for pest control. Understanding the mechanism of sex determination will contribute to improving efficiency of this technique. In this study, we identified the transformer (tra) gene in Z. tau. One female-specific and two male-specific isoforms of tra were found in Z. tau, and the male-specific splicing pattern of tra was found to occur 5 h after egg laying. We performed transcriptome sequencing at 1 h (E1), 5 h (E5), and 9 h (E9) after egg laying and obtained high-quality transcriptome libraries of early embryo development. We identified 13297 and 11713 differentially expressed genes (DEGs) from E5 versus E1 and E9 versus E1 comparisons, respectively. To explore the potential functions of the DEGs during embryonic development, Gene Ontology, Clusters of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes analyses were performed. Twenty-six genes potentially involved in sex determination or differentiation, including Maleness-on-the-Y (MoY), were identified in Z. tau. To verify the transcriptome results, 15 genes were selected for quantitative real-time PCR validation. The results were consistent with the transcriptome sequencing results. Moreover, U2 small nuclear riboprotein auxiliary factor (U2AF-50), female lethal d (fl(2)d), and virilizer (vir) were highly expressed at E5, indicating that they may be related to the sex-specific splicing of tra. Further functional analysis is needed to confirm this speculation. Our data provide an insight into the mechanism underlying sex determination and differentiation in tephritid species.

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression.

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.

Genomic Analysis of Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 From Spanish Hospitals.

Salmonella enterica serovar Kentucky (S. Kentucky) with sequence type (ST) 198 and highly resistant to ciprofloxacin (ST198-CipR) has emerged as a global MDR clone, posing a threat to public health. In the present study, whole genome sequencing (WGS) was applied to characterize all CipR S. Kentucky detected in five Spanish hospitals during 2009–2018. All CipR isolates (n = 13) were ST198 and carried point mutations in the quinolone resistance-determining regions (QRDRs) of both gyrA (resulting in Ser83Phe and Asp87Gly, Asp87Asn, or Asp87Tyr substitutions in GyrA) and parC (with Thr57Ser and Ser80Ile substitutions in ParC). Resistances to other antibiotics (ampicillin, chloramphenicol, gentamicin, streptomycin, sulfonamides, and tetracycline), mediated by the blaTEM–1B, catA1, aacA5, aadA7, strA, strB, sul1, and tet(A) genes, and arranged in different combinations, were also observed. Analysis of the genetic environment of the latter resistance genes revealed the presence of multiple variants of SGI1 (Salmonella genomic island 1)-K and SGI1-P, where all these resistance genes except catA1 were placed. IS26 elements, found at multiple locations within the SGI1 variants, have probably played a crucial role in their generation. Despite the wide diversity of SGI1-K- and SGI1-P-like structures, phylogenetic analysis revealed a close relationship between isolates from different hospitals, which were separated by a minimum of two and a maximum of 160 single nucleotide polymorphisms. Considering that S. enterica isolates resistant to fluoroquinolones belong to the high priority list of antibiotic-resistant bacteria compiled by the World Health Organization, continuous surveillance of the S. Kentucky ST198-CIPR clone is required.

Parental RNAi Silencing of the transformer-2 Gene in a Species of the Anastrepha fraterculus Complex of Cryptic Species (Diptera, Tephritidae)

In genera Anastrepha, Bactrocera and Ceratitis of the tephritid fruit flies the auto-regulatory function of gene transformer is assumed to be activated by maternal derived mRNA or the proteins of the gene transformer (tra-2 mat) and transformer-2 (tra-2mat). However, this maternal effect was not yet been demonstrated. The objective of the present study was to test the effect of absence of tra-2mat in the eggs on the sex determination of A. sp.1 affinis fraterculus. This was achieved by silencing gene tra-2 in the parental females via the pRNA interference. The data showed that tra-2 was transiently silenced in the female for three weeks period. The progenies sex ratio produced by these females during the silencing of tra-2 depart from 1:1 in favor of males. The excess of males was due to the transformation of a fraction of genotypical female XX embryos into XX males, the so-called pseudomales, Individual F1 males from the offspring of treated females crossed to females from the stock, revealed that majority of them showed regular mating behavior and were fertile. However, no offspring was produced in the crosses by a fraction of males that have produced sperms, showed regular mating behavior but did not transfer sperms to the females. The data allow the conclusion that the absence of tra-2mat in the eggs had impaired the self-regulation of the embryonic gene tra resulting in the transformation of XX embryos into pseudomales and also that these pseudomales are sterile. This effect may be useful improve more sustainable technologies for fruit fly control such as SIT.

Predominance of blaTEM and tetA genes in antibiotic-resistant Escherichia coli isolates from Laguna Lake, Philippines

Abstract Lakes are one of the sinks of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs); however, information on ARB and ARGs in lakes in the Philippines is scarce. In this study, Escherichia coli was isolated from the largest freshwater lake in the Philippines, Laguna Lake, to detect antibiotic resistance and the presence of ARGs. Broth microdilution assay (BMA) and molecular identification of five environmentally prevalent ARGs (strA, blaCTX-M, blaSHV, blaTEM, and tetA) were performed. The majority (75.70%) of the isolates harbored at least one of the targeted antibiotic genes. Multiplex PCR detected about 49.07% of the isolates had genes for extended-spectrum β-lactamases (ESBL), which were mostly represented by blaTEM (47.66%). The genes strA and tetA were observed in this study with detection frequencies of 29.91 and 45.33%, respectively. About 95.69% of thermotolerant E. coli isolates were non-susceptible to six different antibiotics using BMA. Nearly 37% of the isolates were found to be multidrug-resistant (MDR) with most isolates resistant to ampicillin (81.72%). Furthermore, the occurrence of ESBL genes was significantly correlated with tetA genes (P = 0.013). To date, this study is the first to report on the presence of MDR and thermotolerant E. coli in Laguna Lake, Philippines.

Antimicrobial Resistance Phenotypes and Genotypes of Salmonella spp. Isolated from Commercial Duck Meat Production in Thailand and Their Minimal Inhibitory Concentration of Disinfectants.

Salmonella is an important foodborne bacterium that has become increasingly resistant to critical antimicrobial and disinfectant agents. The aim of this study was to characterize antimicrobial and disinfectant resistance of Salmonella spp. isolated from ducks raised for meat in Nakhon Pathom province, Thailand. A total of 694 fecal samples from ducks were collected in 2018. Of which, 85 samples were positive for Salmonella (12.2%), and 12 Salmonella serovars were identified from 125 Salmonella isolates. The Altona serovar was the predominant serotype found in this study (36.5%). All isolates showed resistance to at least one class of antimicrobial, and 23.2% displayed multidrug resistance (MDR) phenotype. The blaTEM, aadA2, strA, and dfrA12 genes were detected in antibiotic-resistant strains of Salmonella, whereas the genes within a plasmid-borne qnr family that presented in fluoroquinolone-susceptible Salmonella strains were qnrB (3.8%) and qnrS (1.5%). The minimum inhibitory concentrations of benzalkonium chloride (BKC), cetylpyridium chloride (CPC), and hexadecyltrimethyl ammonium bromide (CTAB) ranged between 128 and 512 μg/mL, while that of didecyldimethylammonium chloride (DDAC) was between 32 and 128 μg/mL. The presences of qacEΔ1, mdfA, sugE(c), sugE(p), and ydgE genes were less prevalent (0.8-1.6%). Taken together, our results indicate that duck is an important source of Salmonella with antimicrobial resistance in food-producing animals. Active surveillance programs for antimicrobial and disinfectant resistance in duck production are needed for an early detection of resistance strains of public health importance.

IDENTIFICATION OF ANTIBIOTIC RESISTANCE OF THE CATTLE PATHOGEN Histophilus somni

Histophilus somni is a Gram-negative bacterium of the Pasteurellaceae family which is a component of the Bovine Respiratory Disease Complex and the pathogen, causing a multisystem disease — histophilosis. For the treatment of diseases caused by Pasteurellaceae bacteria, aminoglycosides, sulfonamides, beta-lactams, tetracyclines and macrolides are most often used, therefore the formation of resistance of H. somni to antibiotics of these groups can be expected. This work is the first study of antibiotic resistance of H. somni isolated from cattle in the Russian Federation. This work aimed at exploration of the antibiotic resistance of circulating H. somni strains by phenotypic and genotypic methods and the evaluation of the PCR method applicability for the prediction H. somni resistance to antimicrobial agents. We studied 18 cultures of H. somni, the causative agent of histophilosis, isolated in 2018-2019 from biological material (parenchymal organs, washes, sperm) of 145 animals of different breed, age, sex and physiological groups using microbiological method. The cultures were studied using the disk diffusion method for sensitivity to 13 antibiotics of aminoglycosides, beta-lactams, tetracyclines and sulfonamides classes. All obtained isolates were tested by PCR for the presence of genetic determinants of antibiotic resistance, most often found in H. somni: tetH (resistance to tetracyclines), blaOXA-2 (resistance to penicillins), aadA25, strA, strB, aphA1 (resistance to aminoglycosides), sul2 (sulfonamide resistance). Resistance to aminoglycoside group was most prevalent, i.e., resistance to streptomycin was 50 %, and resistance to neomycin exceeded 40 %. Resistance genes aadA25, strA, strB and aphA1 were found in the resistant samples. A total of 33 % isolates showed resistance to sulfonamides, all this samples were positive for the sul2 gene in PCR. The sensitivity to penicillins was quite high (~ 75 %), the sensitivity to beta-lactams approached 100 %. The sensitivity to antimicrobials of the tetracycline group was higher than 80 %. However, neither tetracyclines (tetH) nor penicillins (blaOXA-2) resistance genes were identified during the study. Two isolates were multidrug resistant with resistance to aminoglycosides, beta-lactams and tetracyclines. Also, four samples were resistant to antimicrobial agents of two different groups, i.e., two samples were resistant to aminoglycosides and sulfonamides with strA, strB, aadA25, aphA1, and sul2 genes found, and two samples were resistant to aminoglycosides and beta-lactams with only aminoglycoside resistance genes aadA25 and strA identified. With the exception of samples resistant to tetracyclines and beta-lactams, in which the expected genes were not detected, all observed phenotypes of antimicrobial resistance were consistent with the PCR test results. The combination of genotypic and phenotypic methods for determining antibiotic resistance is necessary for understanding of the resistance mechanisms and increases the efficiency of antibiotic resistance monitoring programs.

ВЫЯВЛЕНИЕ УСТОЙЧИВОСТИ К АНТИБИОТИКАМ У ВОЗБУДИТЕЛЯ ГИСТОФИЛЕЗА КРУПНОГО РОГАТОГО СКОТА Histophilus somni

Histophilus somni is a Gram-negative bacterium of the Pasteurellaceae family which is a component of the Bovine Respiratory Disease Complex and the pathogen, causing a multisystem disease — histophilosis. For the treatment of diseases caused by Pasteurellaceae bacteria, aminoglycosides, sulfonamides, beta-lactams, tetracyclines and macrolides are most often used, therefore the formation of resistance of H. somni to antibiotics of these groups can be expected. This work is the first study of antibiotic resistance of H. somni isolated from cattle in the Russian Federation. This work aimed at exploration of the antibiotic resistance of circulating H. somni strains by phenotypic and genotypic methods and the evaluation of the PCR method applicability for the prediction H. somni resistance to antimicrobial agents. We studied 18 cultures of H. somni, the causative agent of histophilosis, isolated in 2018-2019 from biological material (parenchymal organs, washes, sperm) of 145 animals of different breed, age, sex and physiological groups using microbiological method. The cultures were studied using the disk diffusion method for sensitivity to 13 antibiotics of aminoglycosides, beta-lactams, tetracyclines and sulfonamides classes. All obtained isolates were tested by PCR for the presence of genetic determinants of antibiotic resistance, most often found in H. somni: tetH (resistance to tetracyclines), blaOXA-2 (resistance to penicillins), aadA25, strA, strB, aphA1 (resistance to aminoglycosides), sul2 (sulfonamide resistance). Resistance to aminoglycoside group was most prevalent, i.e., resistance to streptomycin was 50 %, and resistance to neomycin exceeded 40 %. Resistance genes aadA25, strA, strB and aphA1 were found in the resistant samples. A total of 33 % isolates showed resistance to sulfonamides, all this samples were positive for the sul2 gene in PCR. The sensitivity to penicillins was quite high (~ 75 %), the sensitivity to beta-lactams approached 100 %. The sensitivity to antimicrobials of the tetracycline group was higher than 80 %. However, neither tetracyclines (tetH) nor penicillins (blaOXA-2) resistance genes were identified during the study. Two isolates were multidrug resistant with resistance to aminoglycosides, beta-lactams and tetracyclines. Also, four samples were resistant to antimicrobial agents of two different groups, i.e., two samples were resistant to aminoglycosides and sulfonamides with strA, strB, aadA25, aphA1, and sul2 genes found, and two samples were resistant to aminoglycosides and beta-lactams with only aminoglycoside resistance genes aadA25 and strA identified. With the exception of samples resistant to tetracyclines and beta-lactams, in which the expected genes were not detected, all observed phenotypes of antimicrobial resistance were consistent with the PCR test results. The combination of genotypic and phenotypic methods for determining antibiotic resistance is necessary for understanding of the resistance mechanisms and increases the efficiency of antibiotic resistance monitoring programs.

Multidrug Resistance of Escherichia coli Strains Isolated From Bovine Feces and Carcasses in Northeast Mexico.

In this work, the antimicrobial resistance profile of Escherichia coli strains (n = 248) isolated from bovine feces and carcass samples from Tamaulipas, Mexico, was evaluated. Susceptibility to 12 antibiotics conventionally used in human and veterinary treatments was determined according to Clinical and Laboratory Standards Institute guidelines. Genes encoding resistance to tetracycline (tetA and tetB), streptomycin (strA), aminoglycoside (aadA), and β-lactamase (blaTEM and blaSHV) were investigated by PCR. Also, stx1, stx2, eae, bfp, and hlyA encoding virulence factors were determined. Of the isolates, 85.9% were confirmed as E. coli strains. Among the 213 E. coli isolates tested, 94.8% (202/213) showed resistance for at least one antimicrobial, mainly ampicillin (83.0%; 177/213), cephalothin (76.0%; 162/213), and tetracyclines (69.0%; 147/213). In all the other antibiotics tested, the resistance percentage was below 36%. A multidrug-resistant phenotype was found in 72.7% of the tested strains. The presence of the tet gene (tetA or tetB) was detected in 43.1% of the isolates, the strA gene in 17.3%, and aadA1 in 51.6%. The blaTEM and blaSHV genes were found in 10.3 and 0.4% of the isolates, respectively. stx1 was detected in 4.2% of isolates, stx2 in 7.0, and hlyA in 2.8%. The virulence genes, eae and bfp, were not detected in any strain. These results indicate that Tamaulipas food products of bovine origin can be a source of multiresistant E. coli strains for the environment and exposure for consumers.

Characterisation of IncI1 plasmids associated with change of phage type in isolates of Salmonella enterica serovar Typhimurium

BackgroundAcquisition of IncI1 plasmids by members of the Enterobacteriaceae sometimes leads to transfer of antimicrobial resistance and colicinogeny as well as change of phage type in Salmonella Typhimurium. Isolates of S. Typhimurium from a 2015 outbreak of food poisoning were found to contain an IncI1 plasmid implicated in change of phage type from PT135a to U307 not previously reported. The origin of the changes of phage type associated with this IncI1 plasmid was investigated. In addition, a comparison of its gene composition with that of IncI1 plasmids found in local isolates of S. Typhimurium typed as U307 from other times was undertaken. This comparison was extended to IncI1 plasmids in isolates of phage types PT6 and PT6 var. 1 which are thought to be associated with acquisition of IncI1 plasmids.ResultsAnalysis of IncI1 plasmids from whole genome sequencing of isolates implicated a gene coding for a 1273 amino acid protein present only in U307 isolates as the likely source of change of phage type. The IncI1 plasmids from PT6 and PT6 var. 1 isolates all had the ibfA gene present in IncI1 plasmid R64. This gene inhibits growth of bacteriophage BF23 and was therefore the possible source of change of phage type. A fuller comparison of the genetic composition of IncI1 plasmids from U307 isolates and PT6 and PT6 var. 1 isolates along with two IncI1 plasmids from S. Typhimurium isolates not showing change of phage type was undertaken. Plasmids were classified as either ‘Delta’ or ‘Col’ IncI1 plasmids according to whether genes between repZ and the rfsF site showed high identity to genes in the same location in R64 or ColIb-P9 plasmids respectively. Comparison of the tra gene sets and the pil gene sets across the range of sequenced plasmids identified Delta and Col plasmids with almost identical sequences for both sets of genes. This indicated a genetic recombination event leading to a switch between Delta and Col gene sets at the rfsF site. Comparisons of other gene sets showing significant variation among the sequenced plasmids are reported. Searches of the NCBI GenBank database using DNA and protein sequences of interest from the sequenced plasmids identified global IncI1 plasmids with extensive regions showing 99 to 100% identity to some of the plasmids sequenced in this study indicating evidence for widespread distribution of these plasmids.ConclusionTwo genes possibly associated with change of phage type were identified in IncI1 plasmids. IncI1 plasmids were classified as either ‘Delta’ or ‘Col’ plasmids and other sequences of significant variation among these plasmids were identified. This study offers a new perspective on the understanding of the gene composition of IncI1 plasmids. The sequences of newly sequenced IncI1 plasmids could be compared against the regions of significant sequence variation identified in this study to understand better their overall gene composition and relatedness to other IncI1 plasmids in the databases.

Co-dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955.

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. The At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologs of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR is preferentially biased toward its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

Molecular studies on antimicrobial resistant genes in predominant staphylococci and E. coli isolates from bovine mastitis in different regions of Karnataka

Bovine mastitis is a persistent and major problem in the dairy industry globally. Staphylococci and Escherichia coli are predominantly responsible for this condition. The aim of this study was to look for the genes associated with antimicrobial resistance in these predominant bacteria isolated from bovine mastitis in different regions in Karnataka. In all, 211 bacterial isolates were recovered from bovine mastitis cases in Karnataka. Of these, 85, 23 and 47 were Staphylococcus aureus, S. epidermidis and E. coli, respectively, as confirmed by PCR. Among S. aureus, blaZ was detected in 61.17% (52/85), mecA and tetK detected in 3.52% (3/85), aacA-aphD, ermC and strA genes detected in 4.7% (4/85), tetM was detected in 16.4% (14/85) and strB in 7.05% (6/85) isolates. None of the isolates contained tetO and ermA genes and 30.58% (26/85) isolates did not reveal any of the genes. Among S. epidermidis, blaZ was detected 43.47% (10/23), mecA and tetM detected in 30.43% (7/23), aacA-aphD and ermC detected in 13.04% (3/23), tetK detected in 8.69% (2/23), strA detected in 4.34% (1/23) and strB detected in 21.73% (5/23) isolates. None of them had tetO and ermA genes and 13.04% (3/23) isolates did not reveal any of the genes screened. Among E. coli, blaTEM and tetA genes were detected in 100% (47/47), blaSHV and strA were detected in 31.9% (15/47), blaOXA 30 detected in 12.76% (6/47), tetM and tetB detected in 2.12% (1/47), and strB was detected in 51.06% (24/47). None of the isolates contained aacA-aphD, mecA, tetO, tetK, ermA and ermC genes. This genetic profile of prevalence of antimicrobial genes for varied antibiotics/antimicrobials indicates the possible wider antimicrobial resistance among the predominant species of bacteria causing bovine mastitis. Further, phenotypic characterisation of these isolates with respect to their antimicrobial resistance would be of significance.

Antibiotic resistance genes and bacterial community on the surfaces of five cultivars of fresh tomatoes.

Antibiotic resistance genes and bacteria (ARGs and ARB) in vegetable or fruit pose risks to ecological environment health. However, the assessment of ARGs and ARB from one popular vegetable, fresh tomato, has not been carried out before. In this study, high-throughput quantitative PCR and 16S rRNA gene Illumina sequencing technology were used to explore the antibiotic resistance characteristics of bacteria on five common cultivars of fresh tomatoes from supermarket. A total of 191 ARGs and 10 mobile genetic elements (MGEs) were detected on the tomato surfaces. The distribution profile of ARGs and MGEs was different among samples, with the organic tomatoes showing more ARGs and MGEs number and relative abundance. Aminoglycoside resistance genes strA and strB, sulfonamide resistance gene sul1, and multidrug resistance gene qacΔ1-01 were the predominant ARGs. Dominant MGEs were transposase genes, which might promote horizontal gene transfer (HGT) of ARGs. Network analysis indicated that fifteen bacterial families might be the potential hosts of ARGs, and the detected MGEs might have positive correlation with ARGs. These results revealed the bacterial ARGs and MGEs from fresh tomato, which might help guide human to pay more attention to ecological environment impacts of ARGs and ARB on the surfaces of vegetable or fruit.

Occurrence of Antibiotic-Resistant Bacteria and Genes in Two Drinking Water Treatment and Distribution Systems in the North-West Province of South Africa.

With the increasing spread of antimicrobial resistance, there is growing attention to the contribution made by drinking water systems. The potential health impact of two drinking water treatment and distribution systems (A and B) in the North-West Province of South Africa was determined by investigating the water quality and occurrence of antimicrobial-resistant heterotrophic bacteria and genes in the raw and treated water over four seasons. Most of the physicochemical parameters except for electrical conductivity were within permissible limits. Coliform bacteria reduced from raw to potable water except for counts higher than the threshold recorded in Summer and Winter. A total of 203 heterotrophic bacterial isolates were recovered on chromogenic R2A medium and subjected to susceptibility testing to twelve antibiotics. Most of the isolates were resistant to β-lactam antibiotics and Trimethoprim, whereas they were susceptible to Ciprofloxacin, Erythromycin, and Neomycin. The proportions of Cephalothin and Kanamycin-resistant isolates were significantly higher (p < 0.05) after treatment for site A, compared to significantly lower β-lactam, Oxytetracycline, and Trimethoprim-resistant isolates for B. Over 50% of isolates were of high risk, indicating their origin from high antibiotic-use sources. Seventy-one (35%) isolates were multidrug-resistant, out of which the majority (53.5%, n = 38) possessed the strA gene, followed by strB 21 (29.6%), dfrB 13 (18.3%), aadA 11 (15.5%), blaCTX-M 5 (7.0%), and tetA 3 (4.2%). The 16S rRNA gene sequences of the isolates revealed strains belonging to eight bacterial families, some of which are clinically important.

Molecular Characterization and Expression Profiling of Three Transformer-2 Splice Isoforms in the Redclaw Crayfish, Cherax quadricarinatus.

Sex determination/sex differentiation is determined by genetics, environmental factors, or the interactions of the two. The Transformer-2 (Tra-2) gene plays an important role in the sex determination cascade signal pathway in insects. In this study, the Tra-2 gene was isolated and characterized from the cDNA library of gonad tissues in the redclaw crayfish, Cherax quadricarinatus. Three splice variants were identified, designated as CqTra-2A, CqTra-2B, and CqTra-2C, and sequence analysis showed that they had a highly conserved RRM domain. Phylogenetic analysis was performed by the NJ method, and the results revealed that the Tra-2 protein of the redclaw crayfish was very closely related to those of Macrobrachium rosenbergii, Fenneropenaeus chinensis, and Macrobrachium nipponense. Real-time PCR analysis showed that the three isoforms were predominantly expressed in the ovary and gradually increased with embryonic development. Additionally, the expression pattern of CqTra-2 at different developmental stages was analyzed by qPCR and revealed that the phase of having a body length of 3 cm may be the key period for the sex differentiation of C. quadricarinatus. RNAi-targeting gene silencing further confirmed the function of CqTra-2 in sexual differentiation in redclaw crayfish. Our experimental data will contribute to understanding the mechanism of sex determination in crustaceans.

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.

Characterisation of Escherichia coli Recovered from Wild Animals Kept at Bikaner Zoo for Their Antibiotic Resistance

In the present study, a total of 41 E. coli isolates obtained from Boselaphus tragocamelus (5), Antelope cervicapra (18) and Gazella gazelle (18) maintained at Bikaner (Rajasthan) zoo were subjected to antibiogram determination against 15 antibiotics belonging to four different classes and were also genotyped for detecting presence of blaTEM, sul-2, strA and aadA genes. Antibiogram study revealed highest efficacy of ciprofloxacin (90.2%) followed by nalidixic acid (75.6%) and chloramphenicol (68.4%) and high resistance to β lactam, Sulfamethoxazole and Aminoglycoside antibiotics. The overall presence of blaTEM, sul-2, strA and aadA genes was detected in 95.12%, 80.48%, 60.97% and 87.8% in isolates.

Characterization of an IMP-4-Producing Klebsiella pneumoniae ST1873 Strain Recovered from an Infant with a Bloodstream Infection in China.

PurposeImipenemase (IMP), an Ambler class B metallo-β-lactamase, is an important carbapenemase that confers resistance to almost all β-lactams. In this study, we characterized the genomic feature of an IMP-4-producing Klebsiella pneumoniae ST1873 strain, a rare sequence type (ST) isolated from an infant with a bloodstream infection in China.Patients and MethodsK. pneumoniae strain, BKP19, was collected from a bloodstream infection in an infant who was hospitalized at the department of paediatrics. The whole genome sequence of the strain was sequenced using the Illumina NovaSeq 6000 platform and long-read MinION sequencer. Multilocus sequence typing, antimicrobial resistance gene identification, plasmid and phylogenetic relationship analysis of the strain were analysed by various bioinformatics approaches.ResultsK. pneumoniae BKP19 was resistant to multiple antimicrobials, including carbapenems. Eleven antimicrobial resistance genes corresponding to beta-lactam resistance, quinolone resistance, phenicol resistance and fosfomycin resistance could be identified in the genome. The carbapenem resistance gene blaIMP-4 was located in an IS26-associated class 1 integron of an IncN-type plasmid with 39,033 bp (pIMP-4-BKP19). Sequence alignment revealed that pIMP-4-BKP19 is closely related to the common plasmid carrying IMP-4 in K. pneumoniae (pIMP-HZ1-like plasmid) but is smaller, lacking the quinolone resistance gene qnrS1 and multiple tra gene orthologs. Conjugation experiment revealed that pIMP-4-BKP19 is a non-conjugative plasmid. According to in silico MLST analysis, K. pneumoniae strain BKP19 belongs to a sporadic clone ST1873.ConclusionIn summary, our study reports the first genome sequence of a K. pneumoniae ST1873 strain harbouring the class B β-lactamase blaIMP-4 in an IncN-type plasmid recovered from an infant with a bloodstream infection in China. Considering the global emergence of IMP-4 carbapenemase, more attention must be paid to prevent its future prevalence.

Clonal Dynamics and Publicness of CMV-Specific TCR Repertoire after Allogeneic Stem Cell Transplantation

[Background] Cytomegalovirus (CMV) disease is a major complication after allogeneic stem cell transplantation (SCT). However, mechanisms of adoptive immunity against CMV are not fully elucidated. Recently, high-throughput next-generation sequencing (NGS) technology made it possible to shed light on the detailed and comprehensive landscape of T-cell receptor (TCR) repertoire. In this study, we analyzed TCR repertoire of CMV-specific cytotoxic T-cells (CMV-CTLs) in patients who suffered from CMV reactivation after SCT to clarify the diversity and dynamics of CMV-specific T-cell immunity. [Methods] We sequentially collected peripheral blood mononuclear cells from patients with HLA-A*24:02 who received SCT in our institution. Samples were collected weekly or every two weeks from their neutrophil engraftment until approximately 100 days after SCT. CMV reactivation was evaluated weekly with CMV antigenemia test. CD8 and CMV pp65 tetramer positive cells were sorted and unbiased next-generation sequencing-based analyses of TRBV/TRBJ gene segments were performed at the timing of CMV reactivation in 16 patients, and TRA gene segments were also analyzed in 10 patients. In addition, TCR beta repertoires after 2-4 weeks were analyzed in 12 patients. In the 12 patients, the dynamics of TCR repertoire diversity and proportional changes of each clone were assessed. We evaluated the diversity by Shannon-Weber index, and we defined TCR beta clonotypes found in two or more patients using the same TRBV/TRBJ gene segments and CDR3 amino acid sequences as public. This study was approved by the ethics committee of Tokyo Metropolitan Komagome Hospital. [Results] Among 16 patients, 11 received bone marrow, 3 received peripheral blood stem cells, and 2 received cord blood transplant. Underlying diseases were acute myeloid leukemia in 7, acute lymphoblastic leukemia in 7, and myelodysplastic syndromes in 2 patients. Median age at SCT was 50 years old (range: 20-71). Median duration from SCT to first CMV reactivation was 39 days (range: 16-55) and 7 patients (43.8%) were administered systemic corticosteroid at the time of reactivation (prednisolone 10-30mg/day). Median peripheral blood CMV-CTLs count at that time was 29.47/uL (range: 4.65-229.6). In most patients TCR beta repertoire of CMV-specific CTLs when CMV reactivated was highly skewed (median Shannon-Weber index was 1.44 [range: 0.542-3.164]). TCR alpha and beta were analyzed together in 10 patients and their diversity correlated well (p&lt;0.001). Interestingly, 12 of 16 patients (75.0%) had at least one public TCR and, in all 12 patients with public TCR, the most frequent TCRs were public. Diversity of TCR repertoire was significantly lower in patients with public TCR compared with those in patients without (p=0.039). There was no obvious association between presence of public TCR and multiple events of CMV reactivation (p=0.57). When we evaluated clonal transition in 12 patients whose TCR beta repertoires were analyzed sequentially, the public clonotypes were continuously detected in 10 of 11 patients with public TCR and remained dominant in 9, while in one patient frequency of the most frequent public clone declined massively (from 50.25% among assigned reads to 6.63%). In addition, there were two patterns of subsequent clonal behavior in TCR repertoire of CMV-CTLs. In 10 patients, prevailing clones persisted and TCR repertoires of CMV-CTLs remained oligoclonal (Figure A). However, in other two patients, TCR repertoires of CMV-CTLs became more diverse (Shannon-Weber index at the time of reactivation and a few weeks after was 0.558/2.958 in one patient and 1.471/3.86 in another), major clones lost, and new private clones appeared afterwards (Figure B). Polyclonal pattern (TCR repertoire of CMV-CTLs was diverse at the time of reactivation or after a few weeks) was detected in 3 patients out of 16 patients, and the pattern was exclusively seen in patients who were administered corticosteroid when CMV reactivated (42.9% vs 0.0%, p=0.063). [Conclusion] TCR repertoire of CMV-CTLs at the time of CMV reactivation after SCT is highly oligoclonal and frequently shared among different patients, but can dynamically change in a short period in some patients. Functional analyses of the dominant TCRs to understand their reactivity against CMV epitope and elucidation of the clinical significance and developmental mechanisms of clone shift are strongly warranted. Figure Disclosures Kitaura: Repertoire Genesis Inc.: Employment. Suzuki:Repertoire Genesis Inc.: Equity Ownership.

TRAV gene segments further away from the TRAJ gene segment cluster appear more commonly in human tumor and blood samples

We considered the possibility that the greater the distance between an immune receptor V and J, the more likely the V usage. Such a hypothesis is supported by results from mouse experiments. And, such a hypothesis is consistent with the fundamental nature of recombination and genomic distance: the further the distance, the greater the chance of a DNA break. Thus, we exploited the vast dataset of V and J recombination reads available for the human TRA gene, particularly from cancer and blood specimens, to assess the frequency of TRAV usage with respect to distance from the TRAJ cluster. Results indicated that, indeed, over the entire TRAV cluster, there is a greater chance of V usage the further the distance from the J cluster. These results do not address causation, and are not consistent for certain individual V gene segments, but the results do indicate that overall, the larger the distance between the V and J gene segment cluster, the more likely the appearance of at least a subset of TRAV segments, particularly among tumor infiltrating lymphocytes. With a similar approach, the distal TRAV gene segments were also found to be more commonly associated with a subset of distal TRAJ segments. These results have implications for restrictions on the apparent TRA repertoire in disease settings.