aadA Gene Research Articles

Plastid Transformation in Poplar: A Model for Perennial Trees.

Poplar (Populus) is an important forest tree and considered model for perennial trees. Here we describe a method for poplar plastid transformation, which involves preparation of explants, vector construction, biolistic bombardment, regeneration and selection of transplastomic poplar plants. The young leaves of 4-week-old poplar plants are used for biolistic bombardment and aadA gene as selectable marker. Homoplasmic transplastomic lines are obtained after regeneration and several rounds of selection with spectinomycin over 10months. Homoplasmy is further confirmed by Southern blot. The establishment of a plastid transformation system for Populus is likely to make a significant contribution to tree genetic improvement.

Prevalence of Aminoglycoside Resistance and Aminoglycoside Modifying Enzymes in Acinetobacter baumannii Among Intensive Care Unit Patients, Ismailia, Egypt

BackgroundAcinetobacter baumannii is an opportunistic pathogen that rapidly develops antibiotic resistance against commonly prescribed antimicrobial agents in hospitalized patients worldwide. Aminoglycosides are commonly used in the treatment of A. baumannii health care-associated infections (HAIs). Aminoglycosides resistance mechanisms are varied and commonly involve production of aminoglycoside-modifying enzymes (AME) and efflux systems.AimThis study aimed to provide an insight into the frequency of genes encoding AME in A. baumannii strains isolated from different clinical specimens in intensive care units (ICU).MethodologyA total of 52 multidrug-resistant (MDR) A. baumannii strains were isolated from ICU, Suez Canal University Hospitals. Species identification and antibiotics susceptibility testing were done by the automated system VITEK 2. The genes encoding AME were detected by PCR.ResultsAminoglycosides resistance (amikacin, gentamicin and tobramycin) was observed in 35 isolates (67.3%). We found that aacC1 gene was the predominant AME resistance gene among A. baumannii isolates, detected in 14 isolates (40%), aphA6 in 11 isolates (31.4%) and addA1 in 5 isolates (14.2%). We found 5 isolates containing 2 AME genes, 3 of them with aacC1 and aphA6 and the remaining 2 with both aacC1 and aadA1 genes. Nearly, 5 isolates (14.2%) were negative for all AME resistance genes.ConclusionOur study indicated that AME encoding genes are predominant in A. baumannii strains in our region which stressed on the importance of preventive measures to control spreading of resistance genes.

Characterization of the Chloroplast Genome Facilitated the Transformation of Parachlorella kessleri-I, A Potential Marine Alga for Biofuel Production.

Introduction The microalga Parachlorella kessleri-I produces high biomass and lipid content that could be suitable for producing economically viable biofuel at a commercial scale. Sequencing the complete chloroplast genome is crucial for the construction of a species-specific chloroplast transformation vector.Methods In this study, the complete chloroplast genome sequence (cpDNA) of P. kessleri-I was assembled; annotated and genetic transformation of the chloroplast was optimized. For the chloroplast transformation, we have tested two antibiotic resistance makers, aminoglycoside adenine transferase (aadA) gene and Sh-ble gene conferring resistance to spectinomycin and zeocin, respectively. Transgene integration and homoplasty determination were confirmed using PCR, Southern blot and Droplet Digital PCR.Results The chloroplast genome (109,642 bp) exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region. The genome encodes 116 genes, with 80 protein-coding genes, 32 tRNAs and 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species-specific vector that facilitated the transformation of P. kessleri-I chloroplast. The transgenic algal colonies were retrieved on a TAP medium containing 400 mg. L-1 spectinomycin, but no transgenic was recovered on the zeocin-supplemented medium. PCR and Southern blot analysis ascertained the transgene integration into the chloroplast genome, via homologous recombination. The chloroplast genome copy number in wildtype and transgenic P. kessleri-I was determined using Droplet Digital PCR.Conclusion The optimization of stable chloroplast transformation in marine alga P. kessleri-I should open a gateway for directly engineering the strain for carbon concentration mechanisms to fix more CO2, improving the photosynthetic efficiency and reducing the overall biofuels production cost.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases

ABSTRACT The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase – (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5ʹ UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5ʹ UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

Acquisition of a genomic resistance island (AbGRI5) from global clone 2 through homologous recombination in a clinical Acinetobacter baumannii isolate.

To reconstruct the evolutionary history of the clinical Acinetobacter baumannii XH1056, which lacks the Oxford scheme allele gdhB. Susceptibility testing was performed using broth microdilution and agar dilution. The whole-genome sequence of XH1056 was determined using the Illumina and Oxford Nanopore platforms. MLST was performed using the Pasteur scheme and the Oxford scheme. Antibiotic resistance genes were identified using ABRicate. XH1056 was resistant to all antibiotics tested, apart from colistin, tigecycline and eravacycline. MLST using the Pasteur scheme assigned XH1056 to ST256. However, XH1056 could not be typed with the Oxford MLST scheme as gdhB is not present. Comparative analyses revealed that XH1056 contains a 52 933 bp region acquired from a global clone 2 (GC2) isolate, but is otherwise closely related to the ST23 A. baumannii XH858. The acquired region in XH1056 also contains a 34 932 bp resistance island that resembles AbGRI3 and contains the armA, msrE-mphE, sul1, blaPER-1, aadA1, cmlA1, aadA2, blaCARB-2 and ere(B) resistance genes. Comparison of the XH1056 chromosome to that of GC2 isolate XH859 revealed that the island in XH1056 is in the same chromosomal region as that in XH859. As this island is not in the standard AbGRI3 position, it was named AbGRI5. XH1056 is a hybrid isolate generated by the acquisition of a chromosomal segment from a GC2 isolate that contains a resistance island in a new location-AbGRI5. As well as generating ST256, it appears likely that a single recombination event is also responsible for the acquisition of AbGRI5 and its associated antibiotic resistance genes.

Screening brinjal (Solanum melongena) accessions conserved in the National Genebank collected from states adjoining Bangladesh for adventitious presence of EE1 event

Systematic management of Plant Genetic Resources (PGRs) is the key to sustainable agriculture for food and nutritional security and in mitigating climate change. The National Genebank (NGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, acts as a repository of PGRs for future use. This study aimed at screening for the adventitious presence of transgenes in brinjal (Solanum melongena L.) accessions conserved in the NGB. The study targeted the collections made during 2007-2016 from areas adjacent to Bangladesh (Assam, Meghalaya, Mizoram, Tripura and West Bengal), where field trials of Bt brinjal event EE1 were conducted during 2005-2012 and commercial cultivation of this event was permitted in 2013.There could be an apprehension of both unintentional introgression and transboundary movement through borders. Adventitious presence of transgenes was checked in a set of 96 accessions of brinjal employing Polymerase Chain Reaction (PCR) and real-time PCR assays. As event EE1 carries cry1Ac gene for insect resistance with Cauliflower Mosaic Virus (CaMV) promoter (P-35S) and marker genes (nptII and aadA), so these genetic elements were targeted for qualitative GM testing. Based on the test results, transgenes were not detected in brinjal accessions conserved in NGB. Our study showed that brinjal and wild species collected from adjoining areas of Bangladesh, post field trials and release, do not contain the event EE1. The study presents an efficient and reliable method to ensure conservation of GM-free germplasm in the NGB.

Co-occurrence of three different plasmids in an extensively drug-resistant hypervirulent Klebsiella pneumoniae isolate causing urinary tract infection

ObjectivesA single carbapenem-resistant, hypervirulent Klebsiella pneumoniae strain has attracted major public concern. The aim of the present study was to better understand the antimicrobial resistance and genetic characteristics of Klebsiella pneumoniae strain XJ-K1. MethodsKlebsiella pneumoniae strain XJ-K1 was isolated from a urine specimen of a 69-year-old male patient in a teaching hospital in Shanghai, China, in January 2018. Antimicrobial susceptibility testing, string test, whole-genome sequencing, bioinformatics analysis and phylogenetic analysis were performed in this study. ResultsKlebsiella pneumoniae XJ-K1 was an extensively drug-resistant (XDR) hypervirulent strain that showed high-level resistance to antibacterial agents. Three novel plasmids were discovered in strain XJ-K1, including a 207,409-bp IncHI1B-type rmpA2-bearing pLVPK-like virulence plasmid, a 130,628-bp Col156/IncFIB/IncFII-type aadA2-, sul1-, mph(A)- and dfrA12-bearing MDR plasmid, and a 99,408-bp IncFII/IncR-type blaKPC-2-, blaTEM-1-, blaCTX-M-65-, blaSHV-12-, rmtB- and fosA3-bearing MDR plasmid. Sequence analysis of the chromosome revealed that the aadA2, fosA and sul1 genes were harboured by XJ-K1. Multilocus sequence typing (MSLT) showed that XJ-K1 was ST11. ConclusionsA large number of resistance genes and a pLVPK-like virulence plasmid carried by Klebsiella pneumoniae strain XJ-K1 might be the main reasons leading to the XDR and hypervirulent phenotype. To the best of our knowledge, this is the first report in China on the co-occurrence of a pLVPK-like virulence plasmid and two MDR plasmids in a single ST11 XDR and hypervirulent Klebsiella pneumoniae isolated from patient urine, which is a serious concern for its further spread.

Prevalence and Characterisation of Class 1 and 2 Integrons in Multi-drug Resistant Staphylococcus Aureus Isolates from Pig Farms in Chongqing, China.

IntroductionIntegrons are mobile DNA elements that allow for acquisition and dissemination of antibiotic-resistance genes among pig farm-derived bacteria. Limited information is available on integrons of Staphylococcus aureus from pig farms. The aim of this study was to characterise and investigate the prevalence of class 1 and 2 integrons in multi-drug resistant (MDR) S. aureus isolates from pig farms.Material and MethodsA total of 724 swabs were collected from 12 pig farms in Chongqing, China, and examined by conventional microbial and molecular methods.ResultsIn total, 68 isolates were S. aureus, 57 of which were methicillin resistant (MRSA). All 68 isolates were MDR strains and carried integrons, of which 88.2% (60/68) harboured both class 1 and 2. In addition, 85.3% (58/68) of the class 2 integron-positive isolates carried the β-lactam resistance gene (blaTEM-1), and 66.7% (40/60) of the class 1 integron–positive isolates carried the aadA1c, aadA1 or dfrA1 gene for respective streptomycin and spectinomycin or trimethoprim resistance.ConclusionsClass 1 and 2 integrons are common among the pig farm-derived S. aureus isolates. On account of their significance for public health, the prevalence of the integrons and their associated resistance genes in pig farm-derived S. aureus isolates should be paid special attention.

Characteristics and dynamics of Salmonella diversity and prevalence of biomarker genes in Port Blair Bays, South Andaman, India

Salmonella is a major cause of morbidity and mortality in humans worldwide, and the infection with multidrug-resistant strains can cause severe diseases. Many coastal cities around the world discharge their wastewaters into the marine environment. These wastewaters contain a variety of pathogenic microorganisms that may have a role in the contamination of this ecosystem and have potential risks for public health. Using an environmental approach, the present study investigated the presence of Salmonella in sediment and water samples collected from Port Blair Bays. In this environmental approach, the provided information about the diversity of the Salmonella serovars, antibiotic resistance and the prevalence of virulence factors in Salmonella, especially from the coastal waters of Port Blair Bays. The occurrence of Salmonellae was significantly higher in water column samples (2.9%) than in those taken from the marine sediments (0.7%). Of the 133 positive Salmonella strains, 22 different serovars were identified. Salmonella enterica serovar Senftenberg was the predominant serovar, being represented by 54 isolates (42.5%), followed by serovar Typhimurium (19 isolates [15%]) and serovar Agona (12 isolates [9.4%]). The presence of virulence genes (filC, sitC, hilA, invA, sipC, hilD, hilC, invF, invE, invH, sipF, aadA, pare, gyrA, spaP and parC) and susceptibility studies with 10 selected antibiotics were also performed. The results of this study revealed that all Salmonella isolates were positive for targeted virulence genes and were resistant to at least one antibiotic. Antibiotic susceptibility studies revealed the presence of multidrug resistant Salmonella strains in coastal water, which usually from land base sources end up in the marine environment and may pose a significant risk on public health.

WGS-Based Prediction and Analysis of Antimicrobial Resistance in Campylobacter jejuni Isolates From Israel.

Rapid developments in the field of whole genome sequencing (WGS) make in silico antimicrobial resistance (AMR) a target within reach. Campylobacter jejuni is a leading cause of foodborne infections in Israel with increasing rates of resistance. We applied WGS analysis to study the prevalence and genetic basis of AMR in 263 C. jejuni human and veterinary representative isolates retrieved from a national collection during 2003–2012. We evaluated the prediction of phenotypic AMR from genomic data. Genomes were screened by the NCBI AMRFinderPlus and the BioNumerics tools for acquired AMR genes and point mutations. The results were compared to phenotypic resistance determined by broth microdilution. The most prevalent resistant determinants were the multi-drug efflux transporter gene cmeABC (100%), the tetracycline resistance tet(O) gene (82.1%), the quinolone resistance gyrA T861 point mutation (75.7%), and the aadE streptomycin resistance gene. A variety of 12 known β lactam resistance genes (blaOXA variants) were detected in 241 (92%) isolates, the most prevalent being blaOXA−193, blaOXA−461, and blaOXA−580 (56, 16, and 7%, respectively). Other aminoglycoside resistance genes and the macrolide resistance point mutation were rare (<1%). The overall correlation rate between WGS-based genotypic prediction and phenotypic resistance was 98.8%, sensitivity, specificity, positive, and negative predictive values being 98.0, 99.3, 99.1, and 98.5%, respectively. wgMLST-based phylogeny indicated a high level of clonality and clustering among the studied isolates. Closely related isolates that were part of a genetic cluster (single linkage distance ≤ 15 alleles) based on wgMLST phylogeny mostly shared a homogenous AMR determinant profile. This was observed in 18 of 20 (90.0%) clusters within clonal complex-21, suggesting clonal expansion of resistant isolates. Strong association to lineage was noted for the aadE gene and the various blaOXA genes. High resistance rates to tetracycline and quinolones and a low resistance rate to macrolides were detected among the Israeli C. jejuni isolates. While a high genotypic-phenotypic correlation was found, some resistance phenotypes could not be predicted by the presence of AMR determinants, and particularly not the level of resistance. WGS-based prediction of antimicrobial resistance in C. jejuni requires further optimization in order to integrate this approach in the routine workflow of public health laboratories for foodborne surveillance.

Independent translation of ORFs in dicistronic operons, synthetic building blocks for polycistronic chloroplast gene expression.

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5'-untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High-level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.

Alpha-tubulin promoter from Chlorella vulgaris allows genetic transformation of green coccoid microalga

Microalgae have become a feasible platform for high-value recombinant protein production. Although diverse sets of genetic tools for microalgae transformation have been developed, some critical elements, such as endogenous promoters, need to be improved for increasing transgene expression levels after genetic transformation. This work aims to evaluate a sequence from the 5′ upstream region of the alpha-tubulin gene from Chlorella vulgarisas a promoter for the expression of an antibiotic resistant gene in Chlorella sorokiniana. Using in silicoanalysis it was possible to identify a proximal promoter corresponding to 281 nucleotides upstream of the ATG start codon, which possessed 9 potential cis-regulatory element, including TATA Box and CAAT Box. The proximal promoter sequencewas used to drive the expression of a streptomycin-resistance gene (aada), previous optimization of its codon usage. The codon optimization of the aadasequence allowed it to obtain a GC content of 69.8% (compared to 53% of the original sequence), which increases its similarity with Chlorellasp. genomes (67.2%). Both genetic elements were cloned into the pUC57-Kan vector and transformed into C. sorokiniana cells by electroporation. The microalgae transgenic colonies were identified through culture in selective medium and PCR. Our results proved the capacity of the Chlorella vulgaris alpha-tubulin promoter to express a foreign antibiotic-resistance gene in C. sorokinianacells. Research of endogenous promoting sequences is essential in order to accomplish an efficient heterologous gene expression, especially in microalgae used for industrial production like those from Chlorella genus.

Prevalence of multidrug resistance genes in Escherichia coli isolates from patients attending four hospitals in Minna, Nigeria

Objectives: This study determined the prevalence, antibiotic susceptibility and resistance genes among multidrug resistant Escherichia coli isolates from diarrheic patients in four hospitals within Minna, Nigeria. Methods: Stool samples from one hundred and seven (107) diarrheic patients were collected and analysed to check for E. coli using spread plate techniques. The resistance pattern of the E. coli isolates to ten (10) antibiotics using disc diffusion techniques was determined. Five isolates with multidrug resistant index ≥ 0.5 were screened for antibacterial resistant genes (Tn3bla, GyrA, ParC, aadA2 and Sul1) using polymerase chain reaction. Results: Seventy (70) samples representing 65.4% were E. coli positive while fifty-five (55) were resistant to at least one antibiotic. The highest level of resistance was against ampicillin (38.57%) while the least was against cefalexin (4.29%). Multidrug resistant isolates were 58.7% (41/70), while 28.6% (20/70) were resistant to three or more antibiotics. The result of the molecular characterization identified the five multidrug resistant isolates to be E. coli strain RAD34, E. coli strain CUSMBN2, E. coli strain CAU3471, and E. coli strain BYPFP. Tn3bla/GyrA/ParC resistant genes were detected in all the five isolates. E. coli isolate RAD34, E. coli strain CUSMBN2, E. coli strain CAU3471 and E. coli strain BYPFP possessed Sul1 gene. However, E. coli strain BYPFP, E. coli strain CAU3471 and E. coli strain CUSMBN2 had aadA2 gene. Conclusion: The result of this study established the active circulation of Tn3bla, GyrA, ParC, aadA2 and Sul1 resistant genes among E. coli in the study area.

Identification of drug resistance genes and drug resistance analysis of Salmonella in the duck farm environment of Zhanjiang, China.

This study investigated and identified the distribution of drug resistance genes in feces, soil, and water of duck farms in Zhanjiang, China, and analyzed the drug resistance of Salmonella in the duck farm environment. PCR was used to assess the distribution of 25 resistance genes that are common in the duck farm environment. The isolation, biochemical identification, PCR identification of Salmonella, and the minimum inhibitory concentration (MIC) of 22 drugs were measured by micro-broth double dilution. In water, 25 drug resistance genes were detected, 24 in soil, and 23 in feces. Among them, the detection rate of the aadA1 gene in soil reached 100%, 13 drug resistance genes had a detection rate above 80%, and five species had a detection rate below 50%. In water, the detection rate of the floR and aadA1 genes was 100%, 12 drug resistance genes had a detection rate above 80%, and eight genes had a detection rate below 50%. In feces, nine drug resistance genes had a detection rate of 100%, nine genes had a detection rate above 80%, and one gene had a detection rate below 50%. In addition, 92 strains of Salmonella were isolated and identified, and their resistance rate to nine drugs was as high as 100%. All isolated Salmonella can tolerate at least nine drugs, 55.43% (51/92) of the strains can tolerate more than 16 drugs, and 4.35% (4/92) of the strains were resistant to up to 21 drugs. In conclusion, the present experiment suggested that drug resistance genes were ubiquitous in the duck farm environment in Zhanjiang and that these drug resistance genes may spread horizontally between feces, soil, and water. Moreover, drug resistance and multi-drug resistance were found for 92 isolated Salmonella strains from the duck farm environment. The government should consequently strengthen the regulation of antimicrobial drug use in duck farms.

Biofilm formation and transfer of a streptomycin resistance gene in enterococci from fermented pork

ObjectivesMultidrug-resistant (MDR) enterococci are found extensively in food samples. This study characterized the phenotypic virulence factors and the ability of horizontal gene transfer of a streptomycin resistance gene among enterococci isolated from fermented pork. MethodsThirty-six MDR enterococci were subjected to screening of gelatinase, biofilm formation at various temperatures (4 °C, 25 °C and 37 °C), clumping ability and conjugation. ResultsAll gelatinase-positive and clumping-positive strains were Enterococcus faecalis (41.7% and 38.9%, respectively). None of Enterococcus faecium and Enterococcus hirae demonstrated both phenotypes. Moderate and strong biofilm formations were found mostly at optimal temperatures in all the three species tested. However, moderate and weak biofilm formations could be found in 52.8% at 4 °C. No association was observed between biofilm formation and asa1, efaA, gelE and esp genes. Surprisingly, our data revealed evidence of the streptomycin resistance gene (aadE) being transferred among meat E. faecalis isolates as characterized by the pheromone-clumping response. ConclusionsHere we report the co-existence of some virulence factors and MDR enterococci from fermented pork. Our data demonstrated for the first time that the aadE gene could be transferred via conjugation among enterococci isolated from meat, contributing to streptomycin resistance. This study highlights the importance of horizontal gene transfer within the food chain reservoir and that transfer to humans might be possible, causing harm or untreatable diseases.

Deadly Puppy Infection Caused by an MDR Escherichia coli O39 bla CTX-M-15, bla CMY-2, bla DHA-1, and aac(6)-Ib-cr - Positive in a Breeding Kennel in Central Italy.

Antimicrobial consumption in veterinary medicine has led to the spread of multi drug-resistance in clinically important bacteria, with the companion animals and their environment involved as emerging reservoirs. While CTX-M-15 and CMY-2 acquired β-lactamases have been widely detected in the bacterial population of companion and breeding animals in European area, DHA-1 enzymes have been rarely reported in veterinary medicine. The aim of the study was to characterize the Escherichia coli associated with mortality of a litter of Bulldog puppies in a breeding kennel located in Pesaro area, Central Italy. The E. coli strains O39 serotype were resistant to 3rd/4th generation cephalosporins, chloramphenicol, aminoglycosides, trimethoprim-sulfamethoxazole, and ciprofloxacin, retaining susceptibility to carbapenems, colistin, fosfomycin, and levofloxacin (by Microscan Autoscan4, EUCAST clinical breakpoints). Pulse field gel electrophoreses (PFGE-XbaI) on five E. coli strains revealed the presence of a single profile. Whole genome sequencing (WGS) analysis revealed a complex resistome, harboring blaTEM–1b, blaCTX–M–15, blaOXA–1, aph(6)-Ib, aac(6′)Ib-cr, aac(3)-Ila, aph(6)-Id, aadA1, qnrB1, sul2, catA1, catB3, tetA, and dfrA14 genes located on a 302597 bp IncHI2/HI2A plasmid. Moreover, blaDHA–1, qnrB4, mph(A), sul1, and dfrA17 determinants were carried on an 83,429 bp IncFII plasmid. A blaCMY–2 determinant was carried on a 90,249 bp IncI1 plasmid. Two IncX1 and IncX4 plasmids without antimicrobial resistance genes were also detected. The presence of lpfA, iss, astA, and gad virulence factors was highlighted. This is the first report in Italy on an invasive infection in eight 2-weeks old dogs caused by the same MDR E. coli O39 blaCTX–M–15, blaCMY–2, blaDHA–1, and aac(6′)-Ib-cr positive strain. The above MDR E. coli clone caused the death of the entire litter, despite amoxicillin-clavulanate and enrofloxacin administration. The tank for storage of the water used to prepare the milk-based meal for the litter was the suspected reservoir.

Identification of risk factors associated with antimicrobial resistance in equine fecal Escherichia coli isolates

Antimicrobial resistance is a growing global problem that will need a multinational collaborative effort to overcome this serious challenge. The aim of the study is to investigate the potential risk factors associated with the prevalence and distribution of antimicrobial-resistance genes (ARGs) of Escherichia coli isolates obtained from equine fecal samples. One hundred and eighteen horses from different geographical locations and management systems were enrolled in the study and a questionnaire containing information about each individual horse was designed and filled. The enrolled horses belonged to 2 main categories (Hospitalized horses (n = 31), and Non hospitalized horses (n = 87)). In total, 103 E. coli isolates were collected from the 118 horse fecal samples. Genes that are responsible for resistance to β-lactams, tetracyclines, aminoglycosides, and trimethoprim were detected using uniplex and multiplex polymerase chain reaction (PCR). The prevalence of antimicrobial resistance was significantly higher in hospitalized horses compared to non-hospitalized ones (p ≤ .05), particularly against (trimethoprim, doxycycline, oxytetracycline, and amoxicillin-clavulanic acid). The most prevalent antimicrobial-resistant genes were aminoglycoside resistant genes (strA, strB, and aadA) in percentages; 89%, 85%, and 84%, respectively. Statistical analysis revealed a significant association between risk factors and occurrence of ARGs (p ≤ .05). Significant risk factors include the last treatment and history of antimicrobial administration, breed of horses, use of horses, type of diet fed for horses, practice management and history of last illness. Tetracycline-resistance gene (tetA) was 23 times more likely to be found in the Arabian and local breeds of horses compared to English and warmblood breed. TetA is also 8 times more likely to be found in horses that were fed a natural diet compared to other horses that were fed manufactured/ processed feed. In conclusion, E. coli bacterium can harbor high resistance to different classes of antimicrobials which increases the risk of potential uncontrolled transmission of the multi-drug resistant E. coli bacterium to veterinarians and horse handlers, as well as to the equine population itself.

Molecular characterization of virulence and drug resistance genes-producing Escherichia coli isolated from chicken meat: Metal oxide nanoparticles as novel antibacterial agents

Escherichia coli is a major global foodborne pathogen, infecting a wide range of animals and contaminating their meat products. E. coli, can lead to high morbidity and mortality with a huge economic loss especially if foodborne diseases are associated with multidrug resistant (MDR)- and multivirulent-producing pathogens. Due to the increased resistance to common antimicrobials used to treat livestock animals and human infections, the discovery of new and innovative nanomaterials are in high demand. Recently, metal oxides can be considered as effective inorganic agents with antimicrobial features. Hence, this study might be the first to evaluate the efficiency of metal oxide nanoparticles (MO-NPs) as novel antibacterial agents against MDR/multivirulent E. coli pathogens isolated from chicken meat. The occurrence of pathogenic E. coli was determined in fresh warm chicken meat parts (breast, thigh, liver and gizzard). Ninety-one of 132 (69%) chicken meat parts were Escherichia -positive with E. coli as the only species isolated. Out of identified 240 E. coli strains, 72.5% (174/240) were classified as MDR E. coli strains. Fifty-five profile patterns were obtained. From each pattern, one strain was randomly selected for further analysis of virulence and resistance genes. Extracted DNA was assessed for the presence of antibiotic resistance genes (blaIMP-7, blaIMP-25, blaTEM, blaSHV, blaOXA-2, tetA, aadA, and aac(3)-IV) and virulence genes (stx1, stx2, hlyA, eaeA, aggR, eltB, estIb, papA, afa and hlyD). Clustering analyses revealed that 10 E. coli harboring the highest number of virulence and resistance genes were shifted together into one cluster designated as cluster X. The average activities of zinc peroxide nanoparticles (ZnO2-NPs) were higher than that of zinc oxide nanoparticles (ZnO-NPs) and titanium dioxide nanoparticles (TiO2-NPs) by 20% and 29%, respectively. The anti-inflammatory activity of ZnO2-NPs in comparison with aspirin was assessed using membrane stabilization, albumin denaturation, and proteinase inhibition methods. Significant anti-inflammatory activity of ZnO2-NPs was achieved at concentration levels of 500–1000 μg/ml. It seems that MO-NPs are effective alternative agents, since they exhibited a competitive antibacterial capability against MDR/multivirulent-producing E. coli pathogens isolated from chicken meat. Hence, ZnO2-NPs are a promising nanoparticles-based material for controlling foodborne pathogens, thereby valued for food safety applications.

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.

Genomic and Clinical Characterization of IMP-1-Producing Multidrug-Resistant Acinetobacter bereziniae Isolates from Bloodstream Infections in a Brazilian Tertiary Hospital.

Acinetobacter baumannii is the main species of the Acinetobacter genus; however, non-baumannii Acinetobacter (NBA) species causing infections have been described for the past years, as well as antimicrobial resistance. In this study, we describe the occurrence of two multidrug-resistant (MDR) IMP-1-producing Acinetobacter bereziniae isolates recovered from bloodstream infections in different patients but in the same intensive care unit among 134 carbapenem-resistant Acinetobacter screened. Antimicrobial susceptibility testing revealed resistance to carbapenems, extended spectrum, and antipseudomonad cephalosporins, amikacin, and trimethoprim-sulfamethoxazole. Both A. bereziniae isolates shared the same ApaI-pulsed-field gel electrophoresis (PFGE) pattern. Whole-genome sequencing of both isolates revealed that blaIMP-1 was embedded into an In86 Class I integron carrying also sul1, aac(6')-31, and aadA genes. A new sequence type (ST1309 Pasteur) was deposited. The virulence genes lpxC and ompA, seen in A. baumannii, were detected in the A. bereziniae strains. Recognition of A. bereziniae causing invasive MDR infection underscores the role of NBA species as human pathogens especially in at-risk patients.