Toxicological evaluation of Alpinia oxyphylla-derived molecule (PD-00105): In vitro genotoxicity studies and 90-day oral toxicity study in rats.

PD-00105 corresponds to a compound initially identified in the fruit of Alpinia oxyphylla Miq., obtained by chemical synthesis and proposed to be use in dietary supplements for its potential neuroprotective properties. The aim of this study was to perform a toxicological evaluation of PD-00105 in accordance with the testing strategy recommended by food regulatory authorities. All studies were conducted in accordance with Good Laboratory Practice (GLP), and followed the Organization for Economic Co-operation and Development (OECD) test guidelines for chemicals. Studies included a bacterial reverse mutation test, one in vitro micronucleus test in mammalian cells, and a repeated dose 90-day oral toxicity study. No sign of toxicity was observed in the two genotoxicity tests. The test item induced a significant liver and kidney toxicity at high doses (50 and 100 mg/kg BW/day), highlighted by significant increases in liver and kidney absolute and relative weights, associated with histopathological findings and concomitant changes in hematology and clinical chemistry. Increases in alanine aminotransferase, alkaline phosphatase, total protein, albumin, globulin, cholesterol, LDL, and HDL have been measured in these two groups. However, findings observed in the low-dose group (10 mg/kg BW/day) were considered as minimal and non-adverse, and were limited to an increase in liver weight in males and in kidneys weight in females, without concomitant changes in blood chemistry. The No Observed Adverse Effect Level (NOAEL) of PD-00105 was established as 10 mg/kg BW/day under the conditions of this study. This study substantiates the use of PD-00105 in dietary supplements at doses of 10 mg/day, taking into account a safety margin factor for dose conversion to humans.

Plasmid-Mediated Colistin Resistance (mcr-1) in Escherichia coli from Non-Imported Fresh Vegetables for Human Consumption in Portugal.

In this study, we report the presence of the plasmid-mediated colistin resistance (PMCR)-encoding gene mcr-1 in an Escherichia coli isolate, INSali25, recovered from lettuce produced and marketed in Portugal. Colistin MIC from the vegetable E. coli isolate-determined by microdilution broth method according to EUCAST guidelines-revealed a non-wild-type phenotype of colistin (MIC 16 mg/L). To understand the genetic background of E. coli INSali25, we performed whole genome sequencing. Plasmid sequencing was also performed after plasmid DNA extraction from the transconjugant TcINSali25 (mcr-1). Directed bioinformatics analysis identified the mcr-1 gene in a 39,998 bp length contig, with an upstream region including the antibiotic resistance gene blaTEM-1 in a partial transposon Tn2, truncated by the insertion sequence IS26 and showing >99% identity with previously described mcr-1-harboring IncHI2 plasmids. Further in silico analysis showed the presence of additional genes conferring resistance to ß-lactams (blaTEM-1), aminoglycosides (aadA1, aph(4)-Ia, aph(6)-Id, aac(3)-Iv), macrolides (mdf(A)-type), phenicol (floR-type), tetracycline (tetA), and sulphonamides (sul2). INSali25 isolate belonged to the ST1716 lineage and showed the fimH54 and fumC27 alleles. Lettuce is a vegetable that is commonly consumed fresh and not subjected to any cooking process, which may amplify human food safety risks. Moreover, the occurrence of plasmid-mediated colistin resistance in a sample that was not imported and was acquired in a large retail store reinforces the widespread distribution of mcr-1.

Genetic Background and Expression of the New qepA4 Gene Variant Recovered in Clinical TEM-1- and CMY-2-Producing Escherichia coli.

A new QepA4 variant was detected in an O86:H28 ST156-fimH38 Escherichia coli, showing a multidrug-resistance phenotype. PAßN inhibition of qepA4-harboring transconjugant resulted in increase of nalidixic acid accumulation. The qepA4 and catA1 genes were clustered in a 26.0-kp contig matching an IncF-type plasmid, and containing a Tn21-type transposon with multiple mobile genetic elements. This QepA variant is worrisome because these determinants might facilitate the selection of higher-level resistance mutants, playing a role in the development of resistance, and/or confer higher-level resistance to fluoroquinolones in association with chromosomal mutations.

Quantitative proteome analysis of an antibiotic resistant Escherichia coli exposed to tetracycline reveals multiple affected metabolic and peptidoglycan processes.

Tetracyclines are among the most commonly used antibiotics administrated to farm animals for disease treatment and prevention, contributing to the worldwide increase in antibiotic resistance in animal and human pathogens. Although tetracycline mechanisms of resistance are well known, the role of metabolism in bacterial reaction to antibiotic stress is still an important assignment and could contribute to the understanding of tetracycline related stress response. In this study, spectral counts-based label free quantitative proteomics has been applied to study the response to tetracycline of the environmental-borne Escherichia coli EcAmb278 isolate soluble proteome. A total of 1484 proteins were identified by high resolution mass spectrometry at a false discovery rate threshold of 1%, of which 108 were uniquely identified under absence of tetracycline whereas 126 were uniquely identified in presence of tetracycline. These proteins revealed interesting difference in e.g. proteins involved in peptidoglycan-based cell wall proteins and energy metabolism. Upon treatment, 12 proteins were differentially regulated showing more than 2-fold change and p<0.05 (p value corrected for multiple testing). This integrated study using high resolution mass spectrometry based label-free quantitative proteomics to study tetracycline antibiotic response in the soluble proteome of resistant E. coli provides novel insight into tetracycline related stress. SIGNIFICANCE: The lack of new antibiotics to fight infections caused by multidrug resistant microorganisms has motivated the use of old antibiotics, and the search for new drug targets. The evolution of antibiotic resistance is complex, but it is known that agroecosystems play an important part in the selection of antibiotic resistance bacteria. Tetracyclines are still used as phytopharmaceutical agents in crops, selecting resistant bacteria and changing the ecology of farm soil. Little is known about the metabolic response of genetically resistant populations to antibiotic exposure. Indeed, to date there are no quantitative tetracycline resistance studies performed with the latest generation of high resolution mass spectrometers allowing high mass accuracy in both MS and MS/MS scans. Here, we report the proteome profiling of a soil-borne Escherichia coli upon tetracycline stress, so that this new perspective could provide a broaden understanding of the metabolic responses of E. coli to a widely used antibiotic.

Draft Genomic Analysis of an Avian Multidrug Resistant Morganella morganii Isolate Carrying qnrD1.

Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3 Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with colisepticemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages, and pathogenicity factors were also detected, improving our understanding toward this human and animal opportunistic pathogen.

Architecture of Class 1, 2, and 3 Integrons from Gram Negative Bacteria Recovered among Fruits and Vegetables.

The spread of antibiotic resistant bacteria throughout the food chain constitutes a public health concern. To understand the contribution of fresh produce in shaping antibiotic resistance bacteria and integron prevalence in the food chain, 333 antibiotic resistance Gram negative isolates were collected from organic and conventionally produced fruits (pears, apples, and strawberries) and vegetables (lettuces, tomatoes, and carrots). Although low levels of resistance have been detected, the bacterial genera identified in the assessed fresh produce are often described not only as environmental, but mostly as commensals and opportunistic pathogens. The genomic characterization of integron-harboring isolates revealed a high number of mobile genetic elements and clinically relevant antibiotic resistance genes, of which we highlight the presence of as mcr-1, qnrA1, bla GES-11, mphA, and oqxAB. The study of class 1 (n = 8), class 2 (n = 3) and class 3 (n = 1) integrons, harbored by species such as Morganella morganii, Escherichia coli, Klebsiella pneumoniae, led to the identification of different integron promoters (PcW, PcH1, PcS, and PcWTNG-10) and cassette arrays (containing drfA, aadA, cmlA, estX, sat, and bla GES). In fact, the diverse integron backbones were associated with transposable elements (e.g., Tn402, Tn7, ISCR1, Tn2 (*), IS26, IS1326, and IS3) that conferred greater mobility. This is also the first appearance of In1258, In1259, and In3-13, which should be monitored to prevent their establishment as successfully dispersed mobile resistance integrons. These results underscore the growing concern about the dissemination of acquired resistance genes by mobile elements in the food chain.

QnrS1- and Aac(6')-Ib-cr-Producing Escherichia coli among Isolates from Animals of Different Sources: Susceptibility and Genomic Characterization.

Salmonella enterica and Escherichia coli can inhabit humans and animals from multiple origins. These bacteria are often associated with gastroenteritis in animals, being a frequent cause of resistant zoonotic infections. In fact, bacteria from animals can be transmitted to humans through the food chain and direct contact. In this study, we aimed to assess the antibiotic susceptibility of a collection of S. enterica and E. coli recovered from animals of different sources, performing a genomic comparison of the plasmid-mediated quinolone resistance (PMQR)-producing isolates detected. Antibiotic susceptibility testing revealed a high number of non-wild-type isolates for fluoroquinolones among S. enterica recovered from poultry isolates. In turn, the frequency of non-wild-type E. coli to nalidixic acid and ciprofloxacin was higher in food-producing animals than in companion or zoo animals. Globally, we detected two qnrS1 and two aac(6')-Ib-cr in E. coli isolates recovered from animals of different origins. The genomic characterization of QnrS1-producing E. coli showed high genomic similarity (O86:H12 and ST2297), although they have been recovered from a healthy turtle dove from a Zoo Park, and from a dog showing symptoms of infection. The qnrS1 gene was encoded in a IncN plasmid, also carrying bla TEM-1-containing Tn3. Isolates harboring aac(6')-Ib-cr were detected in two captive bottlenose dolphins, within a time span of two years. The additional antibiotic resistance genes of the two aac(6')-Ib-cr-positive isolates (bla OXA-1, bla TEM-1,bla CTX-M-15, catB3, aac(3)-IIa, and tetA) were enclosed in IncFIA plasmids that differed in a single transposase and 60 single nucleotide variants. The isolates could be assigned to the same genetic sublineage-ST131 fimH30-Rx (O25:H4), confirming clonal spread. PMQR-producing isolates were associated with symptomatic and asymptomatic hosts, which highlight the aptitude of E. coli to act as silent vehicles, allowing the accumulation of antibiotic resistance genes, mobile genetic elements and other relevant pathogenicity determinants. Continuous monitoring of health and sick animals toward the presence of PMQR should be strongly encouraged in order to restrain the clonal spread of these antibiotic resistant strains.

New Class 2 Integron In2-4 Among IncI1-Positive Escherichia coli Isolates Carrying ESBL and PMAß Genes from Food Animals in Portugal.

The impact of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (PMAßs) of animal origin constitutes a public health concern. In this study, 179 Escherichia coli from food animals and products were analyzed, among which, 15 cephalosporin-resistant isolates harboring ESBL (CTX-M-1 [n = 8], CTX-M-14 [n = 1], SHV-12 [n = 2]) or PMAß [CMY-2, n = 5]) were identified in poultry and swine, from different farms of distinct regions of Portugal. The multiple sequence-type IncI1-driven spread of ESBLs and PMAßs, flanked by widely disseminated mobile elements, was guaranteed by ST26/IncI1-harboring blaSHV-12, ST12/IncI1-harboring blaCMY-2, ST3 and ST38/IncI1-harboring blaCTX-M-1, and ST1/IncI1-harboring blaCTX-M-14. An IS10-disrupted In2-4, presenting a new attI2 recombination site, was also detected in a SHV-12/CTX-M-1-harboring isolate. This study highlights the fact that animals may act as persistent sources of ESBL- and PMAß-harboring plasmids genes that might be transferred to humans through direct contact or via the food chain.

Influence of agricultural practice on mobile bla genes: IncI1-bearing CTX-M, SHV, CMY and TEM in Escherichia coli from intensive farming soils.

Many calls have been made to address antibiotic resistance in an environmental perspective. With this study, we showed the widespread presence of high-level antibiotic resistant isolates on a collection of non-susceptible Gram-negative bacteria (n = 232) recovered from soils. Bacteria were selected using amoxicillin, cefotaxime and imipenem, from sites representing different agricultural practices (extensive, intensive and organic). Striking levels of non-susceptibility were noticed in intensive soils for norfloxacin (74%), streptomycin (50.7%) and tetracycline (46.6%); indeed, the exposure to intensive agricultural practices constituted a risk factor for non-susceptibility to many antibiotics, multidrug resistance and production of extended-spectrum ß-lactamases (ESBL). Analyses of non-susceptibility highlighted that environmental and clinical bacteria from the same species might not share the same intrinsic resistance patterns, raising concerns for therapy choices in environment-borne infections. The multiple sequence-type IncI1-driven spread of penicillinases (blaTEM-1, blaTEM-135), ESBL (blaSHV-12 and blaCTX-M-1) and plasmid-mediated AmpC ß-lactamases (blaCMY-2), produced by isolates that share their molecular features with isolates from humans and animals, suggests contamination of agricultural soils. This is also the first appearance of IncI1/ST28-harbouring blaCTX-M-1, which should be monitored to prevent their establishment as successfully dispersed plasmids. This research may help disclose paths of contamination by mobile antibiotic resistance determinants and the risks for their dissemination.

Draft Genome Sequence of a Pathogenic O86:H25 Sequence Type 57 Escherichia coli Strain Isolated from Poultry and Carrying 12 Acquired Antibiotic Resistance Genes.

Escherichia coli is a commensal bacterium that is frequently associated with multidrug-resistant zoonotic and foodborne infections. Here, we report the 5.6-Mbp draft genome sequence of an E. coli recovered from poultry, which encodes multiple acquired antibiotic resistance determinants, virulence factors, pathogenicity determinants, and mobile genetic elements.

Assessing the antibiotic susceptibility of freshwater Cyanobacteria spp.

Freshwater is a vehicle for the emergence and dissemination of antibiotic resistance. Cyanobacteria are ubiquitous in freshwater, where they are exposed to antibiotics and resistant organisms, but their role on water resistome was never evaluated. Data concerning the effects of antibiotics on cyanobacteria, obtained by distinct methodologies, is often contradictory. This emphasizes the importance of developing procedures to understand the trends of antibiotic susceptibility in cyanobacteria. In this study we aimed to evaluate the susceptibility of four cyanobacterial isolates from different genera (Microcystis aeruginosa, Aphanizomenon gracile, Chrisosporum bergii, Planktothix agradhii), and among them nine isolates from the same specie (M. aeruginosa) to distinct antibiotics (amoxicillin, ceftazidime, ceftriaxone, kanamycine, gentamicine, tetracycline, trimethoprim, nalidixic acid, norfloxacin). We used a method adapted from the bacteria standard broth microdilution. Cyanobacteria were exposed to serial dilution of each antibiotic (0.0015-1.6 mg/L) in Z8 medium (20 ± 1°C; 14/10 h L/D cycle; light intensity 16 ± 4 µEm(-2)s(-1)). Cell growth was followed overtime (OD450nm /microscopic examination) and the minimum inhibitory concentrations (MICs) were calculated for each antibiotic/isolate. We found that ß-lactams exhibited the lower MICs, aminoglycosides, tetracycline and norfloxacine presented intermediate MICs; none of the isolates were susceptible to trimethoprim and nalidixic acid. The reduced susceptibility of all tested cyanobacteria to some antibiotics suggests that they might be naturally non-susceptible to these compounds, or that they might became non-susceptible due to antibiotic contamination pressure, or to the transfer of genes from resistant bacteria present in the environment.

Current perspectives on the dynamics of antibiotic resistance in different reservoirs.

Antibiotic resistance consists of a dynamic web. In this review, we describe the path by which different antibiotic residues and antibiotic resistance genes disseminate among relevant reservoirs (human, animal, and environmental settings), evaluating how these events contribute to the current scenario of antibiotic resistance. The relationship between the spread of resistance and the contribution of different genetic elements and events is revisited, exploring examples of the processes by which successful mobile resistance genes spread across different niches. The importance of classic and next generation molecular approaches, as well as action plans and policies which might aid in the fight against antibiotic resistance, are also reviewed.

Antimicrobial susceptibility and oxymino-ß-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sources.

The impact of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (PMAßs) of animal origin has been a public health concern. In this study, 562 Salmonella enterica and 598 Escherichia coli isolates recovered from different animal species and food products were tested for antimicrobial resistance. Detection of ESBL-, PMAß-, plasmid-mediated quinolone resistance (PMQR)-encoding genes and integrons was performed in isolates showing non-wild-type phenotypes. Susceptibility profiles of Salmonella spp. isolates differed according to serotype and origin of the isolates. The occurrence of cefotaxime non-wild-type isolates was higher in pets than in other groups. In nine Salmonella isolates, blaCTX-M (n = 4), blaSHV-12 (n = 1), blaTEM-1 (n = 2) and blaCMY-2 (n = 2) were identified. No PMQR-encoding genes were found. In 47 E. coli isolates, blaCTX-M (n = 15), blaSHV-12 (n = 2), blaCMY-2 (n = 6), blaTEM-type (n = 28) and PMQR-encoding genes qnrB (n = 2), qnrS (n = 1) and aac(6')-Ib-cr (n = 6) were detected. To the best of our knowledge, this study is the first to describe the presence of blaCMY-2 (n = 2) and blaSHV-12 (n = 1) genes among S. enterica from broilers in Portugal. This study highlights the fact that animals may act as important reservoirs of isolates carrying ESBL-, PMAß- and PMQR-encoding genes that might be transferred to humans through direct contact or via the food chain.

Diversity of extended-spectrum and plasmid-mediated AmpC ß-lactamases in Enterobacteriaceae isolates from Portuguese health care facilities.

A group of 124 Enterobacteriaceae isolates resistant to third generation cephalosporins, and collected in distinct health care facilities of different Portuguese regions was analysed. The great majority of the isolates were also resistant to fourth generation cephalosporins (83.9%), monobactam (96%), amoxicillin plus clavulanic acid (85.5%), and piperacillin plus tazobactam (66.9%). Overall, 84.7% (105/124) were multidrug resistant. Molecular methods enabled us to identify 86.3% (107/124) extended-spectrum ß-lactamases (ESBL) producers, revealing a diversity of class A ß-lactamases from different families, like TEM (TEM-1, TEM-10, TEM-24, and TEM-52), SHV (SHV-1, SHV-12, and SHV-28), CTX-M (CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15, and CTXM-32), and GES (GES-1). We have also detected class C enzymes like plasmid-mediated AmpC ß-lactamases (PMAßs, DHA-1, and CMY-2) and chromosomal AmpCs in Enterobacter and Citrobacter spp. The PMAß genetic context mapping suggests association with mobile elements, plasmid importation and the potential emergence of these ß-lactamases. The most prevalent ß-lactamase detected was CTX-M-15 (66.1%) and in 41.1% of the isolates it was associated with TEM-, OXA-type ß-lactamases and Aac(6)᾿Ib-cr, which might indicate that the respective genotype has settled in our country. Indeed, CTX-M-15 was distributed amongst distinct clinical settings of several health care facilities (93.5%) from various regions. We provide evidence of a concerning clinical situation that includes vast occurrence of ESBLs, the settling of CTX-M ß-lactamases, and the report of plasmidic and chromosomal AmpC in Portugal.

Vancomycin-resistant enterococci among haemodialysis patients in Portugal: prevalence and molecular characterization of resistance, virulence and clonality.

INTRODUCTION: Vancomycin-resistant enterococci (VRE) among haemodialysis patients has increased rapidly and, to date, there is no report of this incidence in Portugal. METHODS: A total of 121 faecal samples were collected from haemodialysis patients, and then tested for VRE. Antimicrobial resistance, virulence and multilocus sequence typing (MLST) were studied. RESULTS: VRE prevalence was 3.3%. Three VRE isolates, Enterococcus faecium, Enterococcus faecalis and Enterococcus raffinosus, were multi-resistant and vanA-positive. E. faecium and E. faecalis belonged to CC17 and CC2, respectively. CONCLUSION: Haemodialysis patients in Portugal are colonized with virulent, multi-resistant enterococci from high-risk clonal complexes, representing a public health concern.

Occurrence of extended-spectrum ß-lactamases among isolates of Salmonella enterica subsp. enterica from food-producing animals and food products, in Portugal.

A total of 1120 Salmonella spp. isolates, recovered from poultry, swine and food products of animal origin (bovine, swine and poultry) over the period of 2009-2011, were investigated in order to determine their serotype, susceptibility to a panel of eleven antimicrobials (A, ampicillin; Ct, cefotaxime; Cp, ciprofloxacin; Tm, trimethoprim; Su, sulfamethoxazole; C, chloramphenicol; S, streptomycin; G, gentamicin; T, tetracycline; NA, nalidixic acid; Fl, florfenicol), and the presence of resistance determinants of extended-spectrum cephalosporins. Overall, Salmonella Enteritidis was the most common serotype in all three animal species. In 618 isolates of poultry, 32.8% comprised S. Enteritidis, 18.3% Salmonella Havana and 16.5% Salmonella Mbandaka; in 101 isolates of pigs, 21.8% comprised Salmonella Rissen and Salmonella Typhimurium, 10.9% Salmonella Derby and Salmonella London. Salmonella I 4,[5],12:i:- was the most common serotype recovered from pork and beef food products comprising 32.6% and 30% of isolates respectively, followed by S. Rissen (26% and 24%) and S. Typhimurium (18.2% and 19%), respectively. In poultry products, S. Enteritidis was the most frequent serotype (62.7%), followed by S. Mbandaka (10.2%) and S. Derby (8.5%). Susceptibility profiles differed according to the origin of the isolates. Five multidrug resistant isolates (0.45%) were further characterized as extended-spectrum ß-lactamase (ESBL) producers. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of bla(CTX-M-1) (n = 2), bla(CTX-M-14) (n = 1), bla(CTX-M-15) (n = 1) and bla(CTX-M-32) (n = 1); bla(SHV-12) and bla(TEM-1) genes were also detected in two isolates of S. I 4,[5],12:i:-. Four isolates, two S. Havana and two S. I 4,[5],12:i:-, carried class 1 integrons and in three, two S. I 4,[5],12:i:- and one S. Havana, ISEcp1 was identified associated to bla(CTX-M-1), bla(CTX-M-32) and bla(CTX-M-14) genes. Additionally, in one S. I 4,[5],12:i:- isolate, orf477 was identified linked to bla(CTX-M-32). No plasmid mediated quinolone resistance-encoding genes were detected. Here, we report for the first time the presence of bla(CTX-M) genes in Salmonella enterica subsp enterica isolates recovered from poultry and food products of swine origin, in Portugal.

High prevalence of ESBL-producing Escherichia coli isolates among hemodialysis patients in Portugal: appearance of ST410 with the bla(CTX-M-14) gene.

Ten extended-spectrum ß-lactamase-producing Escherichia coli isolates were detected among 121 fecal samples (8.3%) recovered from hemodialysis patients in Portugal. The isolates harbored the bla(CTX-M-15), bla(CTX-M-14a), and/or bla(CTX-M-1) genes. A new sequence type, ST2229, was detected, and this study also reports, for the first time, ST410 CTX-M-14-producing isolates.

Emergence and risk factors of ß-lactamase-mediated resistance to oxyimino-ß-lactams in Enterobacteriaceae isolates.

We studied 193 Enterobacteriaceae isolates presenting diminished susceptibility to oxyimino-cephalosporins recovered in a Portuguese hospital (2004-2008). CTX-M-3 producers, firstly detected in Portugal, were associated with a Klebsiella pneumoniae microepidemic clone. Production of CTX-M-type enzymes (CTX-M-1/-3/-9/-14/-15/-32), age ≥65 years, and nosocomial infection were risk factors for higher nonsusceptibility to oxyimino-ß-lactams. CMY-2 and DHA-1 ß-lactamases were only identified in 1% of isolates.