Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf

The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (n = 227) and E. coli (n = 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The bla_TEM, tet(A) and/or tet(B), and aadA or strA‐strB genes were detected among most ampicillin‐, tetracycline‐ or streptomycin‐resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (n = 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Significance and Impact of the Study

This study shows antimicrobial resistance in commensal bacteria from the free‐range, Portuguese, Iberian wolf population. The results indicate that the Iberian wolf could contribute to the spread of resistant bacteria throughout the environment. Additionally, in case of infection, an increased risk of therapeutic failure due to the presence of multiresistant bacteria may represent a health problem for this endangered species. Future studies must be performed to analyse the possible contamination of these animals through the environment and/or the food chain.     

Stylet penetration activities linked to the acquisition and inoculation of Candidatus Liberibacter solanacearum by its vector tomato potato psyllid

The tomato potato psyllid (TPP), Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is the main vector of the bacterium Candidatus Liberibacter solanacearum (Lso), a major disease of solanaceous crops. Feeding of TPP is associated with Lso transmission. However, very little is known about the stylet penetration activities linked to acquisition and inoculation of Lso. The electrical penetration graph (EPG)‐DC system was used to monitor stylet penetration activities during acquisition and inoculation of Lso by individual TPP on tomato [Solanum lycopersicum L. (Solanaceae)]. Female TPP from Lso‐free and Lso‐infected colonies were used in acquisition and inoculation tests, respectively. In the acquisition tests, TPP were tested for Lso after EPG recording of their stylet penetration activities on Lso‐infected tomato shoots. In the inoculation tests, samples from the tomato plants on which the stylet penetration of Lso‐infected TPP had been recorded were tested for Lso infection. The relationships between qPCR results and the EPG waveforms (C, G, D, E1, and E2) representing the main stylet penetration activities performed by individual insects in inoculation and acquisition tests were investigated. Results confirmed that a single adult TPP is capable of infecting a plant with Lso. Our data suggest that acquisition of the bacteria occurs during phloem ingestion (E2), and inoculation is likely associated with salivation into the phloem sieve elements (E1). The durations of EPG parameters were not significantly different between Lso‐infected and Lso‐free TPP (later shown by qPCR) in acquisition tests. In inoculation tests, the durations of E1 or E2 recorded from TPP on Lso‐infected and Lso‐free plants that were later shown by qPCR were not significantly different. However, C was shorter on Lso‐infected plants than on Lso‐free plants, where TPP performed phloem activities. The minimum plant access period required for Lso transmission by a single TPP was estimated to be ca. 2 h, with an acquisition threshold of about 36 min.     

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono‐ and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl‐MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl‐MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12‐oxo‐phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA‐containing galactolipids in the plant kingdom. While acyl‐MGDG was found to be ubiquitous in green tissue of plants ranging from non‐vascular plants to angiosperms, OPDA‐containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non‐oxidized and OPDA‐containing acyl‐MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl‐MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.     

Cellular Responses and Proteomic Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Exposed to Green Tea Polyphenols

The purpose of this study was to characterize the cellular response and proteomic analysis of Escherichia coli exposed to tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis L). TPP showed a dose-dependent bactericidal effect on E. coli. Analysis of cell-membrane fatty acids of E. coli cultures treated with TPP identified unique changes in saturated and unsaturated fatty acids, whereas scanning electron microscopic analysis demonstrated the presence of perforations and irregular rod forms with wrinkled surfaces in cells treated with TPP. Two-dimensional polyacrylamide gel electrophoresis of soluble protein fractions from E. coli cultures exposed to TPP showed 17 protein spots increased or decreased by TPP. Nine upregulated proteins were identified (including GroEL and proteins involved in cellular defense, such as GyrA, RpoS, SodC, and EmrK), whereas the expression of eight proteins was downregulated by exposure to TPP (including proteins involved in carbon and energy metabolism, such as Eno, SdhA, and UgpQ, as well as those involved in amino-acid biosynthesis, such as GltK and TyrB). These results provide clues for understanding the mechanism of TPP-induced stress and cytotoxicity on E. coli.     

Persistence of Escherichia coli introduced into streambed sediments with goose,deer and bovine animal waste

Aims:  The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli. Methods and Results:  The survival experiments were conducted in flow‐through chambers for 32 days using two sediments (mineral and organic) obtained from a first‐order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert‐18 Quanti‐Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer‐borne E. coli populations in the organic sediment. Conclusion:  These results indicate that indigenous sediment‐borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife. Significance and Impact of the Study:  This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory‐cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality.     

Environmental Escherichia coli: ecology and public health implications—a review

Escherichia coli is classified as a rod‐shaped, Gram‐negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm‐blooded animals, including humans, and is often discharged into the environment through faeces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent faecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extraintestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a faecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long‐term survival. Large‐scale studies of population genetics revealed the diversity and complexity of E. coli strains in various environments, which are affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments with regard to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.     

In situ genotyping of a pooled strain library after characterizing complex phenotypes

In this work, we present a proof‐of‐principle experiment that extends advanced live cell microscopy to the scale of pool‐generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single‐molecule fluorescence time‐lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.     

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.     

New Hydrazides and Hydrazide‐Hydrazones of 2,3‐Dihalogen Substituted Propionic Acids: Synthesis and in vitro Antimicrobial Activity Evaluation

The main aim of this research was the synthesis, spectral identification and in vitro antimicrobial evaluation of new hydrazides and hydrazide‐hydrazones of 2,3‐dihalogen substituted propionic acids. New hydrazides were obtained by the substitution reaction of appropriate ethyl esters of 2,3‐dihalogen substituted propionic acids with hydrazine hydrate. Then obtained hydrazides were subjected to condensation reaction with various aldehydes which yielded with new hydrazide‐hydrazone derivatives. All obtained compounds were identified on the basis of spectral methods (¹H‐NMR, ¹³C‐NMR) and in vitro screened against a panel of bacterial and fungal strains according to EUCAST and CLSI guidelines.     

Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis

Mitochondrial Lon1 loss impairs oxidative phosphorylation complexes and TCA enzymes and causes accumulation of specific mitochondrial proteins. Analysis of over 400 mitochondrial protein degradation rates using ¹⁵N labelling showed that 205 were significantly different between wild type (WT) and lon1‐1. Those proteins included ribosomal proteins, electron transport chain subunits and TCA enzymes. For respiratory complexes I and V, decreased protein abundance correlated with higher degradation rate of subunits in total mitochondrial extracts. After blue native separation, however, the assembled complexes had slow degradation, while smaller subcomplexes displayed rapid degradation in lon1‐1. In insoluble fractions, a number of TCA enzymes were more abundant but the proteins degraded slowly in lon1‐1. In soluble protein fractions, TCA enzymes were less abundant but degraded more rapidly. These observations are consistent with the reported roles of Lon1 as a chaperone aiding the proper folding of newly synthesized/imported proteins to stabilise them and as a protease to degrade mitochondrial protein aggregates. HSP70, prohibitin and enzymes of photorespiration accumulated in lon1‐1 and degraded slowly in all fractions, indicating an important role of Lon1 in their clearance from the proteome.     

Residual concentrations of antimicrobial growth promoters in poultry litter favour plasmid conjugation among Escherichia coli

Considering that plasmid conjugation is a major driver for the dissemination of antimicrobial resistance in bacteria, this study aimed to investigate the effects of residual concentrations of antimicrobial growth promoters (AGPs) in poultry litter on the frequencies of IncFII-FIB plasmid conjugation among Escherichia coli organisms. A 2 × 5 factorial trial was performed in vitro, using two types of litter materials (sugarcane bagasse and wood shavings) and five treatments of litter: non-treated (CON), herbal alkaloid sanguinarine (SANG), AGPs monensin (MON), lincomycin (LCM) and virginiamycin (VIR). E. coli H2332 and E. coli J62 were used as donor and recipient strains, respectively. The presence of residues of monensin, lincomycin and virginiamycin increased the frequency of plasmid conjugation among E. coli in both types of litter materials. On the contrary, sanguinarine significantly reduced the frequency of conjugation among E. coli in sugarcane bagasse litter. The conjugation frequencies were significantly higher in wood shavings compared with sugarcane bagasse only in the presence of AGPs. Considering that the presence of AGPs in the litter can increase the conjugation of IncFII-FIB plasmids carrying antimicrobial resistance genes, the real impact of this phenomenon on the dissemination of antimicrobial resistant bacteria in the poultry production chain must be investigated.     

Escherichia coli biofilm: development and therapeutic strategies

Escherichia coli biofilm consists of a bacterial colony embedded in a matrix of extracellular polymeric substances (EPS) which protects the microbes from adverse environmental conditions and results in infection. Besides being the major causative agent for recurrent urinary tract infections, E. coli biofilm is also responsible for indwelling medical device‐related infectivity. The cell‐to‐cell communication within the biofilm occurs due to quorum sensors that can modulate the key biochemical players enabling the bacteria to proliferate and intensify the resultant infections. The diversity in structural components of biofilm gets compounded due to the development of antibiotic resistance, hampering its eradication. Conventionally used antimicrobial agents have a restricted range of cellular targets and limited efficacy on biofilms. This emphasizes the need to explore the alternate therapeuticals like anti‐adhesion compounds, phytochemicals, nanomaterials for effective drug delivery to restrict the growth of biofilm. The current review focuses on various aspects of E. coli biofilm development and the possible therapeutic approaches for prevention and treatment of biofilm‐related infections.     

Progressive genome‐wide introgression in agricultural Campylobacter coli

Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein‐coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST‐828 and ST‐1150 clonal complexes. The ST‐1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST‐828 complex bacteria have 10–11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have <2%. Thus, the C. coli that colonize agriculture, and consequently cause most human disease, have hybrid origin, but this cross‐species exchange has so far not had a substantial impact on the gene pools of either C. jejuni or nonagricultural C. coli. These findings also indicate remarkable interchangeability of basic cellular machinery after a prolonged period of independent evolution.     

Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans‐acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln‐Sm units are displaced by new pICln‐Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis‐assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.     

The prevalence of antimicrobial‐resistant Escherichia coli in sympatric wild rodents varies by season and host

Aims: To investigate the prevalence and temporal patterns of antimicrobial resistance in wild rodents with no apparent exposure to antimicrobials. Methods and Results: Two sympatric populations of bank voles and wood mice were trapped and individually monitored over a 2‐ year period for faecal carriage of antimicrobial‐resistant Escherichia coli. High prevalences of ampicillin‐, chloramphenicol‐, tetracycline‐ and trimethoprim‐resistant E. coli were observed. A markedly higher prevalence of antimicrobial‐resistant E. coli was found in wood mice than in bank voles, with the prevalence in both increasing over time. Superimposed on this trend was a seasonal cycle with a peak prevalence of resistant E. coli in mice in early‐ to mid‐summer and in voles in late summer and early autumn. Conclusions: These sympatric rodent species had no obvious contact with antimicrobials, and the difference in resistance profiles between rodent species and seasons suggests that factors present in their environment are unlikely to be drivers of such resistance. Significance and Impact of the Study: These findings suggest that rodents may represent a reservoir of antimicrobial‐resistant bacteria, transmissible to livestock and man. Furthermore, such findings have implications for human and veterinary medicine regarding antimicrobial usage and subsequent selection of antimicrobial‐resistant organisms.     

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β‐adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics‐based platform proteome mining tool for antigen discovery, called “proteomics‐based expression library screening” (PELS; Kudva et al., 2006). The E. coli O157 IP (O157‐IP) comprised 91 proteins, and included those identified previously using proteomics‐based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp‐2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157‐IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.