Does Reductive Evolution Correlate with Habitat and Pathotype?

IbeA (invasion of brain endothelium), which is located on a genomic island termed GimA, is involved in the pathogenesis of several extraintestinal pathogenic E. coli (ExPEC) pathotypes, including newborn meningitic E. coli (NMEC) and avian pathogenic E. coli (APEC). To unravel the phylogeny of GimA and to investigate its island character, the putative insertion locus of GimA was determined via Long Range PCR and DNA-DNA hybridization in 410 E. coli isolates, including APEC, NMEC, uropathogenic (UPEC), septicemia-associated E. coli (SEPEC), and human and animal fecal isolates as well as in 72 strains of the E. coli reference (ECOR) collection. In addition to a complete GimA (~20.3 kb) and a locus lacking GimA we found a third pattern containing a 342 bp remnant of GimA in this strain collection. The presence of GimA was almost exclusively detected in strains belonging to phylogenetic group B2. In addition, the complete GimA was significantly more frequent in APEC and NMEC strains while the GimA remnant showed a higher association with UPEC strains. A detailed analysis of the ibeA sequences revealed the phylogeny of this gene to be consistent with that obtained by Multi Locus Sequence Typing of the strains. Although common criteria for genomic islands are partially fulfilled, GimA rather seems to be an ancestral part of phylogenetic group B2, and it would therefore be more appropriate to term this genomic region GimA locus instead of genomic island. The existence of two other patterns reflects a genomic rearrangement in a reductive evolution-like manner

Long-term neuromuscular sequelae of critical illness

In this observational study, we analyzed the long-term neuromuscular deficits of survivors of critical illness. Intensive care unit-acquired muscular weakness (ICU-AW) is a very common complication of critical illness. Critical illness polyneuropathy (CIP) and critical illness myopathy (CIM) are two main contributors to ICU-AW. ICU-AW is associated with an increased mortality and leads to rehabilitation problems. However, the long-term outcome of ICU-AW and factors influencing it are not well known. We analyzed the medical records of 490 survivors of critical illness, aged 18-75years and located in the area of the study center. Intensive care unit (ICU) survivors with comorbidities that might influence neuromuscular follow-up examinations, muscle strength, or results of nerve conduction studies, such as renal or hepatic insufficiency, diabetes mellitus, or vitamin deficiency were excluded. A total of 51 patients were finally included in the study. Six to 24months after discharge from the ICU, we measured the Medical Research Council (MRC) sum score, the Overall Disability Sum score (ODSS), and also performed nerve conduction studies and EMG. For all ICU survivors, the median MRC sum score was 60 (range 47-60) and the median ODSS score was 0 (range 0-8). CIP was diagnosed in 21 patients (41%). No patient was diagnosed with CIM. Patients with diagnosis of CIP showed a higher median ODSS scores 1 (range 0-8) versus 0 (range 0-5); p<0.001 and lower median MRC sum scores 56 (range 47-60) versus 60 (range 58-60); p<0.001. The three main outcome variables MRC sum score, ODSS score and diagnosis of CIP were not related to age, gender, or diagnosis of sepsis. The MRC sum score (r=−0.33; p=0.02) and the ODSS score (r=0.31; p=0.029) were correlated with the APACHE score. There was a trend for an increased APACHE score in patients with diagnosis of CIP 19 (range 6-33) versus 16.5 (range 6-28); p=0.065. Patients with the diagnosis of CIP had more days of ICU treatment 11days (range 2-74) versus 4days (range 1-61); p=0.015, and had more days of ventilator support 8days (range 1-59) versus 2days (range 1-46); p=0.006. The MRC sum score and the ODSS score were correlated with the days of ICU treatment and with the days of ventilator support. The neuromuscular long-term consequences of critical illness were not severe in our study population. As patients with concomitant diseases and old patients were excluded from this study the result of an overall favorable prognosis of ICU-acquired weakness may not be true for other patient's case-mix. Risk factors for the development of long-term critical illness neuropathy are duration of ICU treatment, duration of ventilator support, and a high APACHE score, but not diagnosis of sepsis. Although ICU-AW can be serious complication of ICU treatment, this should not influence therapeutic decisions, given its favorable long-term prognosis, at least in relatively young patients with no concomitant disease

ESBL-plasmids carrying toxin-antitoxin systems can be “cured” of wild-type Escherichia coli using a heat technique

Plasmid-encoded extended-spectrum beta-lactamase (ESBL)-enzymes are frequently produced by Escherichia coli. Several ESBL-plasmids contain genes for toxin- antitoxin (TA) systems, which assure the maintenance of plasmids in bacteria and prevent the cells from "post-segregational killing". These systems limit options to "cure" plasmids of ESBL-wild-type strains due to the death of the bacterial cells. A helpful tool to understand the role of ESBL-plasmids in the dissemination of pandemic multi-resistant E. coli are ESBL- plasmid-"cured"-variants (PCVs) and their comparison to ESBL-wild-type strains. The purpose of this study was to construct PCVs of ESBL-wild-type E. coli strains despite the presence of genes for TA systems. Using enhanced temperatures and brain-heart-infusion broth it was possible to construct viable PCVs of wild-type ESBL-E. coli strains. The occurrence of TA system- genes including hok/sok, srnB/C, vagC/D, pemI/K on ESBL-plasmids of replicon types FIA or FIB was demonstrated by bioinformatic analyses. The loss of the plasmid and the genetic identity of PCV and corresponding wild-type strain was confirmed via different methods including plasmid-profile-analysis, pulsed- field gel electrophoresis and bioinformatics using generated whole genome data of the strains. This short report describes the successful construction of viable PCVs of ESBL-wild-type E. coli strains. The results are hence surprising due to the fact that all "cured" ESBL-plasmids contained at least one complete toxin-antitoxin system, whose loss would normally mean the death of bacterial cells

Genome Sequence of AvianEscherichia coliStrain IHIT25637, an Extraintestinal PathogenicE. coliStrain of ST131 Encoding Colistin Resistance Determinant MCR-1

Sequence type 131 (ST131) is one of the predominant Escherichia coli lineages among extraintestinal pathogenic E. coli (ExPEC) that causes a variety of diseases in humans and animals and frequently shows multidrug resistance. Here, we report the first genome sequence of an ST131-ExPEC strain from poultry carrying the plasmid-encoded colistin resistance gene mcr-1

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

Strategies to reduce economic losses associated with post-weaning diarrhea in pig farming include high-level dietary zinc oxide supplementation. However, excessive usage of zinc oxide in the pig production sector was found to be associated with accumulation of multidrug resistant bacteria in these animals, presenting an environmental burden through contaminated manure. Here we report on zinc tolerance among a random selection of intestinal Escherichia coli comprising of different antibiotic resistance phenotypes and sampling sites isolated during a controlled feeding trial from 16 weaned piglets: In total, 179 isolates from “pigs fed with high zinc concentrations” (high zinc group, [HZG]: n = 99) and a corresponding “control group” ([CG]: n = 80) were investigated with regard to zinc tolerance, antimicrobial- and biocide susceptibilities by determining minimum inhibitory concentrations (MICs). In addition, in silico whole genome screening (WGSc) for antibiotic resistance genes (ARGs) as well as biocide- and heavy metal tolerance genes was performed using an in-house BLAST-based pipeline. Overall, porcine E. coli isolates showed three different ZnCl2 MICs: 128 μg/ml (HZG, 2%; CG, 6%), 256 μg/ml (HZG, 64%; CG, 91%) and 512 μg/ml ZnCl2 (HZG, 34%, CG, 3%), a unimodal distribution most likely reflecting natural differences in zinc tolerance associated with different genetic lineages. However, a selective impact of the zinc-rich supplemented diet seems to be reasonable, since the linear mixed regression model revealed a statistically significant association between “higher” ZnCl2 MICs and isolates representing the HZG as well as “lower ZnCl2 MICs” with isolates of the CG (p = 0.005). None of the zinc chloride MICs was associated with a particular antibiotic-, heavy metal- or biocide- tolerance/resistance phenotype. Isolates expressing the 512 μg/ml MIC were either positive for ARGs conferring resistance to aminoglycosides, tetracycline and sulfamethoxazole-trimethoprim, or harbored no ARGs at all. Moreover, WGSc revealed a ubiquitous presence of zinc homeostasis and – detoxification genes, including zitB, zntA, and pit. In conclusion, we provide evidence that zinc-rich supplementation of pig feed selects for more zinc tolerant E. coli, including isolates harboring ARGs and biocide- and heavy metal tolerance genes – a putative selective advantage considering substances and antibiotics currently used in industrial pork production systems

Comparative Genomics of Escherichia coli Isolated from Skin and Soft Tissue and Other Extraintestinal Infections

Escherichia coli, an intestinal Gram-negative bacterium, has been shown to be associated with a variety of diseases in addition to intestinal infections, such as urinary tract infections (UTIs), meningitis in neonates, septicemia, skin and soft tissue infections (SSTIs), and colisepticemia. Thus, for nonintestinal infections, it is categorized as extraintestinal pathogenic E. coli (ExPEC). It is also an opportunistic pathogen, causing cross infections, notably as an agent of zoonotic diseases. However, comparative genomic data providing functional and genetic coordinates for ExPEC strains associated with these different types of infections have not proven conclusive. In the study reported here, ExPEC E. coli isolated from SSTIs was characterized, including virulence and drug resistance profiles, and compared with isolates from patients suffering either pyelonephritis or septicemia. Results revealed that the majority of the isolates belonged to two pathogenic phylogroups, B2 and D. Approximately 67% of the isolates were multidrug resistant (MDR), with 85% producing extended-spectrum beta-lactamase (ESBL) and 6% producing metallo- beta-lactamase (MBL). The blaCTX-M-15 genotype was observed in at least 70% of the E. coli isolates in each category, conferring resistance to an extended range of beta-lactam antibiotics. Whole-genome sequencing and comparative genomics of the ExPEC isolates revealed that two of the four isolates from SSTIs, NA633 and NA643, belong to pandemic sequence type ST131, whereas functional characteristics of three of the ExPEC pathotypes revealed that they had equal capabilities to form biofilm and were resistant to human serum. Overall, the isolates from a variety of ExPEC infections demonstrated similar resistomes and virulomes and did not display any disease-specific functional or genetic coordinates. IMPORTANCE Infections caused by extraintestinal pathogenic E. coli (ExPEC) are of global concern as they result in significant costs to health care facilities management. The recent emergence of a multidrug-resistant pandemic clone, Escherichia coli ST131, is of primary concern as a global threat. In developing countries, such as India, skin and soft tissue infections (SSTIs) associated with E. coli are marginally addressed. In this study, we employed both genomic analysis and phenotypic assays to determine relationships, if any, among the ExPEC pathotypes. Similarity between antibiotic resistance and virulence profiles was observed, ST131 isolates from SSTIs were reported, and genomic similarities among strains isolated from different disease conditions were detected. This study provides functional molecular infection epidemiology insight into SSTI- associated E. coli compared with ExPEC pathotypes

A combinational approach of multilocus sequence typing and other molecular typing methods in unravelling the epidemiology of Erysipelothrix rhusiopathiae strains from poultry and mammals

Erysipelothrix rhusiopathiae infections re-emerged as a matter of great concern particularly in the poultry industry. In contrast to porcine isolates, molecular epidemiological traits of avian E. rhusiopathiae isolates are less well known. Thus, we aimed to (i) develop a multilocus sequence typing (MLST) scheme for E. rhusiopathiae, (ii) study the congruence of strain grouping based on pulsed-field gel electrophoresis (PFGE) and MLST, (iii) determine the diversity of the dominant immunogenic protein SpaA, and (iv) examine the distribution of genes putatively linked with virulence among field isolates from poultry (120), swine (24) and other hosts (21), including humans (3). Using seven housekeeping genes for MLST analysis we determined 72 sequence types (STs) among 165 isolates. This indicated an overall high diversity, though 34.5% of all isolates belonged to a single predominant ST-complex, STC9, which grouped strains from birds and mammals, including humans, together. PFGE revealed 58 different clusters and congruence with the sequence-based MLST-method was not common. Based on polymorphisms in the N-terminal hyper-variable region of SpaA the isolates were classified into five groups, which followed the phylogenetic background of the strains. More than 90% of the isolates harboured all 16 putative virulence genes tested and only intI, encoding an internalin-like protein, showed infrequent distribution. MLST data determined E. rhusiopathiae as weakly clonal species with limited host specificity. A common evolutionary origin of isolates as well as shared SpaA variants and virulence genotypes obtained from avian and mammalian hosts indicates common reservoirs, pathogenic pathways and immunogenic properties of the pathogen

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.Hessian Ministry for the Environment, Climate Change, Agriculture and Consumer Protection (HMUKLV)Hessian Ministry for the Environment, Climate Change, Agriculture and Consumer Protection (HMUKLV

Comparable High Rates of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli in Birds of Prey from Germany and Mongolia

Frequent contact with human waste and liquid manure from intensive livestock breeding, and the increased loads of antibiotic-resistant bacteria that result, are believed to be responsible for the high carriage rates of ESBL- producing E. coli found in birds of prey (raptors) in Central Europe. To test this hypothesis against the influence of avian migration, we initiated a comparative analysis of faecal samples from wild birds found in Saxony-Anhalt in Germany and the Gobi-Desert in Mongolia, regions of dissimilar human and livestock population characteristics and agricultural practices. We sampled a total of 281 wild birds, mostly raptors with primarily north-to-south migration routes. We determined antimicrobial resistance, focusing on ESBL production, and unravelled the phylogenetic and clonal relatedness of identified ESBL-producing E. coli isolates using multi-locus sequence typing (MLST) and macrorestriction analyses. Surprisingly, the overall carriage rates (approximately 5%) and the proportion of ESBL-producers among E. coli (Germany: 13.8%, Mongolia: 10.8%) were similar in both regions. Whereas blaCTX-M-1 predominated among German isolates (100%), blaCTX-M-9 was the most prevalent in Mongolian isolates (75%). We identified sequence types (STs) that are well known in human and veterinary clinical ESBL-producing E. coli (ST12, ST117, ST167, ST648) and observed clonal relatedness between a Mongolian avian ESBL-E. coli (ST167) and a clinical isolate of the same ST that originated in a hospitalised patient in Europe. Our data suggest the influence of avian migratory species in the transmission of ESBL-producing E. coli and challenge the prevailing assumption that reducing human influence alone invariably leads to lower rates of antimicrobial resistance

Population Biology and Comparative Genomics of Campylobacter Species

The zoonotic pathogen Campylobacter is the leading cause for bacterial foodborne infections in humans. Campylobacters are most commonly transmitted via the consumption of undercooked poultry meat or raw milk products. The decreasing costs of whole genome sequencing enabled large genome-based analyses of the evolution and population structure of this pathogen, as well as the development of novel high-throughput molecular typing methods. Here, we review the evolutionary development and the population diversity of the two most clinically relevant Campylobacter species; C. jejuni and C. coli. The state-of-the-art phylogenetic studies showed clustering of C. jejuni lineages into host specialists and generalists with coexisting lifestyles in chicken and livestock-associated hosts, as well as the separation of C. coli isolates of riparian origin (waterfowl, water) from C. coli isolated from clinical and farm-related samples. We will give an overview of recombination between both species and the potential impact of horizontal gene transfer on host adaptation in Campylobacter. Additionally, this review briefly places the current knowledge of the population structure of other Campylobacter species such as C. lari, C. concisus and C. upsaliensis into perspective. We also provide an overview of how molecular typing methods such as multilocus sequence typing (MLST) and whole genome MLST have been used to detect and trace Campylobacter outbreaks along the food chain.Peer Reviewe