Co-occurrence of dual carbapenemases KPC-2 and OXA-48 with the mobile colistin resistance gene mcr-9.1 in Enterobacter xiangfangensis.

Bacterial infections with the genus Enterobacter are notoriously difficult to treat and often associated with resistance to penicillin, aminoglycosides, fluoroquinolones, and third-generation cephalosporins. Also, Enterobacter species have emerged as the third most common hosts for carbapenemases worldwide, forcing the use of colistin as a "last-resort" antibiotic for the treatment. Studies on the population structure of the genus Enterobacter repeatedly detect E. xiangfangensis as a common clinical species present worldwide. Here, we report on the characteristics of an extreme drug-resistant E. xiangfangensis isolate va18651 (ST88), obtained from a cervical swab of an expectant mother. The isolate was resistant to almost all the classes of antibiotics tested, including ß-lactams (viz., penicillins, carbapenems, cephalosporin, monobactams, and their combinations), quinolone, aminoglycosides, and sulfonamide/dihydrofolate reductase inhibitor, and exhibited heteroresistance towards colistin. Analysis of its complete genome sequence revealed 37 antibiotic resistance genes (ARGs), including mcr-9.1, blaKPC-2 , and blaOXA-48 , encoded on three of the four different plasmids (cumulative plasmidome size 604,632 bp). An unusually high number of plasmid-based heavy metal resistance gene (HRG) clusters towards silver, arsenate, cadmium, copper, mercury, and tellurite were also detected. Virulence genes (VGs) for the lipopolysaccharide and capsular polysaccharide structures, iron acquisition (iroBCDEN, ent/fep/fes, sitABCD, iut, and fur), and a type VI secretion system, together with motility genes and Type IV pili, were encoded chromosomally. Thus, a unique combination of chromosomally encoded VGs, together with plasmid-encoded ARGs and HRGs, converged to result in an extreme drug-resistant, pathogenic isolate with survival potential in environmental settings. The use of a disinfectant, octenidine, led to its eradication; however, the existence of a highly antibiotic-resistant isolate with significant virulence potential is a matter of concern in public health settings and warrants further surveillance for extreme drug-resistant Enterobacter isolates.

Comparison of approaches for source attribution of ESBL-producing Escherichia coli in Germany.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli have been widely described as the cause of treatment failures in humans around the world. The origin of human infections with these microorganisms is discussed controversially and in most cases hard to identify. Since they pose a relevant risk to human health, it becomes crucial to understand their sources and the transmission pathways. In this study, we analyzed data from different studies in Germany and grouped ESBL-producing E. coli from different sources and human cases into subtypes based on their phenotypic and genotypic characteristics (ESBL-genotype, E. coli phylogenetic group and phenotypic antimicrobial resistance pattern). Then, a source attribution model was developed in order to attribute the human cases to the considered sources. The sources were from different animal species (cattle, pig, chicken, dog and horse) and also from patients with nosocomial infections. The human isolates were gathered from community cases which showed to be colonized with ESBL-producing E. coli. We used the attribution model first with only the animal sources (Approach A) and then additionally with the nosocomial infections (Approach B). We observed that all sources contributed to the human cases, nevertheless, isolates from nosocomial infections were more related to those from human cases than any of the other sources. We identified subtypes that were only detected in the considered animal species and others that were observed only in the human population. Some subtypes from the human cases could not be allocated to any of the sources from this study and were attributed to an unknown source. Our study emphasizes the importance of human-to-human transmission of ESBL-producing E. coli and the different role that pets, livestock and healthcare facilities may play in the transmission of these resistant bacteria. The developed source attribution model can be further used to monitor future trends. A One Health approach is necessary to develop source attribution models further to integrate also wildlife, environmental as well as food sources in addition to human and animal data.

Infection in a Young Immunocompetent Male Caused by Streptobacillus felis, a Putative Zoonotic Microorganism Transmitted by Cats.

Rat bite fever (RBF) is predominantly caused by Streptobacillus moniliformis. We report a human infection with Streptobacillus felis. Clinical presentation was consistent with RBF, but serologic testing was negative for S moniliformis. Eventually, S felis-specific sequences were detected in skin lesions of the patient and in the oropharynx of local cats.

Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.

Multidrug-Resistant and Clinically Relevant Gram-Negative Bacteria Are Present in German Surface Waters.

Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored bla VIM- 1. Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.

High resistance to tetracycline and ciprofloxacin in bacteria isolated from poultry farms in Ibadan, Nigeria.

INTRODUCTION: Resistance to ciprofloxacin and tetracycline is increasing in the food chain especially in E. coli strains and more worrisome will be occurrence of extended-spectrum beta-lactamase (ESBL) producers among ciprofloxacin- and tetracycline-resistant isolates. This study was undertaken to investigate the occurrence and mechanism of ciprofloxacin-, tetracycline- and ESBL-resistant bacteria in poultry in Ibadan, Nigeria. METHODOLOGY: Bacteria were isolated from poultry feces in two farms in Ibadan and identified by MALDI-TOF. Antibiotic susceptibility patterns of the isolates were determined by disc diffusion and Minimum Inhibitory Concentration (MIC) using Vitek-2 apparatus. Four tetracycline genes and six plasmids mediated quinolone resistance genes (PMQR) were investigated by PCR. Whole genome sequencing was done for strains that were ESBL producers. RESULTS: Bacterial strains (≥ 105 cfu/mL) were counted on ciprofloxacin and tetracycline supplemented plates. 106 bacteria from 14 different species were identified with high resistance to quinolones, tetracycline and trimethoprim. 49% of the strains were E. coli with 90% resistance for nalidixic acid, moxifloxacin (94%), ciprofloxacin (88%) levofloxacin (78%) and tetracycline (77%). The genes tetA, tetB, qnrB, qnrS and qepA were detected with 37%, 4%, 35%, 4% and 2% prevalence in E. coli respectively. Three ESBL-producing E. coli of the sequence type ST-6359 were found and harboured blaCTX-M-15 located in the chromosome, at the same insertion site. All the ESBL producers harboured mutations in gyrA (S83L/D87N/D678E) and parC (S80I). CONCLUSION: The observed high quinolones and tetracycline resistance with ESBL producers in this study calls for caution in the use of these antibiotics in poultry feeds.

Complete Genome Sequence of blaCTX-M-27-Encoding Escherichia coli Strain H105 of Sequence Type 131 Lineage C1/H30R.

Escherichia coli sequence type 131 (ST131) is the most frequent antimicrobial-resistant lineage of E. coli, propagating extended-spectrum ß-lactamases (ESBL) worldwide. Recently, an alarming rate of increase in isolates of the sublineage C1/H30R-blaCTX-M-27 of ST131 in geographically distant countries was reported. Here, we present the complete genome sequence of the ST131 sublineage C1/H30R E. coli isolate harboring blaCTX-M-27 from Germany.

Circulation of clonal populations of fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST410 in humans and animals in Germany.

Multidrug-resistant Escherichia coli encoding CTX-M-type extended-spectrum ß-lactamases (ESBLs) are isolated in increasing numbers from humans, companion animals and livestock, raising concern regarding the exchange and spread of isolates in these populations. In this study, whole-genome sequencing of CTX-M-15-producing E. coli isolates recently sampled from humans, companion animals, livestock and farm environments was performed. In total, 26 different sequence types (STs) were detected, of which ST410 was the most frequent and was the only ST present in all populations studied. Five clades (designated A-E) were detected within the ST410 isolates. In particular, isolates of clade B were present in all four populations and had core genomes that differed by less than 70 single nucleotide polymorphisms (SNPs). Isolates of clades B and C were also clonally marked, exhibiting identical chromosomal insertions of blaCTX-M-15 at distinct loci. These data provide strong evidence for the clonal dissemination of specific clades of CTX-M-15-producing E. coli ST410 in human and animal populations.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: an approach to quantify the distribution of ESBL types between different reservoirs.

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

Multiresistant extended-spectrum ß-lactamase-producing Enterobacteriaceae from humans, companion animals and horses in central Hesse, Germany.

BACKGROUND: Multiresistant Gram-negative bacteria producing extended-spectrum ß-lactamases (ESBLs) are an emerging problem in human and veterinary medicine. This study focused on comparative molecular characterization of ß-lactamase and ESBL-producing Enterobacteriaceae isolates from central Hesse in Germany. Isolates originated from humans, companion animals (dogs and cats) and horses. RESULTS: In this study 153 (83.6%) of the human isolates (n = 183) and 163 (91.6%) of the animal isolates (n = 178) were confirmed as ESBL producers by PCR and subsequent sequencing of the PCR amplicons. Predominant ESBL subtypes in human and animal samples were CTX-M-15 (49.3%) and CTX-M-1 (25.8%) respectively. Subtype blaCTX-M-2 was found almost exclusively in equine and was absent from human isolates. The carbapenemase OXA-48 was detected in 19 ertapenem-resistant companion animal isolates in this study. The Plasmid-encoded quinolone resistance (PMQR) gene aac('6)-Ib-cr was the most frequently detected antibiotic- resistance gene present in 27.9% of the human and 36.9% of the animal ciprofloxacin-resistant isolates. Combinations of two or up to six different resistance genes (penicillinases, ESBLs and PMQR) were detected in 70% of all isolates investigated. The most frequent species in this study was Escherichia coli (74%), followed by Klebsiella pneumoniae (17.5%), and Enterobacter cloacae (4.2%). Investigation of Escherichia coli phylogenetic groups revealed underrepresentation of group B2 within the animal isolates. CONCLUSIONS: Isolates from human, companion animals and horses shared several characteristics regarding presence of ESBL, PMQR and combination of different resistance genes. The results indicate active transmission and dissemination of multi-resistant Enterobacteriaceae among human and animal populations.

Complete Genome Sequence of Phage-Like Plasmid pECOH89, Encoding CTX-M-15.

A nonconjugative and nontypable plasmid of a clinical Escherichia coli isolate expressing resistance to extended-spectrum cephalosporins (ESCs) was isolated and sequenced. The plasmid pECOH89 contains a CTX-M-15 resistance cassette and comprises 111,741 bp, with strong homology to bacteriophage-like plasmids and to the Salmonella-specific bacteriophage SSU5.

Resistance plasmids in ESBL-encoding Escherichia coli isolates from humans, dogs and cats.

We characterized ESBL-producing Escherichia coli isolates from diseased dog, cat and human sources for their plasmid content. Plasmids with different Inc groups and combinations of resistance genes were detected in these isolates. The pan-genome of the plasmid-associated genes was found to be large, indicating diversity of the gene pool among the plasmids. No commonly occurring plasmids with similar gene content in isolates from dog, cats and humans were detected.

Complete sequences of plasmids from the hemolytic-uremic syndrome-associated Escherichia coli strain HUSEC41.

The complete and annotated sequences of four plasmids from a historical enteroaggregative Shiga toxin-producing Escherichia coli (HUSEC) serotype O104:H4 strain, HUSEC41/01-09591, isolated in 2001 in Germany are reported.

Molecular characterization of Borrelia persica, the agent of tick borne relapsing fever in Israel and the Palestinian Authority.

The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5'end of the 16S rRNA gene to the 5'end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5' fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area.

Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces.

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized beta-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human beta1,4-galactosyltransferase-1 and beta1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His(6)CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.

Matuzumab binding to EGFR prevents the conformational rearrangement required for dimerization.

An increasing number of therapeutic antibodies targeting tumors that express the epidermal growth factor receptor (EGFR) are in clinical use or late stages of clinical development. Here we investigate the molecular basis for inhibition of EGFR activation by the therapeutic antibody matuzumab (EMD72000). We describe the X-ray crystal structure of the Fab fragment of matuzumab (Fab72000) in complex with isolated domain III from the extracellular region of EGFR. Fab72000 interacts with an epitope on EGFR that is distinct from the ligand-binding region on domain III and from the cetuximab/Erbitux epitope. Matuzumab blocks ligand-induced receptor activation indirectly by sterically preventing the domain rearrangement and local conformational changes that must occur for high-affinity ligand binding and receptor dimerization.