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[Display omitted] • 148 carbapenem-resistant Enterobacterales were isolated from treated wastewater. • bla NDM-1 , bla OXA-48 and/or bla KPC-2 were detected in most isolates. • Most identified sequence types (n = 14) were associated with human-associated clones. • Possible patogenicity was detected in all sequenced Klebsiella isolates. • mcr-4.3 was identified for the first time in K. pneumoniae and on plasmid IncHI1B. Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were bla OXA-48 and bla NDM-1 , which frequently occurred together, while bla KPC-2 together with bla NDM-1 was mainly detected in K. oxytoca , E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B , phoP/Q , mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics. [ABSTRACT FROM AUTHOR]

The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 β-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe’s largest hospitals, the General Hospital Vienna, were screened for the presence of 31 β-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of β-lactamase genes was estimated using biostatistical models. The carbapenemase genes bla KPC-2 , bla KPC-3 , and bla VIM -4 were identified in carbapenem-resistant and intermediate susceptible isolates, bla OXA-72 in an extended-spectrum β-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired bla DHA - 1 and bla CMY AmpC β-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all β-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated β-lactamases and other resistance genes in one of Europe’s largest hospitals. [ABSTRACT FROM AUTHOR]

Abstract: The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse β-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the β-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 104 DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of β-lactamase genes in dilutions of 107 cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene. [Copyright &y& Elsevier]

Multidrug-resistant Acinetobacter baumannii (MDR-AB) is an important threat for critically ill patients. It can infect the respiratory tract, blood, soft tissues, urinary tract and central nervous system. Recently, carbapenem-resistance was observed in A . baumannii clinical isolates from Bosnia and Herzegovina. This prompted us to analyze these isolates with regards to genotypic diversity, antibiotic susceptibility and occurrence of acquired carbapenem resistance genes. Twelve carbapenem-resistant isolates were collected at a University hospital during two different periods of 2011 and 2015–2016: four isolates in 2011 and eight isolates 2015–2016 and compared to determine the dynamic changes in carbapenemase resistance mechanisms and population structure. All twelve isolates were positive for intrinsic bla OXA-51-like , nine for bla OXA-40-like and one for the bla OXA-23-like gene. IS Aba1 was found upstream of bla OXA-51 in all and upstream of bla OXA-23-like gene in one isolate. Sequencing of the selected PCR products revealed the presence of OXA-72 β-lactamase (strain 1) and OXA-23 β-lactamase (strain 41). WGS of the selected isolate (AB 5) revealed the presence of bla OXA-72 , chromosomal genes bla OXA-69 and bla ADC . Moreover, the aac ( 3 )-1a and aadA1 genes encoding aminoglycoside resistance, and sul1 encoding sulphonamide resistance were identified. PFGE and rep-PCR revealed two clones containing highly similar isolates positive for OXA-40-like; one from 2011 and the other from 2015 to 2016. Implementation of hospital hygiene measures, screening of the patients on admission for carriage of MDR-AB, and the early and accurate detection, with restriction of antibiotic use should be recommended to control the spread of these important hospital pathogenes. To our knowledge, this is the first report of A . baumannii isolates producing carbapenem-hydrolyzing oxacillinases (CHDL) from Bosnia and Herzegovina. [ABSTRACT FROM AUTHOR]

The polymerase chain reaction is not only essential for many DNA-based diagnostic methods but is also exploited in other molecular methods that require an upstream amplification step. Multiplex PCRs are especially attractive as they reduce the number of individual reactions. However, the multiplexing efficiency is impaired by primer interactions such as the formation of primer dimers. In this study, covalent crosslinking of primers via their 5′-ends was used to avoid those undesired effects. The specificity of the primers as well as the efficiency of the PCR could be increased upon primer crosslinking in reactions containing up to 34 primer pairs targeting the most important antibiotic resistance genes in a single multiplex reaction. • Crosslinking of PCR primers to receive more reliable band patterns is proposed. • The crosslinking avoids elongation of primer dimers and favours perfectly matching sequences. • The resulting amplification products were more specific. [ABSTRACT FROM AUTHOR]

Rat inferior caval vein (ICV) ligation (up to the right ovarian vein (ROV)) commonly represents a recapitulation of Virchow: with ligation leading to vessel injury, stasis, thrombosis and hemodynamic changes. We revealed that BPC 157’s therapy collectively attenuated or counteracted all these events and the full syndrome. Methods We applied BPC 157 (10 μg, 10 ng/kg) as an early regimen or as a delayed therapy. Assessment includes gross assessment by microcamera; microscopy, venography, bleeding, blood pressure, ECG, thermography, MDA and NO-level in plasma and ICV, and gene expression. Results Direct vein injury, thrombosis, thrombocytopenia, prolonged bleeding were all counteracted. Also, rapid presentation of collaterals and redistribution of otherwise trapped blood volume (bypassing through the left ovarian vein (LOV) and other veins), with venous hypertension, arterial hypotension and tachycardia counteraction were shown. BPC 157-rats presented raised plasma NO-values, but normal MDA-values; in ICV tissue reverted low NO-values and counteracted increased MDA-levels. Altered expression of EGR, NOS, SRF, VEGFR and KRAS in ICV, ROV and LOV revealed increased or decreased levels, while some genes continuously remained unchanged. Conclusion As a new insight, BPC 157 application largely attenuated or even completely eliminated all consequences of ICV ligation in rats. [ABSTRACT FROM AUTHOR]

Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein–oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure–activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC). • HRP highly selectively substituted at N-terminus amino group. • Highly selective SPAAC click conjugation to HRP N-terminus controlled by very fine pH regulation. • DNA oligonucleotide conjugated to HRP N-terminus does not impede enzymatic activity. [ABSTRACT FROM AUTHOR]

Colistin is a last-resort antibiotic for the treatment of infections caused by multidrug and carbapenem-resistant Gram-negative bacteria. Colistin resistance has been emerging and multiple outbreaks have been reported in Europe and elsewhere. It has been most frequently reported in carbapenem-resistant K. pneumoniae. In this study, 24 multidrug and colistin-resistant clinical isolates (14 K. pneumoniae , one E. aerogenes , one E. cloacae, and eight A. baumannii) were collected from four hospitals in Croatia from 2013 to 2018, in order to analyse the molecular epidemiology and mechanisms of antibiotic resistance. β-lactamase and carbapenemase genes were detected by PCR. Genotyping was done on selected isolates by rep-PCR. Whole genome sequencing (WGS) was performed to discover possible molecular mechanisms for the observed colistin resistance. All isolates, except two K. pneumoniae isolates, were extensively drug resistant. Ten out of 16 (63%) K. pneumoniae isolates possessed bla OXA-48, which is the most common carbapenem resistance gene in Croatia and in other parts of Europe. All A. baumannii isolates possessed the OXA-23-like carbapenem hydrolysing oxacillinase and five turned out to be pandrug-resistant. Colistin resistance was most likely chromosomally mediated. After sequence analysis, none of the isolates were found to possess any of the mcr gene variants. Several previously reported mutations were found in PmrB, PhoP, PhoQ, and MgrB, which are associated with colistin resistance. In the global phylogenetic analysis, DNA mutations causing mutations in the MgrB protein were present mostly in lineages comprising colistin resistant isolates, and the second most prevalent mutation (K3X) was also encountered in our isolates. In addition, based on genotyping by rep-PCR, the spread of colistin resistance is most likely to be clonal. Most importantly, the presence of colistin resistance together with carbapenemase genes in extensively drug resistant isolates poses real threats in the use of carbapenems and colistin to fight infections. • Genetic analysis of colistin resistant isolates in Croatia. • In total, 66.7% (8/12) Enterobacteriaceae and 71.4% (5/7) A. baumannii isolates were XDR. • No evidence for plasmid-mediated colistin resistance mechanisms. • Global phylogenetic analysis of the mgrB and lpxC genes. [ABSTRACT FROM AUTHOR]