High Prevalence of Plasmid-Mediated Quinolone Resistance among ESBL/AmpC-Producing Enterobacterales from Free-Living Birds in Poland.

In this study, we investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) in extended-spectrum ß-lactamase- (ESBL) and/or AmpC-type ß-lactamase-producing Enterobacterales isolates from free-living birds in Poland. The prevalence of the qnrB19 gene was 63%, and the distribution of isolates in terms of bacterial species was as follows: 67% (22/33) corresponded to Escherichia coli, 83% (5/6) to Rahnella aquatilis, 44% (4/9) to Enterobacter cloacae and 33% (1/3) to Klebsiella pneumoniae. The qnrB19 gene was also found in a single isolate of Citrobacter freundii. The molecular characteristics of qnrB19-positive isolates pointed to extended-spectrum beta lactamase CTX-M as the most prevalent one (89%) followed by TEM (47%), AmpC (37%) and SHV (16%). This study demonstrates the widespread occurrence of PMQR-positive and ESBL/AmpC-producing Enterobacterales isolates in fecal samples from wild birds. In this work, plasmid pAM1 isolated from Escherichia coli strain SN25556 was completely sequenced. This plasmid is 3191 nucleotides long and carries the qnrB19 gene, which mediates decreased susceptibility to quinolones. It shares extensive homology with other previously described small qnrB19-harboring plasmids. The nucleotide sequence of pAM1 showed a variable region flanked by an oriT locus and a Xer recombination site. The presence of a putative recombination site was detected, suggesting that interplasmid recombination events might have played a role in the development of pAM1. Our results highlight the broad geographical spread of ColE-type Qnr resistance plasmids in clinical and environmental isolates of Enterobacterales. As expected from the results of phenotypic susceptibility testing, no resistance genes other than qnrB19 were identified.

Mechanisms Protecting Acinetobacter baumannii against Multiple Stresses Triggered by the Host Immune Response, Antibiotics and Outside-Host Environment.

Acinetobacter baumannii is considered one of the most persistent pathogens responsible for nosocomial infections. Due to the emergence of multidrug resistant strains, as well as high morbidity and mortality caused by this pathogen, A. baumannii was placed on the World Health Organization (WHO) drug-resistant bacteria and antimicrobial resistance research priority list. This review summarizes current studies on mechanisms that protect A. baumannii against multiple stresses caused by the host immune response, outside host environment, and antibiotic treatment. We particularly focus on the ability of A. baumannii to survive long-term desiccation on abiotic surfaces and the population heterogeneity in A. baumannii biofilms. Insight into these protective mechanisms may provide clues for the development of new strategies to fight multidrug resistant strains of A. baumannii.

RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes.

DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential.

Molecular characterization of plasmid pMbo4.6 of Moraxella bovis ATCC 10900.

We report the characterization of a small cryptic plasmid unlike any previously described from Moraxella bovis ATCC 10900, a Gram-negative bacterium belonging to the family Moraxellaceae. The complete nucleotide sequence of the plasmid pMbo4.6 was determined. The plasmid was analyzed and found to be 4658 in size with a G+C content of 38.6 mol %. Computer analysis of the sequence data revealed four major open reading frames encoding putative proteins of 10.1 (ORF1), 64.2 (ORF2), 45.7 (ORF3), and 12.1 kDa (ORF4). ORF1 and ORF2 encode proteins that show a high level of amino acid sequence similarity (44 %) with some mobilization proteins. ORF3 encodes a protein showing a relatively high amino acid sequence similarity (about 40 %) with several plasmid replication initiator proteins. Upstream of ORF3, a 320-bp intergenic region, constituting the putative origin of replication that contained an AT-rich region followed by four direct repeats, was identified. This set of repeated sequences resembles iteron structures and plays an important role in the control of plasmid replication by providing a target site for the initiation of transcription and replication factors (IHF and RepA). Several palindromic sequences, inverted repeats, and hairpin-loop structures, which might confer regulatory effects on the replication of the plasmid, were also noted. ORF4 encodes an uncharacterized protein, conserved in bacteria, belonging to the DUF497 family. Sequence analysis and structural features indicate that pMbo4.6 replicates by a theta mechanism.

Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats.

Wild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates (13,7%). Based on the analysis of the coexistence of 4 genes encoding ESBLs/AmpC (blaCTX-M, blaTEM, blaSHV, blaAmpC) and class 1 and 2 integrons genes (intI1, intI2) a subset of two resistance profiles was observed among the investigated E. coli isolates carrying blaAmpC, blaSHV, and blaCTX-M, blaTEM, class 1 and 2 integrons, respectively. The E. coli isolates were categorized into 4 phylogenetic groups A (39.4%), B2 (24.25%), D (24.25%) and B1 (12.1%). The pathogenic B2 and D groups were mainly typical for the Laridae family. Among the 28 virulence factors (Vfs) detected in pathogenic phylogenetic groups B2 and D, 7 were exclusively found in those groups (sfa, vat, tosA, tosB, hly, usp, cnf), while 4 VFs (fecA, fyuA, irp2, kspMTII) showed a statistically significant association (P≤0.05) with phylogroups A and B1. Our results indicated that strains belonging to commensal phylogroups A/B1 possess extensive iron acquisition systems (93,9%) and autotransporters (60,6%), typical for pathogens, hence we suggest that these strains evolve towards higher levels of virulence. This study, which is a point assessment of the virulence and drug resistance potential of wild birds, confirms the importance of taking wild birds as a reservoir of strains that pose a growing threat to humans. The E. coli analyzed in our study derive from different phylogenetic groups and possess an arsenal of antibiotic resistance genes and virulence factors that contribute to their ability to cause diseases.

Unbalanced restriction impairs SOS-induced DNA repair effects.

The contribution of a type II restriction-modification system (R-M system) to genome integrity and cell viability was investigated. We established experimental conditions which enabled the achievement of hemimethylated and unmethylated states for the specific bases of the recognition sequences of the host's DNA. To achieve this, we constructed the MboII R-M system containing only one (i.e. M2.MboII) out of two functional MboII methyltransferases found in Moraxella bovis. Using the incomplete R-M system we were able to perturb the balance between methylation and restriction in an inducible manner. We demonstrate that upon the SOS-induced DNA repair in the mitomycin C treated cells, restriction significantly reduces cell viability. Similar results for the well-studied wild type EcoRI R-M system, expressed constitutively in Escherichia coli, were obtained. Our data provide further insights into the benefits and disadvantages of maintaining of a type II R-M system, highlighting its impact on host cell fitness.

Low-level expression of the Type II restriction-modification system confers potent bacteriophage resistance in Escherichia coli.

Restriction-modification systems (R-M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R-M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R-M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R-M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R-M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R-M operon. Our data provided further insights into Type II R-M system maintenance and the potential conflict within the host bacterium.

The SfaNI restriction-modification system from Enterococcus faecalis NEB215 is located on a putative mobile genetic element.

A type IIS restriction-modification (R-M) system SfaNI from Enterococcus faecalis NEB215 has been characterized. The sfaNIM gene was cloned by the methylase selection method. Methyltransferase SfaNI, a protein of 695 amino acids, consists of two domains responsible for different DNA-strand recognition and modification, and a putative DNA-binding HTH domain located in the N-terminal part of the protein. The sfaNIR gene, located adjacent to the gene of the cognate modification methyltransferases, encodes a protein of 648 amino acids. The enzyme has been purified to apparent homogeneity and its biochemical characteristics have been described. The R-M system SfaNI is flanked by a transposase gene at its 5(') end, and a cassette chromosome recombinase (ccr) gene complex, encoding serine recombinases CcrA and CcrB, at the 3(') end. Both proteins are specifically involved in genome rearrangement and are widely distributed among staphylococcal species. These results suggested that the R-M system SfaNI is present on the putative mobile element.

Phenotypic and molecular characteristics of an Aeromonas hydrophila strain isolated from the River Nile.

Aeromonas hydrophila, an inhabitant of aquatic ecosystems found in most parts of the world, has considerable virulence potential. The polymerase chain reaction technique was used to assay for the presence of five virulence factor genes: haemolytic toxins aerA and ahh1, elastase ahyB, the enterotoxin act, and the polar flagella flaA/flaB in the A. hydrophila strain isolated from the River Nile. Drug screening showed high levels of resistance to ß-lactam antibiotics and tetracycline. Slime production was determined by the Congo red agar plate test. The isolate produced two restriction enzymes named AehI and AehII which are isoschizomers of XhoI and StuI respectively. The complete nucleotide sequence of the cryptic plasmid pAhy2.5 (2524 bp) from this strain was determined. Sequence analysis revealed the presence of two open reading frames (ORFs) encoding putative proteins. The protein coded by ORF1 is homologous with Rep proteins of plasmids belonging to the pC194 family, which are known to replicate by the rolling-circle mechanism. The putative double-strand origin of replication and a region with palindromic sequences that could function as a single-strand origin were detected in pAhy2.5.

Improvement in the visual discrimination of recombinant clones by size reduction of non-recombinant colonies.

A flexible approach circumventing cloning problems related to incomplete vector double digest is described. DNA methyltransferase gene insertion into MCS of commonly used expression vectors facilitates identification of both: i) the correct linear fragment in agarose gels due to the dilator effect, and ii) recombinant colonies by size and opacity differences resulting from methyltransferase toxicity.

The formation of persister cells in stationary-phase cultures of Escherichia coli is associated with the aggregation of endogenous proteins.

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.

Antibiotics promoting oxidative stress inhibit formation of Escherichia coli biofilm via indole signalling.

Recent studies have revealed that antibiotics can promote the formation of reactive oxygen species which contribute to cell death. In this study, we report that five different antibiotics known to stimulate production of reactive oxygen species inhibited growth of Escherichia coli biofilm. We demonstrated that supression of biofilm formation was mainly a consequence of the increase in the extracellular concentration of indole, a signal molecule which suppresses growth of bacterial biofilm. Indole production was enhanced under antibiotic-mediated oxidative stress due to overexpression of tryptophanase (TnaA), which catalyzes synthesis of indole. We found that DMSO (dimethyl sulfoxide), a hydrogen peroxide scavenger, or the lack of trypthophanase, which catalyzes production of indole, partly restored formation of E. coli biofilm in the presence of antibiotics. In conclusion, these findings confirmed that antibiotics which promote formation of ROS (reactive oxygen species) can inhibit development of E. coli biofilm in an indole-dependent process.

M1.MboII and M2.MboII type IIS methyltransferases: different specificities, the same target.

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction-modification (R-M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5'-GAAGA-3'/3'-CTTCT-5'. M1.MboII modifies the last adenine in the recognition sequence 5'-GAAGA-3' to N(6)-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3'-CTTCT-5', yielding N(4)-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000+/-1000 Da under denaturing conditions. Divalent cations (Ca(2+), Mg(2+), Mn(2+) and Zn(2+)) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R-M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.