Agata Krawczyk-Balska

Influence of Soil Use on Prevalence of Tetracycline, Streptomycin, and Erythromycin Resistance and Associated Resistance Genes

ABSTRACT This study examined differences in antibiotic-resistant soil bacteria and the presence and quantity of resistance genes in soils with a range of management histories. We analyzed four soils from agricultural systems that were amended with manure from animals treated with erythromycin and exposed to streptomycin and/or oxytetracycline, as well as non-manure-amended compost and forest soil. Low concentrations of certain antibiotic resistance genes were detected using multiplex quantitative real-time PCR (qPCR), with tet(B), aad(A), and str(A) each present in only one soil and tet(M) and tet(W) detected in all soils. The most frequently detected resistance genes were tet(B), tet(D), tet(O), tet(T), and tet(W) for tetracycline resistance, str(A), str(B), and aac for streptomycin resistance, and erm(C), erm(V), erm(X), msr(A), ole(B), and vga for erythromycin resistance. Transposon genes specific for Tn916, Tn1549, TnB1230, Tn4451, and Tn5397 were detected in soil bacterial isolates. The MIC ranges of isolated bacteria for tetracycline, streptomycin, and erythromycin were 8 to >256 μg/ml, 6 to >1,024 μg/ml, and 0.094 to >256 μg/ml, respectively. Based on 16S rRNA gene similarity, isolated bacteria showed high sequence identity to genera typical of soil communities. Bacteria with the highest MICs were detected in manure-amended soils or soils from agricultural systems with a history of antibiotic use. Non-manure-amended soils yielded larger proportions of antibiotic-resistant bacteria, but these had lower MICs, carried fewer antibiotic resistance genes, and did not display multidrug resistance (MDR).

Identification of a ferritin-like protein of Listeria monocytogenes as a mediator of β-lactam tolerance and innate resistance to cephalosporins

BackgroundThe food-borne pathogen Listeria monocytogenes is the causative agent of listeriosis. The β-lactam antibiotics penicillin G and ampicillin are the current drugs of choice for the treatment of listerial infections. While isolates of L. monocytogenes are susceptible to these antibiotics, their action is only bacteriostatic and consequently, this bacterium is regarded as tolerant to β-lactams. In addition, L. monocytogenes has a high level of innate resistance to the cephalosporin family of β-lactams frequently used to treat sepsis of unknown etiology. Given the high mortality rate of listeriosis despite rational antibiotic therapy, it is important to identify genes that play a role in the susceptibility and tolerance of L. monocytogenes to β-lactams.ResultsThe hly-based promoter trap system was applied to identify penicillin G-inducible genes of L. monocytogenes. The results of reporter system studies, verified by transcriptional analysis, identified ten penicillin G-inducible genes. The contribution of three of these genes, encoding a ferritin-like protein (fri), a two-component phosphate-response regulator (phoP) and an AraC/XylS family transcriptional regulator (axyR), to the susceptibility and tolerance of L. monocytogenes to β-lactams was examined by analysis of nonpolar deletion mutants. The absence of PhoP or AxyR resulted in more rapid growth of the strains in the presence of sublethal concentration of β-lactams, but had no effect on the MIC values or the ability to survive a lethal dose of these antibiotics. However, the Δfri strain showed impaired growth in the presence of sublethal concentrations of penicillin G and ampicillin and a significantly reduced ability to survive lethal concentrations of these β-lactams. A lack of Fri also caused a 2-fold increase in the sensitivity of L. monocytogenes to cefalotin and cephradine.ConclusionsThe present study has identified Fri as an important mediator of β-lactam tolerance and innate resistance to cephalosporins in L. monocytogenes. PhoP and AxyR are probably involved in transmitting signals to adjust the rate of growth of L. monocytogenes under β-lactam pressure, but these regulators do not play a significant role in susceptibility and tolerance to this class of antibiotics.

The intrinsic cephalosporin resistome of Listeria monocytogenes in the context of stress response, gene regulation, pathogenesis and therapeutics

Intrinsic resistance to antibiotics is a serious therapeutic problem in the case of many bacterial species. The Gram‐positive human pathogen Listeria monocytogenes is intrinsically resistant to broad spectrum cephalosporin antibiotics, which are commonly used in therapy of bacterial infections. Besides three penicillin‐binding proteins the intrinsic cephalosporin resistome of L. monocytogenes includes multidrug resistance transporter transporters, proteins involved in peptidoglycan biosynthesis and modification, cell envelope proteins with structural or general detoxification function, cytoplasmic proteins with unknown function and regulatory proteins. Analysis of the regulation of the expression of genes involved in the intrinsic resistance of L. monocytogenes to cephalosporins highlights the high complexity of control of the intrinsic resistance phenotype. The regulation of the transcription of the intrinsic resistome determinants involves the activity of eight regulators, namely LisR, CesR, LiaR, VirR, σB, σH, σL and PrfA, of which the most prominent role play LisR, CesR and σB. Furthermore, the vast majority of the intrinsic resistome determinants contribute to the tolerance of different stress conditions and virulence. A study indicates that O‐acetyltransferase OatA is the most promising candidate for co‐drug development since an agent targeting OatA should sensitize L. monocytogenes to certain antibiotics, therefore improving the efficacy of listeriosis treatment as well as food preservation measures.

Re-evaluation of the significance of penicillin binding protein 3 in the susceptibility of Listeria monocytogenes to β-lactam antibiotics

BackgroundPenicillin binding protein 3 (PBP3) of L. monocytogenes has long been thought of as the primary lethal target for β-lactam antibiotics due to the excellent correlation between the MICs of different β-lactams and their affinity for this protein. The gene encoding PBP3 has not yet been directly identified in this gram-positive bacterium, but based on in silico analysis, this protein is likely to be encoded by lmo1438. However, studies examining the effects of mutations in genes encoding known and putative L. monocytogenes PBPs have demonstrated that inactivation of lmo1438 does not affect sensitivity to β-lactams.ResultsIn this study, overexpression of lmo1438 was achieved using an inducible (nisin-controlled) expression system. This permitted the direct demonstration that lmo1438 encodes PBP3. PBP3 overexpression was accompanied by slightly elevated PBP4 expression. The recombinant strain overexpressing PBP3 displayed significant growth retardation and greatly reduced cell length in the stationary phase of growth in culture. In antibiotic susceptibility assays, the strain overexpressing PBP3 displayed increased sensitivity to subinhibitory concentrations of several β-lactams and decreased survival in the presence of a lethal dose of penicillin G. However, the MIC values of the tested β-lactams for this recombinant strain were unchanged compared to the parent strain.ConclusionsThe present study allows a reevaluation of the importance of PBP3 in the susceptibility of L. monocytogenes to β-lactams. It is clear that PBP3 is not the primary lethal target for β-lactams, since neither the absence nor an excess of this protein affect the susceptibility of L. monocytogenes to these antibiotics. The elevated level of PBP4 expression observed in the recombinant strain overexpressing PBP3 demonstrates that the composition of the L. monocytogenes cell wall is subject to tight regulation. The observed changes in the morphology of stationary phase cells in response to PBP3 overexpression suggests the involvement of this protein in cell division during this phase of growth.

An update on the transport and metabolism of iron in Listeria monocytogenes: the role of proteins involved in pathogenicity

Listeria monocytogenes is a Gram-positive bacterium that causes a rare but severe human disease with high mortality rate. The microorganism is widespread in the natural environment where it shows a saprophytic lifestyle. In the human body it infects many different cell types, where it lives intracellularly, however it may also temporarily live extracellularly. The ability to survive and grow in such diverse niches suggests that this bacterium has a wide range of mechanisms for both the acquisition of various sources of iron and effective management of this microelement. In this review, data about the mechanisms of transport, metabolism and regulation of iron, including recent findings in these areas, are summarized with focus on the importance of these mechanisms for the virulence of L. monocytogenes. These data indicate the key role of haem transport and maintenance of intracellular iron homeostasis for the pathogenesis of L. monocytogenes. Furthermore, some of the proteins involved in iron homeostasis like Fri and FrvA seem to deserve special attention due to their potential use in the development of new therapeutic antilisterial strategies.

Critical role of a ferritin-like protein in the control of Listeria monocytogenes cell envelope structure and stability under β-lactam pressure.

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenes Δ fri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure.

InlL from Listeria monocytogenes Is Involved in Biofilm Formation and Adhesion to Mucin

The bacterial etiological agent of listeriosis, Listeria monocytogenes, is an opportunistic intracellular foodborne pathogen. The infection cycle of L. monocytogenes is well-characterized and involves several key virulence factors, including internalins A and B. While 35 genes encoding internalins have been identified in L. monocytogenes, less than half of them have been characterized as yet. Focusing on lmo2026, it was shown this gene encodes a class I internalin, InlL, exhibiting domains potentially involved in adhesion. Following a functional genetic approach, InlL was demonstrated to be involved in initial bacterial adhesion as well as sessile development in L. monocytogenes. In addition, InlL enables binding to mucin of type 2, i.e., the main secreted mucin making up the mucus layer, rather than to surface-located mucin of type 1. InlL thus appears as a new molecular determinant contributing to the colonization ability of L. monocytogenes.

Listeria monocytogenes protein p60 affects hemolytic activity and uptake of bacteria by macrophages

Bacillus subtilis strains expressing listeriolysin O (LLO) and simultaneously LLO and p60 protein were constructed. The effect of p60 protein on hemolytic activity and on the invasion of professional phagocytes was demonstrated in the absence of other virulence factors ofL. monocytogenes. The hemolytic activity of LLO in the presence of p60 protein decreased which indicates that p60 promoted adhesion and subsequent invasion of professional phagocytes.

Identification of the Molecular Mechanism of Trimethoprim Resistance in Listeria monocytogenes

Trimethoprim with sulfamethoxazole is a therapeutic agent combination used to treat infections caused by the facultative intracellular foodborne pathogen Listeria monocytogenes. The aim of this study was to assess the frequency of resistance of L. monocytogenes arising due to exposure to trimethoprim and subsequently investigate the molecular mechanisms of resistance. After exposure of a culture of L. monocytogenes ATCC 13932 to trimethoprim at 10-fold the minimal inhibitory concentration spontaneous resistant mutants were recovered, giving a frequency of resistance development of 6.85 ± 0.92 × 10-8. The isolates exhibited a 32-64-fold decrease in susceptibility compared with the parental strain. These results indicate the capacity of L. monocytogenes to develop low-level resistance toward trimethoprim after exposure to the drug. The trimethoprim resistance genes (dhfr) and their promoter regions from all trimethoprim-resistant isolates were amplified and sequenced, leading to the identification of four single amino acid substitutions (Met20-Val, Pro21-Leu, Thr46-Asn, Val95-Leu) and two double substitutions (Met20-Ile+Thr46-Asn and Thr46-Asn+Leu85-Phe) in DHFR. Of the identified mutations, the Thr46-Asn substitution has not been previously reported as the mechanism of resistance to trimethoprim in other bacteria; thus this substitution seems to be unique to L. monocytogenes. The expression of the mutated L. monocytogenes dhfr genes in Escherichia coli led to decreased susceptibility of the heterological host, therefore proving that the identified point mutations in dhfr serve as the molecular mechanism of acquired resistance of L. monocytogenes to trimethoprim.

Rifampicin- and Rifabutin-Resistant Listeria monocytogenes Strains Isolated from Food Products Carry Mutations in rpoB Gene

The aim of this study was to investigate the mechanism of rifampicin resistance in Listeria monocytogenes strains isolated from different types of food and the impact of specific mutations in the rpoB gene on susceptibility to different antimicrobial agents and on fitness cost. Fifteen spontaneous rifampicin-resistant strains were selected. The DNA regions corresponding to clusters I-II, III, and N-terminal end of the rpoB gene of Escherichia coli were amplified and sequenced, leading to the identification of 10 different substitutions, nine of which (Ser466Pro, Gln470Lys Asp473Asn, Gly479Asp, His483Tyr/Arg/Asp, Arg486His, and Leu490Pro) were located in cluster I and one (Pro521Leu) in cluster II. From among these mutations, substitutions at positions 466, 470, 486, 490, and 521 have not been described for L. monocytogenes. Only substitutions at positions 470, 479, 483, and 486 lead to resistance to very high concentrations of rifampicin (minimum inhibitory concentration [MIC] ≥256 μg/mL) and rifabutin (MIC 128 μg/mL). Furthermore, mutations at positions 473, 490, and 521 had different effects on susceptibility to rifampicin compared to other bacterial species. A correlation between rifampicin resistance and susceptibility to a wide range of antimicrobials was determined. Substitutions in RpoB did not change the susceptibility of the mutants to different antimicrobials. The fitness of the mutants was assessed by paired competition experiments. Mutations at positions 470 and 479 were not associated with a reduction in fitness level. There was no correlation between the MIC of rifampicin and fitness cost. The risk of transmission of resistant strains through the food chain highlights the need for monitoring resistance, identifying mutant organisms, their genotypes, and their altered phenotypes to understand their dissemination.