Resistance Genes In Pathogenic Bacteria Research Articles

Penicillin-Binding Proteins, β-Lactamases, and β-Lactamase Inhibitors in β-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms.

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the β-lactam antibiotic cephamycin C, a class A β-lactamase, and the β lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the β-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their β-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel β-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the β-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the β-lactamase in S. clavuligerus may be modulated by the β-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the β-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between β-lactam biosynthesis and β-lactam resistance mechanisms in the producer actinobacteria.

Antibiotic resistance from, and to the environment

<abstract> Antibiotic resistance currently causes hundreds of thousands of deaths worldwide; it is a major and growing public health threat globally. The origins of many resistance genes in pathogenic bacteria can be traced down to the environment; while a staggering number of resistant bacteria and resistance genes, selected for by human activities, are released into the environment. The nature, quantities and fates of this bidirectional flux of organisms and genes are mostly unknown. In order to understand the evolution of resistance within clinical settings, and the impact of the release of resistant bacteria into the environment, it is crucial to assess these questions and to assemble an integrated view of the problem. This review aims at providing an update on related issues previously discussed elsewhere, and to contribute to the comprehensive understanding of the environment as a source, receptacle and reservoir of antibiotic resistance. </abstract>

Screening of antibiotic resistance genes in pathogenic bacteria isolated from tiny freshwater shrimp (Macrobrachium lanchesteri) and "Kung Ten", the uncooked Thai food.

Objective:This study aimed to isolate and identify of pathogenic bacteria in tiny freshwater shrimp (Macrobrachium lanchesteri) and in Kung Ten, which is an unusual Thai cuisine that eaten alive shrimp directly. Antimicrobial susceptibility test and identification of antibiotic resistance genes for isolated bacteria were conducted.Materials and Methods:Eighty of fresh shrimp samples and forty of Kung Ten salads were collected from four fresh markets, which were located in Bangkok and Nonthaburi province (N = 120). The isolation, identification, and antimicrobial susceptibility test of pathogenic bacteria were done following the Clinical and Laboratory Standards Institute guidelines. Antibiotic-resistant bacteria were screened for β-lactamase relating genes, such as AmpC (MOX and ACC genes), blaCTX-M, and Int1 genes.Results:The number of bacterial isolates in tiny freshwater shrimp and Kung Ten salad was 136 and 65, respectively. Aeromonas caviae, A. hydrophilla, Proteus penneri, Proteus vulgaris, and Klebsiella pneumoniae were commonly found. Ampicillin, amoxicillin/clavulanic, cefuroxime, tetracycline, and trimethoprim/sulfamethoxazole resistance were observed, and common antibiotic-resistant bacteria were A. caviae, P. vulgaris, Enterobacter Aerogenes, and K. pneumoniae. A. caviae, P. penneri, K. Pneumoniae, and A. hydrophilla were positive for MOX gene; blaCTX-M, and Int1 genes; ACC and Int1 genes; and ACC gene, respectively.Conclusion:Raw or uncooked shrimps in Kung Ten salad may a risk in foodborne diseases due to positive for pathogenic bacterial isolates. However, hygienic control on food preparation is difficult to apply because of the difficulty of changing in local Thai food behavior.

Epidemic characterization and molecular genotyping of Shigella flexneri isolated from calves with diarrhea in Northwest China

BackgroundThe widespread presence of antibiotics resistance genes in pathogenic bacteria can cause enormous problems. Food animals are one of the main reservoirs of intestinal pathogens that pose a potential risk to human. Analyzing the epidemiological characteristics and resistance patterns of Shigella flexneri in calves is necessary for animal and human health.Methods and resultsA total of 54 Shigella flexneri isolates, including six serotypes (1a, 2a, 2b, 4a, 6 and Xv), were collected from 837 fecal samples obtained from 2014 to 2016. We performed pulsed-field gel electrophoresis (PFGE) and applied the restriction enzyme NotI to analyze the genetic relatedness among the 54 isolates and to categorize them into 31 reproducible and unique PFGE patterns. According to the results of antimicrobial susceptibility tests, all 26 Shigella flexneri 2a serotypes were resistant to cephalosporin and/or fluoroquinolones. The genes blaTEM-1, blaOXA-1, and blaCTX-M-14 were detected in 19 cephalosporin-resistant S. flexneri 2a isolates. Among 14 fluoroquinolone-resistant isolates, the aac(6′)-Ib-cr gene was largely present in each strain, followed by qnrS (5). Only one ciprofloxacin-resistant isolate harbored the qepA gene. Sequencing the quinolone resistance determining regions (QRDRs) of the fluoroquinolone-resistant isolates revealed two point mutations in gyrA (S83 L, D87N/Y) and a single point mutation in parC (S80I). Interestingly, two gyrA (D87N/Y) strains were resistant to ciprofloxacin.ConclusionsThe current study enhances our knowledge of Shigella in cattle, although continual surveillance is necessary for the control of shigellosis. The high level of cephalosporin and/or fluoroquinolone resistance in Shigella warns us of a potential risk to human and animal health.

Phylogenetic Considerations in the Evolutionary Development of Aminoglycoside Resistance Genes in Pathogenic Bacteria

This study revisits antibiotic resistance as a source of evolutionary development in pathogenic bacteria. By taking a molecular phylogenetic approach to this inquiry, I seek to find homologous correlations in antimicrobial resistance gene families across a broad spectrum of bacteria, as to identify the possible acquisition of those genes through divergent events in evolutionary context. In order to test this, I examine the various degrees of genetic similarity in two antimicrobial resistance genomic datasets, namely aadA1 and aadA2 aminoglycoside resistance genes, among bacteria that occur in a multitude of environments. Moreover, the results from phylogenetic analysis suggests that pathogenic antibiotic resistance for aadA1 and aadA2 aminoglycoside resistance genes may have been acquired through evolutionary events with a common ancestor of a soil-dwelling bacterium.

Phylogenetic Considerations in the Evolutionary Development of Aminoglycoside Resistance Genes in Pathogenic Bacteria

This study revisits antibiotic resistance as a source of evolutionary development in pathogenic bacteria. By taking a molecular phylogenetic approach to this inquiry, I seek to find homologous correlations in antimicrobial resistance gene families across a broad spectrum of bacteria, as to identify the possible acquisition of those genes through divergent events in evolutionary context. In order to test this, I examine the various degrees of genetic similarity in two antimicrobial resistance genomic datasets, namely aadA1 and aadA2 aminoglycoside resistance genes, among bacteria that occur in a multitude of environments. Moreover, the results from phylogenetic analysis suggests that pathogenic antibiotic resistance for aadA1 and aadA2 aminoglycoside resistance genes may have been acquired through evolutionary events with a common ancestor of a soil-dwelling bacterium.

Functional Metagenome Mining of Soil for a Novel Gentamicin Resistance Gene.

Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.

Gene Network Analysis of Metallo Beta Lactamase Family Proteins Indicates the Role of Gene Partners in Antibiotic Resistance and Reveals Important Drug Targets.

Metallo Beta (β) Lactamases (MBL) are metal dependent bacterial enzymes that hydrolyze the β-lactam antibiotics. In recent years, MBL have received considerable attention because it inactivates most of the β-lactam antibiotics. Increase in dissemination of MBL encoding antibiotic resistance genes in pathogenic bacteria often results in unsuccessful treatments. Gene interaction network of MBL provides a complete understanding on the molecular basis of MBL mediated antibiotic resistance. In our present study, we have constructed the MBL network of 37 proteins with 751 functional partners from pathogenic bacterial spp. We found 12 highly interconnecting clusters. Among the 37 MBL proteins considered in the present study, 22 MBL proteins are from B3 subclass, 14 are from B1 subclass and only one is from B2 subclass. Global topological parameters are used to calculate and compare the probability of interactions in MBL proteins. Our results indicate that the proteins associated within the network have a strong influence in antibiotic resistance mechanism. Interestingly, several drug targets are identified from the constructed network. We believe that our results would be helpful for researchers exploring MBL-mediated antibiotic resistant mechanisms.

Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

ABSTRACTThe pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netB was insertionally inactivated by the chloramphenicol and thiamphenicol resistance gene catP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and the netB gene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 from C. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained the netB gene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2 gene, which encodes a different pore-forming toxin, beta2 toxin.

Compensatory Evolution of pbp Mutations Restores the Fitness Cost Imposed by β-Lactam Resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.

Genomic evidence for antibiotic resistance genes of actinomycetes as origins of antibiotic resistance genes in pathogenic bacteria simply because actinomycetes are more ancestral than pathogenic bacteria

Although in silico analysis have suggested that the antibiotic resistance genes in actinomycetes appear to be the origins of some antibiotic resistance genes, we have shown that recent horizontal transfer of antibiotic resistance genes from actinomycetes to other medically important bacteria have not taken place. Although it has been speculated in Benveniste and Davies' attractive hypothesis that antibiotic resistance genes of actinomycetes are origins of antibiotic resistance genes in pathogenic bacteria because the actinomycetes require mechanisms such as metabolic enzymes (encoded by the antibiotic resistance genes) to degrade the antibiotics they produce or to transport the antibiotics outside the bacterial cells, this hypothesis has never been proven. Both the phylogenetic tree constructed using 16S rRNA gene sequences and that constructed using concatenated amino acid sequences of 15 housekeeping genes extracted from 90 bacterial genomes showed that the actinomycetes is more ancestral to most other bacteria, including the pathogenic Gram-negative bacteria, Gram-positive bacteria, and Chlamydia species. Furthermore, the tetracycline resistance gene of Bifidobacterium longum is more ancestral to those of other pathogenic bacteria and the actinomycetes, which is in line with the ancestral position of B. longum. These suggest that the evolution of antibiotic resistance genes of antibiotic-producing bacteria in general parallels the evolution of the corresponding bacteria. The ancestral position of the antibiotic resistance genes in actinomycetes is probably unrelated to the fact that they produce antibiotics, but simply because actinomycetes are more ancestral than pathogenic bacteria.

Genes for a beta-lactamase, a penicillin-binding protein and a transmembrane protein are clustered with the cephamycin biosynthetic genes in Nocardia lactamdurans.

Three genes encoding a typical beta-lactamase, a penicillin-binding protein (PBP4) and a transmembrane protein are located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans. The similarity of the N. lactamdurans beta-lactamase to class A beta-lactamases from clinical isolates supports the hypothesis that antibiotic resistance genes in pathogenic bacteria are derived from antibiotic-producing organisms. The beta-lactamase is secreted and is active against penicillins (including the biosynthetic intermediates penicillin N and isopenicillin N), but not against cephamycin C. The beta-lactamase is synthesized during the active growth phase, prior to the formation of three cephamycin biosynthetic enzymes. The PBP of N. lactamdurans is a low-M(r) protein that is very similar to DD-carboxypeptidases of Streptomyces and Actinomadura. The pbp gene product expressed in Streptomyces lividans accumulates in the membrane fraction. By disruption of N. lactamdurans protoplasts, the PBP4 was shown to be located in the plasma membrane. Eight PBPs were found in the membranes of N. lactamdurans, none of which bind cephamycin C, which explains the resistance of this strain to its own antibiotic. A transmembrane protein encoded by the cmcT gene of the cluster also accumulates in the membrane fraction and is probably related to the control of synthesis and secretion of the antibiotic. A balanced synthesis of beta-lactam antibiotics, beta-lactamase and PBP is postulated to be critical for the survival of beta-lactam-producing actinomycetes.