Development of a Rapid, Culture-Free, Universal Microbial Identification System Using Internal Transcribed Spacer Targeting Primers. - Related Documents




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507901.0000Development of a Rapid, Culture-Free, Universal Microbial Identification System Using Internal Transcribed Spacer Targeting Primers. The indiscriminate administration of broad-spectrum antibiotics is a primary contributor to the increasing prevalence of antibiotic resistance. Unfortunately, culture, the gold standard for bacterial identification is a time intensive process. Due to this extended diagnostic period, broad-spectrum antibiotics are generally prescribed to prevent poor outcomes. To overcome the deficits of culture-based methods, we have developed a rapid universal bacterial identification system. The platform uses a unique universal polymerase chain reaction primer set that targets the internal transcribed spacer regions between conserved bacterial genes, creating a distinguishable amplicon signature for every bacterial species. Bioinformatic simulation demonstrates that nearly every bacteria in a set of 45 commonly isolated pathogenic species can be uniquely identified using this approach. We experimentally confirmed these predictions on a representative set of pathogenic bacterial species. We further showed that the system can determine the corresponding concentration of each pathogen. Finally, we validated performance in clinical urinary tract infection samples.202539503259
508010.9999Rapid screening for antibiotic resistance elements on the RNA transcript, protein and enzymatic activity level. BACKGROUND: The emerging threat posed by antibiotic resistance has affected public health systems all over the world. Surveillance of resistant bacteria in clinical settings and identifying them in mixed cultures is of paramount importance and can contribute to the control of their spreading. Culture-independent monitoring approaches are highly desirable, since they yield results much faster than traditional susceptibility testing. However, many rapid molecular methods like PCR only detect the sole presence of a potential resistance gene, do not provide information regarding efficient transcription, expression and functionality and, in addition, cannot assign resistance genes to species level in mixed cultures. METHODS: By using plasmid-encoded TEM β-lactamase mediated ampicillin resistances as a proof of principle system, we (1) developed a fluorescence in situ hybridization-test (FISH) capable to detect the respective mRNAs, (2) implemented an immunofluorescence test to identify the corresponding proteins and (3) compared these two microscopic tests with an established colorimetric nitrocefin assay to assess the enzymatic activity. RESULTS: All three methods proved to be suitable for the testing of antibiotic resistance, but only FISH and immunofluorescence were able to differentiate between susceptible and resistant bacteria on the single cell level and can be combined with simultaneous species identification. CONCLUSIONS: Fluorescence in situ hybridization and immunofluorescence tests are promising techniques in susceptibility testing since they bridge the gap between the slow, but accurate and sound cultural methods and molecular detection methods like PCR with much less functional relevance.201627663856
507720.9999Development of a new integrated diagnostic test for identification and characterization of pathogens. Animal diseases directly cause multi-million dollar losses world-wide. Therefore a rapid, highly specific, cost-effective diagnostic test for detecting a large set of bacterial virulence and antimicrobial resistance genes simultaneously is necessary. Hence, our group, the BCBG (Bacterial Chips Bacterial Genes) group, proposes developing a powerful molecular tool (DNA microarray) to detect a broad range of infectious agents, their endogenous main virulence factors and antibiotic resistance genes simultaneously. Effectively, a 70-mer oligonucleotide microarray capable of detecting the presence or absence of 169 Escherichia coli virulence genes or virulence marker genes as well as their variants, in addition to 30 principal antimicrobial resistance genes previously characterized in E. coli strains was developed by our group. This microarray was validated with a large collection of well characterized pathogenic and reference E. coli strains. Moreover, we are developing a new powerful clinical diagnostic microarray tool, to identify pathogenic bacteria of veterinary interest. The commercialization of this assay would allow same day diagnosis of infectious agents and their antibiotic resistance resulting in early treatment. In addition, this technology is also applicable to microbial quality control of food and water.200617058497
507530.9998Fast and Economic Microarray-Based Detection of Species-, Resistance-, and Virulence-Associated Genes in Clinical Strains of Vancomycin-Resistant Enterococci (VRE). Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.202439409516
418140.9998The place of molecular genetic methods in the diagnostics of human pathogenic anaerobic bacteria. A minireview. Anaerobic infections are common and can cause diseases associated with severe morbidity, but are easily overlooked in clinical settings. Both the relatively small number of infections due to exogenous anaerobes and the much larger number of infections involving anaerobic species that are originally members of the normal flora, may lead to a life-threatening situation unless appropriate treatment is instituted. Special laboratory procedures are needed for the isolation, identification and susceptibility testing of this diverse group of bacteria. Since many anaerobes grow more slowly than the facultative or aerobic bacteria, and particularly since clinical specimens yielding anaerobic bacteria commonly contain several organisms and often very complex mixtures of aerobic and anaerobic bacteria, considerable time may elapse before the laboratory is able to provide a final report. Species definition based on phenotypic features is often time-consuming and is not always easy to carry out. Molecular genetic methods may help in the everyday clinical microbiological practice in laboratories dealing with the diagnostics of anaerobic infections. Methods have been introduced for species diagnostics, such as 16S rRNA PCR-RFLP profile determination, which can help to distinguish species of Bacteroides, Prevotella, Actinomyces, etc. that are otherwise difficult to differentiate. The use of DNA-DNA hybridization and the sequencing of special regions of the 16S rRNA have revealed fundamental taxonomic changes among anaerobic bacteria. Some anaerobic bacteria are extremely slow growing or not cultivatable at all. To detect them in special infections involving flora changes due to oral malignancy or periodontitis, for instance, a PCR-based hybridization technique is used. Molecular methods have demonstrated the spread of specific resistance genes among the most important anaerobic bacteria, the members of the Bacteroides genus. Their detection and investigation of the IS elements involved in their expression may facilitate following of the spread of antibiotic resistance among anaerobic bacteria involved in infections and in the normal flora members. Molecular methods (a search for toxin genes and ribotyping) may promote a better understanding of the pathogenic features of some anaerobic infections, such as the nosocomial diarrhoea caused by C. difficile and its spread in the hospital environment and the community. The investigation of toxin production at a molecular level helps in the detection of new toxin types. This mini-review surveys some of the results obtained by our group and others using molecular genetic methods in anaerobic diagnostics.200616956128
511150.9998Antimicrobial Resistance Prediction for Gram-Negative Bacteria via Game Theory-Based Feature Evaluation. The increasing prevalence of antimicrobial-resistant bacteria drives the need for advanced methods to identify antimicrobial-resistance (AMR) genes in bacterial pathogens. With the availability of whole genome sequences, best-hit methods can be used to identify AMR genes by differentiating unknown sequences with known AMR sequences in existing online repositories. Nevertheless, these methods may not perform well when identifying resistance genes with sequences having low sequence identity with known sequences. We present a machine learning approach that uses protein sequences, with sequence identity ranging between 10% and 90%, as an alternative to conventional DNA sequence alignment-based approaches to identify putative AMR genes in Gram-negative bacteria. By using game theory to choose which protein characteristics to use in our machine learning model, we can predict AMR protein sequences for Gram-negative bacteria with an accuracy ranging from 93% to 99%. In order to obtain similar classification results, identity thresholds as low as 53% were required when using BLASTp.201931597945
511260.9998Genome-Based Prediction of Bacterial Antibiotic Resistance. Clinical microbiology has long relied on growing bacteria in culture to determine antimicrobial susceptibility profiles, but the use of whole-genome sequencing for antibiotic susceptibility testing (WGS-AST) is now a powerful alternative. This review discusses the technologies that made this possible and presents results from recent studies to predict resistance based on genome sequences. We examine differences between calling antibiotic resistance profiles by the simple presence or absence of previously known genes and single-nucleotide polymorphisms (SNPs) against approaches that deploy machine learning and statistical models. Often, the limitations to genome-based prediction arise from limitations of accuracy of culture-based AST in addition to an incomplete knowledge of the genetic basis of resistance. However, we need to maintain phenotypic testing even as genome-based prediction becomes more widespread to ensure that the results do not diverge over time. We argue that standardization of WGS-AST by challenge with consistently phenotyped strain sets of defined genetic diversity is necessary to compare the efficacy of methods of prediction of antibiotic resistance based on genome sequences.201930381421
462870.9998Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection. To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction-modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.202135250902
507680.9998Diagnostic microarray for human and animal bacterial diseases and their virulence and antimicrobial resistance genes. Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure.201020035807
992290.9998De novo acquisition of antibiotic resistance in six species of bacteria. Bacteria can become resistant to antibiotics in two ways: by acquiring resistance genes through horizontal gene transfer and by de novo development of resistance upon exposure to non-lethal concentrations. The importance of the second process, de novo build-up, has not been investigated systematically over a range of species and may be underestimated as a result. To investigate the DNA mutation patterns accompanying the de novo antibiotic resistance acquisition process, six bacterial species encountered in the food chain were exposed to step-wise increasing sublethal concentrations of six antibiotics to develop high levels of resistance. Phenotypic and mutational landscapes were constructed based on whole-genome sequencing at two time points of the evolutionary trajectory. In this study, we found that (1) all of the six strains can develop high levels of resistance against most antibiotics; (2) increased resistance is accompanied by different mutations for each bacterium-antibiotic combination; (3) the number of mutations varies widely, with Y. enterocolitica having by far the most; (4) in the case of fluoroquinolone resistance, a mutational pattern of gyrA combined with parC is conserved in five of six species; and (5) mutations in genes coding for efflux pumps are widely encountered in gram-negative species. The overall conclusion is that very similar phenotypic outcomes are instigated by very different genetic changes. The outcome of this study may assist policymakers when formulating practical strategies to prevent development of antimicrobial resistance in human and veterinary health care.IMPORTANCEMost studies on de novo development of antimicrobial resistance have been performed on Escherichia coli. To examine whether the conclusions of this research can be applied to more bacterial species, six species of veterinary importance were made resistant to six antibiotics, each of a different class. The rapid build-up of resistance observed in all six species upon exposure to non-lethal concentrations of antimicrobials indicates a similar ability to adjust to the presence of antibiotics. The large differences in the number of DNA mutations accompanying de novo resistance suggest that the mechanisms and pathways involved may differ. Hence, very similar phenotypes can be the result of various genotypes. The implications of the outcome are to be considered by policymakers in the area of veterinary and human healthcare.202539907470
3808100.9998Expression Profiling of Antibiotic-Resistant Bacteria Obtained by Laboratory Evolution. To elucidate the mechanisms of antibiotic resistance, integrating phenotypic and genotypic features in resistant strains is important. Here, we describe the expression profiling of antibiotic-resistant Escherichia coli strains obtained by laboratory evolution, and a method for extracting a small number of genes whose expression changes can contribute to the acquisition of resistance.201727873258
4629110.9998Screening and in silico characterization of prophages in Helicobacter pylori clinical strains. The increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we developed a method to identify prophages in H. pylori genomes aiming at their future use in therapy. A polymerase chain reaction (PCR)-based technique tested five primer pairs on 74 clinical H. pylori strains. After the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. This study allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Overall, we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.202539368610
9405120.9998Functional Metagenomic Screening for Antimicrobial Resistance in the Oral Microbiome. A large proportion of bacteria, from a multitude of environments, are not yet able to be grown in the laboratory, and therefore microbiological and molecular biological investigations of these bacteria are challenging. A way to circumvent this challenge is to analyze the metagenome, the entire collection of DNA molecules that can be isolated from a particular environment or sample. This collection of DNA molecules can be sequenced and assembled to determine what is present and infer functional potential, or used as a PCR template to detect known target DNA and potentially unknown regions of DNA nearby those targets; however assigning functions to new or conserved hypothetical, functionally cryptic, genes is difficult. Functional metagenomics allows researchers to determine which genes are responsible for selectable phenotypes, such as resistance to antimicrobials and metabolic capabilities, without the prerequisite needs to grow the bacteria containing those genes or to already know which genes are of interest. It is estimated that a third of the resident species of the human oral cavity is not yet cultivable and, together with the ease of sample acquisition, makes this metagenome particularly suited to functional metagenomic studies. Here we describe the methodology related to the collection of saliva samples, extraction of metagenomic DNA, construction of metagenomic libraries, as well as the description of functional assays that have previously led to the identification of new genes conferring antimicrobial resistance.202134410638
4340130.9998Predicting antimicrobial susceptibility from the bacterial genome: A new paradigm for one health resistance monitoring. The laboratory identification of antibacterial resistance is a cornerstone of infectious disease medicine. In vitro antimicrobial susceptibility testing has long been based on the growth response of organisms in pure culture to a defined concentration of antimicrobial agents. By comparing individual isolates to wild-type susceptibility patterns, strains with acquired resistance can be identified. Acquired resistance can also be detected genetically. After many decades of research, the inventory of genes underlying antimicrobial resistance is well known for several pathogenic genera including zoonotic enteric organisms such as Salmonella and Campylobacter and continues to grow substantially for others. With the decline in costs for large scale DNA sequencing, it is now practicable to characterize bacteria using whole genome sequencing, including the carriage of resistance genes in individual microorganisms and those present in complex biological samples. With genomics, we can generate comprehensive, detailed information on the bacterium, the mechanisms of antibiotic resistance, clues to its source, and the nature of mobile DNA elements by which resistance spreads. These developments point to a new paradigm for antimicrobial resistance detection and tracking for both clinical and public health purposes.202133010049
4393140.9998Mechanisms of Staphylococcus aureus Antibiotics Resistance Revealed by Adaptive Laboratory Evolution. Infection caused by drug-resistant Staphylococcus aureus is a serious public health and veterinary concern. Lack of a comprehensive understanding of the mechanisms underlying the emergence of drug-resistant strains, it makes S. aureus one of the most intractable pathogenic bacteria. To identify mutations that confer resistance to anti-S. aureus drugs, we established a laboratory-based adaptive evolution system and performed 10 rounds of evolution experiments against 15 clinically used antibiotics. We discovered a panel of known and novel resistance-associated sites after performing whole-genome sequencing. Furthermore, we found that the resistance evolved at distinct rates. For example, streptomycin, rifampicin, fusidic acid and novobiocin all developed significant resistance quickly in the second round of evolution. Intriguingly, the cross-resistance experiment reveals that nearly all drug-resistant strains have varying degrees of increased sensitivity to fusidic acid, pointing to a novel approach to battle AMR. In addition, the in silico docking analysis shows that the evolved mutants affect the interaction of rifampcin-rpoB, as well as the novobiocin-gyrB. Moreover, for the genes we got in the laboratory evolution, mutant genes of clinical isolates of human had significant differences from the environmental isolates and animal isolates. We believe that the strategy and data set in this research will be helpful for battling AMR issue of S. aureus, and adaptable to other pathogenic microbes.202539762552
9917150.9998Fluorescence-Based Detection of Natural Transformation in Drug-Resistant Acinetobacter baumannii. Acinetobacter baumannii is a nosocomial agent with a high propensity for developing resistance to antibiotics. This ability relies on horizontal gene transfer mechanisms occurring in the Acinetobacter genus, including natural transformation. To study natural transformation in bacteria, the most prevalent method uses selection for the acquisition of an antibiotic resistance marker in a target chromosomal locus by the recipient cell. Most clinical isolates of A. baumannii are resistant to multiple antibiotics, limiting the use of such selection-based methods. Here, we report the development of a phenotypic and selection-free method based on flow cytometry to detect transformation events in multidrug-resistant (MDR) clinical A. baumannii isolates. To this end, we engineered a translational fusion between the abundant and conserved A. baumannii nucleoprotein (HU) and the superfolder green fluorescent protein (sfGFP). The new method was benchmarked against the conventional antibiotic selection-based method. Using this new method, we investigated several parameters affecting transformation efficiencies and identified conditions of transformability one hundred times higher than those previously reported. Using optimized transformation conditions, we probed natural transformation in a set of MDR clinical and nonclinical animal A. baumannii isolates. Regardless of their origin, the majority of the isolates displayed natural transformability, indicative of a conserved trait in the species. Overall, this new method and optimized protocol will greatly facilitate the study of natural transformation in the opportunistic pathogen A. baumanniiIMPORTANCE Antibiotic resistance is a pressing global health concern with the rise of multiple and panresistant pathogens. The rapid and unfailing resistance to multiple antibiotics of the nosocomial agent Acinetobacter baumannii, notably to carbapenems, prompt to understand the mechanisms behind acquisition of new antibiotic resistance genes. Natural transformation, one of the horizontal gene transfer mechanisms in bacteria, was only recently described in A. baumannii and could explain its ability to acquire resistance genes. We developed a reliable method to probe and study natural transformation mechanism in A. baumannii More broadly, this new method based on flow cytometry will allow experimental detection and quantification of horizontal gene transfer events in multidrug-resistant A. baumannii.201830012729
4624160.9998Deciphering the distance to antibiotic resistance for the pneumococcus using genome sequencing data. Advances in genome sequencing technologies and genome-wide association studies (GWAS) have provided unprecedented insights into the molecular basis of microbial phenotypes and enabled the identification of the underlying genetic variants in real populations. However, utilization of genome sequencing in clinical phenotyping of bacteria is challenging due to the lack of reliable and accurate approaches. Here, we report a method for predicting microbial resistance patterns using genome sequencing data. We analyzed whole genome sequences of 1,680 Streptococcus pneumoniae isolates from four independent populations using GWAS and identified probable hotspots of genetic variation which correlate with phenotypes of resistance to essential classes of antibiotics. With the premise that accumulation of putative resistance-conferring SNPs, potentially in combination with specific resistance genes, precedes full resistance, we retrogressively surveyed the hotspot loci and quantified the number of SNPs and/or genes, which if accumulated would confer full resistance to an otherwise susceptible strain. We name this approach the 'distance to resistance'. It can be used to identify the creep towards complete antibiotics resistance in bacteria using genome sequencing. This approach serves as a basis for the development of future sequencing-based methods for predicting resistance profiles of bacterial strains in hospital microbiology and public health settings.201728205635
9918170.9998Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR. The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.202134282723
4791180.9998Detection of tetracycline resistance genes by PCR methods. Rapid, accurate, and sensitive determination of antibiotic resistance profiles of various human and animal pathogens becomes a vital prerequisite for successful therapeutic intervention in the face of the increased occurrences of drug-resistant bacterial infections. The current methods, which are dependent on cultivation of pathogens and phenotypic expression of antibiotic resistance, usually require excessive time, special microbiological equipment, and qualified personnel. However, even with all these requisites, for example, no bacteria can be grown from more than 80% of all clinical samples sent to clinical microbiology laboratories. Besides the cultivation limitations, the cultivation-based determination of an antibiotic resistance profile lacks the genotypic information, which is essential for understanding the epidemiology and routes of transmission of antibiotic resistance genes. These genes often reside on mobile genetic elements and can move freely between commensal and pathogenic microbiota, occurring even between taxonomically distant clinical and environmental microbiota. Therefore, development of genotyping methods for detection of antibiotic resistance genes is highly desirable for fast, accurate, and sensitive detection of antibiotic resistance genes in a broad range of pathogenic and commensal bacteria in both clinical and environmental samples. As a model for our studies we have chosen the genes conferring resistance to tetracyclines. Tetracyclines belong to a family of broad-spectrum antibiotics that include tetracycline, chlortetracycline, oxytetracycline, demeclocycline, methacycline, doxycycline, minocycline, and a number of other semisynthetic derivatives. These antibiotics inhibit protein synthesis in Gram-positive and Gram-negative bacteria by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit. The antibiotics of this group were introduced in the late 1950s and since then have been widely used in clinical and veterinary medicine, as well as for prophylaxis and growth promotion in food animals. Because of the possible misuse and overuse of these drugs, resistance to this class of antibiotics is widespread among many clinical isolates, thus limiting the utility of tetracyclines in treating infections. Despite this shortcoming, antibiotics of this class still remain in the active arsenal for dermatologists to treat skin infections such as acne and rosacea.200415156014
4178190.9998Efficacy and food safety considerations of poultry competitive exclusion products. Competitive exclusion (CE) products are anaerobic cultures of bacteria that are applied to poultry hatchlings to establish a protective enteric microbiota that excludes intestinal colonization by human food-borne pathogens. For safety of the poultry flock and human consumers, the identities of bacteria in CE products need to be known. A CE product is a culture of intestinal contents from adult chickens. It may be microbiologically defined by analysis of bacteria isolated from the culture, but many bacteria are hard to reliably isolate, identify, and characterize with conventional techniques. Sequence analysis of 16S ribosomal RNA (rRNA) genes may be more reliable than conventional techniques to identify CE bacteria. Bacteria in CE products may contain antimicrobial drug resistance and virulence mechanisms that could be transferred to the enteric bacteria of the food animal and to the human consumer. Detection methods for specific antimicrobial drug resistance and virulence genes and the integrase genes of conjugative transposons, mostly utilizing PCR technology, are being developed that can be applied to assess these risks in CE bacteria. With improvements in efficacy, bacterial identification, and detection and control of the possible risks of gene transfer, CE product technology can be made a more effective food safety tool.200617039457