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Screening Helicobacter pylori genes induced during infection of mouse stomachs  [cached]
Aparna Singh,Nathaniel Hodgson,Ming Yan,Jungsoo Joo
World Journal of Gastroenterology , 2012, DOI: 10.3748/wjg.v18.i32.4323
Abstract: AIM: To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host. METHODS: In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes. RESULTS: In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cagA, vacA, lpxC, murI, tlpC, trxB, sodB, tnpB, pgi, rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P < 0.05). tlpC, cagA, vacA, sodB, rbfA, infB, tnpB, lpxC and murI were also significantly upregulated (P < 0.01). These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal. CONCLUSION: The positive identification of these genes demonstrates that our IVET systems are powerful tools for
The antibiotic resistance “mobilome”: searching for the link between environment and clinic  [PDF]
Julie A. Perry,Gerard D. Wright
Frontiers in Microbiology , 2013, DOI: 10.3389/fmicb.2013.00138
Abstract: Antibiotic resistance is an ancient problem, owing to the co-evolution of antibiotic-producing and target organisms in the soil and other environments over millennia. The environmental “resistome” is the collection of all genes that directly or indirectly contribute to antibiotic resistance. Many of these resistance determinants originate in antibiotic-producing organisms (where they serve to mediate self-immunity), while others become resistance determinants only when mobilized and over-expressed in non-native hosts (like plasmid-encoded β-lactamases). The modern environmental resistome is under selective pressure from human activities such as agriculture, which may influence the composition of the local resistome and lead to gene transfer events. Beyond the environment, we are challenged in the clinic by the rise in both frequency and diversity of antibiotic resistant pathogens. We assume that clinical resistance originated in the environment, but few examples of direct gene exchange between the environmental resistome and the clinical resistome have been documented. Strong evidence exists to suggest that clinical aminoglycoside and vancomycin resistance enzymes, the extended-spectrum β-lactamase CTX-M and the quinolone resistance gene qnr have direct links to the environmental resistome. In this review, we highlight recent advances in our understanding of horizontal gene transfer of antibiotic resistance genes from the environment to the clinic. Improvements in sequencing technologies coupled with functional metagenomic studies have revealed previously underappreciated diversity in the environmental resistome, and also established novel genetic links to the clinic. Understanding mechanisms of gene exchange becomes vital in controlling the future dissemination of antibiotic resistance.
Assessment of Bacterial Antibiotic Resistance Transfer in the Gut  [PDF]
Susanne Schj rring,Karen A. Krogfelt
International Journal of Microbiology , 2011, DOI: 10.1155/2011/312956
Abstract: We assessed horizontal gene transfer between bacteria in the gastrointestinal (GI) tract. During the last decades, the emergence of antibiotic resistant strains and treatment failures of bacterial infections have increased the public awareness of antibiotic usage. The use of broad spectrum antibiotics creates a selective pressure on the bacterial flora, thus increasing the emergence of multiresistant bacteria, which results in a vicious circle of treatments and emergence of new antibiotic resistant bacteria. The human gastrointestinal tract is a massive reservoir of bacteria with a potential for both receiving and transferring antibiotic resistance genes. The increased use of fermented food products and probiotics, as food supplements and health promoting products containing massive amounts of bacteria acting as either donors and/or recipients of antibiotic resistance genes in the human GI tract, also contributes to the emergence of antibiotic resistant strains. This paper deals with the assessment of antibiotic resistance gene transfer occurring in the gut.
Pediatric Fecal Microbiota Harbor Diverse and Novel Antibiotic Resistance Genes  [PDF]
Aimée M. Moore, Sanket Patel, Kevin J. Forsberg, Bin Wang, Gayle Bentley, Yasmin Razia, Xuan Qin, Phillip I. Tarr, Gautam Dantas
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0078822
Abstract: Emerging antibiotic resistance threatens human health. Gut microbes are an epidemiologically important reservoir of resistance genes (resistome), yet prior studies indicate that the true diversity of gut-associated resistomes has been underestimated. To deeply characterize the pediatric gut-associated resistome, we created metagenomic recombinant libraries in an Escherichia coli host using fecal DNA from 22 healthy infants and children (most without recent antibiotic exposure), and performed functional selections for resistance to 18 antibiotics from eight drug classes. Resistance-conferring DNA fragments were sequenced (Illumina HiSeq 2000), and reads assembled and annotated with the PARFuMS computational pipeline. Resistance to 14 of the 18 antibiotics was found in stools of infants and children. Recovered genes included chloramphenicol acetyltransferases, drug-resistant dihydrofolate reductases, rRNA methyltransferases, transcriptional regulators, multidrug efflux pumps, and every major class of beta-lactamase, aminoglycoside-modifying enzyme, and tetracycline resistance protein. Many resistance-conferring sequences were mobilizable; some had low identity to any known organism, emphasizing cryptic organisms as potentially important resistance reservoirs. We functionally confirmed three novel resistance genes, including a 16S rRNA methylase conferring aminoglycoside resistance, and two tetracycline-resistance proteins nearly identical to a bifidobacterial MFS transporter (B. longum s. longum JDM301). We provide the first report to our knowledge of resistance to folate-synthesis inhibitors conferred by a predicted Nudix hydrolase (part of the folate synthesis pathway). This functional metagenomic survey of gut-associated resistomes, the largest of its kind to date, demonstrates that fecal resistomes of healthy children are far more diverse than previously suspected, that clinically relevant resistance genes are present even without recent selective antibiotic pressure in the human host, and that cryptic gut microbes are an important resistance reservoir. The observed transferability of gut-associated resistance genes to a gram-negative (E. coli) host also suggests that the potential for gut-associated resistomes to threaten human health by mediating antibiotic resistance in pathogens warrants further investigation.
Pseudomonas aeruginosa acquisition on an intensive care unit: relationship between antibiotic selective pressure and patients' environment
Alexandre Boyer, Adéla?de Doussau, Rodolphe Thiébault, Anne Ga?lle Venier, Van Tran, Hélène Boulestreau, Cécile Bébéar, Frédéric Vargas, Gilles Hilbert, Didier Gruson, Anne Marie Rogues
Critical Care , 2011, DOI: 10.1186/cc10026
Abstract: An open, prospective cohort study was carried out in a 16-bed medical ICU where P. aeruginosa was endemic. Over a six-month period, all patients without P. aeruginosa on admission and with a length of stay >72 h were included. Throat, nasal, rectal, sputum and urine samples were taken on admission and at weekly intervals and screened for P. aeruginosa. All antibiotic treatments were recorded daily. Environmental analysis included weekly tap water specimen culture and the presence of other patients colonized with P. aeruginosa.A total of 126 patients were included, comprising 1,345 patient-days. Antibiotics were given to 106 patients (antibiotic selective pressure for P. aeruginosa in 39). P. aeruginosa was acquired by 20 patients (16%) and was isolated from 164/536 environmental samples (31%). Two conditions were independently associated with P. aeruginosa acquisition by multivariate analysis: (i) patients receiving ≥3 days of antibiotic selective pressure together with at least one colonized patient on the same ward on the previous day (odds ratio (OR) = 10.3 ((% confidence interval (CI): 1.8 to 57.4); P = 0.01); and (ii) presence of an invasive device (OR = 7.7 (95% CI: 2.3 to 25.7); P = 0.001).Specific interaction between both patient colonization pressure and selective antibiotic pressure is the most relevant factor for P. aeruginosa acquisition on an ICU. This suggests that combined efforts are needed against both factors to decrease colonization with P. aeruginosa.Pseudomonas aeruginosa infections on the ICU are a constant concern [1]. Colonization with this organism often precedes infection [2] and its prevention is, therefore, extremely important. P. aeruginosa colonization has been reported to originate from exogenous sources such as tap water [3], fomites and/or patient-to-patient transmission, or as an endogenous phenomenon related to antibiotic use. Some studies have highlighted the importance of exogenous colonization during hospitalization (50 to 70% o
Food and human gut as reservoirs of transferable antibiotic resistance encoding genes  [PDF]
Jean-Marc Rolain
Frontiers in Microbiology , 2013, DOI: 10.3389/fmicb.2013.00173
Abstract: The increase and spread of antibiotic resistance (AR) over the past decade in human pathogens has become a worldwide health concern. Recent genomic and metagenomic studies in humans, animals, in food and in the environment have led to the discovery of a huge reservoir of AR genes called the resistome that could be mobilized and transferred from these sources to human pathogens. AR is a natural phenomenon developed by bacteria to protect antibiotic-producing bacteria from their own products and also to increase their survival in highly competitive microbial environments. Although antibiotics are used extensively in humans and animals, there is also considerable usage of antibiotics in agriculture, especially in animal feeds and aquaculture. The aim of this review is to give an overview of the sources of AR and the use of antibiotics in these reservoirs as selectors for emergence of AR bacteria in humans via the food chain.
Metagenomic Profiling of Antibiotic Resistance Genes and Mobile Genetic Elements in a Tannery Wastewater Treatment Plant  [PDF]
Zhu Wang, Xu-Xiang Zhang, Kailong Huang, Yu Miao, Peng Shi, Bo Liu, Chao Long, Aimin Li
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0076079
Abstract: Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP). Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet) were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.
Antibiotic Resistance: A Concern to Veterinary and Human Medicine  [PDF]
Sitaram Aryal
Nepal Agriculture Research Journal , 2000, DOI: 10.3126/narj.v4i0.4873
Abstract: Bacterial resistance to antibiotics occurs even without the use of antibiotics. Antibiotic use exerts a selective pressure to the bacterial flora that help in the emergence and development of antibiotic resistance. Antibiotics are used worldwide both in veterinary and human medicine. The wide spread use of antibiotics in human and animal has raised the concern about the development of resistant and multi resistant bacteria that possess a potential danger to animals and men, as resistance may cause treatment failure. Resistance may be natural or acquired. Acquired resistance is due to transfer of extrachromosomal genetic material (R-plasmids) and is very important. The R-plasmids are spread to other bacterial cells by transformation, transduction, conjugation and transposition. Transmitted antibiotic resistance in disease causing bacteria may cause zoonotic infections and resistant non-infectious bacteria may serve as a reservoir of R- plasmids for the pathogenic organism(s). This paper highlights the mechanism of development of resistance in bacteria and means to minimize it. Key words: Antibiotic resistance; Bacteria; Extrachromosomal material; Resistance; R-plasmids DOI: http://dx.doi.org/10.3126/narj.v4i0.4873 Nepal Agriculture Research Journal Vol. 4&5, 2001/2002 Page: 66-70 Uploaded date : 9 June, 2011
Resistencia a antimicrobianos en aislamientos de Escherichia coli de origen animal Antimicrobial resistance of Escherichia coli isolated from animals  [cached]
G. Carloni,A. Pereyra,G Denamiel,E. Gentilini
InVet , 2011,
Abstract: Se determinó el perfil de susceptibilidad a antimicrobianos de 100 aislamientos de E.coli provenientes de diversas patologías en bovinos, equinos, caninos y felinos, siguiendo metodología del Clinical and Laboratory Standards Institute y detectando la aparición de aislamientos multiresistentes. El panel de antibióticos ensayados incluyó amicacina, ampicilina/sulbactama, cefotaxima, ciprofloxacina, cloranfenicol, colistina, estreptomicina, gentamicina, nitrofurantoína, tetraciclina, trimetoprima/ sulfametoxazol. El mayor porcentaje de resistencia (R) se detectó frente a tetraciclina en aislamientos de todas las especies animales (entre 34% en los de origen felino y 75% de origen equino). En las cepas de origen canino y felino se encontraron porcentajes considerables frente ampicilina/ sulbactama (27% de caninos y 53% de felinos) y ante ciprofloxacina (30% y 67% respectivamente). En estos aislamientos también, se detectó el mayor porcentaje de multiresistencia (29% en caninos y 67% en felinos). La presión selectiva originada por la aplicación inadecuada de antibióticos puede resultar un factor, aunque no el único, responsable de la aparición de R. Además existe la posibilidad de que E.coli pueda constituirse en un eslabón de transmisión de genes de R a antimicrobianos, aunque no se conoce hasta el momento, el origen de ellos, humano o animal y, su permanencia en el tiempo. Antimicrobial susceptibility tests were determined in 100 isolates of E.coli from differents patologies in cattle, horses, dogs and cats, according to Clinical and Laboratory Standards Institute. Multiresistance isolates were detected in this assay. The antibiotics selected were amikacin, ampicillin /sulbactam, cefotaxime, ciprofloxacin, chloramphenicol, colistin, gentamicin, nitrofurantoin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole. The antibiotic with the highest resistance was tetracycline (34% in cats and 75% in dogs). In isolated strains from dogs and cats it was also found considerable percentages of resistance to ampicilline/ sulbactam (27% in dogs and 53%in cats) and to ciprofloxacine (30%in dogs and 67% in cats). In these isolates it was also found the highest percentage of multiresistance (29% in dogs and 67% in cats). Selective pressure originated from the inadecuate use of antibiotics can be responsible for the appearance of resistance, eventhough it is not the only one. It may be possible that E. coli could transmit genes of resistance to antimicrobials agents, although it is unknown if the presence of these genes are permanent or transitory.
Antibiotic resistance via the food chain: Fact or fiction?
Linda A. Bester,Sabiha Y. Essack
South African Journal of Science , 2010, DOI: 10.4102/sajs.v106i9/10.281
Abstract: The mechanisms that bacteria use to acquire additional genetic material, including genes coding for antibiotic resistance, are principally the secondary pathways that have been described as transformation and conjugation pathways. The farming industry often is reported as a hotspot for antibiotic-resistance reservoirs. In this review, we consider the exposure of food animals during the course of their lifespans to preventative, therapeutic or prophylactic treatment with antibiotic agents. In this context, zoonotic bacteria are commonly recognised as a potential threat to human health, with therapeutic treatment of pathogenic organisms on farms increasing the likelihood of selective antibiotic pressure influencing the commensal flora of the intestines. Existing literature indicates, however, that the effective impact on human health of such interventions in the food production process is still subject to debate.
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