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A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and ?-lactam resistance genes in the gut microbiota.

ABSTRACT: BACKGROUND:The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and ?-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes. RESULTS:The strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and ?-lactam (including blaOXA, blaTEM, blaSHV and blaCTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes. CONCLUSIONS:This study has demonstrated that the gut can be a significant source of aminoglycoside and ?-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes.

SUBMITTER: Fouhy F

PROVIDER: S-EPMC3917905 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and β-lactam resistance genes in the gut microbiota.

Fouhy Fiona F Ross R Paul RP Fitzgerald Gerald F GF Stanton Catherine C Cotter Paul D PD

BMC microbiology 20140205

<h4>Background</h4>The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would ...[more]

PMID: 24499167

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Project description:β-Lactam antibiotics are a class of antibiotics commonly used to treat bacterial infections. However, the effects of β-lactam antibiotics on term neonatal intestinal flora have not been fully elucidated. Hospitalized full-term newborns receiving β-lactam antibiotics formed the antibiotic group (n = 67), while those without antibiotic treatment comprised the non-antibiotic group (n = 47). A healthy group included healthy full-term newborns (n = 16). Stool samples were collected for 16 S rDNA sequencing to analyze gut microbiota variations. Further investigation was carried out within the β-lactam antibiotic group, exploring the effects of antibiotic use on the newborns' gut microbiota in relation to the duration and type of antibiotic administration, delivery method, and feeding practices. The antibiotic group exhibited significant difference of microbial community composition compared to the other groups. Genera like Klebsiella, Enterococcus, Streptococcus, Alistipes, and Aeromonas were enriched, while Escherichia-Shigella, Clostridium sensu stricto 1, Bifidobacterium, and Parabacteroides were reduced. Klebsiella negatively correlated with Escherichia-Shigella, positively with Enterobacter, while Escherichia-Shigella negatively correlated with Enterococcus and Streptococcus. Regardless of neonatal age, β-lactam antibiotics induced an elevated abundance of Klebsiella and Enterococcus. The impact on gut microbiota varied with the duration and type of antibiotic (cefotaxime or ampicillin/sulbactam). Compared to vaginal delivery, cesarean delivery after β-lactam treatment heightened the abundance of Klebsiella, Enterobacteriaceae_Unclassified, Lactobacillales_Unclassified, and Pectobacterium. Feeding patterns minimally influenced β-lactam-induced alterations. In conclusion, β-lactam antibiotic treatment for neonatal pneumonia and sepsis markedly disrupted intestinal microbiota, favoring Klebsiella, Enterococcus, Streptococcus, Alistipes, and Aeromonas. The impact of β-lactam varied by duration, type, and delivery method, emphasizing heightened disruptions post-cesarean delivery.

Project description:Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to dissemination of antibiotic resistance genes (ARGs) in the gut microbiota. The gut microbiota often suffers from various disturbances. It is not clear whether and how disturbed microbiota may affect ARG mobility under antibiotic treatments. For proof of concept, in the presence or absence of streptomycin pre-treatment, mice were inoculated orally with a β-lactam-susceptible Salmonella enterica serovar Heidelberg clinical isolate (recipient) and a β-lactam resistant Escherichia coli O80:H26 isolate (donor) carrying a blaCMY-2 gene on an IncI2 plasmid. Immediately following inoculation, mice were treated with or without ampicillin in drinking water for 7 days. Faeces were sampled, donor, recipient and transconjugant were enumerated, blaCMY-2 abundance was determined by quantitative PCR, faecal microbial community composition was determined by 16S rRNA amplicon sequencing and cecal samples were observed histologically for evidence of inflammation. In faeces of mice that received streptomycin pre-treatment, the donor abundance remained high, and the abundance of S. Heidelberg transconjugant and the relative abundance of Enterobacteriaceae increased significantly during the ampicillin treatment. Co-blooming of the donor, transconjugant and commensal Enterobacteriaceae in the inflamed intestine promoted significantly (P<0.05) higher and possibly wider dissemination of the blaCMY-2 gene in the gut microbiota of mice that received the combination of streptomycin pre-treatment and ampicillin treatment (Str-Amp) compared to the other mice. Following cessation of the ampicillin treatment, faecal shedding of S. Heidelberg transconjugant persisted much longer from mice in the Str-Amp group compared to the other mice. In addition, only mice in the Str-Amp group shed a commensal E. coli O2:H6 transconjugant, which carries three copies of the blaCMY-2 gene, one on the IncI2 plasmid and two on the chromosome. The findings highlight the significance of pre-existing gut microbiota for ARG dissemination and persistence during and following antibiotic treatments of infectious diseases.

Dataset Information

A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and ?-lactam resistance genes in the gut microbiota.

Publications

A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and β-lactam resistance genes in the gut microbiota.

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