ABSTRACT
In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with blaCMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.
Subject(s)
Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , Klebsiella pneumoniae/genetics , Plasmids/genetics , beta-Lactamases/genetics , Escherichia coli/isolation & purification , Feces/microbiology , Humans , Klebsiella pneumoniae/isolation & purification , Multilocus Sequence TypingABSTRACT
OBJECTIVES: Antimicrobial resistance genes (ARGs) can be transferred by means of mobile genetic elements, which play a critical role in the dissemination of resistance in the bacterial community. ARG transmission within mobile genetic elements has been reported in plasmids and transposons but less frequently in bacteriophages. Here, the bacteriophage fraction of seven human faecal samples was purified and deep-sequenced to detect the presence of ARGs in the phage particles. METHODS: Seven faecal samples (five from healthy individuals and two from a patient before and after receiving ciprofloxacin treatment) were used to extract phage DNA, which was purified and then sequenced in a MiSeq (Illumina). Generated reads were checked for quality and assembled, and then the generated contigs analysed with Kraken, PHASTER, VirSorter and Prokka. Some genes were also validated by quantitative PCR. RESULTS: Analysis of the purified phage DNA by Kraken identified from 4 to 266 viruses in the samples. The viral fraction corresponded mainly to the order Caudovirales, including phages from the Siphoviridae and Myoviridae families. Bacterial genes associated with antimicrobial resistance were detected in the viral DNA, as confirmed by quantitative PCR. Higher densities of ARG-carrying phage particles were observed in the post- versus pre-ciprofloxacin treatment sample. CONCLUSIONS: The finding of ARGs in phage particles supports the description of phages as mobile elements contributing to the dissemination of bacterial antibiotic resistance and suggests ciprofloxacin treatment may play a role in the release of ARG-carrying particles, thereby increasing resistance.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Bacteriophages/isolation & purification , Ciprofloxacin/administration & dosage , Drug Resistance, Bacterial , Feces/virology , Genes, Bacterial , Healthy Volunteers , Adult , Aged , Bacteriophages/classification , Bacteriophages/genetics , Biota/drug effects , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/isolation & purification , High-Throughput Nucleotide Sequencing , Humans , Middle Aged , Myoviridae/classification , Myoviridae/genetics , Myoviridae/isolation & purification , Real-Time Polymerase Chain Reaction , Siphoviridae/classification , Siphoviridae/genetics , Siphoviridae/isolation & purificationABSTRACT
Phage particles have emerged as elements with the potential to mobilise antibiotic resistance genes (ARGs) in different environments, including the intestinal habitat. This study aimed to determine the occurrence of ARGs in phage particles present in faecal matter and induced from strains isolated from faeces. Nine ARGs (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, blaOXA-48, qnrA, qnrS, mecA, sul1 and armA) were quantified by qPCR in the phage DNA fractions of 150 faecal samples obtained from healthy individuals who had not received antibiotic treatment or travelled abroad in the 3 months prior to sample collection. On the suspicion that the detected particles originated from bacterial flora, 82 Escherichia coli and Klebsiella pneumoniae isolates possessing at least one identified ARG (blaTEM, blaCTX-M-1 group, blaCTX-M-9 group, armA, qnrA, qnrS and sul1) were isolated and their capacity to produce phage particles carrying these ARGs following induction was evaluated. Of 150 samples, 72.7% were positive for at least one ARG, with blaTEM and blaCTX-M-9 group being the most prevalent and abundant. Of the 82 isolates, 51 (62%) showed an increase in the number of copies of the respective ARG in the phage fraction following induction, with blaTEM, blaCTX-M-1 group, blaCTX-M-9 group and sul1 being the most abundant. Phages induced from the isolates were further purified and visualised using microscopy and their DNA showed ARG levels of up to 1010 gene copies/mL. This study highlights the abundance of phage particles harbouring ARGs and indicates that bacterial strains in the intestinal habitat could be source of these particles.
Subject(s)
Bacteriophages/genetics , DNA, Viral/genetics , Drug Resistance, Bacterial , Escherichia coli/virology , Feces/microbiology , Genes, Bacterial , Klebsiella pneumoniae/virology , Adolescent , Adult , Aged , Aged, 80 and over , Bacteriophages/isolation & purification , Child , Child, Preschool , Escherichia coli/isolation & purification , Female , Healthy Volunteers , Humans , Klebsiella pneumoniae/isolation & purification , Male , Middle Aged , Real-Time Polymerase Chain Reaction , Young AdultABSTRACT
OBJECTIVES: The objectives were to determine the prevalence of colistin resistance in clinical isolates of Enterobacteriaceae, and to gain knowledge on the epidemiological and clinical features of the patients. METHODS: All colistin-resistant Enterobacteriaceae consecutively isolated from clinical samples in our institution from 2012 to 2015, were included in this cross-sectional study. Intrinsic-resistant species were excluded. Minimum inhibitory concentration was performed by gradient diffusion. Detection of plasmid-encoded colistin resistance genes mcr-1 and mcr-2 was performed by amplification. Epidemiological and clinical features were reviewed. RESULTS: Of 13579 Enterobacteriaceae isolates, 91 were colistin-resistant. The overall prevalence of colistin resistance was 0.67%. The rates were higher in Enterobacter cloacae (4.2%) than Escherichia coli (0.5%) and Klebsiella pneumoniae (0.4%). One third of the isolates were multi-drug resistant (MDR). While mcr-2 was not detected, mcr-1 was detected only in E. coli. Regarding these infections, 23% were community-acquired. 89% of the patients had not received colistin previously. There were no significant differences between infections caused by mcr-1 and non-mcr-1-carrying isolates. CONCLUSIONS: Colistin resistance was not restricted to MDR isolates and to clinical settings. Most patients had no record of previous administration of colistin.
Subject(s)
Anti-Bacterial Agents/pharmacology , Colistin/pharmacology , Drug Resistance, Bacterial , Enterobacteriaceae Infections/drug therapy , Enterobacteriaceae Infections/epidemiology , Enterobacteriaceae/drug effects , Adolescent , Adult , Aged , Aged, 80 and over , Analysis of Variance , Anti-Bacterial Agents/therapeutic use , Child , Colistin/therapeutic use , Cross-Sectional Studies , Enterobacteriaceae/isolation & purification , Female , Humans , Male , Microbial Sensitivity Tests , Middle Aged , Prevalence , Prospective Studies , Retrospective Studies , Spain/epidemiology , Tertiary Care Centers , Young AdultABSTRACT
Colistin resistance was detected in 53 of 10,011 Escherichia coli (0.5%) by prospective phenotypic testing of consecutive clinical isolates in a single hospital in Barcelona, Spain (2012-15). The mcr-1 gene was retrospectively identified by PCR and sequencing in 15 of 50 available isolates. Each isolate had a unique PFGE pattern except for two. This clonal diversity supports the hypothesis of horizontal dissemination of the mcr-1 gene in the local study population.
