Molecular characterization of antibiotic resistance in cultivable multidrug-resistant bacteria from livestock manure☆
Graphical abstract
Introduction
The evolution of antibiotic-resistance genes (ARGs) and their spread among bacterial pathogens have important clinical significance. Multiple antibiotic resistant bacteria (MARB) have traveled around the world, leading to concerns about potential untreatable infections. Antibiotic-resistant bacteria (ARB), especially MARB, are also found in animals (Yang et al., 2016), increasingly due to antibiotic compounds widely used in animal husbandry. ARB, MARB, and ARGs present in animal manure can be transferred to soil and even to plants when it is used as fertilizer, resulting in a high prevalence of antibiotic resistance in those environments (Wang et al., 2015). Horizontal transfer of ARGs from the environment to human pathogens has been confirmed, which threatens human health despite the low transfer rates (Sommer, 2014). Therefore, ARGs are considered pollutants, with animal manure as an important reservoir of both ARB and ARGs.
Two widely accepted sources of antibiotic resistance are: genetic mutations that occur at low frequency, and acquisition of exogenous resistance genes. Resistance genes are regarded as the major contributor to ARG dissemination. Resistance genes can be spread by vertical proliferation or horizontal gene transfer via MGEs such as transposons, integrons, and plasmids (Deng et al., 2015). Class 1 integrons are the most ubiquitous and commonly reported MGEs among clinical bacteria and are indicators of horizontal gene transfer (Ma et al., 2011). However, class 1 integrons can move only in association with Tn402-like transposons or the Tn3 transposon family (Deng et al., 2015). Transposons often serve as vehicles for intraspecies and interspecies transmission of resistance genes via site-specific recombination. Insertion sequence common region (ISCR1) is a novel gene-capture system, notable for close association with a wide variety of antibiotic resistance genes (Toleman et al., 2006). Therefore, different types of mobile genetic elements were investigated simultaneously to determine horizontal transfer of ARGs.
Various molecular methods such as PCR and functional metagenomic screening can detect large numbers of and general characteristics of ARGs in the environment (Zhu et al., 2013). Nevertheless, previous studies rarely provide a link between resistance genes and resistant bacteria isolated from manure. Neither the distribution nor the transfer mechanism of ARGs among ARBs has been fully studied. There are even fewer reports on the co-occurrence and even co-transfer of various ARGs among different species of MARB from animal manure.
In a previous study, we found that up to 11 classes of antibiotics were used in Chinese chicken and swine farms. Chicken manure contained much higher concentrations of various antibiotics and higher percentages of ARB including MARB than swine manure because of more frequent and intensive use of antibiotics in chicken farming (Yang et al., 2016). In the present study, the comprehensive profiles of antibiotic resistance, ARGs, and MGEs were investigated in detail for MARB to illuminate the molecular characteristics of antibiotic resistance in different MARB species and their possible risks to human health.
Section snippets
Chicken and swine manure samples and antibiotics
Multiple livestock feedlots were selected to investigate the occurrence and molecular characterization of MARB in manure. Feedlots included three chicken farms (designated C1–C3) and three swine farms (designated S1–S3) in the suburbs of Xinxiang City, Henan Province, China. All feedlots were concentrated livestock feeding operations. More than 5000 chickens were housed on each chicken farm and more than 1000 pigs on each swine farm. Information on antibiotic applications in the livestock
Distribution and diversity of cultivable MARB in chicken and swine manure
Numeration results indicated that counts of THCB were (3.23 ± 4.25) × 109− (3.17 ± 2.05) × 1010 cfu g−1 dry weight in three chicken samples and (7.8 ± 4.45) × 109−(4.92 ± 3.49) × 1010 cfu g−1 dry weight in three swine manure samples. Counts of MARB3 resistant to combinations of any three of the eight types of antibiotics and MARB8 resistant to all tested eight types of antibiotics were 1–2 orders of magnitude lower than THCB for each manure sample: (4.01 ± 1.08) × 108 − (3.08 ± 4.64) × 109 cfu g
Discussion
In this study, LB, MH and SS media were used to detect and isolate MARB. LB is suitable for the majority of heterotrophic bacteria. MH is generally used to detect antibiotic resistance of bacteria due to its low salinity. SS is suitable for intestinal microorganisms. Application of these three types of media would be helpful for obtaining more species of MARB, although we were not able to obtain all the MARB in the chicken manure. The greatest abundance of heterotrophic, cultivable bacteria and
Conclusions
In this study, 33 MARB representing the predominant aerobic, cultivable multiple antibiotic-resistant bacterial community were isolated from chicken manure. Their antibiotic-resistance profiles, antibiotic-resistance genes, and mobile genetic elements were investigated. Chicken manure was a reservoir of diverse and abundant multidrug-resistant bacteria, ARGs and MGEs, in which species of human pathogens or opportunists were present. The MARB obtained in this study from chicken manure generally
Acknowledgments
This work was supported by the National Natural Science Foundation of China [NSFC 21477035 and NSFC 21277041].
References (41)
- et al.
Resistance integrons: class 1, 2 and 3 integrons
Ann. Clin. Microbiol. Antimicrob.
(2015) - et al.
Antibiotic resistance gene spread due to manure application on agricultural fields
Curr. Opin. Microbiol.
(2011) Natural antibiotic resistance and contamination by antibiotic resistance determinants: the two ages in the evolution of resistance to antimicrobials
Front. Microbiol.
(2012)Efflux-mediated multiresistance in gram-negative bacteria
Clin. Microbiol. Infect. Official Publ. Eur. Soc. Clin. Microbiol. Infect. Dis.
(2004)- et al.
Diversity of class 1 and 2 integrons detected in Escherichia coli isolates from diseased and apparently healthy dogs
Vet. Microbiol.
(2016) - et al.
Changes of resistome, mobilome and potential hosts of antibiotic resistance genes during the transformation of anaerobic digestion from mesophilic to thermophilic
Water Res.
(2016) - et al.
Antibiotic resistance genes in manure-amended soil and vegetables at harvest
J. Hazard Mater
(2015) - et al.
Resistance class 1 integron in clinical methicillin-resistant Staphylococcus aureus, strains in southern China, 2001-2006
Clin. Microbiol. Infect.
(2011) - et al.
Characterisation of class 1 integron among Escherichia coli isolated from Mansoura University hospitals in Egypt
Adv. Microbiol.
(2015) - et al.
Molecular mechanisms of antibiotic resistance
Nat. Rev. Microbiol.
(2015)
QIIME allows analysis of high-throughput community sequencing data
Nat. methods
Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample
Proc. Natl. Acad. Sci. U. S. A.
Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement; M100–S23
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB
Appl. Environ. Microbiol.
UPARSE: highly accurate OTU sequences from microbial amplicon reads
Nat. methods
The ecology, epidemiology and virulence of enterococcus
Microbiology
Comparative genomics of multidrug resistance in Acinetobacter baumannii
PLoS Genet.
Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment
ISME J.
The evolution of class 1 integrons and the rise of antibiotic resistance
J. Bacteriol.
Increasing imipenem resistance and dissemination of the ISAba1-associated blaOXA-23 gene among Acinetobacter baumannii isolates in an intensive care unit
J. Med. Microbiol.
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This paper has been recommended for acceptance by Klaus Kummerer.
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Tiantian Tian and Tianqi Niu contributed equally to this work.