Molecular characterization of antibiotic resistance in cultivable multidrug-resistant bacteria from livestock manure

doi:10.1016/j.envpol.2017.05.073

Environmental Pollution

Volume 229, October 2017, Pages 188-198

https://doi.org/10.1016/j.envpol.2017.05.073 Get rights and content

Highlights

•
The distribution of ARGs and MGEs among MARBs from chicken manure was studied.
•
Each MARB carried multiple plasmids in cell and 27–36 different types of tested ARGs.
•
12 types of MGEs were identified among MARBs with class 1 integrons most prevalent.
•
9 different ARG cassettes were identified with dfrA12 + orfF + aadA2 most prevalent.
•
Co-occurrence of multiple MGEs or ARG cassettes in a strain was widely found in MARBs.

Abstract

Diverse antibiotic-resistance genes (ARGs) are frequently reported to have high prevalence in veterinary manure samples due to extensive use of antibiotics in farm animals. However, the characteristics of the distribution and transmission of ARGs among bacteria, especially among different species of multiple antibiotic-resistant bacteria (MARB), have not been well explored. By applying high-throughput sequencing methods, our study uncovered a vast MARB reservoir in livestock manure. The genera Escherichia, Myroides, Acinetobacter, Proteus, Ignatzschineria, Alcaligenes, Providencia and Enterococcus were the predominant cultivable MARB, with compositions of 40.6%–85.7%. From chicken manure isolates, 33 MARB were selected for investigation of the molecular characteristics of antibiotic resistance. A total of 61 ARGs and 18 mobile genetic elements (MGEs) were investigated. We found that 47 ARGs were widely distributed among the 33 MARB isolates. Each isolate carried 27–36 genes responsible for resistance to eight classes of antibiotics frequently used in clinic or veterinary settings. ARGs to the six classes of antibiotics other than streptogramins and vancomycin were present in all 33 MARB isolates with a prevalence of 80%–100%. A total of 12 MGEs were widely distributed among the 33 MARB, with intI1, IS26, ISaba1, and ISEcp1 simultaneously present in 100% of isolates. In addition, 9 gene cassettes within integrons and ISCR1 were detected among MARB isolates encoding resistance to different antibiotic classes. This is the first report revealing the general co-presence of multiple ARGs, various MGEs and ARG cassettes in different species of individual MARB isolates in chicken manure. The results highlight a much higher risk of ARGs spreading through livestock manure to humans than we expected.

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)

Y. Deng et al.
Resistance integrons: class 1, 2 and 3 integrons
Ann. Clin. Microbiol. Antimicrob.
(2015)
H. Heuer et al.
Antibiotic resistance gene spread due to manure application on agricultural fields
Curr. Opin. Microbiol.
(2011)
J.L. Martínez
Natural antibiotic resistance and contamination by antibiotic resistance determinants: the two ages in the evolution of resistance to antimicrobials
Front. Microbiol.
(2012)
K. Poole
Efflux-mediated multiresistance in gram-negative bacteria
Clin. Microbiol. Infect. Official Publ. Eur. Soc. Clin. Microbiol. Infect. Dis.
(2004)
A.K. Siqueira et al.
Diversity of class 1 and 2 integrons detected in Escherichia coli isolates from diseased and apparently healthy dogs
Vet. Microbiol.
(2016)
Z. Tian 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)
F.H. Wang et al.
Antibiotic resistance genes in manure-amended soil and vegetables at harvest
J. Hazard Mater
(2015)
Z. Xu et al.
Resistance class 1 integron in clinical methicillin-resistant Staphylococcus aureus, strains in southern China, 2001-2006
Clin. Microbiol. Infect.
(2011)
M.M. Adel El-Sokkary et al.
Characterisation of class 1 integron among Escherichia coli isolated from Mansoura University hospitals in Egypt
Adv. Microbiol.
(2015)
J.M. Blair et al.
Molecular mechanisms of antibiotic resistance
Nat. Rev. Microbiol.
(2015)

J.G. Caporaso et al.

QIIME allows analysis of high-throughput community sequencing data

Nat. methods

(2010)

J.G. Caporaso et al.

Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample

Proc. Natl. Acad. Sci. U. S. A.

(2011)

F.R. Cockerill et al.

Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement; M100–S23

(2013)

T.Z. Desantis et al.

Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB

Appl. Environ. Microbiol.

(2006)

R.C. Edgar

UPARSE: highly accurate OTU sequences from microbial amplicon reads

Nat. methods

(2013)

K. Fisher et al.

The ecology, epidemiology and virulence of enterococcus

Microbiology

(2009)

P.E. Fournier et al.

Comparative genomics of multidrug resistance in Acinetobacter baumannii

PLoS Genet.

(2006)

W.H. Gaze et al.

Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

ISME J.

(2011)

M. Gillings et al.

The evolution of class 1 integrons and the rise of antibiotic resistance

J. Bacteriol.

(2008)

C. He et al.

Increasing imipenem resistance and dissemination of the ISAba1-associated bla_OXA-23 gene among Acinetobacter baumannii isolates in an intensive care unit

J. Med. Microbiol.

(2011)

Cited by (69)

Electrokinetic technology enhanced the control of antibiotic resistance and the quality of swine manure composting
2024, Chemical Engineering Journal
Aerobic composting is the primary way to utilize animal manure. However, the frequently detected antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in manure pose high risks to both the environment and human health. Based on our previous work on the effective removal of ARB and ARGs from water and soil using electrokinetic (EK) technology, it was introduced in traditional composting in this work to investigate its potentiality for quality improvement as well as antibiotic resistance control. The results showed that temperature, total nitrogen (TN), germination index, and humus index significantly increased in the EK treatment, while the abundances of ARB and ARGs decreased by 33.52–90.66 % and 68.60–85.24 % after 30-d composting, respectively. Moreover, the maturation period was reduced by 5 days compared to control. The electric field strongly affected the bacterial community structure. On one hand, the abundance of Chryseolinea increased and, accordingly, the nitrogen fixation capacity (the TN content) increased by 9.60 % in the EK treatment. Meanwhile, the increased abundance of Bacillus, Thermobacillus and Halocella accelerated the maturation and humification of the compost. On the other hand, the potential ARG host bacteria changed and especially that for sul1 decreased. In detail, the relative abundances of Actinobacteria and Firmicutes decreased by 45.89 % and 30.00 % in the EK treatment, respectively, which was closely associated with ARGs’ removal. Functional prediction using PICRUSt revealed enhanced energy metabolism and membrane transport in response to antibiotic stress in the electric field. This study provides a potential solution to achieve both efficient and safe composting with the EK technology.
Antibiotic resistance genes associated with size-segregated bioaerosols from wastewater treatment plants: A review
2024, Environmental Pollution
The antibiotic-resistant pollution in size-segregated bioaerosols from wastewater treatment plants (WWTPs) is of increasing concern due to its public health risks, but an elaborate review is still lacking. This work overviewed the profile, mobility, pathogenic hosts, source, and risks of antibiotic resistance genes (ARGs) in size-segregated bioaerosols from WWTPs. The dominant ARG type in size-segregated bioaerosols from WWTPs was multidrug resistance genes. Treatment units that equipped with mechanical facilities and aeration devices, such as grilles, grit chambers, biochemical reaction tanks, and sludge treatment units, were the primary sources of bioaerosol antibiotic resistome in WWTPs. Higher enrichment of antibiotic resistome in particulate matter with an aerodynamic diameter of <2.5 μm, was found along the upwind-downwind-WWTPs gradient. Only a small portion of ARGs in inhalable bioaerosols from WWTPs were flanked by mobile genetic elements. The pathogens with multiple drug resistance had been found in size-segregated bioaerosols from WWTPs. Different ARGs or antibiotic resistant bacteria have different aerosolization potential associated with bioaerosols from various treatment processes. The validation of pathogenic antibiotic resistance bacteria, deeper investigation of ARG mobility, emission mechanism of antibiotic resistome, and development of treatment technologies, should be systematically considered in future.
Antibiotic resistance genes and heavy metals in landfill: A review
2024, Journal of Hazardous Materials
Landfill is reservoir containing antibiotic resistance genes (ARGs) that pose a threat to human life and health. Heavy metals impose lasting effects on ARGs. This review investigated and analyzed the distribution, composition, and abundance of heavy metals and ARGs in landfill. The abundance ranges of ARGs detected in refuse and leachate were similar. The composition of ARG varied with sampling depth in refuse. ARG in leachate varies with the distribution of ARG in the refuse. The ARG of sulI was associated with 11 metals (Co, Pb, Mn, Zn, Cu, Cr, Ni, Sb, As, Cd, and Al). The effects of the total metal concentration on ARG abundance were masked by many factors. Low heavy metal concentrations showed positive effects on ARG diffusion; conversely, high heavy metal concentrations showed negative effects. Organic matter had a selective pressure effect on microorganisms and could provide energy for the diffusion of ARGs. Complexes of heavy metals and organic matter were common in landfill. Therefore, the hypothesis was proposed that organic matter and heavy metals have combined effects on the horizontal gene transfer (HGT) of ARGs during landfill stabilization. This work provides a new basis to better understand the HGT of ARGs in landfill.
Transfer of antibiotic resistance genes from soil to wheat: Role of host bacteria, impact on seed-derived bacteria, and affecting factors
2023, Science of the Total Environment
The transfer of antibiotic resistance genes (ARGs) from soils to plants is poorly understood, especially the role of host bacteria in soils and its impact on seed-derived bacteria. Wheat (Triticum aestivum L.) was thus used to fill the gap by conducting pot experiments, with target ARGs and bacterial community analyzed. Results showed that the relative abundances of target ARGs gradually decreased during transfer of ARGs from the rhizosphere soil to root and shoot. Host bacteria in the rhizosphere soil were the primary source of ARGs in wheat. The 38, 21, and 19 potential host bacterial genera of target ARGs and intI1 in the rhizosphere soil, root, and shoot were identified, respectively, and they mainly belonged to phylum Proteobacteria. The abundance of ARGs carried by pathogenic Corynebacterium was reduced in sequence. During transfer of ARGs from the rhizosphere soil to root and shoot, some seed-derived bacteria and pathogenic Acinetobacter obtained ARGs through horizontal gene transfer and became potential host bacteria. Furthermore, total organic carbon, available nitrogen of the rhizosphere soil, water use efficiency, vapor pressure deficit, and superoxide dismutase of plants were identified as the key factors affecting potential host bacteria transfer in soils to wheat. This work provides important insights into transfer of ARGs and deepens our understanding of potential health risks of ARGs from soils to plants.
The vertically-stratified resistomes in mangrove sediments was driven by the bacterial diversity
2023, Journal of Hazardous Materials
Early evidence has elucidated that the spread of antibiotic (ARGs) and metal resistance genes (MRGs) are mainly attributed to the selection pressure in human-influenced environments. However, whether and how biotic and abiotic factors mediate the distribution of ARGs and MRGs in mangrove sediments under natural sedimentation is largely unclear. Here, we profiled the abundance and diversity of ARGs and MRGs and their relationships with sedimental microbiomes in 0–100 cm mangrove sediments. Our results identified multidrug-resistance and multimetal-resistance as the most abundant ARG and MRG classes, and their abundances generally decreased with the sediment depth. Instead of abiotic factors such as nutrients and antibiotics, the bacterial diversity was significantly negatively correlated with the abundance and diversity of resistomes. Also, the majority of resistance classes (e.g., multidrug and arsenic) were carried by more diverse bacterial hosts in deep layers with low abundances of resistance genes. Together, our results indicated that bacterial diversity was the most important biotic factor driving the vertical profile of ARGs and MRGs in the mangrove sediment. Given that there is a foreseeable increasing human impact on natural environments, this study emphasizes the important role of biodiversity in driving the abundance and diversity of ARGs and MRGs.
Two microbial consortia obtained through purposive acclimatization as biological additives to relieve ammonia inhibition in anaerobic digestion
2023, Water Research
Ammonia inhibition is a challenging issue in the anaerobic digestion (AD) of nitrogen-rich substrates and hinders the energy recovery from organic wastes. Bioaugmentation is promising strategy to stabilize AD systems with high ammonia concentration. The composition of microbial consortia often determines their effectiveness in bioaugmentation. Up to now, the effect of various microbial consortia as biological additives on the AD systems is not fully understood. In this study, two microbial consortia (syntrophic microbial consortium, MC, and hydrogenotrophic methanogen consortium, SS) were obtained through two domestication methods, and were applied in a nitrogen-rich AD system. The results showed that the MC and SS treatments could restore AD performance within 21 days and 83 days, respectively. The recovery of digestion performance depended on the methanogenic archaea Methanospirillum, Methanothermobacter, and Methanoculleus in the early and later stages. Analysis of the ¹³C isotope indicated that both MC and SS enhanced the hydrogenotrophic pathway. The KEGG analysis showed that the MC not only promoted the key enzyme genes in the hydrogenotrophic pathway but also had a positive effect on the related enzyme genes of propionate and butyrate degradation, which was affected by the abundant short-chain fatty acids degrading bacteria, such as Syntrophomonas, Syntrophobacter, and Tissierella in the MC. After recovery of digestion performance, there was no significant difference (p > 0.05) in methane yield between the MS and SS treatments. Therefore, the best intervention period for bioaugmentation is when the digestion performance of the AD system is unstable.

View all citing articles on Scopus

^☆: This paper has been recommended for acceptance by Klaus Kummerer.

¹: Tiantian Tian and Tianqi Niu contributed equally to this work.

View full text

Molecular characterization of antibiotic resistance in cultivable multidrug-resistant bacteria from livestock manure☆

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chicken and swine manure samples and antibiotics

Distribution and diversity of cultivable MARB in chicken and swine manure

Discussion

Conclusions

Acknowledgments

Ann. Clin. Microbiol. Antimicrob.

Curr. Opin. Microbiol.

Front. Microbiol.

Clin. Microbiol. Infect. Official Publ. Eur. Soc. Clin. Microbiol. Infect. Dis.

Vet. Microbiol.

Water Res.

J. Hazard Mater

Clin. Microbiol. Infect.

Characterisation of class 1 integron among Escherichia coli isolated from Mansoura University hospitals in Egypt

Adv. Microbiol.

Molecular mechanisms of antibiotic resistance

Nat. Rev. Microbiol.

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.

Increasing imipenem resistance and dissemination of the ISAba1-associated bla_OXA-23 gene among Acinetobacter baumannii isolates in an intensive care unit