novel antibiotic resistance determinants from agricultural soil exposed to antibiotics widely used in

56 Pages·2017·3.37 MB·English

AEM Accepted Manuscript Posted Online 16 June 2017
Appl. Environ. Microbiol. doi:10.1128/AEM.00989-17
© Crown copyright 2017.
The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors
who are government employees. The Crown Copyright is not transferable.
1 NOVEL ANTIBIOTIC RESISTANCE DETERMINANTS FROM AGRICULTURAL
2 SOIL EXPOSED TO ANTIBIOTICS WIDELY USED IN HUMAN MEDICINE AND
3 ANIMAL FARMING
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7 Calvin Ho-Fung Lau,a# Kalene van Engelen,a Stephen Gordon,a Justin Renaud,a and Edward loa
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8 Toppa,b# d

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11 London Research and Development Centre, Agriculture and Agri-Food Canada (AAFC). a
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12 London, Ontario, Canada a; Department of Biology, University of Western Ontario, London, .a
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13 Ontario b .
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16 Running title: Novel antibiotic resistant gene from agricultural soil il 2
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18 # Address correspondence to Edward Topp ([email protected]) and Calvin Lau b
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19 ([email protected]). u
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20 ABSTRACT
21 Antibiotic resistance has emerged globally as one of the biggest threats to human and animal
22 health. Although the excessive use of antibiotics is recognized for accelerating the selection for
23 resistance, there is a growing body of evidence suggesting that natural environments are
24 “hotspots” for the development of both ancient and contemporary resistance mechanisms. Given
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25 that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, w
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26 this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. a
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27 Using functional metagenomics, we interrogated the “resistome” of bacterial communities found d

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28 in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely m

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29 used in human medicine (macrolides) or food animal production (sulfamethazine, tt
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30 chlortetracycline and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes a
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31 (ARGs) recovered, the majority were predicted to encode for (multi)drug efflux systems, while a .a
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32 few share little to no homology with established resistance determinants. We characterized .
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33 several novel gene products, including putative enzymes that can confer high-level resistance /
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34 against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their A
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35 resistance mechanisms and clinical significance. By coupling high-resolution proteomics il 2
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36 analysis with functional metagenomics, we discovered an unusual peptide, PPPAZI 4, encoded 0
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37 within an alternative open-reading frame not predicted by bioinformatics tools. Expression of the b
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38 proline-rich PPPAZI 4 can promote resistance against different macrolides but not other u
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39 ribosomal-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that
40 could be fundamentally linked to the evolutionary design of this peptide.
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41 IMPORTANCE
42 Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial
43 pangenome. Genes and protogenes encoding for specialized and potential resistance mechanisms
44 are abundant in natural environments, but understanding of their identity and genomic context
45 remain limited. Our discovery of several previously-unknown antibiotic resistance genes from
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46 uncultured soil microorganisms indicates that soil is a significant reservoir of resistance w
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47 determinants, which, once acquired and “re-purposed” by pathogenic bacteria, can have serious a
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48 impacts on therapeutic outcomes. This study provides valuable insights into the diversity and d

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49 identity of resistance within the soil microbiome. The finding of a novel peptide-mediated m

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50 resistance mechanism involving an unpredicted gene product also highlights the usefulness of tt
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51 integrating proteomics analysis into metagenomics-driven gene discovery. a
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52 INTRODUCTION
53 Antimicrobial resistance is a serious threat to human and animal health on a global scale
54 with bacteria now resistant to the “last-resort” antibiotics including carbapenems and polymyxins
55 (1-4). It is estimated that, without any international-level efforts to revert our current situation,
56 antibiotic resistance would kill an additional 10 million people and cost the global economy 100
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57 trillion US dollars by 2050 (5). To safeguard the future of medicines required for treating w
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58 otherwise fatal infections, there is an urgent need to advance our understanding of the many a
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59 mechanisms that are exploited by bacteria to promote resistance. d

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60 The ancestral origin of clinical resistance is largely unknown and remains an elusive m

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61 subject to both the medical and research communities. In the perspective of evolutionary tt
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62 genetics, bacteria are capable of adapting to stressful conditions through chromosomal mutations a
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63 and/or acquisition of genetic materials that can provide them with survival advantages. As such, .a
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64 exposure to antibiotics is a key evolutionary driver for the development of various resistance .
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65 mechanisms including, but not limited to, enzymatic drug modification, alteration of drug /
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66 targets, reduced cell membrane permeability, and transporter(s)-mediated drug efflux (6). These A
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67 resistance mechanisms are typically encoded by the so-called antibiotic/antimicrobial resistance il 2
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68 genes (ARGs), and can be disseminated among microorganisms through horizontal gene transfer. 0
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69 The use of antibiotics will accelerate the selection for ARG(s)-carrying bacterial pathogens under b
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70 clinical settings (7). Likewise, anthropogenic inputs of antibiotics into the environment through u
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71 wastewater effluent, agricultural use of manure and biosolids, and aquaculture will increase the
72 diversity and size of environmental ARG reservoir, contributing to the spread of antibiotic
73 resistance (8-10).
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74 Antibiotic resistance is ancient and prevalent in the natural environment, and soil is a
75 significant reservoir of ARGs (11, 12). Soil is a very complex and dynamic environmental
76 matrix, typically containing billions of microorganisms and thousands of bacterial species in
77 each kilogram (13). With such microbial diversity and richness, it is not surprising that soil
78 harbours a large diversity of ARGs (14). In fact, recent studies analyzing soil samples collected
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79 from sites geographically or temporally distant from human activities recovered not only genetic w
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80 material orthologous to known ARGs found in contemporary pathogenic bacteria, but novel a
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81 resistance mechanisms that have potential clinical implications (15-17). d

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82 In the present study, we sought to explore the ARG content of agricultural soil from an m

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83 experimental farm located in London, Canada, by using functional metagenomics. Soil samples tt
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84 collected from antibiotic-amended and control field plots (18, 19) were mined for ARGs that can a
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85 promote resistance against clinically important antimicrobial agents. We report herein the .a
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86 identification of 34 new ARGs and the functional characterization of some of these resistance .
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87 determinants. We uncovered several previously-unknown resistance genes, notably one that /
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88 encodes a novel peptide-associated macrolide resistance mechanism. A
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89 RESULTS
90 To explore the content of antibiotic resistance genes (ARGs) borne by soil
91 microorganisms, three metagenomic fosmid libraries were constructed by cloning soil microbial
92 DNA into Escherichia coli (Table 1). These libraries encompassed over 36 Gb of metagenomic
93 DNA, equivalent to the combined size of about 7,200 E. coli genomes assuming an average
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94 genome size of 5 MB. Phenotypic-selection for resistance against 12 antimicrobial agents w
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95 belonging to six different antibiotic classes recovered ca. 250 fosmid clones from these a
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96 metagenomic libraries. After restriction-pattern analyzes were performed to reduce clonality d

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97 among candidates selected by the same antibiotic, re-transformation confirmed at least 44 m

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98 individual fosmids that are capable of conferring antibiotic resistance. High-throughput fosmid tt
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99 sequencing also revealed selection redundancy of using different and (un)related antibiotics, a
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100 implying that some of the recovered fosmids can promote multidrug resistance. Of the 31 non- .a
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101 redundant fosmids that can confer resistance, 28 had their metagenomic DNA insert sequence .
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102 resolved to completion (ranging 24.2 - 43.9 kb in size), whereas the remaining three were /
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103 partially assembled into separated contigs (8.8 - 26.2 kb) (Dataset S1). A
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104 Based on the re-transformant’s antibiotic susceptibility profiles (Fig 1A & Table S1), the il 2
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105 fosmid-conferred resistance against aminoglycosides, sulfonamides, and most of the observed β- 0
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106 lactams were not associated with co- or cross-resistance against other classes of antibiotics (Fig b
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107 1B). On the contrary, all tetracycline-resistant fosmid clones displayed reduced susceptible u
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108 towards at least one additional class of antibiotic including macrolides (Fig 1B). Among the nine
109 clones that exhibited classic multidrug resistance phenotype (group VI), resistance against a 3rd-
110 generation cephalosporin, ceftazidime (CAZ), was only detected in the MACRO-TET A6 clone
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111 (Fig 1A). Macrolide-specific resistance was observed in six fosmid clones, with MACRO-TYL
112 10 also demonstrating mildly enhanced sensitivity towards an aminoglycoside (Fig 1A).
113 Using random transposon mutagenesis, 36 putative ARGs were identified from the
114 resistance-associated fosmids (Table 2). Functional annotations suggested majority of the
115 identified ARGs encode for efflux-mediated mechanisms, while two known and five presumed
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116 enzymatic-based resistance determinants were also uncovered (Fig 1C). Additional ORFs that w
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117 showed no apparent linkage with any prominent resistance mechanisms were affiliated with a
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118 resistance against macrolides (Table 2). To ascertain their ARG identity and to gain insight into d

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119 the underlying resistance principles encoded by some of these soil-derived resistance m

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120 determinants, a subcloning strategy was employed with the corresponding characterization tt
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121 findings presented in the following sections. a
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122 Efflux-mediated resistance. Individual versus combinational sub-cloning of two putative ATP- .a
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123 binding cassette (ABC) transporter-encoding ORFs identified from fosmid MACRO-TET A7 .
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124 (i.e. orf-45TET A7 and orf-46TET A7) revealed a co-requisite of these two ARGs for conferring /
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125 multidrug resistance (Table 3). The restored antibiotic sensitivity of transposon-mutants that had A
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126 either one of the two transporter-encoding ARGs inactivated supports the notion that these two il 2
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127 ARGs constitute a single functional efflux system. Susceptibility testing against the anti-cancer 0
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128 drugs daunorubicin (DAU) and doxorubicin (DOX) further demonstrated the capability of this b
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129 ABC multidrug efflux pump to protect the bacteria host against the anthracycline class of u
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130 antibiotics (Fig 2A). It was discovered that homologues of this two-component efflux system of
131 MACRO-TET A7 were present in seven other multidrug resistant fosmid clones (Table 2), all of
132 which displayed similar reduction in susceptibility towards both DAU and DOX (Fig 2B).
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133 Enzymatic resistance mechanisms: drug- or target- modifications. ARGs identical to the well-
134 characterized resistance determinants aph-3’, which encodes an aminoglycoside O-
135 phosphotransferase (20), and sul2, which encodes a type II sulfonamide-resistant variant of
136 dihydropteroate synthase (DHPS, also known as FolP) (21), were uncovered (Table 2). Both
137 ARGs were identified within genomic neighbourhoods where elements implicated in lateral gene
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138 transfer (e.g. insertion sequence IS903, transposase, integrase, conjugative transfer protein) and w
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139 homologous recombination (e.g. DNA helicases, restriction endonuclease) were in close a
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140 proximity (Dataset S1 & Table S2). d

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141 Expression of orf-7KAN 4 derived from the fosmid clone SCT-KAN 4 was confirmed to m

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142 promote resistance against three clinically-significant aminoglycosides in not only E. coli, but tt
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143 also the opportunistic pathogen Pseudomonas aeruginosa (Table 4). Given its sequence a
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144 similarity (~60% shared identity) to an aminoglycoside 6’-N-acetyltransferase (Table 2), these .a
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145 results suggest that orf-7KAN 4 encodes for a potentially new member of this aminoglycoside- .
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146 modifying enzyme family, which confers antibiotic resistance through drug-inactivation. /
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147 The broad-spectrum β-lactam resistant determinants recovered from the meropenem- A
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148 selected fosmid clones (Fig 1A, group III) were both inhibited by EDTA, but not by the classic il 2
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149 serine-type β-lactamase inhibitor clavulanic acid (Table 5), in agreement with their predicted 0
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150 functional entities as metallo-β-lactamases (Table 2). The extensive β-lactam resistance b
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151 phenotype was also recapitulated in E. coli and P. aeruginosa strains carrying the sub-cloned orf- u
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152 27MEM A1, which was found proximal to additional β-lactamase-related ARGs-like genetic t
153 elements in MACRO-MEM A1 (Dataset S1 & Table S2).
154 A novel sulfonamide resistance gene hereinafter designated as folPSMZ B27 was discovered
155 from the fosmid clone UTC-SMZ B27. FolPSMZ B27 shares 35% sequence identity with the E. coli
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156 DHPS (22) but, reminiscent of the two sul2-carrying fosmid clones, folP SMZ-B27-expressing E.
157 coli cells were recalcitrant to the bacteriostatic effect of sulfonamide antibiotics (Fig 3), which is
158 mediated through the inhibition of dihydropteorate synthase (DHPS)-dependent folate
159 biosynthesis. Based on its ability to confer growth competence to a thymidine-auxotrophic folP-
160 knockout E. coli strain (23) under a thymidine-deficient condition (Fig 4B, C), it was verified
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161 that folP SMZ-B27 encodes a functional DHPS. The ability to withstand the antagonistic effect of w
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162 sulfonamides specific to DHPSs (Fig 4D, E) also implies that FolP SMZ-B27 represents an loa
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163 insensitive variant of these enzymes. d

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164 By selecting for MACRO-TET A6-derived transposon mutant with restored CAZ m

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165 sensitivity, a new cephalosporin resistance determinant, ORF#11 (hereinafter designated as tt
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166 pbpTET A6), was identified adjacent to an ABC multidrug transporter-encoding operon (i.e. a
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167 ORF#12-13) (Table 2). PBPTET A6 consists of putative N-terminal transglycosylase and C- .a
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168 terminal transpeptidase domains, resembling the bifunctional class A penicillin-binding proteins .
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169 (PBPs) (24). Subcloning of pbpTET A6 confirmed the ability of this ARG-encoding product in /
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170 rendering E. coli resistant to selected 3rd- and 4th- generation cephalosporins (Table 6). A
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171 Introduction of pbpTET A6 into P. aeruginosa further demonstrated its versatility and potential in il 2
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172 promoting resistance against the same spectrum of antibiotics in a clinically-significant pathogen 0
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173 (Table 6). b
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174 Unknown macrolide resistance mechanisms. The MACRO-TYL 10-derived ORF-16TYL 10 has u
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175 64% sequence identity of the GTPase HflX of a Chlamydia-related bacterium, Simkania
176 negevensis, and promoted mild resistance against macrolides when sub-cloned into different E.
177 coli hosts (Table 7). Intriguingly, expression of ORF-16TYL 10 in strain AG100A, a macrolide-
178 sensitive derivative of AG100 that is devoid of the significant multidrug Resistance-Nodulation-
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179 Division (RND) efflux system component AcrAB (25), did not result in any appreciable level of
180 macrolide resistance. Consistent with this, the MACRO-TYL 10-associated reduction of
181 macrolide susceptibilities was also compromised by the RND pump inhibitor phenyl-arginine-β-
182 naphthylamide (PAβN). Yet, orf-16TYL 10 expression did not provide any resistance against other
183 ribosome-targeting antibiotics including tetracycline, lincomycin and chloramphenicol (Fig 1A
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184 & Table 7), despite some of them being excellent substrates of the AcrAB efflux pump (26). w
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185 Collectively, these observations rule out a general up-regulation of AcrAB’s efflux activity in the a
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186 orf-16TYL10 -expressing cell, and indicate that ORF-16TYL10 constitutes a new macrolide-specific d

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187 resistance mechanism, which is somehow dependent on the macrolide extrusion mediated by m

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188 AcrAB. tt
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189 From the macrolide-resistant fosmid MACRO-AZI 4, transposon-mutagenesis identified a
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190 the in-silico predicted orf-28AZI 4, orf-29AZI 4 and orf-30AZI 4 as potential resistance determinants .a
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191 (Fig 5A). Blastx homology search against the NCBI non-redundant (nr) protein database using .
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192 these three theoretical ORFs as queries resulted in no significant similarity found for orf-28 AZI 4 /
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193 and two hypothetical proteins for orf-29AZI 4 and orf-30AZI 4. Through constructing a series of A
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194 pCF430-based subclones (pCL043 - pCL047, Fig 5A), it was deduced that the macrolide il 2
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195 resistance, which is independent of the RND pump-mediated efflux, was attributed solely to the 0
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196 region encompassing orf-28AZI 4 (Table 8). Compared to the control strain (Fig 5B), the pCL047- b
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197 transformant was clearly less susceptible to the growth-inhibitory effect of azithromycin (Fig u
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198 5C). Furthermore, the growth-deficiency associated with MACRO-AZI 4 (Fig 5D) and only
199 certain sub-clones (Fig 5E) failed to correlate with the generally observed macrolide-resistant
200 phenotype (Fig 5A & Table 8).
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