Chemical treatment of poultry litter affects the conjugation of plasmid-mediated extended-spectrum beta-lactamase resistance genes in E. coli

Ó 2019 Published by Elsevier Inc. on behalf of Poultry Science Association Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Chemical treatment of poultry litter affects the conjugation of plasmid-mediated extended-spectrum beta-lactamase resistance genes in E. coli Mauro M.S. Saraiva,*,§ Alexandre L.B. Moreira Filho,† Priscylla C. Vasconcelos,* Pavlos V. Nascimento,* Paulo Sergio Azevedo,* Oliveiro C. Freitas Neto,‡ Patrícia E.N. Givisiez,* Wondwossen A. Gebreyes,§,# and Celso J.B. Oliveira*,#,1 *

Department of Animal Science, Center for Agricultural Sciences, Federal University of Paraiba (CCA/UFPB), Areia, PB 58397-000, Brazil; †Department of Animal Science, Federal University of Rondonia (UNIR), Presidente Médici, RO 76916-000, Brazil; ‡ Department of Preventive Veterinary Medicine, Veterinary School, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil; §Department of Preventive Veterinary Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA; and #Global One Health Initiative (GOHi), The Ohio State University, Columbus, OH 43210, USA Primary Audience: Food Safety Specialists, Researchers and Veterinarians, Representatives of the Poultry Industry, Managers of Poultry Production Units SUMMARY There is a general consensus among healthcare leaders that animal production industries should improve practices aiming to mitigate antimicrobial resistance, a significant global threat to public health. However, the epidemiology of antimicrobial resistance in animal production systems is very complex as a result of innumerous sources and dissemination routes of resistant bacteria. Furthermore, antimicrobial resistance can be transferred among bacteria by mobile elements, such as plasmids, which play a major role in the dissemination of antimicrobial resistance. In broiler production, litter contaminated by feces during the production cycle can serve as a potential reservoir for bacteria harboring antimicrobial resistance genes that can then be disseminated to susceptible bacteria. This study reports the effect of 1) different materials used as litter and 2) different litter recycling protocols on the conjugation frequencies of IncIII plasmids harboring extended-spectrum beta-lactamase–resistant genes among Escherichia coli isolates. Results found that, compared with cane bagasse litter, the use of wood shavings as litter decreased the conjugation frequency among E. coli isolates (P , 0.001). In regard to the recycling protocols, the presence of chemical residues in both types of litter materials was associated with a decreased conjugation frequency, with lowest frequencies observed for quicklime and superphosphate. Our findings suggest that the type of material used as poultry litter as well as litter recycling procedures, distinguished by means of chemical compounds, may affect plasmid conjugation among E. coli isolates. Key words: cane bagasse, mobile ESBL genes, plasmid transfer, poultry, wood shavings 2019 J. Appl. Poult. Res. -:-–https://doi.org/10.1016/j.japr.2019.10.006

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Corresponding authors: [email protected]; [email protected]

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DESCRIPTION OF PROBLEM The emergence of multidrug-resistant bacteria poses a major global public health concern. There has been a great deal of debate over the actual impact of the use of antimicrobial drugs in food animals and the emergence of multidrug-resistant infections in humans [1–3]. Although there are increasing numbers of reports on the occurrence of multidrug-resistant bacteria in food animals [4–6], there is a lack of studies focusing on the dynamics of antimicrobial resistance genes at the animal–environment interface. In intensive broiler production systems, litter is composed of a mixture of bedding, such as wood shavings or alternative materials, with poultry excreta, feed residues, and water that accumulate during farming [7], thus favoring the multiplication of microorganisms. Around 90% of the antimicrobials used in poultry production are not absorbed and are therefore eliminated into the litter through animal excreta [8, 9]. Optimal conditions for microbial growth in combination with the constant presence of subinhibitory concentrations of antimicrobial residues may increase the selection of resistant bacteria, making litter serve as a potential bioreactor. As a result, Escherichia coli isolated from broilers and poultry products have been found to harbor b-lactam resistance genes, including those encoding for extendedspectrum beta-lactamases such as blaTEM, blaCTX-M, blaSHV, and blaCMY [10–12]. These resistance genes have also been found in bacteria associated with critical infections in humans [13]. Litter is a major economical factor in the broiler industry. Recycling of litter by means of chemical treatment between consecutive flocks is a common practice to reduce production costs and the environmental impact associated with poultry production [14]. Considering the highly complex epidemiology of antimicrobial resistance [13], in which not only bacteria but also antimicrobial resistance genes can be disseminated among animals, humans, and the environment [15, 16], we still have a poor understanding of the role that coselection factors in animal production play in relation to the emergence and spread of antimicrobial resistance.

Bacterial conjugation is a major mechanism for the dissemination of plasmid-encoded genes conferring resistance against antimicrobials that are highly critical to human medicine, such as third-generation cephalosporins [17], carbapenems [18], and polymixyn [19]. Conjugation involves production of a pilus (encoded by the conjugative plasmid) that attaches to a recipient cell and facilitates the transfer of the conjugative element. It has been suggested that chemically blocking conjugation factors could limit the spread of plasmid-encoded antimicrobial resistance genes among bacteria [20, 21], therefore contributing to the mitigation of antimicrobial resistance. The major objective of this study was to investigate the effects of chemical residues in different materials used as poultry litter on the conjugation frequencies of a plasmid carrying extended-spectrum beta-lactamase resistance genes among E. coli strains.

MATERIAL AND METHODS Bacterial Strains Used in the Conjugation Assay The human-originated E. coli H2332 strain harboring the plasmid pH2332-166 was used as the donor bacterium in the conjugation assays. As previously described [22], this IncFII-IncFIB plasmid harbors the b-lactamase resistance blaTEM-1 gene as well as genes conferring resistance against amphenicol (catA1), aminoglycosides (aadA1b; strAB), macrolides (mphB), tetracyclines (tetR; tetA), trimethoprim (dfrA1), and sulfonamides (sul1; sul2). A mutant E. coli J62 strain, lacking the lactose metabolizing gene (Lac2) but resistant to both nalidixic acid (Nal1) and rifampicin (Rif1) [17], was used as a recipient strain. Both donor and recipient bacteria were cultured in LuriaBertani broth [23] under orbital shaking at 37  C for 24h and then transferred to peptone water [23]. Concentrations were adjusted to 1 3 107 and 3 3 107 CFU/mL and then kept at room temperature for 1h. Experimental Design A 2 3 5 factorial arrangement on a completely randomized design including 2 types of litter

SARAIVA ET AL: BACTERIAL PLASMID CONJUGATION IN POULTRY LITTER material (cane bagasse and wood shavings) and 5 chemical treatments consisting of T1, litter material only (control); T2, litter material plus 300 mg of quicklime (CaO); T3, litter material plus 100 mg of superphosphate; T4, litter material plus 1 mg of copper sulfate (CuSO4); and T5, litter material plus 1 mg of aluminum sulfate (Al2(SO4)3), resulting in a total of 10 treatments with 15 repetitions each. The concentration of the chemical residues was determined by applying the litter recycling protocols currently used under field conditions [24]. Litter samples (10 g) were placed in sterile plastic bags [25] and gently mixed with peptone water (10 mL) containing the recipient strain J62 (3 3 107 cfu/mL). The bags were kept at room temperature for 1h. Excess broth was discharged, and a 10 mL aliquot of peptone water containing the donor strain H2332 (107 cfu/mL) was added. The bags were kept at room temperature for 1h. After discharge of excess broth, the bags were incubated at 25 C for 24h. Conjugation Frequency Determination and Statistical Analysis After incubation, sterile saline (90 mL) was added into the bags; the materials were homogenized by means of a stomacher [26] and then serially diluted (1:10) in sterile saline. Three aliquots (20 mL) from each dilution were placed onto MacConkey [27] agar plates supplemented with 70 mg of nalidixic acid plus 40 mg of ceftriaxone in parallel with MacConkey [27] agar plates supplemented with 70 mg of nalidixic acid only, to recover transconjugant bacteria and total recipient bacteria, respectively. MacConkey [27] agar plates were incubated at 37  C for 24h. E. coli Lac2 colonies were enumerated on both agar plates, and the conjugation frequency was calculated by dividing the number of transconjugant bacteria by the number of recipient bacteria. The bacterial counts (recipient and transconjugant E. coli) and the conjugation frequency data across treatments were analyzed by a two-way ANOVA with a Bonferroni multiple comparison test (P , 0.05). Statistical analyses were performed using the GraphPad Prism, version 6, software [28].

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RESULTS AND DISCUSSION No differences in the recipient E. coli counts were observed across the treatments (Table 1), except in T5, in which no bacterial growth was observed in wood shavings containing aluminum sulfate (P , 0.001). These results demonstrate that the model used in the present study is appropriate to evaluate the putative effects of the treatments on the conjugative plasmid transfer among donor and recipient E. coli, as these treatments had no bactericidal effect on the recipient bacteria that could compromise the interpretation of the conjugation efficiency. The plasmid conjugation frequencies among E. coli in the 2 litter materials under the 5 treatment groups are shown in Table 2. Interestingly, a significantly lower conjugation frequency occurred in cane bagasse than in wood shavings. A conclusive explanation for this finding is difficult considering the high biological complexity of the bacterial plasmid conjugation mechanisms which are affected by intrinsic plasmid regulatory factors [29] and extrinsic environmental factors such as the presence of ions [30], diversity of recipient bacteria [31], and predation by other microorganisms [32]. Considering the experimental conditions in the present study, which controlled for factors such as microbial diversity, we strongly believe that observed differences are due to mating conditions associated with the different physical characteristics of the materials. Plasmid conjugation efficiency is higher in solid surfaces than in liquid surfaces, possibly because of the facilitated cell contact, especially at low cell densities [33]. Considering the types of materials tested in our trial, media absorption in cane bagasse was significantly higher than in wood shavings. We hypothesize that the increased superficial area of cane bagasse decreases bacterial mating compared with wood shavings in a weight-based comparison as a result of its porosity. However, cane bagasse and wood shavings differ considerably in terms of chemical composition as well. Further studies need to be carried out to gain information on the effects of different materials used for bedding purposes on the transfer of

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Table 1. Logarithmically transformed counts (log cfu/g) of recipient and transconjugant E. coli obtained from the In Vitro conjugation assays performed in cane bagasse and wood shavings only (T1) or in the presence of chemical compounds (treatments T2–T5). Recipient E. coli counts P-value

Type of litter material Treatments T1 T2 T3 T4 T5 Mean

Cane bagasse (log cfu/g) aB

9.13214 9.16301aB 9.07655aB 9.07423aB 8.17852A 8.92489

SD 0.429 0.075 0.095 0.105 0.215

Wood shavings (log cfu/g) aA

9.44535 9.24036aA 9.09204aA 9.06051aA NC 7.36765

SD 0.060 0.036 0.117 0.123 -

Treatment means (cfu/g) 9.28888 9.20169 9.08430 9.06737 -

T

L

T*L

,0.001

,0.001

,0.001

Transconjugant E. coli counts Litter materials Treatments T1 T2 T3 T4 T5 Mean

Cane bagasse (cfu/g) aC

5.71229 3.11797aB 2.77423aA 3.00000aAB 2.81475A 3.48385

SD 0.230 0.186 0.136 0.025 0.152

P-value

Wood shavings (cfu/g) aC

5.84486 4.22787bA 4.47958bB 4.26930bAB NC 3.76432

SD 0.076 0.197 0.144 0.163 -

Treatment means (cfu/g) 5.77858 3.67292 3.62691 3.63465 -

T

L

T*L

,0.001

,0.001

,0.001

Means with different uppercase letters in the columns or different lowercase letters in the lines differ significantly by the two-way ANOVA with Bonferroni multiple comparison test at P , 0.05. Abbreviations: T, difference among treatment means; L, difference between litter material means; T*L, interaction between treatments and liter material; SD, standard deviation; NC, no bacterial growth; T1, control group; T2, quicklime (CaO) group; T3, superphosphate group; T4, copper sulfate (CuSO4) group; T5, aluminum sulfate (Al2(SO4)3) group.

conjugative plasmids in bacteria or even on the survival of bacteria in the environment. For instance, cane bagasse has high moisture and carbohydrate content [34, 35], both of which favor bacterial growth. Although bacteriostatic properties have been described in cane bagasse [36], a wide range of bactericidal compounds have been identified in various wood types [37– 39]. Higher conjugation frequencies were observed in the control groups (no chemical residues) regardless of the litter material used. No bacterial growth was observed in wood shavings containing aluminum sulfate. This result was unexpected considering overall higher conjugation frequencies (P , 0.001) occurred in wood shavings, except for the control group in which no statistical difference (P = 0.149) was observed (Table 2). This finding indicates higher bactericidal activity of aluminum sulfate residues on wood shavings than cane bagasse, possibly related to an

interaction effect associated with the differences in physical properties between wood shavings and cane bagasse. Aluminum sulfate is commonly used to treat poultry litter to reduce ammonia in poultry houses. Therefore, aluminum-treated litter is associated with improved performance and reduced mortality of broilers [40], as well as decreased E. coli contamination [41]. Quicklime residues in both types of materials led to a reduced conjugation frequency compared with the control groups (Table 2). Quicklime is considered a first option to reduce Enterobacteriaceae contamination in poultry litter [42] because of its capacity to reduce moisture and water activity [43]. Calcium oxide reduced pathogenic bacteria by about 2 log, but more efficient results have been observed when used in association with other sanitizers [44]. The lowest conjugation frequencies were observed in litter containing superphosphate and copper sulfate residues. Superphosphate is a

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Table 2. Plasmid conjugation frequency between donor (E. coli H2332) and recipient (E. coli J62) E. coli bacteria in cane bagasse and wood shavings only (T1) or in the presence of chemical compounds (treatments T2–T5). Litter materials Treatments T1 T2 T3 T4 T5 Means (CFU/g)

Cane bagasse (CFU/g) SD 23.39910 25.94615aB 26.30237aA 26.07435aB 25.36394C 25.73703 aD

0.185 0.196 0.134 0.074 0.123

Wood shavings (CFU/g) SD 23.60025 25.01063bA 24.61261bB 24,79119bAB NC 24,68,204 aC

0.060 0.167 0.140 0.152 -

Means (CFU/g)

P-value T

L

T*L

23,49,968 ,0.001 ,0.001 ,0.001 25,56,344 25,53,430 25,35,257

Means with different uppercase letters in the columns or different lowercase letters in the lines differ significantly by the two-way ANOVA with Bonferroni multiple comparison test at P , 0.05. Abbreviations: T, difference among treatment means; L, difference between litter material means; T*L, interaction between treatments and liter material; SD, standard deviation; NC, no bacterial growth; T1, control group; T2, quicklime (CaO) group; T3, superphosphate group; T4, copper sulfate (CuSO4) group; T5, aluminum sulfate (Al2(SO4)3) group.

compound formed by combining rock phosphate with sulfuric acid or phosphoric acid and is used worldwide as an agricultural fertilizer. In animal production systems, superphosphate is used to reduce ammonia concentrations [45]. Phosphate compounds can reduce pathogenic E. coli by decreasing bacterial deposition and biofilm forming [46]. Copper sulfate is broadly used as a growth promoter in intensive poultry farms [47–49] and has been described to be very efficient at reducing E. coli either used alone [50] or together with lactic acid [51]. Although we reported a decreased conjugation frequency in litter containing copper sulfate, copper sulfate and other heavy metals might play an important role as coselection factors for antimicrobial resistance [52]. For example, copper resistance has been found in bacteria carrying tcrB gene on R-plasmids [47, 48]. Bacteria harboring Rplasmids have been reported in both humans and animals worldwide [53, 54]. Our study indicates that residual concentrations of chemicals used to recycle poultry litter can reduce the conjugation frequencies among E. coli isolates in litter. However, the experimental model used in our study has some limitations. First, this model does not consider complex microbial communities of the poultry litter in field conditions. For instance, some studies have been performed using an in vitro gut model [54], allowing for the consideration of variables such as nutrient availability and other conditions that might favor cell-to-cell interactions. Second, and probably more importantly, the model used herein was not able

to assess the effects of the chemical residues individually on each of the 2 components that determine the conjugation dynamics, the conjugation frequency and the subsequent growth of transconjugants, that is, post-transfer selection [55]. A novel experimental approach to decouple these 2 components and then assess the effects of subinhibitory concentrations of antibiotics on the conjugation efficiency has been recently proposed [56]. In spite of limitations, the findings of this study suggest that litter management practices could affect the dissemination of antimicrobial resistance genes in poultry production systems.

CONCLUSIONS AND APPLICATIONS 1. We observed that residues of chemical compounds used to recycle poultry litter can reduce the plasmid conjugation frequencies among E coli isolates under in vitro conditions. 2. The effect of the chemical residues on plasmid conjugation frequencies among E. coli isolates may be affected by the type of materials used as litter.

REFERENCES AND NOTES 1. Economou, V., and P. Gousia. 2015. Agriculture and food animals as a source of antimicrobial-resistant bacteria. Infect. Drug. Resist. 8:49–61. 2. Jindal, A. K., K. Pandya, and I. D. Khan. 2015. Antimicrobial resistance: a public health challenge. Med. J. Armed Forces India 71:178–181.

6 3. Van Boeckel, T. P., C. Brower, M. Gilbert, B. T. Grenfell, S. A. Levin, T. P. Robinson, A. Teillant, and R. Laxminarayan. 2015. Global trends in antimicrobial use in food animals. PNAS 112:5649–5654. 4. Fernández, J., B. Guerra, M. Rodicio, J. Fernández, B. Guerra, and M. R. Rodicio. 2018. Resistance to carbapenems in non-typhoidal Salmonella enterica serovars from humans, animals and food. Vet. Sci. 5:1–13. 5. Kim, Y. B., H. J. Seo, K. W. Seo, H. Y. Jeon, D. K. Kim, S. W. Kim, S. K. Lim, and Y. J. Lee. 2018. Characteristics of high-level ciprofloxacin-resistant Enterococcus faecalis and Enterococcus faecium from retail chicken meat in Korea. J. Food Prot. 81:1357–1363. 6. Martins, B. T. F., C. V. Botelho, D. A. L. Silva, F. G. P. A. Lanna, J. L. Grossi, M. E. M. Campos-Galvão, R. S. Yamatogi, J. P. Falcão, L. S. Bersot, and L. A. Nero. 2018. Yersinia enterocolitica in a Brazilian pork production chain: Tracking of contamination routes, virulence and antimicrobial resistance. Int. J. Food Microbiol. 276:5–9. 7. Ljubojevic, D., N. Puvaca, M. Pelic, D. Todorovic, M. Pajic, D. Milanov, and M. Velhner. 2016. Epidemiological significance of poultry litter for spreading the antibiotic-resistant strains of Escherichia coli. World’s Poult. Sci. J. 72:485–494. 8. Sarmah, A. K., M. T. Meyer, and A. B. A. Boxall. 2006. A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 65:725–759. 9. Heuer, H., H. Schmitt, and K. Smalla. 2011. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr. Opin. Microbiol. 14:236–243. 10. Blaak, H., A. H. A. M. van Hoek, R. A. Hamidjaja, R. Q. J. van der Plaats, L. K. Heer, A. M. R. Husman, and F. M. Schets. 2015. Distribution, numbers, and diversity of ESBL-producing E. coli in the poultry farm environment. PLoS One 10:e0135402. 11. Maamar, E., S. Hammami, C. A. Alonso, N. Dakhli, M. S. Abbassi, S. Ferjani, Z. Hamzaoui, M. Saidani, C. Torres, and I. Boutiba-Ben Boubaker. 2016. High prevalence of extended-spectrum and plasmidic AmpC betalactamase-producing Escherichia coli from poultry in Tunisia. Int. J. Food Microbiol. 231:69–75. 12. Dame-Korevaar, A., E. A. J. Fischer, A. Stegeman, D. Mevius, A. van Essen-Zandbergen, F. Velkers, and J. van der Goot. 2017. Dynamics of CMY-2 producing E. coli in a broiler parent flock. Vet. Microbiol. 203:211–214. 13. Birgand, G., J. R. Zahar, and J. C. Lucet. 2018. Insight into the complex epidemiology of multidrugresistant Enterobacteriaceae. Clin. Infect. Dis. 66:494–496. 14. Tasho, R. P., and J. Y. Cho. 2016. Veterinary antibiotics in animal waste, its distribution in soil and uptake by plants: a review. Sci. Total Environ. 563–564: 366–376. 15. Huddleston, J. R. 2014. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infect. Drug Resist. 7:167–176. 16. Liu, Y. Y., Y. Wang, T. R. Walsh, L. X. Yi, R. Zhang, J. Spencer, Y. Doi, G. Tian, B. Dong, X. Huang, L. F. Yu, D. Gu, H. Ren, X. Chen, L. Lv, D. He, H. Zhou, Z. Liang, J. H. Liu, and J. Shen. 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect. Dis. 16:161–168.

JAPR: Research Report 17. Niero, G., V. Bortolaia, M. Vanni, L. Intorre, L. Guardabassi, and A. Piccirillo. 2018. High diversity of genes and plasmids encoding resistance to third-generation cephalosporins and quinolones in clinical Escherichia coli from commercial poultry flocks in Italy. Vet. Microbiol. 216:93–98. 18. Pulss, S., I. Stolle, I. Stamm, U. Leidner, C. Heydel, T. Semmler, E. Prenger-Berninghoff, and C. Ewers. 2018. Multispecies and clonal dissemination of OXA-48 carbapenemase in Enterobacteriaceae from companion animals in Germany, 2009—2016. Front Microbiol. 9:1265. 19. Yin, W., H. Li, Y. Shen, Z. Liu, S. Wang, Z. Shen, R. Zhang, T. R. Walsh, J. Shen, and Y. Wang. 2017. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli. mBio 8:e00543-17. 20. Lin, A., J. Jimenez, J. Derr, P. Vera, M. L. Manapat, K. M. Esvelt, L. Villanueva, D. R. Liu, and I. A. Chen. 2011. Inhibition of bacterial conjugation by phage m13 and its protein g3p: quantitative analysis and model. PLoS One 6:e19991. 21. Getino, M., R. Fernández-López, C. Palencia-Gándara, J. Campos-Gómez, J. M. Sánchez-López, M. Martínez, A. Fernández, and F. I. Cruz. 2016. Tanzawaic acids, a chemically novel set of bacterial conjugation inhibitors. PLoS One 11:e0148098. 22. Wang, J., R. Stephan, K. Power, Q. Yan, H. Hächler, and S. Fanning. 2014. Nucleotide sequences of 16 transmissible plasmids identified in nine multidrug-resistant Escherichia coli isolates expressing an ESBL phenotype isolated from food-producing animals and healthy humans. J. Antimicrob. Chemother. 69:2658–2668. 23. BD Difco. BeCton, Dickerson and Company. 1 Becton Drive. Franklin Lakes, NJ 07417-1800, EUA. 24. Palhares, J. C. P., and A. Kunz. 2011. Manejo ambiental na avicultura. Concordia – Embrapa Suínos e Aves – Procedures 149:175–200. Accessed April 2019. https://ainfo.cnptia.embrapa.br/digital/bitstream/item/57055/ 1/manejo-ambiental-na-avicultura.pdf ISSN 0101- 6245. 25. Whirl-pack - Nasco. 901 Janesville Avenue. Fort Atkinson, WI 53538-0901, EUA. 26. Marconi - Laboratory Equipment. 260 Rafael Ducatti – Algodoal. Piracicaba, SP 13405-455, Brazil. 27. Acumedia. 620 Lesher Place. Lansing, MI 48912, EUA. 28. GraphPad Software, San Diego, CA. 29. Frost, L. S., and G. Koraimann. 2010. Regulation of bacterial conjugation: balancing opportunity with adversity. Future Microbiol. 5:1057–1071. 30. Shintani, M., K. Matsui, T. Takemura, H. Yamane, and H. Nojiri. 2008. Behavior of the IncP-7 carbazoledegradative plasmid pCAR1 in artificial environmental samples. Appl. Microbiol. Biotechnol. 80:485–497. 31. Sakuda, A., C. Suzuki-Minakuchi, K. Matsui, Y. Takahashi, K. Okada, H. Yamane, M. Shintani, and H. Nojiri. 2018. Divalent cations increase the conjugation efficiency of the incompatibility P-7 group plasmid pCAR1 among different Pseudomonas hosts. Microbiology 164:20–27. 32. Cairns, J., M.i. Jalasvuori, V. Ojala, M. Brockhurst, and T. Hiltunen. 2016. Conjugation is necessary for a bacterial plasmid to survive under protozoan predation. Biol. Lett. Biol. Lett. 12:20150953.

SARAIVA ET AL: BACTERIAL PLASMID CONJUGATION IN POULTRY LITTER 33. Yanagida, K., A. Sakuda, C. Suzuki-Minakuchi, M. Shintani, K. Matsui, K. Okada, and H. Nojiri. 2016. Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities. Biosci. Biotechnol. Biochem. 80:1020–1023. 34. Teixeira, A. S., M. C. Oliveira, J. F. Menezes, B. M. Gouvea, S. R. Teixeira, and A. R. Gomes. 2015. Poultry litter of wood shavings and/or sugarcane bagasse: animal performance and bed quality. Rev. Colom. Cienc. Pecua. 28:238–246. 35. Bezerra, T. L., and A. J. Ragauskas AJ. 2016. A review of sugarcane bagasse for second-generation bioethanol and biopower production. Biofuels Bioprod. Biorefin. 10:634–647. 36. Zhao, Y., M. Chen, Z. Zhao, and S. Yu. 2015. The antibiotic activity and mechanisms of sugarcane (Saccharum officinarum L.) bagasse extract against food-borne pathogens. Food Chem. 185:112–118. 37. Kavian-Jahromi, N., L. Schagerl, B. Dürschmied, S. Enzinger, C. Schnabl, T. Schnabel, and A. Petutschnigg. 2015. Comparison of the antibacterial effects of sapwood and heartwood of the larch tree focusing on the use in hygiene sensitive areas. Eur. J. Wood Prod. 73:841–844. 38. Salido, S., M. Pérez-Bonilla, R. P. Adams, and J. Altarejos. 2015. Phenolic components and antioxidant activity of wood extracts from 10 main Spanish olive cultivars. J. Agric. Food Chem. 63:6493–6500. 39. Alejo-Armijo, A., N. Glibota, M. P. Frías, J. Altarejos, A. Gálvez, E. Ortega-Morente, and S. Salido. 2017. Antimicrobial and antibiofilm activities of procyanidins extracted from laurel wood against a selection of foodborne microorganisms. Int. J. Food Sci. Technol. 52:679–686. 40. Moore, P. A., T. C. Daniel, and D. R. Edwards. 2000. Reducing phosphorus runoff and inhibiting ammonia loss from poultry manure with aluminum sulfate 1. J. Environ. Qual. 29:37–49. 41. Line, J. E. 2002. Campylobacter and Salmonella populations associated with chickens raised on acidified litter. Poult. Sci. 81:1473–1477. 42. Lopes, M., F. L. Leite, B. S. Valente, T. Heres, A. M. Dia Prá, E. G. Xavier, and V. F. B. Roll VFB. 2015. An assessment of the effectiveness of four in-house treatments to reduce the bacterial levels in poultry litter. Poult. Sci. 94:2094–2098. 43. Ahn, H. K., T. L. Richard, and T. D. Glanville. 2008. Laboratory determination of compost physical parameters for modeling of airflow characteristics. Waste Manag. 28:660–670. 44. Ngnitcho, P. F. K., I. Khan, C. N. Tango, M. S. Hussain, and D. H. Oh. 2017. Inactivation of bacterial pathogens on lettuce, sprouts, and spinach using hurdle technology. Innov. Food Sci. Emerg. Technol. 43:68–76. 45. Soliman, E. S., and R. A. Hassan. 2017. Evaluation of superphosphate and meta-bisulfide efficiency in litter treatment on productive performance and immunity of broilers exposed to ammonia stress. Adv. Anim. Vet. Sci. 7:253–259. 46. Wang, L., S. Xu, and J. Li. 2011. Effects of phosphate on the transport of Escherichia coli O157:H7 in saturated quartz sand. Environ. Sci. Technol. 45:9566–9573.

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47. Amachawadi, R. G., H. M. Scott, C. Aperce, J. Vinasco, J. S. Drouillard, and T. G. Nagaraja. 2015a. Effects of in-feed copper and tylosin supplementations on copper and antimicrobial resistance in faecal enterococci of feedlot cattle. J. Appl. Microbiol. 118:1287–1297. 48. Amachawadi, R. G., H. M. Scott, J. Vinasco, M. D. Tokach, S. S. Dritz, J. L. Nelssen, and T. G. Nagaraja. 2015b. Effects of in-feed copper, chlortetracycline, and tylosin on the prevalence of transferable copper resistance gene, tcrb, among fecal enterococci of weaned piglets. Foodborne Pathog. Dis. 12:670–678. 49. Lin, Y., W. Zhao, Z. D. Shi, H. R. Gu, X. T. Zhang, X. Ji, X. T. Zou, J. S. Gong, and W. Yao. 2017. Accumulation of antibiotics and heavy metals in meat duck deep litter and their role in persistence of antibiotic-resistant Escherichia coli in different flocks on one duck farm. Poult. Sci. 96:997–1006. 50. Vincent, M., R. E. Duval, P. P. Hartemann, and M. Engels-Deutsch. 2018. Contact killing and antimicrobial properties of copper. J. Appl. Microbiol. 124:1032–1046. 51. Ibrahim, S. A., H. Yang, and C. W. Seo. 2008. Antimicrobial activity of lactic acid and copper on growth of Salmonella and Escherichia coli O157:H7 in laboratory medium and carrot juice. Food Chem. 109:137–143. 52. Gullberg, E., L. M. Albrecht, C. Karlsson, L. Sandegren, and D. L. Andersson. 2014. Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals. mBio 5:e01918-14. 53. Been, M., V. F. Lanza, M. Toro, J. Scharringa, W. Dohmen, Y. Du, J. Hu, Y. Lei, N. Li, A. Tooming-Klunderud, D. J. J. Heederik, A. C. Fluit, M. J. M. Bonten, R. J. L. Willems, F. I. Cruz, and W. van Schaik. 2014. Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages. Plos Genet. 10:e1004776. 54. Card, R. M., S. A. Cawthraw, J. Nunez-Garcia, R. J. Ellis, G. Kay, M. J. Pallen, M. J. Woodward, and M. F. Anjum. 2017. An in vitro chicken gut model demonstrates transfer of a multidrug resistance plasmid from Salmonella to commensal Escherichia coli. mBio 8:e00777-17. 55. Sørensen, S. J., M. Bailey, L. H. Hansen, N. Kroer, and S. Wuertz. 2005. Studying plasmid horizontal transfer in situ: a critical review. Nat. Rev. Microbiol. 3:700–710. 56. Lopatkin, A. J., T. A. Sysoeva, and L. You. 2016. Dissecting the effects of antibiotics on horizontal gene transfer: analysis suggests a critical role of selection dynamics. Bioessays 38:1283–1292.

Acknowledgments This study was financed in part by the Coordination for the Improvement of Higher Education Personnel (CAPES), Finance code 001. The Brazilian National Council for Scientific and Technological Development (CNPq; proc. 311793/2016-9) and the Institute of International Education (IIE; proc. S-ECAGD-13-CA149). The bacterial strains used in this study were kindly provided by Paul Barrow from the University of Nottingham, UK. The authors wish to express their appreciation to Laura Binkley, the Ohio State University, for her suggestions and carefully reading of the manuscript.