Antimicrobial resistance and genetic relationship 2 among enterococci from siblings and non-siblings 3 Heliconius erato phyllis caterpillars

1 Department of Microbiology, Immunology and Parasitology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 2 Department of Microbiology, Health Sciences Federal University, Porto Alegre, Rio Grande do Sul, Brazil 3 Department of Veterinary Preventive Medicine, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 4 Genetic Department, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil 5 Food Science Institute, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil

78 are also able to synthesize cyanogenic glycosides and transfer them to their eggs (Hay- Roe & 79 Nation, 2007). 80 It has been recognized for a long time that microorganisms play key roles in various 81 physiological functions of animal hosts. The gut microbial community promotes an especially 82 diverse range of benefits for insects, e.g., by improving nutrition via synthesis of vitamins and/or 83 establishment of metabolic pathways, actively participating in degradation of xenobiotic 84 compounds, and contributing to the defense against invading pathogens and immune system 85 modulation (Douglas, 2015;Shao et al., 2017). The presence of microorganisms in the 86 gastrointestinal (GI) tract of insects can be explained by environmental bacteria ingested with 87 food and/or acquired by maternal transfer (Engel & Moran, 2013). A growing number of studies 88 have addressed the importance of the microbiota in the GI tract of insects (Engel & Moran,89 2013; Chen et al., 2016;Douglas, 2018), and Enterococcus is one of the most frequent bacterial 90 genera present in the gut microbiota at different life stages of Lepidoptera ( 93 The Enterococcus genus is often found in the GI tract of humans and animals, as well as 94 in the guts of insects (Shao et al., 2017). Hammer et al. (2014) reported that Enterococcus was 95 the most abundant genus found in immature stages and adults of H. erato from Panama. 96 Furthermore, it has also been reported in insects of other orders, such as Coleoptera (Kim et al., 97 2017), Hymenoptera (Audisio et al., 2011), and Diptera . A characteristic of 98 this genus is its intrinsic resistance to several antimicrobial agents and a great ability to transfer 99 and acquire resistant genes (Hollenbeck & Rice, 2012). Despite the environmental resistome, the 100 intense use of antimicrobials and anthropogenic activities, such as animal husbandry, agronomic 101 practices, and wastewater treatment, play an important role in the emergence and spread of 102 resistant-enterococci and/or antibiotic resistance genes in the environment, especially in soil, 103 water, wastewater, and food (Gothwal & Shashidhar, 2014;Singer et al., 2016). 104 Antimicrobial resistance is one of the most serious public health problems, because of the 105 spread of resistant bacteria leading to persistent infections, which are difficult to treat, and 106 contamination of natural environments (Watkins & Bonomo, 2016;Ferri et al., 2017;Aslam et 107 al., 2018). Insects have a wide distribution and can move freely between different environments; 108 they may play an important role as reservoirs of drug-resistant strains and as their disseminators 109 between animals and humans, especially when in contact with organic waste, livestock and their 110 surrounding environment, and hospital facilities Mohammed et al., 2016;111 Schaumburg et al., 2016;Zhang et al., 2017;Onwugamba et al., 2018). In relation to insects 112 caring antibiotic-resistant bacterial strains, studies have identified flies (Ahmad et al., 2011;113 Usui et al., 2015;Mohammed et al., 2016;Schaumburg et al., 2016;Zhang et al., 2017;114 Onwugamba et al., 2018) and cockroaches (Ahmad et al., 2011;Pai, 2013;Moges et al., 2016) 115 as hosts of extended-spectrum beta-lactamase-and carbapenemase-producing 116 Enterobacteriaceae, vancomycin-resistant E. faecium (VRE), and methicillin-resistant 117 Staphylococcus aureus (MRSA). Insects collected from food establishments and in association 118 with stored products were also found to be colonized by antimicrobial-resistant 119 bacteria (Macovei & Zurek, 2006;Channaiah et al., 2010;Mohammed et al., 2016). Despite 120 their importance, few studies have addressed the concern of insects carrying resistant enterococci 121 (Allen et al., 2009;Channaiah et al., 2010;Ahmad et al., 2011;Lowe & Romney, 2011). 122 As previously mentioned, the Enterococcus genus is often found in the GI tract in   136 The fecal samples used in the present study were collected from fifth instar caterpillars. 137 The caterpillars were sourced from three different populations of Heliconius erato phyllis 138 butterflies and consisted of sibling from the same female. The H. erato phyllis females (HE) 139 were captured with entomological nets in Rio Grande do Sul, South Brazil. The first female 140 (HEAB2) was collected in a forest fragment located in Águas Belas Agronomical Station (30° 141 02' 18.1" S; 51° 01' 23.0" W), the second female (HEV2) from a population in an intense urban 142 area in Viamão (30° 09' 40.5" S; 50° 55' 01.5" W) and the third female (HES2) in a forest 143 fragment located in São Francisco de Paula (29° 26' 34.1" S; 50° 36' 48.8" W).

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Butterflies were kept individually in open-air insectaries with dimensions of 2.3 m x 3 m 145 x 3 m (width, length, height) approximately. Insectaries had many plants for simulation of 146 natural conditions, including P. suberosa, used by females for oviposition. The butterflies were 147 fed daily with a mixture containing water, honey and pollen.

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A total of 12 eggs were collected (five from HEAB2, five from HEV2 and two from 149 HES2) with the assistance of a paintbrush. The eggs were transported to the laboratory, and 150 caterpillars were grown individually in cylindrical plastic pots. Immatures were fed exclusively 151 with P. suberosa leaves (Fig. 1). Fecal samples were collected from each caterpillar individually 152 after 48 h of molting to the fifth instar (n = 12), with the aid of a disposable plastic spoon, stored 153 in 1.5 mL microtubes and maintained at -80 °C until processing. The oviposition dates are shown 154 in Table S1. 155 This study was carried out in accordance with the recommendations of Chico Mendes 156 Institute for Biodiversity Conservation (ICMBio). The protocol was approved by Information 157 Authorization System in Biodiversity (SISBIO) number 33404-1. This study has the Council for 236 Eight six enterococci strains isolated from siblings and non-sibling caterpillars were 237 selected for PFGE analysis according to the following criteria: maternal origin (females HEAB2, 238 HEV2 or HES2), hatched larvae, enterococcal species and antimicrobial profile. Chromosomal 239 DNA extraction and electrophoresis conditions were prepared according to Murray et al. (1990) 240 and Saeedi et al. (2002). The restriction enzyme used was SmaI (Invitrogen®). The 241 electrophoresis was carried out using a clamped homogeneous electric field (CHEF-DRII device; 242 Bio-Rad Laboratories, Richmond, Calif.), with ramped pulse times recommended by Saeedi et 243 al. (2002) at 11 °C. Lambda Ladder PFG Marker (New England Biolabs) was used. The gels 244 were stained with ethidium bromide (0.5 μg/mL for 20 min). The PFGE patterns were interpreted 245 using the program GelCompar II v. 11 6.6, with 1.0% of tolerance, and the percentage of 246 similarity was estimated using the Dice coefficient. The pulsotypes were clustered using the 247 unweighted pair group whit arithmetic averages (UPGMA). A dendrogram was generated to 248 examine the relatedness of PFGE patterns for selected isolates, and cutoff level of 80% applied 249 to this dendrogram (Tenover et al., 1995).   257 A total of 178 strains of Enterococcus were isolated from fecal samples from fifth-instar 258 caterpillars (Table 2). Enterococcus casseliflavus was the most common species identified 259 (74.15%; n = 132), followed by E. mundtii (21.34%; n = 38) and E. faecalis (1.12%; n = 2). Six 260 strains (3.37%) could not be identified to species level.

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Differences in the composition of enterococci were detected between the three groups of 262 caterpillars, as shown in Table 2. The Simpson's diversity index was different between the three 263 populations, with higher diversity of enterococci species from fecal samples of caterpillars from 264 HES2 (1 -D = 0.68), followed by HEV2 (1 -D = 0.49) and HEAB2 (1 -D = 0.27). 265 Antimicrobial susceptibility 266 One hundred and twenty (67.41%) enterococci were resistant to at least one evaluated 267 antimicrobial agent. The frequency of antibiotic-susceptible strains is shown in Table 3. The 268 rifampicin-resistance phenotype was the most commonly observed (56%; n = 100), followed by 269 erythromycin (31%; n = 55). Eight strains (4%) were resistant to norfloxacin and five (3%) to 270 ciprofloxacin. All investigated strains were susceptible to ampicillin, vancomycin, tetracycline, 271 nitrofurantoin, chloramphenicol, gentamicin, and streptomycin.

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The band patterns for E. casseliflavus (n = 32) isolates from sibling caterpillars (6, 7, 10 298 11 and 14) of the HEAB2 female showed six PFGE patterns (P5, P7, P8, P9, P11 and P12) and 299 three singletons. Three PFGE patterns (P7, P8, and P9) included 18 of the 32 strains that were 300 isolated from sibling caterpillars 6, 7, 10, and/or 11, with low levels of genetic variability. P5 and 301 P11 each contained two isolates, and P12 with eight isolates showed genetic variation; the 302 remaining three E. casseliflavus isolates were singletons and represented unique PFGE patterns. 303 All E. mundtii isolates from caterpillar 14 were genetically closely related and were clustered 304 into one pattern (P10), as were the two E. faecalis (P3) isolates from caterpillar 6. These results 305 demonstrate that strains may originate from a single lineage.

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The seven E. casseliflavus strains from caterpillars 3 and 17 (offspring of the HES2 314 female) showed two distinct patterns (P1 and P2) with 100% of genetic similarity between them. 315 P1 contained four isolates and P2 had three isolates. Of the six E. mundtii isolates from 316 caterpillar 3, five showed 100% similarity and were clustered in the P14 pattern, suggesting that 317 these strains may be progeny from a single lineage. One strain had distinct and unrelated PFGE 318 by the criteria of Tenover et al. (1999). In addition, most of the patterns were shared by isolates 319 with the same antimicrobial profile.  Enterococcus is associated with the environment and a wide range of organisms, 332 including plants (Müller et al., 2001;Byappanahalli et al., 2012;Sánchez Valenzuela et al., 333 2012). In the present study, the most abundant Enterococcus species in the feces of H. erato 334 phyllis caterpillars was E. casseliflavus. The diet of insects constitutes an additional source of 335 microorganisms in the GI tract. Enterococci in the GI tract of caterpillars can play an important 336 role in the protection against other pathogens, since these bacteria are able to produce lactic acid 337 (causing a decrease in pH) and enterocins (peptides with antimicrobial activity). Since E. 338 casseliflavus is frequently a part of microbial communities on plants (Byappanahalli et al., 2012;339 Micallef et al., 2013;Ong et al., 2014), the predominance of this species in fecal samples of H. 340 erato phyllis caterpillars could be explained by the plant diet of juveniles, resulting in the 341 dominance of this species. As noted by Chen et al. (2016), enterococci stably colonize the larval 342 gut of Spodoptera littoralis (Lepidoptera: Noctuidae), and may act beneficially in caterpillars by 343 promoting nutrient supplementation (metabolism of terpenoids and polyketides) and host defense 344 (production of antimicrobials by E. mundtii). However, further studies are needed to understand 345 the role of these bacteria as members of the GI tract in H. erato phyllis caterpillars.

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Resistant enterococcal strains isolated from fecal samples of caterpillars are a matter of 347 concern, since these larvae had not been exposed to antibiotics. Studies that highlight the 348 resistance profile of enterococci isolated from insects in natural environments are scarce. 349 Channaiah et al. (2010) described enterococci isolated from insect pests of stored products; they 350 were resistant to tetracycline, streptomycin, erythromycin, kanamycin, ciprofloxacin, ampicillin, 351 and chloramphenicol; this suggests that these animals can be potential vectors in disseminating 352 antibiotic-resistant strains. Ahmad et al. (2011) reported MDR enterococci isolated from house 353 flies and cockroaches in a confined swine production environment, and have suggested that these 354 insects may be potential vectors and/or reservoirs of resistant enterococci. Despite the occurrence 355 and spread of resistant strains intensifying due to the use of antimicrobials, the isolation of 356 resistant enterococci in the present study could be related to the resistance that occurs naturally 357 in the environment (environmental resistome) and/or to anthropogenic activities (contamination 358 of the environment) (Martínez, 2008;Allen et al., 2010). 359 The most widespread mechanism of resistance to macrolides in enterococci is mediated 360 by the erm and msrC genes (Aarestrup et al., 2000;Santestevan et al., 2015;Prichula et al., 361 2016). Nevertheless, none of these genes was detected in the present study. It is possible that 362 these strains harbored other erythromycin-resistance genes, such as ermD, E, and F, and other 363 efflux pump genes such as msrA. A low percentage of virulence genes was detected in 364 enterococci of H. erato phyllis caterpillars. Although these genes are related to pathogenicity of 365 clinical enterococcal strains, their presence in strains in fecal samples from caterpillars may be 366 associated with the maintenance of cells of the GI tract, and consequently with microorganism 367 and host interactions.

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From the analysis of the PFGE fingerprint, E. casseliflavus, E. faecalis, E. mundtii, and 369 Enterococcus sp. isolated from fecal samples of sibling and non-sibling caterpillars demonstrated 370 unrelated or related patterns based on maternal origin. The unrelated patterns found in P1, P2, 371 P3, P4, P5, P6, P10, P11, P12, P14, and P15 demonstrated genetic diversity among these strains. 372 The genetic variation in these strains may be associated with genetic events, such as mobile 373 elements or mutation, a common characteristic of enterococci (Lebreton et al., 2014). The related 374 patterns observed among strains isolated from sibling caterpillars (P7, P8, P9, and P13) may be 375 associated with a common source, e.g., diet (herbivory) and/or vertical transmission (through the 376 egg surface). Since the fecal samples used in the present study were collected from fifth-instar 377 caterpillars, the last stage before the pupa, the results present here may be suggest vertical 378 transmission of enterococci that are being replaced from the diet. Plants are a food source for 379 bacteria present in the GI tract of insects; these bacteria improve the quality of diets poor in 380 nutrients and take part in development and maturation of the immune system to protect the host 381 against pathogenic microorganisms (Dillon & Dillon, 2004;Engel & Moran, 2013). Therefore, it 382 is likely that herbivory provides an abundant supply of enterococci throughout the larval stage of 383 H. erato phyllis. Considering that Passiflora leaves are the only food of the caterpillars, those 384 leaves could be the source of Enterococcus sp. in their GI tracts.