Widespread occurrence of Enteropathogenic Escherichia coli and novel bfpA variants in Australian fruit bats

doi:10.21203/rs.3.rs-1652157/v1

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Widespread occurrence of Enteropathogenic Escherichia coli and novel bfpA variants in Australian fruit bats

https://doi.org/10.21203/rs.3.rs-1652157/v1

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published 29 Aug, 2023

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Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC is characterised by specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp; encoded by the bfp operon, including the bfpA gene for the major pilus subunit). By definition, Bfp are only present in typical EPEC (tEPEC). Humans are the only known natural host of tEPEC. Here we investigated the occurrence and genetic characteristics of EPEC from an Australian fruit bat species, the grey-headed flying-fox. Whole genome sequencing of 61 E. coli isolates from fruit bat faeces revealed that 13 were tEPEC, 11 of which had novel bfpA variants. HEp-2 cell adhesion assays indicated that every tEPEC strain carried a functional bfp operon that encoded localised adherence to human-derived epithelial cells in vitro. Additional analysis of faecal samples using an EPEC-specific multiplex PCR, identified tEPEC in fruit bats from three geographic areas in eastern Australia, with sampling conducted on six occasions over a four-year period (mean occurrence 19.1%, range 1.3–87.0%). This is the first report of the widespread occurrence of tEPEC in a non-human host over extensive geographical and temporal scales. Our findings suggest that fruit bats are a natural host of tEPEC and that host-specific lineages may have evolved in bats to comprise part of their commensal microbiome.

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoea and mortality in infants in low- and middle-income countries^{1, 2}, and prolonged diarrhoea in children in high-income countries^{3, 4}. EPEC are characterised by their ability to cause attaching and effacing lesions of the intestinal epithelium, resulting in microvilli destruction and diarrhoea^{5, 6}. All EPEC carry specific virulence factors, most notably a ~ 35 kb chromosomal locus for enterocyte effacement (LEE) pathogenicity island which encodes the an outer membrane protein, intimin (encoded by the eae gene), a type 3 secretion system, and several type-3 secreted effectors, including a translocated intimin receptor (tir gene), EspA, EspB, EspH and Map ^{7, 8}. In addition to the LEE-encoded genes, there are numerous non-LEE encoded (Nle) type 3 secreted effectors⁸ and adhesins⁹ which may be associated with increased pathogenicity⁸.

EPEC strains which carry the EPEC adherence factor plasmid (pEAF) are classified as typical EPEC (tEPEC) and those lacking this plasmid are known as atypical EPEC (aEPEC)⁶. pEAF carries the bundle forming pilus (bfp) operon and plasmid encoded regulators (per), all of which are required for full virulence of tEPEC in humans¹⁰. Bundle-forming pili facilitate the formation of stable microcolonies on epithelial cell monolayers in vitro, giving rise to a pattern known as localised adherence, which is characteristic of tEPEC⁵.

LEE has multiple evolutionary variants, with distinct EPEC lineages being associated with specific LEE insertion sites and eae subtypes^{11, 12}. Multiple variants of the EAF plasmid and bfpA allele also exist, but unlike LEE, they are not highly conserved within tEPEC lineages ¹³. EPEC lineages have traditionally been characterised by O and H antigens (O:H serotype), with 12 classical EPEC O-serogroups listed by the World Health Organization in 1987¹⁴, and multiple non-classical O-serogroups identified subsequently^{13, 15}. The continuing emergence of new EPEC O-serogroups indicates that the LEE (and pEAF in tEPEC) can be acquired independently by diverse E. coli lineages^{13, 12}.

Humans, especially children, are the primary reservoir of EPEC⁵. Rarely have tEPEC been detected in non-human hosts with only a few reports in domestic animals^16–19 and wildlife^{20, 21, 18, 22–25}, suggesting humans are the only natural reservoir for tEPEC^{5, 18}. In contrast, numerous studies have reported the presence of aEPEC in domestic animals^{16, 26, 17, 20, 18, 27} and wildlife^{28–30, 21, 18, 31, 22–25, 27, 32}.

Although reports of the carriage of enteric bacterial pathogens by bats are sparse^{33–38, 24, 39}, both tEPEC^{37, 24} and aEPEC^{37, 38, 24} have been identified in diverse bat species, suggesting bats may be an important wildlife group associated with EPEC carriage. Bats belong to the order Chiroptera which is divided into two suborders, Yinpterochiroptera (predominantly fruit bats/flying-foxes) and Yangochiroptera (microbats)⁴⁰. In Australia, fruit bats are highly urban-adapted and form large colonies in city parklands^{41, 42}. One species of fruit bat, the grey-headed flying-fox (Pteropus poliocephalus) is widely distributed across eastern Australia⁴³ from North Queensland (QLD), through New South Wales (NSW), Victoria (VIC) and into South Australia (SA). Grey-headed flying-foxes, hereafter referred to as flying-foxes, occupy tree roosts in colonies that can number in the tens of thousands⁴³. This study investigated the occurrence of tEPEC in flying-foxes and examined the genetic characteristics and evolutionary relationships between flying-fox tEPEC strains and human EPEC strains using whole genome sequence (WGS) data and analysis of key virulence genes.

E. coli isolation and WGS analysis of EPEC from flying-foxes

E. coli isolates were cultured from 262 of 468 (56.0%) flying-fox faecal samples collected from five colonies across eastern Australia, with four located in NSW and one in SA (Table S1). A representative subset of flying-fox E. coli isolates (n=61) was selected based on phylogroup distributions for further characterisation using WGS (Table S2). Analysis of WGS data identified 33 EPEC isolates, with 13 being tEPEC (eae and bfp positive) and 20 being aEPEC (eae positive and bfp negative) (Table S2). The majority (31 of 33) of flying-fox EPEC isolates were phylogroup B2, which included all tEPEC (Table S2). None of the 33 flying-fox EPEC carried acquired antimicrobial resistance genes.

Phylogeny of Australian phylogroup B2 EPEC from flying-foxes, humans and other animals

Phylogenetic analysis of all 47 phylogroup B2 E. coli isolated from flying-foxes and a collection of 109 Australian-sourced phylogroup B2 E. coli isolates from humans and animals placed all EPEC strains from flying-foxes, humans and other animals, into one of four broad clades, designated Clades A, B, C and D (Fig. S1). The majority of flying-fox tEPEC were clustered in Clade A (n=10), whereas Clade B contained only aEPEC (n=11), and Clades C and D contained seven aEPEC and three tEPEC (Fig. S1). The largest proportion (n=12) of flying-fox phylogroup B2 EPEC isolates were serogroup O non-typable (ONT), followed by O45 (n=5), O51 (n=3) and O132 (n=3). The most frequent H-antigens were H49 (n=11), H34 (n=5) and H1 (n=5) (Table S2). The 13 flying-fox tEPEC isolates belonged to eight different sequence types (STs), of which seven were novel STs (Table S2). The 18 flying-fox phylogroup B2 aEPEC isolates also belonged to eight different sequence types (STs), with only four STs being novel (Table S2). Nine (50%) of phylogroup B2 aEPEC isolates belonged to ST589 (Fig. S2).

Phylogeny of Australian and globally sourced phylogroup B2 tEPEC isolates

Six of ten flying-fox tEPEC in Clade A belonged to a broad cluster designated ST7799 ONT:H51, which also contained one human aEPEC sourced from the United Kingdom (Fig. 1a). Of the four remaining flying-fox tEPEC in Clade A, three ST2346 isolates were clustered with an aEPEC of Australian mammal origin and were related to tEPEC of the classical serotype, O142:H34 (Fig. 1a). The tenth flying-fox tEPEC in Clade A (ST8437) belonged to a cluster containing predominantly human sourced aEPEC and one human sourced tEPEC of serotype O33:H34 (Fig. 1a).

The two flying-fox tEPEC in Clade C belonged to distant clusters. One isolate exhibited a novel ST (ST7796) and was clustered with a flying-fox aEPEC of the same ST and serotype. The other isolate was clustered with predominantly human sourced aEPEC (Fig. 1b). The single flying-fox tEPEC isolate in Clade D exhibited a novel ST (ST9731) and was also clustered with a flying-fox aEPEC of the same ST and serotype (Fig. 1c). Overall, only three of 13 of flying-fox sourced tEPEC isolates shared STs, serotypes and/or phylogenetic clusters with human associated EPEC (Fig. 1).

Single nucleotide polymorphism (SNP) whole genome analysis of all GHFF tEPEC and the two closely related aEPEC found little variation between closely clustered isolates (0-5 SNP counts between genomes) and large variations between tEPEC from different clades (9,506-12,191 SNP counts between genomes) (Fig. S4). All flying-fox tEPEC had SNP counts between 8,443 and 10,006 against the reference genome tEPEC E2348/69 (Fig. S4).

Virulence factors of phylogroup B2 tEPEC Clades

The LEE-encoded genes, eae, espABDH, map and tir, were present in almost all phylogroup B2 EPEC isolates in Clades A, C and D, whereas Nle-effectors and adhesins were variable (Fig. 1 and Table S3). Of the 13 tEPEC isolates from flying-foxes, cif was present in six, followed by nleB2 in three and nleB2 plus paa in one (Fig. 1 and Table S3). perA was detected in all 13 flying-fox tEPEC, whereas efa1, iha, lpfA, nleB1, nleE and toxB were absent from these isolates (Fig. 1 and Table S3).

Other virulence factors (astA, cdtAB, vat, ibeA, fyuA/irp, malX) showed variable distributions in flying-fox and human EPEC but were somewhat conserved within closely related clusters (Fig. 1). Of the 13 flying-fox tEPEC, astA was present ten, ibeA and malX were both present in two, and cdtAB and vat were present in one isolate each (Fig. 1). None of the 13 flying-fox tEPEC isolates carried shiga toxin stx genes or yersinabactin (fyuA/irp) (Fig. 1).

eae phylogenetic comparison of phylogroup B2 EPEC Clades

Twelve eae subtypes were identified across the four phylogroup B2 EPEC clades, with each subtype largely conserved within clades and closely related clusters (Fig. S3). Nine of the 12 eae subtypes were detected in the 13 flying-fox tEPEC isolates from the phylogroup B2 Clades A, C and D, with the most frequent eae subtypes being e8 (n=3) and m (n=3) (Fig. 2). Of the 13 flying-fox tEPEC eae sequences, 11 showed between 99.65% to 100% identity to reference sequences. One sequence showed 97.45% identity while another sequence exhibited <94% identity and was designated a novel eae subtype (Table S4). The chromosomal insertion site of eae and the associated LEE pathogenicity island was tRNA-Sec (selC) for all 13 flying-fox tEPEC (Fig. 2).

bfp phylogenetic comparison of flying-fox tEPEC

In contrast to the eae gene, bfpA allele type was not conserved within clades or clusters (Fig. 2). Twelve of 13 flying-fox bfpA allele sequences formed genetically distinct clusters (Fig. 2). Only two flying-fox bfpA alleles were close matches to human sourced alleles. FF802 showed 99.15% identity with the bfpA b1 reference allele (GenBank accession No. AF304471) and FF1172 was a 98.63% match to a non-reference human bpfA variant designated b14 (GenBank accession No. AB247933) (Table S5). All other flying-fox bfpA sequences (n=11) were ≤94.84% matches to reference bfpA alleles and considered to be five novel bfpA variants, hereby designated Novel-1 to Novel-5 (Fig. 2). An identical novel bfpA variant (Novel-1) was detected in four flying-fox tEPEC isolates from two different phylogenetic lineages that were collected at two flying-fox colonies located approximately 1200 Km apart (Fig. 2). Isolates FF781 and FF1136 shared an almost identical (99.49%) novel bfpA variant (Novel-4), but they belonged to different lineages and were collected at different sampling locations (Fig. 2). All three ST2346 flying-fox isolates shared the Novel-5 bfpA variant (Fig. 2).

Sequence variations in bfp operons from flying-fox tEPEC correlated with bfpA variant type (Fig. 2). For flying-fox tEPEC with the same bfpA variant, little variation (1-5 SNPs) was observed in the full length bfp operon sequences (Table S6). In GenBank Blastn searches, all full length flying-fox bfp operon sequences showed 96.96% to 97.78% identity to bfp operons from human associated tEPEC (Table S6).

bfpA sequence variations in flying-fox and reference alleles

Alignment of all flying-fox bfpA nucleotide sequences with seven of the most closely related human bfpA alleles identified 135 positions of nucleotide variation across the 20 bfpA sequences (Fig. 3a). The translated flying-fox bfpA amino acid sequences showed highly conserved regions, particularly in the first third (N-terminal) and highly variable regions in the last two-thirds (C-terminal) (Fig. 3b). These conserved and variable regions in flying-fox bfpA alleles correlated with amino acid polymorphisms in the human-associated bfpA reference alleles (Fig. 3b).

HEp-2 adhesion assays

All 13 flying-fox tEPEC and two paired flying-fox aEPEC isolates were investigated in in vitro adhesion assays with HEp-2 cells. Two aEPEC isolates (FF772 and FF1173) were also examined because they shared almost identical genomes to two tEPEC isolates (five and one SNPs with FF802 and FF1172 respectively) (Fig. S4). All 13 flying-fox tEPEC isolates showed the characteristic localised adherence (LA) pattern associated with tEPEC after 3 h incubation with HEp-2 cells (Fig. 4a). Three isolates (FF773, FF812 and FF1136) showed very strong LA that did not correlate to a specific bfpA variant or tEPEC lineage (Fig. 4b). Both paired flying-fox aEPEC (FF772 and FF1173) that lacked the bfp operon, were non-adherent after 3 h incubation (Fig. 4c).

PCR determination of EPEC prevalence in all cultured E. coli isolates

PCR screening for the defining EPEC virulence factors, eae and bfpA, was performed with E. coli isolates cultured from 468 flying-fox faecal samples (Table S7). WGS and/or PCR of cultured E. coli isolates from five colonies, showed an overall tEPEC occurrence of 10.0% across all 10 sampling timepoints (Fig. 5c, Table S7). The frequency of isolation of tEPEC varied in accordance with location and time of sampling, with the highest frequency (45.7%) in the Adelaide Botanic Park (ABP) colony in February 2017 and the lowest occurrence (0.0%) at four sampling timepoints in four separate locations, namely Ku-ring-gai Flying-Fox Reserve (KFFR) in 2015, ABP in July 2018, Blackalls Park (BP) in December 2017 and Camellia Gardens (CG) in January 2018 (Fig. 5, Table S7).

PCR determination of EPEC prevalence in flying-fox faecal DNA

For six sampling timepoints from three colonies, flying-fox faecal DNA was extracted, which enabled screening directly for tEPEC in faecal samples (n=369) (Table S7). Faecal DNA was screened for for eae, bfpA and chuA (the last to confirm the presence of phylogroup B2 E. coli). The combination of E. coli culture and faecal DNA PCR screening in flying-fox faecal samples (n=409) revealed an overall occurrence of tEPEC of 19.1% across the six sampling timepoints (Table S7). The ABP colony had the highest frequency of tEPEC (87.0%), with the lowest frequency of 1.3% at the Blackalls Park (BP) colony in December 2017 (Fig. 5, Table S7). Using both the E. coli culture and faecal DNA screening methods, we also determined that four faecal samples contained both aEPEC and tEPEC (Fig. 5, Table S7).

In this study, we found that grey-headed flying-foxes in Australia carry diverse lineages of tEPEC at variable frequencies across colonies. The high occurrence of tEPEC in flying-foxes was unexpected given that tEPEC are almost exclusively associated with human hosts^{5, 18}. Typical EPEC has been previously reported in two fruit bat species from the Republic of Congo at a frequency of 8%²⁴. In Australian grey-headed flying-foxes, the frequency of tEPEC over a four-year period was highly variable, ranging from 0–87% across five colonies, with a mean occurrence of 10.0% using culture methods alone and 19.1% using a combination of culture and faecal DNA analysis. Such an extremely high frequency of tEPEC in flying-foxes has not been previously reported for any wildlife or domestic animal species. The consistent detection of tEPEC in flying-foxes over a four-year period and across a large geographical distance (> 1200 Km), suggests that flying-foxes are natural hosts of tEPEC and carriage is not sporadic zooanthroponotic transmission as suggested for sporadic cases in other non-human hosts^{16, 28, 17, 20, 21, 18, 19, 22–25}.

The inferred phylogeny of flying-fox associated tEPEC and human-associated EPEC indicates shared evolutionary lineages, with the largely conserved nature of eae subtypes within EPEC clusters and a shared selC LEE insertion site providing further support¹². Around four in five flying-fox tEPEC belonged to Clade A, with eae subtyping indicating that Clade A flying-fox tEPEC shared phylogenetic lineages with several human aEPEC lineages, but not the classical tEPEC O142:H34 which is also within Clade A¹⁴. The presence of multiple novel bfpA allele variants in Clade A flying-fox tEPEC suggests that they acquired bfp operons or EAF plasmids independently,⁴⁴ and then evolved as flying-fox specific lineages. Additionally, the presence of almost identical novel bfp operon variants in flying-fox tEPEC from different phylogenetic lineages and different flying-fox colonies suggests that these bfp operons, and probably EAF plasmids, are circulating within flying-fox microbiomes. The two colonies sharing novel bfpA allele and bfp operon variants in phylogenetically distinct tEPEC are located over 1200 Km apart. Flying-foxes exhibit seasonal and migratory movements between colony sites, but it is rare for an individual flying-fox to travel 1200 Km between New South Wales (eastern Australia) to South Australia^45–47. These findings suggest that these novel bfpA variants may be host specific to flying-foxes and have been circulating within the flying-fox metapopulation for a long time.

Phylogenetic analysis and eae subtyping of the flying-fox tEPEC isolates in Clades C and D also suggested that they share lineages with human associated aEPEC rather than tEPEC. Two of these flying-fox tEPEC belonged to divergent clusters void of human associated EPEC, but harboured bfpA alleles that were closely related to human bfpA variants. Interestingly, each of these divergent flying-fox clusters contained paired isolates with almost identical genomes, but one was aEPEC and the other tEPEC, indicating that either the tEPEC strains had recently acquired the bfp operon or EAF plasmid, or more likely that the aEPEC had lost the bfp operon⁴⁴. Loss of pEAF by the prototypical tEPEC strain, E2348/69, during the course of infection in humans has been reported previously¹⁰.

The conserved regions of the flying-fox BfpA proteins are consistent with conserved regions of human BfpA, which are predicted to be domains with critical roles in the structural integrity of Bfp⁴⁸. The variable regions in flying-fox BfpA proteins also correlate with amino acid polymorphisms observed in human BfpA and are predicted to present diverse surface regions to the host immune system⁴⁸. The characteristic LA to HEp-2 cells by all 13 flying-fox tEPEC, including all those with novel bfpA variants, indicates that flying-fox tEPEC harbour functional bfp operons, as predicted by sequence analysis and presence of other key virulence factors. Additionally, the absence of a bfp operon in the paired aEPEC isolates was correlated with their inability to adhere to HEp-2 cells in vitro, in contrast to the LA observed with their respective, closely related bfp positive tEPEC strains, further indicating that bfp operons are responsible for the characteristic LA. Although these novel tEPEC may be host specific to flying-foxes, their adherence to HEp-2 cells in vitro suggests they may be able to adhere to human intestinal epithelial cells and should be considered potential zoonotic pathogens.

The presence of novel tEPEC lineages and bfpA variants, plus the widespread geographical and temporal distribution of tEPEC in flying-foxes, suggests that flying-fox tEPEC may constitute part of their commensal microbiome. Flying-foxes have a short intestinal tract relative to body length, a simple hindgut with no microbial fermentation chamber and a rapid gastrointestinal transit time of 18–32 min⁴⁹. A study of Australian mammals suggested that in the absence of a modified hindgut, E. coli can only establish intestinal populations in large bodied species⁵⁰. Additionally, E. coli densities in the intestinal microbiome decrease as the gastrointestinal transit time of ingested food increases⁵¹. Laboratory models using bio-reactors also indicate that bacterial populations are less likely to stablish in the gut when there is a fast transit time^52–54. However, bacterial strains which can adhere to the intestinal mucosa are more likely to establish, regardless of gut length or transit time^52–54. Thus, the presence of adhesion factors, such as Bfp in tEPEC, may allow these bacteria to persist in the flying-fox intestinal tract, despite the rapid gastrointestinal transit time and simple hindgut²⁴. In addition, the attaching and effacing capacity of all varieties of EPEC may allow them to remain in the intestinal tract for extended periods, giving EPEC an evolutionary advantage over other enteric bacteria.

Carriage of aEPEC in flying-foxes was not an unexpected finding, as diverse aEPEC have previously been associated with diverse mammalian wildlife species^{30, 18, 22, 27, 32} including bats^{37, 38, 24}. The flying-fox sourced aEPEC isolates reported here typically shared STs, serotypes and/or phylogenetic clusters with human associated aEPEC and were detected in four flying-fox colonies geographically located up to 1200 Km apart over a four-year period. These findings suggest widespread acquisition of diverse human associated aEPEC by flying-foxes, indicating they may also be reservoirs for potentially zoonotic aEPEC.

The occurrence and distribution of tEPEC and aEPEC varied considerably in colonies from different locations and at different sampling timepoints, which may relate to variables including exposure levels to sources of EPEC in the environment^55,56, horizontal transmission rates between individuals⁵⁷, climate⁵⁸ or diet⁵⁰. The largest variations in frequency were observed within the ABP colony, with high tEPEC detection rates in summer 2017 (87.0%), which fell to only 8.2% six months later (August 2017). This observed reduction of around 90% suggests that prevalence of tEPEC fluctuates in flying-fox colonies and carriage may not be long-term.

We do not know if EPEC are pathogenic for flying-foxes but the high prevalence of tEPEC observed in apparently healthy adults sampled in the ABP colony suggests that this is not the case. Interestingly, the tEPEC peak in adult flying-foxes observed during summer 2017 coincided with their post-birthing season⁴³ and a study of EPEC in young flying-foxes may be warranted given that EPEC are pathogenic for human infants but infected adults are typically asymptomatic⁵.

Strains of EPEC obtained from grey-headed flying-foxes do not belong to the classical O:H serotypes associated with human infections¹³, but instead carry novel tEPEC lineages and bfpA alleles, suggesting that flying-foxes tEPEC have evolved in a host-specific manner. HEp-2 adhesion assays indicated that these novel flying-fox tEPEC isolates harbour functional bfp operons. This study is the first to report the consistent detection of tEPEC in a non-human host, over extensive geographical and temporal scales. The high frequencies of apparently virulent tEPEC in large flying-fox colonies located in urban environments has the potential for significant zoonotic and public health risks. Future studies are warranted to examine the potential infectivity of flying-fox EPEC for humans, mechanisms of adherence, microbiological role and dynamics of tEPEC in flying-fox populations and elucidate the evolutionary origin of human and flying-fox tEPEC.

Grey-headed flying-fox faecal sampling

Faecal samples were collected from wild flying-foxes at five colony locations in two Australian states; New South Wales (NSW) (4 colonies) and South Australia (SA) (1 colony). A total of 601 faecal samples were collected: Camellia Gardens (CG), NSW (n = 50); Centennial Park (CP), NSW (n = 52); Ku-ring-gai Flying-Fox Reserve (KFFR), NSW (n = 30); Blackalls Park (BP), NSW (n = 280); Adelaide Botanic Park (ABP), SA (n = 188) (Table S1). ABP was sampled on four occasions, BP on three occasions and CG, CP and KFFR were sampled once (Table S1). Faecal samples were collected over a four-year period, between March 2015 and April 2019.

For NSW sites, faecal samples were collected opportunistically by placing between six and 16 plastic drop sheets measuring 2.6 m x 3.6 m, under roosting animals throughout the flying-fox colony. Between five and 10 discrete faecal samples were selected from each sheet, avoiding samples positioned close together to minimise the risk of repeated sampling from the same individual. For sampling in SA, flying-foxes were trapped using mist nets, placed in clean cotton holding bags and transferred to a veterinary hospital for processing. Trapped flying-foxes were anaesthetised and rectal swabs taken from individuals or faeces collected from the clean holding bags. Faecal samples for culture were transferred using a sterile swab into a FecalSwab™ tube (COPAN, Brescia, Italy) or into sterile faecal jars for those being used for DNA only analysis. All faecal samples were stored at 4˚C and cultured within 72 hours of collection.

Isolation and phylotyping of E. coli from grey-headed flying-fox faecal samples

E. coli strains were isolated by inoculating FecalSwab media directly onto Chromocult Coliform Agar (Merck Millipore, Massachusetts, USA) followed by incubation overnight at 37˚C in air. E. coli colonies were identified by their dark blue to violet appearance. Where morphologically different E. coli colonies (based on colony and halo colour) were present on the same plate, a representative colony of each type was selected. Otherwise a single colony was randomly selected.

DNA was prepared by culturing isolates in 10 mL Luria-Bertani (LB) broth, Miller (Difco Laboratories, Detroit, USA) at 37˚C overnight. Following centrifugation at 12,000g for 10 min, E. coli pellets were resuspended in 0.1 mL sterile PCR water, heated to 95˚C for 10 min, centrifuged at 12,000g for 10 min, and the supernatant used in phylotyping PCRs.

E. coli isolates were assigned to phylotypes the using the Clermont quadruplex PCR method⁵⁹. E. coli strains were assigned to one of eight phylogroups (A, B1, B2, C, D, E, F, or cryptic clade I) based on the combination of four genes (chuA, yjaA, tspE4.C2 and arpA)⁵⁹. PCR amplification was performed using GoTaq® Green Master Mix (Promega, Madison, WI, USA), with all primers at 1 µM each, and cycling conditions of 94°C 4 min; 35 cycles 94°C 20 sec, 59°C 20 sec, 72°C 20 sec; 72°C 4 min.

E. coli whole genome sequencing

Based on phylotype data, a representative subset of E. coli isolates (n = 61) underwent WGS. Isolates were grown in 5 mL LB broth at 35˚C overnight with shaking at 150 r.p.m., after which DNA was extracted using the ISOLATE II Genomic DNA Kit (Bioline, London, UK). The concentration of gDNA was determined using a Qubit dsDNA BR assay kit (Invitrogen, Waltham, USA). Libraries were prepared for WGS using a Nextera XT DNA sample preparation kit (Illumina, San Diego, USA) and the Nextera XT index kit (Illumina) with 0.5 ng of input DNA. WGS was performed on an Illumina MiSeq platform using a 600-cycle MiSeq Nextera XT reagent kit v3 (2 x 300-bp paired-end reads). Raw sequence reads were assembled as de novo genome sequences using CLC Genomics Workbench 8 (CLC Bio, Aarhus, Denmark).

Analysis of assembled DNA sequences

Assembled DNA sequences were screened for virulence factors (VFs) using VirulenceFinder 2.0 ⁶⁰ (available at https://cge.cbs.dtu.dk/services/) and Abricate VFDB (https://github.com/tseemann/abricate) ⁶¹ in Galaxy Australia (available at https://usegalaxy.org.au/), including VFs associated with EPEC; eae, esp genes, map, tir, bfpA, perA and Nle-effectors (cif, nleB, nleE) and adhesins (efa1, iha, lpfA, paa, toxB). Additional screening for VFs which may be associated with EPEC included toxins (astA, cdtAB, stx, vat), invasion protein ibeA, yersiniabactin (fyuA, irp1, irp2) and miscellaneous VFs (iss, malX).

The eae and bfpA sequences were extracted using Geneious Prime 2022.0.2 (Biomatters Limited, Auckland, New Zealand) and BLASTn searches performed (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against eae allele reference sequences (listed in Table S8)^{62, 63} and human bfpA allele reference sequences (listed in Table S9)^{48, 64, 13}. ResFinder 3.2 ⁶⁵ and PointFinder 3.1.0⁶⁶ (both available at https://cge.cbs.dtu.dk/services/) were used to screen sequences for antimicrobial resistance. Serotypes (O:H) were assigned using SeroTypeFinder 2.0⁶⁷. Sequence type (ST) was determined using Multi-Locus Sequence Typing (MLST) 2.0⁶⁸, and fimH type using FimTyper 1.0 (all available at https://cge.cbs.dtu.dk/services/).

The LEE sequence was extracted from assembled DNA for all bfp positive isolates in Clades A to D using Geneious Prime 2022.0.2 (Biomatters Limited, Auckland, New Zealand) and BLASTn searches performed (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the LEE chromosomal insertion sites. Assembled DNA sequences were mapped to an EAF plasmid reference sequence from E. coli O127:H6 E2348/69 (plasmid pMAR2, GenBank FM180569) using Geneious Prime 2022.0.2 (Biomatters Limited, Auckland, New Zealand) to acquire the full length bfp operon sequences of flying-fox tEPEC. BLASTn searches of flying-fox bfp operons were performed (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the closest matches in GenBank. Full length flying-fox bfp operon sequences have been uploaded to GenBank. Accession numbers are provided in Table S6.

Phylogenetic analysis and comparison of flying-fox, human and animal EPEC

Phylogenetic analysis was used to examine the relationship between all E. coli phylogroup B2 isolates (EPEC and non-EPEC) from flying-foxes and other Australian sources (collection supplied by David Gordon) (Supplementary data 2). Based on the distribution of EPEC in the Australian sourced isolates tree, four EPEC clade trees incorporating closely related EPEC identified in EnteroBase by ST searches (http://enterobase.warwick.ac.uk/species/index/ecoli) were constructed (Supplementary data 3). The eae and bfp phylogenetic trees were also constructed using B2 EPEC from all four clades. The B2, EPEC clades, eae and bfpA phylogenetic trees were constructed using the Harvest suite (https://www.cbcb.umd.edu/software/harvest)⁶⁹. All trees were annotated using Interactive Tree of Life v5 (iTOL) (https://itol.embl.de)⁷⁰. Reference sequences and GenBank accession numbers for eae subtypes and bpfA allele variants are described in Tables S7 and S8. Whole genome single nucleotide polymorphism (SNP) analysis of GHFF tEPEC and paired aEPEC isolates (FF772 and FF1173) was performed using CSI Phylogeny 1.4 (available at https://cge.cbs.dtu.dk/services/)⁷¹. Paired raw sequencing reads were uploaded and SNP analysis performed using default settings against the reference genome EPEC E2348/69 variety 2 (GenBank LT827011). A SNP matrix was constructed using SNP counts between all genomes.

HEp-2 adhesion assays with flying-fox tEPEC isolates

In vitro patterns of adherence by 13 flying-fox tEPEC isolates to HEp-2 cells were determined using a modified Center for Vaccine Development (CVD) method⁷². Two flying-fox aEPEC isolates (FF772 and FF1173) that lacked the bfp operon, but otherwise shared almost identical genomes to two flying-fox tEPEC isolates (FF802 and FF1172 respectively) (Fig. S4), were also included in these assays. Three human clinical isolate controls were included; E2348/69 (tEPEC control), W1056 (aEPEC control) and HS (non-pathogenic commensal control). Bacteria were grown in Antibiotic Medium No. 3 Assay Broth (Oxoid, Basingstoke, UK) for 16 hours, without shaking at 37ºC. Approximately 10⁷ bacteria were incubated with HEp-2 cell monolayers grown to ~ 70% confluence in the presence of 0.5% D-mannose at 37ºC in 5% CO₂. After 3 h, the monolayers were washed three times with PBS to remove non-adherent bacteria and cells were then fixed with 3% formalin in PBS before staining with 10% Giemsa stain. Assays were performed in triplicate and adherent bacteria were observed by brightfield microscopy using a Zeiss Axio Imager Z2 microscope. Nine representative images (x400 magnification) for each isolate were captured using a Zeiss Axiocam 506 digital camera and examined blind for adherence patterns; localised adherence (LA), aggregative adherence (AA), diffuse adherence (DA), indeterminate adherence (IA) and no adherence (NA), where non-adherence was defined as < 10 of 200 HEp-2 cells having 5 or more adherent bacteria.

EPEC multiplex PCR of E. coli and faecal DNA

To enable rapid screening for EPEC virulence factors in E. coli isolates, two primer sets targeting eae and bfpA were designed using sequences obtained from the WGS of flying-fox EPEC. The primers were eaeF 5′-GGCTGGCATGAGTCATACAATA-3′ and eaeR 5′-CGTAACGGCTGCCTGATAAT-3′ which give a product size of 373 bp and bfpAF 5′-CTGTATATTGGGCAGACCAGTT-3′ and bfpAR 5′-CGTTGCAAGACTAACACATGC-3′ with a product size of 219 bp. A multiplex PCR for both genes was performed using GoTaq® Green Master Mix (Promega, Madison, WI, USA), eae and bfpA primers at 0.5 µM each and 2 µL E. coli isolate DNA (using the boil preparation method as described for the phylotyping PCRs). The cycling conditions were 94°C 3 min; 35 cycles 94°C 30 sec, 60°C 30 sec, 72°C 1 min 30 sec; 72°C 5 min. Primer pairs were validated by correlating PCR results from 31 flying-fox E. coli isolates with WGS results (Table S10).

The multiplex PCR was then adapted further with the addition of chuA primers (ChuA.1b and ChuA.2) ⁵⁹ to confirm the presence of E. coli from phylogroup B2 or D, with all primers at 0.4 µM each and performed using the E. coli phylotyping PCR conditions as previously described. The faecal multiplex PCR was validated by correlating faecal DNA PCR results (n = 20) to the corresponding E. coli PCR and WGS results (Table S10). The EPEC multiplex PCR was used to screen faecal DNA extracted using the Bioline ISOLATE Fecal DNA Kit following the manufacturer’s instructions.

Ethical approval and scientific permits

All sample collections were conducted under approval from Macquarie University Animal Ethics Committee (No. 2017/013), the University of Adelaide Animal Ethics Committee (No. S-2015-028), and under NSW Government Scientific Licence (No. SL101898) and SA Department of Environment and Water Wildlife Scientific Permit (M-23671-1,2 and 3).

Data availability

Assembled sequences for all flying-fox E. coli isolates are available in EnteroBase (Supplementary data 1 and Supplementary data 2). Novel strains were assigned an ST according to the Achtman seven gene MLST scheme (http://enterobase.warwick.ac.uk/species/index/ecoli). Raw sequence reads for all EPEC isolates were uploaded to NCBI Sequence Read Archive (SRA) under BioProject ID PRJNA613581. EnteroBase and BioSample accession numbers for all flying-fox EPEC isolates and associated metadata are provided in a supplementary excel file (Supplementary data 1). Annotated sequences for novel flying-fox bfp operon variants have been uploaded to GenBank (accession numbers are listed in Table S6). Source data for Fig. 5c is provided.

Acknowledgements

This project was funded by a Holsworth Wildlife Research Endowment from the Equity Trustees Charitable Foundation & the Ecological Society of Australia (to Fiona McDougall), a Lake Macquarie Council Environmental Research Grant awarded to Michelle Power and Fiona McDougall. an Ian Potter Foundation (grant #20140156, to Michelle Power), and a Macquarie Research Development Grant (to Michelle Power).

We wish to thank Terry Reardon, Ian Smith, Kathy Burbidge and the staff at the Animal Health Centre (Adelaide Zoo) for support in collecting samples from the Adelaide Botanic Park flying-foxes, and Tim Pearson, Juliane Schaer, Daniel Russell and Claudia Aguilar-Solis for their assistance collecting samples from flying-fox colonies in New South Wales.

We acknowledge the Wallumattagal clan of the Dharug Nation as the original custodians of the Macquarie University land, the Boon Wurrung and Wurundjeri People as the original custodians of the Doherty Institute and Murdoch Children’s Research Institute lands, and the Ngunnawal people as original custodians of the lands where the Australian National University campus is built. We collected samples from the lands of the Kaurna, Dharug, Eora, Tharawal and Awabakal Nations.

Author Contributions

F.M., M.P., D.G. and R.R-B. designed and instigated this study. W.B. F.M. and M.P. sampled bats and collected faecal samples. F.M. and M.P. performed the isolate culture, and F.M and D.G. performed whole genome sequencing, PCR analysis and the bioinformatics analyses. R.R-B and V.B-W. conducted the HEp-2 adhesion assay studies. F.M. wrote the draft manuscript. Manuscript revision was performed by F.M., M.P., D.G., R.R-B., V.B-W. and W.B. All authors have read and approved the manuscript.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Supplementary Figures - Fig. S1 to Fig. S4

Supplementary Tables - Table S1 to Table S10

Supplementary data

Supplementary data 1 - SRA BioProject PRJNA613581 and Enterobase accessions

Supplementary data 2 - Australian B2 E coli metadata

Supplementary data 3 - Clade trees isolate metadata

Source data

Source data Fig. 5c – Raw data associated with Fig. 5c

Source data Fig. 5c – Unprocessed gels associated with Fig. 5c

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There is NO Competing Interest.

GHFFEPECSupplementaryinformationFiguresS1toS4TablesS1toS10.pdf
Supplementary Figures and Tables
GHFFEPECSupplementarydata1BioProjectPRJNA613581andEnterobaseaccessions.xlsx
Dataset 1
GHFFEPECSupplementarydata2AustralianB2Ecolimetadata.xlsx
Dataset 2
GHFFEPECSupplementarydata3Cladetreesisolatemetadata.xlsx
Dataset 3
GHFFEPECSourcedataFig.5cRawdataassociatedwithFig.5c.xlsx
Source data associated with Figure 5c
GHFFEPECSourcedataFig.5cUnprocessedgelsassociatedwithFig.5c.pdf
Source data associated with Figure 5c

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Widespread occurrence of Enteropathogenic Escherichia coli and novel bfpA variants in Australian fruit bats

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Journal Publication

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Abstract

Figures

Introduction

Results

E. coli isolation and WGS analysis of EPEC from flying-foxes

Phylogeny of Australian phylogroup B2 EPEC from flying-foxes, humans and other animals

Phylogeny of Australian and globally sourced phylogroup B2 tEPEC isolates

Virulence factors of phylogroup B2 tEPEC Clades

eae phylogenetic comparison of phylogroup B2 EPEC Clades

bfp phylogenetic comparison of flying-fox tEPEC

bfpA sequence variations in flying-fox and reference alleles

HEp-2 adhesion assays

PCR determination of EPEC prevalence in all cultured E. coli isolates

PCR determination of EPEC prevalence in flying-fox faecal DNA

Discussion

Conclusion

Methods

Grey-headed flying-fox faecal sampling

E. coli whole genome sequencing

Analysis of assembled DNA sequences

Phylogenetic analysis and comparison of flying-fox, human and animal EPEC

HEp-2 adhesion assays with flying-fox tEPEC isolates

Declarations

Ethical approval and scientific permits

Data availability

Acknowledgements

Author Contributions

Ethics declarations

Competing interests

Supplementary information

Supplementary data

Source data

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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