Background
Escherichia coli is the predominant aero-anaerobic Gram-negative species of the normal microflora colonizing the gastrointestinal tract of humans and animals [
1]. Most
E. coli strains are commensal and rarely cause clinically-relevant disease. However, some strains carry virulence genes that enable selected
E. coli strains to cause intestinal and extra-intestinal infections [
2,
3]. Based on a phylogenetic assay,
E. coli can be classified into four main phylogenetic groups (A, B1, B2 and D) [
4‐
7]. Pathogenic strains which often encode virulence factors belong to group B2 and D, while most fecal
E. coli strains belonging to group A and B1 lack virulence factors [
8,
9]. A group of cytotoxins, including colibactin, cytotoxic necrotizing factors (CNFs), cytolethal distending toxin (CDT) and cycle inhibiting factor (CIF), are classified as cyclomodulins and are genotoxic and/or modulate cellular differentiation, apoptosis and proliferation [
10‐
12].
Colibactin is a cytotoxic hybrid polyketide/nonribosomal peptide produced by several species of
Enterobacteriaceae. It was first identified in 2006 in an extra-intestinal pathogenic
E. coli (ExPEC) strain isolated from a case of neonatal meningitis [
12]. This secondary metabolite, colibactin, is produced by the
clbA-
S genes present in the 54-kb pathogenicity
pks island, a genetic island encoding a non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line [
12,
13]. In vitro studies have shown that
pks+
E. coli strains induce enlargement of cells and nuclei without mitosis (megalocytosis), cause G2 cell cycle arrest, and DNA double strand breaks [
12]. In animal model experiments, a
pks+
E. coli strain (NC101), isolated from specific pathogen free wild-type mice induced inflammation in the cecum in interleukin 10 knockout (IL10
−/−) mice after a 3 week monoassociation period [
14]. Studies also demonstrated that monoassociation with NC101 promotes invasive carcinoma in IL10
−/− mice treated with azoxymethane (AOM). The promotion effect was dependent on expression of the
pks island [
15]. In a previous study from our laboratory, 88% of
E. coli isolates from laboratory mice encoded
pks genes and belonged to phylogenetic group B2 [
16]. Our findings indicated that colibactin-encoding
E. coli commonly colonize laboratory mice and may induce clinical and subclinical disease that may impact in vivo experimental results [
16].
Escherichia coli strains that produce CNF belong to the pathotype necrotoxigenic
E. coli (NTEC) and are associated with intestinal and extra-intestinal infection in both humans and animals [
2]. The majority of CNFs include chromosomally encoded
cnf1 [
17] and plasmid-encoded
cnf2 [
18]. CNF1 is a 115 kDa protein toxin which activates Rho GTPases, leading to cytoskeletal and cell cycle alterations with subsequent macropinocytosis and formation of megalocytic, multinucleated cells. CNF1-producing and β-hemolytic
E. coli strains most notably cause urinary tract and meningeal infections in humans [
19]. These strains are also isolated from healthy and diseased animals. In our laboratory,
cnf1+
E. coli strains were isolated from ferrets with diarrhea and extra-intestinal infections [
20] and from healthy macaques [
21].
cnf1-encoding
E. coli strains have also been isolated from cats [
22], dogs [
23,
24], pigs [
25], and birds [
26].
The prevalence of pks+ E. coli in rhesus macaques is not known, nor is there published evidence that E. coli strains encoding both pks and cnf genes colonize macaques. The purpose of the present study was to characterize rectal E. coli isolates, as well as extra-intestinal isolates, from macaques for the presence of specific virulence genes (pks and cnf) and demonstrate their in vitro toxin activities.
Discussion
Escherichia coli is a normal inhabitant of the gastrointestinal tract of macaques and several different serotypes of bacteria have been isolated from asymptomatic rhesus macaques [
32]. To our knowledge this is the first reported isolation of colibactin-encoding
E. coli strains from macaques. Colibactin was first identified in several
E. coli strains by Oswald and co-workers in 2006 [
12]. Colibactin, encoded by a 54 kb gene cluster (the
pks island), is a genotoxin which causes DNA double strand breaks and activation of the DNA damage checkpoint pathway, leading to cell cycle arrest and eventually cell death. The role of colibactin-encoding
E. coli has been explored in human colorectal cancer and investigated in different types of mouse models including IL10
−/− mice treated with azoxymethane (AOM), C57BL/6J-ApcMin/J mice treated with AOM and dextran sodium sulfate (DSS), and nude mice with xenografts [
11,
15,
33,
34]. These studies have established a role of
pks-encoding
E. coli in inflammation and cancer. The increase in
E. coli growth and colonization may be due to inflammation-generated nitrate, as
E. coli, a facultative anaerobe, can produce energy through the use of nitrate,
S-oxides and
N-oxides as terminal electron acceptors for anaerobic respiration and thus outcompete obligate anaerobes, the major colonizers of the lower bowel [
35].
Among the
E. coli isolates from macaques, 30.1% of isolates were
pks+ strains and colibactin activities were confirmed in selected isolates. The prevalence of
pks+
E. coli colonization in macaques was similar to the 25% prevalence of
pks+ B2
E. coli colonization in humans [
36]. Several publications have revealed the higher prevalence of
pks+
E. coli strains in biopsies from colorectal cancer (CRC) patients (66.7% in CRC patients, 40% in IBD patients and 20.8% in no IBD/no CRC controls) [
11,
15,
37].
The 20.9% prevalence of
cnf1+
E. coli in this study was consistent with our previous findings [
21]. Close correlation was observed between the
cnf1 gene and β-hemolysis in
E. coli isolates. This association was reported in our previous studies [
20,
21] and by other authors [
19,
38]. In the
E. coli J96 strain and other strains, the
cnf1 gene is located downstream of hemolysin (
hlyCABD) in pathogenicity island II (PAI II), and the expression of
cnf1 is regulated by the hemolysin promoter [
39‐
41]. Likewise, our draft genome sequences of macaque
E. coli strains indicate that hemolysin (
hlyCABD) is located directly upstream of the
cnf1 gene. These two toxins could therefore be associated with enhanced virulence. In our in vitro cytotoxicity assays, infection with the
pks−/
cnf1+ isolates (S3, S9, S10) caused ~ 50% of HeLa cells to die, and the surviving cells exhibited an elongated morphology; CNF1 cytotoxicity by these isolates was only observed with sonicate and supernatant treatment. The draft genome sequence data showed that only
pks−/
cnf1+ isolates (S3, S6, S9, and S10) also contained an annotated secreted autotransporter toxin (
sat), which is expressed by some UPEC strains and reported to cause cell elongation in vitro [
42]. We speculate that expression of
sat by these isolates may have caused HeLa cells to adopt an elongated morphology. Interestingly, we also identified
E. coli strains co-harboring the
pks and
cnf1 genes. Thirty-one (13.0%)
E. coli isolates were positive for both
pks and
cnf1 (
pks+/
cnf1+). The
E. coli strains co-harboring these two toxins have also been reported in human samples [
10,
11]. The authors noted that 15% of
E. coli isolates from urosepsis patients and from the feces of healthy individuals were double-positive for
pks and
cnf1 (
pks+/
cnf1+). In our in vitro cytotoxicity assays, the isolates
pks+/
cnf1+ (S1, S2, and S14) exhibited severe toxicity to HeLa cells, given all cells were dead when treated with these live isolates at MOI 5, 25 and 100 (Fig.
5).
Yasuda and co-workers characterized biogeographic relationships in the rhesus macaque intestinal microbiome and found that stool microbiota was highly representative of the colonic lumen and mucosa, which were respectively enriched in obligate and facultative anaerobes [
43]. In our study the
E. coli strains were isolated from rectal swab samples and may, by analogy, be representative of the
E. coli strains colonized in the macaque gastrointestinal tract. Moreover, among
E. coli isolates in the present study, two isolates which were β-hemolytic and
pks+/
cnf1+ were isolated from a cephalic recording chamber and implant-margin skin of two macaques. This raises the concern that these
E. coli isolates may cause meningitis in macaques used in neurobiological research based on the previous reports that
pks+
E. coli were isolated and related to meningitis in humans and animals [
44].
For the
E. coli isolates from macaques in this study, the predominant phylogenetic group was B2 group including B2
1 subgroup (60.1%) followed by B1 (26.0%), A (13.3%), and D group (0.6%). The distribution of phylogenetic groups of macaque
E. coli strains were similar to distribution of
E. coli isolates from healthy humans [
10,
11]. The distribution of cyclomodulin-encoding genes (
pks, cnf,
cdt and
cif) in relation to the phylogenetic background in
E. coli isolates from urosepsis patients and healthy individuals indicated that strains
pks+ and/or
cnf
1+ strongly associated with the B2 group [
10]. In another study, the prevalence of
E. coli producing cyclomodulins and genotoxins in colon cancer had a higher prevalence of the B2 phylogenetic group
E. coli harboring the
pks gene (55.0%) and
cnf1 gene (39.5%) in biopsies of patients with colorectal cancer than that in patients with diverticulosis (19.3%
pks+ and 12.9%
cnf1+) [
11]. In both studies, the percentage of
E. coli strains harboring
cdt and
cif genes were much lower (1–6%). Representative isolates in our study were negative for
cdt and
cif according to PCR, in vitro cytotoxicity assay, and genome analysis results. We found that
pks+,
cnf1+, and β-hemolytic
E. coli strains belonged to group B2. 49.0% (51 out of 104) of the isolates belonging to B2 (including the B2
1 subgroup) were
pks+, 40.4% (42 out of 104) were
cnf1+, and 37.5% (39 out of 104) were β-hemolytic. Seven
pks+
E. coli isolates belonged to the B2
1 subgroup, which is a highly virulent phylogenetic subgroup among extra-intestinal pathogenic
E. coli B2 strains [
7,
44].
The serotype data in the present study revealed that the serotype of selected
E. coli strains corresponded to their toxin-harboring content. Of the isolates serotyped, those
pks+/
cnf1+ (S1 and S2) were the O88:H4 serotype. The
pks−/
cnf1+ isolates (S3, S6, S9, S10, S11) were O25:H4, the
pks+/
cnf1− isolates (S4 and S5) were O7:H7, the
pks−/
cnf1− isolates (S7 and S8) were OM:H14 or OM:H16. The O7:H7:K1 serotype belonging to phylogenetic group B2 was cultured from IL10
−/− and wild-type mice [
45]. In these experiments, cecal and colonic inflammation observed in IL10
−/− mice was accompanied by diminished intestinal microbial diversity and a higher number of
E. coli organisms compared to wild-type mice [
45]. Serotype O7:H7
E. coli strains were also found among Shiga-toxin-producing
E. coli strain isolated from calves in Brazil [
46]. UPEC O25:H4 strains were reported in patients with urinary tract infection [
47] and patients with cystitis or prostatitis [
48]. These strains belonging to group B2 had multiple antibiotic drug resistance. By analyzing the draft genome sequences of our macaque
E. coli isolates, putative multi-antibiotic resistance genes were identified exclusively in
cnf1+ strains (serotype O25:H4). These included resistance genes to the tetracycline (
tet(A)), phenicol (
catB3-
like), fluoroquinolone and aminoglycoside (
aac(6′6′)Ib-
cr), and β-lactam (
blaCTX-
M-
15,
blaOXA-
1) classes of antibiotics, consistent with our previous antibiotic resistance findings of
cnf1+
E. coli strains in macaques [
21]. Other studies have noted that antibiotic-susceptibility is inversely related to the number of virulence factor genes present in extra-intestinal
E. coli strains [
49]. Similarly, in this study isolates that were
pks−/
cnf1+ had the second fewest number of virulence factor genes and also were the only isolates with putative antibiotic resistance genes detected.
Different virulence factor gene profiles appear to be present depending on whether the isolates were
pks+/
cnf1+,
pks+/
cnf1−,
pks−/
cnf1+, or
pks−/
cnf1−. The number and prolife of these virulence genes agrees with in vitro cytotoxicity to HeLa cells in that the isolates showing pronounced cytotoxicity (
pks+ and/or
cnf1+ strains) had substantially more virulence factor genes present in their genomes compared to the less cytotoxic
pks−/
cnf1− isolates. Except for
lpfA, the
pks−/
cnf1−
E. coli isolates had the same virulence factor genes as K12, suggesting that along with the in vitro cytotoxicity results, these isolates likely have attenuated pathogenic potential. Interestingly,
pks+ strains harboring the bacteriocin synthesis genes for colicin E2 and microcin H47 also had the most virulence genes. This agrees with other studies reporting that
E. coli strains expressing bacteriocins are statistically more likely to co-associate with more virulence factor genes in their genomes compared to strains that lack bacteriocin potential [
49‐
51]. Likewise, bacteriocin genes are found more frequently in
E. coli strains belonging to the pathogenic B2 or D phylogroup, such as our monkey isolates [
49,
50,
52]. In particular, microcin H47 is predominantly found in the UPEC strains [
53]. It is hypothesized that bacteriocin activity by pathogenic
E. coli may provide a competitive survival and colonization advantage against commensal organisms, especially when availability of essential nutrients, like iron, is scarce [
51].
In summary, E. coli strains encoding colibactin, CNF1, or both were identified in macaques. The pks+ and/or cnf1+ isolates belonged to phylogenetic group B2 and induced cytotoxic effects to HeLa cells in vitro. The genomic data supports the presence of virulence factor and antibiotic resistance genes in these isolates and suggests that they may have the pathogenic potential to influence clinical and subclinical disease. The impact of these strains on the health of macaques is unclear as analysis of medical records did not allow an association of clinical events and isolation of E. coli. Given colibactin and CNF1-encoding E. coli has been isolated from human and animals populations, there is a concern about potential zoonotic spread. The presence of colibactin and CNF1-producing E. coli strains in primates used in neurobiology emphasizes the importance of appropriate personnel protection and hygiene practices when handling these primates.
Authors’ contributions
YF performed PCR, cell cultures, assisted in data analysis, and was a major contributor in writing the manuscript. AM performed genome sequencing and comparative analysis, cell culture assays, assisted in data analysis, and was a major contributor in writing the manuscript. CMM performed bacterial culture, isolation, and identification, as well as data management. AGS collected samples, assisted in bacterial culture, DNA extraction, and PCR, as well as data interpretation. CT assisted with statistical analyses by evaluating data from an NHP survey of pks+ E. coli samples collected from the colonies at MIT, assisted in bacterial culture, DNA extraction, and PCR. CB assisted in bacterial culture, DNA extraction, and PCR. RPM analyzed medical records for survey, assisted in project oversight, and contributed to writing the manuscript. JGF was responsible for project conception and design, provided project oversight, data interpretation and analysis, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.