Identification of Virulence-Associated Markers in Escherichia Coli Isolated from Captive Red-Browed Amazon Parrot (Amazona Rhodocorytha)

ABSTRACT

Due to the genetic similarity of pathogenic Escherichia coli isolated from birds and pathotypes of human origin, it is suggested that they have a common ancestor and may exchange virulence-associated genes. This study aimed to detect virulence-associated genes in E. coli strains isolated from the Red-browed Amazon parrot (Amazona rhodocorytha) kept at a conservation institute in Brazil. High genetic variability in virulence was observed, since 12 virulence profiles were found among 14 strains. The number of virulence-associated genes of single strains ranged from 5 to 22 out of 33 genes tested, and only one strain did not present any virulence genes. Regarding adhesion genes, most strains presented from two to five genes, and crlA (85.7%) and fimC (85.7%) were the most frequent. Frequencies were similar for invasion and iron acquisition genes. Variations among genes were observed for serum resistance and toxin-related genes. Some of the E. coli strains isolated from parrots presented virulence genes that are commonly associated with pathotypes of human origin, including newborn meningitis E. coli, uropathogenic E. coli, and sepsis-associated E. coli. It is noteworthy that some of these genes were present in the majority of the analyzed strains. Our results indicate that these strains detected in clinically healthy parrots can be potential reservoirs of several virulence-associated genes. These genes can be transmitted to other E. coli strains, including those that affect humans. These E. coli strains present a high pathogenic potential of virulence-associated genes in extraintestinal pathogenic E. coli strains.

Keywords:
Escherichia coli; parrot; public health; virulence genes

INTRODUCTION

The Psittacidae family comprises approximately 350 species, most of which are found in the tropics (British Trust for Ornithology, 2021). Brazil has the greatest diversity of species belonging to this family, ranging from small species such as parakeets to its largest representatives, the macaws (Sigrist, 2006). The Red-browed Amazon (Amazona rhodocorytha), also known as Chauá-Parrot, is a species endemic to the Atlantic Forest in eastern Brazil. It is threatened by both habitat loss and trapping for illegal trade (BirdLife International 2021).

Escherichia coli is a part of both the animal and human commensal flora and can be found in several environments. Pathogenic Escherichia coli can cause intestinal and extraintestinal infections. Gastrointestinal infections are related to diarrheagenic E. coli (DEC), while extraintestinal infections, including sepsis, urinary tract infections and meningitis, are associated with extraintestinal pathogenic E. coli (ExPEC) (Cunha et al., 2017; Saka et al., 2019). Despite comprising many lineages, only specific ExPEC isolates are responsible for the majority of infections (Manges et al., 2019). Among the ExPEC isolates, avian pathogenic E. coli (APEC) is responsible for colibacillosis in poultry, an economically important disease that threatens food safety and avian welfare worldwide. (Cunha et al., 2017). Avian colibacillosis is a multifaceted syndrome that can be associated with respiratory disease, septicemia, swollen head syndrome, yolk-sac infection, omphalitis, and cellulitis (Kim et al., 2020; Rocha et al., 2021).

ExPEC strains present a wide range of virulence-associated markers and an important plasticity of the genome. Their virulence traits include adhesins, invasins, iron acquisition and serum resistance factors, toxins, and proteases. These virulence factors are encoded by genes found in pathogenicity islands, plasmids, and other mobile genetic elements (Sarowska et al., 2019). Studies have shown genetic similarity between APEC and pathotypes of human origin, suggesting a common ancestral origin and an ability of potentially pathogenic strains to compromise human health (Ewers et al., 2007).Close similarities in serogroup, virulence factors, phylogenetic group, and sequence type have been shown between APEC and human ExPEC (Cunha et al., 2017), suggesting the important role of APEC strains to be a reservoir of virulence genes for ExPEC strains in humans (Rocha et al., 2017). It is especially important in those species with close contact with humans such as captive birds.

In this context, this study aimed to detect virulence-associated genes in E. coli strains isolated from the Red-browed Amazon of a conservation institute in Brazil.

MATERIALS AND METHODS

Ethical approval

All experimental procedures were approved by the Chico Mendes Institute for Biodiversity Conservation (ICMBio) under protocol number 38772-1. All methods were carried out in accordance with ICMBio relevant guidelines and regulations.

Red-browed Amazon parrot (Amazona rhodocorytha)

The birds selected for this study were maintained captive in an enriched vivarium at the National Institute of the Atlantic Forest (Santa Teresa, ES, Brazil). The parrots kept at the Institute come from illegal trade and cannot be returned to their habitat. Husbandry, feed, and water were provided by the employees of the Institute. A total of 14 clinically healthy parrots of different ages and both sexes were randomly selected for this study.

Escherichia coli strains

A total of 14 stool samples were collected using a cloacal swab, individually identified, and stored in a cool box for conservation during transportation to the laboratory. In the laboratory, swabs were individually inoculated into brain heart infusion broth (BHI; Oxoid, Basingstoke, UK) and aerobically incubated at 37 ºC. After 24 hr, the content was plated onto eosin methylene blue agar (Oxoid) and aerobically incubated for 24 hr at 37 ºC. Colonies morphologically consistent with those of E. coli (dark metallic green sheen with a low, convex, and smooth shape) were selected for biochemical tests (Lee et al., 2008). Biochemical tests included Gram stain (Gram negative rods), oxidase (negative), indol (positive), H₂S production (negative), glucose (positive), lactose (positive), lysine (positive), and urease (negative). The confirmed isolates were stored at -20 ºC in BHI broth supplemented with 15% glycerin (Synth, Diadema, Brazil) until use. Isolates were reactivated on MacConkey agar (Oxoid) and incubated at 37 ºC for 24 hr. A bacterial colony was solubilized in 200 µL ultra-pure water for DNA extraction.

Detection of virulence-associated genes

DNA extraction was carried out by heat treatment as described by Borsoi et al. (2009) and stored at -20 ºC until use. Isolates were screened for the presence of 33 virulence genes using four multiplex PCRs (Ewers et al., 2007): multiplex-1 (kpsMTII, hlyA, pic, fimC, hrlA, iha, neuC, afa/draB, malX, and sfa/foc), multiplex-2 (chuA, ibeA, traT, sitD chr., gimB, iroN, ompA, and sitD ep.), multiplex-3 (EAST-1, iss, irp2, papC, cvi/cva, iucD, tsh, and vat), and multiplex-4 (crlA, ireA, cnf1/2, tia, sat, fyuA, and mat). Genes functions, primer sequences, and references are listed in Table 1. All PCR amplifications were performed in a mixture (25 µL) consisting of 10 × PCR Buffer [3 µL; 200 mM Tris-HCl (pH 8.4), 500 mM KCl] (2.5 µL), Taq thermostable DNA polymerase (0.3 µL, 5U/µL), MgCl₂ (1.25 µL, 25 mM), dNTPs (0.5 µL each, 2.5 mM), extracted template DNA (5 µL) and each of the primers (2 µL each, 20 pmol/L). Sterile Milli-Q water was added in sufficient quantity to achieve a volume of 25 µL. Reaction mixtures were subjected to the following conditions in a thermal cycler (Biocycler Peltier Thermal Cycler MJ96+/MJ96G, Hercules, USA): 3 min at 94 ºC, 25 cycles of 30 s at 94 ºC, 30 s at 58 ºC, and 3 min at 68 ºC, with a final cycle of 10 min at 72 ºC, followed by a hold at 10 ºC (Ewers et al., 2007). For visualization of the PCR products, 10 µL aliquots were subjected to electrophoresis in a 2% agarose gel in Tris-acetylated EDTA buffer. DNA bands were stained with ethidium bromide for 2 hr at 100 V, viewed under an ultraviolet transilluminator, and photographed. Positive controls included four international E. coli reference strains [IMT5155 (APEC), IMT2470 (APEC), IMT7920 (uropathogenic E. coli - UPEC), and IMT9267 (neonatal meningitis E. coli - NMEC)] and three APEC strains isolated from broiler sources that belong to our stock collection (TK3, CC192, and CC158). Negative control included a mixture of all constituents of the PCR mixed without the addition of extracted DNA.

Table 1
Genes: functions, primers sequences, and primer references.

Function-related group	Gene	Primer (5’  3’)	Amplicon size (bp)	Primer reference
Adhesion	afa/draB	F: TAAGGAAGTGAAGGAGCGTG R: CCAGTAACTGTCCGTGACA	810	Ewers et al., 2007
	crlA	F: TTTCGATTGTCTGGCTGTATG R: CTTCAGATTCAGCGTCGTC	250	Maurer et al., 1998
	fimC	F: GGGTAGAAAATGCCGATGGTG R: CGTCATTTTGGGGGTAAGTGC	477	Janssen et al., 2001
	hrlA	F: TCACTTGCAGACCAGCGTTTC R: GTAACTCACACTGCTGTCACCT	537	Ewers et al., 2007
	iha	F: TAGTGCGTTGGGTTATCGCTC R: AAGCCAGAGTGGTTATTCGC	609	Ewers et al., 2007
	papC	F: TGATATCACGCAGTCAGTAGC R: CCGGCCATATTCACATAAC	501	Janssen et al., 2001
	sfa/focD	F: GTCCTGACTCATCTGAAACTGCA R: CGGAGAACTGGGTGCATCTTA	1242	Ewers et al., 2007
	tsh	F: ACTATTCTCTGCAGGAAGTC R: CTTCCGATGTTCTGAACGT	824	Ewers et al., 2007
	mat	F: TATACGCTGGACTGAGTCGTG R: CAGGTAGCGTCGAACTGTA	899	Ewers et al., 2007
Invasion	gimB	F: TCCAGATTGAGCATATCCC R: CCTGTAACATGTTGGCTTCA	736	Ewers et al., 2007
	ibeA	F: TGGAACCCGCTCGTAATATAC R: CTGCCTGTTCAAGCATTGCA	342	Ewers et al., 2007
	tia	F: AGCGCTTCCGTCAGGACTT R: ACCAGCATCCAGATAGCGAT	512	Ewers et al., 2007
Iron aquision	chuA	F: GACGAACCAACGGTCAGGAT R: TGCCGCCAGTACCAAAGACA	278	Clermont et al., 2000
	fyuA	F: GCGACGGGAAGCGATGACTTA R: CGCAGTAGGCACGATGTTGTA	774	Schubert et al., 2002
	ireA	F: ATTGCCGTGATGTGTTCTGC R: CACGGATCACTTCAATGCGT	384	Ewers et al., 2007
	iroN1	F: ATCCTCTGGTCGCTAACTG R: CTGCACTGGAAGAACTGTTCT	847	Ewers et al., 2007
	irp2	F: AAGGATTCGCTGTTACCGGAC R: TCGTCGGGCAGCGTTTCTTCT	413	Schubert et al., 2002
	iucD	F: ACAAAAAGTTCTATCGCTTCC R: CCTGATCCAGATGATGCTC	714	Janssen et al., 2001
	sitD chr.	F: ACTCCCATACACAGGATCTG R: CTGTCTGTGTCCGGAATGA	554	Ewers et al., 2007
	sitD ep.	F: TTGAGAACGACAGCGACTTC R: CTATCGAGCAGGTGAGGA	1052	Ewers et al., 2007
Serum resistance	cvi/cva	F: TCCAAGCGGACCCCTTATAG R: CGCAGCATAGTTCCATGCT	598	Ewers et al., 2007
	iss	F: ATCACATAGGATTCTGCCG R: CAGCGGAGTATAGATGCCA	309	Ewers et al., 2007
	neuC	F: GGTGGTACATTCCGGGATGTC R: AGGTGAAAAGCCTGGTAGTGTG	676	Ewers et al., 2007
	kpsMT II	F: GCGCATTTGCTGATACTGTTG R: CATCCAGACGATAAGCATGAGCA	280	Genbank accession number X53819
	ompA	F: AGCTATCGCGATTGCAGTG R: GGTGTTGCCAGTAACCGG	919	Ewers et al., 2007
	traT	F: GTGGTGCGATGAGCACAG R: TAGTTCACATCTTCCACCATCG	430	Ewers et al., 2007
Toxins	cnf1/2	F: TCGTTATAAAATCAAACAGTG R: CTTTACAATATTGACATGCTG	446	Ewers et al., 2007
	sat	F: TGCTGGCTCTGGAGGAAC R: TTGAACATTCAGAGTACCGGG	667	Ewers et al., 2007
	astA	F:TGCCATCAACACAGTATATCC R: TAGGATCCTCAGGTCGCGAGTGACGGC	116	Yamamoto et al., 1996
	vat	F: TCCTGGGACATAATGGTCAG R: GTGTCAGAACGGAATTGTC	981	Ewers et al., 2007
	hlyA	F: GTCCATTGCCGATAAGTTT R: AAGTAATTTTTGCCGTGTTTT	352	Ewers et al., 2007
Autotransporter serine protease	pic	F: ACTGGATCTTAAGGCTCAGG R: TGGAATATCAGGGTGCCACT	409	Ewers et al., 2007
Pathogenicity island associated gene	malX	F: GGACATCCTGTTACAGCGCGCA R: TCGCCACCAATCACAGCCGAAC	922	Johnson et al., 2001

• Legend: Base pair (bp); Forward (F); Reverse (R).

Statistical Analysis

The obtained data were subjected to statistical analysis using the PASW Statistics software. Descriptive statistics were used to determine the frequency distribution. Chi-square and Fisher’s exact tests were used to compare the frequencies of virulence-associated genes. Statistical significance was defined at p<0.05 and Bonferroni correction was applied to adjust the confidence intervals for multiple hypothesis testing.

RESULTS AND DISCUSSION

Previous studies have shown that there are differences in the gut microbiota between captive and wild birds. Wild birds usually have a small number of Gram-negative bacteria in the gut microbiota, most of which are transient in healthy birds (Lopes et al., 2016). In contrast, captive birds usually present abundant amounts of E. coli in their microbiota (Saidenberg et al., 2017). In these birds, intestinal colonization by E. coli is dependent on the animal’s health status as well as the environment in which they are kept, because the birds that live in the same enclosure usually use the same feeders and drinkers (Lopes et al., 2016).

The frequency of detection of virulence-associated genes according to their function is shown in Table 2. E. coli was present in all parrots. However, the detection of this microorganism does not characterize enteric disturbance or disease in the animal since the birds appeared healthy (Calaça et al., 2020). Of the 33 genes analyzed, only four genes were not detected (tsh, ireA, sitD ep., astA). The number of virulence-associated genes of single strains ranged from 5 to 22, except for one strain (#5) that did not contain any virulence genes. Our results indicate high genetic variability in virulence since we found a total of 12 virulence profiles among 14 strains (Table 3). Profiles I and VII represent two strains each. In Profile XII, no virulence gene was detected. In Profile XI, only five genes were detected, and VI is the profile with the highest number of detected genes (22 of 33 genes). Other profiles presented, from 9 to 19 virulence markers.

Table 2
Relative (%) and absolute frequencies (n) of virulence genes, according to the function-related groups, and individual results.

Function-related group	Gene	Frequencies % (n)1	Strain identification
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
Adhesion	afa/draB	21.4 (3)b	1	1	0	0	0	0	0	0	0	1	0	0	0	0
	crlA	85.7 (12)a	1	1	1	1	0	1	1	1	1	1	0	1	1	1
	fimC	85.7 (12)a	1	1	1	1	0	1	1	1	1	1	0	1	1	1
	hrlA	14.3 (2)b	0	0	0	0	0	0	1	0	0	0	1	0	0	0
	iha	14.3 (2)b	0	0	0	0	0	0	1	0	0	0	1	0	0	0
	papC	14.3 (2)b	0	0	1	0	0	0	0	0	0	1	0	0	0	0
	sfa/focD	7.1 (1)b	0	0	0	0	0	0	0	0	0	0	1	0	0	0
	tsh	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	mat	71.4 (10)a	1	1	1	1	0	0	1	1	1	1	0	1	0	1
	Total of detected genes		4	4	4	3	0	2	5	3	3	5	3	3	2	3
Invasion	gimB	21.4 (3)a	1	1	0	0	0	0	0	0	0	0	1	0	0	0
	ibeA	50.0 (7)a	0	0	1	0	0	1	1	1	1	0	0	1	1	0
	tia	57.1 (8)a	0	0	1	1	0	1	0	1	1	1	0	1	1	0
	Total of detected genes		1	1	2	1	0	2	1	2	2	1	1	2	2	0
Iron aquision	chuA	57.1 (8)a	0	0	1	1	0	1	1	1	1	0	0	1	1	0
	fyuA	42.9 (6)a	0	0	1	0	0	1	0	1	1	0	0	1	1	0
	ireA	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	iroN1	42.9 (6)a	0	0	1	0	0	1	1	1	0	1	0	0	0	1
	irp2	42.9 (6)a	0	0	1	0	0	1	0	1	1	0	0	1	1	0
	iucD	57.1 (8)a	1	1	1	1	0	0	1	1	1	0	0	1	0	0
	sitD chr.	35.7 (5)a	1	1	0	1	0	1	0	0	0	1	0	0	0	0
	sitD ep	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Total of detected genes		2	2	5	3	0	5	3	5	4	2	0	4	3	1
Serum resistance	cvi/cva	78.6 (11)a	1	1	1	1	0	0	1	1	1	1	1	1	1	0
	iss	21.4 (3)b	0	0	0	0	0	1	1	1	0	0	0	0	0	0
	neuC	71.4 (10)ab	1	1	1	1	0	1	0	1	1	0	1	1	1	0
	kpsMTII	64.3 (9)ab	1	1	1	1	0	0	1	1	1	1	0	1	0	0
	ompA	85.7 (12)a	1	1	1	1	0	1	1	1	1	1	0	1	1	1
	traT	42.9 (6)ab	0	0	0	0	0	1	1	1	1	0	0	1	1	0
	Total of detected genes		4	4	4	4	0	4	5	6	5	3	2	5	4	1
Toxins	cnf1/2	85.7 (12)a	1	1	1	1	0	1	1	1	1	1	1	1	1	0
	sat	28.6 (4)b	0	0	0	1	0	1	0	1	0	1	0	0	0	0
	astA	0.0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	vat	50.0 (7)ab	0	0	1	0	0	1	1	1	1	0	0	1	1	0
	hlyA	42.9 (6)ab	0	0	0	0	0	0	1	1	1	1	1	1	0	0
	Total of detected genes		1	1	2	2	0	3	3	4	3	3	2	3	2	0
Autotransporter serine protease	pic	42.9 (6)	0	0	1	0	0	1	1	1	0	0	1	0	1	0
Pathogenicity island associated gene	malX	78.6 (11)	1	1	1	1	0	1	1	1	1	1	0	1	1	0

• ¹
  Different letter indicates significant differences among genes within function-related group (Fisher’s exact test; adjusted p-value).

Table 3
Virulence profiles identified among Escherichia coli strains (n=14).

Profile identification	Detected genes	Strain identification
I	afa/draB, crlA, fimC, mat, gimB, iucD, sitD chr., cvi/cva, neuC, kpsMT, ompA, cnf1/2, malX	1 and 2
II	crlA, fimC, papC, mat, beA, tia, chuA, fyuA, iroN1, irp2, iucD, cvi/cva, neuC, kpsMT, ompA, malX	3
III	crlA, fimC, mat, tia, chuA, iucD, sitD chr., cvi/cva, neuC, kpsMT, ompA, cnf/12, sat, malX	4
IV	crlA, fimC, ibeA, tia, chuA, fyuA, iroN1, irp2, sitD chr., iss, neuC, ompA, traT, cnf1/2, sat, vat, pic, malX	6
V	crlA, fimC, hrlA, iha, mat, ibeA, chuA, iroN1, iucD, cvi/cva, iss, kpsMT, ompA, traT, cnf1/2, vat, hlyAm pic, malX	7
VI	fimC, hrlA, mat, ibeA, tia, chuA, fyuA, iroN1, irp2, iucD, cvi/cva, iss, neuC, kpsMT, ompA, traT, cnf1/2, sat, vat, hlyA, pic, malX	8
VII	crlA, fimC, mat, ibeA, tia, chuA, fyuA, irp2, iucD, cvi/cva, neuC, kpsMT, ompA, traT, cnf1/2, vat, hlyA, malX	9 and 12
VIII	afa/draB, crlA, fimC, papC, mat, tia, iroN1, sitD chr., cvi/cva, kpsMT, ompA, cnf1/2, sat, hlyA, malX	10
IX	hrlA, iha, sfa/focD, gimB, cvi/cva, neuC, cnf1/2, hlyA, pic	11
X	crlA, fimC, ibeA, tia, chuA, fyuA, irp2, cvi/cva, neuC, ompA, traT, cnf1/2, vat, pic, malX	13
XI	crlA, fimC, mat, iroN1, ompA	14
XII	No virulence genes detected	5

Considering the nine genes associated with adhesion (afa/drab, crlA, fimC, hrlA, iha, papC, sfa/focD, tsh, and mat), only one strain (#5) did not present any gene; other strains presented from two to five genes. The presence of crlA (85.7%) and fimC (85.7%) genes was significantly higher (p<0.007) than afa/draD (21.4%), hrlA (14.3%), iha (14.3%), papC (14.3%), and sfa/focD (7.1%). Invasion genes (gimB, ibeA, and tia) presented similar (p>0.05) frequencies of detection among strains, and two strains (#5 and #14) did not present any genes. Only two strains (#5 and #11) did not present any iron acquisition genes (chuA, fyuA, ireA, iroN1, irp2, iucD, sitD chr., and sitD ep.) and the frequencies of detection were similar (p>0.05) among the strains. The presence of serum resistance genes cvi/cva (78.6%) and ompA (85.7%) was significantly higher (p<0.008) than iss (21.4%). Two strains (#5 and #14) did not present any toxin-related genes. The cnf1/2 (85.7%) gene showed significantly higher (p<0.010) detection than sat (28.6%). The autotransporter serine protease associated gene (pic) and the pathogenicity island associated gene (malX) were not detected in eight (#1, #2, #4, #5, #9, #10, #12, and #14) and three (#5, #11, and #14) strains, respectively. It is likely that the absence of some specific genes does not reflect a decrease in virulence, since they may be replaced by other functionally related groups (Beceiro et al., 2013).

Of the 33 analyzed genes, 12 genes were frequently foundin E. coli isolated from birds (fimC, papC, crlA, ibeA, iucD, iroN1, sitD ep., sitD chr., traT, iss, cvi/cva, vat). However, the others are commonly found in E. coli of human origin, including newborn meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), and sepsis-associated E. coli (SEPEC) (Sarowska et al., 2019). Some of these genes were present in the majority of the analyzed strains, including cnf1/2(85.7%), neuC (71.4%), mat (71.4%), kpsMTII (64.3%), and chuA (57.1%). According to Robins-Browne et al. (2016), the mobility of most of the genes that encode virulence is common and may often occur. However, our results indicate that these strains detected in clinically healthy parrots may be opportunistic pathogens and may cause disease in immunosuppressed birds (Calaça et al., 2020). It is a concerning result, since poultry may be a vehicle or even a reservoir for human ExPEC strains, and can be considered potential zoonotic agents (Ewers et al., 2007; Sarowska et al., 2019). This is of special concern in captive parrots because of their direct contact with humans.

The strains showed high genetic variability in virulence. Our results indicate that these strains detected in clinically healthy parrots can be potential reservoirs of several virulence-associated genes and can be transmitted to other E. coli strains, including those that affect humans (UPEC, NEMEC, and SEPEC). These E. coli strains present a high pathogenic potential of virulence-associated genes in ExPEC strains. The use of techniques to group similar bacteria together is helpful to understand the distribution of virulence markers. Further studies can include the whole genome sequencing (WGS) of E. coli strains. The combination of WGS techniques and clinical and epidemiological data would allow the determination of which strains within a subtype are more virulent than others.

ACKNOWLEDGEMENT

The authors thank Dr. Fabiana Horn (UFRGS), who kindly provided reference strains used for positive controls.

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Publication Dates

• Publication in this collection
  08 Aug 2022
• Date of issue
  2022

History

• Received
  23 Feb 2022
• Accepted
  13 Apr 2022