Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand

Pramualchai Ketkhao; Fuangfa Utrarachkij; Nattaya Parikumsil; Kritchai Poonchareon; Anusak Kerdsin; Peeraya Ekchariyawat; Pawarut Narongpun; Chie Nakajima; Yasuhiko Suzuki; Orasa Suthienkul

doi:10.1371/journal.pone.0307544

. 2024 Jul 25;19(7):e0307544. doi: 10.1371/journal.pone.0307544

Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand

Pramualchai Ketkhao ¹, Fuangfa Utrarachkij ¹, Nattaya Parikumsil ², Kritchai Poonchareon ³, Anusak Kerdsin ⁴, Peeraya Ekchariyawat ¹, Pawarut Narongpun ⁵, Chie Nakajima ^5,⁶, Yasuhiko Suzuki ^5,^6,^7,^*, Orasa Suthienkul ^8,^*

Editor: Kwame Kumi Asare⁹

PMCID: PMC11271939 PMID: 39052658

Abstract

Extraintestinal pathogenic Escherichia coli (ExPEC), especially uropathogenic E. coli (UPEC) are responsible for urinary tract infections (UTIs), while diarrheagenic E. coli (DEC) cause foodborne illnesses. These pathogenic E. coli are a serious threat to human health and a public concern worldwide. However, the evidence on pork E. coli (PEC) harboring UPEC virulence-associated genes is currently limited. Therefore, this study aimed to determine the phylogroups, virulence genes, and their association between PEC and UPEC from UTI patients. In this study, 330 E. coli were obtained from archived stock culture isolated from pork (PEC; n = 165) and urine of patients with UTIs (UPEC; n = 165) during 2014–2022. Phylogroups, UPEC- and diarrheagenic E. coli (DEC) associated virulence genes were assessed using PCR assays. The results showed that phylogroups A (50.3%), and B1 (32.1%) were commonly found among PEC whereas phylogroups B2 (41.8%), and C (25.5%) were commonly detected in the UPEC. PEC and UPEC carried similar virulence-associated genes with different percentages. The most frequent UPEC virulence-associated gene among UPEC, and PEC strains was fimH, (93.3%, and 92.1%), followed by iucC (55.2%, and 12.7%), papC (21.8%, and 4.2%), afaC (22.4%, and 0%), hlyCA (17%, and 0.6%), cnf (16.4%, and 0.6%), and sfa/focDE (8.5%, and 4.8%). Additionally, 6 of 27 UPEC virulence-associated gene patterns were found in both PEC and UPEC strains regardless of phylogroups. Furthermore, the DEC virulence-associated genes were found in only 3 strains, one from PEC harboring eae, and two from UPEC carried fimH-bfpA or afaC-CVD432 indicating hybrid strains. Cluster analysis showed a relationship between PEC and UPEC strains and demonstrated that PEC harboring UPEC virulence-associated genes in pork may be associated with UPEC in humans. Food safety and hygiene practices during pork production chain are important procedures for minimizing cross-contamination of these strains that could be transmitted to the consumers.

Introduction

Pathogenic Escherichia coli are important Gram-negative bacteria that cause intestinal and extraintestinal diseases in humans and animals worldwide [1,2]. The pathogenicity of E. coli is determined by numerous virulence factors such as adhesion, invasion, colonization, and toxin secretion [3]. Intestinal E. coli are composed of non-pathogenic E. coli that are typically lacking virulence factors without causing gastroenteritis, and intestinal pathogenic E. coli (InPEC) or diarrheagenic E. coli (DEC) that carry various virulence factors causing a variety of gastrointestinal tract infections [4]. Extraintestinal pathogenic E. coli (ExPEC) carry virulence factors that commonly cause infections outside the intestinal tract and are mostly associated with urinary tract infections (UTIs). ExPEC can colonize the intestinal tract without causing gastroenteritis [5].

At present, InPEC or DEC are classified into eight pathotypes causing different types of gastroenteritis [4]. Enteropathogenic E. coli (EPEC) contain the intimin (eae) with or without bundle forming pili (bfp) for typical (tEPEC) or atypical (aEPEC) and are primarily associated with diarrhea in infants [6]. Enterotoxigenic E. coli (ETEC) cause illness through the production of heat-stable (st) and heat-labile (lt) enterotoxins causing traveler’s diarrhea [7]. Shiga toxin-producing E. coli (STEC) produce Shiga toxins encoded by the stx1 and stx2 genes, while enterohaemorrhagic E. coli (EHEC) are defined by acquiring an additional intimin (eae) gene and a hemolysin (hlyA) gene. Both pathotypes are responsible for abdominal pain, bloody diarrhea, and are the primary causes of hemorrhagic colitis and hemolytic uremic syndrome (HUS) [8,9]. Enteroinvasive E. coli (EIEC) carry the ipaH gene, which is associated with an invasive plasmid antigen causing bloody diarrhea in infants [10]. Enteroaggregative E. coli (EAEC) contain the aagR gene, which encodes a transcriptional activator and is a major contributor to acute diarrhea in both children and adults globally [11]. Diffusely adherent E. coli (DAEC) possess the afa and dra genes, encoding afimbrial adhesins, and involved in sporadic cases of diarrhea, especially in pediatric patients [12]. Adherent invasive E. coli (AIEC) are associated with inflammatory bowel diseases, like Crohn’s disease. However, AIEC are identified based on its phenotype due to the lack of a specific molecular markers [13]. Diarrheal diseases caused by DEC are a significant cause of morbidity and mortality in developing countries, especially in infants and young children [3]. ETEC, EPEC, and EHEC produce the same and different virulence factors in humans causing diarrheal diseases and also causing several diseases in industrious swine that resulted in the death of piglets [1]. Consequently, these pathotypes are possibly contaminated in animal-origin products including pork causing human diarrhea [4].

ExPEC are classified into four pathotypes namely uropathogenic E. coli (UPEC), newborn meningitis E. coli (NMEC), sepsis-associated E. coli (SEPEC), and avian pathogenic E. coli (APEC) [14]. UPEC are the majority of pathogens among ExPEC pathotypes. This pathotype can disseminate and colonize the urinary tracts of the hosts causing UTIs, affecting approximately 150 million cases globally each year [15,16]. Approximately 80% of UPEC infections cause community-acquired UTIs, but the sources of these UPEC remain unknown [17]. Two groups of essential virulence factors for UPEC to colonize and persist in the urinary tract are bacterial cell surface and secreted virulence factors. Virulence factors associated with bacterial cell surface include type 1 fimbriae (fimH), P fimbriae (pap), S fimbriae, and F1C fimbriae (sfa/foc). Hemolysin (hlyCA), and cytotoxic necrotizing factor 1 (cnf1) are secreted virulence factors [15]. Moreover, hybrid pathogenic E. coli harboring both UPEC and DEC virulence-associated genes isolated from UTI patients have been reported, which may increase the potential severity of intestinal and extraintestinal infections [18].

Tracing the origin of ExPEC causing UTIs has still been discussed. Several studies have demonstrated that human UPEC have potentially originated from animals [19,20]. Some evidence demonstrated that UPEC virulence genes such as fimH, pap, sfa/foc, ibeA (invasin), and iutA, iroN (iron acquisition) from poultry and domestic companion animals were similar to human UPEC [19,21]. Recently, swine ExPEC causing UTIs in swine are increasingly found in the production and supply chain of pork in several countries [22–24]. Various virulence factors of the swine and pork ExPEC such as fimH, iutA, sfa/foc, and kpsMTII (capsular antigens) were similar to the human UPEC [22,24–27]. Furthermore, the same pulse-field gel electrophoresis (PFGE) patterns of E. coli strains from human UTI patients and pork have been reported, suggesting an increased risk of zoonotic infections [27]. These findings showed that swine and their products; pork may serve as a transmission source of UPEC and potentially cause UTIs in humans resulting in public health concerns. However, the information on the genetic virulence determinants related to swine and pork, human UPEC, and hybrid UPEC in Thailand was limited and has to be further elucidated.

Phylogroup identification by Clermont et al. [28] is essential and useful for epidemiological studies of E. coli. This method was applied to classify the various origins of E. coli into eight phylogroups: A, B1, B2, C, D, E, F, and cryptic clade I. The phylogroups have correlated with the presence of common virulence factors associated with ExPEC such as papC, sfa/foc, cnf, and afa (afimbriae adhesins). Most of the ExPEC/UPEC from humans were classified into phylogroups B2 and D while commensal E. coli belongs to phylogroups A and B1 [29,30]. However, there are very limited studies on the phylogroup classification of E. coli both from human UTIs, animals, and pork in Thailand.

Additional information regarding the contamination of pork with E. coli carrying UPEC virulence-associated genes, as well as hybrid UPEC strains, needs to be updated in Thailand. Therefore, this study aimed to elucidate the sharing status of UPEC and DEC virulence characteristics between pork E. coli (PEC) and UPEC from UTI patients. Additionally, the prevalence, distribution, virulence-associated genes, phylogroups, hybrid UPEC strains, and their association between PEC and UPEC from UTI patients were conducted to update the current situation in Thailand.

Materials and methods

Selection and isolation of E. coli

A total of 330 E. coli strains was obtained from the preserved stock culture collection of the Department of Microbiology, Faculty of Public Health, Mahidol University, during 2014–2022. All the strains in this study were retrieved from our stock cultures from two different sources: I) pork (n = 165) from local markets; and II) urine of UTI patients (n = 165) with counts number of E. coli ≥ 10⁵ CFU/ml from hospitals. The studied stock pork E. coli (PEC) and E. coli from UTI patients or UPEC were collected in many provinces. All archived E. coli strains were revived on MacConkey agar (Merck, Darmstadt, Germany), and biochemical confirmation using IMViC (indole, methyl red, Voges-Proskauer, and citrate) was described elsewhere [31].

Genomic DNA extraction

All E. coli strains were extracted their genomic DNA by using the boiled-lysate method as previously described [32]. Briefly, 1 ml of overnight grown E. coli in Luria Bertani (LB) broth (BD Biosciences, CA, USA) was centrifuged at 12,000 xg for 5 min. Then, the supernatant was discarded and 500 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) was added to re-suspend the pellet. The suspension was centrifuged at 12,000x g for 5 min and discarded the supernatant. One hundred microliter TE buffer was added to dissolve the pellet. The suspensions were boiled at 100°C for 10 min then placed on ice for 10 min and centrifuged at 16,000x g for 5 min. The clear gDNA supernatant was obtained and kept as DNA templates at -20°C for further analysis.

Determination of phylogroups

Eight E. coli phylogroups including A, B1, B2, C, D, E, F, and clade I, were determined using quadruplex and singleplex PCR assays as previously described by Clermont et al. (2013). Genes for quadruplex PCR amplification included chuA, yjaA, TspE4.C2, and arpA. Primer sequences and PCR conditions were followed as previously described [28] (S1 Table). Both quadruplex and singleplex PCR mixtures contained 5 μl of 5X GoTaq reaction buffer (1.5 mM MgCl₂), 0.5 μl of dNTP mix (200 μM each), and 0.5 μl of 0.5 μM each forward and reverse primers, 0.125 μl of 0.125 units GoTaq DNA polymerase (Promega, WI, USA), 1 μl of the DNA template and adjusted to a final volume of 25 μl with deionized water.

Thermal cycling was performed in Thermo Hybaid Px2 Thermal Cycler (Thermo Fisher Scientific, MA, USA). Amplification conditions were as follows: initial denaturation at 95°C for 2 min, 30 cycles of denaturation at 95°C for 30 sec, annealing at the appropriate temperature of each primer pair for 30 sec, extension at 72°C for 30 sec, and final extension at 72°C for 5 min. Each specific temperature condition is described in S1 Table. The PCR products were analyzed by electrophoresis on 2% agarose gel in 1X TBE buffer and the amplicons were visualized under Blue Light LED Transilluminator (Dyne Bio, Seongnam, South Korea). The phylogroups were interpreted by the presence and absence of specific amplicons as shown in S2 Table.

Detection of virulence-associated genes

Seven UPEC-associated virulence genes including fimH, papC, sfa/focDE, afaC, hlyCA, cnf, and iucC, and 10 DEC-associated virulence genes used for the detection of STEC (stx1, stx2), EPEC (eae, bfpA), ETEC (lt, st), EIEC (ial), EIEC (ipaH), EAEC (CVD432) and DAEC (daaE) were determined using single and multiplex PCR assays. The primer sequences of studied virulence genes and PCR conditions were as previously described [33–36] (S1 Table).

Each PCR mixture of both single and multiplex PCR contained 5 μl of 5X GoTaq reaction buffer (1.5 mM MgCl₂), 0.5 μl of dNTP mix (200 μM each), and 0.5 μl of 0.5 μM each forward and reverse primers, 0.125 μl of 0.125 units GoTaq DNA polymerase (Promega), 1 μl of the DNA template, and adjusted to a final volume of 25 μl with deionized water. Thermocycling was carried out in Thermo Hybaid Px2 Thermal Cycler (Thermo Fisher Scientific) as follows: 95°C for 2min followed by 30 cycles of 95°C for 30sec, the appropriate annealing temperature for each primer pair for 30s, and 72°C for 1min followed by a final extension step of 72°C for 5min (S1 Table). Amplicons were analyzed as described above.

Cluster analysis of virulence genes and phylogroups

To perform cluster analysis, the presence or absence of UPEC and DEC virulence-associated genes and phylogroups were converted to a zero-one matrix. Then, the analysis was constructed with UPGMA (Unweighted Pair Group Method for Arithmetic Averages) clustering based on Dice’s correlation coefficients with a default bootstrap value of 100 using the DendroUPGMA [37]. A dendrogram of all strains was visualized using Interactive Tree Of Life (iTol) version 6.8.1 [38].

Statistical analysis

The prevalence of phylogroups and virulence-associated gene distribution were expressed as numbers and percentages. The data were analyzed using Statistical Package for Social Sciences software version 26 (SPSS, IL, USA). The prevalence of phylogroups, virulence-associated genes, and their association between PEC and UPEC were compared using the Chi-square or Fisher’s exact. The p-value < 0.05 was considered statistically significant.

Results

Distribution of phylogroups

The phylogroups were determined in 165 PEC strains and 165 UPEC strains. Among 165 PEC strains positive for phylogroup A (50.3%; 83/165) were found in the highest prevalence, followed by B1 (32.1%; 53/165). The low detection rates ranging from 0.6% to 5.5% were found for phylogroups C, D, F, clade I, and B2 (Fig 1). In contrast, the most common phylogroup among 165 UPEC was B2 (41.8%; 69/165), followed by C (25.5%; 42/165) (Fig 1). The clade I group was not found in all tested UPEC strains. When compared to the detection rates between PEC and UPEC strains in phylogroups, phylogroups A and B1 in PEC strains were significantly higher than those in UPEC (p<0.05) while phylogroups B2 and C in UPEC were significantly higher than those in PEC strains (p<0.05), and phylogroups D and F in both strains were not significantly different (p>0.05).

Virulence-associated genes

Amplifying seven UPEC virulence-associated genes, including fimH, papC, sfa/focDE, afaC, hlyCA, cnf, and iucC, in 330 E. coli strains showed that all the genes were found in both PEC and UPEC strains, except for afaC gene that was not found in all PEC strains. The most frequent gene was fimH (92.7%; 306/330), followed by iucC (33.9%; 112/330) (Fig 2). The similar detection rates of fimH were found for PEC (92.1%; 152/165) and UPEC (93.3%; 154/165). The frequencies of PEC and UPEC strains positive for at least one UPEC virulence gene were 92.7% (153/165) and 98.2% (162/165), respectively. In contrast, significant differences between PEC and UPEC strains that harbored UPEC virulence-associated genes were found in this study (p<0.05). Moreover, the detection rates of PEC strains positive for the following virulence-associated genes were significantly lower than those in UPEC strains: iucC (12.7% < 55.2%), papC (4.2% < 21.8%), hlyCA (0.6% < 17%), and cnf (0.6% < 16.4%) (p<0.05) (Fig 2).

Most of the strains (99.1%; 327/330) were negative for all DEC virulence-associated genes, namely stx1, stx2, bfpA, eae, lt, st, ial, ipaH, CVD432, and daaE. Only one PEC strain harbored eae for aEPEC. Additionally, two UPEC strains were positive for bfpA (aEPEC), and CVD432 (EAEC) genes. These two UPEC strains were considered hybrid strains. A UPEC strain carried fimH-bfpA gene and belonged to phylogroup B2 (chuA, TspE4C2) while another UPEC harbored afaC was positive for CVD432 gene and belonged to phylogroup D (arpA, chuA, TspE4C2).

Association between the phylogroups and UPEC virulence-associated genes

The association between the phylogroups and the seven UPEC virulence-associated genes was investigated in this study (Table 1). Among PEC strains, phylogroups were not significantly associated with the presence of most UPEC virulence-associated genes (p>0.05). However, fimH was found the association with phylogroups C and clade I (p = 0.0035, p = 0.0015, respectively) while papC was significantly associated with phylogroup F (p = 0.0214). Moreover, PEC strains belonging to phylogroups B1 and F were significantly associated with iucC (p = 0.0044, p = 0.0243, respectively) (Table 1). Among UPEC strains, the results showed that phylogroup B1 was significantly associated with sfa/focDE (p = 0.0304). Phylogroups B2 and C were significantly associated with iucC, papC, and afaC (p<0.05) (Table 1).

Table 1. Frequencies of virulence factors associated with uropathogenic Escherichia coli (UPEC) in the phylogroups among Escherichia coli isolated from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

Virulence gene	No. (%) of strain positive for phylogroup
	A		B1		B2		C		D		F		Clade I
	PEC (n = 83)	UPEC (n = 21)	PEC (n = 53)	UPEC (n = 9)	PEC (n = 1)	UPEC (n = 69)	PEC (n = 9)	UPEC (n = 42)	PEC (n = 9)	UPEC (n = 16)	PEC (n = 6)	UPEC (n = 8)	PEC (n = 4)	UPEC (n = 0)
fimH	78 (94)	17 (81)	52 (98.1)	9 (100)	1 (100)	69 (100) ^a	5 (55.6) ^a	42 (100)	9 (100)	13 (81.3)	6 (100)	4 (50)	1 (25) ^a	0
iucC	0	9 (42.9)	12 (22.6) ^a	3 (33.3)	1 (100)	58 (84.1) ^a	3 (33.3)	7 (16.7) ^a	2 (22.2)	7 (43.8)	3 (50) ^a	7 (87.5)	0	0
papC	0	2 (9.5)	3 (5.7)	1 (11.1)	1 (100)	29 (42) ^a	1 (11.1)	1 (2.4) ^a	0	2 (12.5)	2 (33.3) ^a	1 (12.5)	0	0
afaC	0	3 (14.3)	0	0	0	5 (7.2) ^a	0	24 (57.1) ^a	0	4 (25)	0	1 (12.5)	0	0
hlyCA	0	0	1 (1.9)	0	0	28 (40.6) ^a	0	0	0	0	0	0	0	0
cnf	0	0	1 (1.9)	0	0	27 (39.1) ^a	0	0	0	0	0	0	0	0
sfa/focDE	0	1 (4.8)	5 (9.4)	3 (33.3) ^a	1 (100)	9 (13)	1 (11.1)	1 (2.4)	0	0	1 (16.7)	0	0	0

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^asignificantly associated with UPEC virulence-associated genes (p<0.05).

UPEC virulence-associated gene patterns distribution among phylogroups of PEC and UPEC strains

Based on the distribution of various UPEC virulence-associated genes, the studied strains were divided into 27 virulence gene patterns (Table 2). Of 27 patterns, pattern No. 25 (sfa/focDE⁺ cnf⁺ hlyCA⁺ fimH⁺ iucC⁺), was only found among PEC strains (0.6%; 1/165) belonging to phylogroup B1, while 19 patterns were found only in UPEC strains (45.5%; 75/165). Six patterns were found both in PEC (92.1%; 152/165) and UPEC strains (52.7%; 87/165). The pattern No. 4 (fimH⁺) was the most commonly found in PEC strains (80%; 132/165) while the pattern No. 12 (fimH⁺ iucC⁺) was the major pattern found in UPEC strains (25.5%; 42/165). Moreover, the pattern No. 4 (fimH⁺) was found in all phylogroups (80%; 132/165) except for B2 among PEC strains while phylogroup A was the most frequent phylogroup (47.3%; 78/165) among PEC strains. The pattern No. 12 (fimH⁺ iucC⁺), the most commonly found among UPEC strains, was also found in all phylogroups except for clade I and B2 (15.2%; 25/165) (Table 2).

Table 2. Uropathogenic Escherichia coli (UPEC) virulence gene patterns among phylogroups of 330 Escherichia coli from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

Pattern No.	Virulence gene							Phylogroup (No. of strain (PEC/UPEC))							No. of strain (%)
Pattern No.	afaC	sfa/focDE	papC	cnf	hlyCA	fimH	iucC	A	B1	B2	C	D	F	Clade I	PEC n = 165	UPEC n = 165	Total N = 330
1								5/1	1/-		3/-	-/2		3/-	12 (7.3)	3 (1.8)	15 (4.5)
2								1				1			0	2 (1.2)	2 (0.6)
3								1							0	1 (0.6)	1 (0.3)
4								78/8	40/4	-/3	3/11	7/5	3/1	1/-	132 (80)	32 (19.4)	164 (49.7)
5													3		0	3 (1.8)	3 (0.9)
6											22	1			0	23 (13.9)	23 (7)
7													1		0	1 (0.6)	1 (0.3)
8									1		1				0	2 (1.2)	2 (0.6)
9								1	1		1				0	3 (1.8)	3 (0.9)
10								-/1			1/-				1 (0.6)	1 (0.6)	2 (0.6)
11										1					0	1 (0.6)	1 (0.3)
12								-/6	5/1	-/25	1/5	2/3	1/2		9 (5.5)	42 (25.5)	51 (15.5)
13								2		4	2	2			0	10 (6.1)	10 (3)
14										1					0	1 (0.6)	1 (0.3)
15									3/2		1/-				4 (2.4)	2 (1.2)	6 (1.8)
16									2/-	-/5		-/2	1/1		3 (1.8)	8 (4.8)	11 (3.3)
17										1					0	1 (0.6)	1 (0.3)
18									1/-	1/2			1/-		3 (1.8)	2 (1.2)	5 (1.5)
19										1					0	1 (0.6)	1 (0.3)
20										1					0	1 (0.6)	1 (0.3)
21										1					0	1 (0.6)	1 (0.3)
22										4					0	4 (2.4)	4 (1.2)
23										1					0	1 (0.6)	1 (0.3)
24										-/4					0	4 (2.4)	4 (1.2)
25									1						1 (0.6)	0	1 (0.3)
26										14					0	14 (8.5)	14 (4.2)
27										1					0	1 (0.6)	1 (0.3)

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Cluster analysis of virulence genes and phylogroups

The UPGMA cluster analysis was used to categorize individual strains into larger clusters and generate a dendrogram illustrating the relationships between PEC and UPEC strains. Based on the presence or absence of DEC and UPEC virulence genes in each strain and phylogroups, eight main clusters and several sub-clusters were found in this study (Fig 3). The most predominant groups were cluster III (31.2%; 103/330) and cluster VIII (21.2%; 70/330). Among the cluster III, 80.6% (83/103) were PEC while 19.4% (20/103) were UPEC. All strains in this cluster belonged to phylogroup A and had fewer UPEC virulence-associated genes. A single UPEC virulence gene, fimH, was commonly found in this cluster. Moreover, an aEPEC strain from PEC (eae) was found in this cluster. In contrast, most of the strains in the cluster VIII were from UPEC (98.6%; 69/70) which belonged to phylogroup B2 and carried multiple UPEC virulence-associated genes. However, only one PEC (1.4%; 1/70) was found. Quintuple and sextuple UPEC virulence-associated genes were frequently found in cluster VIII. A hybrid strain (fimH-bfpA) from UPEC was also found in cluster VIII (Fig 3).

Fig 3 — The dendrogram was constructed based on phylogroups and presence/absence of uropathogenic E. *coli* (UPEC) and diarrheagenic E. *coli* (DEC) virulence-associated genes. Asterisks indicate PEC and UPEC were found in the same lineage. Black or white circles represent strains from pork or urine, respectively. Groups of the same profile strains are collapsed and shown proportionately to the number of strains in that lineage. White or black boxes indicate the absence or presence of virulence genes, respectively. No cut-off value was defined; clustering was based on total differences found.

Discussion

To our knowledge, this is the first study on the determination of E. coli phylogroups and virulence genes between PEC and UPEC in Thailand. Phylogroup analysis data in this study showed that phylogroups A (50.3%) and B1 (32.1%) were commonly found among PEC while phylogroups B2 (41.8%) and C (25.5%) were the most prevalent among UPEC (Fig 1). It is known that E. coli from phylogroups A and B1 are often commensal [28], while human UPEC strains causing UTIs belong to phylogroups B2 and D [39,40]. Therefore, most of the PEC and UPEC in this study were commensal and ExPEC, respectively. However, the frequency of phylogroup C (25.5%) among UPEC strains in the present study was higher than phylogroup D (9.7%). This result differed from previous Thai studies of human UPEC which belonged to phylogroups B2 and D [41,42]. From the phylogenetic tree analysis of several E. coli, phylogroup C was close to phylogroup B1 [28]. However, a low frequency of phylogroup B1 (5.5%) was found in our study indicating that phylogroup C may dominate among UPEC in Thailand instead of phylogroup D. Moreover, phylogroups F and clade I were found in PEC. Phylogroup F is related to phylogroup B2 [28] while clade I is frequently found in human and animal feces [43]. The presence of these new phylogroups might resulted from using the new quadruplex PCR assay [28] which could be a more accurate assignment than the former assay [44].

All seven UPEC virulence-associated genes could be found in UPEC strains in our study (Fig 2). More than half (55.2%) of UPEC strains in the present study carried iucC essential for the survival of UPEC inside urinary tracts under an iron-limiting environment [15]. Several studies from different countries including Thailand were also reported high prevalences of this gene [29,41,45]. Much higher frequencies of hlyCA (17.0%) and cnf (16.4%) were found among UPEC than PEC in this study. However, those from UPEC observed in this study were higher than in the previous study from Thailand (7% and 1%, respectively) [40]. These genes are involved in tissue damage and have been linked to patients with pyelonephritis rather than cystitis [46,47]. Although low frequencies of these genes were found among PEC, this strain may have the ability to cause UTIs. Regarding fimbrial adhesins among UPEC, the prevalences of afaC and papC in our study were 22.4% and 21.8%, respectively. These frequencies were lower than the previous report (79%) in Thai UPEC [48]. The papC and afaC are more associated with upper urinary tract infections due to their ability to promote UPEC attach more specifically to kidneys and renal tubules [49]. Therefore, the discrepancy between our results and previous reports from Thailand may be isolated from the patients with different types of UTIs.

All the UPEC virulence-associated genes were found among PEC, except for afaC gene (Fig 2). It is worth noting that these PEC strains shared several UPEC virulence-associated genes with human strains [34]. In contrast, high frequencies of UPEC virulence genes namely papC (58%), cnf (23.2%), and afa (13.4%) were found in the urine of diseased sows in China [23]. Slaughtering and dressing are critical steps for cross-contamination of these strains from urine to pork that may serve as potential sources of the UPEC strains. Several PEC strains shared similar UPEC virulence-associated gene patterns to UPEC strains in this study (Table 2). Moreover, multiple UPEC virulence-associated genes were found among these PEC strains (highlighted in grey in Table 2). The UPEC strains obtained from patients with pyelonephritis had a greater number of virulence genes than those isolated from patients with cystitis [46]. Various UPEC virulence-associated genes such as fimH, sfa, foc, afa, hly, and cnf are located on pathogenicity islands and could be transferred to other strains through horizontal transfer [50]. Moreover, healthy human intestines colonized by several UPEC strains have been reported [51]. Therefore, gut commensal strains might have gained UPEC virulence-associated genes from PEC with those, making them a possible cause of UTIs. Therefore, consuming undercooked and contaminated pork may increase the risk of colonization of these PEC carrying UPEC virulence genes followed by accidental infections in urinary systems [52].

Only three strains in the present study carried the DEC virulence-associated genes and two of these strains were considered as hybrid strains due to the presence of both UPEC and DEC virulence-associated genes. The UPEC in this study harbored fimH-bfpA (UPEC/aEPEC) while the other UPEC carried afaC-CVD432 (UPEC/EAEC). The UPEC/EAEC was more frequently found among UTI patients with diarrhea and associated with a higher risk of developing pyelonephritis than those without diarrhea [53]. These strains may have acquired UPEC properties and become a potential cause of either UTIs, diarrhea in humans, or both. Females have a higher risk of developing these infections due to the short distance between the rectum and the urethra [54]. However, this finding needs to be further investigated due to limited information on the hybrid strains in Thailand.

To investigate the possibility of an association between phylogroups and UPEC virulence-associated genes, E. coli strains from phylogroup B2 had the highest frequency of virulence-associated genes (Table 1). This result is in good agreement with previous studies in Thailand [48] and Mexico [45]. However, several UPEC virulence-associated genes were found in the PEC belonging to phylogroups A and B1 in this study suggesting that phylogroups A and B1 regarded as commensal strains might be the reservoirs of the UPEC virulence-associated genes. Moreover, these strains could be opportunistic pathogens for the development of UTIs in humans.

The UPGMA clustering results showed that the PEC or UPEC strains in this study were grouped into eight main clusters (Fig 3). Some of the PEC and UPEC strains were found in the same clusters and some strains showed similar genotypes of UPEC virulence-associated genes and phylogroups. For example, 77 PEC and 8 UPEC belonging to phylogroup A and carrying fimH were found in the same lineage (with asterisks indicated in Fig 3). This finding may imply that several UPEC strains in this study were closely related to PEC as previously reported in some human UPEC and animal ExPEC [17,18,52]. The PEC harbored UPEC virulence-associated genes may pose a zoonotic risk and may have a relationship to UPEC in humans. These PEC strains may have potentially caused UTIs in humans after colonizing the outside of gastrointestinal tracts. To confirm this hypothesis, a more comprehensive investigation into the molecular epidemiology of these strains is necessary. This would involve analyzing genetic similarities and evolutionary relationships using both basic and advanced molecular techniques.

Conclusions

To our knowledge, this is a novel study that provides information on phylogroups, UPEC and DEC virulence-associated genes, and the association between E. coli from UTI patients and pork in Thailand. Several UPEC virulence-associated genes were found in PEC strains and shared similar sets of virulence genes to human UPEC strains. Our study highlights that PEC carrying UPEC virulence-associated genes may serve as reservoirs of virulence-associated genes and further transmit to humans causing UTIs. Additional studies are needed to further determine the relationship of genetic clones by the molecular epidemiology among these strains. However, this finding is raising more attention to great concern for food safety, and public health perspectives.

Supporting information

S1 Table. PCR primers used in this study.

(PDF)

pone.0307544.s001.pdf^{(183.1KB, pdf)}

S2 Table. Interpretation of amplified PCR products for phylogroups of Escherichia coli.

(PDF)

pone.0307544.s002.pdf^{(63.8KB, pdf)}

Acknowledgments

We would like to acknowledge the great contributions of the late Prof. Lee Riley, Chair of the Infectious Disease and Vaccinology Division, UC Berkeley School of Public Health, for his valuable and constructive suggestions during the planning and development of this research.

Data Availability

All relevant data are within the manuscript.

Funding Statement

This work was supported, in part, by a grant from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, for the Joint Research Program of the Hokkaido University Research Center for Zoonosis Control toY.S., and, in part, by Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP233fa627005 and JP20wm0125008 to Y.S.

References

1.Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2:123–140. doi: 10.1038/nrmicro818 [DOI] [PubMed] [Google Scholar]
2.Khairy RM, Mohamed ES, Abdel Ghany HM, Abdelrahim SS. Phylogenic classification and virulence genes profiles of uropathogenic Escherichia coli and diarrhegenic Escherichia coli strains isolated from community acquired infections. PLoS One. 2019;14:e0222441. doi: 10.1371/journal.pone.0222441 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013; 26:822–80. doi: 10.1128/CMR.00022-13 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Gomes TAT, Elias WP, Scaletsky ICA, Guth BEC, Rodrigues JF, Piazza RMF, et al. Diarrheagenic Escherichia coli. Braz J Microbiol. 2016;47:3–30. doi: 10.1016/j.bjm.2016.10.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Smith JL, Fratamico PM, Gunther NW. Extraintestinal pathogenic Escherichia coli. Foodborne Pathog Dis. 2007;4:134–63. doi: 10.1093/infdis/jiv429 [DOI] [PubMed] [Google Scholar]
6.Hernandes RT, Elias WP, Vieira MAM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2016;297:137–149. doi: 10.1111/j.1574-6968.2009.01664.x [DOI] [PubMed] [Google Scholar]
7.Fleckenstein JM, Kuhlmann FM. Enterotoxigenic Escherichia coli Infections. Curr Infect Dis Rep. 2019;21. doi: 10.1007/s11908-019-0665-x [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Welinder-Olsson C., Kaijser B. Enterohemorrhagic Escherichia coli (EHEC), Scand J Infect Dis. 2005;37:405–416. doi: 10.1080/00365540510038523 [DOI] [PubMed] [Google Scholar]
9.Askari BM, Taban H, Nemati A, Fernando DSL. Molecular serotyping of Shiga toxin-producing Escherichia coli (STEC) of animal origin in Iran reveals the presence of important non-O157 seropathotypes. Vet Res Forum. 2023;14:267–274. doi: 10.30466/vrf.2022.550618.3416 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.van den Beld MJC, Reubsaet FAG. Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur J Clin Microbiol Infect Dis. 2012;31:899–904. doi: 10.1007/s10096-011-1395-7 [DOI] [PubMed] [Google Scholar]
11.Weintraub A. Enteroaggregative Escherichia coli: epidemiology, virulence and detection. J Med Microbiol. 2007;56:4–8. doi: 10.1099/jmm.0.46930–0 [DOI] [PubMed] [Google Scholar]
12.Le Bouguénec C, Servin AL. Diffusely adherent Escherichia coli strains expressing Afa/Dr adhesins (Afa/Dr DAEC): hitherto unrecognized pathogens. FEMS Microbiol Lett. 2006;256:185–194. doi: 10.1111/j.1574-6968.2006.00144.x [DOI] [PubMed] [Google Scholar]
13.Camprubí-Font C, Martinez-Medina M. Why the discovery of adherent-invasive Escherichia coli molecular markers is so challenging?. World J Biol Chem. 2020;11:1–13. doi: 10.4331/wjbc.v11.i1.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-Ploskonska G, et al. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathog. 2019;11:10. doi: 10.1186/s13099-019-0290-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. 2015;13:269–84. doi: 10.1038/nrmicro3432 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Terlizzi ME, Gribaudo G, Maffei ME. Uropathogenic Escherichia coli (UPEC) infections: virulence factors, bladder responses, antibiotic, and non-antibiotic antimicrobial strategies. Front Microbiol. 2017; 8:1566. doi: 10.3389/fmicb.2017.01566 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Manges AR. Escherichia coli and urinary tract infections: the role of poultry-meat. Clin Microbiol Infect. 2016;22:122–9. doi: 10.1016/j.cmi.2015.11.010 [DOI] [PubMed] [Google Scholar]
18.Tanabe RHS, Dias RCB, Orsi H, de Lira DRP, Vieira MA, dos Santos LF, et al. Characterization of uropathogenic Escherichia coli reveals hybrid isolates of uropathogenic and diarrheagenic (UPEC/DEC) Escherichia coli. Microorganisms. 2022;10:645. doi: 10.3390/microorganisms10030645 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Mora A, López C, Dabhi G, Blanco M, Blanco JE, Alonso MP, et al. Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution. BMC Microbiol. 2009;9:132. doi: 10.1186/1471-2180-9-132 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Ramchandani M, Manges AR, DebRoy C, Smith SP, Johnson JR, Riley LW. Possible animal origin of human-associated, multidrug-resistant, uropathogenic Escherichia coli. Clin Infect Dis. 2005;40:251–7. doi: 10.1086/426819 [DOI] [PubMed] [Google Scholar]
21.Aslam M, Toufeer M, Narvaez BC, Lai V, Rempel H, Manges A, et al. Characterization of extraintestinal pathogenic Escherichia coli isolated from retail poultry meats from Alberta, Canada. Int J Food Microbiol. 2014;177:49–56. doi: 10.1016/j.ijfoodmicro.2014.02.006 [DOI] [PubMed] [Google Scholar]
22.Khan SB, Zou G, Cheng YT, Xiao R, Li L, Wu B, et al. Phylogenetic grouping and distribution of virulence genes in Escherichia coli along the production and supply chain of pork around Hubei, China. J Microbiol Immunol Infect. 2017;50:382–5. doi: 10.1016/j.jmii.2016.03.006 [DOI] [PubMed] [Google Scholar]
23.Spindola MG, Cunha MPV, Moreno LZ, Amigo CR, Silva APS, Parra BM, et al. Genetic diversity, virulence genotype and antimicrobial resistance of uropathogenic Escherichia coli (UPEC) isolated from sows. Vet Q. 2018;38:79–87. doi: 10.1080/01652176.2018.1519321 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Zhu Y, Dong W, Ma J, Yuan L, Hejair HMA, Pan Z, et al. Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China. BMC Vet Res. 2017;13:94. doi: 10.1186/s12917-017-0975-x [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Bok E, Kożańska A, Mazurek PJ, Wojciech M, Baldy CK. Extended phylogeny and extraintestinal virulence potential of commensal Escherichia coli from piglets and sows. Int J Environ Res Public Health. 2020;17:366. doi: 10.3390/ijerph17010366 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Ma J, Cheng Z, Bai Q, Zhao K, Pan Z, Yao H. Screening virulence factors of porcine extraintestinal pathogenic Escherichia coli (an emerging pathotype) required for optimal growth in swine blood. Transbound Emerg Dis. 2020;68:2005–2016. doi: 10.1111/tbed.13848 [DOI] [PubMed] [Google Scholar]
27.Jakobsen L, Garneau P, Bruant G, Harel J, Olsen SS, Porsbo LJ, et al. Is Escherichia coli urinary tract infection a zoonosis? Proof of direct link with production animals and meat. Eur J Clin Microbiol Infect Dis. 2012; 31:1121–1129. doi: 10.1007/s10096-011-1417-5 [DOI] [PubMed] [Google Scholar]
28.Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5:58–65. doi: 10.1111/1758-2229.12019 [DOI] [PubMed] [Google Scholar]
29.Dadi BR, Abebe T, Zhang L, Mihret A, Abebe W, Amogne W. Distribution of virulence genes and phylogenetics of uropathogenic Escherichia coli among urinary tract infection patients in Addis Ababa, Ethiopia. BMC Infect Dis. 2020;20:108. doi: 10.1186/s12879-020-4844-z [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Yazdanpour Z, Tadjrobehkar O, Shahkhah M. Significant association between genes encoding virulence factors with antibiotic resistance and phylogenetic groups in community acquired uropathogenic Escherichia coli isolates. BMC Microbiol. 2020;20:241. doi: 10.1186/s12866-020-01933-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Feng P, Weagant SD. BAM Chapter 4A: Diarrheagenic Escherichia coli. 2020. [cited 3 July 2023]. In: Laboratory Methods (Food) [Internet]. The United States Food and Drug Administration (FDA). Available from: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4a-diarrheagenic-escherichia-coli. [Google Scholar]
32.Chonsin K, Siriphap A, Changkaew K, Noisumdaeng P, Suthienkul O. Virulence genes of Vibrio parahaemolyticus isolated from acute hepatopancreatic necrosis disease associated with shrimp and grow-out pond water. J Med Health Sci. 2021;28:17–27. [Google Scholar]
33.Le Bouguenec C, Archambaud M, Labigne A. Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction. J Clin Microbiol. 1992;30:1189–93. doi: 10.1128/jcm.30.5.1189–1193.1992 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J. Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. J Glob Infect Dis. 2013;17:e450–e3. doi: 10.1016/j.ijid.2013.01.025 [DOI] [PubMed] [Google Scholar]
35.Usein CR, Damian M, Tatu-Chitoiu D, Capusa C, Fagaras R, Tudorache D, et al. Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases. J Cell Mol Med. 2001;5:303–10. doi: 10.1111/j.1582-4934.2001.tb00164.x [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Fialho OB, de Souza EM, de Borba Dallagassa C, de Oliveira Pedrosa F, Klassen G, Irino K, et al. Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol. J Clin Lab Anal. 2013;27:155–61. doi: 10.1002/jcla.21578 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Garcia-Vallvé S, Palau J, Romeu A. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol Biol Evol. 1999;16:1125–34. doi: 10.1093/oxfordjournals.molbev.a026203 [DOI] [PubMed] [Google Scholar]
38.Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:293–6. doi: 10.1093/nar/gkab301 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Halaji M, Fayyazi A, Rajabnia M, Zare D, Pournajaf A, Ranjbar R. Phylogenetic group distribution of uropathogenic Escherichia coli and related antimicrobial resistance pattern: a meta-analysis and systematic review. Front Cell Infect Microbiol. 2022;12:790184. doi: 10.3389/fcimb.2022.790184 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Mostaghimi T, Pournajaf A, Bijani A, Mohammadi M, Rajabnia M, et al. Phylogenetic analysis, biofilm formation, antimicrobial resistance and relationship between these characteristics in Uropathogenic Escherichia coli. Mol Biol Rep. 2024; 51:327. doi: 10.1007/s11033-023-09031-x [DOI] [PubMed] [Google Scholar]
41.Parikumsil N, Prapasawat W, Siriphap A, Chonsin K, Theethakaew C, Sukolrattanamaetee N, et al. Virulence factors and molecular epidemiology of uropathogenic Escherichia coli isolated from paired urine and rectal swab samples of patients with urinary tract infections in Thailand. Southeast Asian J Trop Med Public Health. 2017;48:1029–41. [Google Scholar]
42.Themphachana M, Kongpheng S, Rattanachuay P, Khianngam S, Singkhamanan K, Sukhumungoon P. Molecular characterization of virulence and antimicrobial susceptibility profiles of uropathogenic Escherichia coli from patients in a tertiary hospital, southern Thailand. Southeast Asian J Trop Med Public Health. 2015;46:1021. [PubMed] [Google Scholar]
43.Walk ST. The "Cryptic" Escherichia. EcoSal Plus. 2015;6(2):10.1128/ecosalplus.ESP-0002-2015. doi: 10.1128/ecosalplus.esp-0002-2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol. 2000;66:4555–8. doi: 10.1128/AEM.66.10.4555–4558.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Belmont-Monroy L, Ribas-Aparicio RM, González-Villalobos E, Pérez-Ramos JA, Aparicio-Ozores G, Eslava-Campos CA, et al. Molecular typification of Escherichia coli from community-acquired urinary tract infections in Mexico. Int J Antimicrob Agents. 2022;60:106667. doi: 10.1016/j.ijantimicag.2022.106667 [DOI] [PubMed] [Google Scholar]
46.Firoozeh F, Saffari M, Neamati F, Zibaei M. Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis. J Glob Infect Dis. 2014;29:219–22. doi: 10.1016/j.ijid.2014.03.1393 [DOI] [PubMed] [Google Scholar]
47.Tabasi M, Karam MR, Habibi M, Mostafavi E, Bouzari S. Genotypic characterization of virulence factors in Escherichia coli isolated from patients with acute cystitis, pyelonephritis and asymptomatic bacteriuria. J Clin Diagn Res. 2016;10:dc01–dc7. doi: 10.7860/jcdr/2016/21379.9009 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Tewawong N, Kowaboot S, Pimainog Y, Watanagul N, Thongmee T, Poovorawan Y. Distribution of phylogenetic groups, adhesin genes, biofilm formation, and antimicrobial resistance of uropathogenic Escherichia coli isolated from hospitalized patients in Thailand. PeerJ. 2020;8:e10453. doi: 10.7717/peerj.10453 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney Int. 2007;72:19–25. doi: 10.1038/sj.ki.5002230 [DOI] [PubMed] [Google Scholar]
50.Desvaux M, Dalmasso G, Beyrouthy R, Barnich N, Delmas J, Bonnet R. Pathogenicity factors of genomic islands in intestinal and extraintestinal Escherichia coli. Front Microbiol. 2020;11:2065. doi: 10.3389/fmicb.2020.02065 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Matsui Y, Hu Y, Rubin J, de Assis RS, Suh J, Riley LW. Multilocus sequence typing of Escherichia coli isolates from urinary tract infection patients and from fecal samples of healthy subjects in a college community. MicrobiologyOpen. 2020;9:e1032. doi: 10.1002/mbo3.1032 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Manges AR, Johnson JR. Food-borne origins of Escherichia coli causing extraintestinal infections. Clin Infect Dis. 2012;55:712–9. doi: 10.1093/cid/cis502 [DOI] [PubMed] [Google Scholar]
53.Martínez-Santos VI, Ruíz-Rosas M, Ramirez- Peralta A, Zaragoza García O, Resendiz-Reyes LA, Romero-Pineda OJ, et al. Enteroaggregative Escherichia coli is associated with antibiotic resistance and urinary tract infection symptomatology. PeerJ. 2021;9:e11726. doi: 10.7717/peerj.11726 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Magruder M, Sholi AN, Gong C, Zhang L, Edusei E, Huang J, et al. Gut uropathogen abundance is a risk factor for development of bacteriuria and urinary tract infection. Nat. Commun. 2019;10:5521. doi: 10.1038/s41467-019-13467-w [DOI] [PMC free article] [PubMed] [Google Scholar]

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PONE-D-23-44006Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in ThailandPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: -The introduction should provide more context and background information on the significance of studying the transmission of virulence genes between pork and urinary pathogenic E. coli strains.

-In materials and methods, Were there any specific criteria used for selecting pork samples and UTI patient samples for inclusion in the study?

-The utilization of UPGMA clustering for analyzing the presence of virulence-associated genes and phylogroups is appropriate. However, the manuscript lacks clarity regarding the rationale for selecting this specific clustering method. Were alternative methods considered, and if so, why were they deemed less suitable? Additionally, the authors should provide more details regarding the interpretation of the dendrogram generated from the clustering analysis to aid readers in understanding the relationships between strains.

-We suggest that you refer to new research article doi: 10.3389/fcimb.2022.790184. and doi: 10.1007/s11033-023-09031-x in the discussion section.

Reviewer #2: Dear Authors,

I would like to say this study is very neat and has informative data that will be of interest for international readers of the journal.

Before publication, I recommend following my comment below:

Line 60: This paragraph provide some information about different E. coli pathotypes, I would suggest to use more references and not only one. I suggest to use this study: 10.30466/vrf.2022.550618.3416 as the second references for this paragraph.

Reviewer #3: Comments

*What were the aim and conclusion of your study, exactly? It is not clear to me the aim of the study on sharing virulence genes between PEC and UPEC. Did you aim to demonstrate that pork may serve as one possible source of UPEC? If so, how did you demonstrate that in the present work?

[Cluster analysis … demonstrated that pork contaminated with E. coli harboring UPEC virulence genes may serve as one possible source of UPEC in humans],[This finding may imply…. UPEC strains in this study were closely related to PEC]. [Our study highlights that PEC carrying UPEC virulence-associated genes may………further transmit to humans causing UTIs].

* Genotyping of isolates is needed to obtain accurate data and true prevalence. It is possible that your strains originated from a clone.

*[A total of 330 E. coli strains was obtained from the preserved stock culture collection]:

did you have exactly the same number of PEC and UPEC in your collection or you include 165 isolates (each group) from this collection in your study?

*What was your definition for UPEC?

*Grammatical errors and writing issues were seen in the manuscript.

*For abbreviations, provide their full term for the first mention.

*Information on the isolates is not provided. How were urines collected? What was the demographic data of patients?

* FimH is not only for UPEC. It is found in other E. coli types and in E. coli belonging to normal flora.

*Which tests were used to identify E. coli? Mention them.

*Did you test the antibiotic susceptibility of isolates?

*Some references such as reference 1 were referred to in the manuscript too much. Use other references instead of referring to 1.

*Figure titles need to be revised.

*Remove Table 1 and embed its data within the text

*Table 2: number of isolates is not enough to make a p-value, in some rows.

Best

Reviewer #4: Major concerns:

Please include the criteria used to characterize strains as UPEC, PEC and DEC in the Methods. A description of the approach is given in the Introduction (Lines 55 and 110), but the specific genomic markers used to make the determination should be listed. This impacts the finding on lines 184-185 that “165 isolates PEC and 165 ones UPEC” The collection criteria stated on lines 117-124 only select for an isolation source and make no distinction between UPEC, E.coli commensal or environmental contaminant. If the authors were able to subset the UPEC strains from this dataset, please provide the selection criteria. Otherwise, it would be more accurate to claim that the 330 isolates clustered with at least eight different groups of isolates of UPEC and PEC source. Further, were any tests performed to determine if isolates E1-165 were the causative agents of infection? If not, then additional justification is needed to categorize them as UPEC.

Reviewer #5: Firstly, I would like to thank you for the opportunity and trust in my work to review this scientific article. Below, I provide some comments:

Line 124:

I believe it would be interesting to describe the biochemical tests for E. coli characterization. Was there also molecular confirmation of the species?

The dendrogram image is of low quality, and it was not possible to analyze it.

Although there is a phylogenetic analysis in the paper (represented by the dendrogram that could not be analyzed), I believe that epidemiological data from the samples obtained would be necessary for the inferences made.

The work is interesting, but it should focus on the actual evidence obtained. The samples were obtained from a previous isolate bank. For the conclusions reached, it would be necessary to analyze epidemiological data that were not presented.

I suggest that epidemiological data be presented or that the conclusions and discussions obtained be reviewed.

I also suggest that a higher quality image of the dendrogram be provided.

Reviewer #6: Comment to the authors

Abstract

Have only the specific genes of UPEC been examined? The text raises this suspicion. If not, please change the text of the first lines in the abstract.

Line 36 – 40: The percentage of the genes in the UPEC is higher than in PEC, so it is better to present its name and its percentage first.

The conclusion of the abstract section is not based on the results and should be changed

Introduction

Line 56-59: First introduce EXPEC more. Mention and explain the virulence genes or pathogenic factors of this bacterium that cause urinary infections.

Line 67: Please write in full version for “STEC” for the first time.

Line 76: State the pathotypes of EXPEC here with more details along with its pathogenic genes.

Line 107-108: Please remove this sentence as a novelty, the authors have mentioned it several times in the text.

Material and method

More data should be provided on the sources of isolation of these bacteria, especially on human samples.

Does the study have a code of ethics, mention it.

The bootstrap value should be stated at the level of phylogenetic analysis.

How many times the same branch is observed when repeating the generation of a phylogenetic tree on a resampled set of data? The percentage of times each interior branch is given a value of 1 should be noted.

Reviewer #7: The paper entitled “Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand” has good and important subject, good writing, and interesting results. However some comments are as follow:

The phylogenetic method designed by Clermont et al has different protocol. Why did the authors change the PCR protocol?

In material and method, the authors described that the cluster analysis was performed according to phtlogenetic group and absence or presence of virulence genes, while in the fig 3. the strains with similarity profiles put in different clusters. I think the clustering is drawn according to phylogenetic groups only.

Page 23 Line 293. What is the reason for this difference?

Page 24 Lines 318 -319 are repeated.

Page 24 Lines 347-348 “Females have a higher risk of developing these infections due to the short distance between the rectum and the urethra” What is the connection between this sentence and the previous sentences?

Reviewer #8: The present manuscript evaluated the diversity of phylogenetic groups and virulence genes of E. coli isolates from pork (PEC) and human patients with urinary tract infection (UPEC) in Thailand. The objectives of the work were to compare the virulence genotype of UPEC and PEC. The authors reported that the PEC strains were grouped predominantly in groups A and B1 (commensal strains), while the UPEC strains, which carry a greater number of virulence genes, were classified in phylogroups B2 and C. In conclusion, they pointed to pork as a reservoir of genes associated with UTI, reporting a major public health concern.

The paper has some important limitations that prevent it from being accepted in this format. The main ones are:

1- There is no coherence between the results and the conclusions. How can we say that this is a major public health risk if the profile of the strains in the two groups was different? How can the risk be measured? It would be more appropriate to use more modern analysis tools such as sequencing types or dendrograms generated by comparing WGS data. The frequency of genes between the two groups is important information, but on its own it doesn't serve to support the hypothesis or measure the risk.

2- The Clermont classification used is outdated. Currently, 9 phylogroups are considered. See Clermont et al. (2019) - Group G.

3- There is no hybrid of DAEC and UPEC, as the fimH gene is not a UPEC marker. On the contrary, it is a highly prevalent fimbria in enterobacteria that can be detected in non-pathogenic strains of the microbiota. This information needs to be reviewed and is considered a lack of definition of the concept of hybrid strains.

4- The statistics between the frequency of virulence genes and phylogroups is completely unnecessary information, given that the concept of phylogroups is based on the evolutionary process of acquiring virulence genes in the B2 group. So this result is to be expected and has no significance whatsoever in light of the proposal.

5- The results of group C are of local epidemiological data, bute the importance should be understood in more in-depth studies in Thailand.

So, I think this paper needs to be modified prior the publication.

**********

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Reviewer #1: Yes: Mehrdad Halaji

Reviewer #2: Yes: Mahdi Askari Badouei

Reviewer #3: No

Reviewer #4: Yes: Hami Kaboosi

Reviewer #5: No

Reviewer #6: No

Reviewer #7: No

Reviewer #8: Yes: Terezinha Knöbl

**********

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PLoS One. 2024 Jul 25;19(7):e0307544. doi: 10.1371/journal.pone.0307544.r002

Author response to Decision Letter 0

14 Apr 2024

Responses to Reviewer #1

Thank you for giving us your valuable comments. I am sure our manuscript will be much improved by these.

Comment 1: The introduction should provide more context and background information on the significance of studying the transmission of virulence genes between pork and urinary pathogenic E. coli strains.

Response 1: More background information was added to the introduction part. Please go through it.

Comment 2: In materials and methods, Were there any specific criteria used for selecting pork samples and UTI patient samples for inclusion in the study?

Response 2: The E. coli strains were randomly selected from four regions in Thailand. Both pork and urine strains were selected in the same amount in each region.

Comment 3: The utilization of UPGMA clustering for analyzing the presence of virulence-associated genes and phylogroups is appropriate. However, the manuscript lacks clarity regarding the rationale for selecting this specific clustering method. Were alternative methods considered, and if so, why were they deemed less suitable? Additionally, the authors should provide more details regarding the interpretation of the dendrogram generated from the clustering analysis to aid readers in understanding the relationships between strains.

Response 3: The dendrogram was constructed by zero-one matrix data (binary data) from the phylogroups and virulence associated genes profiles using the unweighted pair group method with arithmetic averages (UPGMA) method via an online tool (www.genoms.urv.cat/upgma). Alternative methods such as sequence-based methods have been considered, but this study did not perform it due to the limited data. Therefore, the UPGMA was suitable for the available data in this study. More details of cluster analysis were added to materials and methods at Lines 181-186.

Comment 4: We suggest that you refer to new research article doi: 10.3389/fcimb.2022.790184. and doi: 10.1007/s11033-023-09031-x in the discussion section.

Response: The research article “10.3389/fcimb.2022.790184.” already mentioned in the discussion. Moreover, the article “10.1007/s11033-023-09031-x” was added to this section accordingly.

Responses to Reviewer #2:

Thank you for giving us your valuable comment. I am sure our manuscript will be much improved by this.

Comment 1: Line 60: This paragraph provide some information about different E. coli pathotypes, I would suggest to use more references and not only one. I suggest to use this study: 10.30466/vrf.2022.550618.3416 as the second references for this paragraph.

Response 1: More information about different E. coli pathotypes was added into the introduction section (Line 60-82). Moreover, the suggested article was used as a reference.

Responses to Reviewer #3: Comments

Thank you for giving us your valuable comments. I am sure our manuscript will be much improved by these.

Comment 1: What were the aim and conclusion of your study, exactly? It is not clear to me the aim of the study on sharing virulence genes between PEC and UPEC. Did you aim to demonstrate that pork may serve as one possible source of UPEC? If so, how did you demonstrate that in the present work?

Response 1: Yes, this study aimed to demonstrate that pork may serve as one possible source of UPEC in humans. The presence of virulence genes in pork strains in this study may imply that these strains may serve as a possible source of UPEC in humans. Moreover, the results from cluster analysis showed that some strains of pork and urine samples were clustered in the same branch of the tree. However, a molecular epidemiology study of these strains will be confirmed in further study.

Comment 2: Genotyping of isolates is needed to obtain accurate data and true prevalence. It is possible that your strains originated from a clone.

Response 2: Our 330 E. coli strains were obtained from different pure stock cultures. A single colony of each E. coli strain was selected for the analysis. These strains must have originated from different clones. However, the similar profiles of pork strains and urine strains found in this study may or may not be the same clone. Therefore, the whole genome sequencing of these strains needed to be performed in further study.

Comment 3: [A total of 330 E. coli strains was obtained from the preserved stock culture collection]: did you have exactly the same number of PEC and UPEC in your collection or you include 165 isolates (each group) from this collection in your study?

Response 3: The 330 E. coli strains were selected from different 330 samples which included 165 pork samples and 165 urine samples.

Comment 4: What was your definition for UPEC?

Response 4: The definition of UPEC in this study was E. coli that isolated from urine of UTI patients with the count number of the E. coli ≥ 105 CFU/ml.

Response 5: Grammatical errors and writing issues were seen in the manuscript.

Response 5: Some grammatical errors and writing issues have been corrected.

Comment 6: For abbreviations, provide their full term for the first mention.

Response 6: The abbreviations have been corrected.

Comment 7: Information on the isolates is not provided. How were urines collected? What was the demographic data of patients?

Response 7: The demographic data of patients could not be provided because all urine strains were selected from stock cultures and human ethics were not available for this study.

Comment 8: FimH is not only for UPEC. It is found in other E. coli types and in E. coli belonging to normal flora.

Response 8: Our results agree with that fimH is not only for UPEC. However, several studies included this gene for UPEC virulence-associated gene determination. It is one of the essential genes for attaching to epithelial cells and has been closely associated with the early development of UTIs (10.1073/pnas.0902179106, 10.1111/j.1758-2229.2010.00166.x, 10.1086/315258). Therefore, fimH was used to determine UPEC virulence-associated genes in this study. Moreover, E. coli originally isolated from the urine of patients with UTIs with counts number of E. coli ≥ 105 CFU/ml were considered UPEC strains in this study.

Comment 9: Which tests were used to identify E. coli? Mention them.

Response 9: The identification of E. coli was determined using morphological of colonies on MacConkey agar and confirmed by biochemical tests (IMViC; indole, methyl red, Voges-Proskauer, and citrate). Moreover, the PCR method was double-confirmed at the step of virulence gene determination using the PCR amplification of the 16S rRNA gene of E. coli (S1 Table). The detail of E. coli identification was added to the manuscript at Lines 135-136.

Comment 10: Did you test the antibiotic susceptibility of isolates?

Response 10: The antibiotic susceptibility of isolates will be performed in further study. This study focused on the phylogenetic diversity and virulence gene profiles of two sources of isolates and needed to emphasize the relationship between the strains from pork and urine.

Comment 11: Some references such as reference 1 were referred to in the manuscript too much. Use other references instead of referring to 1.

Response 11: The other references were used instead of using only reference 1.

Comment 12: Figure titles need to be revised.

Response 12: Fig 1 title was changed to “Distribution of the phylogroups among Escherichia coli isolated from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.” and Fig 3 title was changed to “Fig 3. UPGMA clustering analysis of 330 Escherichia coli from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.”.

Comment 13: Remove Table 1 and embed its data within the text

Response 13: Table 1 has been removed and the data was embedded into the text at Lines 232-234.

Comment 14: Table 2: number of isolates is not enough to make a p-value, in some rows.

Response 14: The data in Table 2 has been corrected.

Responses to Reviewer #4

Thank you for giving us your valuable comments. I am sure our manuscript will be much improved by these.

Comment 1: Please include the criteria used to characterize strains as UPEC, PEC and DEC in the Methods.

Response 1: In this study, UPEC were the E. coli originally isolated from the urine of patients with UTIs with counts number of E. coli ≥ 105 CFU/ml. For PEC, these E. coli were isolated pork meat. The DEC strains were identified using specific DEC virulence-associated genes. The details were added into the Selection and isolation of E. coli section (Line 132-133).

Comment 2: A description of the approach is given in the Introduction (Lines 55 and 110), but the specific genomic markers used to make the determination should be listed.

Response 2: The specific genomic markers used to make the determination were listed into introduction (Lines 61-74 for DEC virulence-associated genes and Lines 90-93 for UPEC virulence-associated genes).

Comment 3: This impacts the finding on lines 184-185 that “165 isolates PEC and 165 ones UPEC” The collection criteria stated on lines 117-124 only select for an isolation source and make no distinction between UPEC, E.coli commensal or environmental contaminant. If the authors were able to subset the UPEC strains from this dataset, please provide the selection criteria. Otherwise, it would be more accurate to claim that the 330 isolates clustered with at least eight different groups of isolates of UPEC and PEC sources. Further, were any tests performed to determine if isolates E1-165 were the causative agents of infection? If not, then additional justification is needed to categorize them as UPEC.

Response 3: UPEC were the E. coli originally isolated from the urine of patients with UTIs with counts number of E. coli ≥ 105 CFU/ml while PEC were the E. coli isolated pork meat obtained from markets (The more information was added into the Selection and isolation of E. coli section at Lines 130-133). Eight clusters in this study were constructed based on phylogroups and virulence genes. Therefore, the sources of isolates were not used for this analysis. The UPEC Pork strains that exhibited similar phylogroups and virulence gene profiles to urine strains in UTI patients could not be confirmed as the causative agents of UTIs in this study. However, the genetic relationships between these strains will be performed using WGS in further study.

Responses to Reviewer #5

Thank you for giving us your valuable comments. I am sure our manuscript will be much improved by these.

Comment 1: Line 124: I believe it would be interesting to describe the biochemical tests for E. coli characterization. Was there also molecular confirmation of the species?

Response 1: Details of the biochemical test were added to the materials and methods (Lines 135-136). Moreover, the molecular confirmation of E. coli was also used at step virulence-associated gene determination by PCR amplification 16S rRNA of E. coli (S1 Table).

Comment 2: The dendrogram image is of low quality, and it was not possible to analyze it.

Response 2: The new high quality dendrogram image was re-submitted. The epidemiological data from samples was not available for this study because all strains were selected from stock cultures.

Comment 3: The work is interesting, but it should focus on the actual evidence obtained. The samples were obtained from a previous isolate bank. For the conclusions reached, it would be necessary to analyze epidemiological data that were not presented. I suggest that epidemiological data be presented or that the conclusions and discussions obtained be reviewed. I also suggest that a higher quality image of the dendrogram be provided.

Response 3: The revised manuscript included the reviewer's suggestion.

Responses to Reviewer #6

Thank you for giving us your valuable comments. I am sure our manuscript will be much improved by these.

Abstract

Comment 1: Have only the specific genes of UPEC been examined? The text raises this suspicion. If not, please change the text of the first lines in the abstract.

Response 1: Several UPEC virulence-associated genes have been examined, such as csgA, csgE, fimA, fimH, papA, papC, papGIII, kpsmII, iutA, cnf, hlyD, sfaS, focG, and afa (https://doi.org/10.1111/tbed.13848; https://doi.org/10.1016/j.jmii.2016.03.006). However, there have been few studies examining these virulence genes among E. coli from pork samples.

Comment 2: Line 36 – 40: The percentage of the genes in the UPEC is higher than in PEC, so it is better to present its name and its percentage first.

Response 2: The revised manuscript included the reviewer's suggestion (Lines 37-40).

Comment 3: The conclusion of the abstract section is not based on the results and should be changed

Response 3: “These strains may pose health risks for the development of UTIs in humans” (I think this sentence should be removed according to the comment). The revised manuscript included the reviewer's suggestion (Lines 45-46).

Introduction

Comment 4: Line 56-59: First introduce EXPEC more. Mention and explain the virulence genes or pathogenic factors of this bacterium that cause urinary infections.

Response 4: The virulence genes and virulence factors causing UTIs in humans were described at Lines 83-85 and 90-93.

Comment 5: Line 67: Please write in full version for “STEC” for the first time.

Response 5: The full version of Shiga toxin-producing E. coli (STEC) was added in to the manuscript at Line 65.

Comment 6: Line 76: State the pathotypes of EXPEC here with more details along with its pathogenic genes.

Response 6: The revised manuscript included the reviewer's suggestion (Lines 83-85).

Comment 7: Line 107-108: Please remove this sentence as a novelty, the authors have mentioned it several times in the text.

Response 7: The revised manuscript included the reviewer's suggestion (Lines 118-119).

Material and method

Comment 8: More data should be provided on the sources of isolation of these bacteria, especially on human samples. Does the study have a code of ethics, mention it.

Response 8: The urine and pork strains in this study were selected from bacterial stock cultures. Therefore, human and animal ethics are not available for this study.

Comment 9: The bootstrap value should be stated at the level of phylogenetic analysis.

Response 9: The dendrogram was constructed using a bootstrap value of 100 based on the default settings of the program. The details of cluster analysis have been added into materials and methods (Lines 182-186).

Comment 10: How many times the same branch is observed when repeating the generation of a phylogenetic tree on a resampled set of data? The percentage of times each interior branch is given a value of 1 should be noted.

Response 10: The times of same branch were not available because this dendrogram is constructed using a bootstrap value of 100 based on the default settings of the program.

Responses to Reviewer #7

Thank you for giving us your valuable comments. I am sure our manuscript will be much improved by these.

Comment 1: The phylogenetic method designed by Clermont et al has different protocol. Why did the authors change the PCR protocol?

Response 1: We followed the original protocol for phylogrouping from Clermont et al. (2013) and did not change the PCR protocol.

Comment 2: In material and method, the authors described that the cluster analysis was performed according to phtlogenetic group and absence or presence of virulence genes, while in the fig 3. the strains with similarity profiles put in different clusters. I think the clustering is drawn according to phylogenetic groups only.

Res

PLoS One. doi: 10.1371/journal.pone.0307544.r003

Decision Letter 1

Kwame Kumi Asare

10 May 2024

PONE-D-23-44006R1Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in ThailandPLOS ONE

Dear Dr. Suzuki,

Please submit your revised manuscript by Jun 24 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Kwame Kumi Asare, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

Reviewer #6: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #6: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: N/A

Reviewer #6: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #6: No

**********

6. Review Comments to the Author

Reviewer #1: This article is accepted in this form

This article is accepted in this form

Reviewer #2: The authors addressed all the questions properly, and I would like to recommend for acceptance of this article.

Reviewer #3: The authors made an extensive effort to improve the manuscript. There are still some ambiguities.

*Sharing virulence genes between E. coli groups is important data, but it can’t support your hypothesis. The dendrogram and cluster analysis of virulence genes also can’t be used to support the aim of the present study. Therefore, please change and revise the corresponding lines.

[Comment 1: What were the aim and conclusion of your study, exactly? ….…. Response 1 : Yes, this study aimed to demonstrate that pork may serve as one possible source of UPEC in humans. The presence of virulence genes in pork strains in this study may imply that these strains may serve as a possible source of UPEC in humans. Moreover, the results from cluster analysis showed that some strains of pork and urine samples were clustered in the same branch of the tree. However, a molecular epidemiology study of these strains will be confirmed in further study.]

*Some simpler and cheaper methods than WGS could be used to analyze the relationships between the isolates. Furthermore, the genotyping may help to support the hypothesis that :pork may serve as one possible source of UPEC in humans

*Please add more references to the first paragraph in Introduction: lines 51-59.[Comment 11: Some references were referred to in the manuscript too much. Use other references instead of referring to 1. Response 11: The other references were used instead of using only reference 1].

Reviewer #4: The authors have responded to the reviewer queries adequately and implemented the changes that have improved the accuracy and readability of the manuscript. I am satisfied with the changes implemented by the authors in this revised manuscript. However there are some more needs to revise the manuscript. First, Fig. 2 should be transferred to supplementary file. Second, in dendrogram it is unknown that what is the tolerance and optimization rate in analysis. There are some strains whose patterns are the same with 100 % similarity. But the dendrogram clustered them into different patterns. Like strains U20 and U19, also strains U21 and U22. Or some bands have been missed between isolates like strains U16 and U35 between P39 and P30 (U30). It seems that the authors should analyze the bands again with new tolerance and less sensitivity. So that after analysis these strains will got into the same pattern. After re-analysis the dendrogram all the text should be revised based on the results.

Reviewer #6: At first, the authors did not adequately answer some questions. For example, one of the questions was why the researchers used the method of UPGMA clustering for cluster analysis and why the other clustering methods were not suitable. They suggested that due to the limited facilities, it was not possible to perform other tests such as a complete genome examination, which was a wrong answer to this question. In fact, they should have explained other clustering methods, and why they did not use those methods. Another example is my question “Have only the specific genes of UPEC been examined?” they didn’t answer it correctly. They stated that the purpose of their study was to examine virulence genes related to UPEC isolates, but they have other types of E. coli isolates. In this section, writing should have been done in such a way that the importance of all isolate types was also shown.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Mahdi Askari Badouei

Reviewer #3: Yes: Safoura Derakhshan

Reviewer #4: Yes: Hami Kaboosi

Reviewer #6: No

**********

PLoS One. 2024 Jul 25;19(7):e0307544. doi: 10.1371/journal.pone.0307544.r004

Author response to Decision Letter 1

22 May 2024

Reviewer #1:

Comment: This article is accepted in this form

Response: Thank you for your acceptance. We appreciate much.

Reviewer #2:

Comment: The authors addressed all the questions properly, and I would like to recommend for acceptance of this article.

Response: Thank you for your acceptance. We appreciate much.

Reviewer #3:

Comment 1: What were the aim and conclusion of your study, exactly? ….…. Response 1 : Yes, this study aimed to demonstrate that pork may serve as one possible source of UPEC in humans. The presence of virulence genes in pork strains in this study may imply that these strains may serve as a possible source of UPEC in humans. Moreover, the results from cluster analysis showed that some strains of pork and urine samples were clustered in the same branch of the tree. However, a molecular epidemiology study of these strains will be confirmed in further study.

Response: The conclusion of this study in the abstract section was revised to “Cluster analysis showed a relationship between PEC and UPEC strains and demonstrated that PEC harboring UPEC virulence-associated genes in pork may be associated with UPEC in humans” (Line 44-46). Moreover, the conclusion in the discussion part of cluster analysis was revised to “The PEC harbored UPEC virulence-associated genes may pose a zoonotic risk and may have a relationship to UPEC in humans.” (Lines 370-371).

Comment 2: Some simpler and cheaper methods than WGS could be used to analyze the relationships between the isolates. Furthermore, the genotyping may help to support the hypothesis that :pork may serve as one possible source of UPEC in humans

Response: The suggested methods for analyzing the relationship between the isolates were revised (Lines 373-375).

Comment 3: Please add more references to the first paragraph in Introduction: lines 51-59.[Comment 11: Some references were referred to in the manuscript too much. Use other references instead of referring to 1. Response 11: The other references were used instead of using only reference 1].

Response: References were accordingly in the Introduction (Line 51-59)

Reviewer #4:

Comment: Fig. 2 should be transferred to supplementary file. Second, in dendrogram it is unknown that what is the tolerance and optimization rate in analysis. There are some strains whose patterns are the same with 100 % similarity. But the dendrogram clustered them into different patterns. Like strains U20 and U19, also strains U21 and U22. Or some bands have been missed between isolates like strains U16 and U35 between P39 and P30 (U30). It seems that the authors should analyze the bands again with new tolerance and less sensitivity. So that after analysis these strains will got into the same pattern. After re-analysis the dendrogram all the text should be revised based on the results.

Response: Thank you for your insightful suggestions. Our responses are as below.

1. Regarding your suggestion to transfer Fig. 2 to the supplementary file, we understand your suggestion. However, we would like to demonstrate the importance of Fig. 2 in illustrating the distribution of UPEC virulence-associated genes among pork and urine strains in Thailand. Given its significance for readers' understanding of the main study's findings, we believe it would be beneficial to keep Fig. 2 in the main manuscript. I hope you will consider this rationale.

2. In this study, the UPGMA cluster analysis did not rely on gel banding from typing methods such as enterobacterial repetitive intergenic consensus (ERIC)-PCR, pulse field gel electrophoresis (PFGE), or restriction fragment length polymorphism (RFLP). We analyzed based on data from 1) Clermont phylogroup classification from PCR analysis that could identify each strain to be Clermont group (A, B, C, D, etc) and 2) UPEC and DEC virulence-associated gene profiles. These profiles were converted to binary data (presence <1> or absence <0>) for the UPGMA cluster analysis. Therefore, tolerance and optimization rate analysis were not applicable in this study. U20 and U19, U21 and U22 exhibited 100% similarity in virulence-associated gene profiling, suggesting they should be clustered together. However, they belonged to different phylogroups, resulting in their placement into separate groups. For example, U20 belonged to phylogroup C while U19 belonged to phylogroup B1. Consequently, these strains were assigned to different clusters despite sharing identical virulence-associated gene profiles due to their distinct phylogroups. Therefore, re-analysis of the dendrogram may not be necessary.

I hope you understand our response.

Reviewer #6:

Comment: At first, the authors did not adequately answer some questions. For example, one of the questions was why the researchers used the method of UPGMA clustering for cluster analysis and why the other clustering methods were not suitable. They suggested that due to the limited facilities, it was not possible to perform other tests such as a complete genome examination, which was a wrong answer to this question. In fact, they should have explained other clustering methods, and why they did not use those methods. Another example is my question “Have only the specific genes of UPEC been examined?” they didn’t answer it correctly. They stated that the purpose of their study was to examine virulence genes related to UPEC isolates, but they have other types of E. coli isolates. In this section, writing should have been done in such a way that the importance of all isolate types was also shown.

Responses: Thank you for your valuable comments. Please fine our responses below.

1. The reason to use the UPGMA clustering method for cluster analysis is because our data came from phylogrouping and virulence-associated genes determining which were binary data [absence (0) or presence (1)]. For binary data, hierarchical clustering methods like UPGMA and hierarchical agglomerative clustering (HAC) are commonly used to construct dendrograms. These two methods are suitable for binary data because they can utilize appropriate similarity index, such as the Jaccard index or Dice coefficient, to quantify the similarity between samples based on their binary profiles. UPGMA treats binary data more naturally by considering the presence or absence of traits. It computes distances between samples without evolutionary assumptions. In contrast, HAC may require additional preprocessing or transformation of binary data to compute meaningful distances between samples. Moreover, UPGMA produces dendrograms with a uniform branch length, which can be easier to interpret visually while HAC dendrograms may exhibit varying branch lengths depending on the chosen linkage criterion, offering more detailed insights into the clustering structure. In addition, UPGMA is relatively simple to implement and computationally efficient, especially for moderate-sized data sets compared to HAC that suitable for small to medium-sized data sets. Therefore, the UPGMA was suitable for clustering in this study.

2. In this study, we also determined the presence of virulence-associated genes associated with diarrheagenic E. coli in all 330 E. coli strains. Additional details regarding this aspect have been included in the abstract to provide further clarity on the scope of this study (Lines 26-30).

PLoS One. doi: 10.1371/journal.pone.0307544.r005

Decision Letter 2

Kwame Kumi Asare

9 Jul 2024

Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand

PONE-D-23-44006R2

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PLoS One. doi: 10.1371/journal.pone.0307544.r006

Acceptance letter

Kwame Kumi Asare

17 Jul 2024

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. PCR primers used in this study.

(PDF)

pone.0307544.s001.pdf^{(183.1KB, pdf)}

S2 Table. Interpretation of amplified PCR products for phylogroups of Escherichia coli.

(PDF)

pone.0307544.s002.pdf^{(63.8KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript.

[pone.0307544.ref001] 1.Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2:123–140. doi: 10.1038/nrmicro818 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref002] 2.Khairy RM, Mohamed ES, Abdel Ghany HM, Abdelrahim SS. Phylogenic classification and virulence genes profiles of uropathogenic Escherichia coli and diarrhegenic Escherichia coli strains isolated from community acquired infections. PLoS One. 2019;14:e0222441. doi: 10.1371/journal.pone.0222441 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref003] 3.Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013; 26:822–80. doi: 10.1128/CMR.00022-13 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref004] 4.Gomes TAT, Elias WP, Scaletsky ICA, Guth BEC, Rodrigues JF, Piazza RMF, et al. Diarrheagenic Escherichia coli. Braz J Microbiol. 2016;47:3–30. doi: 10.1016/j.bjm.2016.10.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref005] 5.Smith JL, Fratamico PM, Gunther NW. Extraintestinal pathogenic Escherichia coli. Foodborne Pathog Dis. 2007;4:134–63. doi: 10.1093/infdis/jiv429 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref006] 6.Hernandes RT, Elias WP, Vieira MAM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2016;297:137–149. doi: 10.1111/j.1574-6968.2009.01664.x [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref007] 7.Fleckenstein JM, Kuhlmann FM. Enterotoxigenic Escherichia coli Infections. Curr Infect Dis Rep. 2019;21. doi: 10.1007/s11908-019-0665-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref008] 8.Welinder-Olsson C., Kaijser B. Enterohemorrhagic Escherichia coli (EHEC), Scand J Infect Dis. 2005;37:405–416. doi: 10.1080/00365540510038523 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref009] 9.Askari BM, Taban H, Nemati A, Fernando DSL. Molecular serotyping of Shiga toxin-producing Escherichia coli (STEC) of animal origin in Iran reveals the presence of important non-O157 seropathotypes. Vet Res Forum. 2023;14:267–274. doi: 10.30466/vrf.2022.550618.3416 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref010] 10.van den Beld MJC, Reubsaet FAG. Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur J Clin Microbiol Infect Dis. 2012;31:899–904. doi: 10.1007/s10096-011-1395-7 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref011] 11.Weintraub A. Enteroaggregative Escherichia coli: epidemiology, virulence and detection. J Med Microbiol. 2007;56:4–8. doi: 10.1099/jmm.0.46930–0 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref012] 12.Le Bouguénec C, Servin AL. Diffusely adherent Escherichia coli strains expressing Afa/Dr adhesins (Afa/Dr DAEC): hitherto unrecognized pathogens. FEMS Microbiol Lett. 2006;256:185–194. doi: 10.1111/j.1574-6968.2006.00144.x [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref013] 13.Camprubí-Font C, Martinez-Medina M. Why the discovery of adherent-invasive Escherichia coli molecular markers is so challenging?. World J Biol Chem. 2020;11:1–13. doi: 10.4331/wjbc.v11.i1.1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref014] 14.Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-Ploskonska G, et al. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathog. 2019;11:10. doi: 10.1186/s13099-019-0290-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref015] 15.Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. 2015;13:269–84. doi: 10.1038/nrmicro3432 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref016] 16.Terlizzi ME, Gribaudo G, Maffei ME. Uropathogenic Escherichia coli (UPEC) infections: virulence factors, bladder responses, antibiotic, and non-antibiotic antimicrobial strategies. Front Microbiol. 2017; 8:1566. doi: 10.3389/fmicb.2017.01566 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref017] 17.Manges AR. Escherichia coli and urinary tract infections: the role of poultry-meat. Clin Microbiol Infect. 2016;22:122–9. doi: 10.1016/j.cmi.2015.11.010 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref018] 18.Tanabe RHS, Dias RCB, Orsi H, de Lira DRP, Vieira MA, dos Santos LF, et al. Characterization of uropathogenic Escherichia coli reveals hybrid isolates of uropathogenic and diarrheagenic (UPEC/DEC) Escherichia coli. Microorganisms. 2022;10:645. doi: 10.3390/microorganisms10030645 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref019] 19.Mora A, López C, Dabhi G, Blanco M, Blanco JE, Alonso MP, et al. Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution. BMC Microbiol. 2009;9:132. doi: 10.1186/1471-2180-9-132 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref020] 20.Ramchandani M, Manges AR, DebRoy C, Smith SP, Johnson JR, Riley LW. Possible animal origin of human-associated, multidrug-resistant, uropathogenic Escherichia coli. Clin Infect Dis. 2005;40:251–7. doi: 10.1086/426819 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref021] 21.Aslam M, Toufeer M, Narvaez BC, Lai V, Rempel H, Manges A, et al. Characterization of extraintestinal pathogenic Escherichia coli isolated from retail poultry meats from Alberta, Canada. Int J Food Microbiol. 2014;177:49–56. doi: 10.1016/j.ijfoodmicro.2014.02.006 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref022] 22.Khan SB, Zou G, Cheng YT, Xiao R, Li L, Wu B, et al. Phylogenetic grouping and distribution of virulence genes in Escherichia coli along the production and supply chain of pork around Hubei, China. J Microbiol Immunol Infect. 2017;50:382–5. doi: 10.1016/j.jmii.2016.03.006 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref023] 23.Spindola MG, Cunha MPV, Moreno LZ, Amigo CR, Silva APS, Parra BM, et al. Genetic diversity, virulence genotype and antimicrobial resistance of uropathogenic Escherichia coli (UPEC) isolated from sows. Vet Q. 2018;38:79–87. doi: 10.1080/01652176.2018.1519321 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref024] 24.Zhu Y, Dong W, Ma J, Yuan L, Hejair HMA, Pan Z, et al. Characterization and virulence clustering analysis of extraintestinal pathogenic Escherichia coli isolated from swine in China. BMC Vet Res. 2017;13:94. doi: 10.1186/s12917-017-0975-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref025] 25.Bok E, Kożańska A, Mazurek PJ, Wojciech M, Baldy CK. Extended phylogeny and extraintestinal virulence potential of commensal Escherichia coli from piglets and sows. Int J Environ Res Public Health. 2020;17:366. doi: 10.3390/ijerph17010366 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref026] 26.Ma J, Cheng Z, Bai Q, Zhao K, Pan Z, Yao H. Screening virulence factors of porcine extraintestinal pathogenic Escherichia coli (an emerging pathotype) required for optimal growth in swine blood. Transbound Emerg Dis. 2020;68:2005–2016. doi: 10.1111/tbed.13848 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref027] 27.Jakobsen L, Garneau P, Bruant G, Harel J, Olsen SS, Porsbo LJ, et al. Is Escherichia coli urinary tract infection a zoonosis? Proof of direct link with production animals and meat. Eur J Clin Microbiol Infect Dis. 2012; 31:1121–1129. doi: 10.1007/s10096-011-1417-5 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref028] 28.Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5:58–65. doi: 10.1111/1758-2229.12019 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref029] 29.Dadi BR, Abebe T, Zhang L, Mihret A, Abebe W, Amogne W. Distribution of virulence genes and phylogenetics of uropathogenic Escherichia coli among urinary tract infection patients in Addis Ababa, Ethiopia. BMC Infect Dis. 2020;20:108. doi: 10.1186/s12879-020-4844-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref030] 30.Yazdanpour Z, Tadjrobehkar O, Shahkhah M. Significant association between genes encoding virulence factors with antibiotic resistance and phylogenetic groups in community acquired uropathogenic Escherichia coli isolates. BMC Microbiol. 2020;20:241. doi: 10.1186/s12866-020-01933-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref031] 31.Feng P, Weagant SD. BAM Chapter 4A: Diarrheagenic Escherichia coli. 2020. [cited 3 July 2023]. In: Laboratory Methods (Food) [Internet]. The United States Food and Drug Administration (FDA). Available from: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4a-diarrheagenic-escherichia-coli. [Google Scholar]

[pone.0307544.ref032] 32.Chonsin K, Siriphap A, Changkaew K, Noisumdaeng P, Suthienkul O. Virulence genes of Vibrio parahaemolyticus isolated from acute hepatopancreatic necrosis disease associated with shrimp and grow-out pond water. J Med Health Sci. 2021;28:17–27. [Google Scholar]

[pone.0307544.ref033] 33.Le Bouguenec C, Archambaud M, Labigne A. Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction. J Clin Microbiol. 1992;30:1189–93. doi: 10.1128/jcm.30.5.1189–1193.1992 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref034] 34.Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J. Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. J Glob Infect Dis. 2013;17:e450–e3. doi: 10.1016/j.ijid.2013.01.025 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref035] 35.Usein CR, Damian M, Tatu-Chitoiu D, Capusa C, Fagaras R, Tudorache D, et al. Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases. J Cell Mol Med. 2001;5:303–10. doi: 10.1111/j.1582-4934.2001.tb00164.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref036] 36.Fialho OB, de Souza EM, de Borba Dallagassa C, de Oliveira Pedrosa F, Klassen G, Irino K, et al. Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol. J Clin Lab Anal. 2013;27:155–61. doi: 10.1002/jcla.21578 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref037] 37.Garcia-Vallvé S, Palau J, Romeu A. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol Biol Evol. 1999;16:1125–34. doi: 10.1093/oxfordjournals.molbev.a026203 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref038] 38.Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49:293–6. doi: 10.1093/nar/gkab301 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref039] 39.Halaji M, Fayyazi A, Rajabnia M, Zare D, Pournajaf A, Ranjbar R. Phylogenetic group distribution of uropathogenic Escherichia coli and related antimicrobial resistance pattern: a meta-analysis and systematic review. Front Cell Infect Microbiol. 2022;12:790184. doi: 10.3389/fcimb.2022.790184 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref040] 40.Mostaghimi T, Pournajaf A, Bijani A, Mohammadi M, Rajabnia M, et al. Phylogenetic analysis, biofilm formation, antimicrobial resistance and relationship between these characteristics in Uropathogenic Escherichia coli. Mol Biol Rep. 2024; 51:327. doi: 10.1007/s11033-023-09031-x [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref041] 41.Parikumsil N, Prapasawat W, Siriphap A, Chonsin K, Theethakaew C, Sukolrattanamaetee N, et al. Virulence factors and molecular epidemiology of uropathogenic Escherichia coli isolated from paired urine and rectal swab samples of patients with urinary tract infections in Thailand. Southeast Asian J Trop Med Public Health. 2017;48:1029–41. [Google Scholar]

[pone.0307544.ref042] 42.Themphachana M, Kongpheng S, Rattanachuay P, Khianngam S, Singkhamanan K, Sukhumungoon P. Molecular characterization of virulence and antimicrobial susceptibility profiles of uropathogenic Escherichia coli from patients in a tertiary hospital, southern Thailand. Southeast Asian J Trop Med Public Health. 2015;46:1021. [PubMed] [Google Scholar]

[pone.0307544.ref043] 43.Walk ST. The "Cryptic" Escherichia. EcoSal Plus. 2015;6(2):10.1128/ecosalplus.ESP-0002-2015. doi: 10.1128/ecosalplus.esp-0002-2015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref044] 44.Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol. 2000;66:4555–8. doi: 10.1128/AEM.66.10.4555–4558.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref045] 45.Belmont-Monroy L, Ribas-Aparicio RM, González-Villalobos E, Pérez-Ramos JA, Aparicio-Ozores G, Eslava-Campos CA, et al. Molecular typification of Escherichia coli from community-acquired urinary tract infections in Mexico. Int J Antimicrob Agents. 2022;60:106667. doi: 10.1016/j.ijantimicag.2022.106667 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref046] 46.Firoozeh F, Saffari M, Neamati F, Zibaei M. Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis. J Glob Infect Dis. 2014;29:219–22. doi: 10.1016/j.ijid.2014.03.1393 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref047] 47.Tabasi M, Karam MR, Habibi M, Mostafavi E, Bouzari S. Genotypic characterization of virulence factors in Escherichia coli isolated from patients with acute cystitis, pyelonephritis and asymptomatic bacteriuria. J Clin Diagn Res. 2016;10:dc01–dc7. doi: 10.7860/jcdr/2016/21379.9009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref048] 48.Tewawong N, Kowaboot S, Pimainog Y, Watanagul N, Thongmee T, Poovorawan Y. Distribution of phylogenetic groups, adhesin genes, biofilm formation, and antimicrobial resistance of uropathogenic Escherichia coli isolated from hospitalized patients in Thailand. PeerJ. 2020;8:e10453. doi: 10.7717/peerj.10453 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref049] 49.Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney Int. 2007;72:19–25. doi: 10.1038/sj.ki.5002230 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref050] 50.Desvaux M, Dalmasso G, Beyrouthy R, Barnich N, Delmas J, Bonnet R. Pathogenicity factors of genomic islands in intestinal and extraintestinal Escherichia coli. Front Microbiol. 2020;11:2065. doi: 10.3389/fmicb.2020.02065 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref051] 51.Matsui Y, Hu Y, Rubin J, de Assis RS, Suh J, Riley LW. Multilocus sequence typing of Escherichia coli isolates from urinary tract infection patients and from fecal samples of healthy subjects in a college community. MicrobiologyOpen. 2020;9:e1032. doi: 10.1002/mbo3.1032 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref052] 52.Manges AR, Johnson JR. Food-borne origins of Escherichia coli causing extraintestinal infections. Clin Infect Dis. 2012;55:712–9. doi: 10.1093/cid/cis502 [DOI] [PubMed] [Google Scholar]

[pone.0307544.ref053] 53.Martínez-Santos VI, Ruíz-Rosas M, Ramirez- Peralta A, Zaragoza García O, Resendiz-Reyes LA, Romero-Pineda OJ, et al. Enteroaggregative Escherichia coli is associated with antibiotic resistance and urinary tract infection symptomatology. PeerJ. 2021;9:e11726. doi: 10.7717/peerj.11726 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0307544.ref054] 54.Magruder M, Sholi AN, Gong C, Zhang L, Edusei E, Huang J, et al. Gut uropathogen abundance is a risk factor for development of bacteriuria and urinary tract infection. Nat. Commun. 2019;10:5521. doi: 10.1038/s41467-019-13467-w [DOI] [PMC free article] [PubMed] [Google Scholar]

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Phylogenetic diversity and virulence gene characteristics of Escherichia coli from pork and patients with urinary tract infections in Thailand

Pramualchai Ketkhao

Fuangfa Utrarachkij

Nattaya Parikumsil

Kritchai Poonchareon

Anusak Kerdsin

Peeraya Ekchariyawat

Pawarut Narongpun

Chie Nakajima

Yasuhiko Suzuki

Orasa Suthienkul

Roles

Abstract

Introduction

Materials and methods

Selection and isolation of E. coli

Genomic DNA extraction

Determination of phylogroups

Detection of virulence-associated genes

Cluster analysis of virulence genes and phylogroups

Statistical analysis

Results

Distribution of phylogroups

Fig 1. Distribution of the phylogroups among Escherichia coli isolated from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

Virulence-associated genes

Fig 2. The distribution of uropathogenic Escherichia coli (UPEC) virulence-associated genes of E. coli from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

Association between the phylogroups and UPEC virulence-associated genes

Table 1. Frequencies of virulence factors associated with uropathogenic Escherichia coli (UPEC) in the phylogroups among Escherichia coli isolated from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

UPEC virulence-associated gene patterns distribution among phylogroups of PEC and UPEC strains

Table 2. Uropathogenic Escherichia coli (UPEC) virulence gene patterns among phylogroups of 330 Escherichia coli from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

Cluster analysis of virulence genes and phylogroups

Fig 3. UPGMA clustering analysis of 330 Escherichia coli from pork (PEC) and urine (UPEC) of patients with urinary tract infections (UTIs) in Thailand.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Kwame Kumi Asare

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Author response to Decision Letter 0

Decision Letter 1

Kwame Kumi Asare

Roles

Author response to Decision Letter 1

Decision Letter 2

Kwame Kumi Asare

Roles

Acceptance letter

Kwame Kumi Asare

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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