Characterization of O25b-ST131 Escherichia coli Clone Producing CTX-M-15,DHA-4,and CMY-42 in Urinary Tract Infections in a Tunisian Island

Abstract

The dissemination of extended-spectrum β-lactamases encoding genes in Escherichia coli, especially in the uropathogenic O25b-ST131 E. coli clone, constitutes a real concern. We aimed to identify the molecular mechanisms of resistance to cephalosporins among E. coli clinical isolates and to estimate the prevalence of the uropathogenic O25b-ST131 clone in our study. Forty-two cephalosporin-resistant E. coli implicated in urinary tract infections were collected from the Regional Hospital of a southeastern Tunisian Island from April 2015 to August 2016. Molecular screening of β-lactamases encoding genes by PCR and sequencing showed that the majority of our isolates harbored bla_CTX-M gene (bla_CTX-M-15 [n = 36], bla_CTX-M-14 [n = 2]). Nevertheless, the bla_SHV, bla_TEM, and bla_OXA-1 genes were not detected. Various class C β-lactamases encoding genes were observed in association or not with bla_CTX-M genes and were as follows: bla_ampC (n = 14), bla_CMY-42 (n = 7), bla_CMY-2 (n = 1), and bla_DHA-4 (n = 1). The research of O25b-ST131 clone was carried out by duplex PCR (pabB and trpA genes) and revealed that most of our isolates (n = 30) belonged to this clone. We also noted that the majority of our isolates belonged to the B2 phylogenetic group (n = 32), five isolates to the B1 phylogenetic group, three isolates to the D phylogenetic group, and only two isolates belonged to the A phylogenetic group. Our study provides new epidemiological information about E. coli clinical isolates in this area. Indeed, this is the first report of CTX-M-14 producing O25b-ST131 E. coli in our country and the first report of DHA-4 and CMY-42 producing E. coli in Tunisia.

Introduction

Escherichia coli bacilli belong to the large family of Gram-negative bacteria, Enterobacteriaceae, and constitute a part of the normal bowel flora. This germ is also pathogenic because it has caused several types of infections, such as intestinal infections, neonatal meningitis, bloodstream infections, and urinary tract infections.¹ Furthermore, it was reported that E. coli is the main cause of urinary tract infections, noting a very high rate equal to 80%.² Moreover, it was well demonstrated that ST131 E. coli is becoming the predominant uropathogenic clone, also qualified as a worldwide pandemic clone, causing widespread antibiotic resistance determinants.^3–5 Besides, this E. coli ST131 clone was first described in 2008 in North America, Europe, and Asia.⁶ Then, it has been dramatically disseminated throughout the globe, in health care settings and community, posing a serious therapeutic problem.^5,7 The ST131 E. coli clone is frequently resistant to the most β-lactam antibiotics in addition to other antibiotic families such as aminoglycosides, fluoroquinolones, and trimethoprim–sulfamethoxazole.³ Thus, multidrug-resistant uropathogenic ST131 E. coli clone is usually associated with high morbidity and mortality rates and prolonged hospital stays.^3,5

The ST131 E. coli clone is usually involved in the successful spread of extended-spectrum β-lactamases (ESBLs), mainly the most widespread CTX-M-15 enzyme.⁸ Thus, a high rate of ESBLs production was found in the ST131 E. coli clone compared with non-ST131 isolates.⁷ Moreover, it was shown that ST131 E. coli isolates are more pathogenic than non-ST131 E. coli.⁹ ST131 E. coli isolates usually belong to the B2 phylogenetic group and the O25b or O16 serotypes.^5,10 On the contrary, the dissemination of this clone has also been reported in Tunisia, posing a real concern in northern Tunisian hospitals, because of its association with the spread of the CTX-M-15 enzyme.¹¹ This enzyme, first described in 2001, was derived from CTX-M-3 by only one amino acid substitution (Asp-Gly), which increases the catalytic efficiency against ceftazidime.¹²

In this study, we aimed to identify β-lactamases encoding genes implicated in resistance to cephalosporins among E. coli clinical isolates collected from a regional hospital of southeastern Tunisian Island, Djerba, known as an attractive tourist destination. This 514 km² Mediterranean Island has a latitude and longitude of 33°52'29” N and 10°51'33” E, respectively. Besides, we shed light on the existence of the ST131 E. coli clone in this hospital. To our knowledge, this is the first epidemiological study that focuses on β-lactamases production among E. coli in this area.

Materials and Methods

Bacterial isolates

A total of 42 cephalosporin-resistant E. coli clinical isolates were collected from the Regional Hospital of Djerba, a southeastern Tunisian Island during a period extended from April 2015 to August 2016. All our isolates were obtained from urine samples using a selective medium, eosin methylene blue agar (EMB-Agar). Moreover, bacterial identification was performed by biochemical test kits (bioMerieux API 10S), then verified by amplification and sequencing of 16S rRNA.

Antibiotic susceptibility testing

The antimicrobial susceptibility of the 42 E. coli clinical isolates was carried out by the disk diffusion method using Mueller–Hinton agar medium. Fourteen antibiotics of diverse antimicrobial families were tested: (amoxicillin [25 μg], ticarcillin [75 μg], amoxicillin + clavulanic acid [20 + 10 μg], cefoxitin [30 μg], cefotaxime [30 μg], ceftazidime [30 μg], ertapenem [10 μg], imipenem [10 μg], nalidixic acid [30 μg], ofloxacin [5 μg], ciprofloxacin [5 μg], trimethoprim–sulfamethoxazole [1.25 + 23.75 μg], amikacin [30 μg], and gentamicin [10 μg]). The obtained results were analyzed according to the French Committee recommendations, CA-SFM 2015 (www.sfm-microbiologie.org/). The screening of ESBLs producing isolates was carried out by the double-disk synergy test.

Molecular characterization of β-lactamase genes

The detection of the different Ambler class A β-lactamases encoding genes was performed by PCR using specific primers for bla_SHV, bla_TEM, and bla_CTX-M as previously described.^13,14 Besides, the presence of other β-lactamase genes coding for class C (bla_CMY, bla_ampC, bla_DHA, bla_CIT, and bla_EBC) and class D (bla_OXA-1) was also tested.^15–18

Sequencing was performed for positive isolates to determine the variant of each detected gene. Resulting sequences were compared with those available in the GenBank.

Phylogenetic group determination

The four main phylogenetic groups of E. coli (A, B1, B2, and D) were determined by PCR method based on the absence or presence of three genes (chuA, yjaA, and tspE4.C2) as previously described.¹⁹ In fact, the chuA gene is present only among B2 and D phylogenetic groups and codes for outer membrane hemin receptor. The yjaA gene that codes for conserved stress-induced protein (www.uniprot.org/uniprot/J7QJ88) was tested to discriminate between B2 and D phylogenetic groups. However, the tspE4.C2, an anonymous DNA fragment with unknown function, is present only among B1 E. coli phylogenetic group.¹⁹

O25b-ST131 E. coli clone detection

The duplex polymerase chain reaction method was performed using specific primers for amplification of a 347 bp fragment of the pabB gene and 427 bp fragment of the trpA gene to detect the O25b-ST131 clone, as described in a previous report.²⁰

Results

Bacterial isolates and phenotypic characterization of antibiotic resistance

In total, 42 cephalosporin-resistant E. coli clinical isolates were collected from April 2015 to August 2016. All our clinical isolates were obtained from urine samples and were resistant to cefotaxime. Thirty-eight of these isolates were resistant to ceftazidime and only 14 were resistant to cefoxitin. As described in Table 1, our isolates showed a multidrug resistance pattern. Indeed, we have noted resistance to several antibiotic families, including β-lactams, aminoglycosides, quinolones, and sulfonamides.

Table 1.

Characterization of Cephalosporin-Resistant Escherichia coli Clinical Isolates of a Tunisian Island

Isolates	Origins	Phylogenetic group	O25b-ST131 clone detection	Antibiotic resistance pattern	Production of β-lactamases
1	Urine	B2	+	AMX, AMC, CTX, CAZ, TIC, SXT, NAL, OFX, GMI, TMN	CTX-M-15
2	Urine	B2	+	AMX, AMC, CTX, CAZ, TIC, SXT, NAL, OFX, GMI, TMN	CTX-M-15
3	Urine	D	−	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, SXT, NAL, OFX	CTX-M-15/AmpC
4	Urine	B2	+	AMX, AMC, CTX, CAZ, SXT, NAL, OFX, TMN	CTX-M-15
5	Urine	B2	+	AMX, AMC, CTX, CAZ, ETP, FOX, CIP, TIC, NAL, OFX, TMN	CTX-M-15/AmpC
6	Urine	B2	+	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX, GMI, TMN	CTX-M-15/AmpC/CMY-42
7	Urine	B2	+	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX, GMI, TMN	CTX-M-15/AmpC/CMY-42
8	Urine	B2	+	AMX, CTX, CAZ, CIP, TIC, NAL, OFX	CTX-M-15
9	Urine	B2	+	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX, AKN	CTX-M-15/AmpC/CMY-42
10	Urine	B2	+	AMX, CTX, TIC, SXT	CTX-M-15
11	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, NAL, OFX	CTX-M-15
12	Urine	B2	+	AMX, AMC, CTX, CIP, TIC, SXT, NAL, OFX	CTX-M-15
13	Urine	B2	+	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX	CTX-M-15/AmpC/CMY-42
14	Urine	B2	+	AMX, CTX, CAZ, CIP, TIC, NAL, OFX	CTX-M-15
15	Urine	B2	+	AMX, CTX, CIP, TIC, SXT, NAL, OFX	CTX-M-14
16	Urine	B2	+	AMX, CTX, CAZ, CIP, TIC, SXT, NAL, OFX	CTX-M-15
17	Urine	B2	+	AMX, CTX, CAZ, CIP, TIC, SXT, OFX, NAL, GMI, TMN	CTX-M-15
18	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX, GMI	CTX-M-15
19	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX	CTX-M-15
20	Urine	B2	+	AMX, CTX, CAZ, CIP, TIC, NAL, OFX	CTX-M-15
21	Urine	B2	+	AMX, AMC, CTX, CIP, TIC, SXT, NAL, OFX	CTX-M-15
22	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX, GMI	CTX-M-15
23	Urine	B1	−	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX, GMI	CTX-M-15/AmpC
24	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX, GMI	CTX-M-15
25	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, NAL, OFX, GMI	CTX-M-15
26	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, NAL, OFX, GMI	CTX-M-15
27	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX	CTX-M-15
28	Urine	A	−	AMX, AMC, CTX, CAZ, FOX, TIC, OFX	CTX-M-15/AmpC
29	Urine	B2	+	AMX, CTX, CAZ, FOX, CIP, TIC, NAL, OFX	CTX-M-15/AmpC
30	Urine	D	−	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, SXT, NAL, OFX, TMN	CTX-M-15/AmpC/DHA-4
31	Urine	A	−	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, OFX, GMI, TMN	CTX-M-15
32	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX, GMI	CTX-M-15
33	Urine	B2	−	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX, GMI	CTX-M-15
34	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, NAL, OFX, GMI	CTX-M-15
35	Urine	B1	−	AMX, AMC, CTX, CAZ, TIC, SXT, OFX	CTX-M-15
36	Urine	B2	−	AMX, AMC, CTX, CAZ, CIP, TIC, SXT, FOX	CTX-M-15
37	Urine	D	−	AMX, AMC, CTX, CAZ, TIC, SXT	CTX-M-14
38	Urine	B2	+	AMX, AMC, CTX, CAZ, CIP, TIC, NAL, OFX, TMN	CTX-M-15
39	Urine	B1	−	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, SXT, OFX, GMI, TMN	AmpC/CMY-2
40	Urine	B1	−	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX	AmpC/CMY-42
41	Urine	B2	+	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX	AmpC/CMY-42
42	Urine	B1	−	AMX, AMC, CTX, CAZ, FOX, CIP, TIC, NAL, OFX, TMN	AmpC/CMY-42

Molecular characterization of β-lactamase genes

The results of β-lactamase genes identified in our study are shown in Table 1. Several β-lactamases encoding genes were detected among our microbial drug resistance (MDR) E. coli clinical isolates, noting the dominance of the bla_CTX-M-15 gene (n = 36). In contrast, the bla_CTX-M-14 gene was observed in only two isolates. On the contrary, the class C β-lactamases encoding genes (bla_ampC, bla_DHA-4, bla_CMY-2, and bla_CMY-42) were found in our cefoxitin-resistant isolates. Moreover, an association between the bla_CTX-M-15 gene and other class C β-lactamases encoding genes was noted in some isolates (bla_CTX-M-15 + bla_ampC [n = 5]; bla_CTX-M-15 + bla_ampC + bla_CMY-42 [n = 4]; bla_CTX-M-15 + bla_DHA-4 + bla_ampC [n = 1]). Besides, an association was observed between bla_ampC and bla_CMY-2 in one isolate, and between bla_ampC and bla_CMY-42 in three isolates. However, bla_SHV, bla_TEM, and bla_OXA-1 genes were not detected in this study.

Phylogenetic group

As described in Table 1, the majority of our MDR isolates belonged to the phylogenetic group B2 (n = 32), all of them harbored the bla_CTX-M-15 gene except two isolates, one carried the bla_CTX-M-14 gene and the second harbored only the class C β-lactamases encoding genes, bla_ampC and bla_CMY-42. Whereas only five isolates belonged to the phylogenetic group B1, of which three were negative for bla_CTX-M-15 gene and they harbored only the class C β-lactamases encoding genes (bla_ampC + bla_CMY-2 [n = 1], bla_ampC + bla_CMY-42 [n = 2]). Furthermore, our results revealed that three isolates belonged to the D phylogenetic group, of which two isolates carried the bla_CTX-M-15 gene in association with bla_DHA-4 and/or bla_ampC. However, the remaining other isolate harbored only the bla_CTX-M-14 gene. We have also noted that only two isolates belonged to the A phylogenetic group and were identified as CTX-M-15 producers.

O25b-ST131 E. coli clone detection

As given in Table 1, a high prevalence of O25b-ST131 E. coli clone was noted in this study. In fact, the majority of our MDR E. coli B2 isolates belonged to the ST131 clone and the O25b serotype (30/32). Moreover, all our O25b-ST131 isolates carried the bla_CTX-M-15 gene, except two isolates, one harbored only the bla_CTX-M-14 gene, and the second carried bla_ampC and bla_CMY-42 genes coding for class C β-lactamases.

Discussion

The dissemination of ESBLs encoding genes in Enterobacteriaceae, especially in E. coli, constitutes a major health public concern. Indeed, the emergence of ESBLs among E. coli leads to the reduction of therapeutic options because of their resistance to several antibiotic families, which allows them to be qualified as multidrug-resistant bacteria.^21,22 Thus, E. coli is the most common pathogen involved in urinary tract infections and constitutes one of the leading causes of morbidity.^21,23 Recently, it has been shown that 63% of urinary tract infections are associated with E. coli and 67% of them are resistant to third-generation cephalosporins.²⁴ Besides, the predominance of E. coli ST131 in urinary tract infections has been clearly shown. Thus, it was qualified as an uropathogenic and pandemic clone worldwide. This clone was also implicated in the successful widespread of antibiotic resistance determinants and the ESBLs.^3,5,8 In our study, we have reported the dominance of O25b-ST131 E. coli clone detected in clinical urine samples. This clone usually belongs to the B2 phylogenetic group and the O25b serotype,²⁰ and it is considered as a high-risk clone.¹⁰ Moreover, a global surveillance study revealed that ST131 accounts for 50% to 60% of ESBL-producing E. coli isolates.²⁵

In our study, we have noted that only two B2 E. coli isolates did not belong to the ST131 clone. However, we have recorded that our non-ST131 isolates belonged to the other phylogenetic groups A, B1, and D. To the best of our knowledge, virulent extraintestinal isolates belonged mainly to B2 and D groups, whereas the majority of commensal isolates were attributed to the A and B1 phylogenetic groups.¹⁹

Several ESBL types have been reported in E. coli clinical isolates, including CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14, and CTX-M-15.^8,26–28 Besides, it has been shown that E. coli ST131 clone was strongly implicated in the spread of CTX-M-15 enzyme.⁶ In this study, we have detected only two CTX-M types, CTX-M-15 and CTX-M-14. Most of our isolates carried the bla_CTX-M-15 gene, whereas the bla_CTX-M-14 gene was detected only in two isolates. Similarly, several other studies have shown that the prevalence of the CTX-M enzyme has increased over time with the dominance of CTX-M-15 type in the most regions of the world except few countries such as China, Southeast Asia, South Korea, Japan, and Spain where CTX-M-9, particularly the CTX-M-14 enzyme, is the most dominant, and South America where the CTX-M-2 is the most prevalent.²⁹

A previous Tunisian study showed the dominance of the bla_CTX-M-15 gene among E. coli clinical isolates, in contrast to the bla_CTX-M-14 gene, which was scarcely reported in Tunisia.³⁰ In fact, it was first detected in 2007 among E. coli isolated from food samples.³¹ Then, it was described among E. coli clinical isolates in 2011 in our country.³² To our knowledge, this study is the first report of CTX-M-14 producing O25b-ST131 E. coli in Tunisia. Indeed, this clone is commonly detected as a CTX-M-15 producer,^11,20 but there are few data describing the production of CTX-M-14 enzyme by this clone that has been recently reported in northern-eastern Europe³³ and Taiwan.³⁴

Moreover, the β-lactamases encoding genes, detected among our E. coli clinical isolates, are less diversified compared with those detected among Klebsiella pneumoniae species in this regional hospital, as described in our two recent previous data.^35,36 In fact, in this study, we have noted the absence of the bla_SHV, bla_TEM, and bla_OXA-1 genes among our isolates. However, we have detected other genes coding for class C β-lactamases, which are bla_ampC, bla_CMY-2, bla_CMY-42, and bla_DHA-4. The bla_ampC gene is frequently detected in E. coli, and it is characterized by an attenuated promoter that causes the production of this enzyme with a low level. Nevertheless, some clinical isolates carrying this gene became resistant to various β-lactam antibiotics by hyperproduction of this enzyme because of mutations in its promoter.¹⁵

In addition, CIT type enzymes, including the CMY-2 variant, are generally plasmid-borne and originate from Citrobacter freundii and Enterobacter spp.³⁷ Moreover, CMY-2 is considered the most widespread ESBL belonging to the CIT group enzymes. Indeed, it has been found in various Gram-negative species, including E. coli, K. pneumoniae, and Pseudomonas aeruginosa.^38–40 However, in this study, we have detected only one CMY-2-producing isolate. Besides, the remaining CMY producers harbored the CMY-42 encoding gene. The CMY-2 differs from CMY-42 enzyme by only one amino acid substitution that leads this latter to hydrolyze third-generation cephalosporins more efficiently than CMY-2 enzyme.³⁵ The CMY-42 enzyme is uncommon in Tunisia. It has been recently reported for the first time in our country among only one K. pneumoniae clinical isolate recovered in this area,³⁵ and it has not been hitherto detected in any other region of this country. Our study reported the first detection of CMY-42-producing E. coli in Tunisia.

Of interest, we have revealed the bla_DHA-4 gene in one isolate. This gene was derived from Morganella morganii,³⁷ but it was also detected among other Enterobacteriaceae species. In Tunisia, this gene is still scarcely reported.³⁸ To the best of our knowledge, the DHA-4 variant is never detected in our country. Thus, this study is the first report of this enzyme in Tunisia.

This geographical variation of resistance mechanisms could be explained by the variability of epidemiological factors, antibiotic use policies, and hospital hygiene measures between the different Tunisian health care settings. This study allowed us to discover the different genes involved in resistance to cephalosporins among E. coli clinical isolates in the southeast of Tunisia. Thus, we have noted a different molecular resistance pattern compared with those of the other regions of our country. Indeed, several β-lactamases encoding genes were identified among our clinical isolates recording the dominance of the O25b-ST131 E. coli clone implicated in urinary tract infections. The increasing dissemination of ESBL production by this clone is a major public health problem. Therefore, preventive strategies should be established to minimize the spread of ESBL-producing bacteria in our health care units.

Footnotes

Acknowledgments

The authors thank the staff of the laboratory of the regional hospital of Djerba for their helpful assistance to obtain the clinical isolates.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was partially supported by the Tunisian Ministry of Higher Education and Scientific Research.

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