High Prevalence of CTX-M-15-Producing O25b-ST131 Escherichia coli Clone in Bulgarian Hospitals

Abstract

According to the European Antimicrobial Resistance Surveillance System project results, Bulgaria has become one of the European countries with dramatically increasing rates of extended-spectrum beta-lactamase (ESBL) producers. The aim of this work was to investigate the epidemiology of ESBL-producing Escherichia coli clinical isolates in Bulgaria, collected from seven clinical centers in three towns, during two study periods: 2002–2003 and 2006–2009. For 193 ESBL-producing E. coli isolates random amplified polymorphic DNA (RAPD) analyses, phylogenetic typing, and screening for O25b-ST131 isolates were carried out. Antimicrobial susceptibility, ESBL-type and transferability of resistance determinants were analyzed. Four different ESBL-types, namely TEM-139, SHV-12, CTX-M-3, and CTX-M-15 were found. CTX-M-15 dominated, being found in 88% of the isolates. RAPD-typing revealed 35 types, among which type A dominated, comprising 65% of the isolates. Sixty-eight percent of the 193 isolates belonged to the O25b-ST131 clone, to the phylogenetic group B2, mostly showed RAPD-type A (92%) and were found in all participating hospitals. O25b-ST131 isolates predominantly produced CTX-M-15 (96%), and less SHV-12 (n=3) or TEM-139 (n=2). In conclusion, this study demonstrated for the first time the country-wide dissemination of a highly resistant B2 O25b-ST131 CTX-M-15 producing E. coli clone in Bulgaria.

Introduction

Since the end of the 20th century CTX-M-type extended-spectrum beta-lactamases (ESBLs) have replaced TEM- and SHV-variants in many countries.^4,9,15,23 The first CTX-M-type ESBLs were characterized by a 4 to 16 times higher activity against cefotaxime than against ceftazidime,³ but recently CTX-M-type beta-lactamases with enhanced activity against ceftazidime were detected. Among them, CTX-M-15, first described by Karim et al.,¹³ became the most frequent ESBL and has been found worldwide.^3,9,15

Recently, it has been reported, that CTX-M-15 producing Escherichia coli isolates most often belong to the globally disseminated, multiresistant and highly virulent O25b-ST131 lineage and are responsible for a wide range of community- and hospital-acquired infections, causing significant morbidity and mortality rates.^20–22,25 This lineage has been detected in Canada, Asia,^10,24,25 the United States,¹² South America,²⁶ South Africa²² and in Europe.^21,25

In Bulgaria, similar to the worldwide trend, the prevalence of CTX-M producing E. coli has increased since 2001.^16,17 The European Antimicrobial Resistance Surveillance System (EARSS) annual report 2008 (www.ecdc.europa.eu/en/activities/surveillance/EARS-et/Documents/2008_EARSS_Annual_Report.pdf) for Bulgaria showed increased rates of clinically significant E. coli isolates, mostly from bacteremia, resistant to oxyimino-cephalosporins from 7% in 2001 to 29% in 2008. This worrying finding prompted more detailed investigations.

The aim of the present work was to investigate ESBL-producing E. coli, isolated from seven clinical centers in three of the most populated Bulgarian towns–Sofia, Varna, and Pleven, during two time periods–2002–2003 and 2006–2009, regarding their spectrum of ESBLs, phylogenetic types, genetic relatedness, and affiliation to the O25b-ST131 lineage.

Materials and Methods

Bacterial isolates

A total of 55 clinically significant and nonduplicate E. coli isolates, presumptively identified as ESBL-producers by the participating centers using the double disk synergy method,¹¹ were collected between 2006 and 2009. The ESBL-production was confirmed by the CLSI ESBL disk confirmatory method.⁸ Another 138 strains, which were collected between 2002 and 2003 and previously studied for their antibiotic susceptibility, type of ESBL, and transferability of resistance determinants,¹⁷ were further characterized in this study for epidemiological purposes. The isolates were recovered from patients of seven teaching hospitals located in three Bulgarian towns: 1- Pleven (no. of isolates: 88); 2- Sofia I (32); 3- Sofia II (27); 4- Sofia III (19); 5- Varna (16); 6- Sofia IV (6); 7- Sofia V (5). The isolates were from urine (no. of isolates: 104), respiratory tract secretion (18), wounds (48), blood and central venous catheter (16), and others (7). They were collected from patients hospitalized in the following wards: urology/nephrology (no. of isolates: 85), intensive care units (30), neonatology (10), surgery (25), pediatrics (10), internal wards (35); nine isolates have been recovered from ambulatory patients.

Species identification was done by API 20E or Mini API 20E (bioMérieux, Marcy l'Etoile, France) or Crystal Enteric/NF identification systems (Becton Dickinson, Sparks, MD).

Antimicrobial susceptibility testing and conjugation experiments

Antimicrobial susceptibility was determined by the disc diffusion method according to the CLSI guidelines.⁸ Eighty nine E. coli isolates, representing different centers, susceptibility types, and beta-lactamase groups, were selected for conjugational transfer experiments on solid media as previously described.¹⁷

Identification of ESBLs

Beta-lactamase production was analyzed by isoelectric focusing (IEF) followed by a bioassay as previously described.^1,18

Polymerase chain reaction (PCR) was performed to detect the beta-lactamase genes of the SHV-, TEM-, CTX-M, and OXA-groups.¹⁷ Nineteen isolates (6 from 2006 to 2009 and 13 from 2002 to 2003) representative for hospital centers, beta-lactamase groups, and random amplified polymorphic DNA (RAPD)-types were selected for sequencing of their bla genes as previously described.¹⁷

Typing of isolates and screening for the O25b-ST131 lineage

Whole-cell DNA was prepared using the GFX Genomic DNA Purification Kit (Amersham Biosciences, Little Chalfont, United Kingdom) for RAPD analysis with ERIC 1 and ERIC 2A primers.²⁹ Strains showing up to two band differences were interpreted as clonally related.

The main phylogenetic groups (A, B1, B2, and D) were detected by multiplex PCR,⁶ using the following temperature regimen: 94°C for 5 min; 30 cycles of 94°C for 10 sec, 58°C for 10 sec, 72°C for 30 sec, and a final extension step of 5 min at 72°C.

Screening for isolates of the O25b-ST131 lineage was done by PCR, using the primers O25pabBspe.F and O25pabBspe.R, which amplify a 347 bp fragment of the pabB gene specifically present in isolates belonging to the O25b-ST131 clone according to Clermont et al.⁶

Statistical analysis

Differences in the resistance rates of O25b-ST131 E. coli versus the rest of the isolates were assessed with chi-square test or Fisher's exact test.

Results

Characteristics of ESBLs

IEF revealed beta-lactamases with isoelectric points (pIs) of 5.4, 6.3, 7.4, 8.2, 8.4, and 8.8. Taking into consideration their ceftazidime- or cefotaxime-hydrolyzing capacity, as determined by bioassay, the enzymes were assigned into different groups of beta-lactamases. The ESBL-group-specific PCR and sequencing revealed the presence of four different ESBLs: SHV-12 (pI 8.2), TEM-139 (pI 6.3), CTX-M-3 (pI 8.4), and CTX-M-15 (pI 8.8). The majority of the isolates produced additionally TEM-1 (pI 5.4). The beta-lactamase with pI 7.4 was identified as OXA-1 and was found in almost all CTX-M-15 producers; however, it was rarely present in E. coli producing CTX-M-3, SHV-12, or TEM-139. Most of the isolates (88%) were CTX-M-15-producers (Tables 1 and 2) which persisted during the entire study period and which were present in all seven centers, accounting for 84% to 93% of the isolates (data not shown). SHV-12 and CTX-M-3 producers sporadically appeared in five centers and TEM-139 producing E. coli were limited to two centers and to the first part of the study period (2002–2003) (Table 2).

Table 1.

Characteristics of Random Amplified Polymorphic DNA-Types

RAPD-type ^a	ESBL-type ^a	Center of isolation	Years of isolation of O25b-ST131 PCR positive isolates	Phylogenetic type ^a
A (121/126)	CTX-M-15 (103/107)	1–7	2002,2003,2006–2009	B2 (103/106); A (0/1)
	CTX-M-15 + SHV-12 (14/14)	2, 3, 4	2002,2003	B2 (14/14)
	CTX-M-3 (0/1)	2		B2 (0/1)
	SHV-12 (2/2)	2	2003	B2 (2/2)
	TEM-139 (2/2)	1, 6	2003	B2 (2/2)
B (0/9)	CTX-M-15 (0/9)	1		A (0/9)
D (0/5)	CTX-M-15 (0/4)	3		A (0/4)
	SHV-12 (0/1)	3		A (0/1)
F (0/5)	CTX-M-15 (0/3)	4		A (0/3)
	CTX-M-15 + SHV-12 (0/1)	4		A (0/1)
	CTX-M-3 (0/1)	4		A (0/1)
S (6/6)	CTX-M-15 (6/6)	2	2008	B2 (6/6)
30 minor RAPD-types (5/42)	CTX-M-15 (2/22)	1–7	2003	B2 (2/6); A (0/15); D (0/1)
	CTX-M-15 + SHV-12 (2/3)	3, 4	2003	B2 (2/2); A (0/1);
	CTX-M-3 (0/9)	1, 2, 4, 5		B2 (0/1); A (0/7); D (0/1)
	SHV-12 (1/6)	2, 3, 5, 6	2003	B2 (1/1); A (0/5);
	TEM-139 + CTX-M-3 (0/2)	6		B2 (0/1); A (0/1);

In parentheses: number of O25b-ST131-isolates (in bold)/number of all isolates.

ESBL, extended-spectrum beta-lactamase; RAPD, random amplified polymorphic DNA; PCR, polymerase chain reaction.

Table 2.

Chronology of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli

Year of isolation ^a	Five major RAPD-types ^a	Phylogenetic type ^a	ESBL-types ^b
2002 (38/55)	A (37/37)	A (0/15)	CTX-M-15 (35/46)
	B (0/3)	B2 (38/39)	CTX-M-15 + SHV-12 (3/5)
	F (0/3)	D (0/1)	CTX-M-3 (0/2)
	D (0/3)		SHV-12 (0/1)
			CTX-M-3 + TEM-139 (0/1)
2003 (62/83)	A (58/61)	A (0/15)	CTX-M-15 (44/57)
	B (0/2)	B2 (62/68)	CTX-M-15 + SHV-12 (13/13)
	F (0/2)		CTX-M-3 (0/6)
			SHV-12 (3/4)
			TEM-139 (2/2)
			CTX-M-3 + TEM-139 (0/1)
2006 (4/10)	A (4/4)	A (0/6)	CTX-M-15 (4/8)
		B2 (4/4)	CTX-M-3 (0/1)
			SHV-12 (0/1)
2007 (2/7)	A (2/2)	A (0/4)	CTX-M-15 (2/6)
	B (0/4)	B2 (2/3)	CTX-M-3 (0/1)
2008 (16/24)	A (10/12)	A (0/5)	CTX-M-15 (16/22)
	D (0/1)	B2 (16/18)	CTX-M-3 (0/1)
	S (6/6)	D (0/1)	SHV-12 (0/1)
2009 (10/14)	A (10/10)	A (0/4)	CTX-M-15 (10/12)
	D (0/1)	B2 (10/10)	SHV-12 (0/2)

In parentheses: number of O25b-ST131-isolates (in bold)/all isolates.

ESBL-types for period 2002–2003 have been previously published.¹⁷

RAPD typing

Epidemiological typing by RAPD identified a dominating pattern (RAPD-type A) in 126 isolates (65%) which persisted from 2002 to 2009 and in all hospitals (Tables 1 and 2). The majority of RAPD-type A isolates (n=121) produced CTX-M-15. Four smaller RAPD-types (B, D, F, and S) comprised 5 to 9 isolates and were restricted to one center each. Additionally, 30 minor types, consisting of one to three isolates were found (Table 1).

Phylogenetic typing

The assignment of the isolates to phylogenetic groups by multiplex PCR revealed the presence of three groups. Seventy-four percent (n=142) of the isolates were positive for the chuA, yjaA, and tspE4.C2 genes classifying them into phylogenetic group B2. All but one isolates with RAPD-pattern A (99%) and all isolates with RAPD-type S belonged to group B2. Members of that group could be found in all centers at rates between 42% and 86% (data not shown), during both study periods and predominantly produced CTX-M-15 (134 of 142) (Table 2). Members of the phylogenetic group A accounted for 25% of the studied strains, they were found in all centers and during the entire investigational period, and belonged to the RAPD-types B, D, and F.

Additionally, two isolates were assigned to the phylogenetic group D (Table 1).

Screening for O25b-ST131 E. coli

Among the 193 ESBL-producing E. coli, 132 (68%) gave positive results with the O25b-ST131-specific PCR. Members of this clonal complex were found over the entire study period at rates ranging from 29% to 75% and were present in all participating centers (Table 1). They were isolated mainly from urine samples (no. of isolates: 74), and also from wound secretions (31), blood and central venous catheters (13), lower respiratory tract secretions (12), and bile (2).

As expected, all of the O25b-ST131 E. coli strains were members of the phylogenetic group B2. Conversely, 10 of the B2 isolates and the isolates of the phylogenetic group A and D, did not belong to the O25b-ST131 lineage (Table 1).

Almost all E. coli, showing RAPD-type A (121 of 126), were O25b-ST131-positive (Table 1). In addition, the six members of RAPD-type S, and five other isolates with unique RAPD-profiles, were identified as O25b-ST131 E. coli. In contrast, none of the isolates of the RAPD-types B, D, and F, all assigned to the phylogenetic group A, belonged to the O25b-ST131-lineage.

Ninety six percent of the isolates (127 of 132), assigned to the O25b-ST131 lineage, produced CTX-M-15 (16 of them in combination with SHV-12), two harbored bla_TEM-139 and three bla_SHV-12 genes.

Antimicrobial susceptibility and transferability of resistance determinants

Analysis of the susceptibility of all 193 E. coli isolates demonstrated high resistance rates (including resistant and intermediate isolates) for cefotaxime (100%), ceftazidime (92%), amoxicillin/clavulanic acid (78%), tobramycin (90%), gentamicin (80%), ciprofloxacin (83%), and tetracycline (87%). The resistance rates for sulfamethoxazole/thrimethoprim and chloramphenicol were 41% and 40%, respectively. Carbapenems and amikacin were the most active compounds with susceptibility rates of 100% and 81%, respectively. The susceptibility of isolates of both investigational periods, 2002–2003 and 2006–2009, revealed no significant differences (data not shown). Comparison of the resistance rates of O25b-ST131 E. coli versus the rest of the isolates showed statistically significant differences for amoxicillin/clavulanic acid (99% vs. 76%; p<0.0001) and ciprofloxacin (96% vs. 69%; p<0.0001). In contrast, for sulfamethoxazole/thrimethoprim O25b-ST131 E. coli showed lower resistance rates (44% vs. 69%; p=0.006).

The analysis of 66 transconjugants (40 from 2002 to 2003 and 26 from 2006 to 2009) demonstrated that genes conferring resistance to tobramycin, gentamicin, and tetracycline were most often co-transferred with the ESBL-genes, respectively in 63%, 55%, and 53% of the cases. Transfer of resistance determinants for amikacin (29%), sulfamethoxazole/thrimethoprim (23%), and chloramphenicol (17%) was less frequent. Interestingly, for none of the CTX-M-15-producers, resistance determinants for amikacin were co-transferred, while those for tetracycline were exclusively found in CTX-M-15 producing transconjugants. Additionally, the genes encoding OXA-1 were co-transferred with bla_CTX-M-15 in all cases.

Discussion

This study provides epidemiological data on ESBL-producing E. coli, isolated in seven hospital centers in three of the most populated Bulgarian towns–Sofia, Varna, and Pleven.

The ESBL-types identified during the period 2006 to 2009 were identical to those previously described (2002–2003),¹⁷ namely SHV-12, CTX-M-3, and CTX-M-15, except that TEM-139 has been detected during the first period only. CTX-M-15 was by far the dominating ESBL-type found at high rates during both study periods and in all centers.

Among our isolates we observed three phylogenetic groups A, D, and B2, and similar to other studies, the majority of the isolates (74%) belonged to the phylogenetic group B2, which is known to be associated with extraintestinal pathogenic E. coli.^{6,14,20,21,30}

Recent studies reported an ongoing spread of a CTX-M-15 producing O25b-ST131 E. coli clone, belonging to the phylogenetic group B2, which often coproduces the beta-lactamases OXA-1 and TEM-1 and the aminoglycoside acetyltransferase aac(6’)-Ib-cr.^20,21,25 Among our ESBL-producing E. coli the rate of O25b-ST131 isolates was higher (68%) than those found in France (46%),⁷ the Central African Republic (50%),⁷ and in Canada (50.3%)²⁷ and differed greatly from that found in Spain (20% to 23%).²

In the present study, the O25b-ST131 lineage isolates were mostly associated with the dominating RAPD-type A. Only 11 isolates representing five other RAPD-types were also O25b-ST131-PCR-positive. The members of O25b-ST131 lineage among Bulgarian isolates were present over the entire study period and in all centers. Their majority (96%) produced CTX-M-15, which is in concordance with other investigations.^14,20,21,25 However, some O25b–ST131 E. coli produced SHV-12 or the only locally detected TEM-ESBL TEM-139. The linkage of O25b-ST131 E. coli with other ESBL-types, for example, CTX-M-3, CTX-M-14, several TEM-ESBLs, and recently SHV-12, at lower numbers, has been shown by other investigators too.^5,7,19 To the best of our knowledge, the production of TEM-139 by O25b-ST131 E. coli has not been shown before.

In contrast to RAPD-type A, the members of the RAPD-types B, D, and F were assigned to the commensal phylogenetic group A. They did not belong to the O25b–ST131 clone, and appeared at lower numbers and remained restricted to single centers.

Other authors have concluded that bla_CTX-M-15 have a greater potential for mobilization and dissemination.^2,3 From our study, it is likely that the predominance of CTX-M-15 producers among Bulgarian ESBL-positive E. coli is mainly due to the wide dissemination of the O25b-ST131 E. coli clonal complex. Such isolates were detected in all seven studied centers at rates, ranging from 26% to 82% (data not shown). Interestingly, the association of SHV-12 and TEM-139 with this clonal complex was restricted to the first investigational period (up to 2003), and no CTX-M-3 producers belonged to it. In the second study period (2006–2009) O25b-ST131 E. coli exclusively produced CTX-M-15.

The isolates analyzed in this study showed high resistance rates for amoxicillin/clavulanic acid, oxyimino-cephalosporins, tobramycin, gentamicin, ciprofloxacin, and tetracycline. This is in concordance with recent studies demonstrating that most often ESBL-producing E. coli are resistant to different groups of antimicrobials, particularly ciprofloxacin, tetracycline, and aminoglycosides.^24,28 Isolates belonging to the O25b-ST131 lineage displayed higher resistance rates for several compounds in comparison to non-O25b-ST131 isolates, thereby limiting further treatment options. The determinants conferring resistance to tobramycin, gentamicin, and tetracycline were most often co-transferred with the ESBL-gene. These facts imply an increased risk for selecting ESBL-producers by the usage of those antimicrobials besides oxyimino-cephalosporins. In contrast, a significantly lower resistance rate for amikacin was observed for CTX-M-15 producers and no resistance determinants for amikacin were linked with the bla_CTX-M-15 gene.

In conclusion, this study demonstrates for the first time the countrywide, intra- and interhospital dissemination of CTX-M-15 producing B2 O25b-ST131 E. coli strains in Bulgaria. This worrying development underlines the need for continuing surveillance of the local epidemiology and for the implication of strong measures like more active prudent antibiotic policy and control of infections to prevent further spread of ESBL-producers.

Footnotes

Acknowledgment

This work was partially supported by grants from the Medical University, Sofia, Bulgaria (N 23/2009) (R.M.).

Disclosure Statement

No conflict of interest.

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