Molecular characterization of Escherichia coli producing extended-spectrum β-lactamase CTX-M-14 and CTX-M-28 in Mexico

Abstract

The spread of ESBL-producing Escherichia coli has constantly increased in both clinical and community infections. Actually, the main ESBL reported is the CTX-M family, which is widely disseminated between the Enterobacteriaceae family. The epidemiology of the CTX-M family shows the CTX-M-15 variant dominating worldwide, followed by CTX-M-14 and CTX-M-27. The specific ESBL-producing E. coli clones included mainly the sequence types ST131, ST405, and ST648. In this report, we present the molecular characterization of ESBL-producing E. coli clinical isolates from eight hospitals in Mexico. From a collection of 66 isolates, 39 (59%) were identified as bla_CTX-M-14 and bla_CTX-M-27 belonging to the group CTX-M-9. We identified 25 (38%) isolates, producing bla_CTX-M-28 belonging to the group CTX-M-1. bla_CTX-M-2 and bla_TEM-55 were identified in one isolate, respectively. Fourteen isolates (21%) were positive for bla_CTX-M-14 (13%) and bla_CTX-M-28 (7.3%) that were selected for further analyses; the antimicrobial susceptibility showed resistance to ampicillin (> 256 µg/mL), cefotaxime (> 256 µg/mL), cefepime (> 64 µg/mL), and ceftazidime (16 µg/mL). The ResFinder analysis showed the presence of the antimicrobial resistance genes aacA4, aadA5, aac(3)lla, sul1, dfrA17, tet(A), cmlA1, and bla_TEM-1B. PlasmidFinder analysis identified in all the isolates the replicons IncFIB, which were confirmed by PCR replicon typing. The MLST analysis identified isolates belonging to ST131, ST167, ST405, and ST648. The ISEcp1B genetic element was found at 250 pb upstream of bla_CTX-M-14 and flanked by the IS903 genetic element at 35 pb downstream. The IS1380-like element ISEc9 family transposase was identified at 250 pb upstream of bla_CTX-M-14 and flanked downstream by the IS5/IS1182 at 80 pb. Our study highlights the significant prevalence of CTX-M-14 and CTX-M-28 enzymes as the second-most common ESBL-producing E. coli among isolates in Mexican hospitals. The identification of specific sequence types in different regions provides valuable insights into the correlation between ESBL and E. coli strains. This contribution to understanding their epidemiology and potential transmission routes is crucial for developing effective strategies to mitigate the spread of ESBL-producing E. coli in healthcare settings.

Supplementary information

The online version contains supplementary material available at 10.1007/s42770-023-01183-z.

Introduction

Antimicrobial resistance is a global health concern, significantly limiting treatment options for severe infections [1]. The use of broad-spectrum antibiotics, such as cephalosporins, has led to the emergence of resistance-conferring enzymes known as extended-spectrum β-lactamases (ESBLs) [2]. The first ESBL, bla_SHV, capable of hydrolyzing extended-spectrum cephalosporins, emerged in 1983. For several years, some ESBL variants of bla_TEM and bla_SHV types were prevalent among hospital-acquired organisms [3, 4].

In the last two decades, the number of bla_CTX-M variants has increased, with the bla_CTX-M family now widely disseminated within the Enterobacteriaceae family [5, 6]. Currently, there are more than 265 bla_CTX-M variants, primarily clustered into five groups: CTX-M-1, 2, 8, 9, and 25. These variants are often geographically region-specific, and their global distribution remains limited. Nevertheless, the CTX-M group members remain the most commonly reported ESBLs worldwide [7–9].

The epidemiology of the CTX-M family indicates that CTX-M-15 is the most prevalent variant, followed by CTX-M-3 and CTX-M-1 (from the CTX-M-1 group). The CTX-M-14 is the most prevalent variant, followed by the CTX-M-9 and, with increasing frequency, the CTX-M-27 variant (from the CTX-M-9 group). In the case of the CTX-M-2, CTX-M-8, and CTX-M-25 groups, their frequencies are low, and their representative variants are the same as their respective groups [9–11]. Approximately 80% of identified variants belong to CTX-M groups 1 and 9. The high prevalence of these groups, mainly due to specific antibiotic exposures like cefotaxime and ceftazidime in clinical settings, drives the rapid evolution of these enzymes [6, 12].

The bla_CTX-M-15 variant has become the most prevalent within the CTX-M family of ESBL identified in clinical settings. Its high prevalence is often associated with resistance against third-generation cephalosporin, which can complicate the treatment of bacterial infections. In contrast, other CTX-M family members with lower frequencies, such as bla_CTX-M-9, bla_CTX-M-14, bla_CTX-M-27, and bla_CTX-M-28 are less commonly identified but still contribute to the global challenge of antibiotic resistance. They often emerge in specific geographic regions or healthcare settings, emphasizing the importance of surveillance to monitor and mitigate their spread [7–11]. The worldwide spread of ESBL production has continually increased over the last decade, particularly within the Enterobacteriaceae family and Escherichia coli. This is a critical concern for healthcare systems due to the rising number of hospital and community infections caused by multidrug-resistant bacteria [8, 10, 13]. Diverse epidemiology studies have demonstrated the increasing dispersion of specific ESBL-producing E. coli clones in most continents. These include the ST10 clonal complex with the sequence types ST38, ST131, ST315, ST393, ST405, and ST648. However, CTX-M-15 and CTX-M-14, which belong to ST131, ST405, and ST648, remain the most widely distributed enzymes among ESBL-producing E. coli [11, 14]. The primary mechanism for the dissemination of antimicrobial resistance is through plasmids, driven by the evolution of antibiotic resistance. In E. coli, specific plasmids harboring the ESBL CTX-M have been repeatedly identified, suggesting that some plasmids are well adapted to successful spread in specific ST clones, as reported for the pandemic clone ST131 [15, 16]. Several studies have shown a direct relation between the presence of the multidrug-resistant CTX-M-15 and CTX-M-14 genes and the identification of broad-host range plasmid replicon types, including IncF, IncI, IncN, IncHI2, IncM, and IncK [11, 14, 15]. The goal of this study was the molecular characterization of CTX-M ESBL-producing E. coli isolated from various regions of Mexico, excluding those producing the most prevalent bla_CTX-M-15.

Materials and methods

Specimen collection and identification

From a total of 547 ESBL-producing E. coli clinical isolates collected between 2010 and 2018, a subset of 66 isolates that tested negative for the most common ESBL genes by PCR were included in the analysis. The isolates were collected from 8 hospitals from six of the eight regions (NW, NE, W, NC, SC, and SW) of Mexico: Hospital Universitario de Nuevo León, Nuevo León (n = 2); Instituto Nacional de Cancerología, Ciudad de México (n = 16); Hospital Civil de Guadalajara, Jalisco (n = 2); Hospital General de Acapulco, Guerrero (n = 2); ISSSTESON, Sonora (n = 1); Sanatorio Durango, Ciudad de México (n = 9); Hospital Central, San Luis Potosí (n = 21); and Hospital San Juan Tecnológico de Monterrey, Nuevo León (n = 13). The bacterial species identification and susceptibility pattern were initially detected using the Dade MicroScan system and were subsequently re-confirmed as E. coli by the VITEK 2 compact system (BioMérieux, Durham, USA).

Antimicrobial susceptibility testing

The phenotypic profile of ESBL production was detected using the double-disk synergism method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI) (M100-S30) [17]. The minimal inhibitory concentration (MIC) of Ampicillin, Aztreonam, Cefepime, Cefotaxime, Ceftazidime, Levofloxacin, and Gentamicin were determined by micro-dilution in broth, and the results of antimicrobial susceptibility testing were interpreted according to CLSI [17].

Molecular identification of ESBL

The CTX-M-1-group, CTX-M-2-group, CTX-M-8-group, CTX-M-9-group, and CTX-M-25-group were screened by single PCR with specific primers for each CTX-M group and confirmed by nucleotide sequencing [18]. The PCR products specified by nucleotide sequencing were purified by the commercial kit from Roche (Roche, USA) and sequenced by the BigDye Terminator v3.1 Cycle Sequencing Kit in the automated system ABIPrisma 3100 (Applied Biosystem, the USA). The Translate Tool (http://ca.expasy.org/tools/dna.html) was used for each nucleotide sequence to obtain the amino acid sequences, which were compared by BLASTp in the GenBank database (http://www.ncbi.nlm.nih.gov/).

Molecular epidemiology typing

To evaluate genetic diversity among the E. coli-ESBL isolates, we conducted random amplified polymorphic DNA (RAPD) analysis [19]. The similarity correlation was computed using the Dice coefficient, and a dendrogram was generated using the UPGMA clustering method. These analyses were performed within the GelCompar II program. The phylogenetic group of the E. coli isolates was performed using the triplex PCR for the chuA and yjaA genes and the DNA fragment TSPE4.C2, which allows classification into four different groups, as previously described by Clermont et al. [20]. The multilocus sequencing typing (MLST) was performed in the E. coli-ESBL-producing isolates using the MLST tools (https://enterobase.warwick.ac.uk) [21].

Plasmid analysis and mating experiments

Criteria used for isolate selection for mating experiments were ESBL producing, RAPD profile, and phylogroup. Mating assays for the horizontal transfer of ESBL resistance were performed using E. coli J53-2 (Met-, Pro-, Rif^r) as the recipient strain in solid-phase mating as described by Miller [22]. Transconjugants were selected on Luria–Bertani (LB) agar supplemented with rifampin (100 μg/mL) and nalidixic acid (8 µg/mL), ampicillin (100 μg/mL), or cefotaxime (1 μg/mL). All transconjugants were verified according to their auxotrophic requirements (Met- and Pro-), and plasmids were analyzed according to the method described by Kieser [23].

Plasmid replicon typing

Incompatibility groups were detected by PCR replicon typing. Specific primers were used, including HI1, HI2, I1, X, L/M, N, FIA, FIB, W, P, FIC, Y, FIIA, A/C, T, K, B/O, and F previously described by Carattoli et al. [24].

Plasmid sequencing and in silico analysis

Plasmid DNA was obtained from eight transconjugants and subsequently sequenced by the Genome Analyzer IIx System – Illumina. The assembly was obtained using the PHRED-PHRAP-CONSED and Newbler programs. The prediction of open reading frames (ORFs) was identified with the Glimmer3 and RAST programs and compared with the GenBank nr database. In silico plasmid analysis was performed using the Center for Genomic Epidemiology’s tools (https://cge.cbs.dtu.dk/) to identify antimicrobial resistance genes (ResFinder) and plasmid replicons (PlasmidFinder) [25].

Results

A subset of 66 (12%) clinical isolates, initially tested negative for the presence of bla_CTX-M-15, bla_SHV, bla_TEM, and bla_TLA using specific PCR screening, were included in this study. All isolates were re-confirmed as ESBL-producing E. coli; they were obtained from eight hospitals in six of the eight regions of Mexico (NW, NE, W, NC, SC, and SW).

A total of 39/66 (59%) ESBL-producing E. coli isolates were positive for the amplification by PCR of the CTX-M-9 group, while 25/66 (37%) were positive for the CTX-M-1 group. One isolate (1.5%) belongs to the CTX-M-2 group, and one isolate corresponds to the TEM group (1.5%). The genes’ sequences and the phylogenetic groups of the E. coli isolates were determined as part of the analysis. From the isolates positive again for the CTX-M-9 group, 37/39 isolates (94.8%) were identified as having the bla_CTX-M-14 gene, of which 35/37 (94%) were isolates belonging to phylogroup D, 1/37 (2.7%) were isolates belonging to phylogroup A, and 1/37 (2.7%) were isolates belonging to B1, respectively. Only 2/39 (5%) isolates were positive for the bla_CTX-M-27 and belong to phylogroup D. From the isolates positive for the CTX-M-1 group, 25/25 (100%) isolates were identified with the bla_CTX-M-28, with 14/25 (56%) isolates belonging to phylogroup A, 6/25 (24%) isolates to phylogroup B2, and 5/25 (20%) to phylogroup D, respectively. From the rest of the isolates, 1/66 (1.5%) of them had the bla_CTX-M-2, which belongs to phylogroup A, and 1/66 isolates (1.5%) had the bla_TEM-55, which belongs to phylogroup B1. In our analysis, it was observed that 93.8% of the isolates had bla_CTX-M-14 (56%) or bla_CTX-M-28 (37.8%). In order to have a representative characterization of the major CTX-M variants identified, fourteen isolates were selected and analyzed (Table 1). The antimicrobial susceptibility to different β-lactam antibiotics showed a resistance phenotype to ampicillin (> 256 µg/mL), cefotaxime (> 256 µg/mL), cefepime (> 64 µg/mL), ceftazidime (16 µg/mL), and aztreonam (> 16 µg/mL), and all showed susceptibility to gentamicin and levofloxacin. The susceptibility results for β-lactam antibiotics were consistent with the identification of ESBL-producing CTX-M.

Table 1.

Molecular characteristics of E. coli producing ESBL CTX-M-14 and CTX-M-28

Strain	Region	ESBL	MLST	Filogroup	Inc	pMLST	AMP	AZT	FEP	CTX	CAZ	LVX	GEN
01–208	West	CTX-M-14	ST-405Cplx	D	IncFIB	IncF [F31:A-:B6]	> 256	> 16	> 64	> 256	64	0.5	0.5
05–206	Northeast	CTX-M-14	ST-405Cplx	D	IncFIB	IncF [F36:A-:B6]	> 256	<  = 8	> 64	> 256	32	2	2
05–220	Northeast	CTX-M-14	ST-405Cplx	D	IncFIB	IncF [F31:A-:B6]	> 256	> 16	> 64	> 256	16	4	4
10–224	Center	CTX-M-14	ST-405Cplx	D	IncFIB	IncF [F31:A-:B12]	> 256	> 16	> 64	> 256	32	2	2
06–1299	Northeast	CTX-M-14	ST-405Cplx	D	IncFIB	IncF [F31:A-:B6]	> 256	> 16	> 64	> 256	> 256	1	1
14,237	Center	CTX-M-14	ST-405Cplx	D	IncFIB	IncF[F36:A-:B32]	> 256	> 16	> 64	> 256	128	4	4
09–270	Center	CTX-M-14	ST-648	D	IncFIB	IncF[F31:A-:B6]	> 256	> 16	> 64	> 256	32	2	2
03–224	Northwest	CTX-M-14	ST-648	D	IncFIB	IncF[F36:A-:B6]	> 256	> 16	> 64	> 256	> 256	1	1
16,234	Northeast	CTX-M-14	ST-405Cplx	D	IncFIB	NA	> 256	> 16	> 64	> 256	64	2	2
16,235	Northeast	CTX-M-28	ST-167	A	IncFIB	NA	> 256	> 16	> 64	> 256	64	1	1
10–214	Center	CTX-M-28	ST-405Cplx	D	IncFIB	NA	> 256	> 16	> 64	> 256	16	2	2
05–222	Northeast	CTX-M-28	ST-405Cplx	B2	IncFIB	NA	> 256	> 16	> 64	> 256	16	1	1
09–238	Center	CTX-M-28	ST-131Cplx	B2	IncFIB	NA	> 256	> 16	> 64	> 256	128	2	2
09–246	Center	CTX-M-28	ST-167	A	IncFIB	NA	> 256	> 16	> 64	> 256	64	2	2

AMP, ampicillin; AZT, Aztreonam; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; LVX, levofloxacin; GEN, gentamicin; NA, not applicable

According to the criteria used for isolate selection, nine isolates with bla_CTX-M-14 and five bla_CTX-M-28 were selected for mating experiments. Transconjugants containing the ESBL determinants were used for plasmid DNA extraction and complete sequencing. The successful complete nucleotide sequence of eight plasmids from the selected isolates was analyzed in silico by ResFinder, showing different antimicrobial resistance gene profiles in addition to bla_CTX-M-14, including bla_TEM-1B, aadA5, aac(3)lle, sul1, dfrA17, tet(A), and cmlA to confer resistance to B-lactams, aminoglycosides, macrolides, trimethoprim, tetracycline, and chloramphenicol. The resistome of the plasmid sequences in this work as well as the plasmid backbone closely related to our sequenced plasmids were included in Supplementary Table 1.

The PlasmidFinder analysis identified the replicons IncFIB in all isolates; the incompatibility groups IncF were confirmed by PCR replicon typing. The pMLST analysis identified three principal multilocus sequences: [F31:A-:B6], IncF [F31:A-:B12], and IncF [F36:A-:B6] (Table 1).

In order to identify the genetic relationship among E. coli ESBL producer isolates, multilocus sequencing typing was determined. The 9/37 (24%) selected isolates that produce bla_CTX-M-14 belong to phylogroup D, of which 7/9 isolates were identified as belonging to ST405 and 2/9 isolates were identified as belonging to ST648. Within the group of CTX-M-28-producing, from 5/25 (20%) selected isolates, 2/5 isolates belong to ST405, 2/5 to ST167, and one to ST131. We identified ST405 in all hospital samples collected in different years and regions of Mexico (Table 1).

The genetic context of the isolates 01–208, 03–224, 05–206, 06–1299, and 10–224 has the ISEcp1B genetic element at 250 pb upstream of bla_CTX-M-14 and flanked by the IS903 genetic element at 35 pb downstream of the cluster. The IS1380-like element belonging to the ISEc9 family transposase was identified at 250 pb upstream of bla_CTX-M-14 and flanked downstream by the IS5/IS1182 family transposase at 80 pb in the 05–220, 14237, and 09–270 isolates.

Discussion

The high prevalence of CTX-M-producing E. coli isolates is a significant concern, as they are associated with resistance to a wide range of β-lactam antibiotics, leading to limited therapeutic options [26]. While bla_CTX-M-15 is identified as the most common ESBL (88%), the frequency of isolates harboring bla_CTX-M-14 and bla_CTX-M-28 reflects their presence as the second-most common variants in E. coli strains producing ESBL in the analyzed Mexican hospitals. This finding underlines the significant contribution of the less frequent CTX-M variants to antibiotic resistance within specific geographic regions, emphasizing the importance of surveillance for monitoring and mitigating their spread.

Additionally, the phylogenetic group analysis revealed that most CTX-M-14 positive isolates belonged to group D, while CTX-M-28 positive isolates were mainly belonging to group A. These observations are consistent with previous reports, suggesting that specific phylogenetic groups within E. coli are more frequently associated with resistance to antibiotics and the production of ESBLs [27]. All isolates show resistance to multiple β-lactam antibiotics but remain susceptible to gentamicin and levofloxacin. This resistance pattern has been reported in other studies involving ESBL-producing E. coli isolates [28].

The MLST analysis of the isolates revealed the ST405 as the most common sequence type identified, which has been reported in several countries worldwide, suggesting a widespread dissemination of this successful clone [29]. The identification of ST405 in isolates from different years and regions of Mexico further emphasizes its dissemination potential.

The plasmid-mediated nature of ESBL genes is a major factor contributing to their rapid spread among bacterial populations [30]. Plasmid analysis in this study identified various resistance gene profiles, including bla_TEM-1B, aadA5, aac(3)lle, sul1, dfrA17, tet(A), and cmlA with the potential to confer resistance to a wide range of antimicrobial agents (Supplementary Table 1). The presence of multiple resistance genes on these plasmids may facilitate the co-selection and maintenance of resistance determinants in bacterial populations [31]. These findings correlate with the incompatibility group IncF, found in all isolates, highlighting its potential role in the dissemination of ESBL genes between Enterobacteriae [32].

The genetic context of the CTX-M-14-producing isolates revealed the presence of mobile genetic elements flanking the bla_CTX-M-14, such as ISEcp1B, IS903, and IS5/IS1182 family transposases. These mobile elements have been implicated in the mobilization and dissemination of resistance genes among different bacteria [33] . The presence of these genetic elements in the isolates highlights the potential for horizontal gene transfer and the spread of resistance determinants in the hospitals studied.

Conclusions

The identification and molecular characterization of ESBL-producing E. coli isolates analyzed in this work are distinct from the most prevalent CTX-M-15-producing strains. This has provided us with specific insight into how the ESBL correlates with specific sequence types in different regions of Mexico.

This study underlines the prevalence of ESBL-producing E. coli isolates in Mexican hospitals, with CTX-M-14 and CTX-M-28 enzymes being the second-most prevalent in Mexican clinical isolates. The identification of multidrug resistance, along with well-known mobile genetic elements such as ISEcp1B, IS5/IS1182, and successful clones like ST405, highlights the necessity for ongoing surveillance and the implementation of effective infection control measures to limit the spread of ESBL-producing E. coli isolates within healthcare settings. Understanding the molecular characteristics of these isolates is crucial for developing effective strategies to prevent antimicrobial resistance and increase patient outcomes in both clinical and community settings. Further research is required to better understand the molecular mechanisms, epidemiology, and transmission dynamics of specific ESBL-producing E. coli in healthcare settings, which will contribute to the design of effective prevention and control strategies.

Supplementary information

Below is the link to the electronic supplementary material.

Author contribution

Conceptualization: HBC, JSS; methodology: HBC, JDB, LL, FRF; writing–original draft preparation: HBC, JDB; writing–review and editing: HBC, JSS, JDB; funding acquisition: JSS; supervision: HBC.

Declarations

Conflict of interest

The authors declare no competing interests.