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Article

Characterization of ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a Northern Portuguese Hospital: Predominance of CTX-M-15 and High Genetic Diversity

by
Isabel Carvalho
1,2,3,4,5,
José António Carvalho
6,
Sandra Martínez-Álvarez
5,
Madjid Sadi
5,7,
Rosa Capita
8,9,
Carlos Alonso-Calleja
8,9,
Fazle Rabbi
10,
Maria de Lurdes Nunes Enes Dapkevicius
11,12,
Gilberto Igrejas
2,3,4,
Carmen Torres
5 and
Patrícia Poeta
1,4,*
1
Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
2
Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
3
Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, 5000-801 Vila Real, Portugal
4
Laboratory Associated for Green Chemistry (LAQV-REQUIMTE), New University of Lisbon, 2829-516 Monte da Caparica, Portugal
5
Area Biochemistry and Molecular Biology, University of La Rioja, 26006 Logroño, Spain
6
Medical Center of Trás-os-Montes e Alto Douro E.P.E., 5000-508 Vila Real, Portugal
7
Laboratory of Biotechnology Related to Animals Reproduction, Université Saad Dahlab de Blida, Blida 09000, Algeria
8
Department of Food Hygiene and Technology, Veterinary Faculty, University of León, 24071 León, Spain
9
Institute of Food Science and Technology, University of León, 24071 León, Spain
10
Australian Computer Society, Melbourne 3008, Australia
11
Faculty of Agricultural and Environmental Sciences, University of the Azores, 9500-321 Angra do Heroísmo, Portugal
12
Institute of Agricultural and Environmental Research and Technology (IITAA), University of the Azores, 9500-321 Azores, Portugal
 
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*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(9), 1914; https://doi.org/10.3390/microorganisms9091914
Submission received: 30 June 2021 / Revised: 30 August 2021 / Accepted: 3 September 2021 / Published: 9 September 2021
(This article belongs to the Special Issue ß-Lactamases)
Versions Notes

Abstract

:
Background: Enterobacteriaceae are major players in the spread of resistance to β-lactam antibiotics through the action of CTX-M β-lactamases. We aimed to analyze the diversity and genetic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patients in a Northern Portuguese hospital. Methods: A total of 62 cefotaxime/ceftazidime-resistant E. coli (n = 38) and K. pneumoniae (n = 24) clinical isolates were studied. Identification was performed by MALDI-TOF MS. Antimicrobial susceptibility testing against 13 antibiotics was performed. Detection of ESBL-encoding genes and other resistance genes, phylogenetic grouping, and molecular typing (for selected isolates) was carried out by PCR/sequencing. Results: ESBL activity was detected in all 62 E. coli and K. pneumoniae isolates. Most of the ESBL-producing E. coli isolates carried a blaCTX-M gene (37/38 isolates), being blaCTX-M-15 predominant (n = 32), although blaCTX-M-27 (n = 1) and blaCTX-M-1 (n = 1) were also detected. Two E. coli isolates carried the blaKPC2/3 gene. The lineages ST131-B2 and ST410-A were detected among the ESBL-producing blood E. coli isolates. Regarding the 24 ESBL-producing K. pneumoniae isolates, 18 carried a blaCTX-M gene (blaCTX-M-15, 16 isolates; blaCTX-M-55, 2 isolates). All K. pneumoniae isolates carried blaSHV genes, including ESBL-variants (blaSHV-12 and blaSHV-27, 14 isolates) or non-ESBL-variants (blaSHV-11 and blaSHV-28, 10 isolates); ten K. pneumoniae isolates also carried the blaKPC2/3 gene and showed imipenem-resistance. ESBL-positive E. coli isolates were ascribed to the B2 phylogenetic group (82%), mostly associated with ST131 lineage and, at a lower rate, to ST410/A. Regarding K. pneumoniae, the three international lineages ST15, ST147, and ST280 were detected among selected isolates. Conclusions: Different ESBL variants of CTX-M (especially CTX-M-15) and SHV-type (specially SHV-12) were detected among CTX/CAZR E. coli and K. pneumoniae isolates, in occasions associated with carbapenemase genes (blaKPC2/3 gene).

1. Introduction

Escherichia coli is a commensal microorganism of the intestinal microbiota of humans and animals. It is also involved in a great variety of intestinal and extra-intestinal infections as an opportunistic pathogen, including septicemia, as well as urinary and wound infections, among others; this microorganism presents a high facility to acquire antimicrobial-resistant genes [1,2]. In addition, Klebsiella pneumoniae can be found in the intestinal microbiota of healthy humans and animals although may also cause life-threatening infections; it is considered a major opportunistic pathogen implicated in nosocomial infections (as is the case of pneumonia or bloodstream infections), especially in patients of intensive-care units (UCIs) and in immunocompromised hosts with severe underlying diseases. K. pneumoniae strains can accumulate resistance genes, increasing their pathogenicity and causing severe infections [1,3,4,5]. Moreover, it is known that K. pneumoniae spreads easily, mainly in the hospital environment [5].
During recent years, the emergence and rapid dissemination of Enterobacteriaceae carrying genes encoding Extended Spectrum β-lactamases (ESBL), acquired AmpC β-lactamases (qAmpC), or carbapenemases are considered great concerns [6]. In particular, ESBLs of CTX-M-type and qAmpC enzymes (especially the CMY-2 type) were increasingly reported worldwide, specifically among clinical isolates from E. coli [7,8,9] and K. pneumoniae [6,10,11,12]. Moreover, recent studies reported ESBL-producing K. pneumoniae isolates among urinary tract infections and invasive infections in patients in Portugal [4,5,9], as well as among healthy Portuguese students in Lisbon [13]. Furthermore, a previous report created by our group showed the presence of ESBL-producing E. coli among healthy and sick cats [14] and dogs [15] in Portugal.
According to a recent review conducted by Sengodan et al. [16], more than eighty CTX-M variants have been reported worldwide; the majority of them are more active on cefotaxime than on ceftazidime. Particularly, the CTX-M-15 is frequently found among Enterobacteriaceae in humans in Europe [17,18], specifically in both clinical samples and healthy humans in Portugal [19,20,21]. Moreover, the CTX-M-15 β-lactamase is frequently associated with the uropathogenic international E. coli clone ST131 [22,23]. In the last decade, E. coli clonal group ST131 has emerged as a high-risk clone with important clinical health concerns causing multidrug-resistant (MDR) infections worldwide [11]. Particularly in Portuguese territory, ST131 clone was detected among residents of nursing homes [22], sick dogs [15], as well as in UTI clinical isolates from humans [24]. Nevertheless, other high-risk international clones have been detected among ESBL- or carbapenemase-producing E. coli isolates, as is the case of ST410 [25,26,27]. Other high-risk E. coli clones have been extensively reported in extraintestinal human infections [28].
According to Domokos et al. [18], high-risk K. pneumoniae clones, such as ST11 and ST15, have the tenacity and flexibility to accumulate resistance determinants, contributing to the increase of their pathogenicity. Regarding a recent study, the ST258, ST11, ST15, and ST147 clones spread for two decades, and recently, the CTX-M-15-producing ST307 clone in K. pneumoniae emerged globally [29]. Particularly, carbapenems are considered last-resort treatment but K. pneumoniae acquired resistance to this last resort antibiotic worldwide [30].
On the other hand, a great diversity of SHV variants have been reported, being a group of them of ESBL-type [31] being frequently detected among K. pneumoniae isolates, especially the SHV-5 and SHV-12 variants [1,16].
Antimicrobial resistance is commonly related to the spread of plasmids and the acquisition of resistance genes that normally occur by horizontal gene transfer (HGT). Another important aspect is the mutational events, mainly in sequences of genes encoding the target for certain antibiotics. Another mechanism of resistance (mainly in quinolones) is the alteration of the outer membrane proteins associated with active efflux pumps, which finish with the expulsion of the antibiotic out of the bacterial cell [1,16].
To our knowledge, some studies have been performed in Portugal based on the detection of ESBL-producing Enterobacteriaceae in hospitalized patients [3,4,12,19,32,33,34,35,36]. In a previous study performed by our research group, the ESBLs types were determined among invasive K. pneumoniae isolates recovered from blood cultures in a hospital located in Northern Portugal [5]. The purpose of the present study is to expand this previous work by analyzing in the same hospital the ESBLs types and the main associated resistance mechanisms in broad-spectrum cephalosporin-resistant K. pneumoniae isolates obtained from different clinical origins (except blood), and also in Escherichia coli isolates obtained from blood and urine samples; furthermore, the genetic lineages of selected isolates were also the aim of this research.

2. Materials and Methods

2.1. Bacterial Isolates

A collection of 38 cefotaxime/ceftazidime-resistant (CTX/CAZR) E. coli isolates (18 of urine samples and 20 from blood samples) were obtained from hospitalized patients (one isolate/patient) in a Northern Portuguese hospital (Centro Hospitalar de Trás os Montes e Alto Douro, CHTMAD, Vila Real), between December 2016 and August 2018. A collection of 24 CTX/CAZR K. pneumoniae isolates (18 from the urine; three of bronchial secretion and three from pus/biopsy/catheter origins) were also collected from the same hospital between December 2016 and December 2017. The identification of the isolates was confirmed by the matrix-assisted laser desorption-ionization time-of-flight mass spectrometry method (MALDI-TOF MS) [37,38], following the instructions of the manufacturer (Bruker Daltonik, Bremen, Germany).

2.2. Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing was performed by using Kirby–Bauer disk diffusion method on Mueller-Hinton agar, according to Clinical Laboratory Standards Institute guidelines (CLSI, 2019) [39]. The susceptibility of E. coli and K. pneumoniae isolates was tested for the following antibiotics (μg/disk): amoxicillin + clavulanic acid (20 + 10), cefoxitin (30), ceftazidime (30), cefotaxime (30), imipenem (10), tetracycline (30), gentamicin (10), streptomycin (10), tobramycin (10), ciprofloxacin (5) and trimethoprim-sulfamethoxazole (1.25 + 23.75). Moreover, the susceptibility for meropenem (10) and ertapenem (10) was also tested for K. pneumoniae isolates. The plates were incubated for 24 h at 37 °C. E. coli ATCC 25922 was used as a reference strain in susceptibility testing assays.
The screening of phenotypic ESBL production was carried out by the double-disk synergy test using cefotaxime, ceftazidime, and amoxicillin/clavulanic acid discs [39]. Isolates showing resistance to three or more antibiotic classes were considered as MDR.

2.3. DNA Extraction and Quantification

Genomic DNA from CTX/CAZR E. coli isolates was extracted using the boiled method [40], selecting three to five colonies in 1mL of sterile Milli-Q water for 8 min. Moreover, genomic DNA from K. pneumoniae isolates was extracted using the InstaGene Matrix (Bio-Rad).

2.4. Detection of Antibiotic Resistance Genes

E. coli and K. pneumoniae ESBL-positive isolates were screened by PCR/sequencing for the presence of the genes blaCTX-M (different groups) [41], blaSHV [42], blaTEM [41], blaCMY-2, blaDHA-1, blaKPC-2/3, blaVIM, blaVEB, blaOXA-48, and blaNDM [43,44]. The obtained amplicons were sequenced and analyzed by BLAST software, available at the National Center for Biotechnology Information [45]. The isolates were also screened for the presence of the genes encoding resistance for tetracycline (tetA, tetB) [46] and colistin (mcr-1) [47]. Moreover, the presence of the int1 gene (encoding the integrase of class 1 integrons) was analyzed on the E. coli isolates obtained from blood origin [48]. Positive controls of the University of La Rioja were used in each of the PCRs carried out in this work.

2.5. Molecular Typing of Selected E. coli and K. pneumoniae Isolates

Phylogenetic classification of all 38 E. coli isolates was performed as previously reported by Clermont et al. [49], according to the existence of arpA, chuA, yjaA, and TSPE4.C2 genes. The E. coli isolates of blood origin were further typed: (a) isolates of phylogroup B2 were screened for their affiliation to sequence type (ST) 131 by a specific PCR-region of the ST131 genome, as previously reported by Doumith et al. [50]; (b) the remaining blood isolates were typed by multilocus-sequence-typing (MLST) with seven housekeeping genes (fumC, adk, purA, icd, recA, mdh, and gyrB). The protocol described on PubMLST (Public databases for molecular typing and microbial genome diversity) was followed [51], and the allele combination was determined after sequencing of the seven genes to determine the sequence type (ST).
Moreover, the MLST was performed on selected K. pneumoniae isolates (based on the type of beta-lactamase genes they carried) by PCR/sequencing of seven housekeeping genes (gapA, phoE, infB, pgi, rpoB, tonB, and mdh) as previously indicated [52].

3. Results

3.1. Antimicrobial Resistance Phenotype in E. coli and K. pneumoniae Isolates

Regarding the 38 CTX/CAZR E. coli isolates, all of them were ESBL-producers (Table 1). High rates of antibiotic resistance were observed among these isolates for ciprofloxacin (n = 36; 94.7%), tobramycin (n = 27; 71.1%), trimethoprim/sulfamethoxazole (n = 25; 65.8%), tetracycline (n = 22; 57.9%), gentamicin (n = 22; 57.9%) and amoxicillin + clavulanic acid (n = 21; 55.3%). Importantly, two isolates showed imipenem resistance (IMPR). All E. coli isolates were categorized as MDR. The detailed antimicrobial resistance profiles of these isolates are listed in Table 1.
All of the 24 CTX/CAZR K. pneumoniae isolates were ESBL producers (100%) and they were considered for further genetic resistance analysis. Interestingly, 16 ESBL-producing K. pneumoniae isolates were also resistant to ertapenem (66.7%), 17 isolates to imipenem (70.8%), and 14 to meropenem (58.3%) (Table 2). Beside for β-lactam antimicrobials, high resistance levels were recorded towards trimethoprim/sulfamethoxazole (n = 24; 100%), ciprofloxacin (n = 22; 91.6%), tetracycline (n = 18; 75%), gentamicin (n = 18; 75%), amoxicillin + clavulanic acid (n = 17; 70.8%) and cefoxitin (n = 7; 29.2%) (Table 2). Moreover, all of the K. pneumoniae isolates showed a MDR-phenotype.

3.2. Genetic Characteristics of ESBL- or Carbapenemase-Producing E. coli Isolates

Most of the 38 ESBL-producers E. coli isolates carried a blaCTX-M gene, mainly the blaCTX-M-15 gene (n = 32; 84.2%) (Table 1). Furthermore, the blaCTX-M-27 gene was detected in one isolate (blood origin) and the blaCTX-M-1 was positive for another isolate (urine origin); the blaCTX-M variant could not be detected in three additional isolates. Interestingly, two of the ESBL-positive isolates were also IMPR and both of them carried the blaKPC-2/3 gene. Likewise, resistance to tetracycline was conferred, particularly, by the tet(A) (n = 16) or tet(B) (n = 3) genes. Moreover, the mcr-1 gene, conferring colistin resistance, was not detected among our clinical E. coli isolates. The int1 gene was found among most strains from blood origin (n = 12) (Table 1).
ESBL-positive E. coli isolates were ascribed mainly to the phylogenetic group B2 (n = 31; 75.6%), followed by A (n = 3), D (n = 2), and C (n = 1) phylogroups; according to Clermont et al. (2013) [34], the phylogenetic group associated with the remaining isolate was not conclusive. It is important to note that most of the ESBL-positive E. coli isolates from blood origin were typed as ST131/B2 (n = 18/20), although also we could identify two ST410/A isolates (Table 1).

3.3. Genetic Characteristics of ESBL-Producing K. pneumoniae Isolates

Regarding the 24 ESBL-producing K. pneumoniae isolates, the blaCTX-M-15 gene was the most common among these isolates (n = 16 isolates, 66.7%), which 11 of them belong to urine origin (Table 2). Furthermore, the blaCTX-M-55 gene was also found among these isolates (n = 2). A high diversity of SHV variants was detected, specifically SHV-27 (n = 8, ESBL- type), SHV-28 (n = 4), SHV-11 (n = 6, associated with CTX-M-15 or CTX-M-55), and SHV-12 (n = 6, ESBL-type and frequently associated with KPC2/3 gene) (Table 2). Regarding the K. pneumoniae isolates recovered from urine, 8 of them also carried the blaTEM gene. Moreover, the mcr-1 gene was not detected among K. pneumoniae isolates.
Three sequence types (from 5 selected isolates) belonging to major international lineages of human pathogenic β-lactamases-producing K. pneumoniae isolates were identified as follows (sequence-type/associated ESBLs): ST15/CTX-M-15 + SHV-12, ST15/CTX-M-15 + SHV-28, ST280/CTX-M-15 + SHV-27, ST280/SHV-27, and ST147/SHV-12 (Table 2).

4. Discussion

In our study, the ESBL-producing E. coli isolates were mainly associated with the carriage of the blaCTX-M-15 gene (>80%) (Table 1). This finding is according to a previous result obtained in E. coli isolated from nosocomial settings in Portugal [19]. In Germany, this enzyme was also the most prevalent ESBL type (around 50% of the strains investigated) [17]. Recently, the blaCTX-M-15 gene was the most frequently detected among E. coli of both sick and healthy dogs and cats in Portugal [14,15]. Nevertheless, the blaCTX-M-1 was the most common ESBL gene among healthy students in Portugal (Lisbon), followed by the blaCTX-M-15 gene [13].
Other Portuguese reports showed the detection of qAmpC β-lactamase-producing E. coli recovered from clinical settings (DHA and/or CMY-2) [8] and non-clinical isolates (CMY-2) [20]. Furthermore, the CMY-2 encoding gene was recently reported among healthy/sick cats in Portugal [14]; although these genes were tested, they were not found among our isolates. The ESBL-positive E. coli isolates analyzed in this study were ascribed mainly to the phylogenetic group B2 (Table 1). These findings are according to the study conducted by Zhang et al. [53], showing the high prevalence of phylogenetic group B2 among urinary E. coli isolates from human patients in the USA. Similar results were recently obtained among blood isolates in Spain [54], as well as among healthy humans [55] and nursing home residents [22], both studies performed in Portugal. Furthermore, the B2 phylogroup was the most frequent among dogs with UTI in Portugal [56]. Contrastingly, other authors reported a phylogenetic diversity among clinical isolates from humans and/or pets in Germany [17] and Switzerland [11]. The B2 group is frequently associated with virulent extra-intestinal strains of humans/animals, frequently found in human ExPEC infections; the group D is also associated with extra-intestinal infections although at a lower rate. Contrastingly, B1 and A are ubiquitous and more associated with commensal E. coli both in humans and vertebrate animals.
The CTX-M-15 gene is frequently associated with a specific international lineage, the epidemic clone ST131, which has spread worldwide [15,57]. According to Belas et al. (2019) [56], the E. coli ST131/CTX-M-15/B2 is the most disseminated E. coli clonal group worldwide associated with an extensive antimicrobial resistance profile; this lineage was found among 90% of our E. coli clinical isolates (Table 1), which represent a public health concern. Particularly in Portuguese territory, ST131 clone was detected among residents of nursing homes [22], sick dogs [15], as well as in healthy humans [55].
Moreover, the ST410 clone was identified among E. coli isolates in this study (Table 1). This clone was previously reported in clinical dog samples in Liverpool [58], Switzerland [11], France [59], and Portugal [60]. According to a recent report, E. coli ST410 should be classified as a potential new high-risk international clone [25]. Regarding clinical isolates from humans, this clone is widely distributed, mainly in Danish patients [25] and those from Southeast Asia [61].
Considering the ESBL-producing K. pneumoniae isolates, the blaCTX-M-15 gene was the most commonly detected (66.7%) (Table 2), similarly with the results obtained in different countries in clinical samples from humans [3,5,10,62] and pets [63,64,65]. Moreover, this gene was the most commonly detected in K. pneumoniae isolated from septicemias obtained from the same hospital of the present study (CHTMAD, Vila Real, Portugal) [5]. This gene was also detected among non-hospitalized patients in Portugal, according to a recent study [66]. Furthermore, the blaCTX-M-15 gene was the most detected among companion animals in Italy [67] and Germany [17]. These data related with CTX-M-15 producers over-predominating is according with the majority of European countries, and this variant is widely distributed among both humans and pets.
Furthermore, a high diversity regarding SHV variants (ESBL and not ESBL ones) detected among our isolates is according to the results obtained on clinical isolates in Portugal (SHV-11/12), Spain (SHV-12), China (SHV-12/27), India (SHV-28), and Brazil (SHV-27) [4,34,68,69,70,71]. Moreover, Carvalho et al. (2021) [5] detected also different variants of the blaSHV gene (blaSHV-1, blaSHV-11, or blaSHV-27) among K. pneumoniae isolates of blood cultures in the same hospital. It is important to note that a recent previous study showed the detection of blaCTX-M-15 gene (associated in most cases with blaSHV-28 gene) among K. pneumoniae isolated from healthy and sick dogs in Portugal [65]. Curiously, the blaSHV-12 (ESBL-variant) was widely reported in wildlife [31,72,73].
KPC-3-producing bacteria is endemic in many countries, but just recently appears in Portuguese hospitals. This fact is in line with our study and can be explained by the increase in carbapenems consumption. Specifically, according to the European Centre for Disease Prevention and Control (ECDC) [74], Portugal is considered one of the top carbapenem consumers in Europe (10.9% in 2019). The blaKPC-2/3 gene was also detected among Portuguese hospitalized patients [5,32,75], as well as in non-hospitalized patients [66]. According to Rodrigues et al. (2016) [66], the widespread distribution of KPC-3 among K. pneumoniae clinical isolates in Portugal was associated with successful high-risk clones ST147 and ST15, similarly with our results.
Three sequence types belonging to major international lineages of human pathogenic β-lactamases-producing K. pneumoniae were identified in this study. The ST15, associated with CTX-M-15, was detected in two isolates (Table 2). This clone was previously reported in other parts of the world among both hospital and community settings, namely in Portugal [4,5] and the Netherlands [76], indicating their global spread. Particularly, this clone was recently detected in a blood sample from the same hospital, associated with SHV-106/TEM production [5]. Furthermore, recent studies showed the presence of ST15/CTX-M-15 among healthy pets in Portugal [77], as well as sick dogs [65]. A possible explanation is the fact of HGT is caused by the proximity between humans and companion animals.
Curiously, the ST280 was found among two K. pneumoniae isolated from urine samples. This infrequent lineage was found among a patient in Korea [78] and in New York (in this last case, associated with the CTX-M-15 gene) [79].
On the other hand, the ST147 clone was associated with the spread of SHV-12 in a K. pneumoniae urine isolate in our study (Table 1); this lineage was also found among blood/urine samples in Portuguese hospitals, also associated with SHV-12 [12] or SHV-1/11+ KPC-2/3 [5].

5. Conclusions

ESBL-producing Enterobacteriaceae are endemic in different Portuguese clinical settings. In conclusion, the present study revealed the presence of ESBL-producing Enterobacteriaceae among clinical isolates from a hospital located in Northern Portugal, with the dominance of spread of CTX-M-15 co-harboring SHV-type ESBLs, and subsequently KPC-2/3 gene.
This work also showed the diversity of ESBLs associated with high-risk international clones (ST15, ST147, and ST280 clones in K. pneumoniae, and the ST131 and ST410 in E. coli), which play an important role in dissemination in hospital settings, and increased frequency in nosocomial infections with human health impact. A relentless vigilance of the evolution of the ESBL situation and the application of a One Health interdisciplinary approach is necessary to keep this problem under control.

Author Contributions

I.C. conceptualization, sampling, methodology, investigation, resources, data curation, writing and rewriting; J.A.C. sampling, methodology; S.M.-Á. investigation, data curation; M.S. investigation, data curation; R.C. validation, funding acquisition; C.A.-C. validation, funding acquisition; F.R. review and editing; M.d.L.N.E.D. review and editing; G.I. conceptualization, methodology, validation, resources, data curation, writing—review and editing, visualization, supervision, project administration, funding acquisition; C.T. conceptualization, methodology, validation, resources, data curation, writing—review and editing, visualization, supervision, project administration, funding acquisition; P.P. conceptualization, methodology, validation, resources, data curation, writing—review and editing, visualization, supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

I.C. gratefully acknowledges the financial support of “Fundação para a Ciência e Tecnologia” (FCT—Portugal) related to Ph.D. grant, through the reference SFRH/BD/133266/2017 (Medicina Clínica e Ciências da Saúde), as well as MCTES (Ministério da Ciência, Tecnologia e Ensino Superior) and European Union (EU), with reference to Fundo Social Europeu (FSE). The experimental work carried out in the University of La Rioja (Spain) was financed by the project SAF2016-76571-R from the Agencia Estatal de Investigation (AEI) of Spain and FEDER of EU. This work was partially supported by the Ministerio de Ciencia, Innovación y Universidades (Spain; grant number RTI2018-098267-R-C33), the Junta de Castilla y León (Consejería de Educación, Spain; grant number LE018P20) and the Associate Laboratory for Green Chemistry—LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflict of interest. Preliminary data was presented as a poster entitled “Genetic diversity among selected ESBL and Carbapenem-producing Klebsiella pneumoniae isolates from urocultures in a Portuguese hospital” in ECA 2021 Webinar congress. Part of this research was submitted to ESHG 2021 Webinar congress, with the following title “Resistance profile and genetic diversity among selected ESBL-producing Escherichia coli isolates from urocultures in a Portuguese hospital” and ECCMID 2021 Webinar Congress, entitled “Detection of ESBL and Carbapenem-resistant Klebsiella pneumoniae isolated from hospitalized patients”.

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Table
Table 1. Resistance phenotype and genotype in the 38 CTX/CAZR Escherichia coli isolates from clinical samples in a Portuguese hospital.
Table 1. Resistance phenotype and genotype in the 38 CTX/CAZR Escherichia coli isolates from clinical samples in a Portuguese hospital.
SampleOriginDate (Month Year)Resistance Phenotype aESBL
Production b		Β-lactamasesMLST cResistant Genes/Integrons dPG e
X1068BloodMarch 2017AMC, CTX, CAZ, CIP, SXTPCTX-M-15, TEMST131int1B2
X1080BloodJuly 2018AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15, TEMST131int1, tetAB2
X1062BloodDecember 2016AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1063BloodDecember 2016AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1064BloodDecember 2016AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1B2
X1065BloodJune 2017AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1066BloodJune 2017AMC, CTX, CAZ, TOB, CIP, GENPCTX-M-15ST131NDB2
X1067BloodFebruary 2017AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1069BloodMarch 2017AMC, CTX, CAZ, TOB, CIP, GENPCTX-M-15ST131NDB2
X1070BloodMarch 2017AMC, CTX, CAZ, CIP, GENPCTX-M-15ST131NDB2
X1071BloodApril 2017AMC, CTX, CAZ, TET, TOB, CIP, SXTPCTX-M-15ST131int1, tetAB2
X1072BloodApril 2017AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1073BloodApril 2017AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1074BloodApril 2017AMC, CTX, CAZ, TET, TOB, CIP, GEN, SXTPCTX-M-15ST131int1, tetAB2
X1075BloodMay 2017AMC, CTX, CAZ, CIPPCTX-M-15ST410NDA
X1076BloodMay 2017AMC, CTX, CAZ, TET, TOB, CIPPCTX-M-15ST131NDB2
X1105BloodMay 2017AMC, CTX, CAZ, CIPPCTX-M-15ST410NDA
X1079BloodJuly 2018AMC, CTX, CAZ, TOB, CIP, GEN, SXTPCTX-M-15ST131int1B2
X1081BloodAugust 2018AMC, CTX, CAZ, TET, TOB, CIPPCTX-M-15ST131tetAB2
X1078BloodJuly 2018CTX, TET, CIP, SXTPCTX-M-27ST131NDB2
X3158UrineFebruary 2017CTX, CAZ, TET, TOB, SXT, SPCTX-M-15NTNDB2
X3159UrineFebruary 2017CTX, CAZ, TOB, CIP, GEN, SPCTX-M-15NTNDB2
X3160UrineFebruary 2017CTX, CAZ, TET, TOB, CIP, GEN, SPCTX-M-15NTNDB2
X3161UrineMarch 2017CTX, CAZ, TOB, CIP, GEN, SPCTX-M-15NTNDB2
X3162UrineApril 2017CTX, CAZ, TOB, CIP, GEN, SPCTX-M-15NTNDD
X3163UrineMay 2017CTX, CAZ, TET, CIP, SXT, SPCTX-M-15NTtetA, tetBD
X3164UrineMay 2017CTX, CAZ, CIP, SXT, SPCTX-M-15NTNDB2
X3165UrineMay 2017CTX, CAZ, CIP, SPCTX-M-15NTNDB2
X3167UrineJune 2018CTX, CAZ, TET, TOB, CIP, GEN, SXT, SPCTX-M-15NTtetAB2
X3168UrineAugust 2018CTX, CAZ, TET, TOB, CIP, GEN, SXT, SPCTX-M-15NTtetAB2
X3169UrineAugust 2018CTX, CAZ, TOB, CIP, GEN, SXT, SPCTX-M-15NTtetAB2
X3170UrineAugust 2018CTX, CAZ, TET, CIP, SPCTX-M-15NTtetBB2
X3173UrineJune 2018AMC, FOX, CTX, CAZ, IMP, TET, CIP, SXT, SPCTX-M-15, KPC2/3NTtetBA
X3157UrineFebruary 2017CTX, TET, SXT, SPCTX-M-1NTtetAC
X3155UrineDecember 2016CTX, TOB, CIP, SXT, SPCTX-M-variantNTNDB2
X3166UrineMay 2018ERT, TOB, CIP, GEN, SXT, SPCTX-M-variantNTNDB2
X3171UrineDecember 2016CTX, TOB, CIP, SXT, SPCTX-M-variantNTNDNC
X3156UrineJune 2017AMC, FOX, CTX, CAZ, IMP, TET, TOB, CIP, GEN, SXT, SPKPC2/3NTtetAB2
Legend:a AMC: amoxicillin + clavulanic acid; FOX: cefoxitin; CTX: cefotaxime; CAZ: ceftazidime; CHL: chloramphenicol; CIP: ciprofloxacin; TOB: tobramycin; GEN: gentamicin; SXT: trimethoprim + sulfamethoxazole; S: streptomycin; TET: tetracycline; IMP: imipenem; b P—Positive, N—Negative; c MLST—MultiLocus Sequence Typing; d NT: not tested; ND: not detected; NC: Not concluded; e Phylogroups.
Table
Table 2. Resistance phenotype and genotype present in the 24 CTX/CAZR Klebsiella pneumoniae isolates from clinical samples in a Portuguese hospital.
Table 2. Resistance phenotype and genotype present in the 24 CTX/CAZR Klebsiella pneumoniae isolates from clinical samples in a Portuguese hospital.
SampleOriginDate (Month Year)Antimicrobial Resistance Phenotype aESBL
Production b		β-lactamasesMLST cOther Genes/Int d
X2175UrineJune 2017AMC, CTX, CAZ, IMP, MRP, ERT, CIP, SXT, SPCTX-M-15, KPC-2/3, SHV-12, TEMST15ND
X2143UrineDecember 2016AMC, FOX, CTX, CAZ, IMP, MRP, ERT, TET, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-27, TEMST280tetA
X2153UrineJune 2017AMC, FOX, CTX, CAZ, IMP, MRP, ERT, TET, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-27, TEMNTtetA
X2155UrineFebruary 2017AMC, CTX, CAZ, IMP, MRP, ERT, TET, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-27, TEMNTtetA
X2166UrineMay 2017AMC, CTX, CAZ, IMP, MRP, ERT, TET, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-27, TEMNTtetA
X3098UrineDecember 2016CTX, CAZ, TET, CIP, TOB, GEN, SXT, SPCTX-M-15, SHV-27NTtetA
X3100UrineMarch 2017CTX, CAZ, IMP, TET, CIP, TOB, SXT, SPCTX-M-15, SHV-27NTtetA
X3104UrineMay 2017AMC, CTX, CAZ, IMP, MRP, ERT, TET, CIP, TOB, SXT, SPCTX-M-15, SHV-11NTND
X2157UrineApril 2017AMC, FOX, CTX, CAZ, IMP, MRP, ERT, CHF, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-28, TEMST15ND
X3095UrineDecember 2016CTX, CAZ, MRP, TET, CIP, TOB, GEN, SXT, SPCTX-M-15, SHV-28NTtetA
X3105UrineMay 2017AMC, CTX, CAZ, ERT, TET, CIP, TOB, GEN, SXT, SPCTX-M-15, SHV-28NTtetA
X3106UrineMay 2017CTX, CAZ, ERT, TET, CIP, TOB, GEN, SXT, SPCTX-M-55, SHV-11NTtetA
X2142UrineDecember 2016AMC, FOX, CTX, CAZ, IMP, MRP, ERT, CHF, CIP, GEN, SXT, SPSHV-12, KPC-2/3, TEMST147ND
X3092UrineDecember 2016AMC, CTX, CAZ, IMP, TET, TOB, CIP, GEN, SXT, SPSHV-12NTND
X3096UrineDecember 2016CTX, CAZ, IMP, TET, CIP, SXT, SPSHV-12NTtetA
X3097UrineDecember 2016CTX, CAZ, IMP, TET, TOB, CIP, GEN, SXT, SPSHV-12NTtetA
X3107UrineMay 2017AMC, FOX, ERT, CTX, CAZ, TET, CIP, SXT, SPSHV-12NTtetA
X2232UrineJanuary 2017AMC, CTX, CAZ, IMP, MRP, ERT, TET, CIP, GEN, SXT, SPSHV-27, KPC-2/3, TEMST280tetA
X3085Bronchial secretionDecember 2017AMC, FOX, CTX, CAZ, IMP, MRP, ERT, TOB, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-11NTND
X3094PusDecember 2017CTX, CAZ, TET, TOB, GEN, SXT, SPCTX-M-15, SHV-11NTtetA
X3102BiopsyMay 2017AMC, CTX, CAZ, TOB, GEN, SXT, SPCTX-M-15, SHV-11NTND
X3087Bronchial secretionDecember 2016AMC, FOX, CTX, CAZ, IMP, MRP, ERT, TET, TOB, CIP, GEN, SXT, SPCTX-M-15, SHV-27NTtetA
X3088Bronchial secretionJune 2017AMC, CTX, CAZ, IMP, MRP, ERT, TOB, CIP, GEN, SXT, SPCTX-M-15, KPC-2/3, SHV-28NTND
X3101CatheterApril 2017AMC, CTX, CAZ, IMP, MRP, ERT, TET, TOB, CIP, SXT, SPCTX-M-55, SHV-11NTtetA
Legend:a AMC: amoxicillin + clavulanic acid; FOX: cefoxitin; CTX: cefotaxime; CAZ: ceftazidime; CHL: chloramphenicol; CIP: ciprofloxacin; TOB: tobramycin; GEN: gentamicin; SXT: trimethoprim + sulfamethoxazole; S: streptomycin; TET: tetracycline; IMP: imipenem; ERT: ertapenem; MRP: meropenem; b P—Positive, N—Negative; c MLST—MultiLocus Sequence Typing; d NT: not tested; ND: not detected.
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Carvalho, I.; Carvalho, J.A.; Martínez-Álvarez, S.; Sadi, M.; Capita, R.; Alonso-Calleja, C.; Rabbi, F.; Dapkevicius, M.d.L.N.E.; Igrejas, G.; Torres, C.; et al. Characterization of ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a Northern Portuguese Hospital: Predominance of CTX-M-15 and High Genetic Diversity. Microorganisms 2021, 9, 1914. https://doi.org/10.3390/microorganisms9091914

AMA Style

Carvalho I, Carvalho JA, Martínez-Álvarez S, Sadi M, Capita R, Alonso-Calleja C, Rabbi F, Dapkevicius MdLNE, Igrejas G, Torres C, et al. Characterization of ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a Northern Portuguese Hospital: Predominance of CTX-M-15 and High Genetic Diversity. Microorganisms. 2021; 9(9):1914. https://doi.org/10.3390/microorganisms9091914

Chicago/Turabian Style

Carvalho, Isabel, José António Carvalho, Sandra Martínez-Álvarez, Madjid Sadi, Rosa Capita, Carlos Alonso-Calleja, Fazle Rabbi, Maria de Lurdes Nunes Enes Dapkevicius, Gilberto Igrejas, Carmen Torres, and et al. 2021. "Characterization of ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a Northern Portuguese Hospital: Predominance of CTX-M-15 and High Genetic Diversity" Microorganisms 9, no. 9: 1914. https://doi.org/10.3390/microorganisms9091914

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