Characterization of Escherichia coli Cefotaxime-Resistance in Al-Ahsa, KSA: Predominance of CTX-15 and First Report of blaCMY-42 Gene
by
, ,
Melek Ben Aissa
1,*,
Sana Ferjani
2,
Mohamed Salah Abassi
3
,
Nada Al-Suwailem
4 and
Ilhem Boutiba
2,5 1
Applied College in Abqaiq, King Faisal University, Al-Ahsa 31982, Saudi Arabia
2
Laboratory of “Resistance to Antimicrobiens” LR99ES09, College of Medicine, Tunis El Manar University, Tunis 1007, Tunisia
3
Laboratory of Bacteriological Research, Tunisian Institute of Veterinary Research, Tunis El Manar University, Tunis 1006, Tunisia
4
Chemistry Department, College of Science, King Faisal University, Al-Ahsa 31982, Saudi Arabia
5
Laboratory of Microbiology, Charles-Nicolle Hospital, Tunis 1006, Tunisia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2022, 12(19), 9964; https://doi.org/10.3390/app12199964
Submission received: 5 September 2022
/
Revised: 20 September 2022
/
Accepted: 28 September 2022
/
Published: 4 October 2022
Abstract
:We determined an antibiotic resistance mechanism in the eastern region, KSA, and the genetic factor clonal relatedness within Gram-negative bacteria. During our retrospective study, a total number of 29 E. coli ESBL producer strains were isolated for patients visiting King Fahad Hospital, Al-Ahsa, KSA. The bla genes were detected via PCR and identified via sequencing. Associated plasmid-mediated quinolone resistance genes, as well as int1 and int2 genes, were also studied. Phylogenetic groups, the ST131 clone, virulence factors, and PFGE were also checked. The blaCTX-M-9 (3.7%), blaCTX-M-27 (22.2%), and blaCTX-M-15 (77.8%) genes were identified; however, the blaCMY-42 (7.4%) gene was recorded for the first time in KSA. The qnrS1 gene was found in 44.4% of strains, and among them, 50% concomitantly harbored the aac(6′)Ib-cr. The int1 gene was detected in 25.9% strains; nonetheless, the int2 gene was found in 7.4% of isolates. The strains belonged mainly to the B2 and D phylogroups. PFGE showed unrelated patterns. Some isolates belonged to the pandemic clone ST131. We describe a large dissemination of antibiotic resistance to third-generation cephalosporins in the eastern region, KSA, with the occurrence of the blaCMY-42 gene. The clone ST131 seems to be the principal contributor for blaCTX-M-15 gene spread.
1. Introduction
Secondary infections and organ failure are the graver consequences observed worldwide during COVID-19, causing a high mortality rate [1]. Secondary infections specifically associated with SARS-CoV-2 are still poorly known; however, many studies have shown that co-infection factors include Salmonella, Shigella, and E. coli [2,3,4]. On the other hand, human extra-intestinal diseases, including urinary tract infection (UTI), bacteremia, and meningitis can be caused by a group of pathogens such as Escherichia coli [5]. E. coli, a producer of ESBLs, is recorded among the six highest multi-resistant germs that are particularly curable with only a few effective drugs [6]. It is frequently correlated with high healthcare costs, morbidities, and mortalities [7,8]. In 2017, the estimated number of critical cases of infection with pathogens producing ESBLs was about 200,000 with 9100 deaths; however, the total healthcare cost was estimated to be USD 1.2B [9]. Furthermore, numerous investigations have confirmed that the ESBL, exhibited among E. coli, is the major mechanism reported for the β-lactam resistance [10,11]. Over the last two decades, the fast diffusion of ESBLs has emerged globally, with the development of a worldwide outbreak being a powerful menace to human health, while continuous surveys and molecular characterizations of these strains have been conducted in healthcare units.
The discovery of pharmaceutical compounds with new antibacterial properties used to fight these bacterial infections seems to have been dropped, due to the vicious circle of new resistance for each new antibiotic discovered [12]. On the other hand, the high number of intra-travelers and extra-travelers in KSA has contributed to the wide dissemination of antibiotic resistance, making antibiotic prescriptions a complicated process, especially when it is due to bacterial co-infections or complications in healthcare units for critical clinical cases, particularly throughout the COVID-19 pandemic [13].
The spread of ESBLs is related to the diversity of enzymes and transferable genetic elements, and the capacity for a large spread of clones [14]. Indeed, a significantly large amount of published data in different countries [15] has indicated that the ST131 clone producing CTX-15 was associated with noticeable epidemiological changes in hospitals and communities for infectious diseases [16]. In KSA, a few epidemiological data on pathogens producing ESBLs were given, and seemed to be mainly focused on Riyadh and Dammam.
In this observational study, we determined the implication of ESBL genetic factors and plasmids. We assessed the clonal relatedness and virulence factors among a collection of E. coli samples collected between November 2016 and March 2017 in King Fahad Hospital (KFH), Al-Ahsa in KSA.
2. Materials and Methods
2.1. Study Design
The research was observational, cross-sectional, and retrospective. Convenience sampling for 5 months during the winter season between November 2016 and March 2017 was utilized (Figure 1). The sample size was calculated using Statdirect software version 26 (CI 95%). For inclusion criteria, only E. coli strains and positive ESBLs were maintained in the study. However, the exclusion criteria were the high sensitivity to cephalosporin of third-generation (C3G), non-ESBL producers, and not an E. coli strain. EC10 and EC13 were excluded for a loss of stable C3G resistance characteristics after a few generations.
2.2. Antimicrobial Susceptibility Tests and Cefotaxime Resistance Transfer Assays
Antimicrobial susceptibilities were determined by conducting the disk diffusion technique according to the European Committee on Antimicrobial Susceptibility Testing guidance (CA-SFM/EUCAST, 2016). A total of 22 antibiotics (Bio-Rad, Marnes-la-Coquette, France) were used according to the CA-SFM guidelines (amoxicillin/clavulanic acid, ceftazidime, cefotaxime, cefoxitin, cefepime, ertapenem, nalidixic acid, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracycline, minocycline, gentamicin, tobramycin, netilmicin, amikacin, norfloxacin, tigecycline, colistin, nitrofurantoin, fosfomycin, chloramphenicol, and aztreonam). The minimum inhibitory concentration was determined for cefotaxime and ceftazidime using the agar diffusion method, following the Clinical and Laboratory Standards Institute. ESBLs were distinguished using the test of synergy (TS) within amoxicillin/clavulanic acid and ceftazidime, cefotaxime, aztreonam, or cefepime (with and without cloxacillin at 250 mg/L). The control strains used were ATCC700603 and ATCC25922 (EUCAST, 2015). Conjugation experiments utilizing E. coli J53 (Rifampin resistant) as a receiver were performed to assess the transferability of C3G resistance. Our isolates were initially selected on Mueller Hinton (MH) agar (Oxoid Ltd., Basingstoke, UK) plates incorporating rifampicin within 400 mg/L. Resistance transfer experiments were implemented on brain–heart infusion broth (BHI) (Oxoid Ltd., Basingstoke, UK) for 18 h at 37 °C, and the obtained transconjugants were grown on MH agar plates after adding both cefotaxime (2 mg/L) and rifampicin (400 mg/L). The transconjugants’ antimicrobial susceptibilities were tested using a previously mentioned method. The strain origins were recorded (Table 1). Frequencies were calculated using SPSS software version 26.
2.3. Characterization of Antibiotic Resistance Genes
The strains exhibiting positive TS were evaluated via PCR for the occurrence of blaTEM, blaSHV, blaCTX-M, blaPER, blaGES, and blaVEB genes (Table 2). Furthermore, the screenings for cephalosporinases were aimed at blaFOX, blaACC, blaCIT, blaDHA, blaEBC, and blaMOX genes, as described previously (Dallenne et al., 2010). Amplified DNA fragments were sequenced, applying a DNA sequencer (ABI PRISM 3130, Applied Biosystems, Foster City, CA, USA) [17], and then compared in the GenBank database.
2.4. Quinolone Resistance Genes, Integrons, and Plasmid Incompatibility Groups
2.5. Phylogenetic Analysis, Genetic Relatedness, and ST131 Identification
Triplex PCRs for the phylogenetic groups A, B1, B2, and D, respectively, were screened [20]. Furthermore, virulence genotypes encoding toxins, invasins, adhesins, and siderophores were assessed, applying multiplex PCR (Table 3). The control isolates CFT073 and J96 were used [5,21,22]. The XbaI restriction enzyme was used for Pulsed-Field Gel Electrophoresis (PFGE), it was performed to assess the clonal correlation of different isolates, as reported previously [23]. ST131 was researched using an O25b-specific PCR method, with pabB and trpA allele-specific primers for the B2 phylogenetic group strains [24].
2.6. Ethical Approval
The deanship of scientific research at King Faisal University, Al-Ahsa, KSA approved the study (ref. no. EA000528). The information and details collected were confidential. No personal information was shared. The study was a secondary analysis to a routine laboratory test.
3. Results
3.1. Antibiotic Susceptibilities and Cefotaxime Resistance Transfer Assays
During the winter season, the patterns of E. coli ESBL producers were 51.9% female, the mean age was 54.78, with 44.4% aged between 39 and 55 years; and urine was the prevalent specimen type, at 51.9% (Table 1). The multidrug resistance characterization indicated the production of both ESBLs and non-ESBLs among our collection (Table 2). A high resistance rate for the tested antibiotics was observed for amoxicillin/clavulanic acid, cefotaxime, and ceftazidime (Table 2). However, all the strains were susceptible to amikacin, colistin, and carbapenems. The isolates were resistant to gentamicin (18.5%; 5/27), tobramycin (33.3%; 9/27), netilmicin (7.4%; 2/27), nalidixic acid (100%; 27/27), ciprofloxacin (88.8%; 24/27), tetracycline (55.5%; 15/27), minocycline (14.8%; 4/27), fosfomycin (100%; 27/27), chloramphenicol (70.3%; 19/27), and trimethoprim/sulfamethoxazole (85.2%; 23/27) (Table 2). ESBL phenotypes were found in 26 isolates, and plasmidic cephalosporinases (pAmpC) were identified in two strains. However, one strain co-produced both an ESBL and a cephalosporinase. A successful transfer of cefotaxime resistance was detected for only 12 isolates (Table 5).
3.2. Characterization of Antibiotic Resistance
The CTX-15 enzyme was found in 77.8% (21/27) strains; however, CTX-27 was recorded in 22.2% (6/27), and CTX-9 β-lactamase was less frequently presented, with only 3.7% (1/27) of the strains. The blaTEM-1 gene was identified in 74.1% (20/27) strains, while the blaSHV-1 gene was detected in 18.5% (5/27) strains (Table 1). The gene sets blaCMY-42/ blaCTX-M-15/blaSHV-1 and blaCMY-42/blaTEM-1/blaSHV-1 were identified as co-ESBL producers. The blaCTX-M-15/blaTEM-1 gene association was observed for 48.14% (13/27) of the strains, while the blaCTX-M-27/blaTEM-1 was detected in 11.1% (3/27). Both the blaCTX-M-15/blaSHV-1 genes were seen in 7.4% (2/27) of the strains. The association of blaCMY-42/ blaCTX-M-15/blaSHV-1, blaCTX-M-15/blaCTX-M-27/blaTEM-1, blaCTX-M-9/blaCTX-M-27/blaTEM-1, blaCTX-M-15/blaTEM-1/blaSHV-1, and blaCMY-42/blaTEM-1/blaSHV-1 genes was reported in 3.7% for each set (1/27) (Table 3).
3.3. Quinolone Resistance Genes, Integrons, and Plasmid Incompatibility
The qnrS1 gene was found in 44.44% (12/27) of the strains. The aac(6′)Ib-cr variant was observed in 22.22% (6/27) of the strains. The genes int1 and int2 were reported, respectively, in 25.9% (7/27) and 7.4% (2/27) for our isolates (Table 3). A total of 48.14% (13/27) of the strains showed a plasmid replicon type. Only IncF (11.1%; 3/27) and IncFIA (18.5%; 5/27) were identified among our collection; moreover, both co-occurred in 18.5% of isolates (5/27) (Table 3). The transfer of the blaCTX-M-15/blaTEM-1/qnrS1/aac(6′)Ib-cr and int1 genes has been frequently observed (Table 4). Similarly, the IncF and IncFIA plasmids were also successfully transmitted through conjugation (Table 5).
3.4. Genetic Relationship, Phylogenetic Groups, and Identification of the ST131 Clone
The B2 phylogenetic group was identified in 70.4% (19/27); it was considered as the main group in our study. Further, the phylogroups D, A, and B1 were presented, respectively, in 18.5% (5/27), 7.4% (2/27), and 3.7% (1/27) of isolates. Three strains amongst the B2 phylogenetic group were positive for the ST131 clone (Table 4). PFGE (Pulsed-Field Gel Electrophoresis) analysis revealed 21 different DNA profiles (P1 to P21) among the 27 isolates. Each pulsotype, P8, P16, and P19 included two isolates, and the pulsotype P1 contained three B2-ST131 isolates. The remainder of the isolates were unrelated (Table 4, Figure 2).
3.5. Occurrence of Virulence Genes
Virulence genes were distributed as follows: fimH (55.5%, 15/27), papGI (11.1%, 3/27), papGII (74.1%, 20/27), fyuA (3.7%, 1/27), iha (59.3%, 16/27), iutA (37.1%, 10/27), ompT (77.7%, 21/27), traT (25.9%, 7/27), kpsMTII (22.2%, 6/27), malX (55.5%, 15/27), and usp (62.9%, 17/27). The virulence scores varied from 0 to 9 (median: 5).
4. Discussion
The aim of our investigation was to determine the ESBL producer-relevant agents and to decrypt their genetic factors. The ubiquity of ESBLs produced by Enterobacteriaceae, particularly with E. coli (ESBL-EC), has increased worldwide throughout the past two decades [25]. In our investigation, E. coli isolates were collected from various samples. Several cefotaxime-resistant strains were detected. A total of 92.59% (25/27) strains were considered as being ESBL producers. However, 3.7% (1/27) of isolates produced AmpC β-lactamases. Indeed, this strain contained both ESBL and AmpC β-lactamase enzymes. This rate of ESBL-producer isolates was similar to those reported previously [7] from Al-Abha and Riyadh, KSA [26,27]. The prevalence rates of ESBL-EC were 6.5% and 10.3% in 2002 and 2004, sequentially [8]. The preponderance rates were 15.4% and 4.5% for inpatients and outpatients, respectively [28,29]
The multidrug resistance (MDR) was described for all isolates, even for quinolones, aminosides, tetracycline, trimethorprim/sulfamethoxazole, fosfomicin, and chloramphenicol. MDR is frequent in E. coli; however, the high rates of resistance found for fosfomycin in this study are uncommon in such strains. Conversely, fosfomycin susceptibility tests are limited, since this agent is occasionally available in most clinical laboratories. Uncomplicated UTI is commonly treated in many countries with fosfomycin, whereas worldwide attention has been oriented to sparing carbapenems in ESBL-producing strains. Fosfomycin in combination with colistin was recommended for treating Enterobacteriaceae that were resistant to carbapenems. A recent study from China [29] reported uropathogenic ESBL-producing E. coli isolates that are resistant to fosfomycin. Indeed, fosfomycin resistance was encoded by the fosA3 gene carried by a 54.2 Kb transferable plasmid also co-harboring a blaCTX-M gene. The genetic characterization of fosfomycin resistance mechanisms and its possible linkage to ESBL-encoding genes needs further investigation in KSA.
The typical TEM and SHV variants of ESBLs have declined during the past two decades and were interchanged worldwide by the CTX-M group as the predominant ESBL group. Similarly, the ESBLs generated in our strains harbored CTX-M β-lactamases. The blaCTX-M-15 (21/27), blaCTX-M-27 (5/27), and blaCTX-M-9 (1/27) genes were identified. The blaCMY-42 gene was recorded in two strains; moreover, one isolate co-accommodated the blaCTX-M-15 gene. The concomitant occurrence of ß-lactamase coded by the blaSHV-1 or blaTEM-1 genes was also common in these isolates, as reported previously [30]. Our findings support other studies from KSA showing the prevalence of a blaCTX-M15 type [26,31]. However, the blaCTX-M-9 and blaCTX-M-27 genes are prevalent in KSA, and low rates were reported [7,32]. It is also important to know the occurrence of the blaCMY-42 gene, a CMY-2 variant, which was present in two isolates of our collection. The blaCMY-42 was first described in E. coli [33], and was then rarely reported and mainly found to be associated with blaNDM-5, blaSHV-12, blaCTX-M-14, or blaCTX-M-15, as described in one isolate of our study. Presumably, we describe an early case of blaCMY-42 harboring E. coli isolates in KSA [34].
The prevalent plasmid replicon group was found in resistant Enterobacteriaceae segregated from both animals and humans holding incompatibility (Inc) group F (including FIA, FIB, and FII replicons), and A/C, L/M, I1, HI2, and N [35,36]. Different blaCTX-M genes are also combined with specific plasmid replicon types (IncN, I1, FII, and L/M) [36]. The blaCTX-M-15 was primarily found on plasmids that involve FII and FIA replicons, and, to a lesser extent, to IncI1, IncN, and IncA/C, as well as being on pir-type plasmids. Concordantly, IncF (3/27) and IncFIA (5/27) plasmids were identified among our collection, and both co-occurred in five isolates, indicating the possible localization of the blaCTX-M-15 gene on one of these plasmids. The concomitant transfer of the blaCTX-M-15 gene with IncF and/or IncFIA plasmids for these strains supported this hypothesis. However, further experiments (S1-PFGE hybridization) are needed to assess the exact genetic localization of all bla genes. Both integrons belonging to classes 1 and 2, as well as the occurrence of these plasmids, might explain the MDRs of our isolates, as reported previously [31,32]. A significant proportion of our isolates were observed to belong to phylogroups D and B2. This finding concurred with other studies; moreover, most of the ESBL-producing E. coli are considered to be the main leading causes of extra-intestinal infections. Furthermore, three CTX-15-producing E. coli strains positive for the B2 phylogroup belonged to the ST131 clone. The ST131 clone, known as the major pandemic clone, controlled the worldwide dissemination of the β-lactamase CTX-15 type [35,37]. This clone has been also reported previously in KSA [7].
Genetic relatedness assessed by PFGE revealed 21 pulsotypes, where P1 encompassed three isolates, while pulsotypes P8, P16, and P19 contained two isolates. The clonally related isolates were all of pulsotype B2, and all, except two of pulsotype P16, harbored identical β-lactamases. However, antimicrobial susceptibility, PMQR genes, integrons, plasmids, and virulence genes contents were not mainly identical within the related isolates.
Taken together, despite the reduced isolate number, our study showed the dominance of the CTX-15 enzyme among E. coli in KSA, and the occurrence of some clonal isolates spreading within patients from different regions. ST131 was not the main clone in our isolates; other lineages or plasmids contributed significantly to the spread of the CMY-42 enzyme.
5. Conclusions
We describe the first report of CMY-42-producing E. coli isolates in the Kingdom. The CMY-42 enzyme was identified among unrelated E. coli pulsotypes, as well as CTX-M-27, CTX-15, and CTX-9. The ST131 seems to be a significant contributor for the spread of the CTX-15 enzyme among our isolates. The IncF and/or IncFIA plasmids were identified within the blaCTX-M-15 gene, as well as class 1 and class 2 integrons, showing their probable horizontal transmission. The MDR phenotype associated with several virulence genes was observed in our collection, showing a large dissemination of antibiotic resistance in the eastern region. Our research could form a strong guide for clinicians who are interested in the genetic factors of MDR.
The rigorous control of antibiotic therapy and the continuous surveillance of epidemiologic analysis are essential for limiting the resistance diffusion factor in the post-COVID-19 era, as well as fostering a better understanding of their dissemination process for better oriented outcomes. The actual situation is very difficult due to the increased need for non-pharmaceutical (sanitizers) and pharmaceutical (antibiotics) stressors used during the COVID-19 era. Antibiotic resistance management within artificial intelligence and machine learning may be powerful tools for helping decision makers to implement new solutions in healthcare departments and medication prescriptions.
Author Contributions
Conceptualization, M.B.A.; methodology, M.B.A. and S.F.; software, M.B.A. and S.F.; validation, M.B.A. and S.F.; formal analysis, M.B.A. and S.F.; investigation, M.B.A. and N.A.-S.; resources, M.S.A., I.B., and N.A.-S.; data curation, M.B.A.; writing—original draft preparation, M.B.A., S.F., and M.S.A.; writing—review and editing, M.B.A.; visualization, M.B.A.; supervision, M.B.A.; project administration, M.B.A. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by King Faisal University, grant number GRANT593, and APC was funded by the Deanship of Scientific Research of King Faisal University, project number GRANT593.
Institutional Review Board Statement
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Deanship of Scientific Research at King Faisal University (EA000528).
Informed Consent Statement
Patient consent was waived due to retrospective data.
Data Availability Statement
The data presented are available on request from the corresponding author.
Acknowledgments
All the authors are grateful to the Deanship of Scientific Research at King Faisal University, project number GRANT593.
Conflicts of Interest
The authors declare no conflict of interest.
References
- Anas, A.; Sobhanan, J.; Sulfiya, K.; Jasmin, C.; Sreelakshmi, P.; Biju, V. Advances in photodynamic antimicrobial chemotherapy. J. Photochem. Photobiol. C Photochem. Rev. 2021, 49, 100452. [Google Scholar] [CrossRef]
- Bokhary, H.; Pangesti, K.N.A.; Rashid, H.; Abd El Ghany, M.; Hill-Cawthorne, G.A. Travel-Related Antimicrobial Resistance: A Systematic Review. Trop. Med. Infect. Dis. 2021, 6, 11. [Google Scholar] [CrossRef] [PubMed]
- Martinez-Guerra, B.; Gonzalez-Lara, M.; De-Leon-Cividanes, N.; Tamez-Torres, K.; Roman-Montes, C.; Rajme-Lopez, S.; Villalobos-Zapata, G.; Lopez-Garcia, N.; Martínez-Gamboa, A.; Sifuentes-Osornio, J.; et al. Antimicrobial Resistance Patterns and Antibiotic Use during Hospital Conversion in the COVID-19 Pandemic. Antibiotics 2021, 10, 182. [Google Scholar] [CrossRef]
- Saini, V.; Jain, C.; Singh, N.; Alsulimani, A.; Gupta, C.; Dar, S.; Haque, S.; Das, S. Paradigm Shift in Antimicrobial Resistance Pattern of Bacterial Isolates during the COVID-19 Pandemic. Antibiotics 2021, 10, 954. [Google Scholar] [CrossRef] [PubMed]
- Johnson, J.R.; Stell, A.L. Extended Virulence Genotypes of Escherichia coli Strains from Patients with Urosepsis in Relation to Phylogeny and Host Compromise. J. Infect. Dis. 2000, 181, 261–272. [Google Scholar] [CrossRef] [Green Version]
- Scientific Opinion on the public health risks of bacterial strains producing extended-spectrum β-lactamases and/or AmpC β-lactamases in food and food-producing animals|EFSA. Available online: https://www.efsa.europa.eu/it/efsajournal/pub/2322 (accessed on 1 October 2022).
- Al-Agamy, M.H.; Shibl, A.M.; Hafez, M.M.; Al-Ahdal, M.N.; Memish, Z.A.; Khubnani, H. Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: Emergence of CTX-M-15-producing E. coli ST131. Ann. Clin. Microbiol. Antimicrob. 2014, 13, 4. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Alharbi, N.S.; Khaled, J.M.; Kadaikunnan, S.; Alobaidi, A.S.; Sharafaddin, A.H.; Alyahya, S.A.; Almanaa, T.N.; Alsughayier, M.A.; Shehu, M.R. Prevalence of Escherichia coli strains resistance to antibiotics in wound infections and raw milk. Saudi J. Biol. Sci. 2018, 26, 1557–1562. [Google Scholar] [CrossRef] [PubMed]
- Majumder, A.A.; Rahman, S.; Cohall, D.; Bharatha, A.; Singh, K.; Haque, M.; Hilaire, M.G.-S. Antimicrobial Stewardship: Fighting Antimicrobial Resistance and Protecting Global Public Health. Infect. Drug Resist. 2020, 13, 4713–4738. [Google Scholar] [CrossRef]
- Ben Aissa, M.; Fernea, A.H.; Bouraoui, H.; Ben Aissa, R.; Mercuri, P.S.; Manai, M. Molecular characterization of plasmid-encoded ACC-1a4 β-lactamase produced by Salmonella strains isolated from water in a Tunisian hospital. Ann. Microbiol. 2011, 62, 31–36. [Google Scholar] [CrossRef]
- Samarasinghe, S.; Reid, R.; Varnakulasingam, A. The Distribution of ESBL-Producing Enterobacteriaceae: Leicestershire UK Compared to Worldwide. Am. J. Biomed. Sci. Res. 2019, 3, 70–72. [Google Scholar] [CrossRef]
- Nosheen, S.; Bukhari, N.I.; Junaid, K.; Anwar, N.; Ahmad, F.; Younas, S.; Ejaz, H. Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan. Saudi J. Biol. Sci. 2021, 28, 5875–5883. [Google Scholar] [CrossRef] [PubMed]
- Chedid, M.; Waked, R.; Haddad, E.; Chetata, N.; Saliba, G.; Choucair, J. Antibiotics in treatment of COVID-19 complications: A review of frequency, indications, and efficacy. J. Infect. Public Health 2021, 14, 570–576. [Google Scholar] [CrossRef] [PubMed]
- Ojer-Usoz, E.; González, D.; Vitas, A.I. Clonal Diversity of ESBL-Producing Escherichia coli Isolated from Environmental, Human and Food Samples. Int. J. Environ. Res. Public Health 2017, 14, 676. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yasir, M.; Farman, M.; Shah, M.W.; Jiman-Fatani, A.A.; Othman, N.A.; Almasaudi, S.B.; Alawi, M.; Shakil, S.; Al-Abdullah, N.; Ismaeel, N.A.; et al. Genomic and antimicrobial resistance genes diversity in multidrug-resistant CTX-M-positive isolates of Escherichia coli at a health care facility in Jeddah. J. Infect. Public Health 2019, 13, 94–100. [Google Scholar] [CrossRef]
- Zgurskaya, H.I. Molecular analysis of efflux pump-based antibiotic resistance. Int. J. Med Microbiol. 2002, 292, 95–105. [Google Scholar] [CrossRef]
- Sanger, F.; Nicklen, S.; Coulson, A.R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 1977, 74, 5463–5467. [Google Scholar] [CrossRef] [Green Version]
- Ciesielczuk, H.; Hornsey, M.; Choi, V.; Woodford, N.; Wareham, D. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J. Med Microbiol. 2013, 62, 1823–1827. [Google Scholar] [CrossRef]
- Sáenz, Y.; Zarazaga, M.; Briñas, L.; Lantero, M.; Larrea, F.R.; Torres, C. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int. J. Antimicrob. Agents 2001, 18, 353–358. [Google Scholar] [CrossRef]
- Carattoli, A.; Bertini, A.; Villa, L.; Falbo, V.; Hopkins, K.L.; Threlfall, E.J. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 2005, 63, 219–228. [Google Scholar] [CrossRef]
- Cantón, R.; Horcajada, J.P.; Oliver, A.; Garbajosa, P.R.; Vila, J. Inappropriate use of antibiotics in hospitals: The complex relationship between antibiotic use and antimicrobial resistance. Enferm. Infecc. Microbiol. Clin. 2013, 31, 3–11. [Google Scholar] [CrossRef]
- Takahashi, T.; Masuda, T.; Koyama, Y. Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol. Genet. Genom. 2006, 275, 460–470. [Google Scholar] [CrossRef] [PubMed]
- Tenover, F.C.; Arbeit, R.D.; Goering, R.V.; Mickelsen, P.A.; Murray, B.E.; Persing, D.H.; Swaminathan, B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J. Clin. Microbiol. 1995, 33, 2233–2239. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Clermont, O.; Dhanji, H.; Upton, M.; Gibreel, T.; Fox, A.; Boyd, D.; Mulvey, M.R.; Nordmann, P.; Ruppe, E.; Sarthou, J.L.; et al. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J. Antimicrob. Chemother. 2009, 64, 274–277. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wilson, H.; Török, M.E. Corrigendum: Extended-spectrum β-lactamase-producing and carbapenemase-producing Enterobacteriaceae. Microb. Genom. 2018, 4, e000218. [Google Scholar] [CrossRef] [PubMed]
- Alqasim, A.; Abu Jaffal, A.; Alyousef, A.A. Prevalence of Multidrug Resistance and Extended-Spectrum β-Lactamase Carriage of Clinical Uropathogenic Escherichia coli Isolates in Riyadh, Saudi Arabia. Int. J. Microbiol. 2018, 2018, 302685. [Google Scholar] [CrossRef] [Green Version]
- Alzahrani, M.A.; Sadoma, H.H.M.; Mathew, S.; Alghamdi, S.; Malik, J.A.; Anwar, S. Retrospective Analysis of Antimicrobial Susceptibility of Uropathogens Isolated from Pediatric Patients in Tertiary Hospital at Al-Baha Region, Saudi Arabia. Healthcare 2021, 9, 1564. [Google Scholar] [CrossRef]
- Senok, A.; Khanfar, H.S.; Bindayna, K.M.; Botta, G.A. Extended spectrum beta-lactamases (ESBL) in Escherichia coli and Klebsiella pneumoniae: Trends in the hospital and community settings. J. Infect. Dev. Ctries. 2009, 3, 295–299. [Google Scholar] [CrossRef] [Green Version]
- Bi, W.; Li, B.; Song, J.; Hong, Y.; Zhang, X.; Liu, H.; Lu, H.; Zhou, T.; Cao, J. Antimicrobial susceptibility and mechanisms of fosfomycin resistance in extended-spectrum β-lactamase-producing Escherichia coli strains from urinary tract infections in Wenzhou, China. Int. J. Antimicrob. Agents 2017, 50, 29–34. [Google Scholar] [CrossRef]
- Sghaier, S.; Abbassi, M.S.; Pascual, A.; Serrano, L.; De-Alba, P.D.; Ben Said, M.; Hassen, B.; Ibrahim, C.; Hassen, A.; López-Cerero, L. Extended-spectrum β-lactamase-producing Enterobacteriaceae from animal origin and wastewater in Tunisia: First detection of O25b-B23-CTX-M-27-ST131 Escherichia coli and CTX-M-15/OXA-204-producing Citrobacter freundii from wastewater. J. Glob. Antimicrob. Resist. 2019, 17, 189–194. [Google Scholar] [CrossRef] [PubMed]
- Al-Agamy, M.H.; Aljallal, A.; Radwan, H.H.; Shibl, A.M. Characterization of carbapenemases, ESBLs, and plasmid-mediated quinolone determinants in carbapenem-insensitive Escherichia coli and Klebsiella pneumoniae in Riyadh hospitals. J. Infect. Public Health 2017, 11, 64–68. [Google Scholar] [CrossRef]
- Yasir, M.; Ajlan, A.M.; Shakil, S.; Jiman-Fatani, A.A.; Almasaudi, S.B.; Farman, M.; Baazeem, Z.M.; Baabdullah, R.; Alawi, M.; Al-Abdullah, N.; et al. Molecular characterization, antimicrobial resistance and clinico-bioinformatics approaches to address the problem of extended-spectrum β-lactamase-producing Escherichia coli in western Saudi Arabia. Sci. Rep. 2018, 8, 14847. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hentschke, M.; Kotsakis, S.D.; Wolters, M.; Heisig, P.; Miriagou, V.; Aepfelbacher, M. CMY-42, a Novel Plasmid-Mediated CMY-2 Variant AmpC Beta-Lactamase. Microb. Drug Resist. 2011, 17, 165–169. [Google Scholar] [CrossRef] [PubMed]
- Feng, Y.; Yang, P.; Xie, Y.; Wang, X.; McNally, A.; Zong, Z. Escherichia coli of sequence type 3835 carrying blaNDM-1, blaCTX-M-15, blaCMY-42 and blaSHV-12. Sci. Rep. 2015, 5, 12275. [Google Scholar] [CrossRef] [Green Version]
- Carattoli, A. Resistance Plasmid Families in Enterobacteriaceae. Antimicrob. Agents Chemother. 2009, 53, 2227–2238. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Madec, J.-Y.; Haenni, M. Antimicrobial resistance plasmid reservoir in food and food-producing animals. Plasmid 2018, 99, 72–81. [Google Scholar] [CrossRef]
- Mathers, A.J.; Peirano, G.; Pitout, J.D.D. The Role of Epidemic Resistance Plasmids and International High-Risk Clones in the Spread of Multidrug-Resistant Enterobacteriaceae. Clin. Microbiol. Rev. 2015, 28, 565–591. [Google Scholar] [CrossRef]
Figure 1.
Study design flow diagram (records were approved after repeated antibiograms and synergy tests. Results were compared to our inclusion criteria.).
Figure 1.
Study design flow diagram (records were approved after repeated antibiograms and synergy tests. Results were compared to our inclusion criteria.).
Figure 2.
PFGE patterns for E. coli isolates, with their corresponding pulsotypes.
Table 1.
Patients’ demographic data and specimen types included in our study.
| Variables | Numbers | Percentage (%) | |
|---|---|---|---|
| Gender | Male | 13 | 48.1 |
| Female | 14 | 51.9 | |
| Age | ≤37 | 5 | 18.5 |
| 38–55 | 12 | 44.4 | |
| 56–73 | 6 | 22.2 | |
| 74+ | 4 | 14.8 | |
| Specimen | Sputum | 2 | 7.4 |
| Surgical tissue | 2 | 7.4 | |
| Urine | 14 | 51.9 | |
| Wound | 9 | 33.3 |
Table 2.
E. coli strains characteristics and associated antibiotic resistances.
| Strain | β-Lactam Resistance Phenotypes Detected * | Non-β-Lactam Resistance Phenotypes Detected * |
|---|---|---|
| EC 1 | CAZ, CTX, FEP | NAL, CIP, TET, FOS, SXT |
| EC 2 | AMC, CAZ, CTX, FEP | TOB, NAL, CIP, FOS, CHL, SXT |
| EC 3 | AMC, CTX, FEP | NAL, TET, FOS, CHL, SXT |
| EC 4 | AMC, CTX | NAL, CIP, TET, FOS, CHL, SXT |
| EC 5 | AMC, CTX, FEP | NAL, TET, MNO, FOS, CHL, SXT |
| EC 6 | AMC, CAZ, CTX, FEP | NAL, CIP, TET, MNO, FOS, CHL, SXT |
| EC 7 | AMC, CTX | NAL, CIP, FOS |
| EC 8 | AMC, CAZ, CTX, FOX | TOB, NAL, CIP, TET, MNO, FOS, CHL, SXT |
| EC 9 | AMC, CAZ, CTX, FEP | NAL, CIP, FOS, CHL, SXT |
| EC 11 | AMC, CAZ, CTX, FEP | GMN, TOB, NAL, CIP, TET, FOS, SXT |
| EC 12 | AMC, CAZ, CTX, FEP | NAL, FOS, SXT |
| EC 14 | AMC, CAZ, CTX, FEP | GMN, TOB, NET, NAL, CIP, TET, FOS, CHL, SXT |
| EC 15 | AMC, CTX, FEP | NAL, CIP, FOS, CHL, SXT |
| EC 16 | AMC, CAZ, CTX, PEP | GMN, TOB, NAL, CIP, TET, FOS, CHL, SXT |
| EC 17 | AMC, CTX, FEP | NAL, CIP, TET, FOS, CHL, SXT, |
| EC 18 | CAZ, CTX, FEP | NET, NAL, CIP, FOS, CHL, |
| EC 19 | AMC, CTX, FEP | NAL, CIP, FOS |
| EC 20 | CTX, FEP | NAL, CIP, FOS, SXT |
| EC 21 | AMC, CAZ, CTX, FEP | NAL, CIP, TET, MNO, FOS, SXT |
| EC 22 | AMC, CTX | NAL, CIP, TET, FOS, CHL, SXT |
| EC 23 | AMC, CTX | NAL, CIP, FOS, CHL, SXT |
| EC 24 | AMC, CTX | TOB, NAL, CIP, FOS, CHL, SXT |
| EC 25 | AMC, CTX, FEP | TOB, NAL, CIP, FOS, SXT |
| EC 26 | AMC, CTX, FEP | NAL, CIP, TET, FOS, CHL |
| EC 27 | AMC, CAZ, CTX, FEP | GMN, TOB, NAL, CIP, TET, FOS, CHL, SXT |
| EC 28 | AMC, CAZ, CTX, FEP | GMN, TOB, NAL, CIP, TET, FOS, CHL, SXT |
| EC 29 | AMC, CTX, FOX | NAL, CIP, FOS, CHL, SXT |
* AMC: amoxicillin/clavulavic acid (30 µg); CAZ: ceftazidime (10 µg); CTX: cefotaxime (30 µg); FOX: cefoxitine (30 µg); FEP: cefepime (30 µg); NAL: nalidixic acid (30 µg); CIP: ciprofloxacin (5 µg); SXT: sulfamethoxazole/trimethoprim (25 µg); TET: tetracycline (30 µg); MNO: minocycline (30 µg); GMN: gentamicin (10 µg); TOB: tobramycin (10 µg); NET: netilmicin (10 µg); FOS: fosfomycin (50 µg); CHL: chloramphenicol (30 µg).
Table 3.
Molecular characterization, virulence profiles, and virulence scores of E. coli strains.
| Strains | bla and PMQR* Genes Identified | Virulence Profile | Virulence Score |
|---|---|---|---|
| EC 1 | blaCTX-M-15, qnrS1 | fimH-papGII-iha-iutA-traT-malX-usp-ompT | 8 |
| EC 2 | blaCMY-42, blaCTX-M-15, blaSHV-1 | fimH-papGII-iutA-malX | 4 |
| EC 3 | blaCTX-M-15, blaCTX-M-27, blaTEM | papGII-ompT | 2 |
| EC 4 | blaCTX-M-27 | fimH-papGII-iha-malX-usp-ompT | 6 |
| EC 5 | blaCTX-M-15, blaTEM-1 | malX-ompT | 2 |
| EC 6 | blaCTX-M-15, blaTEM-1 | papGII-fyuA-malX-ompT | 4 |
| EC 7 | blaCTX-M-15, qnrS1 | fimH-papGII-iha-iutA-kpsMTII-malX-usp-ompT | 8 |
| EC 8 | blaCMY-42, blaTEM-1, blaSHV-1, qnrS1, aac(6′)Ib-cr | fimH-papGII | 2 |
| EC 9 | blaCTX-M-15, blaTEM-1 | papGII-malX | 2 |
| EC 11 | blaCTX-M-15, blaSHV, qnrS1 | papGII-iha-traT-malX-usp-ompT | 6 |
| EC 12 | blaCTX-M-15, qnrS1, aac(6′)Ib-cr | papGI-iha-traT-malX-usp | 5 |
| EC 14 | blaCTX-M-15, blaTEM-1 | papGIII-malX-usp-ompT | 4 |
| EC 15 | blaCTX-M-15 | papGII-iha-usp-ompT | 4 |
| EC 16 | blaCTX-M-15, blaTEM-1, blaSHV-1 | papGII-iha-iutA-traT-usp-ompT | 6 |
| EC 17 | blaCTX-M-15, blaTEM-1 | fimH-papGII-iha--iutA-ompT | 5 |
| EC 18 | blaCTX-M-15, blaTEM-1 | fimH-iha-iutA-kpsMTII-malX-usp-ompT | 7 |
| EC 19 | blaCTX-M-15, blaTEM-1, qnrS1 | fimH-iha-iutA-kpsMTII-malX-usp-ompT | 7 |
| EC 20 | blaCTX-M-27, blaTEM-1, qnrS1 | fimH-papGII-iha-traT-usp-ompT | 6 |
| EC 21 | blaCTX-M-15, blaTEM-1, qnrS1 | fimH-iha-kpsMTII-usp-ompT | 5 |
| EC 22 | blaCTX-M-27, blaTEM-1 | fimH-papGII-iha-usp-ompT | 5 |
| EC 23 | blaCTX-M-27, blaTEM-1 | papGII-ompT | 2 |
| EC 24 | blaCTX-M-15, blaTEM-1, qnrS1, aac(6′)Ib-cr | - | - |
| EC 25 | blaCTX-M-15, blaSHV-1 | fimH-papGII-iha-iutA-malX-usp-ompT | 7 |
| EC 26 | blaCTX-M-15, blaTEM-1 | fimH-papGII-iutA-ompT | 4 |
| EC 27 | blaCTX-M-15, blaTEM-1, qnrS1, aac(6′)Ib-cr | papGI-papGII-iha-kpsMTII-hlyA traT-usp-ompT | 8 |
| EC 28 | blaCTX-M-15, blaTEM-1-1, qnrS1, aac(6′)Ib-cr | fimH-papGI-iha-iutA-kpsMTII-traT-malX-usp-ompT | 9 |
| EC 29 | blaCTX-M-9, blaCTX-M-27, blaTEM-1, qnrS1, aac(6′)Ib-cr | fimH-papGII-malX-usp | 4 |
* PMQR: plasmid-mediated quinolone resistance.
Table 4.
Identification of associated plasmid-mediated int1 and int2 genes, phylogenetic groups, ST131 clone, and virulence factors and pulsotypes.
Table 4.
Identification of associated plasmid-mediated int1 and int2 genes, phylogenetic groups, ST131 clone, and virulence factors and pulsotypes.
| Strains | int Genes | PRT | PG | PFGE |
|---|---|---|---|---|
| EC 1 | - | F | B2 | P6 |
| EC 2 | int1 | F | D | P2 |
| EC 3 | int2 | FIA | D | P20 |
| EC 4 | - | - | B2 | P11 |
| EC 5 | - | - | A | P7 |
| EC 6 | - | - | B2 | P21 |
| EC 7 | - | - | D | P9 |
| EC 8 | int2 | FIA-F | B1 | P10 |
| EC 9 | int1 | - | B2 | P8 |
| EC 11 | - | F | B2 | P14 |
| EC 12 | int1 | FIA-F | B2 | P19 |
| EC 14 | - | - | B2 | P8 |
| EC 15 | int1 | FIA | B2 | P18 |
| EC 16 | int1 | - | B2 | P19 |
| EC 17 | - | FIA | B2 * | P1 |
| EC 18 | - | FIA | B2 * | P1 |
| EC 19 | - | FIA | B2 * | P1 |
| EC 20 | - | - | B2 | P16 |
| EC 21 | - | - | B2 | P15 |
| EC 22 | - | - | B2 | P16 |
| EC 23 | - | - | D | P12 |
| EC 24 | - | FIA-F | D | P5 |
| EC 25 | - | - | B2 | P4 |
| EC 26 | - | - | A | P13 |
| EC 27 | int1 | FIA-F | B2 | P3 |
| EC 28 | int1 | FIA-F | B2 | P3 |
| EC 29 | - | - | B2 | P1 |
-: Negative character; *: strain belongs to ST131 clone; P: pulsotype; PG: Phylogenetic Group; PRT: Plasmid Replicon Type; PFGE: Pulsed-Field Gel Electrophoresis.
Table 5.
Positive transconjugants compared with donor strains.
| Strains | Non-β-Lactams Resistance Phenotypes Detected | bla and PMQR Genes | int Genes | PRT |
|---|---|---|---|---|
| EC 1 | NAL, CIP, TET, FOS, SXT | blaCTX-M-15, blaTEM, qnrS1 | - | F |
| * Tc EC 1 | TET | blaCTX-M-15 | - | F |
| EC 7 | NAL, CIP, FOS | blaCTX-M-15, qnrS1 | - | - |
| Tc EC 7 | - | blaCTX-M-15 | - | - |
| EC 8 | TOB, NAL, CIP, TET, MNO, FOS, CHL, SXT | blaCMY-42, blaTEM, blaSHV, qnrS1, aac(6′)Ib-cr | int2 | FIA-F |
| Tc EC 8 | TET, MNO | blaCMY-42, blaTEM, qnrS1 | - | FIA |
| EC 11 | GMN, TOB, NAL, CIP, TET, FOS, SXT | blaCTX-M-15, blaSHV, qnrS1 | - | F |
| Tc EC 11 | TET | blaCTX-M-15 | - | F |
| EC 12 | NAL, FOS, SXT | blaCTX-M-15, qnrS1, aac(6′)Ib-cr | int1 | FIA-F |
| Tc EC 12 | - | blaCTX-M-15 | int1 | FIA |
| EC 19 | NAL, CIP, FOS | blaCTX-M-15, blaTEM, qnrS1 | - | FIA |
| Tc EC 19 | - | blaCTX-M-15 | - | FIA |
| EC 20 | NAL, CIP, FOS, SXT | blaCTX-M-27, blaTEM, qnrS1 | - | - |
| Tc EC 20 | SXT | blaCTX-M-27, blaTEM, qnrS1 | - | - |
| EC 21 | NAL, CIP, TET, MNO, FOS, SXT | blaCTX-M-15, blaTEM, qnrS1 | - | - |
| Tc EC 21 | TET, MNO, SXT | blaCTX-M-15, blaTEM, qnrS1 | - | - |
| EC 24 | TOB, NAL, CIP, FOS, CHL, SXT | blaCTX-M-15, blaTEM, qnrS1, aac(6′)Ib-cr | - | FIA-F |
| Tc EC 24 | TOB, CHL, SXT | blaCTX-M-15, blaTEM, qnrS1, aac(6′)Ib-cr | - | FIA-F |
| EC 27 | GMN, TOB, NAL, CIP, TET, FOS, CHL, SXT | blaCTX-M-15, blaTEM, qnrS1, aac(6′)Ib-cr | int1 | FIA-F |
| Tc EC 27 | TET | blaCTX-M-15 | int1 | F |
| EC 28 | GMN, TOB, NAL, CIP, TET, FOS, CHL, SXT | blaCTX-M-15, blaTEM, qnrS1, aac(6′)Ib-cr | int1 | FIA-F |
| Tc EC 28 | TET, SXT | blaCTX-M-15, blaTEM, qnrS1 | int1 | FIA-F |
| EC 29 | NAL, CIP, FOS, CHL, SXT | blaCTX-M-9, blaCTX-M-27, blaTEM, qnrS1, aac(6′)Ib-cr | - | - |
| Tc EC 29 | CHL, SXT | blaCTX-M-9, blaTEM, qnrS1 | - | - |
* Tc: transconjugant; -: negative character; NAL: nalidixic acid; CIP: ciprofloxacin; SXT: trimethoprim-sulfamethoxazole; TET: tetracycline; MNO: minocyclin; GMN: gentamicin; TOB: tobramycin; FOS: fosfomicin; CHL: chloramphenicol; PMQR: plasmid-mediated quinolone resistance; PRT: plasmid replicon type.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Aissa, M.B.; Ferjani, S.; Abassi, M.S.; Al-Suwailem, N.; Boutiba, I. Characterization of Escherichia coli Cefotaxime-Resistance in Al-Ahsa, KSA: Predominance of CTX-15 and First Report of blaCMY-42 Gene. Appl. Sci. 2022, 12, 9964. https://doi.org/10.3390/app12199964
AMA Style
Aissa MB, Ferjani S, Abassi MS, Al-Suwailem N, Boutiba I. Characterization of Escherichia coli Cefotaxime-Resistance in Al-Ahsa, KSA: Predominance of CTX-15 and First Report of blaCMY-42 Gene. Applied Sciences. 2022; 12(19):9964. https://doi.org/10.3390/app12199964
Chicago/Turabian StyleAissa, Melek Ben, Sana Ferjani, Mohamed Salah Abassi, Nada Al-Suwailem, and Ilhem Boutiba. 2022. "Characterization of Escherichia coli Cefotaxime-Resistance in Al-Ahsa, KSA: Predominance of CTX-15 and First Report of blaCMY-42 Gene" Applied Sciences 12, no. 19: 9964. https://doi.org/10.3390/app12199964
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
