Antibiotic Resistance Hotspot: Comparative Genomics Reveals Multiple Strains of Multidrug-Resistant Citrobacter portucalensis in Edible Snails
by
, , ,
Arthur C. Okafor
1,*
,
Adriana Cabal Rosel
1
,
Frank C. Ogbo
2,
Charles O. Adetunji
3,
Odoligie Imarhiagbe
4,
Lukas Gamp
1,
Anna Stöger
1,
Franz Allerberger
1 and
Werner Ruppitsch
1,5,6,* 1
Institute of Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety, 1090 Vienna, Austria
2
Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka PMB 5025, Anambra State, Nigeria
3
Department of Microbiology, Edo State University, Uzairue PMB 04, Edo State, Nigeria
4
Department of Health and Social Science, London School of Science and Technology, Birmingham B6 5RQ, UK
5
Department of Biotechnology, University of Natural Resources and Life Sciences, 1180 Vienna, Austria
6
Faculty of Food Technology, Food Safety and Ecology, University of Donja Gorica, 81000 Podgorica, Montenegro
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(18), 9889; https://doi.org/10.3390/ijms25189889
Submission received: 16 August 2024
/
Revised: 7 September 2024
/
Accepted: 11 September 2024
/
Published: 13 September 2024
(This article belongs to the Section Molecular Microbiology)
Abstract
:The demand for terrestrial snails as a food source is still on the increase globally, yet this has been overlooked in disease epidemiology and the spread of antimicrobial resistance. This study conducted genomic analyses of twenty Citrobacter portucalensis strains isolated from live edible snails traded in two hubs. The isolates were subjected to MALDI-TOF MS, antimicrobial resistance testing, whole genome sequencing, and analyses for in-depth characterization. The findings disclosed that seventeen strains across the two trading hubs were distinct from previously reported ones. Four isolates were found to share the same sequence type (ST881). Genome-based comparison suggests a clonal transmission of strains between snails traded in these hubs. All the isolates across the two hubs harbored similar variety of antimicrobial resistance genes, with notable ones being blaCMY and qnrB. Sixteen isolates (80%) expressed phenotypic resistance to second-generation cephalosporins, while eleven isolates (55%) exhibited resistance to third-generation cephalosporins. This report of multi-drug-resistant C. portucalensis strains in edible snails highlights significant concerns for food safety and clinical health because of the potential transmission to humans. Enhanced surveillance and stringent monitoring by health authorities are essential to evaluate the impact of these strains on the burden of antimicrobial resistance and to address the associated risk.
1. Introduction
Some terrestrial snails are considered nutritious and are commonly available in the market for commercial purposes in most regions of the world. Achatina achatina is a terrestrial gastropod of the family “Achatinidae”, which has been listed among edible snails [1]. The demand for terrestrial snails as a food source is still on the increase among consumers. For instance, the European market size for such snails is estimated to grow to USD 699.72 million by 2028 [2]. The association of edible snails with high counts of viable bacterial pathogens, which can be difficult to eliminate during culinary preparation in domestic kitchens [3,4,5], is a neglected factor in disease epidemiology and the spread of antimicrobial resistance. This represents a serious health concern in countries where the value chain is neither traceable nor controlled.
Citrobacter species are members of the Enterobacteriaceae, which are of rising clinical importance and usually present in soil, water, and the intestines of food animals, such as snails [3,6,7,8]. Pawar et al. concluded that an apparent feature of bacterial communities in snails’ gastrointestinal tract was the abundance of members of the genus Citrobacter [9]. There is no report on the genomic diversity of specific strains of Citrobacter in terrestrial snails destined for human consumption which might aid in assessing the safety of the value chain. The use of conventional tests and MALDI-TOF MS for differentiating species of Citrobacter for recognizing species of clinical significance has been challenging [8,10,11], thereby highlighting the need for more-advanced techniques. The whole genome sequence (WGS)-based characterization of bacterial pathogens offers unique resolution in discriminating even highly related lineages, thereby precluding the use of species-dependent protocols. Genome data provide more information concerning pathogen detection and identification, epidemiological typing, and antimicrobial resistances [12]. Several Citrobacter species have been found to be zoonotic pathogens capable of transmission to humans during the processing of carcasses of food animals; they can survive long periods of time in their hosts and accumulate antimicrobial resistance (AMR) genes [13,14].
C. portucalensis was first isolated from an aquatic sample in Portugal [15]. WGS had been used to detect C. portucalensis in non-clinical sources such as water and vegetables and has been found to possess antimicrobial resistance [15,16]. A pioneer report on a clinical multi-drug-resistant C. portucalensis strain suggests that the prevalence of this species in the clinical environment is significantly underrated. This study emphasized the potential for clinically “rare” species to become reservoirs of drug resistance in the future [17]. To our knowledge, WGS has not been deployed to assess the genomic diversity of multiple strains of C. portucalensis associated with non-clinical sources in the context of food safety.
Hence, our study aimed to understand the diversity and genomic characteristics of twenty C. portucalensis strains recovered from the gut of live terrestrial snails fated for culinary preparations from two popular trading hubs across two cities in Nigeria. Our study contributes specific data for enhancing public health surveillance, thereby engendering efforts to reduce the burden of AMR as per the One Health concept.
2. Results and Discussion
Our study has demonstrated the emergence of diverse strains of C. portucalensis in the gut of terrestrial snails sold as food source in two markets. The twenty C. portucalensis isolates were assigned to seventeen new sequence types (STs) (ST881–ST897) based on the conventional MLST scheme. Four isolates (Nsk1, Nsk2, Igbk10, and Igbk13) were found to share the same ST (ST881) (Table 1). This diversity across the two markets supports our hypothesis that occurrence and spread of C. portucalensis in food snails is underestimated with conventional methods. Previous studies have reported the presence of C. portucalensis in non-clinical sources such as water and vegetables via WGS analysis [15,16]. C. portucalensis was detected in some Nigerian vegetables [16]. Vegetables play a major role in the dietary habit of snails [18] and could be the source of this bacterium in snails.
The genetic relationship between the isolates in our study, determined using conventional MLST and an ad hoc cgMLST scheme, resulted in a cluster between the markets for isolates Nsk1 and Igbk13 (Figure 1 and Table 1). The allelic difference between the two isolates is 1 (Figure 1), which suggests a clonal transmission of strains between snails traded in two markets. Genomic comparisons utilizing cgMLST can be effective for detecting novel transmission [19]. All the strains in our study were found to be unrelated to the GenBank strains.
All strains harbored more than 20 AMR genes, notably blaCMY and qnrB (Table 2). This is a first-time report of the presence of AMR genes in C. portucalensis strains associated with terrestrial snails meant for culinary processing in domestic kitchens. These findings are of clinical significance because a previous study had reported a similar resistant strain of C. portucalensis (strain 3839) recovered from a diabetic patient in China [17]. blaCMY and qnrB encode resistance to beta-lactam and fluoroquinolone antibiotics, respectively. These genes (blaCMY and qnrB) have also been reported in C. portucalensis strains isolated from vegetables, poultry, diabetic patients, and green sea turtles [16,17,20,21]. Thus, our results corroborate the findings of other studies that C. portucalensis is a global multi-drug-resistant bacterium that harbors intrinsic resistance genes, such as blaCMY and qnrB [21], with potential for mobilization to mobile genetic elements [15,22]. Scientific investigations have recognized soil as a principal reservoir for antibiotic resistance genes [23,24,25]. The soil is probably the source of the AMR genes harbored by these snails because their movement is characterized by continuous contact with the soil. Our study also demonstrated that the isolates exhibited phenotypic resistance to certain antibiotics, such as cefoxitin (n = 16), ampicillin (n = 9), amoxicillin/clavulanic acid (n = 12), and cefpodoxime (n = 11), which have food safety and clinical implications. One of our strains (Igbk10) was found to be deceptive because of its inability to express AMR phenotypically despite its possession of functional genes and plasmids (Table 2).
Three virulence genes (traT, irp2, and fyuA) were concurrently detected in the genomes of 4 of the 20 isolates (Table 2) of the same sequence type (ST881) (Table 1). The traT increases serum resistance in the regulation of virulence in bacterial pathogens [26,27], while irp2 and fyuA are iron-uptake genes [28]. Salgueiro et al. [29] reported some virulence genes, such as traT-type, present in strains of C. portucalensis isolated from bivalve samples collected in Portuguese farms. traT was the most-detected virulence gene (9.0%) in 66 Gram-negative bacteria isolated from seabream and bivalve molluscs in the southern region of Portugal. fyuA and irp2 were among the nine virulence genes demonstrated to occur more frequently in avian pathogenic E. coli strains than non-pathogenic strains [30]. In fact, these two genes have been detected by PCR in E. coli strains isolated from poultry birds with clinical signs of colibacillosis in Korea and Brazil [31,32]. Exploring the distribution of virulence genes among C. portucalensis isolates can offer valuable insights into their epidemiology and pathogenic potential.
Two plasmids (col440I and IncFIB(k)) were detected in one of our 20 isolates. Plasmid IncFIB(pHCM2) was found in another isolate (Table 2). IncFIB(K)-type plasmid has been reported in strains of C. portucalensis recovered from gills of fishes in the southern region of Portugal [29]. IncFIB(K)-type and Col440I-type plasmids have been described for strains of C. portucalensis recovered from bivalve samples collected in farms in the southern region of Portugal [29]. The detection of different plasmid types among these C. portucalenesis strains underscores their genetic diversity and adaptability.
3. Materials and Methods
3.1. Isolation of Citrobacter
A total of 50 samples of terrestrial snails (Achatina achatina) were randomly procured from two markets (Nsukka and Igboukwu) in the southeast zone of Nigeria. The intestinal sections of the samples were aseptically collected during the usual evisceration step of culinary preparation in the domestic kitchen, as is common practice in Nigeria. Within an hour, samples were transported to the laboratory for analysis. The intestinal samples (50 g) were homogenized with 450 mL of peptone water (Oxoid, UK). Aliquots (5 mL) of the homogenate were enriched in Selenite F broth (45 mL) for 18 h before plating 0.1 mL on Salmonella–Shigella agar (Oxoid). Strains were identified as Citrobacter using standard bacteriological methods, as previously described [3].
3.2. In Vitro Characterization of Sensitivity to Antimicrobial Agents
The isolates were tested for antimicrobial resistance using the disk diffusion method according to the CLSI M02-A12 Supplement [33]. The following antimicrobial agents (Oxoid) were used: ampicillin (10 µg), imipenem (10 µg), meropenem (10 µg), mecillinam (10 µg), piperacillin/tazobactam (110 µg), amoxycillin/clavulanic acid (30 µg), gentamicin (10 µg), ertapenem (10 µg), levofloxacin (5 µg), ciprofloxacin (5 µg), trimethoprim (5 µg), amikacin (30 µg), cefpodoxime (10 µg), cefpodoxime/clavulanic acid (1 µg), cefoxitin (30 µg), cefotaxime (5 µg), cefepime (30 µg), and ceftazidime (30 µg). The European Committee on Antimicrobial Susceptibility Testing [34] Clinical Breakpoint Table was used for interpretation.
3.3. Identification of Bacteria Using MALDI-TOF MS
Twenty strains that were found to be either hemolytic or resistant to at least one antibiotic were selected for identification using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) based on the manufacturer’s instructions (Bruker Daltonics, Bremen, Germany). Briefly, isolates were sub-cultured on blood agar and incubated at 35 °C for 24 h prior to the MALDI-TOF identification step. Next, a single colony of each isolate was picked from the agar plate and aseptically smeared on a spot of a bar-coded MSP 96 target polished steel plate (Bruker Daltonics, Germany). The smeared spots on the bar-coded steel plate were overlaid with 1 µL matrix solution provided by the manufacturer and allowed to dry at room temperature. The samples were measured with a Bruker Microflex MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) using MBT AutoX. Identification was conducted using the Biotyper software, version 4.1.100, in the default settings. Identification scores of >1.7 or >2.0 indicated a reliable genus or species identification, respectively [35].
3.4. Whole Genome Sequencing (WGS) of the Isolates and Analysis
Genomic DNA was extracted from overnight cultures grown at 37 °C on blood agar using the MagAttract® HMW DNA kit 48 (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of DNA was measured using Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) with the dsDNA HS assay kit (Thermo Fisher Scientific) and DropSense 16 System (Trinean NV, Gentbrugge, Belgium) in accordance with the manufacturer’s manual.
In strict compliance with the manufacturer’s protocol, ready-to-sequence DNA libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, San Diego, USA). WGS (paired end, 2 × 150 bp) was performed using the Illumina Nextseq 2000 instrument. The de novo assembly of raw reads was conducted with SPAdes (version 3.9.0) [36]. The WGS data were analyzed using multi-loci sequence typing (MLST) and core genome (cg)MLST using Ridom SeqSphere+ software v.9.0.8 (Ridom GmbH, Münster, Germany). An ad hoc cgMLST scheme comprising 2962 core target genes was created using C. portucalensis NCTC11104 as the reference genome (GenBank accession NZ_LR134214.1), along with 21 other C. portucalensis genomes from GenBank, as query genomes for comparison of our strains with all C. portucalensis genomes available at NCBI GenBank. The genomic relationship of isolates was depicted using minimum spanning trees.
The presence of resistance genes, virulence genes, and mobile genetic elements were determined with the Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca) (accessed on 14 July 2022) and Centre for Genomic Epidemiology web server tools (https://cge.food.dtu.dk/services/MobileElementFinder/) (accessed on 19 July 2022). All software was used with default parameters.
4. Conclusions
The presence of multi-drug-resistant strains of C. portucalenesis in edible snails traded in two Nigerian hubs, as determined by next-generation sequencing, is of both food safety and clinical concern for areas where the value chain of snails is neither traceable nor controlled. These findings highlight the need for enhanced surveillance and stringent monitoring of edible snails to mitigate potential risks associated with the spread of antimicrobial resistance. Public health authorities should prioritize assessing and managing the impact of these resistant strains to prevent their potential transmission to humans and address the broader implications for antimicrobial resistance.
Author Contributions
Conceptualization, A.C.O., F.C.O. and W.R.; methodology, A.C.O., A.C.R., F.C.O., C.O.A., O.I., L.G., A.S., F.A. and W.R.; software, A.C.O., A.C.R., L.G., A.S. and W.R.; validation, A.C.O., F.C.O., C.O.A., O.I., F.A. and W.R.; investigation, A.C.O., F.C.O., C.O.A., O.I., L.G. and A.S.; resources, A.C.O., A.C.R., F.C.O., L.G., F.A. and W.R.; data curation, A.C.O., F.C.O. and A.S.; writing—original draft preparation, A.C.O., F.C.O., F.A. and W.R.; writing—review and editing, A.C.O., A.C.R., F.C.O., C.O.A., O.I., L.G., A.S., F.A. and W.R.; funding acquisition, A.C.O. and W.R. All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported by a postdoctoral research fellowship (Ernst Mach grant MPC-2021-00071) awarded to Arthur Okafor, financed by the Austrian Federal Ministry of Education, Science, and Research (BMBWF) and administered by Austria’s Agency for Education and Internationalisation (OeAD).
Institutional Review Board Statement
Ethical review and approval were waived for this study because it involved bacterial strains isolated from edible snail samples classified as food items in Nigeria. The samples were not subjected to any experimentation beyond what is standard for food preparation.
Informed Consent Statement
Not applicable.
Data Availability Statement
This Whole-Genome Shotgun project has been deposited at the DDBJ/ENA/GenBank under the BioProject accession no. PRJNA855947. The version described in this paper is the first version.
Conflicts of Interest
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
References
- European Commission [EC]. Regulation EC 853/2004 on Laying Down Specific Rules for Food of Animal Origin. 2004. Available online: https://eur-lex.europa.eu/eli/reg/2004/853/oj (accessed on 8 September 2023).
- Cognitive Market Research. Global Edible Snail Market Report, CMR165668. 2023. Available online: https://www.cognitivemarketresearch.com/edible-snail-market-report (accessed on 8 September 2023).
- Okafor, A.C.; Ogbo, F.C. Occurrence and Enumeration of Multiple Bacterial Pathogens in Edible Snails from South East Nigeria. Food Sci. Technol. 2019, 7, 23–30. [Google Scholar] [CrossRef]
- Okafor, A.C.; Ogbo, F.C. Virulence Potentials of Bacillus Strains Recovered From Edible Snails and Survival During Culinary Preparation. Food Control. 2020, 108, 106834. [Google Scholar] [CrossRef]
- Okafor, A.C.; Ogbo, F.C. Potentials of Common Desliming Agents in Decontaminating Snail Meat During Culinary Processing (Oral Presentation). In Proceedings of the 43rd Annual Conference of Nigerian Society for Microbiology (NSM), University of Port Harcourt, Port Harcourt, Nigeria, 6–10 September 2021; pp. 58–60. [Google Scholar]
- Oberhettinger, P.; Schüle, L.; Marschal, M.; Bezdan, D.; Ossowski, S.; Dörfel, D.; Vogel, W.; Rossen, J.W.; Willmann, M.; Peter, S. Description of Citrobacter cronae sp. nov., isolated from human rectal swabs and stool samples. Int. J. Syst. Evol. Microbiol. 2020, 70, 2998–3003. [Google Scholar] [CrossRef] [PubMed]
- Liu, L.-H.; Wang, N.-Y.; Wu, A.Y.-J.; Lin, C.-C.; Lee, C.-M.; Liu, C.-P. Citrobacter freundii bacteremia: Risk factors of mortality and prevalence of resistance genes. J. Microbiol. Immunol. Infect. 2018, 51, 565–572. [Google Scholar] [CrossRef]
- Okafor, A.C.; Rosel, A.C.; Ogbo, F.C.; Stöger, A.; Allerberger, F.; Ruppitsch, W. Draft Genome Sequence of Citrobacter cronae Awk (Sequence Type 880), Associated with Edible Snails Commercially Available in Awka, Nigeria. Microbiol. Genome Announc. 2022, 11, e0063422. [Google Scholar] [CrossRef]
- Pawar, K.D.; Banskar, S.; Rane, S.D.; Charan, S.S.; Kulkarni, G.J.; Sawant, S.S.; Ghate, H.V.; Patole, M.S.; Shouche, Y.S. Bacterial diversity in different regions of gastrointestinal tract of Giant African Snail (Achatina fulica). Microbiol. Open 2012, 1, 415–426. [Google Scholar] [CrossRef]
- O’Hara, C.M.; Roman, S.B.; Miller, J.M. Ability of commercial identification systems to identify newly recognized species of Citrobacter. J. Clin. Microbiol. 1995, 33, 242–245. [Google Scholar] [CrossRef] [PubMed]
- Kolínská, R.; Španělová, P.; Dřevínek, M.; Hrabák, J.; Žemličková, H. Species identification of strains belonging to genus Citrobacter using the biochemical method and MALDI-TOF mass spectrometry. Folia Microbiol. 2014, 60, 53–59. [Google Scholar] [CrossRef]
- Quainoo, S.; Coolen, J.P.M.; van Hijum, S.A.F.T.; Huynen, M.A.; Melchers, W.J.G.; van Schaik, W.; Wertheim, H.F.L. Whole-genome sequencing of bacterial pathogens: The future of nosocomial outbreak analysis. Clin. Microbiol. Rev. 2017, 30, 1015–1063. [Google Scholar] [CrossRef]
- Lessenger, J.E. Diseases from Animals, Poultry, and Fish. In Agricultural Medicine; Springer: New York, NY, USA, 2006; pp. 367–382. [Google Scholar]
- Pepperell, C.; Kus, J.V.; Gardam, M.A.; Humar, A.; Burrows, L.L. Low-virulence Citrobacter species encode resistance to multiple antimicrobials. Antimicrob. Agents Chemother. 2002, 46, 3555–3560. [Google Scholar] [CrossRef]
- Ribeiro, T.G.; Goncalves, B.R.; da Silva, M.S.; Novais, Â.; Machado, E.; Carrico, J.A.; Peixe, L. Citrobacter portucalensis sp. nov., isolated from an aquatic sample. Int. J. Syst. Evol. Microbiol. 2017, 67, 3513–3517. [Google Scholar] [CrossRef] [PubMed]
- Igbinosa, E.O.; Rathje, J.; Habermann, D.; Brinks, E.; Cho, G.S.; Franz, C.M. Draft genome sequence of multidrug-resistant strain Citrobacter portucalensis MBTC-1222, isolated from uziza (Piper guineense) leaves in Nigeria. Genome Announc. 2018, 6. [Google Scholar] [CrossRef] [PubMed]
- Cao, X.; Xie, H.; Huang, D.; Zhou, W.; Liu, Y.; Shen, H.; Zhou, K. Detection of a clinical carbapenem-resistant Citrobacter portucalensis strain and the dissemination of C. portucalensis in clinical settings. J. Glob. Antimicrob. Resist. 2021, 27, 79–81. [Google Scholar] [CrossRef]
- Chah, J.M.; Inegbedion, G. Characteristics of snail farming in Edo South Agricultural Zone of Edo State, Nigeria. Trop. Anim. Health Prod. 2013, 45, 625–631. [Google Scholar] [CrossRef] [PubMed]
- Leopold, S.R.; Goering, R.V.; Witten, A.; Harmsen, D.; Mellmann, A. Bacterial whole-genome sequencing revisited: Portable, scalable, and standardized analysis for typing and detection of virulence and antibiotic resistance genes. J. Clin. Microbiol. 2014, 52, 2365–2370. [Google Scholar] [CrossRef]
- Hasan, S.; Sultana, M.; Hossain, M.A. Complete Genome Arrangement Revealed the Emergence of a Poultry Origin Superbug Citrobacter Portucalensis Strain NR-12. J. Glob. Antimicrob. Resist. 2019, 18, 126–129. [Google Scholar] [CrossRef]
- Sellera, F.P.; Fernandes, M.R.; Fuga, B.; Fontana, H.; Vásquez-Ponce, F.; Goldberg, D.W.; Monte, D.F.; Rodrigues, L.; Cardenas-Arias, A.R.; Lopes, R.; et al. Phylogeographical landscape of citrobacter portucalensis carrying clinically relevant resistomes. Microbiol. Spectr. 2022, 10, e0150621. [Google Scholar] [CrossRef] [PubMed]
- Porres-Osante, N.; Sáenz, Y.; Somalo, S.; Torres, C. Characterization of beta-lactamases in faecal enterobacteriaceae recovered from healthy humans in Spain: Focusing on AmpC polymorphisms. Microb. Ecol. 2014, 70, 132–140. [Google Scholar] [CrossRef] [PubMed]
- Martínez, J.L. Antibiotics and antibiotic resistance genes in natural environments. Science 2008, 321, 365–367. [Google Scholar] [CrossRef]
- Finley, R.L.; Collignon, P.; Larsson, D.G.J.; McEwen, S.A.; Li, X.-Z.; Gaze, W.H.; Reid-Smith, R.; Timinouni, M.; Graham, D.W.; Topp, E. The scourge of antibiotic resistance: The important role of the environment. Clin. Infect. Dis. 2013, 57, 704–710. [Google Scholar] [CrossRef]
- Fitzpatrick, D.; Walsh, F. Antibiotic resistance genes across a wide variety of metagenomes. FEMS Microbiol. Ecol. 2016, 92, fiv168. [Google Scholar] [CrossRef] [PubMed]
- Chakraborty, S.; Mukhopadhyay, A.K.; Bhadra, R.K.; Ghosh, A.N.; Mitra, R.; Shimada, T.; Yamasaki, S.; Faruque, S.M.; Takeda, Y.; Colwell, R.R.; et al. Virulence Genes in Environmental Strains of Vibrio cholerae. Appl. Environ. Microbiol. 2000, 66, 4022–4028. [Google Scholar] [CrossRef]
- Altalhi, A.D.; Hassan, S.A. Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers. Food Control. 2009, 20, 913–917. [Google Scholar] [CrossRef]
- Paixão, A.C.; Ferreira, A.C.; Fontes, M.; Themudo, P.; Albuquerque, T.; Soares, M.C.; Fevereiro, M.; Martins, L.; de Sá, M.I.C. Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates. Poult. Sci. 2016, 95, 1646–1652. [Google Scholar] [CrossRef]
- Salgueiro, V.; Manageiro, V.; Rosado, T.; Bandarra, N.M.; Botelho, M.J.; Dias, E.; Caniça, M. Snapshot of resistome, virulome and mobilome in aquaculture. Sci. Total. Environ. 2023, 905, 166351. [Google Scholar] [CrossRef]
- Rodriguez-Siek, K.E.; Giddings, C.W.; Doetkott, C.; Johnson, T.J.; Nolan, L.K. Characterizing the APEC pathotype. Veter- Res. 2005, 36, 241–256. [Google Scholar] [CrossRef]
- Oh, J.; Kang, M.; Yoon, H.; Choi, H.; An, B.; Shin, E.; Kim, Y.; Kim, M.; Kwon, J.; Kwon, Y. The embryo lethality of Escherichia coli isolates and its relationship to the presence of virulence-associated genes. Poult. Sci. 2012, 91, 370–375. [Google Scholar] [CrossRef] [PubMed]
- De Carli, S.; Ikuta, N.; Lehmann, F.K.M.; da Silveira, V.P.; Predebon, G.d.M.; Fonseca, A.S.K.; Lunge, V.R. Virulence gene content in Escherichia coli isolates from poultry flocks with clinical signs of colibacillosis in Brazil. Poult. Sci. 2015, 94, 2635–2640. [Google Scholar] [CrossRef]
- Clinical and Laboratory Standards Institute. M02-A12 Performance Standards for Antimicrobial Disk Susceptibility Test, 12th ed.; Approved Standards; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2015. [Google Scholar]
- The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 11.0. 2021. Available online: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_11.0_Breakpoint_Tables.pdf (accessed on 7 June 2022).
- Sogawa, K.; Watanabe, M.; Sato, K.; Segawa, S.; Ishii, C.; Miyabe, A.; Murata, S.; Saito, T.; Nomura, F. Use of the MALDI BioTyper system with MALDI–TOF mass spectrometry for rapid identification of microorganisms. Anal. Bioanal. Chem. 2011, 400, 1905–1911. [Google Scholar] [CrossRef]
- Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef]
Figure 1.
Minimum spanning tree generated from the cgMLST comparison of Citrobacter portucalensis strains associated with edible snails (n = 20) with other previously reported strains (n = 8) available on GenBank. The numbers close to the lines indicate allelic difference.
Figure 1.
Minimum spanning tree generated from the cgMLST comparison of Citrobacter portucalensis strains associated with edible snails (n = 20) with other previously reported strains (n = 8) available on GenBank. The numbers close to the lines indicate allelic difference.
Table 1.
Selected genomic features of Citrobacter portucalensis isolates (n = 20) from snails sold in two Nigerian markets.
Table 1.
Selected genomic features of Citrobacter portucalensis isolates (n = 20) from snails sold in two Nigerian markets.
| Isolate | Source Location | Size (bp) | GC Content (%) | Sequence Type | Average Coverage | % Good Targets cgMLST | Genome Accession Number |
|---|---|---|---|---|---|---|---|
| CP_Nsk1 | Nsukka | 5,252,065 | 51.8 | 881 | 41 | 99.2 | JALGBJ000000000 |
| CP_Nsk2 | Nsukka | 5,170,058 | 52.0 | 881 | 46 | 99.0 | JANBWY000000000 |
| CP_Nsk3 | Nsukka | 4,896,645 | 52.0 | 882 | 53 | 99.2 | JANBWX000000000 |
| CP_Nsk4 | Nsukka | 4,969,172 | 52.0 | 883 | 69 | 98.9 | JANBWW000000000 |
| CP_Nsk5 | Nsukka | 5,136,318 | 52.0 | 895 | 67 | 99.4 | JANBWN000000000 |
| CP_Nsk6 | Nsukka | 5,012,648 | 51.9 | 896 | 54 | 99.2 | JANBWM000000000 |
| CP_Nsk7 | Nsukka | 4,843,198 | 52.0 | 897 | 53 | 99.3 | JANBWL000000000 |
| CP_Igbk1 | Igboukwu | 4,648,030 | 52.0 | 884 | 82 | 99.6 | JANBWV000000000 |
| CP_Igbk2 | Igboukwu | 4,968,643 | 51.8 | 885 | 92 | 99.3 | JANBWU000000000 |
| CP_Igbk3 | Igboukwu | 4,766,390 | 52.0 | 886 | 68 | 98.9 | JANBWT000000000 |
| CP_Igbk4 | Igboukwu | 4,838,025 | 52.1 | 887 | 75 | 99.5 | JANDBI000000000 |
| CP_Igbk5 | Igboukwu | 4,943,623 | 52.1 | 888 | 67 | 98.9 | JANDBH000000000 |
| CP_Igbk6 | Igboukwu | 5,797,490 | 51.8 | 889 | 73 | 99.0 | JANBWS000000000 |
| CP_Igbk7 | Igboukwu | 5,295,614 | 51.7 | 890 | 47 | 98.1 | JANDBG000000000 |
| CP_Igbk8 | Igboukwu | 4,794,523 | 52.1 | 891 | 83 | 99.3 | JANBWR000000000 |
| CP_Igbk9 | Igboukwu | 5,430,528 | 51.5 | 892 | 61 | 98.6 | JANBWQ000000000 |
| CP_Igbk10 | Igboukwu | 5,192,886 | 51.9 | 881 | 82 | 98.8 | JANBWP000000000 |
| CP_Igbk11 | Igboukwu | 4,937,101 | 52.0 | 893 | 49 | 99.3 | JANBWO000000000 |
| CP_Igbk12 | Igboukwu | 4,960,921 | 51.8 | 894 | 77 | 99.2 | JANDBF000000000 |
| CP_Igbk13 | Igboukwu | 5,162,285 | 51.9 | 881 | 38 | 98.6 | JANBWK000000000 |
cgMLST: core genome multi-locus sequence typing.
Table 2.
Antimicrobial resistance, virulence, and plasmids detected in Citrobacter portucalensis isolates (n = 20) from snails sold in two Nigerian markets.
Table 2.
Antimicrobial resistance, virulence, and plasmids detected in Citrobacter portucalensis isolates (n = 20) from snails sold in two Nigerian markets.
| Isolate | Resistance Phenotype | Resistance Genes | Virulence Genes | Plasmid |
|---|---|---|---|---|
| CP_Nsk1 | AMC-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB6, CMY-46. | traT, irp2, fyuA | - |
| CP_Nsk2 | CN-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB6, CMY-46. | traT, irp2, fyuA | - |
| CP_Nsk3 | AMC-CD-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB18, CMY-77, CMY-108. | - | - |
| CP_Nsk4 | AMP-AMC-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, acrB, QnrB57, CMY-124. | - | - |
| CP_Nsk5 | AK | PmrF, mdfA, acrA, rsmA, KpnE, KpnF, KdpE, mdtB, mdtC, baeR, msbA, CRP, marA, H-NS, CMY-46, ampH, emrR, emrB, QnrB17, mdtG, GlpT, UhpT, PBP3, EF-Tu, soxS, marR. | - | - |
| CP_Nsk6 | AMP-AMC-CPD-FOX | KdpE, PmrF, mdfA, ampH, acrB, acrA, CRP, QnrB18, H-NS, mdtC, baeR, marA, msbA, mdtG, KpnE, KpnF, CMY-34, rsmA, emRB, emrR, GlpT, PBP3, UhpT, EF-Tu, soxS, marR, qnrB23, qnrB29, qnrB48, blaCMY-106, blaCMY-108, blaCMY-37 | - | - |
| CP_Nsk7 | AMC-CN-CPD-FOX-CTX | CMY-63, KdpE, QnrB6, acrA, PmrF, KpnF, KpnE, emrB, emrR, rsmA, msbA, CRP, baeR, mdtC, mdtB, H-NS, mdtG, mdfA, marA, ampH, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, qnrB41, blaCMY-46 | - | - |
| CP_Igbk1 | AMP-AMC-CD-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB6, qnrB9, blaCMY-25, blaCMY-124, blaCMY-2 | - | - |
| CP_Igbk2 | AMP-AMC-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB17, CMY-129, | - | - |
| CP_Igbk3 | AMP-AMC-CD-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB6, CMY-35, qnrB9, blaCMY-2 | - | - |
| CP_Igbk4 | AMP-AMC-CD-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB18, CMY-124, qnrB48, qnrB23, qnrB29 | - | - |
| CP_Igbk5 | AMP-CPD-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB6, CMY-71 | - | - |
| CP_Igbk6 | AMP-AMC-CPD-FOX | msbA, kdpE, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB16, CMY-106 | - | - |
| CP_Igbk7 | FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB16, CMY-124 | irp2, fyuA | - |
| CP_Igbk8 | AMP-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB16, CMY-106 | - | - |
| CP_Igbk9 | - | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB17, CMY-77 | - | - |
| CP_Igbk10 | - | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB6, CMY-46 | traT, irp2, fyuA | Col440I, IncFIB(K) |
| CP_Igbk11 | AMC-FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB18, CMY-108, qnrB48, qnrB23, qnrB29 | - | - |
| CP_Igbk12 | FOX | msbA, kdpE, PmrF, baeR, mdtC, mdtB, mdfA, marA, rsmA, KpnE, KpnF, CRP, ampH, emrB, emrR, H-NS, mdtG, UhpT, PBP3, GlpT, EF-Tu, soxS, marR, acrA, QnrB17, CMY-46, qnrB77 | traT | IncFIB(pHCM2) |
| CP_Igbk13 | AMC-CTX | KdpE, acrA, H-NS, mdtB, mdtC, baeR, rsmA, mdfA, CRP, emrB, emrR, PmrF, msbA, marA, KpnE, KpnF, QnrB6, ampH, mdtG, PBP3, UhpT, GlpT, EF-Tu, soxS, marR, qnrB13, blaCMY-46 | irp2, fyuA, traT | - |
AMP: ampicillin; AMC: amoxycillin/clavulanic acid; CN: gentamicin; AK: amikacin; CD: cefpodoxime/clavulanic acid; CPD: cefpodoxime; FOX: cefoxitin; CTX: cefotaxime.
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Okafor, A.C.; Rosel, A.C.; Ogbo, F.C.; Adetunji, C.O.; Imarhiagbe, O.; Gamp, L.; Stöger, A.; Allerberger, F.; Ruppitsch, W. Antibiotic Resistance Hotspot: Comparative Genomics Reveals Multiple Strains of Multidrug-Resistant Citrobacter portucalensis in Edible Snails. Int. J. Mol. Sci. 2024, 25, 9889. https://doi.org/10.3390/ijms25189889
AMA Style
Okafor AC, Rosel AC, Ogbo FC, Adetunji CO, Imarhiagbe O, Gamp L, Stöger A, Allerberger F, Ruppitsch W. Antibiotic Resistance Hotspot: Comparative Genomics Reveals Multiple Strains of Multidrug-Resistant Citrobacter portucalensis in Edible Snails. International Journal of Molecular Sciences. 2024; 25(18):9889. https://doi.org/10.3390/ijms25189889
Chicago/Turabian StyleOkafor, Arthur C., Adriana Cabal Rosel, Frank C. Ogbo, Charles O. Adetunji, Odoligie Imarhiagbe, Lukas Gamp, Anna Stöger, Franz Allerberger, and Werner Ruppitsch. 2024. "Antibiotic Resistance Hotspot: Comparative Genomics Reveals Multiple Strains of Multidrug-Resistant Citrobacter portucalensis in Edible Snails" International Journal of Molecular Sciences 25, no. 18: 9889. https://doi.org/10.3390/ijms25189889
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