Molecular detection, quantification and characterization of extended-spectrum β-lactamase (ESBL)- and AmpC-encoding genes in a cucumber agroecosystem

Abstract

Antimicrobial resistance (AMR) is a major concern in health care, and has more recently been reported in environmental and agricultural settings. The presence of antibiotic resistant bacteria (ARB) (including extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae) in fresh produce, is therefore imperative owing to the global push towards healthier diets with a focus on raw food due to its high nutritious value. In this context, bacteria that are present on the surfaces of fresh produce may be transferred to the consumers and potentially serve as an indirect route of human exposure to ARB. If pathogenic bacteria such as Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are antibiotic resistant (AR) and found on fresh produce that is consumed raw, difficult-to-treat infections may result later. In addition, this may lead to ARB remaining in the consumer’s gut. This may potentially result in transfer of antibiotic resistance genes (ARGs) to other commensal and human pathogenic bacteria using mobile genetic elements (MGEs) such as plasmids. Treatment of the abovementioned infections with currently available antibiotics may thus become ineffective or fail. When this happens, more expensive medicines will have to be utilised and failure to rapidly recover may have severe impact on health care systems. This therefore calls for improved surveillance of AMR in order to develop mitigation strategies to reduce AMR.

Knowledge about AMR in agriculture in general is limited and more so in production systems. Therefore, to address this knowledge gap and explore the AMR status in a model crop system in South Africa, a cucumber production system was studied. The primary focus of the study was on ESBL- and /or AmpC-producing Enterobacteriaceae due to their prevalence in fresh produce systems and the environment as stated by the World Health Organisation (WHO). In addition, plasmid-mediated AmpC β-lactamase (pAmpC)-producing Enterobacteriaceae have been reported clinically to easily transfer their resistance genes to other bacteria through conjugation and transformation mediated by the plasmids. Herein, molecular methods such as endpoint PCR, Sanger sequencing, whole genome sequencing (WGS) and digital droplet PCR (ddPCR) were used to gain knowledge about AMR in agriculture. This knowledge will be useful in improving AMR surveillance and thus reducing drug resistance.

Using chromogenic ESBL media and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis 65.74% of the detected isolates were Enterobacteriaceae from water 76.06% and cucumber 23.94% samples. In addition, the isolates were found to be 39.44% K. pneumoniae; 29.58% E. coli; 26.76% Proteus vulgaris; 2.82% Enterobacter asburiae and 1.41% Citrobacter freundii. Additionally, using disc diffusion 61.97%, 4.23%, 28.17% and 5.63% of the Enterobacteriaceae isolates produced: ESBLs, AmpC β-lactamases, both ESBLs and AmpC β-lactamases and unknown βlactamases. Furthermore, using molecular method (end-point PCR) led to the detection of ESBL encoding genes [blaSHV (83.09%), blaTEM (69.01%), blaOXA (36.62%), blaCTX-M-Group-1 (35.21%) and blaCTX-M-Group-9 (9.86%)] and pAmpC, encoding genes [blaCIT (2.82%), blaEBC (2.82%), blaACC (1.41%), blaMOX (1.41%) and blaDHA (1.41%)] in the Enterobacteriaceae isolates. Thereafter, using Sanger sequencing, the gene family variants included (SHV-206, SHV-18, SHV-228 and SHV-61), (TEM-242 and TEM-1), OXA-1, CTX-M-15 and (CTX-M14 and CTX-M-27).

In addition, β-lactamase encoding genes in clinically relevant and predominant Enterobacteriaceae species (E. coli and K. pneumoniae) isolated in water and cucumber samples, were detected and characterized using WGS. From the WGS data, the specific βlactamase gene family variants of the Enterobacteriaceae (E. coli) were TEM-1, CTX-M-15 and CTX-M-14 while those of Enterobacteriaceae (K. pneumoniae) were TEM-30, TEM-207, TEM-1, SHV-27, SHV-106, SHV-207, CTX-M-15 and CTX-M-14.

The E. coli isolates’ sequence types were: ST752, ST10, unknown (water) and ST58 (cucumber) and those of K. pneumoniae isolates were: ST29, ST3609, ST985, ST873 (water) and ST985 (cucumber). Furthermore, the E. coli isolates’ serotypes were O123:H40/ O186:H40, O101:H9 and H37 (water) and O75:H9 (cucumber) and those of K. pneumoniae were K30, K39, K52 and K117 (water) and K39 (cucumber). The K. pneumoniae strain with MLST ST985 and K39 serotype was present in both water and cucumber samples. IncF plasmid replicons were identified in 46.15% E. coli and 38.46% K. pneumoniae. A total of 48 virulence genes were identified in the selected isolates with 29 detected in E. coli and 19 in K. pneumoniae. Further WGS data analysis predicted all E. coli and K. pneumoniae isolates as human pathogens.

A next generation ddPCR enumeration model was evaluated and optimised to detect and quantify ESBL-type blaSHV genes (ARG example) in irrigation water and sediment samples from a cucumber production system. The model in conjunction with ultrafiltration monitored a large volume of irrigation water sample for the quantity of ESBL-type blaSHV genes. Results here showed that the irrigation water samples contained between 18.04 copies/mL and 24.24 copies/mL of ESBL-type blaSHV genes whilst sediment samples contained an average of 148.30 copies/mL ESBL-type blaSHV genes. From this study’s results we can conclude that molecular methods have the potential to be useful in providing additional information (about the prevalence, characterization and quantification of ARB/ARGs) that is not well known in agriculture/ fresh produce production systems, that phenotypic diagnostic methods cannot provide. Therefore, the evaluation and improvement of molecular diagnostic methods leads to improved AMR surveillance and thus can aid in developing AMR mitigation strategies. Future studies can use these molecular methods to accumulate more information about AMR dissemination, outbreaks, prevalence, characterization and quantification in agriculture and different fresh produce production systems. As more studies of this nature are conducted and AMR surveillance as well as service delivery (for example water and sanitation infrastructure in countries such as South Africa) is improved a more equitable and healthier living environment can be created.