A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum β-Lactamases, Plasmid-Mediated AmpC β-Lactamases, and Integrons in Gram-Negative Bacteria

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases, aac(6′)-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was blaRANH-2, a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant β-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC β-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.


Introduction
Antimicrobial resistance (AMR) is defined as the ability of microorganisms (bacteria or fungi) to overcome the action of the antibiotics that intend to kill them (i.e., bactericidal), or inhibit their growth (i.e., bacteriostatic) [1]. AMR is considered one of the greatest threats to global public health [1]. In the USA, annual data estimate more than 2.8 million infections with antibiotic-resistant bacteria, causing more than 35,000 deaths [1]. Because of antimicrobial-resistant infections, it is estimated that approximately 10 million global deaths, with fiscal losses of > USD 100 trillion, will occur by 2050 [2].
β-lactams are one of the most clinically important and widely prescribed classes of antibiotics used for the treatment of Gram-negative or Gram-positive bacteria [3]. The
DNA sequences of the different genes and their variants were obtained from the Gen-Bank database and aligned using Clustal Omega software (Clustal W) (https://www.ebi.ac. uk/Tools/msa/clustalo/) (accessed on 10 November 2022). Specific primers were manually designed for each family to cover all alleles deposited in the GenBank database as of May 2018 and amplify internal fragments of different sizes (Tables 3 and 4, Figures 1 and 2). For example, CTX-M primers were designed to cover all 214 publicly available CTX-M variants (including those belonging to phylogenetic group 1, 2, 8, and 9), KLUA, KLUC, KLUG, and TOHO-1 to TOHO-3. Table 3. Group-specific oligonucleotides used in this study for eicosaplex PCR.      Example of the primer design for octaplex PCR for the major AmpC families. The catalytic residues for β-lactamases are indicated above the alignment of the amino acid sequences of each βlactamase family with a red color. The forward and reverse primers are highlighted in turquoise and bright green colors, respectively. SPF and SPR indicates the specific forward and reverse primers for each AmpC family in the octaplex, respectively. CSF and CSR indicate the conserved forward and reverse primers for each AmpC family in the eicosaplex PCR, respectively.

Eicosaplex and Octaplex PCR Technique
DNA was prepared using the boiling lysate method, as previously reported [15]. Total DNA (0.2 μL) was subjected to eicosaplex or octaplex PCR in a 10 μL reaction mixture Figure 2. Example of the primer design for octaplex PCR for the major AmpC families. The catalytic residues for β-lactamases are indicated above the alignment of the amino acid sequences of each β-lactamase family with a red color. The forward and reverse primers are highlighted in turquoise and bright green colors, respectively. SPF and SPR indicates the specific forward and reverse primers for each AmpC family in the octaplex, respectively. CSF and CSR indicate the conserved forward and reverse primers for each AmpC family in the eicosaplex PCR, respectively.

DNA Sequencing and Analysis
Amplified genes were purified using the ExoSAP-IT™ PCR product cleanup kit (https: //assets.fishersci.com/TFS-Assets/LSG/manuals/78200b.pdf) (accessed on 10 November 2022) (Applied Biosystems, Foster City, CA, USA) and sequenced in both directions using a 3730xl DNA Analyzer (Applied Biosystems). To identify genes or their variants, a search was performed using the BLAST program accessible on the NCBI BLAST homepage (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 10 November 2022).

Whole Genome Sequencing and Analysis
Some interesting carbapenem-resistant Gram-negative bacteria detected in this study were subjected to WGS using Illumina MiniSeq (Illumina, San Diego, CA, USA) and Oxford MinION Nanopore (Oxford Nanopore Technologies, Oxford, UK). Hybrid assembly was performed as previously reported to produce high-quality sequences [22]. The complete genome sequences of the three isolates and their analyses have been previously published [20].

MER, CTX, CAZ-Resistant Strains from the ANO and AO Substances
We screened 109 strains isolated in this study on LB agar medium supplemented with 4 µg/mL MEM, 4 µg/mL CTX, and 16 µg/mL CAZ. A total of 8, 33, and 15 different Gramnegative strains were resistant to MEM 4, CTX 4, and CAZ 16, respectively, indicating the potential for spreading and infecting human beings. Fresh produce is commonly consumed raw or is under cooked [23]. Subsequently, a significant portion of the latest foodborne outbreaks have been associated with fresh produce polluted by bacteria [24]. Similarly, the spread of antimicrobial resistance in humans can occur through the consumption of vegetables contaminated with multidrug-resistant bacteria [24].

Design of Specific Primers for Eicosaplex and Octaplex PCRs
In this study, we developed two multiplex PCRs, namely eicosaplex and octaplex, for the detection of ESBLs and AmpC β-lactamases in combination with integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons and aac(6')-Ib gene in Gram-negative bacteria. After the optimization of the PCR reaction conditions, the expected target size of each amplicon was obtained using either the primer pairs in a simplex ( Figure 3C,D) or multiplex ( Figure 3A,B) approach. The specificity was good, with amplification of all the expected fragments (Figure 3). For example, every DNA template of bla TEM and bla CTX-M resulted in 602 bp and 509 bp, respectively. Antibiotics 2023, 12, x FOR PEER REVIEW 9 of 17

Evaluation of the Eicosaplex and Octaplex PCRs with Gram-Negative Strains Isolated from Vegetables and Fruits in Japan
To verify the specificity of the system, simplex and multiplex PCRs were performed on 109 Gram-negative strains isolated in this study. All the PCR products were bidirectionally sequenced.
Screening CMY2/MIR/ACT/EC and ACT enzymes were the most frequently detected plasmidmediated AmpC β-lactamases (found in 15 and 11 isolates, respectively); only one variant of MOX-12 AmpC β-lactamase was reported. Carbapenemase-encoding genes were detected using previously published primers and conditions [25]. New Delhi metallo-β-lactamase 1 (NDM-1) was detected in two K. pneumoniae (AO15 and AO22) isolates in this study [20]. The two isolates belonged to the same clone and sequence type 15 (ST15) [20]. Complete genome sequencing and ResFinder (https://cge.food.dtu.dk/services/ResFinder/) (accessed on 10 November 2022) analysis identified that both the isolates carried 19 different antimicrobial resistance genes. bla NDM-1 was carried by a self-conjugable IncFII(K):IncR plasmid of 122,804 bp with other genes conferring resistance to β-lactams (bla CTX-M-15 , bla OXA-9 , and bla TEM-1A ), fluoroquinolones [aac(6 )-Ib-cr], aminoglycosides [aac(6 )-Ib, aadA1, aph(3 )-VI], and quinolones (qnrS1) [20]. In addition, another A. baumannii AO22 isolate identified in our study carried a chromosomal bla OXA-66 and two copies of bla OXA-72 on a 10,880 bp GR2-type plasmid [20]. None of the carbapenemase genes targeted were found in this study in the other five isolates showing decreased susceptibility to meropenem. A complete description of the characteristics of the 109 Gram-negative isolates in this study are presented in Tables 7 and 8.      A recent study from Nepal reported E. coli and Salmonella producing multidrugresistant ESBL in vegetable salads served at restaurants and hotels. Alarmingly, three samples harbored E. coli O157: H7 [26]. Colosi et al. (2020) reported β-lactamase-producing Enterobacterales in 7.9% of raw vegetables retailed in Romania, with 5.5% showing the ESBL or AmpC phenotype and 2.4% associated with carbapenemase producers [27]. Therefore, retail vegetables and fruits might be important reservoirs of multidrug-resistant bacteria that produce ESBLs, AmpC, or carbapenemases. Furthermore, the detection of these organisms in fresh vegetables in Japan, a country with quite low levels of antimicrobial resistance and high-level sanitary standards, is disturbing and poses food safety and public health concerns, as resistant organisms might be transmitted to humans. From a global health perspective, surveillance plans using a fast, low-cost, and effective method (i.e., eicosaplex/octaplex system) are essential at both the international and local levels to offer evidence for risk improvement strategies to reduce the spread of resistance.
The PCR products can be sequenced directly and efficiently, permitting the detection of a large number of β-lactamases. Even if the strain carried two different variants of the same β-lactamase, it could be detected by sequence analysis of the resulting amplicon (for example, the positive control strains LM22-1 carrying bla CTX-M-9 and bla CTX-M-15 , and AO15/22 carrying bla CTX-M-14b and bla CTX-M-15 ). Here, we developed two rapid, easy-to-perform, cost-effective, and reliable multiplex PCRs for the efficient screening of ESBLs, AmpC β-lactamases, and integrons in Gram-negative bacteria. This method allows for the detection of new variants of ESBLs and AmpC β-lactamases. Interestingly, in eicosaplex PCR, we were able to amplify 20 targets in a single tube using Tks Gflex DNA Polymerase (TaKaRa Bio, Shiga, Japan, Cat. #R060A/B).

Conclusions
In this study, we developed two multiplex PCRs, eicosaplex and octaplex, in a single tube (using Tks Gflex™ DNA Polymerase), for the detection of ESBLs, plasmid-mediated AmpC β-lactamases, and class 1, 2, and 3 integrons in Gram-negative bacteria. This PCR assay is a rapid, low-cost, easy-to-perform, and reliable method for screening clinically relevant mobile and disseminated β-lactamases and integrons. This method allows for the detection of new variants of ESBLs and AmpC β-lactamases. For epidemiological surveys in low-and middle-income countries, and for reference laboratories, our technique may be extremely helpful in reducing the time and cost of multiplex PCRs. This PCR system will assist in controlling the emergence and spread of frequently encountered β-lactamases.
In conclusion, our study showed the prevalence of ESBLs and AmpC among our panel of ampicillin-resistant Gram-negative strains isolated from vegetables and fruits in Japan, and the detection of NDM and OXA carbapenemases. This situation is disturbing and poses food safety and public health concerns, as resistant organisms can be transmitted to humans. This multiplex PCR assay is a promising workflow in bacterial diagnosis in high-risk patients providing a great assistance in optimizing and fast choice of antibiotics for treating infections due to ESBLs-and AmpC β-lactamases-producing Gram-negative pathogens.