Prophage and Plasmid-Mediated Beta-Lactamases in Multidrug-Resistant Extraintestinal Escherichia coli

Abstract Objectives  Antibiotic resistance can arise as a mutation to adapt to stress or be mediated by horizontal gene transfer. This study aimed at identifying the resistance determinants present in the mobile genetic elements of prophages and plasmids within multidrug-resistant (MDR) extraintestinal Escherichia coli . Materials and Methods  Thirty-five anonymized MDR E. coli isolates of nonintestinal infections were confirmed for their antimicrobial resistance to six categories of antimicrobials by the disk diffusion test. Genes coding for beta-lactamases and carbapenemases in bacterial genome, plasmid, and prophage fractions were separately determined by polymerase chain reaction. Transducing ability of prophages carrying resistance genes was determined. Results  Twenty-six isolates were positive for the gene bla CTX-M , nine for bla TEM , one each for bla KPC and bla VIM , thirteen for bla NDM, and seven for bla OXA . A majority of these isolates carried these determinants in plasmids and prophage fractions. Twenty-one percent of the prophage fractions (4 of 19) were able to successfully transfer resistance to sensitive isolates. Conclusion  This study indicates bla CTX-M , bla TEM, and bla NDM genes that are reported most frequently in MDR isolates are more frequent in the plasmid and prophage fractions thus supporting for increased mobility.


Introduction
Extraintestinal infections like urinary tract infections (UTIs) and bacteremia, caused by Escherichia coli, have surfaced in recent decades and this organism is now a dreaded pathogen.The impact of virulence is enhanced by the concomitant occurrence of antibiotic resistance.E. coli, the most prevalent pathogen causing UTI, has developed resistance to many antibiotics including the last line beta-lactams and carbape-nems. 1In India, as of 2019, more than 83% of invasive E. coli isolates exhibit resistance to cephalosporin and 41% are resistant to carbapenems. 2 An approximate 10% increase in resistance was observed in a 2-year reporting period. 2 Plasmids have gained attention as prime mediators of antimicrobial resistance (AMR) not only in clinical isolates but in the environment as well. 3,4Bacterial genomes are impregnated with prophages that are speculated to be important mediators of AMR in species that do not generally support transformation and conjugation. 5Prophages may act as reservoirs for AMR genes.The occurrence of resistance genes within prophage elements of multidrug-resistant isolates and the contribution of these prophages to the transfer and spread of AMR is known.However, not well quantified. 6The presence of beta-lactamases and carbapenemases in the genome, plasmid, and prophage fractions of extraintestinal E. coli was detected in this study.The ability and rate of transfer of prophages induced from the drug resistant bacteria to transform sensitive isolates to multidrug resistant ones were determined.Status on resistance patterns, genes that code for AMR in multidrug-resistant (MDR) organisms, and understanding mechanisms of transfer aid in developing strategies to combat and control the spread of AMR effectively.

Materials and Methods
The study was initiated after obtaining institutional ethics committee and biosafety committee approval.All experiments were conducted in triplicates maintaining the necessary controls.

Screening Isolates for Beta-Lactamase Genes
One milliliter of overnight culture was pelleted at 4,000g for 10 minutes and pellet suspended in 100 µL 10mM tris-ethylenediaminetetraacetic acid (EDTA) buffer.The suspension was heated at 98°C for 10 minutes and flash cooled on ice.The resultant solution was used as DNA source.The presence of beta-lactamase (bla) genes bla CTX -M , bla TEM , bla SHV , bla KPC , bla VIM , and bla OXA was detected by polymerase chain reaction (PCR) using specific primers at optimized cycling conditions in a thermocycler (Nexus GX2, Eppendorf; ►Supplementary Table S1).PCR products were resolved by horizontal electrophoresis in a 2% agarose gel incorporated with SYBR safe dye.The gel was visualized and analyzed using a gel documentation system (GelDoc, BioRad).

Detection of Beta-Lactamase Genes in Plasmid
Plasmid was extracted using QIAprep Miniprep kit (Qiagen, United States) 8 and quantified using a nanodrop spectrophotometer (Implen, United States).Presence of plasmid-mediated bla genes was determined using the plasmid as template for PCR.

Detection of Beta-Lactamase Genes in Prophages
Prophages were chemically induced using different reagents.Isolates were grown in 5 mL nutrient broth at 37°C, 180 revolutions per minute (rpm) till OD 600 $ 0.6.The cultures were separately treated with 1% sodium dodecyl sulphate, mitomycin C (1 µg/mL), and nalidixic acid (12.5 µg/mL) and incubation continued for 5 hours.The flasks were observed for visual clearance.The lysate was clarified by centrifugation at 4,000 g for 10 minutes followed by filtration through 0.2 µm filters.The lysate was subjected to ultracentrifugation (Optima XPN-100, Beckman Coulter, United States) at 23,000 g for 3 hours.The pellet was suspended in 1 mL sodium chloride magnesium sulphate and gelatin (SM) buffer to obtain pure concentrated phage fractions.The concentrated phage fraction was treated with 10 units/mL DNase for 37°C for 1 hour to remove any residual bacterial DNA.Phage DNA was extracted by treatment with TENS buffer (50 mMTris-100 mM EDTA: 0.3% sodium dodecyl sulphate (SDS): 100mM NaCl) followed by phenolchloroform extraction. 9Nucleic acid was quantified by spectrophotometry.The presence of bla genes in the prophage was detected by PCR using the phage DNA as template.

Transducing Ability of the Prophages
The ability of prophage elements to induce AMR by transduction was determined as follows.The activity of the phages in the clarified mitomycin C-induced lysates of the MDR-E.coli was determined by spot assay wherein 5 µL of lysate was spotted on a lawn of environmental E. coli (as these were susceptible to all antibiotics tested). 10For lysates that showed activity on the environmental E. coli isolates, prophage induction in 50 mL volume was performed using mitomycin C (1 µg/mL).Mitomycin C-induced lysates of the 9 MDR isolates that were positive for the AMR genes in prophage fraction and showed lytic activity on the environmental isolates (►Supplementary Table S2) were selected for transduction studies.The test contained 1 mL of the sensitive E. coli culture and 100 µL of prophage lysate from MDR isolate, while culture and lysate were separately maintained as controls.The experimental tubes were incubated at 37°C overnight.Cultures were centrifuged at 4,000g for 20 minutes and the supernatant discarded.The cell pellet washed thrice in 1 mL aliquots of sterile saline and suspended in Luria Bertani (LB) broth.The tubes were further incubated at 37°C 180 rpm for 1 hour.One hundred µL from each tube were spread on LB agar and LB AMP agar (LB agar incorporated with 100 µg/mL ampicillin) and incubated at 37°C overnight.Colonies that developed on LB AMP agar were tested for antimicrobial susceptibility using all the antibiotics mentioned earlier and the presence of bla genes tested by PCR.Colonies positive for bla genes were considered as true transformants obtained after transduction.

Characterization of the Beta-Lactamase Genes in Mobile Genetic Elements
DNA sequencing of the gene bla CTX-M from isolates that were positive for this gene in both plasmid and phage DNA fractions was undertaken to determine the presence of the bla CTX-M variant present.Sequencing was performed at Eurofins Genomics India Pvt Ltd, Bengaluru, India.Sequences were subject to NCBI BLAST tool and multiple sequence alignment in Multalin (http://multalin.toulouse.inra.fr/multalin/) to determine the similarity and gene relatedness.

Bacterial Isolates
Thirty-five E. coli of clinical origin and five of environmental origin were included in this study.All 35 clinical isolates were resistant to CTX, 34 (97%) to MRP, CAZ, and CIP, 30 (86%) to COT, 27 (77%) to IPM, 24 (69%) to TET, and 4 (11%) to C. All 35 isolates were resistant to at least three classes of antimicrobials conferring them as MDR.The environmental isolates were sensitive to all antibiotics tested.The resistance pattern observed is shown in ►Table 1.

Beta-Lactamase Genes in Mobile Genetic Elements
The thirty-five MDR E. coli were screened for genes coding for beta-lactamases by PCR.Presence of resistance determinants in the isolates as tested from crude DNA is shown in ►Table 1.The number of plasmid DNA and induced phage fraction that carried the bla genes is shown in ►Table 2. Genes encoding multiple resistance determinants were detected in several isolates.Concomitant resistance to carbapenems IMP and MRP in the absence of the carbapenemases tested was observed in isolates U5663 and P5943.The χ 2 test of association compared presence in plasmid and phage for individual resistance determinants.For the resistance determinants bla CTX-M , bla TEM, and bla NDM , the concomitant presence in plasmid and prophage fractions was found to be associated (χ 2 (1) ¼ 3.313, p < 0.05; χ 2 (1) ¼ 0.1042, p < 0.05; and χ 2 (1) ¼ 0.0925, p < 0.05, respectively).

Transducing Ability of the Prophages
Prophages were easily induced on all chemical treatments used.Nineteen prophage lysates were screened for active phages by spot assay on the anitbiotics susceptible environmental isolates.Nine lysates showed lytic activity (►Supplementary Table S2).Transduction experiments were carried out using lysate-isolate combinations as per ►Supplementary Table S2.Transformants that grew on LB AMP plates were screened for resistance genes that were present in the respective phage lysates.To supplement the genotype information, antimicrobial susceptibility test was performed.Transformants of transduction from four lysates showed phenotypic resistance to antimicrobials identical to their respective parent cultures U2368, U5663, B5773, and U2642 (►Table 3).

Characterization of the Beta-Lactamase Genes in Mobile Genetic Elements
The bla CTX-M amplicon of six isolates B5773, U6040, U2368, U5133, U5663, and IRU1123, which were positive for the gene in both plasmid and phage DNA, was sequenced.Alignment of our sequences revealed that all the sequences were identical to bla CTX-M 15 .The sequences have been deposited in Genbank (accession numbers MN692192 to MN692197).

Discussion
E. coli has gained the attention of the healthcare sector over the past five decades as a pathogen with increased resistance to antimicrobial agents.Reports of extraintestinal E. coli infections are increasing and the prevalence of extended spectrum beta-lactamases (ESBLs) and carbapenemases among them are alarming.Among the beta-lactamases, bla CTX-M has superseded the variants bla TEM and bla SHV to become a global dominant menace.To date, more than 175 variants of the bla CTX-M gene have been reported with bla CTX-M-15 being highly prevalent among clinical isolates of E. coli.The carbapenemases bla KPC , bla VIM , bla IMP , bla NDM, and bla OXA-48 type are regarded as the most effective in carbapenem hydrolysis and geographical spread. 11Modified human activities, poor access to good sanitary facilities, and poor hygiene support the spread of CTX-M and NDM genes. 12he nature of these resistance genes and an understanding of triggers for transfer would aid in complementing antimicrobial stewardship practices by developing intervention strategies to combat the spread of the AMR pandemic.
Reports from healthcare settings differ in the resistance patterns and the occurrence of resistance determinants.Beta-lactamases and carbapenemases appear in multiple combinations, thereby conferring resistance to virtually all β-lactam antibiotics. 3,12,13In our study, the bla CTX-M , bla TEM , and bla NDM were the most common bla genes encountered.The phenotypic resistance to carbapenems in isolates U5663 and P5943 but absence of resistance genes tested in this study highlights the increasing adaptability of the ARDs and the presence of other resistance mechanisms that were not included in this study.
ESBLs bla CTX-M , bla TEM and the carbapenemases bla KPC , bla VIM , bla NDM , and bla OXA are often reported as plasmid mediated.5][16] In our study, bla CTX-M , bla TEM , and bla NDM occurred frequently as plasmids and in the prophage fractions.Procedural limitations of genomic DNA contamination in the plasmid and prophage DNA preparations are a concern while interpreting these results.However, since the results mentioned herein are the consensus results of three biological replicates, we consider the percentage of carriage of the bla genes in plasmids and phages to accurately represent the samples.Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages.Phage-like plasmids carry the resistance gene KPC-2. 17Many genes encoding resistance determinants are found in specific phage-plasmids and these phage-plasmids contribute widely to the dissemination of AMR. 18We speculate that the detection of the AMR genes in both plasmids and phages may be due to the presence of phage-plasmids.Genomic data of these MDR E. coli isolates would clarify if these elements are indeed phage-plasmids.
From a total of 19 phage lysates that carried resistance genes, 9 were selected for the transduction study and of these, 4 were capable of successfully transferring resistance via transduction as screened on LB AMP .The use of ampicillin The bla CTX-M of isolates positive for the gene in both plasmid and phage DNA were identical to bla CTX-M-15 as determined by sequencing.This concomitant presence in multiple mobile elements in the same isolate reinforces and provides an insight for bla CTX-M-15 being highly prevalent among clinical isolates of E. coli and probably as phage-plasmids. 18he limited number of isolates included in the study and the few resistance genes tested have narrowed the spectrum of this study, but provide insights into the probability of the role of many more AMR genes as phages.As this study focused on prophage mediated AMR dissemination, the ability of the plasmids to transfer the AMR genes by conjugation was not determined.
Shedding of infectious organisms from diseased personnel into the environment is unavoidable in cases where quarantine is not possible.Cleaning products that contain certain antimicrobial compounds are speculated to accelerate the development and spread of AMR. 20Many components of cleaning products like detergents act as plasmid and prophage inducers.This study reiterates that genes like bla CTX-M , bla TEM , and bla NDM that occur frequently in both the mobile genetic elements, plasmids and prophages, easily get induced on exposure to cleaning agents and thus disseminate faster than other genes.However, these findings do not hinder phage therapy as phage therapy specifically used lytic phages only.