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.

Keywords

beta-lactamase - carbapenemases - mobile genetic elements - MDR E. coli

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 carbapenems.[1] In 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] [4] Bacterial genomes are impregnated with prophages that are speculated to be important mediators of AMR in species that do not generally support transformation and conjugation.[5] Prophages 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.[6] The 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.

Bacterial Cultures

MDR-E. coli cultures (n = 35) previously isolated from blood and urine were anonymized using secondary identifiers and revived from −80°C glycerol stocks to be included in this study. Environmental isolates of E. coli (n = 5) were also included. Standard quality control strain E. coli ATCC 25922 was maintained as control. Cultures were maintained on nutrient agar. The isolates were confirmed for antibiotic resistance by the disc diffusion method as per Clinical and Laboratory Standards Institute (CLSI) guidelines (2019).[7] Antibiotics include cefpodoxime (CAZ 30 µg), ceftazidime (CTX 30 µg), meropenem (MRP 10 µg), imipenem (IMP 10 µg), chloramphenicol (C 30 µg), co-trimethoxazole (COT 25 µg), ciprofloxacin (CIP 5 µg), and tetracycline (TE 30 µg).

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 OD₆₀₀ ∼ 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 phenol-chloroform extraction.[9] Nucleic 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).[10] For 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.

Results

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].

Table 1 Multidrug resistance and the beta-lactamases observed in extra-intestinal E. coli isolates

Isolates	Class of antimicrobial						Beta-lactamase gene present
	CEP	CARB	TET	FLO	C	FPI	CTX-M	TEM	SHV	KPC	NDM	VIM	OXA
U6451	R	R	S	R	S	S		+			+
B5308	R	R	S	R	S	S	+	+
B5773	R	R	S	R	S	S	+
U6106	R	R	S	S	S	R					+
U5546	R	R	S	R	S	R							+
IRU2553	R	R	S	R	S	R
B6127	R	R	S	R	S	R		+			+
U2368	R	R	S	R	S	R	+	+
U5265	R	R	S	R	S	R
U3659	R	R	R	R	S	S	+						+
U5801	R	R	R	R	S	S	+				+		+
U2592	S	R	R	R	S	R
IRU1123	R	R	R	R	S	R	+					+
U5663	R	R	R	R	S	R	+
B3263	R	R	R	R	S	R	+				+		+
U5849	R	R	R	R	S	R	+
U6320	R	R	R	R	S	R				+
IRU1070	R	R	R	R	S	R
U5620	R	R	R	R	S	R	+				+
U5133	R	R	R	R	S	R	+				+
U5861	R	R	R	R	S	R
U6487	R	R	R	R	S	R	+	+			+
U5389	R	R	R	R	S	R	+						+
U6040	R	R	R	R	S	R	+	+			+
U3842	R	R	R	R	S	R	+						+
IRU1729	R	R	R	R	S	R	+	+
U3731	R	R	R	S	S	R	+	+			+
U5926	R	R	S	R	S	R	+
U5187	R	R	R	R	R	R	+
U2642	R	R	R	R	R	R
U6306	R	R	R	R	S	R	+				+
U3542	R	R	R	R	S	R	+						+
U5716	R	R	R	R	S	R	+				+
U3239	R	R	R	R	R	R
P5943	R	R	R	R	R	R	+

Abbreviations: +:gene present; C, chloramphenicol; CARB, carbapenem; CEP, cephalosporin; E. coli, Escherichia coli; FLO, fluoroquinolone; FPI, folate pathway inhibitor TET, tetracycline.

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 χ² 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 (χ² (1) = 3.313, p < 0.05; χ² (1) = 0.1042, p < 0.05; and χ²(1) = 0.0925, p < 0.05, respectively).

Table 2 Distribution of antibiotic resistance genes in various genetic fractions

Genes	No. of isolates
	Crude DNA	Plasmid only	Phage only	Plasmid and phage
CTX-M	26	7	10	9
TEM	9	4	0	5
SHV	0	0	0	0
KPC	1	1	0	0
NDM	13	5	1	7
VIM	1	0	0	1
OXA	7	5	0	2

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]).

Table 3 Phenotype and genotype of transformants obtained from transduction

Antibiotic sensitive host	Characteristics of prophage source	Characteristics post-transduction
EC153	U5663: TER,CIPR,COTR,CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M	COTR,CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M
EC69	U2642: TER, CIPR,COTR,CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M	CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M
EC69	B5773 CIPR,CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M	CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M
EC69	U2368 CIPR,COTR,CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M,bla TEM	COTR,CTXR,CAZR,IPMRMRPR Genotype: bla CTX-M,bla TEM

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.[11] Modified human activities, poor access to good sanitary facilities, and poor hygiene support the spread of CTX-M and NDM genes.[12] The 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] [13] In 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. Phages are important vehicles for horizontal gene exchange between and within different bacterial species and are important agents for antimicrobial gene transfer.[14] [15] [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.[17] Many genes encoding resistance determinants are found in specific phage-plasmids and these phage-plasmids contribute widely to the dissemination of AMR.[18] We 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 selects for resistance to third-generation cephalosporins, and vice-versa and hence can be used for screening transformants.[19] Since the hosts were sensitive to all antibiotics, the use of ampicillin could screen for cephalosporin and carbapenem resistance. The transformants obtained after transduction were resistant to several other antibiotics as determined by the disk diffusion method and included the use of 8 antibiotics. The genes tested for the transduction study include only the bla _CTX-M and bla _TEM. However, it is possible that genes encoding resistance to other classes of antibiotics but not screened for in this study were present as a single cassette, and thus horizontally transferred by the phage. Hence, the antibiogram could be established for different classes of antibiotics but genotype only for two genes.

The blaCTX-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.[18]

The 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.[20] Many 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.

Conflict of Interest

None declared.

Approvals Statement

The study was approved by the Institutional Research Advisory Committee via order INST/RAC/2017-18/9 and the Institutional Ethics Committee via sanction order INST.EC/2017-18/001 dated 22.01.2018.

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Publication History

Article published online:
24 August 2023