Emergence of Carbapenem Resistant Enterobacteriaceae co-harboring bla OXA-78 and bla OXA-58 from India

doi:10.21203/rs.3.rs-3022723/v1

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Research Article

Emergence of Carbapenem Resistant Enterobacteriaceae co-harboring bla OXA-78 and bla OXA-58 from India

https://doi.org/10.21203/rs.3.rs-3022723/v1

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published 07 Sep, 2023

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Background

Carbapenem Resistant Enterobacteriaceae (CRE) has been categorized as pathogens of critical priority by both CDC and WHO as they pose significant threat to global public health. Carbapenemase production considered as the principal resistance mechanism against carbapenems and with the recent surge and expansion of carbapenemases and its variants among clinically significant bacteria in India, the present study reports expansion bla_OXA-78 and bla_OXA-58 of in CRE of clinical origin.

Methods

Bacterial isolates were collected from a tertiary referral hospital and identified through VITEK® 2 Compact automated System (Biomerieux, France). Rapidec® Carba NP (Biomerieux, France) was used to investigate carbapenemase production followed by antibiotic susceptibility testing through Kirby-Bauer Disc Diffusion method and agar dilution method. Class D carbapenemase genes were targeted through PCR assay followed by investigation of horizontal transmission of bla_OXA-58 and bla_OXA-78. Whole genome sequencing was carried out using Illumina platform to investigate the genetic context of bla_OXA-58 and bla_OXA-78 genes and further characterization of the CRE isolates.

Results

The carbapenem resistant Escherichia coli (BJD_EC456) and Serratia marcescens (BJD_SM81) received during the study from the tertiary referral hospital were isolated from sputum and blood samples respectively. PCR assay followed by whole genome sequencing revealed that the isolates co-harbor bla_OXA-58 and bla_OXA-78, a variant of bla_OXA-51. Horizontal transfer of bla_OXA-58 and bla_OXA-78 genes were unsuccessful as these genes were located on the chromosome of the study isolates. Transposon Tn6080 was linked to bla_OXA-78 in the upstream region while the insertion sequences ISAba26 and ISCfr1 were identified in the upstream and downstream region of bla_OXA-58 gene respectively. In addition, both the isolates were co-harboring multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines. BJD_EC180 belonged to ST2437 while BJD_SM81 was of an unknown sequence type. The nucleotide sequences of bla_OXA-78 (OQ533021) and bla_OXA-58 (OQ533022) have been deposited in GenBank.

Conclusions

The study provides a local epidemiological information regarding carbapenem resistance aided by transposon and insertion sequences associated bla_OXA-78 and bla_OXA-58 genes associated and warrants continuous monitoring to prevent their further dissemination into carbapenem non-susceptible strains thereby contributing to carbapenem resistance burden which is currently a global concern.

Antimicrobial resistance

carbapenem resistant enterobacteriaceae

blaOXA-78

blaOXA-58

Tn6080

ISAba26

Centre for Disease Control and Prevention (CDC) and World Health Organization (WHO) has recognized Carbapenem Resistant Enterobacteriaceae (CRE) as a significant threat to public health owing to its rate of infection, high mortality rates and widespread transmission potential and categorized them as pathogens of “critical priority” and also has issued guidelines to check their dissemination in healthcare settings [1, 2]. Carbapenemase production is considered as the prime resistance mechanism against carbapenem antibiotics and the genes encoding carbapenemases are usually associated with mobile genetic elements such as plasmids, transposons which helps in their intercellular and intracellular dissemination, maintenance and expression [3, 4].

OXA-78, a variant of OXA-51 has emerged in recent periods within diverse species of Enterobacteriaceae and other non-fermenters [5, 6, 7]. Similarly, like OXA-51, its variant bla_OXA−78 gene exhibits weak hydrolytic activity against carbapenems, however, provided a strong transcriptional promoter in the upstream region of the gene associated with mobile genetic elements can contribute to carbapenem resistance thereby compromising therapeutic options [7, 8, 9]. In 2005, another class D carbapenemase, bla_OXA−58 was reported in France within a carbapenem-resistant Acinetobacter baumannii [10, 11]. The gene was plasmid-borne and the enzyme hydrolyses imipenem, and gradually were reported in pathogens of clinical priority worldwide associated with several outbreaks [12].

Carbapenem resistance determinants aided by diverse mobile genetic elements can confer high level of clinical resistance to carbapenems thereby increasing antibiotic resistance burden which is at present is a global concern. Besides intra and inter specific dissemination; these mobile elements under exposure to selective carbapenem pressure also contributes to the maintenance and expression of carbapenemase genes within bacterial host [3, 4, 13, 14]. With the surge and expansion of carbapenem hydrolyzing class D beta-lactamases (CHDLs) among clinically significant bacteria in India and the paucity of information available; and carbapenems being considered as last therapeutic options against infection caused by multidrug resistant gram-negative bacteria, the present study reports expansion of bla_OXA−78 and bla_OXA−58 in clinical isolates of Escherichia coli and Serratia marcescens.

Bacterial isolates

This study was conducted in the Department of Microbiology, Assam University, Silchar. Two ertapenem non-susceptible Enterobacteriaceae isolates were received in between January and December 2019 from Silchar Medical College and Hospital, a tertiary referral hospital in Silchar, Assam, India. The isolates were recovered from sputum and blood samples of patient admitted to the medicine ward of the tertiary referral hospital. The isolates were identified at the species level by VITEK® 2 Compact automated System (Biomerieux, France) and were investigated for carbapenemase production via Rapidec® Carba NP (Biomerieux, France) as per manufacturer’s instructions using Escherichia coli ATCC 25922 as negative control.

Antibiotic susceptibility testing

According to the break point recommended by the Clinical Laboratory Standard Institute, 2022 (CLSI, 2022) for Enterobacteriaceae using Escherichia coli ATCC 25922 as quality control strain [15]. Susceptibility of the isolates were tested against ampicillin (30 µg), cefepime (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), aztreonam (30 µg), ertapenem (10 µg), imipenem (10 µg), meropenem (10 µg), amikacin (10 µg), gentamicin (10 µg) and ciprofloxacin (5 µg) (HiMedia, India) via Kirby-Bauer Disc Diffusion method. Minimum Inhibitory Concentration (MIC) for ertapenem (MSD, France), imipenem (Merck, France) and meropenem (AstraZeneca, UK) were determined through agar dilution method (concentration range : 1–64 µg/ml).

Detection of class D carbapenemases

Total DNA was extracted from the isolates using boiling centrifugation method [16]. The presence of class D carbapenemase genes, namely bla_OXA-23, bla_OXA-48, bla_OXA-51 and bla_OXA-58 were detected through PCR assay using previously described primers and reaction conditions and the amplified products were confirmed by sequencing [17, 18]. PCR assay was performed in Veriti™ 96-Well Fast Thermal Cycler (Applied Biosystems™, USA) with each single reaction volume of 25 µl containing 2 µl of template DNA (~ 100 ng/µl), 1 µl of each primer (10 pmol/µl), 12.5 µl of Go Taq green Master Mix 2X DNA polymerase (Promega, Madison, USA) and nuclease free water.

Horizontal gene transferability assay of bla_OXA-78 and bla_OXA-58

To assess the genetic location of bla_OXA-78 and bla_OXA-58 in the genome, transformation and conjugation assays were performed. Plasmids were extracted using QIAprep Spin Miniprep Kit (Qiagen, Germany) as per manufacturer’s instructions and were transformed into recipient strain Escherichia coli DH5α by heat shock method and transformants were selected on Luria Bertani agar (HiMedia, India) supplemented with 0.5 µg/ml of imipenem (Merck, France) [19]. For conjugation assay, an azide-resistant Escherichia coli J53 was used as recipient strain and transconjugants were selected on Luria Bertani agar (HiMedia, India) medium supplemented with a combination of imipenem (0.5 µg/ml) and sodium azide (100 µg/ml).

Whole genome sequencing and assembly

Whole genome sequencing was carried out using Illumina platform (outsourced to Bionivid Technology Private Limited, Bengaluru, India). Quality control and data filtering was done using Fastp version 0.20.0 with standard parameters [20]. De novo assembly and scaffolding after quality trimming of the reads was conducted using SPAdes version 3.13.0 [21]. The 16s rRNA gene sequence was predicted using Metaerg version 1.2.0 tool and the nearest genome reference was identified using NCBI BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Genomes were oriented and rearranged using web-based tool MeDuSa using default web-interface parameters [22]. Genomes were annotated using Prokka version 1.11.1 software [23]. Antimicrobial resistance genes were identified through ResFinder 4.1 (https://cge.food.dtu.dk/services/ResFinder/). Additionally, mobile genetic elements and their relation to resistance determinants were identified through MobileElementFinder version 1.0.3 (https://cge.food.dtu.dk/services/MobileElementFinder/) while plasmids and their possible location in the bacterial genome were screened using NCBI BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). PathogenFinder 1.1 was used for finding pathogenicity of the isolates towards human hosts (https://cge.food.dtu.dk/services/PathogenFinder/).

Escherichia coli (BJD_EC456) was isolated in 24.01.2019 from sputum sample of a female patient while Serratia marcescens (BJD_SM81) was isolated in 27.12.2019 from blood sample of a male patient and both the specimen were collected from the medicine ward of the tertiary referral hospital. Both the isolates were co-harboring bla_OXA-78 and bla_OXA-58 genes and were resistant to all the tested antibiotics and were having MIC above breakpoints (≥ 32 µg/ml) for carbapenems (Table 1). Attempt to transfer the class D carbapenemase genes bla_OXA-78 and bla_OXA-58 from BJD_EC456 and BJD_SM81 by transformation and conjugation was not successful. Whole genome sequenced data revealed that these class D carbapenemase genes were chromosomally located in both the isolates and were associated with mobile genetic elements which might have helped in their acquisition and integration in the bacterial genome. Transposon Tn6080 was associated with the carriage of bla_OXA-78 gene. In case of bla_OXA-58, two insertion sequences were identified in the upstream and downstream region of the gene, ISAba26 in the upstream region while in the downstream region ISCfr1 was present. Additionally, BJD_EC456 co-harbored multiple resistance genes, namely, aph(3')-VI, aph(3')-IIa, aac(6')-Ib, aac(6')-Ic, aadA1, bla_SST-1, bla_OXA-9, bla_SHV-59, bla_TEM-1, bla_NDM-1, bla_CTX-M-15, qacE, fosA, catA1, qnrS1, aac(6')-Ib-cr, sul1 and tet(41) along with five plasmids viz. Col440I, IncFII(pKPX1), IncFIB(K), IncFIB(pKPHS1) and IncM1 (Fig. 1). BJD_SM81 contains Col(MG828) plasmid and carried the following genes; aph(3')-IIa, aac(3)-IId, aac(6')-Ic, bla_SST-1, bla_TEM-116, bla_NDM-1, bla_ADC-25, OqxB_1, msr(E), mph(E), catA1 and tet(41) (Fig. 2). Multi Locus Sequence Typing (MLST) results showed that BJD_EC180 belonged to Escherichia coli sequence type ST2437, while BJD_SM81 belonged to an unknown sequence type. The nucleotide sequences of bla_OXA-58 and bla_OXA-78 have been deposited in GenBank under the accession numbers OQ533022 and OQ533021 respectively, and the profiles of BJD_SM81 and BJD_EC456 have been summarized in table 2.

Carbapenem antibiotics are the most potent among all clinically available beta-lactam antibiotics and are used as last resort drugs to treat infection caused by multidrug resistant Gram-negative bacteria. However, with the emergence of CRE has threatened this class of antibiotics and pose a serious threat to global public health. In India also, reports of CRE isolates have been increased significantly over the years [24, 25, 26, 27]. In this study, we reported the co-carriage of bla_OXA-78, a variant of bla_OXA-51 and bla_OXA-58 genes in two CRE isolates (Escherichia coli and Serratia marcescens) obtained from a tertiary referral hospital in northeastern part of India. The finding of our study is in congruence with a recent study conducted in 2022, that reported the co-occurrence of bla_OXA-51-like and bla_OXA-58 genes in Enterobacteriaceae isolates recovered from urine samples of UTI patients from a hospital of Tehran, Iran [5]. Similarly, study conducted by Leski and his team in 2013 also reported the co-existence of bla_OXA-51-like and bla_OXA-58 genes within Enterobacteriaceae isolates obtained from Mercy Hospital, Bo, Sierra Leone [6]. In India, the co-carriage of bla_OXA-51-like and bla_OXA-58 genes was reported in 2015 in carbapenem-resistant Acinetobacter baumannii isolated from various clinical specimens obtained from a university teaching hospital [24]. However, to the best of our knowledge this is the first report of co-occurrence of bla_OXA-78 and bla_OXA-58 genes from India.

In the present study, the chromosomally located bla_OXA-78 and bla_OXA-58 genes were found associated with diverse mobile genetic elements. Tn6080 transposon was observed with the carriage of bla_OXA-78 gene in both the isolates. This finding is in accordance with previous studies that reports this transposon as a carrier of bla_OXA-51 genes and its variants [14, 28, 29]. In case of bla_OXA-58 gene, ISAba26, a single nucleotide variant belonging to the ISAba256 family in the upstream region and ISCfr1 belonging to IS1182 family in the downward region were found associated with the gene. These carbapenem resistance determinants associated with transposon and insertion sequences possess a serious health hazard as transposition of these mobile genetic elements can alter bacterial gene expression thereby increasing antibiotic resistance burden which is at present is a global concern [14]. These mobile elements facilitate mobilization of carbapenemase genes thereby aiding in their intra and inter specific dissemination; and acquisition of such elements by susceptible phenotypes results into the evolution of resistant ones [3, 4, 14]. Selective pressure induces adaptive response and led to the emergence and expansion of antibiotic resistance thereby contributing to resistance burden which is at present a serious threat to global public health due to its limiting effect on therapeutic options. It is evident from previous studies that insertion sequences play a major role in conferring clinical resistance to carbapenems as their insertion upstream of bla_OXA genes provides a strong outward promoter thereby aiding in better expression of otherwise silent bla_OXA genes encoding carbapenemases [14, 30]. Selective antibiotic pressure also contributes in the maintenance of transposon within the host genome that carry antibiotic resistance genes [14]. And in accordance, in our study also it was observed that the bla_OXA-78 and bla_OXA-58 genes were maintained within such unnatural hosts by their respective insertion sequences and transposons and also the isolates co-harboring them exhibited high MICs for carbapenem antibiotics. These findings highlight the role of positive selection pressure generated by the surge in usage of carbapenems within the study center that aid in the maintenance of mobile genetic elements carrying bla_OXA-78 and bla_OXA-58 genes and also in the expression of these resistance determinants conferring clinical resistance to carbapenems, antibiotic of last resort.

Studies suggests that isolates harboring carbapenemase encoding genes often carry additional resistance genes that confer resistance to other beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines and other antibiotics [31, 32, 33]. And in accordance, our study isolates also co-harbored multiple resistance genes elucidating their multidrug resistant nature correlating with the observations of antibiotic susceptibility testing. As heavy carbapenem use in hospitals especially of developing countries is already an established risk factor for emergence of carbapenem resistant organisms, and considering the fluid nature of carbapenemase genes, bla_OXA-78 and bla_OXA-58 genes conferring resistance towards carbapenems aided by mobile genetic elements possess a serious health hazard as potential source and vehicle of future dissemination and warrants urgent monitoring as they pose a threat to the control of antimicrobial resistance and endangering our fight against antimicrobial resistance.

Antimicrobial resistance, at present is a global concern and with the increase in incidence of class D carbapenemases and its variants among clinically significant gram-negative bacteria, the findings of the present study, provide a local epidemiological information regarding carbapenem resistance and mobile genetic elements associated dissemination of bla_OXA−78 and bla_OXA−58 genes in carbapenem resistant isolates of Escherichia coli and Serratia marcescens of clinical origin. Since carbapenems are regarded as antibiotic of last resort for the treatment of infections caused by multi-drug resistant gram-negative bacteria, the findings of the present study warrant continuous monitoring of these carbapenem resistance determinants considering the fluid nature of carbapenemase genes aided by transposon and insertion sequences along with a scope to design and assess strategies to prevent the spread and emergence of carbapenem resistance determinants and accordingly optimize clinical therapy to avoid treatment failure.

CDC : Centre for Disease Control and Prevention
WHO : World Health Organization
CRE : Carbapenem Resistant Enterobacteriaceae
CHDLs : Carbapenem Hydrolyzing Class D Beta-lactamases
bla : Beta-lactamases
OXA : Oxacillinase
CLSI : Clinical Laboratory Standard Institute
ATCC : American Type Culture Collection
MIC : Minimum Inhibitory Concentration
DNA : Deoxyribonucleic Acid
ng : Nanogram
μl : Microlitre
pmol : Picomole
μg : Microgram
ml : Millilitre
NCBI : National Center for Biotechnology Information
BLAST : Basic Local Alignment Search Tool
Tn : Transposon
IS : Insertion Sequence
Inc : Incompatibility
MLST : Multi Locus Sequence Typing
ST : Sequence Type
UTI : Urinary Tract Infection

Nucleotide sequence accession number

The nucleotide sequences of bla_OXA-58and bla_OXA-78 have been deposited in GenBank under the accession numbers OQ533022 and OQ533021 respectively.

Ethical approval

The study was approved by Institutional Ethics Committee, Assam University, Silchar vide Agenda No. 3, Resolution Serial No. 4 in the meeting held on 9^th April 2018.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that the study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

The study was supported by Department of Biotechnology (DBT), Government of India DBT-NER Twinning order no. BT/PR242/NER/95/716/2017 dated 28.09.2018 and Indian Council of Medical Research, India (ICMR) for awarding Senior Research Fellowship (ICMR-SRF) to Bhaskar Jyoti Das vide letter no. 2020-7955/CMB-BMS dated 09.03.2021. However, the funding body has no role in analysis and interpretation of data and in writing the manuscript.

Authors’ contributions

Bhaskar Jyoti Das: Investigation, Formal Analysis, Data Curation, Writing - Original Draft. K Melson Singha: Resources and formal analysis. Jayalaxmi Wangkheimayum: Formal analysis. Debadatta Dhar Chanda: Methodology and Writing - Review & Editing. Amitabha Bhattacharjee: Conceptualization and Supervision. All authors ensured that this is the case. All the authors read and approved the final manuscript.

Acknowledgements

The authors would also like to thank Department of Biotechnology (DBT) project BT/PR242/NER/95/716/2017 dated 28.09.2018 and Indian Council of Medical Research (ICMR) ICMR-SRF vide letter no. 2020-7955/CMB-BMS dated 09.03.2021.

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Table 1: Antibiogram of BJD_EC456 and BJD_EC81 co-harboring bla_OXA-78 and bla_OXA-58

Isolates ID	Organism	Isolation date	Specimen	Resistance profiles	MICs (µg/ml)
Isolates ID	Organism	Isolation date	Specimen	Resistance profiles	ERT	IMP	MEM
BJD_EC456	E. coli	24.01.2019	Sputum	AMP, FEP, CRO, CAZ, ATM, ERT, IMP, MEM, AMK, GEN, CIP	≥64	≥32	≥64
BJD_SM81	S. marcescens	27.12.2019	Blood	AMP, FEP, CRO, CAZ, ATM, ERT, IMP, MEM, AMK, GEN, CIP	≥64	≥32	≥64

AK: amikacin, AMP: ampicillin, AT: aztreonam, CPM: cefepime, CTX: Cefotaxime,

CTR: ceftriaxone, CIP: ciprofloxacin, CAZ: ceftazidime, GEN: gentamicin,

ETP: ertapenem, IPM: imipenem, MRP: meropenem

Table 2: Profiles of BJD_EC456 and BJD_SM81

Isolates ID

bla_OXA-51variant

Carbapenemases

β-lactamases

Aminoglycoside resistance genes

Other resistance genes

Virulence genes

Mobile genetic elements

Sequence type

BJD_EC456

bla_OXA-78

bla_OXA-58,

bla_NDM-1

bla_OXA-9,

bla_SHV-59,

bla_TEM-1,

bla_CTX-M-15,

bla_SST-1

aph(3')-VI,

aph(3')-IIa,

aac(6')-Ib,

aac(6')-Ic,

aac(6')-Ib-cr

aadA1,

qacE,

fosA,

catA1,

qnrS1,

sul1,

tet(41)

nlpl,

mrkA,

iutA,

fimH,

gad,

clpK1

Col440I, IncFII(pKPX1), IncFIB(K), IncFIB(pKPHS1), IncM1,

ISAba26,

ISCfr1I,

Tn6080

ST2437

BJD_SM81

bla_OXA-78

bla_OXA-58,

bla_NDM-1,

bla_SST-1,

bla_TEM-116,

bla_ADC-25

aph(3')-IIa,

aac(3)-IId,

aac(6')-Ic

OqxB_1,

msr(E),

mph(E),

catA1,

tet(41)

clpK1

Col(MG828)

ISAba26,

ISCfr1,

Tn6080

Unknown

No competing interests reported.

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Emergence of Carbapenem Resistant Enterobacteriaceae co-harboring bla OXA-78 and bla OXA-58 from India

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Abstract

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Background

Methods

Antibiotic susceptibility testing

Detection of class D carbapenemases

Whole genome sequencing and assembly

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Additional Declarations

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