Emergence of multidrug-resistant ST11 bla KPC-2 producing Klebsiella pneumoniae coharboring bla CTX-M and bla SHV in Pakistan

Introduction: Carbapenemases are primarily responsible for the intensified spread of multidrug-resistant (MDR) K. pneumoniae by virtue of antibiotics overuse. Therefore, frequent investigation of high-risk clones especially from developing world is crucial to curtail global spread. Methodology: In this observational study, 107 K. pneumoniae were retrieved and confirmed genotypically from April 2018 to March 2020 from tertiary care hospitals in Lahore, Pakistan. Carbapenemases and extended-spectrum β -lactamases were verified by Polymerase Chain Reaction and Sanger sequencing. Multilocus sequence typing and plasmid replicon typing were used to assign clonal lineages and plasmid replicons. Results: Among the K. pneumoniae , 72.9% (78/107) strains were carbapenem resistant (CR) with 65.4% (51/78) exhibiting carbapenemase producing phenotype. Among CR K. pneumoniae 38.5% (30/78) strains exhibited the following carbapenemase genotypes: bla NDM-1 (26.7%, 8/30), bla OXA-48 (26.7%, 8/30), bla KPC-2 (20.0%, 6/30), bla VIM (10.0%, 3/30), bla NDM-1 /bla OXA-48 (10.0%, 3/30), bla OXA-48 /bla VIM (3.3%, 1/30) and bla OXA-48 /bla IMP (3.3%, 1/30). Tigecycline and polymyxin-B retained susceptible profile. β -lactam drugs showed intermediate to high resistance. The occurrence of CR K. pneumoniae infections was significantly associated with wound (39.7%, p = 0.0007), pus (38.5%, p = 0.009), general surgery (34.6%, p = 0.002) and intensive-care unit (26.9%, p = 0.04). bla KPC-2 producing K. pneumoniae coharboring bla CTX-M / bla SHV (66.7%) and bla CTX-M (33.3%) exhibited sequence type (ST) 258 ( n = 4) and ST11 ( n = 2) sequence types with IncFII, IncN, IncFIIA, IncL/M and IncFIIK plasmids. Conclusions: This is the first report describing the emergence of MDR bla KPC-2 producing K. pneumoniae ST11 coharboring bla CTX-M and bla SHV in Pakistan.


Introduction
Over the years, evolutionary changes have resulted in the emergence of successful epidemic bacterial clones that ultimately lead to the development of multidrug resistance (MDR) attributable to selection pressure and misuse of antibiotics.Clinically, the Enterobacteriaceae family accommodates many such high-risk MDR clones, thereby posing one of the most frightening issues of deteriorating efficacy of antibiotics.As a result, life expectancy is dramatically reduced compared to the pre-antibiotic era [1].Carbapenem-resistant K. pneumoniae (CRKP) has turned out to be an exceptionally diverse MDR species associated with a variety of healthcare and community-based infections and features successful accumulation of survival and resistant determinants.Such high-risk clones are of great clinical concern due to the presence of ever evolving β-lactamases that present the hardest challenge to all the β-lactams by hydrolyzing the βlactam bond, thus rendering them non-functional [2].
K. pneumoniae carbapenemases (KPCs) present one of the most critical genetic mechanisms of acquired carbapenem resistance among the clinically important transmissible carbapenemases including blaNDM-1, blaOXA-48, blaVIM and blaIMP.Since the first identification of KPC-producing K. pneumoniae in the United States, its spread has been reported in several countries like Israel, India, China, several European countries, Vietnam, Taiwan, China, and Pakistan demonstrating its remarkable potential for universal distribution [3][4][5][6][7][8][9].However, distinct resistance patterns exist in different regions depicting the mixing of resistance clones due to globalization, traveling and different regional infection control measures.This is evidenced from reports mentioning the coexistence of blaKPC-2 with other resistance genes in K. pneumoniae such as blaKPC/blaOXA-48 from Taiwan, blaKPC/blaNDM-1/blaSHV/blaCTX-M from Turkey, blaKPC/blaSHV/blaCTX-M from India and China, blaKPC/blaGES from Iran, blaKPC/blaNDM-1 from China and Greece and the coexistence of blaKPC and blaNDM-1 has been reported in Pakistan [4,10].
This multidimensional spread of blaKPC-harboring CRKP strains is facilitated through several mechanisms.However clonal spread is considered as one of the important modes of transmission responsible for the growing pervasiveness of CRKP strains globally.Among CRKP, the prominent clonal group is CC258 with varying diversity of sequence types (ST) geographically.These include ST258 which is the most prevalent clone in the US and ST11 in China.Sporadic reports of other STs among blaKPC-2 bearing CRKP strains has been described such as ST15, ST660, ST48 from China, ST15 from Turkey, ST48, ST340 from Thailand, ST307, ST48 from South Korea and ST101 from India [11].However, only ST258 has previously been identified in Pakistan demonstrating the transmission capacity of selected clonal groups in relation to different regions [12].Therefore, it is critical to understand the molecular mechanisms that ascertain the emergence and distribution of clinically fit lineages of blaKPC-2 harboring CRKP variants worldwide.
There has been worrisome rise in antimicrobial resistance globally and there is only limited information available regarding the prevalence and underlying population structure of blaKPC-2 harboring CRKP strains in Pakistan [4,12].In view thereof, the present study was intended to encompass the prevalence, genetic variability and clonal expansion of MDR blaKPC-2 harboring K. pneumoniae in Pakistan.

Identification of clinical isolates
In this observational study, 107 MDR K. pneumoniae strains were collected from April 2018 to March 2020, from the Pathology sections of tertiary care hospitals in Lahore, Pakistan.Phenotypic and biochemical characterization of the strains were carried out by analyzing colony morphology, Gram staining and API-20E (bioMerieux, Marcy-I'Etoile, France).

Phenotypic identification of β-lactamases
The frequency of extended spectrum β-lactamases (ESBLs) among all CRKP strains was determined by double-disc synergy test using AMC alone and in combination with CAZ in accordance with CLSI guidelines.Carbapenem inactivation method (CIM) was performed for the phenotypic screening of carbapenemase production as per CLSI guidelines.

Molecular identification of strains and carbapenemases
Genomic DNA was isolated from bacterial cultures using the heat lysis technique.Briefly, 2 to 3 colonies of bacterial culture were mixed with 500 μL sterile distilled water and heated at 98 °C for 10 min at 300 rpm (ThermoMixer, Fischer Scientific, Waltham, MA, USA).Tubes were centrifuged at 1000 rpm for 10 min and the supernatant collected in newly labeled tube.DNA was stored at -80 °C.Selected carbapenemase encoding genes such as blaKPC-2, blaNDM-1, blaVIM, blaIMP and blaOXA-48, selected ESBLs including blaSHV, blaTEM and blaCTX-M were amplified and molecular identification of Klebsiella were performed by standard Polymerase Chain Reaction (PCR).The PCR reaction mixture contained 25 μL of 2x PCR Master Mix (Cat # K0171, ThermoScientific, Waltham, MA, USA), 1 μL of each primer, 2 μL of DNA and dH2O upto 50 μL in a thermal cycler (Proflex, Applied Biosystems, ThermoFischer Scientific, Waltham, MA, USA).Amplicons were resolved by agarose gel electrophoresis (1-1.5%).The details of the primer sequences are listed in Table 1.

Sequencing of carbapenemase genes
Carbapenemases were further analyzed by gold standard Sanger's sequencing technique at Advance Research Center for Biomedical Sciences, King Edward Medical University, Lahore.Cycle sequencing was performed by BigDye Terminator v3.1 kit with 10  1.

Statistical Analysis
Statistical analysis was performed on SPSS 24.0 (Chicago, IL, USA).Normality of the data were analyzed by the Kolmogorov-Smirnov test.Categorical data were evaluated by χ 2 test or Fisher's exact test where applicable.The p value of < 0.05 was considered statistically significant.

Ethics approval and informed Consent
The study was approved by institutional review board of the University of Health Sciences, Lahore, Pakistan and King Edward Medical University, Lahore, Pakistan.Informed consent was obtained from the participants for inclusion in study.
Of all the clinical strains, 87.9% (n = 94) were detected as ESBL producers with 66.0% resistant to all the third generation cephalosporins (3GCs), 28.7% were resistant against two 3GCs, while 5.3% strains were resistant to one of the 3GCs.Association analysis established that 69.1% ESBL producers were surfaced from the wound/pus samples.

Discussion
The unregulated use of antibiotics has resulted in the dispersal of high-risk CRKP clinical strains worldwide, thus narrowing the available treatment options leading to a staggering burden on healthcare facilities.It is critical to trail the spread of resistant species in recalcitrant infections in order to prevent them.This challenge is amplified due to the insufficient available data, especially from developing countries.
In the current study, we detected high prevalence of carbapenem resistance with 72.8% CRKP and 65.4% CPKP strains highlighting resistance up trends compared to previous reports from Pakistan that documented up to 82.8% resistance and other reports form South Asia [14][15][16][17].This clearly indicates that carbapenem resistance has increased in the study population over the years due to the lack of stewardship and unrestrained use of antimicrobials.The occurrence of CRKP infection was found significantly associated with wound (39.7%, n = 31, p value = 0.0007), pus (38.5%, n = 30, p value = 0.009), general surgery (34.6%, n = 27, p < 0.002) and ICU (26.9%, n = 21, p < 0.04).This is the first study reporting such associations in Pakistan; however, these were consistently observed worldwide [14,18].Therefore, the emergence of CRKP in general surgery and ICU underscores their possible contribution in the successful survival of high-risk MDR K. pneumoniae.
The successful dissemination of KPCs has been attributed to ST258 K. pneumoniae and its single locus variant ST11.It has been reported that ST258 was identified mainly from Europe and US while ST11 was commonly spotted in the Asian region [11].In the current study, we identified novel blaKPC-2 producing K. pneumoniae ST11 coharboring blaSHV and blaCTX-M which to the best of our knowledge has not been previously reported in Pakistan.ST11 was linked to blaNDM and blaOXA-48 CRKP in Pakistan and several other countries such as Europe, China, USA, Thailand, Australia, UAE, Malaysia and Iran depicting the potential of ST11 to retain resistance with the acquisition of different carbapenemases [11,20].While ST11 harboring blaKPC-2 CRKP has been shown to become a dominant strain in China as a result of progressive evolutionary changes in the genetic makeup [27].On the other hand, ST258 among blaKPC-2 CRKP has been described from Pakistan [12] but we observed ST258 CRKP isolates coharbored blaKPC-2/blaSHV and blaCTX-M.Determination of ST11 bearing blaKPC-2/blaSHV and blaCTX-M in our study demonstrated that the heterogeneous ST11 genome has started emerging in Pakistan.
Thereafter, we identified IncFII, IncN, IncFIIA, IncL/M and IncFIIK replicon types among blaKPC-2 CRKP isolates.It has been shown that IncFII-like plasmids are largely responsible for the dissemination of blaKPC-2 among K. pneumoniae especially in ST11 due to the presence of Tn1721 transposon.Other reported replicon types among K. pneumoniae isolates responsible for the spread of carbapenemases included IncR, IncFIB, IncX3, IncA/C and IncL/M IncFIIK [11].

Conclusions
We report the emergence of blaKPC-2 producing K. pneumoniae ST11 coharboring blaSHV and blaCTX-M in Pakistan.The emergence of such high-risk clones with several resistance genes due to misuse and selection pressure of antimicrobials is quite alarming and warrants immediate attention.Therefore, we need to understand resistance patterns and the uniqueness of such high-risk clones in order to decipher their inexorable fate.

Table 1 .
Primer sequences used for PCR and Sequencing.