Molecular epidemiology of carbapenem resistant Enterobacteriaceae in Sri Lanka: First report of KPC-producing Klebsiella pneumoniae CURRENT STATUS: POSTED

Background Extended spectrum β-lactamase producing Enterobacteriaceae (ESBL-PE) and carbapenamase producing Enterocacteriaceae (CPE) are widely disseminated globally creating a huge public health threat. Even though incidence of multidrug-resistant Enterobacteriaceae is rapidly growing, the epidemiological data regarding the occurrence of CRE in Sri Lanka is scarce. In this study, we determined the prevalence of ESBP-PE and CRE and the genetic determinants of CPE. Methods A total of 593 clinically significant Enterobacteriaceae was isolated from different clinical samples (urine, pus/wound, respiratory, blood, and other sterile specimens) at a tertiary care hospital in Sri Lanka from December 2017 – February 2018. Antimicrobial susceptibility and identification of ESBL-PE, CRE were done by disc diffusion method. CRE were identified to species level using a rapid identification kit and carbapenemase production was determined by modified carbapenem inactivation method. The presence of blaKPC, blaNDM, blaOXA-48-like genes were detected by PCR.

variable, but high rates of ESBL-PE (> 50%) and CRE (> 10%) have been reported ( 9,(11)(12)(13). Though the occurrence of CRE has increased dramatically during the last decade in neighboring India ( 12), a little is known about the prevalence of CRE in Sri Lanka, which appears to be growing ( [14][15][16]. Additionally, the CPE in Sri Lanka was first reported in 2013 and the existence of several different carbapenemase types has been revealed thereafter (17)(18)(19)(20). Sri Lanka has always been at a greater risk of being affected by ESBL-PE and CPE outbreaks due to the liberal antibiotic usage in the country and the increased travel from MDR Enterobacteriaceae endemic countries like India, China, Middle East, etc. ( 21-23). Hence, baseline epidemiological data of MDR Enterobacteriaceae is essential to streamline the national antibiotic policy and the infection control protocols. Present study sought to determine the prevalence of MDR Enterobacteriaceae with the emphasis on ESBL-PE and CRE at a tertiary healthcare facility in Sri Lanka and to explore the genetic determinants of carbapenem resistance.

Study design
A laboratory based, prospective cross-sectional study was conducted on clinically significant Enterobacteria isolated from patients attended at the Colombo North Teaching Hospital (CNTH), Sri Lanka over a ten week period from December 2017 to February 2018. CNTH is a major public tertiary care hospital with 1477 beds located in the suburbs of Colombo, the capital of Sri Lanka.
Enterobacteriaceae were isolated from variety of clinical specimens (blood, urine, other sterile fluids, respiratory specimens, and wound/pus swabs) received consecutively to the microbiology laboratory at CNTH.

Bacterial isolates and antimicrobial susceptibility testing
In order to isolate Enterobacteria, clinical materials were cultured on blood agar. Each isolated organism was screened by standard microbiological procedures including Gram staining, oxidase test, lactose fermentation and other characteristics on Kligler Iron Agar. Antimicrobial susceptibility was performed by disc diffusion method on Muller-Hinton agar according to M100-S28 of Clinical and Laboratory Standards Institute (CLSI) guidelines. Antibiotic discs were from Oxoid, UK. Any isolate resistant to at least one antibiotic in 3 or more antimicrobial classes was considered as MDR ( 24). ESBL-PE and CRE were identified by disk diffusion method as per CLSI guidelines.
Accordingly, Enterobacteria, resistant to cefotaxime were further tested for ESBL production by double disc synergy test using cefotaxime (30 μg) and amoxicillin-clavulanic acid (20/10 μg) and by combined disc containing ceftazidime-clavulanic acid (30/10 μg). Organisms resistant to meropenem and / or imipenem were considered as CRE. ESBL-PE and CRE isolates were stored in peptone broth containing 20% glycerol at -80 °C until used. Before further experiments, the frozen bacterial samples were recovered on blood agar and were subsequently sub cultured on Muller-Hinton agar (Oxoid, UK).

Phenotypic and genotypic characterization
All CRE isolates were identified up to species level by Rap ID One system Enterobacteriaceae identification kit (Remel, Thermo scientific) according to manufacturer's instructions. Phenotypic carbapenemase production in CPE was determined by modified carbapenem inactivation method

Epidemiology of multidrug-resistance Enterobacteriaceae
A total of 593 clinically significant Enterobacteriaceae was isolated from the consecutive patient samples received to the microbiology laboratory at a tertiary care hospital in Sri Lanka. Of them, 328 (55.3%) were resistant to cefotaxime and 154 (26.0%) were identified as ESBL-PE, whereas 57 (9.6%) were identified as CRE. Table 1 summarizes the epidemiological characteristics of the study sample.
The modified carbapenem inactivation method (mCIM) was used to explore whether the carbapenem resistance is conferred by the carbapenem degrading enzymes. The carbapenemase production was detected in 54 (94.7%) of 57 CRE isolates. Two K. pneumoniae and one P. rettgeri isolates were of non CPE. Moreover, Eleven out of 12 (91.7%) CRE isolated from ICU samples were carbapenamase producers. All the Enterobacteriaceae identified as CPE had a zone diameter of 6 mm for the meropenm disc incubated with the test organism in the mCIM study.

Discussion
In this prospective study, we determined the prevalence of ESBL-PE and CRE in one of the major public tertiary care hospitals in Sri Lanka based on 593 clinically significant Enterobacteriaceae isolated during December 2017 -February 2018. The patients involved in the study group comprised of nearly equal number of males and females with the age ranged from 4 days to 89 years.
Enterobacteria were recovered from a diverse spectrum of microbiological samples from patients (Table 1), the majority being from urine (52.4%) and pus/wound swabs (27.7%). Yet, this is in parallel with the types of infections caused by Enterobacteriaceae in clinical settings.
Probing into the antibiotic resistance data in the present study, the incidence of ESBL-PE was 26.0%.
Although the ESBL-PE occurrence was highest (30.8%) among urinary coliforms (Figure 1 This study identified the overall prevalence of CRE as 9.6%, which was comparable to the CRE prevalence reported in many regions of South Asia ( 13). Further, sample-wise analysis noted that the occurrence of CRE was highest among respiratory specimens (20.8%) and that was lowest among the Enterobacteriaceae isolated form pus/wound swabs (6.7%) (Figure 1). The prevalence of CRE among the urinary Enterobacteriaceae isolates in our study was found to be 8.7%, which is in agreement with the rate of meropenem resistance (9.0%) reported in the coliforms isolated from urine cultures in a multi-center study conducted in the Western Province of Sri Lanka ( 14). Additionally, Fernando et. al. has documented that the prevalence of meropenem resistance among ESBL producing uropathogenic Enterobacteriase as 4.9% ( 16). However, the carbapenem resistance was not detected within the ESBL-PE cohort of the present study.
Intensive care units (ICUs) are known reservoirs for multi-drug resistant bacteria in healthcare settings due to the presence of high antibiotic pressure and higher propensity for cross-contamination ( 30,31). Considering the antibiotic resistance in the cohort of Enterobacteriaceae isolated from the clinical samples of the ICUs, we found that high overall prevalence of CRE (38.7%; 12/31), which was significantly higher than the prevalence of ESBL-PE (22.6%; 7/31) detected in the same group.
However, this difference need to be further explored with the antibiotic usage pattern of the relevant ICU settings since frequent use of third-generation cephalosporins drives the emergence of ESBL-PE whereas CRE selection is driven by the use of carbapenems. Further, six out of 10 (60.0%) of respiratory CRE were found to be of ICU origin and the prevalence of CRE among the ICU respiratory Enterobacteriaceae isolates was 33.3%. These observations may at least partly be associated with the frequent usage of carbapenems and ventilator equipment in ICU settings. Similarly, high rate of respiratory CRE (38.1%; 2/7 E.coli and 6/14 K. pneumoniae) has been reported in the ICUs of a tertiary care hospital in the Central Province of Sri Lanka ( 30). Additionally, in the present study, an alarmingly high prevalence of CRE (83.3%) was found among the blood culture Enterobacteriaceae isolates of ICU origin. However, these observations need to be confirmed by further studies with larger sample sizes.
In this study, we encountered surprising species diversity within CRE isolates. Enterobacteriaceae is a diverse family of Gram negative rods which includes diverse spectrum of human pathogens with K. pneumoniae being the most frequent CRE species found globally ( coli, and E. cloacae in Sri Lanka ( 18,19,30). In addition to the previously documented Enterobacteriaceae species in the country, carbapenem resistance was also recognized in C. freundii, P. rettgeri, and E. aerogens in our study ( Figure 2). Further, K. pneumoniae was the predominant CRE detected and was isolated as the only CRE species from respiratory and blood samples. In contrast, E. coli has been reported as the predominant organism among urinary ESBL-PE in Sri Lanka ( 16,27).
Investigation of the ability of CRE to inactivate meropenem by carbapenem inactivation method, we found that the majority of CRE were carbapenemase producers (94.7%). This is in agreement with the fact that carbapenemase production is the most common mechanism of carbapenem resistance in Enterobacteriaceae ( 32). Further, genomic identification of CPE by polymerase chain reactions found that KPC, NDM, and OXA-48-like carbapenemases as the determinants of carbapenem resistance in our CPE cohort ( Table 2). Of them, OXA-48-like was identified as the most common carbapenemase (88.9%).
Previously, OXA-181 has been reported as the predominant carbapenemase type in Sri Lanka (  18,19). In addition, 7.4% of CPE in our study were found to coproduce NDM and OXA-48-like enzymes. Similarly, K. pneumoniae carrying bla NDM-1 and bla OXA-181 genes have been previously documented in Sri Lanka by Hall et. al. ( 19). Most interestingly, the existence of the bla KPC gene was found in two CRE isolates as the first such finding in Sri Lanka. Of the two KPC harboring K. pneumonia, one was recovered from a sputum sample of 71 year old female patient and the other was isolated from a urine sample of 55 year old male patient. KPC has been reported internationally and has been identified as the most common carbapenemase type in China ( 22). Yet, other two major carbapenemases; IMP and VIM have not been reported in Enterobacteriaceae in Sri Lanka to date and in the present study as well.
A number of limitations were recognized in our study. The initial screening of CRE isolates was done using only imipenem and/or meropenem depending on the availability of antibiotic discs, which might have left out some of the CRE according to the 2015 CDC CRE surveillance definition (i.e. any Enterobacteriaceae resistant to imipenem, meropenem, doripenem, or ertapenem are considered as CRE) ( 34). Additionally, the DNA sequence analysis was not performed to determine the exact genotype of identified carbapenamase genes. Prevalence of ESBL-PE and CRE were not compared in all sample types due to small sample number.

Conclusions
Our study emphasizes the burden of multidrug-resistant Enterobacteriaceae in a tertiary healthcare setting in the country by providing baseline data for overall and the sample-wise prevalence of the ESBL-PE and CRE. K. pneumoniae was identified as the predominant CPE organism. Carbapenemase production was found to be the main mechanism behind carbapenem resistance and bla KPC , bla NDM , and bla OXA-48-like genes were identified as the molecular determinants. To our knowledge, this is the first document, which shows the occurrence of KPC type carbapenemase in Sri Lanka. Since carbapenemase encoding genes are transmissible and prevalent, implementation of preventive measures are urged to minimize the spread of CRE that could otherwise lead to future epidemics of difficult to treat infections in the country. The ethical approval for this study was granted (P/215/08/2017) by the ethics review committee, Faculty of Medicine, University of Kelaniya, Sri Lanka. During the initial screening, the organisms were collected from the microbiology laboratory at the CNTH. The data (age and gender) were obtained from the laboratory request form and no personal identifiers were collected. The demographic and clinical data were collected from patients, who were found to have infected with ESBL-PE and CRE after obtaining informed written consent. In case of children (<18 years) or critically ill patients, informed written consent was obtained from their parents/guardians.    Figure 1 Distribution of ESBL-PE and CRE in each sample type