The Inc FII Plasmid and its Contribution in the Transmission of blaNDM-1 and blaKPC-2 in Klebsiella pneumoniae in Egypt

The emergence of blaKPC-2 and blaNDM-1 producing Klebsiella pneumoniae represents a great problem in many Egyptian hospitals. One hundred and twenty-six K. pneumoniae isolates from patients admitted to Assiut University Hospital were identified by an API20E kit. Carbapenemase-producing K. pneumoniae (CPKP) was detected by the modified carbapenem inactivation method (mCIM), the EDTA-modified carbapenem inactivation method (eCIM), and an E-test. Based on the polymerase chain reaction, all isolates were negative for bla-VIM-1 and bla-IMP-1, fifteen of these isolates were positive for both blaKPC-2 and blaNDM-1, two isolates were positive for blaKPC-2 only, and twenty-eight isolates were positive for bla-NDM-1 only. Although one isolate was positive for the string test, all CPKP isolates were negative for capsular genes. Only 71.1% of CPKP transferred their plasmids to their corresponding transconjugants (E. coli J53). The resistance patterns of the clinical isolates and their transconjugates were similar, except for 12 isolates, which showed differences with their transconjugates in the resistance profile of four antibiotics. Molecular typing of the plasmids based on replicon typing showed that Inc FIIK and FII plasmids predominated in isolates and their transconjugants carrying blaKPC-2 and/or blaNDM-1. Conjugative Inc FII plasmids play an important role in the spread of CPKP, and their recognition is essential to limit their spread.


Introduction
K. pneumonia is a common bacterial pathogen that can cause several life-threatening infections, such as blood stream infections, pneumonia, as well as urinary tract, post-surgical, and intensive care-related infections [1,2]. Such infections result in significant morbidity and mortality. K. pneumonia also significantly increases medical hospital costs [3]. It can spread well and survive in the hospital environment and frequently causes nosocomial infections and outbreaks [4]. K. pneumonia's major route of infection is aspiration of the oropharyngeal secretions of the patients colonized by it. The gastrointestinal tract of patients may also play a secondary role. Further, contaminated medical equipment and the hands of health care workers may contribute to sustaining colonization and creating an increased risk of infection [5]. Additionally, antibiotic therapy can suppress normal bacterial flora and lead to an over-growth of multidrug resistant K. pneumoniae [6].

PCR for Carbapenemase Genes and KI and K2 Capsular Genes
All K. pneumoniae isolates were tested for carbapenemase genes and K1 and K2 capsular genes. It was found that all non CPKP were negative for these genes. On the other hand, Carbapenemase-encoding genes were founded in 45/77 (58.44%) of carbapenemase-producing K. pneumoniae isolates. Bla NDM-1 was present in 43/77 (55.84%) isolates and bla KPC-2 in 17/77 (22.07%) isolates.

Comparison of Phenotypic and Genotypic Methods for Detection of CPKP
Isolates were divided into four groups according to their positivity for the tested genes ( Table 1). The isolates negative for all genes (Group 1) were negative for mCIM. Out of the 28 isolates (Group 2) that tested positive for bla NDM-1 and mCIM, nine isolates were metallo-β-lactamases (MBL) producers (positive for eCIM), while the two isolates (KP8, KP17) positive for blaKPC-2 (Group 3) were positive for the mCIM and eCIM test (Table 1). In addition, the five isolates of Group 4, which were positive for bla NDM1 and bla KPC2 and mCIM, were positive for eCIM and were considered to be MBL producers. Table 1. Phenotypic and genotypic methods for the detection of CPKP.

Genotypic Method
No

Plasmid Transfers
Conjugation experiments were carried out for all bla KPC-2 and/or bla NDM-1 carrying K. pneumoniae isolates (n = 45). Only 32 of these were found to have the ability to transfer their plasmids by conjugation to their corresponding transconjugants (E. coli J53). These transconjugants were tested for bla KPC-2 and/or bla NDM-1 genes by the PCR method. All thirty-two transconjugants were found to harbor bla KPC-2 and/or bla NDM-1 genes as their parent cells.
All transconjugants were observed to exhibit Multi-drug resistance (MDR) phenotypes like those of the donor K. pneumoniae isolates, except 12 of the transconjugants were found to have different antibiotic resistance profiles. The difference in the resistance profiles among these isolates was observed with the following antibiotics: Tetracycline, Gentamicin, Sulfamethoxazole/trimethoprim, and Levofloxacin. The resistance profiles of the 12 tested isolates and transconjugates against these four antibiotics are listed in Table 2. Table 2. Resistance patterns of the 12 isolates and transconjugates showing different resistance profiles.

PCR-Based Replicon Typing of bla KPC2 and/or bla NDM1 Encoding K. pneumoniae
PCR-based replicon typing (PBRT) showed typing for only 29/32 of the self-transmissible bla KPC-2 and/or bla NDM-1 encoding K. pneumoniae isolates. The replicon types identified were FIIK, FII, FIB, FIC, L, and Inc M. It was found that FIIK and FII were highly distributed among bla KPC-2 and/or bla NDM-1 encoding K. pneumoniae isolates, followed by the FIB, FIC, Inc L, and Inc M groups. Furthermore, PBRT failed to detect the replicon types of the plasmids of the remaining three isolates (Kp5, Kp6, Kp22) ( Table 3). Table 3. Relation between the plasmid replicon type and the transfer of resistant genes.

Strain Code
Carbapenemase Gene PBRT of Isolates PBRT of Transconjugant

Transfer of Plasmids with a Specific Replicon Type to the Transconjugants
PCR-based replicon typing of the transconjugants (E. coli J53) carrying bla KPC-2 and/or bla NDM-1 encoding genes (n = 32) revealed that nine (28.1%) of the strains were positive for Inc FIIK alone, while three (9.3%) of the transconjugants were positive for all FII, FIIK, and FIB incompatibility types. Two (6.25%) of the strains carried plasmids belonging to the Inc FII group only, whereas Inc FIB was found in one (3.1%) of them. On the other hand, PBRT confirmed the transfer of Inc FII and FIIK plasmids to two transconjugants and the transfer of Inc FIIK and FIB groups to other two transconjugants. The remaining strains (n = 13) could not be typed by the PBRT method (Table 3). Also, it was observed that Inc L, M, and FIC plasmids did not show the ability to transfer to the recipient cells.

Discussion
Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most widespread pathogens among hospital-acquired multi-drug resistant pathogen associated infections [7]. Although many reports exhibited the emergence of carbapenem resistant K. pneumoniae in Egypt [12,23] in the last decades, the data on plasmid epidemiology and which plasmid types are mainly associated with the spread of bla KPC2 and/or bla NDM1 plasmids in Egypt are rare. Thus, in this study, we attempted to determine the plasmids responsible for transferring the determinants of carbapenem-resistance among K. pneumoniae isolates in this country.
In the present study, carbapenemase phenotypic activity was detected in 77/126 (61.1%) of the K. pneumoniae isolates by an mCIM test. Although the rate we observed was lower than that in a study in the USA [24], it was higher than the previous reports in Egypt. The prevalence of CPKP was found to be 33.3% among K. pneumoniae isolates in a study performed by Moemen and Masallat [23]; 44.3% at the Suez Canal University Hospitals in a study performed by El-Sweify et al. [10]; and 13.9% in the Egyptian National Cancer Institute in a study performed by Ashour and El-Sharif [25]. Increasing rates of CPKP isolates may be due to the misuse of carbapenems, as carbapenems are considered the best choice for the treatment of serious infections caused by multi-drug resistant pathogens in our hospitals, where there is presently no implementation of an antimicrobial stewardship program [26].
Our results show that the mCIM negative isolates were also negative for both bla KPC2 and/or bla NDM1 , and there was compatibility between the results of the mCIM and PCR results among carbapenemase producing isolates. The sensitivity of this test was 100% compared to PCR, the gold standard test, suggesting that this test can be used as a primary test for the detection of CPKP. On the contrary, the eCIM test did not identify isolates that were positive for both bla KPC2 and bla NDM1 in accordance with the CLSI [27] report.
The capsular serotypes K1 and K2 of K. pneumoniae are the predominant virulent strains that contribute to the high mortality rate associated with K. pneumoniae infections [28]. Although there was only one isolate (Kp27) positive for the string test, all CPKP isolates were negative for the K1 and K2 capsular gene. This may be due to the fact that antibiotic resistance is not dependent from the hypervirulence of KP [29], and isolates were recovered from hospital-acquired infections, not from community-acquired infections [30,31]. In addition, the incidence of bla NDM1 producers among the CPKP in our hospital (55.84%) was much higher than that previously reported by Yan et al. [8]. Bla NDM1 was first detected in Egypt from one K. pneumoniae isolate in 2013 by Abdelaziz et al. [32] and then from two Pseudomonas aeruginosa isolates [33], as well as in Acinetobacter baumannii isolates [34], which indicates that this gene may spread among Enterobacteriaceae by transferable plasmids [35,36].
In Egypt, the plasmid types that are mainly associated with the spread of bla KPC2 and/or bla NDM1 have not been identified, except for the eight carbapenem-resistant NDM1-producing K. pneumoniae isolates carried by non-transferrable plasmids (either IncR or untypeable), which were reported by Gamal et al. [11].
Our study showed that most bla KPC2 and/or bla NDM1 plasmids were successfully transferred by conjugation and that bla KPC2 and/or bla NDM1 were commonly carried on the Inc FII plasmid. Similar results were reported by [37], who reported that bla KPC-2 was located on Inc FII plasmids, but in another study performed by Jin et al. [38], bla KPC2 was found to be located on non-self-transmissible plasmids or on the chromosome.
Our results showed the predominance of both Inc FIIK and FII plasmids among the isolates and their transconjugants carrying bla KPC2 and/or bla NDM1 , suggesting that these types of plasmids mediate horizontal transmission and contribute to the dissemination of bla KPC2 and/or bla NDM1 in the environment of the Egyptian hospitals, which represents a great challenge and an important factor in the dissemination of resistance and treatment failure in cases of severe infections. Agyekum et al. [39] reported that FIIK is the most common plasmid replicon found in K. pneumoniae. Another study done by Al-Marzooq et al. [40] reported that the most common plasmid replicons were IncR and IncL/M. Moreover, a high incidence of conjugative FIIK (69%) and L/M plasmids (66%) found in CPKP isolates were reported in Saudi Arabia [41].
Based on the previous findings, the Inc F plasmid is the most frequently described plasmid type to be associated primarily with resistance genes in humans [42,43].
In our study, Inc L, M, and FIC plasmids were not detected in the recipient cells, indicating that these types of plasmid were non-conjugative, and their role in the spread of carbapenem resistance among CPKP isolates is not clear; it is possible that these plasmids have no role in the transfer of resistance in our area. The prevalence of certain plasmid types is different even among samples collected from different sources within the same city and between different countries [42].
The antibiotic sensitivity tests of transconjugants showed that all transconjugants were resistant to Levofloxacin-like donor cells, except for KP19 and KP25, which can be explained by the co-existence and the transfer of quinolone resistant genes with carbapenem resistant determinants in the same strain [8].
Based on the previous results, diverse clones of multidrug-resistant (MDR) K. pneumoniae can spread between patients and the environment in the same hospital, especially with the increase of the number of patients and the inappropriate application of infection control guidelines among hospitals in Egypt. Therefore, the horizontal transmission of carbapenem-resistant plasmids among admitted patients may encourage the dissemination of carbapenem resistance to other species of Enterobacteriaceae, thereby leading to the maintenance of carbapenem-resistant clones in patients and/or the environment [7,40]. As a result, it is necessary to limit the spread of CPKP carrying highly conjugative Inc F plasmid types within healthcare settings and to implement effective infection control measures.
To the best of our knowledge, this is the first report on the identification of the conjugative Inc F plasmid that circulates and spreads in NDM-1 and KPC-2-producing CPKP isolates from a single hospital in Egypt.

Klebsiella pneumoniae Isolation and Identification
One hundred and twenty-six Klebsiella pneumoniae were isolated from patients admitted to different departments at Assiut University Hospital over a period of 14 months, from April 2018 to May 2019. Klebsiella pneumoniae were isolated from endotracheal aspirate samples, sputum samples, blood samples, urine samples, wound swabs, and throat swabs. Isolates were identified by an API20E kit (BioMerieux, Marcy L Etoile. France).

Antimicrobial Susceptibility Testing
The antimicrobial susceptibility of the isolated K. pneumoniae was tested by the Kirby-Bauer disc diffusion method according to recommendations of the clinical laboratory standards institute [27]. . The E. coli ATCC 25922 strain was used for quality control. The test was repeated twice for each isolate. In addition, the minimum inhibitory concentrations (MIC) of imipenem against the tested K. pneumoniae were tested using Imipenem E-test strips (bioMérieux, Solna, Sweden).

Modified Carbapenem Inactivation Methods (mCIM) for the Suspected Carbapenemase Producing Isolates
Modified carbapenem inactivation methods (mCIM) were performed as described by Pierce et al. [44] for suspected Carbapenemase Producing K. pneumoniae isolates. The inhibition zones for meropenem were determined after incubation at 35 • C for 18 or 24 h. A test is considered negative for carbapenemase production if the zone diameter is ≥19 mm and is considered positive if the zone diameter is 6 to 15 mm or features the appearance of pinpoint colonies within a 16 to 18 mm zone.

EDTA-Modified Carbapenem Inactivation Method (eCIM) for Detection of MβL Enzymes
If the mCIM test is positive, the EDTA-modified carbapenem inactivation method (eCIM) should be applied to differentiate between metallo-β-lactamases (MBL; Class B carbapenemases) and serine carbapenemases (Class A and D carbapenemases) in the K. pneumoniae isolates.
Bacterial isolates showing a positive mCIM test were cultured on Trypticase soy agar with sheep blood (TSAB). The EDTA-modified carbapenem inactivation method (eCIM) was performed according to Sfeir et al. [45]. The isolates were considered positive for MBL production if the zone diameter of meropenem disc increased by ≥5 mm in comparison to the zone diameter observed for the mCIM and were considered negative for MBL production if the increase in the zone diameter was ≤4 mm.

Detection of Hypermucoviscocity Phenotyping
The hypermucoviscocity phenotypes of the CPKP strains were checked by using the string test. The formation of a viscous filament of ≥5 mm was observed after stretching a Klebsiella spp colony with a loop cultured on an agar plate. CPKP strains positive on string tests may be considered hypervirulent CPKP (hvCPKP) [46].

Plasmid Transfers
Conjugation experiments were performed with modifications according to Hardiman et al. [17]. For broth culture mating, the donor and recipient (E. coli J-53, azide resistant strain) cultures were mixed at a 1:4 ratio in fresh TSB and incubated at 37 • C without agitation for 20 h. The selection of transconjugants was done on a MacConkey agar containing azide (100 µg/mL) and meropenem (0.5 µg/mL). Transconjugants were tested against the same antibiotic discs used against the donor isolates. Then, the transconjugants were tested for bla-NDM1 and/or bla-KPC2 by PCR, as mentioned above.

Conclusions
Molecular typing of plasmids based on replicon typing showed that Inc FIIK and FII plasmids were predominant among isolates in our area and their transconjugants carrying bla KPC-2 and/or bla NDM-1 . Also, conjugative Inc FII plasmids were found to have an important role in the spread of CPKP, and their recognition is essential to limit their spread. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.