Investigation on the Genetic Signatures of Antibiotic Resistance in Multi-Drug-Resistant Klebsiella Pneumoniae Isolates From National Guard Hospital, Riyadh

Introduction Despite a large number of antibiotics available to treat Klebsiella (K.) pneumoniae (KP), resistance against these antibiotics is ever-increasing and has now become a global threat to human life. The most frequently observed resistant genes in Klebsiella pneumoniae are CTX-M, OXA-48, IMP, and NDM; some are clone-specific while others form a reservoir for infection. Methods Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was employed for the identification of the pathogens and automated VITEK-2 (bioMérieux, Marcy-l'Étoile, France) was used for minimum inhibitory concentration (MIC) determination, followed by polymerase chain reaction (PCR) amplification of target genes and Sanger sequencing of amplicons. Results Forty-three out of 50 isolates (86%) were OXA gene-positive, and 49 out of 50 (98%) isolates were CTX-M gene positive. Two phenotypes of OXA were identified in 33 samples sequenced, OXA-505 (70%) and OXA-232 (30%). Sixteen isolates (32%) were positive for NDM-1. Twelve isolates were positive for both OXA and NDM. Multilocus sequence typing (MLST) on these isolates showed that they were distributed in 12 sequence types (STs). Thirty-six out of 50 were grouped in four clonal complexes. ST-14 was the predominant genotype. Conclusion This study has revealed that CTX-M-15 is the most common extended-spectrum beta-lactamase (ESBL) present in almost all isolates. The study also shows the presence of OXA as the main carbapenemase gene, alone or in combination with other carbapenemases such as NDM-1. Multilocus sequence typing revealed the incidence of polyclonal KP pool with ST-14, ST-29, ST-307, and ST-15 being the predominant ones.


Introduction
Resistance against antibiotics is ever-increasing and has now become a global threat to human life [1][2]. Bacterial pathogens have developed resistance against many of these antibiotics, and some countries have reported strains that are resistant to all antibiotics available, i.e. pan-drug resistance [3]. Various mechanisms involving a battery of diversified genes are described for these resistances. Some of these genes are found on the bacterial chromosome, whereas others are found on episomal deoxyribonucleic acid (DNA) fragments called plasmids [4]. These plasmid-mediated genes can transfer resistance horizontally from one bacterium to another by inter-species or intra-species conjugations. The most frequently observed resistant genes in Klebsiella (K.) pneumoniae (KP) are TEM, SHV, CTX-M, OXA-48, IMP, VIM, NDM, and KPC. Some are clone-specific while others are transferred independently. Carbapenems are supposed to be the last line of drugs available against these pathogens. However, carbapenems have now also been reported to be ineffective due to the emergence of new resistant genes, such as KPC, OXA, NDM, VIM, and IMP, against this antibiotic [5]. Although the KPC gene is not much prevalent in Saudi Arabia, OXA-48 is widely reported in carbapenem-resistant Klebsiella pneumonia [6]. The New Delhi metallo-beta-lactamase 1 (NDM-1) gene is also being reported regularly from different parts of Saudi Arabia [7][8]. Few studies are available on the molecular basis of antibiotic resistance, virulence factors present, plasmid profiles carrying resistance genes, and the clonal diversity of these drug-resistant Klebsiella pneumoniae in Saudi Arabia. However, the true picture of antibiotic resistance in Klebsiella pneumoniae from the Kingdom in general and the National Guards Hospital, Riyadh, in particular still remains unclear. The aim of the present research is to understand the mechanisms of antibiotic resistance in multi-drug Carbapenem-resistant isolates of Klebsiella pneumoniae from the national guard hospital by investigating the genetic signatures of these resistant genes. We would also determine the genotypes of these strains that are predominant in this region to facilitate the control programs. All of this could help improve the infection control intervention and promote/support effective antibiotic stewardship programs in the National Guard Hospital, Saudi Arabia.

Materials And Methods
The study was conducted at the infectious diseases laboratory of King Abdullah International Medical Research Center (KAIMRC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was employed for the identification of the pathogens and automated VITEK-2 (bioMérieux, Marcy-l'Étoile, France) was used for minimum inhibitory concentration (MIC) determination. The isolates, which showed elevated MIC for carbapenem and beta-lactam antibiotics and were designated as resistant by the microbiology department, National Guard Hospital, were the subject of this present study. Detection of resistance gene targets, such as CTX-M, OXA-48, and NDM, was done by polymerase chain reaction (PCR) using their specific primers. The sequencing of the gene products was done by the Sanger method to look for their variants (novel, if any). Multilocus sequence typing (MLST) was performed on these isolates using seven loci scheme proposed by Diancourt et al. [9] to determine their clonality and sequence type (ST).

Data collection process
Bacterial isolates were derived from patients on routine lab investigations. The sources of these isolates are given in Table 1. The identification was done by the MALDI-TOF technique in the microbiology lab. MICs were determined by VITEK-2. The KP isolates with elevated MIC were grown on blood agar medium. A single colony was selected and grown in a liquid broth medium. Fifty isolates of multi-drug carbapenem-resistance of Klebsiella pneumonia were investigated. DNA was extracted and PCR amplification of genes of antibiotic resistance (CTX-M, NDM, OXA) was done using the primers specific for these genes. These amplified products were visualized on an agarose gel by electrophoresis. Positive and negative controls were included. The confirmed positive PCR products were processed for sequencing by the Sanger Sequencing method. The DNA sequences were aligned and analyzed using Lasergene software. Mega 5 software was used for detecting single nucleotide polymorphisms (SNPs) and for phylogenetic analysis [10]. Each sequence was blasted against either the National Center for Biotechnology Information (NCBI) gene bank or a specific database for phenotype designation of these genes. The various phenotypes of the resistance genes of CTX-M, NDM, and OXA were determined and reported. Five researchers worked on the data collection process and the study took two years.

Demography
The sources of these isolates are given in Table 1  The clinical sources of the specimen for isolation of KP was as follows: respiratory (n = 10); surgical wound (n = 10); QC (n = 1); urine (n = 14); blood (n = 14); and Others (n = 1).

Multilocus sequence typing
The multi-locus sequence typing (MLST) method using seven loci) showed 38 isolates being distributed in 12 STs. Twelve isolates could not be typed due to technical reasons. The distribution of STs was as follows:

Discussion
Multi-drug resistance among KP has become a world phenomenon, nullifying the benefits of high-profile clinical skills such as open-heart and transplantation surgeries [11]. Several reports have projected Saudi Arabia as a growing pool for such multi-drug resistant pathogens because of several reasons [11][12][13]. In addition to common ESBLs, the incidence of CTX-M and other carbapenems are on the increase [14]. Here, we report that all our isolates were positive for CTX-M-15, underlying the multi-drug resistant profiles of these isolates. CTX-M-15 has previously been reported to be dominant among KP isolates from Saudi Arabia and has also been reported among outbreak isolates elsewhere, where 55% of Klebsiella pneumoniae isolates showed the phenotypic characterization of high ESBL [14][15]. The Klebsiella pneumoniae-producing ESBL were positive for the SHV, TEM, and CTX-M β-lactamase genes; the CTX-M genes that were more prevalent were CTX-M-1 and CTX-M-9-like genes [14][15]. CTX-M-15 was present in isolates from all STs, indicating its epidemicity. The OXA-48 gene has previously been reported in MDR-KP isolates from National Guard Health Affairs (NGHA), Riyadh. Our present study also showed the presence of OXA-505 (a member of the OXA-48 family) in as many as 70% of studied isolates. OXA-232 is reported for the first time in our KP isolates [6,8,11]. These findings need further investigations in a larger number of samples. The phylogenetic relationship between those two phenotypes shows clear isolation between these two groups ( Figure 1).
The incidence of NDM in as much as 32% of isolates is in agreement with the previous reports from Saudi Arabia [8,11,16]. The coexistence of OXA and NDM genes also has been previously reported and underpins the elevated MIC levels of Carbapenem for these isolates. None of the resistance genes is clone specific and evenly distributed among all sequence types of KP ( Table 1).  [19]. The large numbers of certain sequence types might be due to their clonal expansion [20]. Genetic diversity and population structure have inferences, which have been drawn by the MLST tool. This study has shown several genetically related antibiotic-resistant KP STs grouped into at least four clonal complexes based on mono-, di-, and tri-locus variations and are designated as C1-C4 for our study ( Table 2). In members of complex C1, there is a single locus variation in infB among their isolates, whereas in complex C2 and C3, there is a tri-locus variation involving the mdh, pgi, phoE, and tonB genes. Finally, complex C4 has a di-locus variation in pgi mad tonB genes. As we see, three complexes have locus variations in tonB, which are C2, C3, and C4.
The main limitation of this study is that these results might not give the true picture of antibiotic resistance in the whole country. A multi-centric study involving a larger number of isolates is needed to reach a meaningful conclusion and a true picture of antibiotic resistance in the Kingdom. Investigations on the plasmids carrying the resistance genes would also shed light on the horizontal transfer of these resistant genes. However, our study forms a base for such investigations.

Conclusions
The study has revealed the presence of CTX-M-15 in almost all isolates. The study also shows the presence of carbapenemase genes, such as OXA and NDM, singularly or in combination. Newer OXA phenotypes, such as OXA-232 and OXA-505, were detected. Multilocus sequence typing revealed the incidence of a polyclonal KP pool, ST14 and ST15 being the predominant ones. Further studies should be focused on these lines to generate comprehensive data to form a solid database of an antibiotic-resistant gene profile among the bacterial pathogens in Saudi Arabia.

Additional Information Disclosures
Human subjects: Consent was obtained by all participants in this study. Institutional Review Board of King Abdulaziz Medical City issued approval H-01-R-005. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.