Dissemination of ST101 blaOXA-48 producing Klebsiella pneumoniae at tertiary care setting

Introduction: The worldwide dissemination of the acquired carbapenemases in Gram-negative bacteria is a strongly expressed demand for the emergence of post antibiotic era. The aim of this study was to test the production of carbapenemase by Klebsiella pneumoniae strains isolated from hospitalized cancer patients and to investigate the genetic relationship of carbapenemase producing carbapenem resistant K. pneumoniae using multilocus sequence typing (MLST). Methodology: Antibiotic susceptibility testing and phenotypic testing for extended spectrum -lactamases (ESBL) and carbapenemases production were performed. PCR amplification of ESBL and carbapenemase genes was performed. MLST was done to detect the genetic relatedness of the isolates. Results: Our data showed all strains were sensitive to colistin. Carba NP test was positive in thirty-one carbapenem resistant K. pneumoniae isolates and 26 out of 34 K. pneumoniae isolates were metallo-beta-lactamases (MBL) positive. All carbapenemase-positive isolates were ESBL CTX-M-1-like positive. blaOXA-48 gene was detected in 25 isolates (80.65%) and 21 isolates (67.75%) produced blaNDM-1 like enzyme. VIM and KPC genes were not identified in this study. Association of blaOXA-48 like and blaNDM-1 like was found in 15 (48.39%) isolates, while the coproduction of OXA-48-like and IMP-1 was revealed in only one K. pneumoniae isolate. MLST revealed ten distinct sequence types (STs). Conclusion: Here we have documented the coexistence of NDM-type and OXA-48-like, and the coproduction of OXA-48-like and IMP in carbapenem resistant K. pneumoniae in patients with cancer. The dominant clone of the OXA-48-like-producing K. pneumoniae isolates from Egypt was ST101 epidemic clone belonging to clonal complex 101, an association that has been reported worldwide. The second most frequent ST was ST383.ST11 was assigned to OXA-48-producing K. pneumoniae.


Introduction
Klebsiella pneumoniae is a significant bacterial pathogen associated with hospital-acquired infections and capable of causing severe morbidity and mortality, especially in immunocompromised patients [1], and the occurrence of carbapenemase expands mortality rates [2].Acquired carbapenemases are encoded by transferable genes located in mobile elements such as plasmids and transposons, which may disseminate between various isolates, carbapenemases mediated resistance represents the major threat in carbapenem resistant K. pneumoniae [3].The existence of either class B (IMP, VIM, NDM) or classes A (KPC) and class D (OXA-48) carbapenemases have been reported world-wide [3,4].
In the Middle East Northern African countries, such as Morocco, Algeria, Tunisia, Libya, Egypt, or Saudi Arabia, OXA-48-producing K. pneumoniae have been predominantly described [5,6].On the other hand, in some regions of the Arabian Peninsula, NDM-1 was the experienced carbapenemases to the greatest extent followed by OXA-48-like carbapenemases [7].Association of NDM and other carbapenemases (NDM-1/OXA-48) in K. pneumoniae has been detected worldwide [8][9][10][11].ST11, ST14, ST101, ST147, and ST258 are major carbapenemase-producing K. pneumoniae clones.Without regard to carbapenemase types; K. pneumoniae clones were observed in diverse carbapenemase-producing K. pneumoniae [12].In this study, the aim was to investigate carbapenemase production among K. pneumoniae isolates collected from hospitalized cancer patients in Egypt and to study the genetic relationship of carbapenemase producing carbapenem resistant K. pneumoniae clinical isolates using Multi-Locus Sequence Typing.

Bacterial strains
Thirty-four consecutive non-duplicate carbapenemresistant K. pneumoniae isolates were obtained from different clinical specimens received at the microbiology lab at the National Cancer Institute (NCI), Cairo, Egypt, from June to August 2015.Ethical Committee of NCI, Cairo University, has approved the study, as the bacterial isolates examined were collected from routine clinical microbiology laboratory tests for patient care and no additional clinical specimens were collected for the purpose of research.VITEK 2 (bioMerieux, Marcy l'Etoile, France) automated machine was used for identification and susceptibility testing of the isolates and further identification was confirmed by 16S rRNA sequencing.The identified isolates were preserved in glycerol broth cultures at -70°C.

Data collection
The data recorded for each patient were age, sex, clinical diagnosis, ward of isolation, department (medical or surgical), hospitalization, prior carbapenem therapy, clinical site of infection, duration of episode and the outcome one month following infection.

Susceptibility testing
The VITEK 2 system and disc diffusion assays (Oxoid ltd., Basin Stoke, Hants, England) on Mueller Hinton agar were used to perform antibiotic susceptibility testing and results were explained as stated in Clinical and Laboratory Standards Institute (CLSI) recommendations [13].Colistin susceptibility was detected by using the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) as there is no breakpoint available for polymyxins for Enterobacteriaceae according to CLSI guidelines [14].The following antimicrobial agents were used: amikacin, ampicillin, ampicillin-sulbactam, cefazolin, cefepime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, colistin, gentamicin, levofloxacin, meropenem, piperacillin-tazobactam, sulfamethoxazole / trimethoprim, and tobramycin.

Phenotypic detection
The Carba NP test (RAPIDEC, bioMérieux, Marcy l'Etoile, France) were in accordance with the manufacturer's instructions to seek the occurence of carbapenemases.MBL production was tested using a combined disk assay [15].Ceftazidme/ceftazidme+clavulanate E test according to manufacturer's instructions was performed to test ESBL production.

Detection of carbapenemases and ESBL genes
An in-house multiplex PCR test using previously described primers was used to test the presence of carbapenemase genes (bla OXA-48 , bla NDM-1 , bla IMP , bla VIM , and bla KPC ) and ESBL CTX-M genes [16,17].All PCR experiments were done in presence of Negative and positive controls.Analysis of reaction mix containing PCR product (5 microliters) were analyzed by electrophoresis in 1 % (w/v) agarose (Fermentas, Waltham,US).The amplicons of bla OXA- 48 were purified using a QIAquick PCR Purification Kit (Qiagen, Crawley, UK) and sequenced in both directions using the ABI Prism 3700 DNA Sequencer (Applied Biosystems, Foster City, CA, USA).The sequence of the gene was compared with sequences in the GenBank nucleotide database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)[18].

Results
A total of (n = 34) non-duplicate carbapenemresistant K. pneumoniae isolates were obtained from different patient samples.Susceptibility testing results are presented in Table 1.Of the 34 carbapenem resistant K. pneumoniae isolates, 31 (91.2%)produced carbapenemase.Twenty-six (83.87%) out of 31 of carbapenemase-positive K. pneumoniae isolates were MBL producers.All carbapenemase positive isolates were ESBL positive.The class D bla OXA-48 gene was found in 25 (80.65%)carbapenemase producing K. pneumoniae isolates.While 21 (67.74%)isolates expressed MBL NDM-type.The MBL bla IMP-1 gene was observed in one K. pneumoniae isolate only.No KPC-or VIM-type βlactamases were detected.Carbapenemase association of OXA-48 like and NDM-type was identified in 15 (48.39%) isolates, while the coproduction of OXA-48 like and IMP-1like was revealed in one K. pneumoniae isolate (Table 1).CTX-M-1-like gene was present in 100% of carbapenemase producing K. pneumoniae isolates.
Table 2 and figure 1 show the results of MLST for OXA-48 producing K. pneumoniae isolates.MLST results presented ten distinct STs which were detected among 25 OXA-48-producing K. pneumoniae isolates.Fifteen out of 25 isolates were clustered in three major STs [(ST101 (n = 8), ST383 (n = 4) and ST147 (n = 3)].The predominant ST was ST101 which was detected in 8 (32%) isolates; 4 of them were retrieved from the same 4 th .floor at the same day and 2 days apart; the other 3 isolates were recovered from the same 3 rd .floor with 2 days apart, the remaining one from different 5 th .floor.The second ST was ST383, which were detected in four isolates followed by ST147, which was detected in three isolates.
Two isolates of each of ST11, ST16, and ST1399 were detected, while only one isolate of each of ST22, ST37, ST785and ST2193 were detected (Table 1 and  2).

Discussion
The increase in carbapenem-resistant K. pneumoniae prevalence may augment the healthcare and economic burden on the society.The current study was carried out to detect carbapenemase production among carbapenem resistant K. pneumoniae collected from Egyptian hospitalized patients with cancer and to examine the genetic relationship of carbapenemase producing carbapenem resistant K. pneumoniae isolates using MLST.The results of this work clearly demonstrates the occurrence of OXA-48 among carbapenemase resistant K. pneumoniae isolates from various origins recovered from patients with cancer having solid tumors and hematology malignancy.The dominant carbapenemase was bla OXA-48 , identified in 25 isolates (80.65%) followed by bla NDM-1 type, observed in 21 isolates (67.74%).In a different way, bla IMP was detected in single isolate only.Reports of OXA-48 carbapenemases have been published worldwide, including Middle East and North Africa countries, for example, Saudi Arabia, Sultanate of Oman, Qatar, United Arab Emirates, Turkey, Egypt, Tunisia, Morocco, Libya and Lebanon [6,7,10,11,[20][21][22].The results presented here provide the first insight into the coproduction of the OXA-48 like carbapenemase encoding gene and NDM-type representing MBL production in K. pneumoniae as well as the association of OXA-48 like and IMP-1 like among hospitalized cancer patients in Cairo, Egypt.Our results revealed that carbapenemase association with OXA-48 like and NDM-type was identified in 15 isolates, while the coproduction of OXA-48 and IMP-1 like was revealed in only one K. pneumoniae isolate.The coexistence of OXA-48 like with NDM-type producers in K. pneumoniae is alarming and demands further investigation regarding its possible endemicity in the region.
The coproduction of carbapenemases producing genes could be described as NCI is a tertiary hospital receiving patients from different regions of Egypt with the high possibility that patients receive treatment before presenting to NCI in other hospitals.Patients with Cancer are hospitalized for long periods, in addition to being immunocompromised thus are predisposed to easily acquiring highly resistant isolates from hospital environment.VIM and KPC gene expression were not identified in our study, indicating that during the period of the study carbapenemases didn't have any role in our hospital.
Our MLST results illustrated ST101 to be the most predominant ST among the carbapenem-nonsusceptible OXA-48-producing K. pneumoniae isolates investigated in the present study, which was detected in 8 isolates.The results of molecular epidemiologic study of OXA-48 in Europe and North Africa in an 11-year (2001-2011) showed that ST101 was the most commonly observed, followed by ST395 and ST15 in K. pneumoniae [23].The high widespread of these STs illustrates their epidemic potential.The second and third most common STs in our study were ST383 and ST147, which were detected in 4 and 3 isolates respectively.ST383 was previously reported for the first time in study in China in 2016 which documented the outbreak of OXAKp ST383 worldwide [24].In this study we document the occurrence of OXA-48producing K. pneumoniae isolates involving an ST101 and ST383 clones in NCI Hospital, Egypt.Clonal dissemination was identified in OXA-48-producing K. pneumoniae isolates in the present study where ST101 was detected in 8 isolates ; 4 of them were detected in the same floor (4th) at the same day and 2 days apart; the other 3 isolates were recovered from the same floor (3 rd ) with 2 days apart, the remaining one from different floor (5 th floor), also out of the 4 ST383 isolates; 3 of them were recovered from the same floor (4 th floor) and one recovered from the fifth floor; However, the dissemination of bla OXA-48 in our hospital is attributed to diverse sequence types of K. pneumoniae in which 10 distinct sequence types were identified.It is noteworthy to recognize the impact of clonal dissemination on the widespread of OXA-48-producing K. pneumoniae, in this study 9/25(36%) patients died (Table 2), most of patients were hospitalized and all of them having solid tumors or hematology malignancy.
Three isolates were assigned to ST147 OXA-48producing K. pneumoniae which is the first time to be detected worldwide; ST147 is a significant clone of VIM-producing K. pneumoniae isolates in Hungary [25], KPC-producing K. pneumoniae isolates in Greece and Canada [26,27] and NDM producing K. pneumoniae isolates in Canada, India, Sweden and the UK [27,28].In addition, also an association of ST147 with IMP-8-producing K. pneumoniae isolates in a Far East country was observed [29].ST11 was found in 2 isolates in this study; K. pneumoniae ST11 was identified in France for the first time in 1997 and it has subsequently been detected across the world involving in the USA, Sweden, Norway, Finland, the Netherlands, Hungry, Poland, the Czech Republic, Portugal, Spain, Italy, Brazil, China and South Korea [30].Only one isolate of each of ST22, ST37, ST785 and ST2193were detected in our study.

Conclusion
In Conclusion, OXA-48 and NDM-1 were the most common carbapenemases, and the most frequently detected sequence types were ST101 and ST383 for OXA-48-producing K. pneumoniae in Cancer patients in Egypt.ST383 was the second most common ST which has previously only been described in China.ST11 was assigned to OXA-48-producing K. pneumoniae for the first time worldwide.Association of NDM-type and OXA-48-like, and the coproduction of OXA-48-like and IMP in carbapenem-resistant K. pneumoniae were also identified in our hospital.A useful addition to classical investigation methods was proved in the study by implementing the molecular epidemiology.The main mode of spread in nosocomial outbreaks might be attributed to reservoirs in healthcare workers, patients, or the hospital environment.

Figure 1 .
Figure 1.Phylogenetic tree of 25 K. pneumoniae strains built based on the maximum likelihood algorithm in the MEGA6 software[30] and the concatenated alleles of 7 housekeeping genes with bootstrap values.The phylogenetic analysis identified ST101, ST383, ST147, ST1399, ST11, ST16, ST 2193, ST 785, ST 37 and ST 22.The numbers in the branches depict the sample ID of K. pneumoniae.