Genomic analysis revealing the resistance mechanisms of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from pig and humans in Malaysia
Golnaz Mobasseri1 & Kwai Lin Thong2 & Cindy Shuan Ju Teh3
Abstract
Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae has been associated with a wide range of infections in humans and animals. The objective of this study was to determine the genomic characteristics of two multiple drug resistant, ESBLs-producing K. pneumoniae strains isolated from a swine in 2013 (KP2013Z28) and a hospitalized patient in 2014 (KP2014C46) in Malaysia. Genomic analyses of the two K. pneumoniae strains indicated the presence of various antimicrobial resistance genes associated with resistance to β-lactams, aminoglycosides, colistin, fluoroquinolones, phenicols, tetracycline, sulfonamides, and trimethoprim, corresponding to the antimicrobial susceptibility profiles of the strains. KP2013Z28 (ST25) and KP2014C46 (ST929) harbored 5 and 2 genomic plasmids, respectively. The phylogenomics of these two Malaysian K. pneumoniae, with other 19strains aroundthe world was determinedbased onSNPs analysis. Overall,the strains were resolved into five clusters that comprised of strains with different resistance determinants. This study provided a better understanding of the resistance mechanisms and phylogenetic relatedness of the Malaysian strains with 19 strains isolated worldwide. This study also highlighted the needs tomonitor the usage ofantibiotics inhospitalsettings, animalhusbandry, and agricultural practices due to the increase of β-lactam, aminoglycosides, tetracycline, and colistin resistance among pathogenic bacteria for better infection control.
Keywords ESBL . Klebsiella pneumoniae . MDR . Whole genome sequencing . Resistancegenes
Introduction
Klebsiella pneumoniae (K. pneumoniae) is an important Gramnegative bacterium belonging to the Enterobacteriaceae family. It causes a wide range of infections in humans and animals, such as pneumonia, septicemia, urinary tract infections, and soft tissue infections (De Oliveira et al. 2008). This organism is associated with multidrug resistance, and its ability to produce extended-spectrum β-lactamases (ESBL) has been reported worldwide (Ali et al. 2018; Paterson and Bonomo 2005).
The first ESBLs-producing K. pneumoniae was reported in 1983 from a patient in Germany. Since then, the cases and outbreaks of infections caused by ESBL-producing organisms were reported worldwide (Ali et al. 2018; Keynan and Rubinstein 2007; Ben Hamouda et al. 2003; Romero et al. 2007). In Malaysia, Palasubramaniam et al. (2005) reported a nosocomial outbreak associated with ESBL SHV-5 K. pneumoniae. Other ESBL types have also been reported later between 2009 and 2019 (CTMX, SHV, TEM) (Lim et al. 2009; Al-Marzooq et al. 2015; Low et al. 2017; Mobasseri et al. 2019b). ESBL-producing K. pneumoniae are resistant to various antibiotics, including aminoglycosides, sulfamethoxazoles, fluoroquinolones, and trimethoprim. These antibiotics have been used in prophylaxis treatment in patients and growth promotion in food animals (HAIAP 2013).
The most frequent ESBL enzymes belong to the CTXM, TEM, and SHV families. CTX-M, the predominant type, is divided into five groups namely CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 according to their amino-acid identities (Ali et al. 2018). Recently, the increasing carbapenem resistance and colistin resistance among K. pneumoniae has drawn worldwide attention. Carbapenem resistance among K. pneumoniae is mainly caused by the production of carbapenemase such as KPC, IMP, NDM, OXA48-like enzymes. In Malaysia, NDM and OXA48 remain the most predominant carbapenemases that are responsible for carbapenemresistant K. pneumoniae (Low et al. 2017; Rodrigues et al. 2014; Yan et al. 2015).
The overuse of colistin has also resulted in the increase of colistin resistance, and this resistance is mainly caused by lipopolysaccharide (LPS) modification which is associated with two-component systems; phoPQ, pmrAB, and the regulator mgrB (Olaitan et al. 2014). In addition, colistin resistance is also believed to be caused by a plasmid-mediated mcr-1 resistance gene (Lui et al. 2016; Hasman et al. 2015; Hu et al. 2016; Falgenhauer et al. 2016; Ye et al. 2016; Mobasseri et al. 2019a).
With the increase of colistin-resistant K. pneumoniae in Malaysia and the advent of next-generation sequencing technology, it would be useful and important to study the whole genomes to have a deeper understanding of the resistance and virulence traits. Therefore, in this study, we aimed to determine the resistance mechanisms of two MDR ESBLs-producing K. pneumoniae isolated from a swine in 2013 and a hospitalized patient in 2014 in Malaysia.
Materials and methods
Bacterial strains
Two Malaysian K. pneumoniae strains were sequenced. One strain (KP2013Z28) was isolated from the rectal swab of a healthy swine from a farm in Peninsular Malaysia in 2013, and another strain (KP2014C46) was from the tissue sample (infected sacral sore) of a hospitalized patient in 2014. Both strains were characterized previously and were identified as ESBL-producer (Mobasseri et al. 2019b). The antibiotic susceptibility of both strains to different antimicrobial agents based on disc diffusion as well as the minimum inhibitory concentration to cefotaxime, ceftazidime, imipenem, meropenem, and colistin were summarized in Table 1.
DNA isolation, whole genome sequencing, assembly, and annotation
Bacterial DNAs were extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s instructions. The quality of extracted genomic DNAs was determined using the NanoDrop spectrophotometer (Eppendorf), and the agarose gel electrophoresis was carried out to check on the integrity of the DNA.
Whole genome sequencing was carried out for two ESBLsproducing K. pneumoniae strains using the Illumina Genome Analyzer (GA2X, pipeline version 1.6, insert size 300), generating > 10 total GB of data. A de novo assembly and annotation were carried out by using the CLC Genomic Workbench version 8.5 (CLC Bio, Aarhus, Denmark) and RAST (Rapid Annotation using Subsystem Technology), Prodigal, and Blast2GO, respectively.
Identification of antimicrobial resistance genes and plasmids
The sequences of known K. pneumoniae antibiotic resistance genes were downloaded from the Antibiotic Resistance Genes Database (ARDB; http://ardb.cbcb.umd.edu/index.html), which is a resource for antibiotic resistance genes of bacterial pathogens. These sequences were used as TBLASTN queries for the KP2013Z28 and KP2014C46 genomes. Identification of the resistance genes and plasmids was determined by using ResFinder (ResFinder threshold of ID = 98%) and PlasmidFinder web from the Center for Genomic Epidemiology (http://www.genomicepidemiology. org) and Resistance genes identifier (RGI) (https://card. mcmaster.ca/analyze/rgi). In silico multilocus sequence typing (MLST) Housekeeping genes for multilocus sequence typing (MLST) of K. pneumoniae were identified and submitted to the MLST database for sequence type (ST) assignment (http://bigsdb. web.pasteur.fr/klebsiella/klebsiella.html).
Phylogenetic analysis
Nineteen global genome sequences of MDR K. pneumoniae strains were included in the analysis [Genbank accession numbers :KPNIH32 (NZ_CP009775.1), 30684/NJST258-2 (NZ_CP006918), JM45 (NC_022082), CG43 (NC_0225566.1), ZYST1 (CP031613.1), KPN06 (CP012992), CAV1344 (NZ_CP011624), CAV1193 (CP013322), KP617 (CP012753), KP13 (NZ_CP003999), HKUOPLC (NZ_CP012300), KP-1 (CP012883), KPN01 (CP0120987), HK787 (NZ_CP006738), DMC1097 (NZ_CP011976), PMK1 (NZ_CP008929), XH209 *CFP cefoperazone, ATM aztreonam, AMK amikacin, SCF sulbactam cefoperazone, MEM meropenem, CFM cefixime, AMC amoxicillin-clavulanate, CIP ciprofloxacin, AMP ampicillin, TZP tazobactam, IMP imipenem, CAZ ceftazidime, CT colistin, CTX cefotaxime, GEN gentamycin TET and tetracycline (NZ_CP009461.1), KP52.145 (NZ_F0837906), and KCTC (CP002911)]. The downloaded draft genomes were submitted to the Reference Sequence Alignment based Phylogeny Builder (RealPhy) for phylogenetic analysis based on singlenucleotide polymorphisms (SNPs) by using the default parameters. The complete genome of HKUOPLC was used as the reference genome.
Accession numbers
Complete genome sequences K. pneumoniae KP2013Z28 and KP2014C46 have been deposited in the NCBI nucleotide database under accession numbers WJHY00000000 and WJHX00000000, respectively.
Results and discussion
WGS analysis and antimicrobial resistance determinants
Whole genome sequencing was performed on two K. pneumoniae strains. Both strains, KP2013Z28 and KP2014C46, with total reads of 2,222,814 and 1,537,485 were assembled into 145 and 167 contigs, respectively. The details of the assembled genomes were summarized in Table 2.
According to the ARDB, RGI, and ResFinder, the genes that were responsible for resistance in KP2013Z28 and KP2014C46 were identified. For KP2013Z28, which was resistant to nine different classes of antibiotics (β-lactam, aminoglycosides, colistin, fluoroquinolones, phenicols, tetracycline, sulphonamides, trimethoprim and bleomycin), 32 antibiotic resistance genes were identified. Strain KP2014C46 which was resistant to seven classes of antibiotics (β-lactam, aminoglycosides, fluoroquinolones, phenicols, tetracycline, sulphonamides, and trimethoprim) harbored 29 antibiotic resistance genes. The identifiable resistance genes are summarized in Table 3.
A total of six β-lactamase genes (bl2b-tem, bl2b-tem1, bl2c-pse1, bl2be-ctxm, bl2be-oxy1, and bl2b-ula) were identified in both KP2013Z28 and KP2014C46 which are resistant to cefotaxime, ceftazidime, and cefexime; and an additional gene, Bl2d-oxa1, was only found in KP2014C46. Based on the findings using RGI and ResFinder, blaSHV-11, blaTEM-176, and blaCTX-M-15 (accession numbers EF035557, GU550123, and DQ302097, respectively) that were responsible for resistance to cephalosporins, monobactams, and penicillins were identified in KP2013Z28. In KP2014C46, blaTEM-1, blaCTX-M-15, and blaOXA-1 (accession numbers JF910132, DQ302097, and J02967, respectively) were identified and has been associated with resistance to cloxacillin, monobactams, penicillins, and cephalosporins. blaTEM and blaSHV (blaTEM-1, blaTEM-176, blaSHV-1, and blaSHV-11) which were previously reported by Lim et al. (2009) were present in both strains. In addition, CTX-M-15, the most prevalent ESBL enzyme worldwide among
Enterobacteriaceae family has also been found in both strains in this study. These results indicate that most of the ESBLencoding genes are located on plasmids which are transmissible, suggesting that the spread of ESBL and other antibiotic resistance determinants is likely to be plasmid-mediated. This finding is in agreement with other reports (Lim et al. 2009; Li et al. 2003; Sompolinsky et al. 2005).
The coexistence of ESBLs and aminoglycosides in Enterobacteriaceae has been reported, and the aminoglycosides-encoding genes have been found on the same plasmids that harbor genes encoding ESBLs. The rate of amikacin resistance was high in strains from Canada, Europe, Latin America, the USA, and the Western Pacific region (Ma et al. 2009). However, most of the strains from Malaysia were still susceptible to amikacin as previously reported (Lim et al. 2009; Al-Marzooq et al. 2014). In contrast, high gentamycin resistance was reported among Malaysian K. pneumoniae strains (Lim et al. 2009; Al-Marzooq et al. 2014). In our present study, antimicrobial susceptibility profiles showed that these two strains were resistant to gentamycin and sensitive to amikacin.
Six aminoglycoside antibiotic resistance genes were identified in the studied K. pneumoniae strains, including aph6id, aph33ib, ant3ia, ant2ia, aac3ia, and aac6ib. Based on RGI and ResFinder, strB, aadA1, strA, aadA2 (accession numbers M96392, JQ414041, M96392, and JQ364967, respectively) were found in KP2013Z28 while strA, aac(6′)Ib-cr, and strB (accession numbers AF321551, DQ303918, and M96392, respectively) were identified in KP2014C46. These resistance genes have been associated with resistance to isepamicin, netilmicin, tobramycin, amikacin, sisomicin, dibekacin, spectinomycin, and streptomycin in these strains. aac(6′)Ib-cr which was found in KP2014C46 is categorized as fluoroquinolone and aminoglycoside resistance gene.
Aminoglycoside-modifying enzymes such as adenylyltransferases, acetyltransferases, and phosphotransferases were identified, suggesting their roles in the modification of amikacin (Ramirez and Tolmasky 2010). In this study, KP2013Z28 was sensitive to amikacin and resistant to gentamycin which had strA, strB, aadA1, and aadA2 aminoglycoside resistance enzymes. While KP2014C46 was sensitive to amikacin and resistant to gentamycin which had strA, strB, and aac(6′)Ib-cr as aminoglycoside resistance genes. However, any one of these enzymes alone cannot confer resistance to all aminoglycosides because of their narrower substrate specificities. Because gentamicin-modifying enzymes have poor activity against amikacin and because amikacin was developed from kanamycin to block the access of a variety of kanamycin-modifying enzymes to their target sites, a relatively low prevalence of amikacin resistance is usually observed among members of the family Enterobacteriaceae (Ma et al. 2009).
Four tetracycline genes including tetA, tetC, tetD, and tetE which are the major facilitator superfamily transporter of tetracycline efflux pumps weredetected. The efflux systems play an important role to extrude a broad spectrum of substrates, including various antimicrobial agents. Tetracycline is used in disease prophylaxis and therapeutics in food animals. Hence, high resistance (90–100%) among zoonotic strains have been reported from Portugal, Spain, and Malaysia, (Amador et al. 2019; Marchant et al. 2013; Mobasseri et al. 2019a). The tetracycline resistance trends of clinical K. pneumoniae in Malaysia have also been reported at 22% in 2009 by Lim et al. (2009) and 88.2% in 2017 by Low et al. (2017).
In Enterobacteriaceae family, the plasmid-mediated chloramphenicol acetyltransferase gene (cat), other non-enzymatic resistance plasmidic genes, floR and cmlA, encoding efflux pumps have been identified as the most common mechanism (Amador et al. 2019). In this study, three chloramphenicol resistance genes catb2, catb3, and catb4 were found in KP2014C46 (clinical) while four chloramphenicol resistance genes, cml-e3, cm-e1, cmlA1, and floR were found in KP2013Z28 (zoonotic). Chloramphenicol is no longer use in human and animal medicine due to serious side effects of this drug such as fatal aplastic anemia, irreversible, new-born infants, and gray baby syndrome in premature. In Malaysia and elsewhere, the trend of chloramphenicol resistance is this still increasing; this could be due to the determinant genes via plasmid and integrin without high selective drug pressure which have been reported previously (Amador et al. 2019; Lim et al. 2009; Marchant et al. 2013; Sood 2016; Nitzan et al. 2015).
Fluoroquinolone-resistant gene qnrs, which belongs to the pentapeptide repeat family and protects DNA gyrase from the inhibition of quinolones and oqxAB (oqxA and oqxB) as quinolone efflux pump were found in KP2013Z28 (zoonotic) while qnrb, aac(6′)Ib-cr, and qnrB66 were found in KP2014C46 clinical strain. Both the strains were resistant to ciprofloxacin. In Malaysia, the resistance rate of fluoroquinolone remains high compared to other strains from China, South Korea, the UK, and France (Al-Marzooq et al. 2014; Crémet et al. 2011; Shin et al. 2009; Yang et al. 2008; Dashti et al. 2006). Resistance to quinolone in Enterobacteriaceae is mainly due to mutations in the quinolone target enzymes (DNA gyrase and topoisomerase IV) encoded by gyrA and parC. Besides, plasmid-mediated quinolone resistance genes also confer low levels of quinolone resistance (Hopkins et al. 2005; Robicsek et al. 2006; Huang et al. 2015).
The K. pneumoniae zoonotic strain KP2013Z28 harbors mcr-1 (accession number KP347127) as an acquired colistin resistance gene according to RGI and ResFinder analysis. The plasmid-mediated colistin resistance gene, mcr-1 which was detected in KP2013Z28 (MIC > 16) was first reported by Liu et al. (2016) from animal and humans in China. Since then, mcr-1 has been detected around the world such as in Belgium (Xavier et al. 2016), Portugal (Kieffer et al. 2017), and Italy (Di Pilato et al. 2016). Recently, we have reported blaMCR-1 among K. pneumoniae from swine farms in Malaysia(Mobasseri et al. 2019a).
On the other hand, based on WGS analysis, the trimethoprim resistance in KP2013Z28 and KP2014C46 due to DHFR-coding genes (dfra12, dfra17, dfra14, and dfra5) have also been identified. These genes belong to antibiotic group A drug-insensitive DHFR that cannot be inhibited by trimethoprim (Brolund et al. 2010). Trimethoprim resistance mostly reported with other resistance genes which cause the possibility for co-selection of trimethoprim resistance and plasmids carrying both dfr-genes and other resistance genes using other antibiotic classes (Brolund et al. 2010). Three sulfonamide resistance genes sul1, sul2, and sul3 were found in KP2014Z28 while in KP2014C46, sul 2 was the only gene detected. These genes code for the synthesis of sulfonamideresistant dihydropteroate. Kumar et al. (2011) reported the presence of sulfonamide resistance gene sul1, encoding the sulfonamide resistance protein in a clinical K. pneumoniae strain in the USA. According to ARDB, KP2013Z28 has a blable a bleomycin resistance gene, while KP2014C46 did not harbor any bleomycin resistance gene. Previous studies reported that bleomycin resistance genes mostly flanking all blaNDM variants in clinical and swine K. pneumoniae strains (Ahmad et al. 2019; Chen et al. 2014).
Plasmid analysis
Previously, plasmid profiling of these two MDR-resistant K. pneumoniae strains was performed using S1-nucleasedigestedplasmid DNA, followedby separationby pulsed field gel electrophoresis (PFGE). This analysis revealed that KP2013Z28 harbored three plasmids, with the size of ~ 2500, ~ 6000, and ~ 10000 bp, while KP2014C46 had two plasmids, with~ 2500 and ~ 4000 bp(Mobasseri et al. 2019b). However, in this study, five plasmids have been identified in KP2013Z28, of which three carried a total of 29 antimicrobial resistance genes based on PlasmidFinder (Table 3). The plasmid which harbored mcr-1 was identical to IncI2 plasmid (accession number KP347127). The resistance genes such as blaCTX-M, blaTEM, sul 2, sul3, AaadA1, AaadA2, dhfrA12, dhfrA17, ant2ia, ant3ia, tet, strA, strB, floR, cmIAI, and qnrsI were identified on plasmid IncF IB and IncF II (accession numbers CP000966 and EU370913, respectively). The data obtained from both studies suggested that the two other plasmids that were found by PlasmidFinder might be plasmid replicons. The most common plasmid replicons in K. pneumoniae are the incompatibility (inc) group FIIK, incL/M, and incR. These plasmids have ability to stably coexist with other plasmids and are transferred together (Arabaghian et al. 2019). On the other hand, the β-lactamase genes, oqxA, oqxB, and blaSHV-11 were located on the chromosome.
Only two plasmids were identified in KP2014C46, which carried a total of 28 antimicrobial resistance genes (Table 3).
The resistance genes such as blaCTX-M-15, blaTEM-1, blaOXA-1, sul 2, dhfrA5, dhfrA14, dhfrA16, dhfrA17, ant2ia, ant3ia, aac (6’), tet, strA, strB, and qnrb were identified on plasmid IncF IB and IncF II (accession numbers JN233709 and CP000648, respectively). Only one gene, blaSHV-1 is located on the chromosome.
The IncF plasmid group is one of the most common plasmid types associated with ESBL-producing strains (mostly CTX-M-15) and the spread of other antimicrobial resistance genes responsible for β-lactam, aminoglycoside, and quinolone resistance around the world (Al-Marzooq et al. 2015; Villa et al. 2010; Dolejska et al. 2013). In this study, the IncFII and IncFIB plasmids harbored most of the antimicrobial resistance genes such as blaCTX, blaTEM, sul, AaadA, dhfrA, ant, tet, str, flo, cmI, and qnrs which cause resistance to several classes of antimicrobial agents including β-lactam, aminoglycoside sulphonamide, trimethoprim, tetracycline, chloramphenicol, and fluoroquinolone. Our findings concurred with the previous reports in Malaysia and elsewhere that IncF group could carry multiple resistance genes and remained the most common plasmid among Enterobacteriaceae strains from human and animal sources (Al-Marzooq et al. 2015). IncI was only detected in KP2013Z28, the zoonotic colistin-resistant strain. This concurred with previous studies that reported the presence of mcr1 gene on IncI2, IncHI2, and IncX4 worldwide (Li et al. 2016; Cui et al. 2017; Doumith et al. 2016; Zhang et al. 2017).
Multilocus sequence types (MLST) of K. pneumoniae
The sequence types of KP2013Z28 and KP2015C46 were ST25 and ST929, respectively. Worldwide, ST25 has been reported from human and animal sources. Two K. pneumoniae ST25 which harbored mcr-1 on their Inc I 2 plasmid have been reported in 2017 from patients in China (Zhang et al. 2017). In addition, Bowring et al. (2017) in Australia reported the outbreaks of septicaemia caused by strains of ST25 among pigs. Bidewell et al. (2018) also reported another outbreak of septicaemia due to MDR K. pneumoniae ST25 from pig farms between 2011 and 2014 in England (Bowring et al. 2017; Bidewell et al. 2018). K. pneumoniae ST929 previously reported by Yan et al. (2015) was detected as ESBL-producing strain in Taiwan which harbored blaCTX-X-15.
Phylogenetic analysis
The phylogenetic relationship based on SNPs of the two Malaysian genomes and 19 K. pneumoniae strains from other countries were studied. The 19 selected genomes were mostly isolated from human and pig sources and were resistant to multiple antibiotics. Five clusters were generated (Fig. 1). Cluster I comprisedoftwogroups;group1containedPMK1fromtheUK which exhibits close homology with KP617 from South Korea. The second group in cluster I consisted the Malaysian clinical strain (KP2014C46) which was identified as ST929 and two strains from Taiwan, HK787 and CG43, which were both identified as ST86 (clonal complex 86). Although these strains were subtypedasdifferentSTs,the 100%homology suggested acommon ancestor (Zhou et al. 2015; Bialek Davenet et al. 2014). In fact, the result obtained in this study is expected to be more accurate and reliable as the phylogenetic tree was generated based on the SNP of the whole genome while MLST was only focused on 7 housekeeping genes (Liu et al. 2020). In addition, cluster II, mainly ST25 comprised of strains from France (KP52.145), China (ZYST1), South Korea (KCTC 2242), and Malaysia (KP2013Z28). All the strains in this cluster were resistant to at least one antimicrobial drug in three or more antimicrobial categories and identified as MDR. Cluster III contained strains from China and Singapore with high similarity while cluster IV consists of two strains from Canada. Finally, cluster V comprised of two groups of strains from the USA, China, and
In conclusion, this study describes the genomic analysis of the draft genome sequence of two MDR K. pneumoniae strains from clinical and swine sources with different multilocus sequence type (ST25 and ST929). Several antimicrobial resistance genes which were associated with resistance to β-lactams (including blaSHV-11, blaCTX−M−15, blaTEM−176, blaTEM−1b, blaOXA-1), aminoglycosides, colistin, fluoroquinolones, phenicols, tetracycline, sulfonamides, and trimethoprim (such as acc(6′)-lb-cr, strA, strB, catB3, sul2, sul3, tetA, dfrA14, dfrA12, cmIA1, floR, aph(3’)Ia, aadA1, aadA2, mcr-1, oxqA, oqxB, qnrbS1, and qnrb66) have been identified on plasmids (IncFIB, IncFII, IncI2). The findings suggested the potential spread of these resistant determinants which poses a threat to public health and treatment failure (Lim et al. 2009; Al-Marzooq et al. 2015; Low et al. 2017; Mobasseri et al. 2019a). Therefore, the authority needs to monitor the usage of antibiotics in hospital settings, animal husbandry, and agricultural practices to control and prevent antimicrobial resistance issue.
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