Phenotypic and Genotypic Characterization of Pan-Drug Resistant Klebsiella pneumoniae Isolated in Qatar

1. Introduction

Carbapenem-resistant Enterobacterales (CREs) are resistant gram-negative bacteria of recognized public health priority with significant clinical and economic outcomes (1, 2). At its forefront, Klebsiella pneumonia is responsible for a wide spectrum of presentations including intraabdominal, urinary, and respiratory tracts infections frequently presenting as community as well as hospital-associated infections with significant morbidity and mortality (3, 4). The pathogen frequently affects vulnerable patients such as those with chronic comorbidities, immunocompromised or with critical diseases particularly following invasive surgery or procedures at critical care units (3, 5). Globally, the past decades witnessed alarming challenges of K.pneumoniae due to its recognized capabilities as a prime community and nosocomial infection, ability to accumulate extensive resistance phenotypes with special attention directed to its hypervirulent, spreading multidrug-resistant (MDR) and extensively drug-resistant (XDR) clones (6).

To evade antimicrobials, isolates of K. pneumoniae exhibit various complex resistance mechanisms which are acquired through random genetic mutations or horizontally through mobile genetic elements which can be subsequently amplified through upregulation of resistance genes or continuously disseminated (7). Studies conducted in this field, describe a large group of antimicrobial resistance genes (ARGs), that impede therapeutic function of broad spectrum antimicrobials such as carbapenems as well as novel βlactam-βlactamase Inhibitors (BLBLIs) which are considered as the ultimate management of serious infections secondary to Gram-negative bacteria (GNB) (8, 9).

Worryingly, recent reports highlighted the emergence of extensively and Pan-drug resistant (PDR) strains of K. pneumoniae from different countries limiting further treatment options (10-13). Due to regional variations, in the Gulf countries including Qatar, there is lack of data to correspond pathogens phenotypic with genotypic characteristics to obtain a reliable molecular epidemiology of AMR in the region. In the current study, we outline clinical, phenotypic, and genotypic characteristics of three PDR K. pneumoniae isolates aiming to describe the underlying resistance mechanisms using observations obtained from antimicrobial susceptibility tests (AST) as well as whole-genome sequencing (WGS).

2. Results

All three cases were hospital-acquired isolated from patients admitted to critical care units who had multiple risk factors for MDR infections, including recent systemic antimicrobial therapy (3/3), hospitalization or outpatient hospital attendance within the previous 90 days (2/3), invasive medical devices (2/3), and history of MDR infection or colonization within the past 90 days prior to hospitalization (2/3) as shown in patients’ demographics in Table 1. The most frequent underlying comorbidities were diabetes mellitus (2/3), and immune suppression following transplant (1/3). Meropenem, tigecycline, and colistin were used to treat the patient with KP1 isolate with new signs of infection, while the two colonized patients (isolates KP2 and KP3) did not receive specific therapy as evaluated as colonization.

The ASTs for the isolates are outlined at Table 2 demonstrating resistant to all conventional anti-GNB agents from different antimicrobial classes. Additional ASTs for novel antimicrobials revealed susceptibility to cefiderocol 3/3, while 2/3 isolates were susceptible to eravacycline, omadacycline and ceftazidime/avibactam, and none to plazomicin, ceftolozane/tazobactam, imipenem/relebactam and meropenem/vabrobactam.

The genomic size of the three PDR K. pneumoniae isolates KP1, KP2, KP3 were 5,617,763 base pair (bp), 5,617,835 bp, and 5,677,828 bp respectively. Two of the three PDR K. pneumoniae isolates belonged to sequence type ST231 while the last to ST383. Genomic data of the three PDR K. pneumoniae isolates revealed that they possessed 7 different β-lactamase genes from all classes; Class A ESBL (CTX-M-15 and TEM-1) in all isolates (3/3), while class A β-lactamase (CTX-M-14 and SHV-1), class B β-lactamase (NDM-5) and Class D β-lactamase (OXA-232) were present in (2/3) (Table 3). Different enzyme encoding genes highlighting antibiotics target alteration such as mutant gyrA, penicillin-binding protein (PBP3), and16S rRNA methyltransferase (rmtF) (G1405) and K. pneumoniae mutated outer membrane porin with reduced permeability (OmpK37) were detected in all isolates in addition to active efflux pump complexes such as emrB, baeR as depicted in Table 4 and Table 5.

In order to understand the underlying mechanisms that derive antimicrobial resistance (AMR), a detailed evaluation of extensive drug resistant (XDR) and pan-drug resistant (PDR) organisms is of paramount importance (16). The problem of AMR is more evident in Gram negative bacteria (GNB) because of accumulated and diverse resistance mechanism that peaks at carbapenem resistance (CR) pathogens (17). The subset of CREs represented by K. pneumoniae is one of the leading challenges implicated in healthcare-associated infections (HAIs) with significant negative clinical and economic outcomes (3). Resistance in K. pneumoniae is increasingly reported particularly in patients with chronic comorbidities, immune-suppressions, previous antimicrobial exposure and prolonged hospital or critical care length of stay (18). The described cases are from patients with all risk factors for AMR and HAIs, namely, prolonged hospital stay, prior antimicrobial therapy, invasive devices, and chronic comorbidities including immune suppression (19).

The outlined microbiological and genomic studies of the three cases of PDR K. pneumoniae demonstrate extensive resistance to conventional antimicrobials such as all third and fourth generations cephalosporins, monobactams, carbapenems, aminoglycosides, quinolones as well as polymyxins represented by colistin (Table 1 and Table 2). Upon genomic characterization, two of the three isolates belong to the notorious epidemic clones: K. pneumoniae ST231 which was first described in South-East Asia and has been associated with healthcare outbreaks as well as demonstrated diverse ARGs spanning major classes, particularly blaOXA-48 (20). In a previous surveillance study from Qatar of 149 CREs, 8.6% of isolates were of the same epidemic clone which explains its endemicity (21). Additionally, Class A extended-spectrum β-lactamases (ESBLs) were represented by blaCXM-14 and blaCXM-15, blaSHV-1_, and blaTEM-1. Globally, blaCXM-15 is the most widely distributed ARGs in GNB including the Gulf countries (22-24). Furthermore, the worrying carbapenemase blaNDM-5 of class B metallo-beta-lactamase (MBL), is present in KP1 isolate. This notorious ARGS is an offspring of blaNDM that was first described in 2012 which is carried horizontally through mobile genetic elements (MGEs) being capable of producing carbapenamses that can destroy all basic and advanced β-lactamases including cephalosporins and carbapenems but remain susceptible to monobactams, one of the only few therapeutic agents that are capable to overcome MBLs albeit critically vulnerable to other ESBLs. The notorious blaNDM-1 ARG is endemic in the region being one of the most prevalent genes amongst CREs (25, 26).Nevertheless, blaNDM-5 has been reported mainly in CRE E. coli from Greece, Japan, China, and Latin America usually associated with highly virulent strains (27-29).. The ARGs were also been isolated from companion animals which emphasize the concept of one health approach integrating the impact of interactions between humans, animal, and the environment (30). The introduction of novel antimicrobials βlactam βblactamase inhibitors (BLBLIs) agents in the region such as ceftazidime-avibactam and ceftlozane-tazobactam that has activity against OXA-type CREs but remains vulnerable to MBLs such as NDM might be responsible for the shifting epidemiology that need further supporting surveillance over coming years.

Characteristically, the first (KP1) showed utmost resistance to novel combinations including β-lactam/β-lactamase inhibitors (BLBLIs) such as ceftazidime-avibactam, ceftolozane-tazobactam as well as meropenem-vaborbactam while the other two were susceptible to ceftazidime-avibactam but resistant to ceftolozane-tazobactam. Avibactam is a potent BLBLI that inhibits class A, C, and D including OXA-types but is incapable of inactivating enzymes encoded by class B β-lactamases such blaNDM and blaVIM as in KP1, while ceftolozane is an advanced cephalosporin that overcomes many enzymes encoded by ARGs but remains susceptible to broad spectrum ones including OXA-types ARGs (31). The fact that the two other isolates KP2 and KP3 lacks blaNDM but harbour blaOXA 232 demonstrating broad BLBLIs discordance (KP2 and KP3 were sensitive to ceftazidime-avibactam but both resistant to ceftolozane-tazobactam), postulates that the ARG blaOXA 232 can be suppressed by avibactam but not tazobactam as previously observed (32). Similarly both Meropenem/vaborbactam and Imipenem/relebactam were inferior supporting the observation that the novel agent does not cover blaNDM and blaOXA type isolates as previously described (33).

The recommended management of CRE harbouring class B MBLs such as blaNDM is usually complex involving combination therapy with agents that are capable to overcome the genotypic resistance such as aztreonam-avibactam or cefiderocol with or without the addition of additional antimicrobials such as polymyxins, tigecycline, or eravacycline (34). It is also intriguing to notice that all PDR isolates were susceptible to cefiderocol one of the novel antimicrobials that demonstrated potent in vitro activity against XDR and PDR Gram-negative strains spanning all β-lactamase classes but needed supporting data towards clinical efficacy (35, 36). Cefidercol is a novel synthetic siderophore antimicrobial that hijack bacterial iron transporting mechanisms to traverse microbial cell wall and eventually lead to cell lysis through interference with cell wall synthesis. Its distinct ability to resist cell wall based βlactamases including class A, B, C and D in addition to its unique ability to overcome bacterial efflux pumps and porin channels making it a promising a trojan horse capable of overcoming different mechanism of bacterial AMR. The antimicrobial is of significant potential interest since it has remarkable in vitro antimicrobial activity particularly for notorious organisms such as MDR-pseudomonas aeruginosa and carbapenem resistant Acinetobacter baumannii as well as Stenotrophomonas maltophila. Nevertheless, translation of the ASTs needs supporting clinical data since in some observational studies, cefidercol was linked to increased mortality such as the one observed with carbapenem resistant Acinetobacter baumannii Similarly, both eravacycline and omadacycline showed activity against two of the three PDR strains (KP2 and KP3 at Table 2). These advanced and novel derivatives agents of the tetracycline group are of focused interest particularly for the coverage of resistant strains from intrabdominal infections albeit are limited for invasive diseases such as blood stream infections based on its pharmaco-kinetic and pharmacodynamic characteristics (36-38).

While polymyxins resistance is extremely rare in CRE (less than 5% in most regions), intriguingly the three isolates are colistin resistance (9). Polymyxins are not natural targets for ARGs since they act primarily and independently at cell wall levels of GNB destroying structural lipopolysaccharides. Reported resistance is mainly driven through plasmid-mediated mobile colistin resistance (mcr resistant gene) first described in China in 2015 in E. coli then described in most Enterobacterales (39). The fact that no mcr-related genes had been identified in the isolates, points toward other outer membrane resistance mechanisms. Previous studies of colistin-resistant K. pneumoniae lacking the mcr resistance gene explored the role of mutations in the two-component membrane system of the PhoPQ and PmrAB (40). Additionally, all isolates harbored Rifampin ADP-ribosyl transferase (Arr) which confers rifamycin resistance when used as an adjuvant therapy albeit not tested in the study. Furthermore, all the isolates harbored OXA-type carbapenemases such as >blaOXA-48 and it is closely related to resistant gene blaOXA-232 which has five point mutations from bla_OXA48 and both are similarly capable of inactivating carbapenems (41). It is worth pointing that OXA-type together with class B NDM carbapenemases are the most frequent carbapenemases in the Middle East and Gulf regions (21, 23).

Distinctively genomic characterization revealed mutations at the outer membrane porin permeability channels with the expressions of K. pneumoniae associated OMK35 that leads to decrease permeability to several hydrophilic antimicrobials such as β-lactams including carbapenems, fluoroquinolone, tetracycline, and its derivatives glycylcycline represented by tigecycline as well as rifamycin as well as being associated with increased virulence. Previous studies demonstrated restoration of the porin outer cellular mutations leads to restoring of lost antimicrobial activities (42).

In addition to the common ARGs, genotypic characterization revealed occurrence of other mechanism such as aminoglycosides modifying enzymes (AMEs), macrolide phosphotransferase (MPT), and fosfomycin thiol transferases resistance genes that might be linked to reported phenotypic patterns.For PDR- GNB, the co-occurrence and transmission of multiple resistance mechanisms is a well-recognized phenomenon that frequently leads to propagation of AMR (43).

Despite the study tries to elucidate underlying mechanism of resistance focusing on Klebsiella pneumoniae, it has some limitations which should be taken into considerations for the validity of scientific evaluation. It must be highlighted that two of the Klebsiella pneumoniae isolates were evaluated as colonizer rather than a true infection which limits its clinical evaluation particularly options for therapeutic interventions. Additionally, the genomic study focused on ARGs rather than combing that with virulence factor which was plausible not explored because of the small collection and paucity of invasive disease. Nevertheless, the relationship between bacterial AMR and virulence proved to be elusive even for comprehensive studies. Despite these limitations, the study continues to expand our knowledge for the fascinating study of mechanism of resistance particularly for GNB that should pave the way to overcome its challenges.

3. Material and methods

4.1. Identification and susceptibility testing

Phenotypic characterizations were performed using BD Phoenix^TM automated system while bacterial identification and confirmation was performed using Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) of Bruker Daltonics MALDI Biotyper (Billerica, MA, USA) according to the manufacturer’s recommendations. Additional ASTs for fosfomycin, cefiderocol, plazomicin, omadacycline, eravacycline, doxycycline, meropenem/vaborbactam, ceftazidime/avibactam, imipenem/relebactam, and ceftolozane/tazobactam were performed using MIC Test Strips (Liofilchem®, Diagnostics, Italy), while broth microdilution was used for colistin susceptibility testing (ComASP Colistin, Liofilchem, Roseto degli Abruzzi, Italy). Escherichia coli ATCC 25922, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 were used as controls. Susceptibility reporting was based recommendations of the CLSI at the time (14). Since, there were no intermediate susceptibility category available for ceftazidime/avibactam against Enterobacteriaceae, isolates were therefore described as susceptible if the MIC was ≤ 8 mg/L and as non-susceptible if the MIC was >8 mg/L (14). PDR K. pneumoniae isolates were defined as having in vitro non-susceptibility to all routinely and conventionally tested anti- Gram-negative antimicrobial agents excluding additional advanced susceptibility tests (15). WGS was performed by Eurofins GATC Biotech GmbH, Konstanz, Germany using the Illumina HiSeq 2000 system (Illumina, San Diego, CA, USA).

4.2. Ethical approval

The study was approved by the Medical Research Centre (MRC) and Research and Ethics Committee (Protocol: MRC-04-22-522) at Hamad Medical Corporation, Doha, Qatar which abides by the local and international standards of ethics in medical research including, patients consent, data anonymity and management.

5. Conclusion:

The clinical, phenotypic, and genotypic characterization of three PDR K. pneumoniae revealed multiple risk factors for acquisition of healthcare associated MDR pathogens resulting in extensive resistance profiles while examining genomic studies revealed multiple underlying ARGs associated with phenotypic patterns. For PDR- K. pnuemoniae, novel antimicrobial agents such as cefiderocol, omadacycline and eravacycline are potential therapeutic agents that need further clinical evaluation.