OXA-181-Like Carbapenemases in Klebsiella pneumoniae ST14, ST15, ST23, ST48, and ST231 from Septicemic Neonates: Coexistence with NDM-5, Resistome, Transmissibility, and Genome Diversity

Neonatal sepsis is a leading cause of neonatal mortality in low- and middle-income countries (LMICs). Treatment of sepsis in this vulnerable population is dependent on antimicrobials, and resistance to these life-saving antimicrobials is worrisome.

N eonatal sepsis is one of the primary causes of neonatal deaths (23%) in middleand low-middle-income countries (1). Multidrug-resistant bacteria complicate the treatment of sepsis in this vulnerable population (2). Klebsiella pneumoniae, belonging to the Enterobacteriaceae family, is one such species that has high rate of acquisition of resistance compared to other bacteria of this family (3). In addition, K. pneumoniae is also the leading cause of neonatal sepsis in developing countries (4). With escalating resistance to all available b-lactam antibiotics for neonates (penicillins, monobactam, cephalosporins, etc.), use of carbapenems has gradually increased, ultimately leading to a global upsurge of carbapenem-resistant K. pneumoniae (CR-Kp) in the last 2 decades (1,3). According to the Centre for Disease Dynamics, Economics & Policy (CDDEP), there has been an increase in CR-Kp from 24% (2008) to 59% (2017) in India (1), a country that bears the burden of one-fourth of all neonatal deaths that occur globally each year (5).
Though NDM has gained prominence, oxacillinase (OXA)-48-like carbapenemases (OXA-48), first reported from Turkey in K. pneumoniae (2001) (6), has now spread to different genera of Enterobacteriaceae. Outbreaks and case reports throughout Europe, North Africa, the Middle East, and South Asian countries are increasingly documented (11)(12)(13). Reports of emergence or outbreak in neonatal units from Middle Eastern countries have also surfaced (14). Detection of OXA-48-producing microorganisms is not limited to clinical settings and is often detected in environmental surface samples, companion animals, livestock, production animals, and wild animals (11,15,16).
To date, 39 variants of OXA-48 have been reported (https://www.ncbi.nlm.nih.gov/ pathogens/isolates#/refgene/oxa-48). Currently, OXA-181 and OXA-232 constitutes the 2nd and 3rd most common global OXA-48-like derivatives after OXA-48 (14). OXA-181 was first reported from India (17) and differed from OXA-48 by four amino acid substitutions (T104A, N110D, E168Q, S171A) but did not evolve from it. On the other hand, OXA-232 first reported from France is a derivative of OXA-181 with a single amino acid substitution at R214S (14). OXA-48-like enzyme hydrolyzes penicillins and narrow-spectrum cephalosporins efficiently but does not hydrolyze extended-spectrum cephalosporins and exhibits poor activity toward meropenem while also showing the highest known catalytic efficiency for imipenem (6). Therefore, OXA-48 producers often remain undetected during surveillance because they are categorized as susceptible to carbapenems according to CLSI and EUCAST (6,14). Like other carbapenemases, OXA-48-like carbapenemases are not inhibited by conventional b-lactamase inhibitors, but nowadays, use of avibactam (a non-b-lactam b-lactamase inhibitor) has been put forward. However, increasing reports of resistance toward avibactam have been documented (18). Hence, specific phenotypic detection of class D carbapenemases is still confusing. High-level resistance to temocillin (MIC, .64 mg/liter) has been suggested as a criterion to screen OXA-48-like carbapenemase; however, due to a similar resistance profile toward KPC and other metallo-b-lactamases (19), this is not suitable. This further emphasizes the difficulty in the identification of OXA-48, which inevitably leads to poor tracking of emergence and spread, and infection control measures. Carriage of such resistance markers on plasmids is often associated with international clones such as sequence type 11 (ST11), ST14, ST15, ST63, ST147, ST231, etc., which aided in their rapid dissemination across boundaries (10,14,20).
Studies focusing on the epidemiology and genomic characterization of isolates harboring OXA-48-like genes particularly in neonatal septicemic cases are rare, with few reports of outbreaks or sporadic infections (13,14). This study, however, monitors the presence of these genes in a neonatal unit over a period of 4 years (2013 to 2016) and evaluates the isolates in terms of their STs, production of multiple carbapenemases, their transmissibility, and associated mobile genetic elements. We performed core genome analysis incorporating isolates in this study in a global context with other OXA-48-like carbapenemase-harboring genomes, including those from other neonatal studies, to explore the genomic epidemiology and variability of carbapenemase lineages, focusing on the context of neonatal sepsis.
Detailed characterization of OXA-48-like carbapenemase-producing strains. All the OXA-48-like producers were Klebsiella pneumoniae (Kp1 to Kp11). Some of the neonates from whom the K. pneumoniae was isolated did not survive, and most were "outborns" referred from some other hospitals (data not shown).
Two types of OXA-48-like carbapenemases namely, bla OXA-181 and bla OXA-232 , were found among the study strains, henceforth called bla OXA-181-like . bla NDM-5 was the only class B carbapenemase detected and was found in four of the bla OXA-181-like positive strains. All 11 bla OXA-181-like strains harbor bla CTX-M-15 along with different b-lactamases and aminoglycoside resistance and quinolone resistance genes in various combinations ( Table 2).
Strains were found to possess virulence genes ( Table 2) such as iut, ent, fep, fes, ybt, irp, iro, etc. (iron-chelators). The occurrence of serum resistance and antiphagocytosis capsular factors along with different K-and O-loci were found in the strains. Strains also possessed various integrative conjugative elements. The presence of rmpA, rmpA2, and magA responsible for hypermucoidy and hypervirulence was found in Kp6, which has already been reported in a separate study (16).
Most of the TCs/TFs with only bla OXA-181-like showed the presence of similar plasmid scaffolds, i.e., ColKP3, except for one (Kp5) with IncFIIK (Table 1). WGS data also specified the association of ColKP3 with the bla OXA-181-like (Table 2). On the other hand, bla NDM-5 was present on IncFII ( Table 1).
The MIC of the TCs/TFs for different antimicrobials were assessed (Table 1). TCs/TFs with only bla OXA-181-like exhibited high MICs for imipenem followed by ertapenem compared to meropenem. However, TCs where coexistence of bla NDM-5 and bla OXA-181-like were observed showed higher MIC for meropenem.
Analysis of mobile genetic elements (MGEs). The genetic environment of bla OXA-181-like revealed the presence of a mobilization relaxosome (mobA, mobB, mobC, and mobD) upstream, and DlysR (transcription regulator), DereA (erythromycin esterase), and Col replicase (repA) downstream, respectively ( Fig. 2a and b). Deletion of ISEcp1 was found with varying stretches of its right-end extremity except for Kp1 and Kp5 (Table 1). All study strains were found in truncated Tn2013.
On the other hand, bla NDM-5 was bracketed between truncated ISAba125 and bleomycin resistance genes (ble MBL ) found upstream and downstream, respectively. ISAba125 is preceded by a truncated transposase of the IS30 family and truncated IS26, while ble MBL is succeeded by N-(59-phosphoribosyl) anthranilate isomerase (trpF), twinarginine translocation pathway signal sequence protein (tat), and the truncated transposase of IS91 (Fig. 2c). Kp3 and Kp9-Kp11 have similar genetic environments with truncated Tn125.
A phylogenetic global comparison of OXA-48-like genomes and K. pneumoniae isolated from neonates. The maximum likelihood core genome phylogenetic tree was constructed with 197 K. pneumoniae from (i) a global collection of OXA-48-like and NDM carbapenemase-carrying isolates and (ii) published genomic data of septicemic neonatal K. pneumoniae (Fig. 4). As few neonatal studies with published sequence data (either GenBank NCBI or ENA-EMBL) were available, all possible sequences were incorporated, irrespective of carbapenem resistance.
bla OXA-48-like K. pneumoniae detected from 21 countries and 20 sample sources, including human, animal, and environmental samples, were remarkably diverse, with 40 different STs identified.
The diversity at the core genome level of the strains within this study was vast, spanning multiple lineages, showing both diversity among themselves as causative agents of neonatal sepsis and varying levels of relatedness compared to strains from different parts of the world. EN5153 (Kp1) showed similarities with strains from Tanzania and Ghana; EN5218 (Kp5), with strains from China, Spain, and Norway; EN5275 (Kp6), with distantly related strains from Romania; EN5338 (Kp8), with strains from Thailand, Pakistan, the United States, and Switzerland; and EN5199 (Kp3) and EN5339 (Kp9), with strains from the United Kingdom, the United States, South Korea, Pakistan, Thailand, and Tanzania. Also, EN5199, EN5338, and EN5339 showed  Isolates are colored at the endpoint according to country, and the outer ring abbreviation is labeled according to the sample source. The additional two outer rings denote the presence of bla NDM and bla OXA-48-like antibiotic resistance genes. Clades containing isolates from this study are highlighted in teal, and light blue clade highlights indicate K. pneumoniae neonatal sepsis isolates from other studies. The year of sample collection for isolates in this study has been added external to the tree phylogeny. similarities with strains reported from various parts of India. When genomes of bacteria causing neonatal infections are compared, EN5153, EN5199, and EN5339 showed similarities with neonatal strains from Tanzania and Ghana. Interestingly, core genome single nucleotide polymorphism (SNP) phylogeny of EN5153 suggests that all ST48 neonatal isolates sit within the same cluster, and the additional ST48 with the greatest similarity from the NCBI database (an isolate from a rectal swab in London from 2018) sits on a single branch (Fig. 5).
Six variants of bla OXA-48-like were identified in the collective core genome phylogeny, of which only bla OXA-181 or bla OXA-232 were detected from neonatal K. pneumoniae in both Ghana and this study. Apart from these, none of the neonatal strains harbor carbapenem-resistant genes.

DISCUSSION
In this study, we characterized bla OXA-181-like -producing K. pneumoniae in a neonatal setting over 4 years, showing the diversity of the genomes. We identified 11 bla OXA-181/232 carbapenemases-producing K. pneumoniae. bla NDM-5 was found in some of the strains. OXA-48-like carbapenemases have been found to be the most common carbapenemases among Enterobacteriaceae family pathogens in certain parts of the world, such as Europe, the Middle East, North America, etc., while NDM carbapenemases are endemic to India and Southeast Asia (10,14,20). The presence of bla OXA-181/OXA-232 along with bla NDM-5 has been reported in patients from South Korea, the United States, Chad, and Nepal, having travel history from India or the Indian subcontinent (8,(21)(22)(23). The existence of dual carbapenemases (bla OXA-181/232 and bla NDM-5 ) among the strains reduced their susceptibility to all carbapenems (imipenem, ertapenem, and meropenem), thereby making them extremely drug resistant. Infection with these organisms is dreadful, especially in neonates with limited therapeutic options. Following an extensive PubMed search for reports of bla OXA-181/OXA-232 along with bla NDM-5 in neonates, we found no matches; however, bla OXA-232 has been reported in neonatal infections from China (20). Hence, to the best of our knowledge, this is the first study to report the coexistence of bla OXA-181/OXA-232 with bla NDM-5 in septicemic neonates.
A plethora of resistance and virulence genes were identified among the strains, which supports the survival of the pathogen in antibiotic-laden environments of health care settings as well as their successful colonization in the host. The occurrence of resistance genes on plasmids and virulence genes on integrative conjugative elements instigates the spread of these genes in the community. Hence, the presence of drug-resistant virulent strains of K. pneumoniae in neonates can cause severe infection leading to critical consequences.
In the current study, two specific plasmid scaffolds were seen to be associated with the studied carbapenemases genes. bla OXA-181-like were found on a nonconjugative ColKP3 plasmid on a truncated Tn2013, as reported previously (14,30,31). bla OXA-232 has always been reported in Tn2013, but bla OXA-181 has been found in Tn2013 and in other transposons, such as Tn6360 (14). Deletion of ISEcp1 from the upstream of bla OXA-181/232 was noted among the strains, which must have restricted its transposase activity, resulting in stabilization of bla OXA-181/OXA-232 on pKP3/pOXA232-like plasmids (30,31). bla NDM-5 was found in a conjugative IncFII plasmid within truncated Tn125 with a comparable plasmid background reported from a nontraveler in Spain (32), although the association of bla NDM-5 is predominantly reported in IncX3, but they have also been found in IncFII (32). This study also indicated the presence of bla OXA-181/OXA-232 and bla NDM-5 on separate plasmids, suggesting two independent events of gene acquisition by the organism. The majority of previous reports have proposed that the spread of bla OXA-181-like is through clonal dissemination, but this study corroborated the results from few earlier reports (14,30,31), describing the involvement of a helper plasmid (bla NDM-5 ) that facilitated conjugal transfer of bla OXA-181-like , reinforcing the role of helper plasmids in their transmission. Such a phenomenon underlines the threat these carbapenemases pose when present with bla NDM , not only in terms of increased resistance and further treatment limitations, but also in the ease of transfer.
WGS analysis of neonatal strains is largely limited to outbreak cases, and studies of isolates collected over longer periods are rare. This study is probably the first to incorporate a global collection of K. pneumoniae harboring OXA-48-like and NDM carbapenemases with special reference to septicemic neonatal strains. Strains of this study belonged to diverse sequence types, which ruled out clonal spread of bla OXA-181-like -carbapenemases and were similar to outbreak strains from neonates in Tanzania, Ghana, and Austria (33)(34)(35). Genomes were diverse, but the plasmid scaffold (ColKP3) harboring bla OXA-181-like was similar across the study strains as also reported by other studies (14,30,31). Diversity among the isolates studied here could be, in part, due to many neonatal referrals from other hospitals within this study, and therefore neonates were exposed to both different health care and environmental factors.
Although there are limitations of short read sequencing with respect to plasmid assembly, holistic understanding of the genomes and their spread across the globe and in specific populations or patients is possible. The presence of carbapenem-resistant K. pneumoniae in low-middle-income countries (LMIC) such as India, where neonatal deaths amount to nearly 0.75 million per year (5), is a serious concern which requires rapid investigation. With increasing WGS facilities and decreasing cost of sequencing, short read sequencing is an extremely useful tool to aid routine antimicrobial resistance (AMR) surveillance. This study thus gives an insight about such strains not only in a particular setting but also in a wider global context.

MATERIALS AND METHODS
Ethical approval. The study protocol was approved by the Institutional Ethics Committee of the ICMR-National Institute of Cholera and Enteric Diseases (no. A-1-2/2018/IEC). Patient information was anonymized and deidentified prior to analysis.