Emergence of blaNDM-5 and blaOXA-232 Positive Colistin- and Carbapenem-Resistant Klebsiella pneumoniae in a Bulgarian Hospital

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. blaNDM-5 was the only NDM variant, and it was always combined with blaOXA-232. The coexistence of blaOXA-232 and blaNDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All blaNDM-5- and blaOXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed blaOXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for blaNDM-5- and blaOXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for blaOXA-232- or blaKPC-2-positive isolates. All blaOXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation.


Introduction
Klebsiella pneumoniae is an important nosocomial pathogen.It can cause a wide range of nosocomial infections such as the bloodstream, surgical wounds, urinary infections, pneumonia, and catheter-associated infections [1].It also causes necrotizing pneumonia, pyogenic liver abscesses, and endogenous endophthalmitis [1].In the last years, the resistance of isolates from this species has increased mostly due to the rising frequency of extended-spectrum beta-lactamase production and carbapenemase carriers.The most widespread carbapenemases are class A Klebsiella-producing carbapenemase (KPC), class D oxacillinases (OXA)-OXA-48, OXA-181, and their variants-as well as the class B metallobeta-lactamases (MBLs) (New Delhi Metalo beta-lactamase, NDM; imipenemase, IMP; and Verona Integron Metallo-carbapenemase, VIM) [2].NDM-, KPC-and OXA-48-producing enterobacteria, especially Klebsiella pneumoniae, are widely distributed all over the world, involving many international high-risk clones as ST258, ST11, ST15, ST395, and causing serious therapeutic problems [2][3][4].KPC enzymes are endemic in the USA and some European countries such as Greece and Italy [5].OXA-48 and its variants were widely detected in Turkey and some European countries, such as Spain and France; particularly, OXA-232 is a new, emerging, and quite successful variant in Asia and Turkey [2][3][4][5][6].NDMproducing enterobacteria appeared for the first time in India and was then distributed all over the world [2,5].The carbapenemase producers are widely resistant to almost all beta-lactams and many other antibiotics such as colistin (polymixin E), aminoglycosides, quinolones, and tetracyclines [3].Some isolates are extremely resistant or pan-resistant, which is a serious problem for the therapy [3].
The rapid spread of carbapenemase-producing strains has led to renewal of polymyxin use in medicine.The increased and inappropriate colistin (polymyxin E) use was followed, however, by a faster appearance of lipopolysaccharide (LPS) modifications: an addition of 4-amino-4-deoxi-L-arabinose or phosphoethanolamine.These modifications occur due to mutations in pmrCAB and arnBCADTEF-pmrE operons and are associated with an increase in the positive charge in LPSs and, therefore, a decrease in colistin binding [7].They are under the control of the two-component systems, PhoP/PhoQ and PmrA/PmrB.The PhoP/PhoQ system is subjected to negative-feedback regulation by MgrB protein [7,8].In recent years, plasmid-mediated colistin resistance encoded by mcr genes has also been reported [9].The mcr genes encoding phosphoethanolamine transferase add phosphoethanolamine and diminish the negative charge of LPS, thus decreasing the affinity of LPS to colistin.
In addition to the beta-lactams and colistin, another important antibiotic group is that of aminoglycosides.They could be used in combination with beta-lactams or other antibiotics, especially for treatment of infections caused by carbapenemase-producing enterobacteria [10].The main mechanism of resistance to them is the production of aminoglycosidemodifying enzymes (phosphotransferases, adenylyltransferases, nucleotidyltransferases, and acetyltransferases).Each of those enzymes may confer resistance to some aminoglycosides but not to all of them.In the last few decades, 16S rRNA methyltransferase enzymes (16S rRNTases) have increasingly been detected.They methylate 16S rRNA subunit; thus, the aminoglycosides cannot bind to the ribosome, which confer high-level resistance to all clinically relevant aminoglycosides.Many variants of RTNases have been detected (armA, rmtA-H, and NpmA) [11].They are plasmid-encoded, have the possibility to be easily transferred and have often been found in carbapenemase-producing isolates, which, taking into account other mechanisms, can lead to extremely resistant or pandrug-resistant Gram-negative isolates [10][11][12].
This study aimed to collect and genetically characterize colistin-and carbapenemextremely resistant isolates in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria.

Bacterial Isolates and Patients' Susceptibility Testing
Colistin-and carbapenem-resistant Enterobacterales isolates were collected in the Military Medical Academy, Sofia, Bulgaria, from September 2023 to January 2024.Such isolates started to appear in August 2023 [20], and the last isolate was detected at the end of January 2024.Data about the patients such as the month of isolation, sample, ward, prior treatment, and diagnosis are presented in Table 1.Only one isolate per patient was included in this study when the isolates were identical.Routine methods were used to carry out the culti-vation.The samples were obtained during routine therapeutic and diagnostic work.The project was approved by the Medical Science Council of Medical University, Sofia.
The disk diffusion method (30 µg disk, Liofilchem, Roseto degli Abruzii, Italy) was used for ceftazidime/avibactam susceptibility testing.Minimal inhibitory concentrations (MICs) of eravacycline and plazomicin were detected by E-tests (Liofilchem, Roseto degli Abruzii, Italy), and cefiderocol and colistin susceptibility was determined by a broth microdilution method (ComASP Cefideracol with iron-depleted broth, Liofilchem, Italy and ComASP Colistin, Liofilchem, Italy, respectively).The analysis of the results was based on the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), 2024 [21] and the Food and Drug Administration (FDA) for plazomicin and eravacycline [22,23].FDA breakpoints of ≤0.5 mg/L for susceptibility and ≥1 mg/L for non-susceptibility were considered for eravacycline [22].For plazomicin, the manufacturers/FDA breakpoints were ≤2 mg/L for susceptibility, 4 mg/L for intermediate susceptibility, and ≥8 mg/L for resistance [23].Multidrug resistance (MDR) was defined according to their non-susceptibility to at least one agent in ≥3 antibiotic classes.Extensive drug resistance (XDR) was found when isolates were non-susceptible to one or more agents in all but two or fewer antimicrobial categories [24].

Molecular Typing
Whole-cell DNA was prepared by a DNA Swab Isolation Spin Kit (Illustra Bacteria Genomic DNA Prep Kit (GE Healthcare, Little Chalfont, UK).ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR was performed with ERIC 1 and ERIC 2A primers as previously described [14].ERIC fingerprints were used to observed genetic similarity using the simple clustering method, UPGMA (unweighted-pair group method with arithmetic mean), and the Dice coefficient as a similarity measure (http://genomes.urv.cat/UPGMA/accessed on 1 June 2024).A clone was defined when the coefficient of similarity of the isolates was above 0.8 (http://genomes.urv.cat/UPGMA/accessed on 1 June 2024).

Replicon Typing
We performed replicon typing by the PCR-based replicon typing scheme described by Carattoli et al. [31] using whole-cell DNAs extracted from the isolates.

Bacterial Isolates and Patients' Susceptibility Testing
From September 2023 to January 2024, we collected 14 colistin-resistant and carbapenemnon-susceptible Klebsiella pneumoniae in the Military Medical Academy, Sofia, Bulgaria.
To obtain an insight into the distributed clones and enzymes, we included six randomly selected colistin-susceptible and carbapenem-resistant isolates of K. pneumoniae, detected in January 2024.
The susceptibility of isolates is shown in Table 2.The colistin-and carbapenemresistant isolates were also resistant to all the antimicrobials tested, which makes them pandrug-resistant according to Magiorakos' criteria [24].Additional testing found that only cefideracole susceptibility of the isolates was 100%, while the susceptibility to ceftazidime/avibactam was 35% (7/20) (four colistin-resistant and three colistin-susceptible isolates were susceptible to ceftazidime/avibactam).The isolates were highly resistant to eravacycline and plazomicin.Legend: S-susceptible, I-susceptible increased exposure, R-resistant.
All isolates were positive for the SHV group-specific primers set and almost all showed a presence of bla SHV-28 genes, except two isolates that produced KPC enzymes, and the SHV variant in these isolates was bla SHV-1 .
Among the 17 isolates highly resistant to aminoglycosides, 12 showed the presence of rmtB and three had the presence of armA (Table 1).

Replicon Typing
Sixteen isolates were positive for the colE replicon type.They included all bla OXA-232positive isolates with or without NDM-5 production.KPC-2-producing isolates showed a presence of plasmids of IncFII AS type (Table 1).

mgrB and Plasmid Colistin Resistance
All isolates were negative for mcr-1, -2, -3, -4, and -5.Among the 14 colistin-resistant isolates, a disruption in the mgrB locus with a lack of amplification was found in six, and sequencing revealed an exchange in position a124t in three isolates.This resulted in an exchange of asparagin N with tyrosin Y in position 42.One isolate had a 17 bp deletion from 72 to 89 nucleotides causing frameshift and premature termination.Four isolates displayed wild types.

Discussion
The number of infections caused by multidrug-resistant bacteria has been increasing worldwide.Antibiotic resistance has been considered one of the biggest threats to human health [3].The frequency of carbapenemase-producing K. pneumoniae in Bulgaria has increased from 7.2% in 2018 to 19.3% in 2021 (https://www.bam-bg.net/index.php/bg/bulstar accessed on 15 June 2024).This percentage is nearly twice as high as those for Europe: 10.9% for 2022 [33].This sharp increase in carbapenem-resistant isolates in the country reflects the increase in invasive K pneumoniae isolates.In Europe, the frequency of carbapenem-resistant invasive K. pneumoniae isolates rose sharply (by 49.7%) from 2019 to 2022 [33].The same trend was observed in Bulgaria, where the frequency of these isolates increased from 0% in 2013 to 21.2% in 2018 and 47.3% in 2022, with the biggest increase during the last two years [33].This coincides with the huge increase in total antibiotic consumption in Bulgaria during the last few years.Bulgaria was in third place in Europe in 2022 for community antibiotic usage with 25.2 DDD per 1000 inhabitants per day, and around 1.49 DDD per 1000 inhabitants per day (hospital usage) with a higher prevalence of cephalosporines and carbapenems [34].
Interestingly, the current work revealed a complete change in the spectrum of carbapenemases in Bulgaria.bla NDM-5 was the only one NDM variant in the current study, and in every case, it was combined with bla OXA-232 .
NDM-5 was first identified in the ST648 Escherichia coli isolate in 2011 from a patient with a hospitalization history in India [38].It differed from bla NDM-1 by two point mutations at positions 262 (G →T) and 460 (A →C) responsible for the two amino acid substitutions 88 (Val→Leu) and 154 (Met→Leu) [38].Subsequently, this carbapenemase was detected in K. pneumoniae, and its producers caused sporadic outbreaks worldwide [39].
OXA-232 differs from OXA-181 by one amino acid (aa) substitution (Arg214Ser) and from OXA-48 by five aa substitutions [40].OXA-232 has weaker carbapenem hydrolytic activity, but a stronger penicillin hydrolysing activity when compared with OXA-48 and OXA-181 [41].This observation was proved by our results.We observed three isolates that produced only OXA-232 and had intermediate susceptibility to imipenem.This enzyme was initially described in France (in patients returning from India), and thereafter, it was detected in China, India, South Korea, Singapore, Thailand, and the USA [40,[42][43][44].
The coexistence of bla OXA-232 and bla NDM-5 was observed for 10/14 (72%) of colistinresistant and carbapenem-non-susceptible K. pneumoniae isolates and for three of colistinsusceptible isolates in the current study.For the first time, this combination has been detected in Nepal [45].Similar reports have been published in India [46], Italy [47], and Bangladesh [48].Coproduction of NDM-5 and OXA-232 has been reported for various spectrums of STs types: ST14, ST16, ST147, ST231, ST2497, and ST427 (CC258) [46,47].Interestingly, the isolates found in the current study belonged to the rare ST type (ST6260), which differs from ST101 by only one allele, pho601 (ST6260), instead of pho4 (for ST101).Because of the difference of only one nucleotide between ST101 and ST6260, we hypothesize that ST101 is a possible progenitor of ST6260.To the best of our knowledge, this clone has not previously been reported to carry ESBLs or carbapenemase genes.ST101 is a well-known international clone, which has been reported to carry a wide range of carbapenemases such as OXA-48 [42].It is not clear, however, whether the mutation in the pho allele occurred in the Military Medical Academy or another hospital.Further studies should be performed in more hospitals in Sofia and Bulgaria to reveal the development of this cluster.Ten colistin/carbapenem-resistant and three colistin-susceptible/carbapenem-resistant isolates belonged to the NDM-5-and OXA-232-producing ST6260 K pneumoniae cluster.All the isolates showed the presence of the ColE replicon type, which is in concordance with reports about the bla OXA-232 gene being usually located on a 6-kb nonconjugative ColE-type plasmid [40].In addition, three of our isolates, positive only for bla OXA-232 , showed the presence of the ColE-type plasmid.Except this plasmid type, bla OXA-232 was also located on ColK3 plasmid types, which showed a similarity to ColE plasmids [6,49].
In addition, the isolates from the ST6260 (ST101-like) clone showed a high diversity in mgrB mutations: 10 out of 13 isolates were colistin-resistant, and all of them had mgrB variations.Six isolates showed absence of mgrB (lack of amplifications), one had a 17 bp deletion, causing a frameshift and premature termination, and in three isolates, aa substitutions were found.In total, 71% (10/14) of colistin-resistant isolates displayed mgrB genetic variations.The researchers reported that mgrB mutations are the main reason for colistin resistance [50].In a previous study in Bulgaria, among 27 colistin-resistant isolates, only 9 had mgrB variations, but they were members from the ST11 clone carrying bla NDM-1 [19].The data from the current study showed a high (71%) percentage of isolates with mgrB mutations, especially among ST6260 colistin-resistant isolates (100%).These mutations more probably occurred independently, due to higher colistin usage (almost all patients were treated with colistin).In addition, colistin was used in suboptimal doses (2 × 1 million units, personal information) [20].The implementation of strict infection control measures in the hospital resulted in the disappearance of colistin-resistant isolates (personal information).
It is worrisome that the investigated isolates were extremely resistant, especially the colistin-resistant members of the ST6260 clone.They were susceptible only to cefiderocol, which could cause serious therapeutic problems.At the end of the study period, we observed three isolates from this clone carrying the same carbapenemases, but they were colistin-susceptible.These facts revealed that isolates from this ST101-like (ST6260) clone showed a great potential to spread in the hospital and to cause therapeutic problems.In addition to cefiderocol, ceftazidime/avibactam in combination with aztreonam could be a good alternative for the treatment of such extremely resistant isolates [51] Among 13 members of the ST101-like (ST6260) clone, seven isolates were from blood samples and all these patients died (all of them with colistin-resistant variants).Moreover, some previous reports showed that ST101 K. pneumoniae can cause bloodstream infections with a high mortality rate [52].In general, the cause of this increase in mortality is not possible to establish.It could be due to the increased virulence of these isolates, worsening or complications of infection, or underlying comorbidities.The association of ST101 with increased mortality is pointed out by our observation that the investigated in the current study ST101 isolates (colistin-susceptible) has been isolated from patients with unsuccessful therapy (they died).The ST101 isolates produced different carbapenemase KPC-2, in combination with CTX-M-15 ESBL, and we found the FII AS replicon type.Most probably, the transmission of bla KPC-2 is associated with IncFII plasmids.In the current study, this plasmid type was also detected in the single KPC-2-producing ST258 K pneumoniae isolates (colistin-susceptible) and ST16 colistin-resistant isolate.In previous studies in Bulgaria, IncFII plasmids were observed as the main mobile structure that spreads bla KPC-2 [13,14].
Except the ST101-like (ST6026) clone, among colistin-resistant isolates, we observed three isolates belonging to the ST14 clone.They carried bla OXA-232 and had intact mgrB, which suggests other mechanisms of colistin resistance.PmrA/B, PhoP/Q two-component system, or regulator of PmrAB(Ccr) can influence the negative charge of LPS and thus decrease colistin binding to the LPS [50].ST14 is a member of ST15 CC and is one of the important international high-risk clones that can carry a wide range of carbapenemases [42].ST14 carrying bla OXA-232 has been reported from authors in China [53][54][55].Interestingly, all three ST14 K. pneumoniae isolates were positive for the replicon type ColE, the same as in ST6260, carrying bla OXA-232 and bla NDM-5 .
The isolates in the current study showed high resistance rates, especially ST6260 members.The colistin-and carbapenem-resistant bla OXA-232 -and bla NDM-5 -positive isolates were susceptible only to cefiderocol, and bla OXA-232 -or bla KPC-2 -positive isolates were susceptible to cefiderocol and ceftazidime/avibactam.The current isolates showed increased resistance to newly introduced antimicrobials such as eravacycline and plazomicin (only one isolate was susceptible).This is in contrast with our previous study on carbapenemase-producing isolates in 2018 showing susceptibility to eravacycline and plazomicin in 76.3% and 90.6% of the isolates, respectively [36].The higher resistance of the investigated isolates to aminoglycosides, especially ST101-like (ST6026) isolates, can be explained by the presence of rmtB 16S rRNA methyltransferases among all except one isolate.In ST14 and ST101 isolates carrying bla OXA-232 or bla KPC-2 , we observed ArmA methylases (Table 1).The rRNA methyltransferases cause a higher level of resistance to all aminoglycosides, including the recently introduced plazomicin, which increases therapeutic problems with such strains.These isolates were observed in our previous study in 2018 and had a spectrum similar to those common for this period (2017-2018) for Bulgaria (NDM-1 production in 61% and KPC-2 in 23%).Except for plazomicin and eravacycline, a big difference was observed in respect to cefiderocol, if we compare the current study with a previous one (on NDM-1and KPC-2-producing isolates).Cefiderocol resistance in the previous study was 37.5% according to the EUCAST criteria and 71.8% according to the FDA criteria [36] in comparison with 100% susceptibility in the current study.The method used for susceptibility testing (in our previous study [36]) was the disk diffusion method [36].In the present study, we used broth the microdilution method with iron-depleted broth.The disk diffusion method was also applied in the current study, and the inhibitory zone diameters around cefiderocol disks were in an Area of Technical Uncertainty (ATU) zone of 21 mm-23 mm [21] for all similar isolates in the previous study [36].We can suppose that broth microdilution can give more accurate results.The same findings were detected by other authors [56].Another possibility is different beta-lactamases (carbapenemases NDM-1, AmpC enzyme CMY-4, and extended-spectrum beta-lactamases CTX-M-3/-15) to influence cefiderocol resistance.Some authors associated NDM and AmpC with increased levels of resistance to cefiderocol [57,58].
The appearance of extremely resistant bla NDM-5 -or/and bla OXA-232 -positive isolates in the current study leads us to the pre-antibiotic era.The situation has additionally worsened after the COVID-19 pandemic.In Bulgaria, there was a large increase in general antibiotic use, especially for third-generation cephalosporins and carbapenems.The localization of genes encoding carbapenemases on mobile genetic elements (plasmids, transposons, and integrons), which also carry genes for resistance to other antimicrobial agents, can further select problematic microorganisms.The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution imply and highlight the need for urgent measures to control the situation.

Table 1 .
Characteristics of patients and K. pneumoniae isolates.