First report of a blaNDM-producing extensively drug resistant Klebsiella pneumoniae ST437 in Italy

Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) have recently been observed to spread rapidly worldwide. New Delhi metallo-β-lactamase (NDM) producing clones of Klebsiella pneumoniae (K. pneumoniae) cause a significant healthcare burden, particularly in Indian sub-continent, where this clone is circulating widely. However, in Italy, data on the incidence of these new clones is limited, and an ST437 NDM-producing K. pneumoniae strain has not been reported to date. A sacral ulcer infection caused by a K. pneumoniae strain was identified in an 85-year-old Italian male patient with several comorbidities. Antimicrobial susceptibility testing revealed an extensive resistance to a wide range of antimicrobials, including novel agents such as cefiderocol and ceftazidime/avibactam. Genomic analysis identified the pathogen as an ST437 K. pneumoniae strain harboring bla_NDM-5, bla_OXA-232 and bla_CTX-M-15 genes. Following the identification of this first case, several infection control measures were implemented in healthcare settings, including direct precautions and reinforcement of standard cross-transmission control measures. The emergence of pathogenic microbial clones carrying new genetic determinants, particularly in a little city, requires prompt diagnosis and therapeutic protocols. An effective infection control system for the early detection and/or control of the transmission of NDM-producing Enterobacteriaceae is also needed. Further investigations are required to better understand the potential transmission routes and evolution of these clones.

1 Introduction

Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) have spread rapidly around the world (Guo et al., 2023). The European Antimicrobial Resistance Surveillance Network (EARS-Net) of the European Centre for Disease Prevention and Control (ECDC) have recently reported a rate of 24.9% for carbapenem-resistant Klebsiella pneumoniae (Kp) isolates in Italy (European Centre for Disease Prevention and Control, 2023). New Delhi metallo-β-lactamase (NDM) is able to hydrolyse almost all β-lactams, including carbapenems. Since its 2008 discovery from a Kp strain isolated from a patient repatriated to Sweden after hospitalization in New Delhi, NDM-positive strains have been causing healthcare-associated outbreaks worldwide (Wu et al., 2019).

In Italy, the first outbreak of NDM-producing Kp (November 2018-May 2019) was documented in the northwestern area of Tuscany. This outbreak, involving nine different hospitals and 350 patients, was mostly caused by an NDM-1-producing Kp clone of ST147 (European Center for Disease Prevention and Control, 2019; Di Pilato et al., 2022).

Here, we report the identification of a bla_NDM-5 and bla_OXA-232 producing Kp clone of ST437, isolated for the first time from a patient hospitalized in a healthcare setting in Abruzzo region, Southern Italy.

2 Case presentation

In April 2023, an 85-year-old Italian male patient was admitted to the hospital with a proximal femoral fracture, and underwent emergency surgery. The patient had comorbidities, particularly senile dementia associated with Parkinsonism, arterial hypertension, and a second stage sacral pressure ulcer due to reduced mobility.

On the 9^th day after surgery, the patient developed fever and hypotension due to elevated lactate and procalcitonin levels (73 ng/ml). Blood and urine samples were collected, and empirical antibiotic treatment with meropenem 500 mg Q12H IV and vancomycin 1000 mg Q24H IV (renal impairment dosing) was initiated. On the 48^th hour, a suspected Kp strain was detected in both blood and urinary cultures. The isolate (Kp1) was identified as Klebsiella pneumoniae by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using Vitek MS (Biomérieux, Marcy l’Étoile, France). Carbapenemase genes were screened using the Amplex Eazyplex SuperBug CRE test (Amplex Diagnostics, Gars am Inn, Germany) based on the loop-mediated isothermal amplification (LAMP) method. The bla_KPC and bla_CTX-M1 genes were also detected).

The antimicrobial susceptibility test (AST) was performed using the MicroScan WalkAway system DxM 1096 (Beckman Coulter), and the results were interpreted according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2023, version 13.1). Since the breakpoint of tigecycline against Kp has not been made available by EUCAST, the EUCAST breakpoint for E. coli was used. Cefiderocol (FDC) susceptibility was tested using Minimum Inhibitory Concentration (MIC) Test Strip (Liofilchem, Roseto degli Abruzzi, Italy), and interpreted according to EUCAST clinical breakpoints. FDC is a novel injectable siderophore cephalosporin that is active against most carbapenem-resistant Enterobacteriaceae and is used for the treatment of complicated urinary tract infections, hospital-acquired bacterial pneumonia, and ventilator-associated bacterial pneumonia (Wu et al., 2020).

The results indicated that a modification to the antibiotic therapy may be required (Table 1). Hence, ceftazidime/avibactam (CZA) 2.5 g Q8H IV treatment was initiated against carbapenem-resistant Kp (Tumbarello et al., 2019). The patient’s clinical condition improved rapidly after microbiological eradication (negative blood and urine cultures).

Table 1

Table 1. Minimum inhibitory concentration (MIC) and susceptibility results of Kp1 and Kp2 strains.

On the 12^th day of antibiotic therapy (2 days of meropenem and 10 days of CZA), the patient underwent surgical toileting due to clinical worsening of the sacral ulcer, and vacuum-assisted closure therapy was initiated with gradual improvement of the lesion. Tissue sampling for microbiological examination was performed, and a different Kp strain (Kp2) was isolated and identified from the culture (Table 1). Screening of carbapenemase genes in the Kp2 strain revealed the presence of bla_NDM, bla_OXA-181 and bla_CTX-M1 genes. Considering the microbiological eradication of Kp1, CZA was interrupted and no indication for further antibiotic therapy was given, considering Kp2 to be a colonizing strain of the sacral ulcer. The medical histories of the patients and laboratory analyses of Kp strains are outlined in Figure 1.

Figure 1

Figure 1. Medical history of the patient. Different colored bars in antibiotics treatment represent different antibiotics: light blue bar (Meropenem), green bar (Vancomycin), purple bar (Ceftazidime-avibactam).

Both Kp1 and Kp2 strains were subjected to whole-genome sequencing (WGS). DNA was extracted according to the protocol described by Cornacchia et al. (2022), and WGS was performed using the Illumina platform (Cornacchia et al., 2022). An in-house pipeline was used for the WGS data analysis. The genome assembly quality check was assessed according to Hennart et al. (2022) (n. contigs < 1,000, total length ranged from 4.5 to 6.5 Mbp, GC% < 59%). Species confirmation and K/O locus determination were performed in Kleborate (Lam et al., 2021) hosted on the PathogenWatch platform (Argimón et al., 2021); meanwhile, sequence type (ST) was calculated in silico according to the multilocus sequence typing (MLST) scheme hosted on the Pasteur sdb platform (Diancourt et al., 2005 and Brisse et al., 2009). Chromosomal and acquired resistance genes and outer membrane porin alterations were detected by querying the ResFinder 4.4.2 database (v. 12th December 2023) (Florensa et al., 2022).

3 Results

WGS results confirmed that both strains belonged to species Klebsiella pneumoniae. MLST analysis revealed that Kp1 isolated from the blood and urine was a clone of ST307-CG307, and Kp2 from the sacral ulcer was a clone of ST437-CG10268. Capsular type (KL) and O locus (OL) analyses revealed that Kp1 belonged to KL102, OLO1/O2v2, and the capsular polysaccharide type was wzi173, while Kp2 belonged to KL52, OL101, and the capsular polysaccharide type was wzi50.

We focused on the Kp2 strain due to its phenotypic antimicrobial resistance and the presence of bla_NDM gene, which had never been identified in the healthcare system of our province before. The AST (Table 1) showed that Kp2 was extensively resistant, in particular to the combinations of cephalosporins/β-lactamase inhibitors such as CZA or carbapenem/β-lactamase inhibitor meropenem/vaborbactam (MEM/VAB). Resistance was also observed for FDC. Kp2 was susceptible only to tetracyclines (eravacycline (MIC < 0.5 μg/ml) and tigecycline (MIC < 0.5 μg/ml)) and polymyxins (colistin (MIC <=2 μg/ml)). Based on the resistance phenotype, this strain is classified as extensively drug resistant (XDR) (Magiorakos et al., 2012).

Carbapenem resistance genes bla_NDM-5 and bla_OXA-232 and Extended Spectrum β-Lactamase (ESBL) bla_CTX-M-15 genes were identified through querying the ResFinder 4.4.2 database. The other penicillin resistance genes identified were bla_TEM-1D and bla_SHV-11. Finally, trimethoprim (dfrA12), sulfonamide (sul1), and aminoglycoside (aac(6’)-Ib, aadA2, rmtB, rmtF) resistance genes were also identified. The GyrA-83I and ParC-80I mutations were also detected.

The presence of ompK36 with a glycine-aspartate (GD) L3 insertion was detected. The strain harbored several plasmid replicons (100% identity), including Inc-type (IncFII(K), IncFII, IncFIB(K), IncR) and Col-type (ColKP3, Col(BS512)) replicons. The ST437 strain was shown to have yersiniabactin ybt 14 located within the integrative conjugative element ICEKp5 to specifically overcome host-mediated iron limitation.

4 Discussion

To the best of our knowledge, this is the first report of the identification of an ST437 bla_NDM-5 producing Kp in the Abruzzo region of Italy. The ST437 Kp is a global epidemic clone, previously reported in North and South America (Wang et al., 2013; Tijet et al., 2014; Aires et al., 2020), Europe (Emeraud et al., 2022; Benulič et al., 2020; Palmieri et al., 2020; Fuster et al., 2020; Marti et al., 2017) and Asia (Zhu et al., 2018; Weng et al., 2020; Sahoo et al., 2023).

The ST437 clone is often associated with β-lactamase-producing genes, mainly bla_KPC-2 and bla_CTX-M-15, and more recently with bla_OXA-like (bla_OXA-48 and bla_OXA-245) or bla_NDM-_like (specifically bla_NDM-1, bla_NDM-7 and bla_NDM-23) genes. ST437 bla_KPC-2 and bla_CTX-M-15 expression was previously reported in isolates from Brazil (Andrade et al., 2011; Palmeiro et al., 2019) and bla_KPC-2 and bla_OXA-1 in Ontario, Canada (Tijet et al., 2014).

In Europe, several studies have reported the presence of ST437 clones as well. For instance, bla_OXA-245 producing Kp (Oteo et al., 2013), bla_NDM-7 producing Kp (Seara et al., 2015) and bla_OXA-48 and bla_NDM-1 Kp were identified in Spain (Fuster et al., 2020).

Benulič et al. described the population of carbapenem-resistant Kp detected during an outbreak occurred in Slovenia between 2014–2017, including bla_OXA-48 and/or bla_NDM-1 ST437 strains. Palmieri et al. (2020) reported the presence of ST437, bla_OXA-48 and bla_NDM-1 in clinical Kp isolates from Serbia. In the same year, Weng et al. described for the first time a bla_OXA-232 producing ST437 Kp strain in China. The bla_OXA-232 producing strains are frequently associated with travel to India.

In 2023, bla_NDM-5 ST437 Kp was isolated from a river in eastern India (Sahoo et al., 2023).

In Italy, the ST437 Kp clone is rarely detected, with previous reports in two hospitals in Padua between June 2009 and December 2011, which highlighted the presence of bla_KPC-2 gene.

The ST437 Kp2 clone detected in our hospital was susceptible to colistin and tigecycline, and showed elevated MIC values of all carbapenems, cephalosporins and also novel β-lactamase inhibitor combinations (BLICs) tested. The bla_NDM-5 carbapenemase identified in the ST437 Kp explains all β-lactam resistance observed, with the exception of that to aztreonam, which is not hydrolyzed by metallo-β-lactamases. However, this strain also carries bla_CTX-M-15 as well as another class D carbapenemase, bla_OXA-232, which may contribute to aztreonam resistance (de Man and Limbago, 2016). In this context, the use of avibactam protects aztreonam from hydrolysis, making it effective against NDM (Tamma et al., 2023).

High MICs for tobramycin, amikacin, and gentamicin were also observed, most likely due to the presence of rmtF. Fluoroquinolone resistance is also likely affected by the presence of GyrA-83I and ParC-80I mutations. The ST437 Kp isolate, in addition to bla_NDM-5 and bla_OXA-232, carried several mutated porin genes in ompK36 and ompK37 which are linked to resistance to cephalosporins and carbapenems. The presence of carbapenems and the co-occurrence of other resistance determinants (bla_NDM-5, bla_CTX-M, bla_SHV, and bla_TEM), virulence factors (ybt14), and the capsular serotype (KL52) further highlights its high pathogenic potential.

Among the bla_NDM variants, bla_NDM-5 was first reported in the UK in 2011 and has gained significant attention owing to its enhanced resistance to carbapenems and broad-spectrum cephalosporins (Li et al., 2018).

Infections caused by bla_NDM-5-carrying CP-Kps are associated with high rates of morbidity, mortality, and transmission due to the dissemination ability of NDM-producing Kp strains (Paczosa and Mecsas, 2016; Jia et al., 2022).

In this study, the acquisition of bla_NDM-5-producing Kp by the patient could not be explained by direct contact with a colonized patient. However, the potential involvement of undetected and asymptomatically-colonized patients in transmission routes cannot be excluded. The selective pressure of CZA treatment may have also favored the emergence of this NDM-producing Kp strain.

The case described here had no proven connection with India, Pakistan, the Middle East, the Balkans, Spain in Europe, Brazil, or North America. Therefore, the route through which NDM-producing Kp was introduced into the hospital is unknown. According to European Centre for Disease Prevention and Control (2011), patients with no direct link to countries where this clone is circulating are defined as autochthonous cases, indicating an unknown reservoir of NDM.

Two STs clones with similar virulence potentials and resistance profiles were also identified. Hence, a single dominant clone is not the only one responsible for severe infections, causing repercussions on prognosis and therapeutic treatment (Venturini et al., 2022). Although the number of bacterial isolates belonging to the same species analyzed per sample is determined by response times, increases in testing capacity and improved diagnostic protocols are needed to improve preciseness in the identification procedure. The ST437 Kp2 clone was considered by the infectious disease clinician to be a colonizing strain, in fact after surgical toilet and vacuum-assisted suture therapy the lesion gradually improved and no local or systemic signs of active infection occurred. For these reasons the patient did not receive further antibiotic treatment. Nevertheless, antibiotic pressure plays a decisive role in modifying the susceptibility of microorganisms to various antibiotics (Shields et al., 2017).

Approximately one-third of deaths caused by AMR in Europe occur in Italy (https://www.ecdc.europa.eu/en/publications-data/antimicrobial-resistance-surveillance-europe-2022-2020-data). Italy is one of the countries with a high rate of antibiotic resistance, and where antibiotics are used excessively and often inappropriately, although there are signs of improvement, as reported in the last Ears-NET report (carbapenem-resistant isolates percentage from 26.7% to 24.9%).

Southern regions of Italy have critically higher levels of carbapenem-resistant Kp strains than the northern parts. In Southern Italy, Abruzzo, Apulia and Campania are the regions with the highest levels of prevalence of antimicrobial use (around 45%) (Medicines use in Italy National Report Year 2022 Published on: 01 December 2023).

Pérez-Galera et al. (2023) described several risk factors, which may contribute favoring Carbapenem-resistant Enterobacteriaceae (CRE) infections. These factors include hospitalization in the last three months, previous colonization/infection by CRE, chronic heart failure, dementia, chronic renal failure, central venous and urinary catheters, dialysis, and previous use of antibiotics. Moreover, significant comorbidities and invasive surgeries play a key role in the deterioration of the immune status conditions of hospitalized patients (European Centre for Disease Prevention and Control, 2011; Zuo et al., 2021). Most of the previously described risk factors were identified in the present case.

After the first case of NDM-producing ST437 Kp, several infection control measures were implemented in our healthcare setting, including contact precautions in individual rooms, reinforcement of standard cross-transmission control measures, cleaning procedures, and active surveillance cultures, together with retraining courses for operators. All these measures were adopted to raise awareness of this issue and avoid the spread of this strain associated with high mobility and mortality in our patients (World Health Organization, 2016 and 2017). All control measures activated and surveillance activities (culture swabs combined with molecular methods) were successful, indeed only one positive patient was identified. The patient positive for the presence of the ST437 Kp strain was a 92 years old woman, bedridden, with a long hospital stay and a bladder catheter, therefore with many risk factors for colonization. After the first detection of this strain in the hospital, other rare and sporadic findings of ST437 Kp strain were recorded (6 cases of urinary infections) in the following 7 months. In all cases, infection control measures effectively prevented the spread of the strain among patients. Heterogeneity in coverage across the wards in the hospital and the use of culture and not molecular methods for the identification of carbapenem resistant strains in screening practice, limits the knowledge of the real spread of the ST437 Kp strain in our healthcare setting.

The effectiveness of infection control interventions for the early detection and/or control of the transmission of NDM-producing Enterobacteriaceae has not been proven yet. However, an ECDC risk assessment report demonstrated the effectiveness of active surveillance and control measures, including a dedicated team for all suspected and CPE-positive patients (European Centre for Disease Prevention and Control, 2011). The emergence of clones carrying new genetic determinants, especially in local areas and small regions such as the Abruzzo region, requires prompt diagnosis and therapeutic protocols, as well as further investigations to better understand the potential transmission routes and evolution of these clones. This case report is the snapshot of a restricted geographic area in a limited period of time and its origin is still unknown. For this reason, a more active AMR surveillance involving other hospitals in Abruzzo and the other Italian regions combined with next generation methods could potentially improve Kp infection treatment and patient outcomes. It is time for a more effective and widespread antimicrobial stewardship with a multidisciplinary approach to be implemented in hospitals and outpatients. These strategies should involve local, regional, and central government monitoring, auditing, and feedback.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, SAMN40624209 https://www.ncbi.nlm.nih.gov/genbank/, SAMN40624210.

Ethics statement

Ethical approval was not required for the studies involving humans because The retrospective analysis of the clinical data did not involve procedures that normally require written consent outside of the research context. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements because The retrospective analysis of the clinical data did not involve procedures that normally require written consent outside of the research context. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article because The retrospective analysis of the clinical data did not involve procedures that normally require written consent outside of the research context.

Author contributions

SR: Conceptualization, Formal Analysis, Methodology, Writing – original draft, Writing – review & editing. VF: Methodology, Writing – review & editing. AD’: Methodology, Writing – review & editing. MF: Methodology, Writing – review & editing. VD: Investigation, Methodology, Writing – review & editing. CD: Investigation, Methodology, Writing – review & editing. SD: Methodology, Writing – review & editing. LD: Methodology, Writing – review & editing. MD: Investigation, Methodology, Writing – review & editing. MP: Methodology, Writing – review & editing. ACH: Formal Analysis, Methodology, Writing – original draft, Writing – review & editing. GC: Methodology, Writing – review & editing. CC: Methodology, Writing – review & editing. FP: Funding acquisition, Writing – review & editing. ACO: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by EU funding within the NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT).

Conflict of interest

The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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