A MULTIDISCIPLINARY APPROACH AGAINST ANTIMICROBIAL RESISTANCE: INTEGRATING IMMUNITY, MICROBIOLOGICAL FACTORS AND CLINICAL CARE TO FACE THE THREAT OF KLEBSIELLA PNEUMONIAE-CARBAPENEMASE (KPC)-PRODUCING KLEBSIELLA PNEUMONIAE

BACKGROUND: Klebsiella pneumoniae-carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) accounts for a third of invasive strains of Kp in Italy and is responsible for increased morbidity and mortality. Host and bacterial factors involved in KPC-Kp colonization and/or infection are not yet clearly characterized. METHODS: Observational, prospective, multicenter national study part of the project Ricerca Finalizzata 2016 (GR-2016-02362572). Patients were enrolled from 03/2019 to 08/2021. Patients enrolled in a previous study at one of the participating centers (09/2017-05/2018) were included in the analysis. Plasma, peripheral blood mononuclear cells (PBMC) and faecal samples were collected at the diagnosis of KPC-Kp colonization and/or infection. In a subgroup of patients, KPC-Kp strains were collected for genomic analyses. Soluble markers (pentraxin-3 and soluble IL-1R2) were measured by home-made sandwich ELISAs. Immunophenotyping was conducted on frozen PBMCs by multiparameter flow cytometry analysis. Microbiome was sequenced for V3–V4 region of 16S rRNA amplicons (Illumina MiSeq) and analyzed using QIIME2. KPC-Kp strains were analyzed by whole genome sequencing (Illumina Nextseq 550 System), virulence and resistance genes were characterized with Kleborate software. RESULTS: Overall, 135 patients were included: 89/135 (65.9%) with KPC-Kp colonization (KPCcol), 46/135 (34.1%) with KPC-Kp infection (KPCinf). The two groups differed in time between hospital admittance and enrollment (KPCcol median 18.5 (Q1-Q3: 8-37) days, KPCinf 10 (5-24) days, p 0.004), invasive procedures in the 72 hours prior to KPC-Kp isolation (KPCcol 18%, KPCinf 36.4%, p 0.03), concomitant infection/colonization by multidrug-resistant bacteria other than KPC-Kp (KPCcol 45.2%, KPCinf 25.6%, p 0.038). Conversely, the number of patients with acute medical conditions and with ongoing antibiotic therapy at KPC-Kp isolation resulted similar in both groups at high percentages (KPCcol 85.9%, KPCinf 85.5%; KPCcol 70.6%, KPCinf 76.7%, respectively). Whole genome sequencing was performed in 37 KPC-Kp strains. The most common sequence type (ST) was ST307 (32.4%) followed by ST512 (24.3%) and ST20 (21.7%). Two KPC variants were found, KPC-2 in 13/37 (35.1%) and KPC-3 in 24/37 (64.9%) isolates. For each KPC-Kp strain, major virulence and resistance genes were characterized and virulence and resistance scores were calculated. Core SNP phylogenetic tree analysis including KPC-Kp strains from European hospitals was obtained. Soluble markers were evaluated in 79 KPCcol and 39 KPCinf. No differences between groups were observed (PTX3: KPCcol 14.63 (9.78-23.36), KPCinf 18.87 (10.58-39.51) ng/ml; sIL-1R2: KPCcol 16.01 (11.6-24.2), KPCinf 20.64 (13.63-25.04) ng/ml). PBMCs were evaluated in 37 KPCcol and 21 KPCinf. The two groups differed in percentages of circulating Th1, Th2 and Th17, with higher values in KPCcol compared to KPCinf (Th1: KPCcol 8.48 (4.29-17.1), KPCinf 4.43 (1.16-9.01) % CD4+ T cells, p 0.017; Th2: KPCcol 9.38 (4.29-17.1), KPCinf 4.26 (2.40-9.73) % CD4+ T cells, p 0.037; Th17: KPCcol 16.6 (7.09- 38.8), KPCinf 7.89 (3.55-15.42) % CD4+ T cells, p 0.035 ) Gut microbiome was evaluated in 46 KPCcol and 23 KPCinf. Alpha diversity values differed between groups (Pielou’s eveness index: KPCcol 0.68 (0.6-0.74), KPCinf 0.56 (0.51-0.69), p 0.019) whereas beta diversity values did not. Alpha diversity values differed also comparing patients with and without broad-spectrum antibiotic therapy at KPC-Kp isolation (0.64 (Q1-Q3: 0.51-0.70) vs 0.68 (Q1-Q3: 0.60-0.75), p 0.021) and, within KPCINF, comparing those presenting with and without sepsis at presentation (0.50 (Q1-Q3: 0.48-0.54) vs 0.65 (Q1-Q3: 0.51-0.7), p 0.033). CONCLUSION: In addition to different clinical characteristics, patients with KPC-Kp infection seem to display poorer gut microbiome richness and different circulating cellular immunity as compared to hospitalized subject with KPC-Kp colonization. Although still partial, these results indicate that an integrated approach combining immunological and microbiological analyses could unravel important aspects of the complex host-pathogen interaction.