During the study period, almost all of the CR-KP strains isolated from the patients with BSIs in the Puglia region (Southern Italy) carried the blaKPC gene (96%), confirming that the production of the KPC-type carbapenemase was the most common carbapenem-resistance mechanism, as previously reported in Italy [7,9] and Southern Italy [28]. In our study, the KPC-3 variant was found in all of the analysed KPC-KP isolates, and this is consistent with the finding that this variant is the most prevalent in other KP surveillance studies in Italy [7,10,16,17,28]. We found a higher proportion of blaVIM and blaNDM genes (3.6%) in the BSIs compared with the proportion reported by Iacchini et al. (1.9%) for Italian isolates [9]. Calia et al. identified the blaVIM gene in 5.0% of CR-KP isolates. However, the data reported in the latter study, which was conducted in the Puglia and Basilicata regions (Southern Italy), included strains isolated from any infection site and not BSIs specifically [28]. Moreover, we found one isolate carrying the blaNDM gene, whereas no NDM-KP was reported by Calia et al. [28]. Carriage of multiple carbapenemase genes is rarely reported [9], and our study followed this trend (0.6%). Molecular characterization of such isolates could be of particular interest. Unfortunately, the four isolates carrying multiple carbapenem-resistance genes included in our study were not characterized because they were not available for WGS.
Almost half of the CR-KP isolates included in the present study belonged to ST512. However, over the study period, we found an increasing proportion in BSIs with ST101 and ST307 strains, which accounted for approximately 20% and 18% of all the 231 characterized isolates, respectively. These STs have been rarely reported in Italy, except for in the report of Bonura et al. and in Fasciana et al. in which prevalence of 29% (27/94) and of 12% (3/25) were reported for ST307, respectively [10,16]. Nevertheless, these data included CR-KP strains isolated from any site of infection or colonization and not BSIs specifically. Furthermore, Calia et al. found only five isolates belonging to ST307 (3.3%) [28], which is substantially lower than the prevalence of this ST in the present study. In Milan (Lombardy), ST307 was found in 4.4% of isolates [32], whereas no strains belonging to this ST were found in Sassari (Sardinia) [11]. These data regarding the spread of ST307 in Italy seem to suggest that this ST is replacing the predominant hyperendemic STs belonging to CC258 [33].
ST101 is an emerging nosocomial high-risk clone associated with infections with increased mortality compared with non-ST101 infections [26]. Moreover, ST101 is a high-risk for mediating further spread of carbapenem resistance [34,35]. In Italy, this ST has generally been reported as sporadic [7,10,11,16,17,32], with higher prevalence when the studied strains were isolated from any infection site [34,36,37]. We found that approximately 18% of the characterized strains belonged to ST101, while no isolates belonging to this ST were identified in the report of Calia et al. in Southern Italy [28]. All of the ST101 isolates identified in the present study were KPC-3-producing strains, whereas other Italian studies reported ST101 isolates that only produced the KPC-2 variant [7,17,34].
Our resistome analysis showed that all of the characterized isolates harboured genes conferring resistance to aminoglycosides, beta-lactams, and fluoroquinolones. In particular, the ST307 isolates carried a significantly higher number of resistance genes compared to the numbers in the ST101 isolates. However, no clear correlation between genetic background and phenotypic resistance in these strains could be established [32]. In the present study, three out of five ST307 isolates (57POL, 95POL, and 12ACQ) carried blaCTX-M-15, blaSHV-28 and blaKPC-3. This pattern has been previously reported in Italy[16,27,37]. Furthermore, the three isolates that carried blaCTX-M-15, blaSHV-28 and blaKPC-3 showed the same allelic profile for efflux pump genes, which differed from that identified in the ST101 isolates.
The analysis of virulence genes showed that the two ST101 isolates seemed to be potentially extremely virulent strains, as they carried the siderophore gene (irp1), the ferric uptake system (kfuABC), the yersiniabactin siderophore cluster genes (ybtAEPQSTUX), and the mannose-resistant Klebsiella-like (type III) fimbriae cluster (mrkABCDFHIJ). These virulence factors have been previously reported in only one KP-ST101 isolate identified in Northern Italy in 2013 [32]. All of the isolates characterized in our study harboured at least one heavy metal resistance gene, which, as previously suggested, can contribute to antibiotic-resistance gene dissemination and maintenance [38].
Interestingly, we also identified many plasmids in the characterized isolates, especially IncF-type and ColRNAI plasmids. This finding is consistent with studies reporting that these plasmids are frequently associated with successful dissemination of carbapenemase, CTX-M-type extended-spectrum beta-lactamase, and plasmid-mediated quinolone resistance genes, as well as other virulence determinants in Enterobacteriaceae [39,40].
When treating infections involving antimicrobial resistance (AMR), colistin is the last-resort antibiotic for treatment of patients with Enterobacteriaceae infections. Colistin resistance associated with plasmid-borne mcr genes in KP has been reported in Italy, although rarely [41]. Traditionally, colistin resistance in KP is associated with mutations in chromosomal genes [42]. Among the isolates characterized in our study, two carried the L213M substitutions in the chromosomal pmrB gene, although, according to Lomonaco et al. [37], these mutations seem not to be related to colistin resistance. However, these two isolates showed phenotypic resistance to colistin.
Finally, our findings regarding the K-antigens were consistent with previously reported results for ST307 isolates carrying the wzi173 allele, but not for the ST101 isolates, which carried the wzi149 allele instead of the wzi137 allele [32].
This study has some limitations. First, our data did not cover the entire Puglia region but only three provinces. Nevertheless, the population of the provinces included in this study represents half of the total population of Puglia. A future option would be to implement epidemiological and molecular surveillance of CR-KP infections in the entire region. Second, WGS analysis was conducted on a limited number of isolates. Unfortunately, we could not characterize either the CR-KP strains that carried more than one carbapenem-resistance gene nor all the identified ST307 and ST101 isolates. Nevertheless, we believe that our data could provide important information about the hospital-based dissemination of two emerging high-risk clones, which have, to date, been rarely described in Italy. These clones have characteristics that might lead to increased fitness, persistence in and adaptation to hospital environments and, consequently, an ability to supplant the most frequently reported CC258 clones. Whole-genome sequencing is a promising surveillance tool that will be useful for early identification and characterization of both existing and emerging AMR clones, especially in hospital settings where antibiotic-resistant pathogens pose a great challenge to clinicians due to limited available treatment options.