Molecular characterization of nosocomial infections: an Italian (KPC-producing Klebsiella pneumoniae) and a Spanish (Clostridium difficile ribotype 027) experience

During the last decade, Clostridium difficile and Klebsiella pneumoniae represented two of the most emblematic cause of nosocomial outbreaks, especially following the spread of epidemic variants, able to produce virulent factors and to present higher resistance against several antimicrobial classes. The rapid identification of dangerous pathogens circulating in nosocomial environment has been made possible by the contribution of molecular methods in support to the classical diagnostic techniques. The principal objective of this work is to highlight the effectiveness of different molecular typing techniques in the characterization of nosocomial infections, evidencing the genetic relatedness among isolates and the transmission routes of strains involved in epidemic events. In the first part of the work (Chapter one), the analysis of the principal molecular and phenotypic features of clinical isolates of KPC-producing K. pneumoniae collected in a 27-months period at Ospedale di Circolo e Fondazione Macchi (Varese, Italy) is reported. Of the 16 isolates analyzed, 7 were involved in an outbreak occurred the Intensive Care Unit (ICU). We investigated the presence of genes involved in carbapenemases resistance (i.e. blaKPC, blaIMP, blaNDM, blaVIM, and blaOXA), the expression of genes for virulence factors (pili/fimbriae, capsular antigen, hypermucousviscosity protein, and siderophores), and mutations leading to colistin resistance. We also made a phylogenetic analysis adding all the 16 genomes to 319 genomes that represent the global diversity of K. pneumoniae strains. We found that all isolates analyzed belong to clonal complex CG258. This finding is not surprising, considering previous reports that showed the worldwide diffusion and high prevalence of this clonal group among carbapenem-resistant K. pneumoniae strains. Interestingly, three of the four previously identified groups of Italian isolates of CG258 were found circulating in the hospital, suggesting that several entrance events of the clones may occur over the study period. About resistance genes, we showed that all 16 genomes express blaKPC genes and none of them had other known carbapenem-resistance genes. Interestingly, 10 of the sixteen isolates, which were colistin-resistance, presented IS5-like transposons in mgrB gene, conferring resistance to this drug. The second part of the work (Chapter two) aimed to identify and characterize all the clinical isolates of Clostridium difficile ribotype 027 (here, briefly CD027) collected during a 20-months period in the Hospital General Universitario Gregorio Marañon (Madrid, Spain). The main objective was to characterize the epidemiological links among the CD027 clinical isolates and to defining their transmission routes. To better understand the epidemiological relationships between the strains, all cases identified since January 2014 were added to the analyses. Besides, presence of genetic markers (i.e. mutation in gyrA gene) characterizing the evolution and spread of specific epidemic lineages of CD027 was investigated. During a 20-months period (January 2014-August 2015), 132 first episodes of C. difficile ribotype 027 has been detected, distinguishing 9 different subtypes (MLVA-types), organized in 5 different clonal complexes (CC) and 4 unique MLVA-patterns. Specifically, one of the five clonal groups (named MLVA-type 1) was responsible of an outbreak which involved 111 patients, and quickly spread in the hospital from October 2014. MLVA typing analysis showed the close genetic relationship of all the strains, suggesting the evolution from a common ancestor. All CD027 isolates carried a specific mutation in gyrA genes, indicating the presence of high transmissible clones belonging to CD027 lineages, but this do not provide more information on their dissemination course. Moreover, the clonal complexes showed different capacity of spread, which was evidenced by the dissemination of the only CC of MLVA-type 1 leading to the epidemic event. For this reason, the second objective of Chapter two aimed to evaluate the possible correlation between the transmissibility of the analyzed CD027 clinical isolates and the ability to sporulate. An in vitro protocol has been performed for the evaluation of sporulation rate of epidemic CD027 strain comparing them with those of the ribotype 001, with the objective of determining whether the increased transmissibility of CD027 is due to the greater ability to release endospores into the environment. Comparison between strains belonging to the same ribotype was performed, to evaluate the possible association between sporulation rate and transmissibility. We found that CD027 strains possessed higher capacity (p=0.005) to produce spores respect to isolates belonging to CD001 but surprisingly their germination ability was significantly lower (p=0.0008) compared to that of CD001, leading to reflect on the adaptability of this pathogen to the environment and the complexity of the mechanisms regulating pathogenic capacity. Once characterized the rapid spread of an outbreak due to a particular CD027 clone (MLVA-type 1), a further aim was to highlight the possible differences between the efficiency of sporulation and germination of strains belonging to the same ribotype but with different transmission characteristics, underlying the possible association between the ability of sporulation and the strain transmissibility. Moreover, analysis showed non-significant strain-to-strain variability between C. difficile isolates belonging to the ribotype 027; in particular, comparison was conducted between highly transmissible MLVA-type 1 isolates and the other MLVA-types, showing that neither in sporulation rate (p value =0.72) nor in germination rate (p value=0.24) significant differences exist. Our findings suggest that further studies preferably by analysis of the transcriptome must be performed to clarify the features involved in CD027 pathogenicity. This work shows how is possible, by application of molecular typing, to identify pathogens responsible for epidemic situations and to determine the presence of virulence factors o drug resistance genes associated with pathogenicity of strains. Besides it is possible to obtain useful and additional information as the genetic relationships between analyzed strains and their transmission route. In conclusion, it is showed that active surveillance and characterization of circulating strains at local and national level is crucial to prevent the spread of more virulent variants, with benefits for the patient and the health system.