Mobile genetic elements and clonal dynamics of antibiotic-resistant hospital-acquired bacteria

Over the past decades antimicrobial resistance rates are rapidly growing and have provoked the emergence of multidrug-resistant bacterial pathogens. Of particular concern are nosocomial outbreaks and the clonal spread of antibiotic-resistant bacteria representing a major threat for the healthcare system and especially to susceptible patient populations. One of the aims of this PhD project was to investigate the spread of three bacterial species commonly involved in nosocomial infections: vancomycin-resistant Enterococcus faecium (VREfm) and third-generation cephalosporin-resistant (3GCR) Klebsiella pneumoniae recovered from screening samples of patients admitted to six university hospitals in Germany, and carbapenem-resistant Acinetobacter baumannii (CRAb) from two hospitals in Bolivia. Between 2014 and 2018 a steady increase in the prevalence of VREfm (2014, 0.8%; 2015, 1.2%; 2016, 1.3%; 2017, 1.5%; 2018, 2.6%) was observed in six university hospitals in Germany. In the present cohort the sequence type (ST) 117 was identified as the predominant clone followed by ST80, ST203, ST78 and ST17. Furthermore, vanB was the most common glycopeptide resistance determinant among the colonising isolates while the vast majority of the ST117 isolates were vanB-positive. A remarkable clonal dissemination of the vanB-positive cluster type (CT) 71 subpopulation of the ST117 clone was identified among VREfm. In contrast, other STs, such as ST80, formed mostly local clusters restricted to single study centres. The ST117/CT71/vanB VREfm clone was widespread over six geographically separated centres and was endemic and indicated that the epidemiological profile of VREfm in Germany has changed in the past five years. Conversely, patients on hospital admission in Germany were colonised with a diverse population of 3GCR K. pneumoniae complex isolates. Over the study period an increase in the prevalence of 3GCR K. pneumoniae complex (2016, 0.8%; 2017, 0.9%; 2018, 1.1%) was observed. The clinically relevant K. pneumoniae was the predominant species in the present study, but a few representatives of Klebsiella quasipneumoniae and Klebsiella variicola were also identified. Among 3GCR K. pneumoniae isolates CTX-M-15 was the most common acquired β-lactamase followed by TEM-1B. Carbapenem-resistance was rare among the colonising isolates with only one K. variicola isolate harbouring a carbapenemase, OXA-181. Lastly, no clonal expansion and only small clusters of closely related isolates were identified, suggesting that a diverse K. pneumoniae population is circulating in the participating study centres. The clonal spread of CRAb isolates obtained from two hospitals in Bolivia was demonstrated in the present thesis. Among A. baumannii 51 were identified as carbapenem-resistant. The vast majority of the CRAb isolates were identified as ST25 or a single locus variant of it, ST991. The latter ST was only identified in one of the hospitals while ST25 was predominant in the second hospital. Moreover, all CRAb isolates harboured OXA-23 on a Tn2008 transposon while no other carbapenemase was detected. All but one of the CRAb isolates belonged to the international clone (IC) 7 and formed five transmission clusters. Furthermore, one CRAb isolate was assigned as IC4. These results suggested that several IC7 OXA-23-positive CRAb clones are endemic and are circulating in two hospitals in Bolivia. Horizontal gene transfer and mobile genetic elements (MGEs) are the main drivers of the evolution, diversification and spread of antibiotic resistance genes (ARGs) in bacteria. This thesis analysed and characterised MGEs including plasmids, transposons and insertion sequences (ISs) catalysing the mobilization of ARGs in A. baumannni clinical isolates. For the first time the carbapenemase blaNDM-6, a blaNDM-1 variant, was identified in a CRAb isolate recovered in northern Spain in 2019. The ST85 CRAb clustered together with the recently described and widespread clonal lineage IC9. Furthermore, in the latter isolate NDM-6 was detected in two copies and in a novel genetic environment linked to MGEs. Novel insights into MGEs harbouring ARGs in A. baumannii IC4, IC5 and IC7 representatives from Bolivia were observed. A diverse array of transposons, plasmids and resistance islands, such as strA and strB harboured by a large plasmid in the IC4 CRAb and by a chromosome encoded resistance island in the IC5 isolate, were identified in the present study. Furthermore, MGEs, e.g. a small plasmid or the Tn2008 carrying the blaOXA-23, were found across different ICs. These data reflect the prevalence of MGEs in A. baumannii and the strong link between the spread of ARGs and the mobilome. Bacteria can be clonal but yet different. The extent of clonal heterogeneity associated with MGEs in bacterial populations was also discussed for three extensive drug-resistant K. pneumoniae colonising isolates from Germany. The isolates were assigned to the high-risk clone ST147 and were armed in addition with a wide repertoire of ARGs, including the plasmid encoded carbapenemase OXA-181. Of particular interest was an IncR plasmid harbouring in total 12 resistance determinants and a wide variety of ISs. The K. pneumoniae isolates were closely related but diversity including inversions or deletions in the proximity to MGEs within the IncR plasmid was identified. Nevertheless, this genetic variation did not affect the resistance phenotype of the three K. pneumoniae isolates. These data demonstrated the influence of MGEs on genome plasticity and their contribution to heterogeneity within closely related isolates. Another example of clonal heterogeneity was detected in a CRAb outbreak in a German university hospital. The CRAb isolates harboured the acquired β-lactamase NDM-1 embedded in a transposon adjacent to the aminoglycoside modifying enzyme AphA6. Moreover, a second carbapenemase, OXA-23, was carried by the CRAb isolates. By cgMLST analysis the isolates were closely related and formed a transmission cluster. However, one patient was co-infected with two near identical CRAb isolates, i.e. one NDM-1-positive isolate and one isolate lacking NDM-1 and AphA6. Nevertheless, the NDM-1-negative isolate was still carbapenem-resistant because of the presence of blaOXA-23. The loss of the antibiotic resistance determinants NDM-1 and AphA6 could be attributed to a transposition event. MGEs associated with ARGs can lead to genetic variation during the course of an outbreak but not necessarily alter the resistance phenotype. Also, a co-infection of two closely related but phenotypically different E. faecium isolates was discussed in the present study. Both isolates were obtained from the same blood culture and were identified as ST203. Furthermore, one isolate was VREfm while the second was vancomycin-susceptible E. faecium (VSEfm). Molecular epidemiology of the two isolates revealed that the VREfm and VSEfm were closely related. In the VSEfm isolate, a 12 kb plasmid was detected which was also present in the VREfm isolate. However, in the VREfm the latter plasmid carried also the vanA gene cluster embedded in a Tn1546-type transposon as also the resistance genes ant(6)-Ia, erm(B) and cat-like and two more replicons indicating the presence of a chimeric plasmid. Here, MGEs and clonal diversity led to a heterogenous resistance phenotype within closely related isolates. Taken all together, this thesis documents the clonal spread and diversity of three hospital-acquired bacterial species but also highlights the dynamics of MGEs contributing to genome plasticity and the genetic diversity within closely related isolates