1 National Food Institute, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark3 Division of Epidemiology and Microbial Genomics, National Food Institute, Technical University of Denmark
The family of Enterobacteriaceae is comprised of Gram negative bacteria found in a variety of natural environments as well as in the gastrointestinal (GI) tracts of humans and many animals including diverse mammals, birds and reptiles. Three species of the enteric bacteria are largely responsible for causing infections both in humans and animals worldwide; these are Escherichia coli, Salmonella enterica and Klebsiella pneumoniae; ß-lactams are antimicrobials commonly prescribed to treat uncomplicated as well as severe infections caused by these Enterobacteriaceae and other Gram negative and also Gram positive bacteria. In particular, aminopenicillins, cephalosporins and carbapenems found broad application in treatment of infections caused by the aforementioned enteric species. Recently however, increasing levels of resistance to ß-lactams is observed in these key infectious agents as well as in many other previously susceptible species. This phenomenon has become a major public concern. Antimicrobials including ß-lactams have been often used in heavy amounts in farming, not only to treat the diagnosed infections in individual animals but also as prophylaxis, metaphylaxis and growth promotion. It is believed that these practices lead to the generation of reservoirs of antimicrobial resistance genes in the GI tracts of intensively reared food - production animals like pigs, poultry and cattle. Moreover, it has been previously shown that the bla genes (e.g. genes encoding resistance to ß-lactams) could be transmitted between different bacteria on mobile genetic elements (MGEs) like plasmids and variety of transposons. Evidences were also published indicating that zoonotic bacteria like E. coli or S. enterica resistant to diverse antimicrobials and harbouring plasmids might have been transmitted from farm animals to humans (farm workers, animal caretakers etc.). It has been therefore speculated whether the plasmids with the bla genes found in Enterobacteriaceae in humans could actually originate from the animal sources. The overall aim of this thesis was to verify if indeed related resistance plasmids can circulate between enteric bacteria from humans and food production animals; and if so, then which of these plasmid species are specifically associated with the epidemic types of blaTEM genes in Enterobacteriaceae. Furthermore, the association of the plasmid encoded blaTEM genes with transposable elements is also studied in order to get a broader perspective of which MGEs are involved in mobilization and spread of these bla genes in the diverse reservoirs. Finally, an attempt is made to encompass ecological aspects of plasmid driven transmission of resistance among the enteric bacteria. In the first study the relationship between plasmids harbouring blaTEM-52 genes isolated from humans, poultry and also meat products was examined. Twenty- two plasmids from a collection of E. coli and different serovars of S. enterica were characterized. The study delivered molecular evidences that epidemiologically related plasmids circulated in the diverse species of enteric bacteria and between humans and animals, and the possible transmission route could have been contaminated food products like meat. Two types of epidemic plasmids were detected in isolates of E. coli and S. enterica; namely undistinguishable IncI1 blaTEM-52 plasmids were found in human and poultry isolates of E. coli and S. enterica; also undistinguishable IncX1 plasmids were isolated from E. coli and S. enterica from human infections, poultry and meat products (from poultry, broiler and beef). The strains harbouring these plasmids were confirmed not to be clonally related, hence indicating the transmission of the plasmids between the different bacteria from humans and animals rather than isolation of the same bacterial clones from the different reservoirs. With relation to the study I, a range of other observations was made. On majority of the examined plasmids, the blaTEM-52 genes resided on the Tn3-related transposons. Further analysis of the genetic environment of these blaTEM genes resulted in the conclusion, that it was a defined type of Tn3-like element that harboured the blaTEM-52, namely the Tn2 transposon. This knowledge was later used in the second study to design more discriminatory PCR method that would allow for distinction of which transposon types (Tn1, 2 or 3) or insertion sequences (IS26) could be linked to the blaTEM genes of interest. Moreover, the initial typing of – as realized later the IncX1 –blaTEM-52 plasmids with the use of available standard PCR-based methods for replicon typing (PBRT) was unsuccessful. In the course of this study the whole plasmid from E. coli 2161 was sequenced and deposited in GenBank as plasmid pE001. It became apparent that the standard PBRT method targeted another group of IncX family of replicons, namely the IncX2 plasmids, which so far have been rather rarely detected in humans or animals. The replicon of the pE001 was designated in the published Manuscript I as an IncX1A. The reason to that was the discovery of dissimilarities between the replicon of pE001 and the replicon of an IncX1 plasmid called pOLA52. The latter was published before pE001 and was considered to carry a classical IncX1 replicon. In the study I, an incompatibility assay for the pE001 and the pOLA52 (variant with the deletion in blaTEM gene) was performed. The two plasmids turned out to be compatible, which was surprising considering the high degree of overall similarities between the two sequenced scaffolds. Based on these results it was concluded that the standard incompatibility assays may in some cases give a false reflection of the real relatedness of the examined plasmids. Combining this experience with the knowledge that the PBRT method is often sensitive to the sequence substitutions within the replicon scaffolds, another idea was generated. It was previously reported that the plasmids from Klebsiella pneumoniae often escaped the detection by the classical PBRT methods, which was originally designed based on E. coli replicons. Therefore in the third study, which will be described later in this summary, a novel method was elaborated for rapid detection and sub-classification of plasmids from this species. The blaTEM-52 genes that were the focus of the first sub-project are in fact evolutionary descendants of blaTEM-1 genes. The second study aimed therefore at verifying on which plasmids scaffolds these blaTEM predecessors are usually located in Enterobacteriaceae found in humans and food production animals like pigs, poultry and cattle. In this sub-project the focus was stated on the plasmids from E. coli, which is known to be either an indicator organism colonizing (as a commensal) both the human and animal GI tracts; or it may cause infections to its hosts. Evidences were found in the study II that either undistinguishable or similar blaTEM-1 plasmids circulated in different E. coli from humans and from animal sources in Denmark. Possibly epidemic blaTEM-1 IncI1 and IncB/O plasmids were found in humans and the diverse animals (pigs, poultry and cattle). Moreover, a larger variation of the transposable elements linked to the blaTEM-1 genes was detected on plasmids in the second study compared to blaTEM-52 plasmids. In the second study usually specific alleles of the blaTEM-1 genes resided on either the Tn2 (blaTEM-1b and blaTEM-1c) or Tn3 (blaTEM-1a) transposons. In many cases the insertions of IS26 elements upstream of the blaTEM-1 genes were detected by PCR. These results gave important clues not only regarding which plasmids but also which specific transposons might have served as platforms for mobilization and evolution of the blaTEM-1 to blaTEM-52 genes. In the third sub-project plasmids from K. pneumoniae from human infections and from surface waters (designated as environmental isolates) were typed. In this study the strains were not pre-selected based on the defined resistance markers. The results allowed for evaluating if there are differences in the replicons normally found on plasmids from humans and from the environment in this bacterial species. Additionally, these potentially host specific replicons could have been compared to the replicons of plasmids previously shown to be specifically associated with the resistance genes in the clinically relevant K. pneumoniae. At the time when this project was initiated the standard PBRT method often failed to detect the replicons from K. pneumoniae. Therefore a novel multiplex PCR (mPCR) was designedfor detection of these otherwise untypable plasmids. While this was pursued, updated protocols for PBRT were published by other authors and many of the sequenced plasmids from K. pneumoniae used as references for designing of the mPCR turned out to be the IncFIIk types. However, an interesting observation was made. Namely, in one of the previously sequenced K. pneumoniae strain MGH78578 apparently multiple plasmids with the similar incompatibility determinants IncFIIk were detected; this was opposing to the theory that the plasmids from the same incompatibility groups typically would not be able to co-exist in the same bacterium. In fact, a similar pattern was seen in study III in some of the examined K. pneumoniae strains from human infections and from the environment, where also multiple IncFIIk plasmids were present in the same isolates simultaneously, but these plasmids harboured diverse secondary replicons detected by the mPCR. The conclusion from the third study was that plasmids may acquire secondary replicons in order to persist in the given bacterium and to overcome the incompatibility phenomenon and this seems to be fairly common in K. pneumoniae. Analysis of literature data against the data from the study III resulted also in a conclusion, that the same replicons that are generally predominant in K. pneumoniae (IncFIIk, likely also IncR and yet unknown replicons) are often associated with a variety of the bla genes in the clinical strains of this species. In summary, the combined data from the three studies suggested that often the blaTEM plasmids are generally host specific to the species they were detected in. This is exemplified by IncFII, IncFI, IncB/O and IncI1 replicons in E. coli, IncI1 and likely IncX1 in S. enterica or IncFIIk in K. pneumoniae. Many of the broad host range replicons (IncP, IncA/C, IncR, IncL/M, IncN) were found rather occasionally in these hosts. Although some exceptions were seen, namely the IncP were often found with blaTEM-1 genes in particular in cattle, while IncA/C were often associated with blaTEM variants encoding Extended Spectrum ß- Lactamases in the diverse reservoirs. Evidences presented above indicate that the transmission of plasmids between animal and human Enterobacteriaceae is possible and it is likely that in some cases the resistance plasmids might have been delivered from animal to human strains via food chain. Further studies are needed to determine the chromosomal progenitors of the resistance genes like blaTEM-1. Determination of the very origins of resistance genes is crucial if further mobilization of these genes from the given source is to be prevented. Plasmids undoubtedly play a major role in transmission of bla genes also across the reservoirs. Solutions like whole genome sequencing should be preferentially applied in the future in order to efficiently detect, classify and tract the epidemiology of resistance plasmids in populations of Enterobacteriaceae.