1 Introduction
The therapeutic success of antibiotics is challenged by the spread of
antimicrobial resistance genes (ARGs) among bacteria that cause
healthcare-associated and community-acquired infections, an escalating
global health threat
(Arias & Murray,
2009; Davies & Davies, 2010; WHO, 2014). In 2017, the World Health
Organization released a list of pathogens against which healthcare
systems urgently need new antimicrobial alternatives due to their
commonly extensive antibiotic resistance profiles
(WHO, 2017). These include Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., Serratia spp. and several other taxa
commonly resistant to important beta-lactams, such as carbapenems and
third-generation cephalosporins. Other critical pathogens on this list
are fluoroquinolone-resistant Salmonella spp. and Shigella spp. These pathogens are closely related and belong to the family of Enterobacteriaceae . It is noteworthy that the medically-relevant
ARGs within this family have been primarily found encoded by conjugative
plasmids; mobile genetic elements that frequently mediate their
dissemination among bacteria through horizontal gene transfer (HGT)
(Carattoli, 2009).
The currently known variety of plasmids found within Enterobacteriaceae have been divided into 28 distinct groups
according to their incompatibility (Inc); i.e. their inability to
coexist over time in the same cell-line
(Couturier et al.,
1988; Novick, 1987; Rozwandowicz et al., 2018). In order to understand
the role of specific plasmid groups in the context of ARG dissemination,
it is important to understand their ecology, including the diversity of
their hosts (host range). Generally, plasmid host range denotes the
phylogenetic or taxonomic breadth of organisms that can carry a plasmid;
however, it can be further broken down into i) which recipients the
plasmid can be transferred into; ii) which recipients the plasmid can
replicate within; and iii) whether the plasmid is stably maintained over
time in the cell-line. The two initial stages are known as the transfer - and replication host range, respectively, and
the latter is termed evolutionary host range. Since these stages
occur sequentially, the host range narrows from one stage to the next
(Suzuki et al., 2010). The
evolutionary host range and the classification of plasmids are closely
related (Redondo-Salvo et
al., 2020), thus understanding the host range of archetypes
representing a plasmid group reveals crucial knowledge about plasmid
ecology. Recently, Redondo-Salvo and colleagues defined a host range
scale for plasmids, which grades host range according to the highest
taxonomic rank they distribute in; from I at the species level being
very narrow, e.g. IncFIB, to VI at the phylum level being very broad,
e.g. IncP1 (P1), which was shown to cross phylum level
(Redondo-Salvo et al.,
2020). Nonetheless, it is important to bear in mind that for specific
strains or taxa within the plasmid host range, barriers may exist that
prevent plasmid establishment. For example, host-encoded defense systems
can form effective barriers against HGT by blocking the entry of foreign
nucleic acids. These include restriction/modification (R/M) systems
(Oliveira et al., 2014),
CRISPR-Cas systems (Makarova
et al., 2019), and Wadjet systems
(Doron et al., 2018). In
response, plasmids have developed various systems to evade host defense
systems (anti-defense systems) e.g. Anti-R/M and Anti-CRISPR proteins,
which directly interact with and inhibit R/M and CRISPR-Cas systems,
respectively (Mahendra
et al., 2020; Roy et al., 2020). Thus measurements of distribution
breath, i.e. the taxonomic distance between the hosts in which a given
plasmid is found
(Redondo-Salvo et al.,
2020), only describe the potential host range of a plasmid.
One of the Enterobacteriaceae plasmid groups, the IncHI1A group
(HI1A), comprises conjugative plasmids typically in the size range of 75
to 400 kb (Rozwandowicz et
al., 2018). HI1A plasmids are associated with the dissemination of ARGs
such as the bla NDM-1 gene, which confers resistance towards
carbapenems, a last resort drug used when treating extended spectrum
beta-lactamase (ESBL) producing Enterobacteriaceae spp.
(Carattoli, 2013;
Dolejska et al., 2013). The transfer of some plasmids in the HI1A
group, such as R27, is thermo-sensitive and primarily conjugate below
30° C, a feature that is speculated to promote the transmission of ARGs
in the environment
(Maher & Taylor, 1993;
Sherburne et al., 2000). Additionally, plasmids in the HI1A group carry
genes encoding thick flexible pili, which enable high conjugative
transfer efficiency within both planktonic and surface-associated
bacterial communities, thus emphasizing their potential to disseminate
ARGs across a variety of environments
(Bradley et al., 1980).
Cultivation-based transfer experiments have found that HI1A plasmids can
also be transferred to bacteria belonging to genera outside Enterobacteriaceae, such as Vibrio spp. and Aeromonas spp. (Maher
& Taylor, 1993). Based on such experiments, and supported by
bioinformatic predictions
(Suzuki et al., 2010), HI1A
plasmids are believed to have a wide or intermediate host range.
However, an understanding of the initial host range stages, stage i) and
ii), of HI1A is crucial for an improved understanding of the frequency
and phylogenetic extent of plasmid-mediated HGT in the environment. Some
transfer events may never extend beyond these initial host range stages,
and therefore not result in stable plasmid-host association, however,
still result in transfer of the plasmid from this host. Thus, a
short-term host may function as a crucial stepping-stone for plasmids
reaching into stable hosts.
Wastewater treatment plants (WWTPs) have been suggested to facilitate
HGT of ARGs among bacteria
(Guo et al., 2017; Li et
al., 2018). Indeed, all known antibiotic resistance mechanisms have
been found represented in WWTPs reservoirs
(Rizzo et al., 2013), along
with multiple mobile genetic elements, including plasmids
(Rizzo et al., 2013;
Zhang et al., 2011). Furthermore, studies have shown that the microbial
communities of WWTPs are highly permissive towards broad host range
plasmids (i.e. highly capable of taking up a given plasmid)
(Jacquiod et al., 2017;
Li et al., 2018). Moreover, it has been revealed that a core-permissive
fraction of bacteria, capable of receiving several types of plasmids,
are abundant across WWTPs (Li
et al., 2018). In addition, certain bacterial taxa have repeatedly been
identified as a part of the core-permissive fraction of bacteria across
diverse environments
(Klümper et
al., 2015; Li et al., 2018; Musovic et al., 2014; Pinilla-Redondo et
al., n.d.).
In this study, we investigated the host range, including the initial
host range stages, and transfer efficiency of the HI1A plasmid R27 in
the microbial community of urban residential sewage collected at three
WWTPs located in urban areas of southern Sweden. We utilized a dual
fluorescent reporter gene platform, which previously has been used to
examine transfer of various plasmid groups, including IncI1
(Anjum et al., 2018,
2019) and P1
(Jacquiod
et al., 2017; Klümper et al., 2015; Li et al., 2018; Musovic et al.,
2014; Pinilla-Redondo et al., n.d.). This platform is based on a
plasmid-encoded green fluorescent protein (gfp ) gene under the
control of a lacIq repressible promoter. Donor
cells, which introduce the plasmid to the community, carry a chromosomalmCherry along with lacIq and hence,
although carrying the plasmid, do not express GFP. However, since
indigenous bacteria in the sewage community likely do not encodelacIq , which is a modified version oflacI , transconjugants express GFP constitutively. We used
fluorescence-activated cell sorting (FACS) for high throughput
identification of transconjugants in order to reveal the plasmids
transfer host ranges (henceforth referred to as host range).
Specifically, host range was investigated through post-mating sorting
and 16S rRNA gene amplicon sequencing analysis of taxonomic and
phylogenetic relationships of the transconjugant communities. Although
the permissiveness towards broad host range plasmids of the microbial
community in WWTPs have been assessed utilizing a similar strategy
(Jacquiod et al., 2017;
Li et al., 2018), the capability of the sewage transconjugant community
to further disseminate plasmids to potential pathogens have not been
investigated. Thus, subsequently, we assessed the donor potential of
sorted transconjugants by further enriching this fraction in plasmid
selective media to further track plasmid transfer to a model Enterobacteriaceae pathogen. In parallel, we performed the same
experiments with pB10, a representative of the well-studied P1 group
(Jacquiod
et al., 2017; Klümper et al., 2015; Li et al., 2018; Musovic et al.,
2014; Pinilla-Redondo et al., n.d.), which we hypothesized to represent
the maximal host range grade VI
(Redondo-Salvo et al.,
2020).