Various bacteria can cause human diseases. They spread directly from person to person
or indirectly via various environmental matrixes, such as food and water. Major food-
and waterborne pathogens include Campylobacter, Listeria, Salmonella, Shigella, Shiga
toxin-producing Escherichia coli, Salmonella, Yersinia, and Vibrio (31). Most of these
pathogens spread through the fecal-oral route. Their primary hosts include humans,
farm animals (e.g., cows, pigs, and chickens), and wildlife (e.g., deer, birds). These
hosts contribute to the spread of pathogens. For example, geese and other birds are
known to harbor diverse Campylobacter (29, 40, 59) and Salmonella spp. (40). However,
some of these pathogens also survive for long periods of time and even grow in environments
such as water, soil, sediment, and algae (13, 22, 32), in many cases in association
with or by forming biofilms (35, 36, 52).
Since difficulties are associated with detecting various pathogens in a timely manner,
the microbial quality of food and water has been monitored using so-called fecal indicator
bacteria (FIB), such as E. coli and enterococci (22, 24). Although their primary habitats
are the gastrointestinal tracts of warm-blooded animals (18, 49), some FIB strains
are more adapted to soil or other environments (22, 24, 37). Moreover, alternative
FIB, such as Bacteroides, have been used to identify the occurrence of pathogens and
their potential sources of contamination (28, 59). However, poor correlations have
been reported between pathogen and FIB concentrations (23, 58), which limits the use
of FIB for predicting the occurrence of pathogens.
Some opportunistic pathogens, including Mycobacterium avium and Legionella pneumophila,
are not of a fecal origin. These opportunistic pathogens use environments such as
water distribution systems (11, 14, 15) and showerhead biofilms (12) as their primary
habitats, and occasionally infect humans to cause diseases. Furthermore, various environmental
bacteria have been reported as emerging pathogens. Among these, Arcobacter spp. are
of great interest because this genus is frequently and abundantly detected in many
wastewater treatment plants (10, 50). This genus is phylogenetically closely related
to Campylobacter, but is metabolically more versatile and can grow at relatively low
temperatures and with a wider range of O2 concentrations (9). Some members of Arcobacter
have also been reported to form symbiotic relationships with protists (16). A better
understanding of the ecology of these environmental pathogens is essential for preventing
their occurrence and spread (39, 47).
Antibiotics are one of the most important scientific discoveries to combat pathogens.
Antibiotic treatments save millions of lives each year worldwide. However, nearly
a century of antibiotic use and misuse has resulted in the evolution of the resistance
of bacterial pathogens to most antibiotics approved for medical use (56). In addition
to clinical settings, antibiotic resistance is an issue in environments. Raw sewage
is one of the major reservoirs of antibiotic-resistant bacteria (ARB) and antibiotic
resistance genes (ARGs) (45). Farm animals, including cows, pigs, and horses, can
also harbor various ARB, some of which are pathogenic to humans (8, 33, 55). These
ARB/ARGs contaminate surrounding and downstream environments (3, 30). Wildlife can
also contribute to the spread of ARB/ARGs. Migratory birds are most likely responsible
for the spread of ARB/ARGs to wide areas, including remote Arctic islands (34) and
Antarctica (48).
Horizontal gene transfer (HGT) plays an important role in the spread of ARGs among
diverse bacteria (4, 44). Some ARGs are found on mobile genetic elements, such as
insertion sequences and transposons, and can be transferred between cells when mediated
by plasmids, integrative conjugative elements (ICEs), or bacteriophages (44). The
HGT of ARGs can occur in environments such as wastewater treatment plants (45). In
addition to conjugation (mediated by plasmids and ICEs) and transduction (mediated
by bacteriophages), ARGs can be transferred between cells via extracellular vesicles
(6, 53, 54) as well as when bacteria take up naked DNA (i.e., transformation). A recent
study demonstrated that the grazing activities of Ciliates enhanced the release of
ARGs from bacterial cells into the environment (5). These naked ARGs can be taken
up by bacteria, thereby transforming them to be resistant to antibiotics. To prevent
the spread of ARB/ARGs, it is necessary to understand the mechanisms and frequencies
of ARG acquisition in environments.
The major challenges associated with the study of pathogens and ARB/ARGs in environments
include (i) various types of targets, (ii) low concentrations, (iii) intra-species
diversity, (iv) previously uncharacterized target genes, and (v) the occurrence of
HGT. However, we can see great opportunities in these challenges. Recent technological
advances have provided various tools to explore these opportunities. Three notable
tools are briefly summarized below.
High-throughput quantitative PCR
Quantitative PCR (qPCR) is commonly used to detect pathogens and ARGs. The advantages
of qPCR include its high sensitivity and specificity. However, with conventional qPCR,
a large number of runs are needed to detect many targets. To overcome this issue,
high-throughput qPCR platform has been developed using microfluidic technology. In
microfluidic qPCR (MFQPCR), multiple qPCR assays are simultaneously run for many samples
in nanoliter-volume chambers that are present in high densities on a chip. MFQPCR
technology has been used to quantitatively detect various pathogens and ARGs in many
environmental samples (1, 7, 23, 34, 46, 48, 57). Amplicons generated on the chip
can also be recovered and sequenced for further analyses (41). The MFQPCR approach
is particularly useful when target pathogens/ARGs are known, their concentrations
are low, and many target genes/samples need to be analyzed; therefore, it can overcome
challenges (i) and (ii) described above.
Metagenomics and amplicon sequencing
High-throughput sequencing technology has greatly advanced our understanding of microbial
ecology in various environments (19, 38). Culture-independent, high-throughput sequencing
of the 16S rRNA gene fragment is most commonly performed (21, 60); however, other
applications, such as (meta)genomics and (meta)transcriptomics, are also frequently
used (19, 25, 38). Since pathogenic and non-pathogenic strains are both present within
a genus/species (e.g., pathogenic E. coli), it is difficult to identify the presence
of pathogens in a sample by the high-throughput 16S rRNA gene sequencing approach
alone. However, sequencing the amplicons of pathogen-specific genes is useful for
analyzing the diversities of target pathogens without bacterial isolation (15, 59),
and can be used to rapidly identify the sources of pathogen contamination. This approach
can overcome challenge (iii) described above.
A metagenomic approach is also useful for detecting ARGs (2, 26, 27). Since this approach
is independent of PCR bias, it can detect previously unknown ARGs, and therefore,
overcome challenge (iv) described above. However, it could be difficult to detect
genes that are present at low abundance. Furthermore, it would become expensive when
analyzing many samples.
Single-cell analysis
Various single cell-based approaches have been developed and applied to detect, quantify,
isolate, and identify target pathogens and ARB. For example, a simple improvement
in the fluorescence in situ hybridization (FISH) protocol greatly increased the detection
efficiencies of Enterobacteriaceae cells (17). The combination of direct viable counts,
multi-probe FISH, and solid-phase cytometry allows researchers to quantify viable
Vibrio spp. (13). Fluorescently labeled E. coli O157:H7 cells can be individually
isolated by flow cytometry-fluorescence-activated cell sorting (FACS) for downstream
analyses (43).
In addition, a single-cell PCR or genomics approach can link the phylogeny and function
of microbes. For example, single-cell PCR technology, called epicPCR (emulsion, paired
isolation and concatenation PCR) (51), has been applied to identify ARB in wastewater
samples without cultivation (20). In epicPCR, individual bacterial cells are stochastically
encapsulated in polyacrylamide gel beads, in which fragments of the 16S rRNA gene
and a functional gene, such as ARG, are co-amplified and fused. Amplified fused PCR
products are recovered and sequenced to link functional gene sequence information
with that of phylogenetic markers (51). Although there is room for further technological
refinement (e.g., to make the size of beads even), this approach is particularly useful
for the study of ARB because it has the ability to identify the host of ARGs in relatively
high throughput. With this approach, it would be possible to culture-independently
identify antibiotic-resistant pathogens, which is of the greatest concern for public
health.
Concluding remarks
As briefly summarized in this note, great opportunities are associated with studying
the ecology of pathogens and antibiotic resistance in environments. The approaches
introduced here have their own strengths and weaknesses. Although not explained in
detail, the traditional culture-based method is still important, particularly when
detecting resistance acquired by gene mutations (42). These methods complement each
other and there is no true optimum method. Researchers need to select the most suitable
approaches depending on their scientific objectives and goals.
The ecology of pathogens and antibiotic resistance in environments is one of the hot
topics in environmental microbiology and microbial ecology. This topic is among the
scope of Microbes and Environments, and its editorial board welcomes the submission
of manuscripts on this topic to our journal.