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. 2023 Aug 14;89(8):e00371-23. doi: 10.1128/aem.00371-23

Broad-Host Dissemination of Plasmids Coharboring the fos Operon for Fructooligosaccharide Metabolism with Antibiotic Resistance Genes

Tomas Nohejl a,b, Jana Palkovicova b, Kristina Nesporova b, Adam Valcek b,c,d, Jarmila Lausova a,b, Monika Dolejska a,b,e,f,✉
Editor: Martha Vivesg
PMCID: PMC10467340  PMID: 37578374

ABSTRACT

The fos operon encoding short-chain fructooligosaccharide (scFOS) utilization enables bacteria of the family Enterobacteriaceae to grow and be sustained in environments where they would struggle to survive. Despite several cases of the detection of the fos operon in isolates of avian and equine origins, its global distribution in bacterial genomes remains unknown. The presence of the plasmid-harbored fos operon among resistant bacteria may promote the spread of antibiotic resistance. A collection of 11,538 antimicrobial-resistant Enterobacteriaceae isolates from various sources was screened for the fosT gene encoding the scFOS transporter. Out of 307 fosT-positive isolates, 80% of them originated from sources not previously linked to fosT (humans, wastewater, and animals). The chromosomally harbored fos operon was detected in 163/237 isolates subjected to whole-genome sequencing. In the remaining 74 isolates, the operon was carried by plasmids. Further analyses focusing on the isolates with a plasmid-harbored fos operon showed that the operon was linked to various incompatibility (Inc) groups, including the IncHI1, IncF-type, IncK2, IncI1, and IncY families. Long-read sequencing of representative plasmids showed the colocalization of fos genes with antibiotic resistance genes (ARGs) in IncHI1 (containing a multidrug resistance region), IncK2 (blaTEM-1A), IncI1 [sul2 and tet(A)], and IncY [aadA5, dfrA17, sul2, and tet(A)] plasmids, while IncF-type plasmids had no ARGs but coharbored virulence-associated genes. Despite the differences in the locations and structures of the fos operons, all isolates except one were proven to utilize scFOSs. In this study, we show that the fos operon and its spread are not strictly bound to one group of plasmids, and therefore, it should not be overlooked.

IMPORTANCE It was believed that members of the family Enterobacteriaceae are unable to grow under conditions with short-chain fructooligosaccharides as the only source of carbon. Nevertheless, the first Escherichia coli isolate from chicken intestine was able to utilize these sugars owing to the chromosomally harbored fos operon. Studies on E. coli isolates from horses discovered the horizontal transfer of the fos operon on IncHI1 plasmids along with genes for antibiotic resistance. The first plasmid detected was pEQ1, originating from the feces of a hospitalized horse in the Czech Republic. Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon in the Netherlands, Germany, Denmark, and France among healthy horses. Despite several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The fos operon possibly plays a role in the adaptation of plasmids among resistant bacteria and therefore may promote the spread of antibiotic resistance.

KEYWORDS: Escherichia coli, MinION, scFOS, sequencing

INTRODUCTION

Short-chain fructooligosaccharides (scFOSs) are soluble fibers naturally found in edible plants such as chicory, onion, garlic, asparagus, and wheat. scFOSs, as a mixture of oligosaccharides, are not digestible by enzymes of human or animal origin. Nevertheless, they act as prebiotics and thus can be fermented in the large intestine by several bacterial genera such as Bifidobacterium and Lactobacillus (1). scFOS fermentation enhances the production of short-chain fatty acids, acidifies the large intestine, and therefore provides selective advantages to probiotic bacteria in the intestinal tract that lead to the suppression or displacement of undesirable or pathogenic bacteria. Metabolites produced during scFOS fermentation, especially butyrate, may repair and enhance the barrier of intestinal epithelial cells and maintain host-microbe homeostasis. Butyrate promotes the proliferation of advantageous bacteria (Bifidobacterium and Lactobacillus) and inhibits potentially pathogenic bacteria (Staphylococcus, Escherichia coli, Salmonella, and others) (2, 3).

It was proposed previously that Enterobacteriaceae are unable to grow under conditions where prebiotics are the only sources of carbon (4). Nevertheless, the first report of an avian extraintestinal pathogenic Escherichia coli isolate that utilizes common prebiotics, BEN2908 from chicken gut, was published in 2006 (5). The E. coli BEN2908 isolate was able to utilize scFOSs owing to the fos operon, consisting of seven genes (fosK, fosY, fosGH2, fosX, fosGH1, fosT, and fosR) carried on the chromosome. The fos operon therefore enables a proportion of opportunistic pathogens such as E. coli to grow and be sustained in environments where they would struggle to survive.

Since scFOSs are commonly supplemented in human and animal nutrition as prebiotics (1, 4, 6), the metabolism of these sugars mediated by mobile genetic elements could play a key role in the evolution of bacteria, including antibiotic-resistant pathogens. Previous studies suggested the horizontal transfer of the fos operon onto IncHI1 plasmids along with genes encoding antibiotic resistance (antibiotic resistance genes [ARGs]) (7–9). The first detected fos operon-harboring plasmid was pEQ1, obtained from an E. coli isolate originating from the feces of a hospitalized horse in the Czech Republic (7). Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon among E. coli isolates from healthy horses in the Netherlands, Germany, Denmark, and France. A strong linkage among the fos operon, the multidrug resistance (MDR) region, E. coli sequence type 1250 (ST1250), and horses has been recognized (8, 9).

The horizontal gene transfer of mobile genetic elements, especially plasmids, is crucial for the dissemination of antimicrobial resistance (AMR) as well as other traits among bacteria from humans, animals, and the environment (10). The fos operon acquired by plasmids among resistant strains may promote the spread of AMR in bacterial populations. However, besides several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The goal of this study was to determine the occurrence and genetic context of the fos genes in a large collection of antibiotic-resistant isolates from diverse sources. Furthermore, we aimed to reveal the occurrence and characteristics of plasmids associated with genes for scFOS metabolism along with AMR in order to evaluate the potential risk of fos operon and antibiotic resistance comobilization.

RESULTS

The fosT-positive isolate collection.

We obtained 307 isolates harboring the fosT gene encoding an scFOS transporter. The fosT-positive isolates corresponded to about 2.66% of all screened isolates (307/11,538). Subsequently, 237 representative isolates were selected based on metadata and subjected to short-read whole-genome sequencing (WGS). A total of 87.76% (208/237) of the fosT-positive isolates originated from sources that had not been linked with fosT previously (excluding horse and poultry isolates). These included human (blood; skin and soft tissue; nasal, oral, throat, and genital smears; sputum and urine from infected adult patients; and rectal swabs from healthy newborns), animal (feces of wildlife birds and rectal swabs from food-producing and companion animals), and environmental (swabs from stable environments and wastewater from municipal wastewater treatment plants [WWTPs]) isolates (Fig. 1).

FIG 1.

FIG 1

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Assessment of the location of the fos operon location compared to its isolate source. The 237 representative isolates selected for WGS originated from 178 animal, 35 environmental, and 24 human samples.

Sequence analysis revealed that 151/237 (63.71%) isolates had an MDR profile (defined as resistance to 3 or more different antibiotic groups). The most commonly detected genes were those encoding resistance to aminoglycosides [aph(3″)-Ib and aph(6)-Id], beta-lactams (blaACT-12, blaCTX-M-1, and blaTEM-1), trimethoprim (dfrA17), fosfomycin (fosA2), sulfonamides (sul2), and tetracycline [tet(A) and tet(B)]. The representative isolates consisted mainly of E. coli isolates (180/237; 75.95%), but 57/237 (24.05%) belonged to other species such as Enterobacter cloacae (n = 35), Klebsiella spp. (n = 9), Citrobacter freundii (n = 8), Escherichia vulneris (n = 3), and Buttiauxella spp. (n = 2). A chromosomal location of the fos genes was found in 163 isolates, while in the remaining 74 isolates (all E. coli), the genes were harbored by plasmids (Fig. 1).

The plasmid-located fos operon.

Plasmids carrying the fos operon were from strains of diverse (animal, n = 45; environmental, n = 17; human, n = 12) origins and included the IncHI1 (n = 26), IncF-type (n = 26), IncK2 (n = 14), IncI1 (n = 4), and IncY (n = 4) families (Fig. 2).

FIG 2.

FIG 2

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Assessment of the plasmid-located fos operon compared to its isolate source. The 74 isolates harboring the plasmid-located fos operon originated from 45 animal (“A”), 17 environmental (“E”), and 12 human (“H”) samples.

Complete sequences of plasmids representing each group, including IncF-type (n = 4), IncK2 (n = 4), IncI1 (n = 1), and IncY (n = 4) plasmids, were obtained in this study, while the sequence of the previously reported plasmid pEQ1 (7) was used as a representative of the IncHI1 group. The plasmids within these families were highly similar in coverage and identity (see Table S1 in the supplemental material). All of the fosT-positive isolates (n = 237) were able to utilize the tested scFOS-containing prebiotics and nystose, with the exception of one isolate harboring a fos operon located on an IncK2 plasmid.

Phylogenetic analysis of isolates carrying a plasmid-borne fos operon.

Phylogenetic analysis revealed the vast variability of E. coli isolates (n = 74) harboring a plasmid-located fos operon (Fig. 3 and Fig. S1). The constructed phylogenetic tree was divided into the three main clusters (pink, blue, and green) by plasmid groups harboring a fos operon. The clustering also corresponded to the divergence of the integrity of the fos operon.

FIG 3.

FIG 3

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Genomic comparison of E. coli isolates harboring a plasmid-located fos operon. Colored boxes indicate the presence of a complete fos operon consisting of the seven fos genes fosK, fosY, fosGH2, fosX, fosGH1, fosT, and fosR (dark blue). The numbers of ARGs are depicted via a heatmap. An asterisk in front of an isolate name signifies our selection for long-read sequencing. CZ, Czech Republic; DE, Germany; IC, Ivory Coast; SK, Slovakia.

The spread of specific E. coli lineages, including isolates with high levels of similarity from various sources, carrying unique plasmids was observed in the pink and blue clusters. IncK2 plasmids of sequence type 103 (ST103) with incomplete fos operons were linked mostly to E. coli ST115 in the pink cluster. These strains were collected in different years and originated from diverse sources. Some of the strains were closely related and differed by fewer than 10 single nucleotide polymorphisms (SNPs), such as isolates obtained from kites, WWTPs, and a broiler. However, generally, even the isolates within E. coli ST115 were quite variable as they differed in 2 to 1,873 SNPs. A major part of the blue cluster consisted of E. coli ST93 isolates associated with IncF type [F–:A1:B1] harboring a complete fos operon. Even though E. coli ST93 isolates showed differences of 1 to 1,275 SNPs, more than half of the strains were closely related (difference of 1 to 18 SNPs). The green cluster contained E. coli isolates of various STs carrying diverse plasmids. However, IncHI2/ST9 plasmids containing a complete fos operon and associated with ARGs were the most predominant plasmids in this cluster. Clonal spread of E. coli ST1250 associated with this plasmid was observed, with zero SNP differences; only one strain (T18B_scFOS) was unrelated to the other E. coli ST1250 isolates. Another clone associated with IncHI1 found within the green cluster was E. coli ST224 showing a high level of clonality (0 to 15 SNPs). IncF type [F–A–:B1] was detected in E. coli ST88 and ST1800 isolates. Differences of 275 to 1,320 SNPs were observed in E. coli ST88 isolates, indicating their disparity; meanwhile, E. coli ST1800 isolates showed a relatedness of 0 to 69 SNPs, with the majority representing close relationships (0 to 8 SNPs). The dissemination of IncHI1/ST9 and IncF-type [F–:A1:B1] plasmids across different E. coli clones underlines the epidemiological importance of these plasmids as well as the potential for the horizontal transfer of plasmid-mediated fos operons among unrelated strains.

Integration of the fos operon.

The genetic environments of the fos genes varied among the individual plasmid groups (Fig. 4). A complete fos operon was associated with the insertion sequence (IS) family IS1 among IncHI1, IncY, and IncF-type plasmids. The IncHI1 plasmids mobilized the fos operon by a 24-kb element flanked by IS5 and Tn7-like elements. IS91 mediating rolling-circle transposition was present in the IncK2 and IncI1 groups with an incomplete fos operon accompanied by genes encoding the utilization of rhamnose (rhaM and rhaD). Despite these similarities, the means of fos operon integration were not distinguishable based on a comparison of sequences close to the insertion sequences. However, the integration of the fos operon and its genetic surroundings were similar within the plasmid groups.

FIG 4.

FIG 4

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Linear comparison of the fos operons located on various plasmids. The plasmid sequences obtained in this study and previously reported plasmid pEQ1 (IncHI1) (GenBank accession number KF362121) were compared based on a cluster alignment of the coding sequences. Red represents the fos operon, blue represents insertion sequences, green represents genes encoding the utilization of an unrelated saccharide (rhamnose), and gray represents accessory genes. Δ represents an incomplete gene or sequence. Purple vertical lines indicate 7-bp direct repeats (DR) of the 24-kb element insertion site in the IncHI1 plasmid. Vertical blocks between sequences indicate regions of shared identity of >95%. DMSO, dimethyl sulfoxide; HP, hypothetical protein.

IncHI1 plasmids.

A Basic Local Alignment Search Tool (BLAST) Ring Image Generator (BRIG) comparison of 26 IncHI1 plasmid sequences showed that they are highly conserved, with discrepancies being found mainly in variable regions containing ARGs (Fig. S2). The IncHI1 plasmids originated mainly from horse-related isolates, with the exception of two isolates (p152_K-B_Kac_scFOS and p55_K-B_Kac_scFOS) from food-producing animals, namely, rectal swabs from pigs from the Czech Republic. All of the plasmids harbored identical gene contents, with some differences in the MDR regions, and none of them carried virulence-associated genes (VAGs). The MDR regions contained genes conferring resistance to cefotaxime (blaCTX-M-1), chloramphenicol (catA1), gentamicin [aac(3)-IId], macrolides [mph(A)], aminoglycosides [aph(6)-Id, aph(3″)-Ia, and aph(3″)-Ib], sulfonamides (sul2), tetracycline [tet(B)], and trimethoprim (dfrA17). As described previously (7), the genetic surroundings of the fos operon (24-kb element) consisted of a 9,253-bp fos operon that was flanked by two IS903-like elements from the IS5 family and a Tn7-like transposon downstream from the second IS903-like element. Moreover, the 24-kb element was surrounded by 7-bp direct repeats on both sides, providing evidence of a transposition event.

IncF-type plasmids.

A BRIG comparison of 26 IncF-type plasmid sequences revealed differences in their backbones that separated them by plasmid replicon sequence type (RST) (Fig. S3). They belonged to 5 groups, including [F–:A–:B1] (n = 10), [F–:A1:B1] (n = 9), [F–:A6:B1] (n = 5), [F77:A1:B1] (n = 1), and [F–:A8:B]– (n = 1). All of them lacked ARGs but harbored VAGs. These plasmids carried a virulence region surrounded by IS1A-like elements on each side, which was composed of sitA, sitB, sitC, and sitD, ensuring iron transport; iucA, iucB, iucC, and iucD, providing aerobactin synthesis; and iutA, encoding an aerobactin receptor. All of the IncF-type plasmids except for one (pB0034186_scFOS) also harbored other virulence genes (iss2, iroB, iroC, iroD, iroE, and iroN) commonly associated with avian-pathogenic E. coli and encoding salmochelin production, glucosylation, transport, and processing. In addition, all plasmids contained genes that promote their persistence within bacterial hosts by toxin-antitoxin systems (ccdA-ccdB, relE-parE, and vapB-vapC).

A linear comparison of four IncF-type plasmids subjected to long-read sequencing was performed using the most similar plasmid sequences from GenBank, under accession numbers CP081193 (child feces from China, 158,111 bp, harboring the fos operon, and not harboring ARGs), with a coverage of 99% and an identity of 99.92%, and CP043737 (retail chicken meat from the United States, 179,651 bp, not harboring the fos operon, and harboring ARGs), with a coverage of 90% and an identity of 99.62% (Fig. S4). In these plasmids, the fos operon was incorporated into the plasmid variable region, localized downstream of the truncated genes for the fertility inhibition protein FinO and the conjugal transfer pilus acetylase TraX. Nevertheless, other complete variants of FinO and TraX, together with a recombinase family protein, a hypothetical protein, and an endonuclease, were present within the region between the IS1 sequence and the fos genes. This region probably represents a relic of an earlier transfer of the fos operon via IS1.

IncK2 and IncI1 plasmids.

Fourteen isolates harboring the fos operon on an IncK2 plasmid were obtained, and four of them were subjected to long-read sequencing. Similarly, for fos operons located on an IncI1 plasmid of either ST3 (n = 1) or ST103 (n = 3), one plasmid of ST103 was sequenced on the MinION platform. A BRIG comparison revealed major similarities in the sequences of the IncK2 and IncI1 plasmids (Fig. S5). In comparison to pEQ1 and other plasmid families analyzed in this study, the fos operons of both IncK2 and IncI1 plasmids consisted of only four genes (ΔfosR, fosT, fosGH1, and ΔfosX) and were surrounded by IS91 upstream of incomplete fosR (ΔfosR). Downstream of ΔfosX were genes encoding the metabolism of rhamnose (rafY, rhaM, and rhaD). In the case of pOV-CH82_scFOS, the IS66 family ISCro1-like element was inserted into the rhaD gene. Short-read data analysis of isolate P-88_scFOS showed that IS91 was inserted into the fosT gene. Despite the structural differences in the fos operons compared to those of other plasmid groups, the strains harboring these plasmids were able to utilize scFOSs. The exception was isolate P-88_scFOS, in which fosT was disrupted by the insertion of IS91 and the fosR sequence was completely missing (Fig. S5A). The IncK2 and IncI1 plasmids carried various ARGs, including blaTEM-1A, sul2, or tet(A), but the contents of the particular genes varied across plasmids.

By employing a hybrid assembly of short and long reads, four complete IncK2 plasmid sequences and one IncI1 plasmid sequence were obtained. Compared to the most similar plasmid from GenBank, under accession number CP043035 (human blood from China and 108,364 bp), the obtained IncK2 plasmids showed 78.00% coverage and 96.07% identity (Fig. S6). The obtained IncI1 sequence showed a high level of similarity to a plasmid under GenBank accession number CP057400 (pig feces from the United Kingdom with 87% coverage and 99.99% identity) that harbored a fos operon and a plasmid under GenBank accession number HE654725 (mouse intestine from Switzerland with 79% coverage and 98.88% identity) that did not carry the fos operon (Fig. S7).

IncY plasmids.

A BRIG comparison of four IncY phage-like plasmids revealed high levels of similarity of their sequences (Fig. S8). All of the plasmids carried genes conferring resistance to aminoglycosides (aadA5), sulfonamides (sul2), and trimethoprim (dfrA17). Plasmid p1505h_scFOS also harbored the tet(A) gene conferring tetracycline resistance. IncY plasmids were intertwined with IS1 sequences. Also, together with IS1, an IS5 family element (ISKpn26) was found near the fos operon. The number of sequences of IS1 suggests its possible use for fos operon mobilization; nevertheless, this could not be proven from the WGS data.

Four complete sequences of IncY plasmids obtained by short- and long-read sequencing showed 77% coverage and 99.99% identity to the most similar plasmid from GenBank, under accession number CP086663 (chicken intestine from China, 94,190 bp, and not harboring the fos operon) (Fig. S9).

DISCUSSION

Short-chain fructooligosaccharides, commonly present in plants, are among the prebiotic substances that have been shown to selectively stimulate the growth and activity of certain bacterial species. It is assumed that scFOSs are metabolized by probiotic bacteria of the microbiota present in the intestine, leading to a decrease in the pathogenic bacterial population (6). Plasmid-mediated saccharide metabolism could therefore play a key role in the adaptation of bacteria, including MDR pathogens. As such, the fos operon may contribute to strain fitness in the intestinal tract of the host. Moreover, it may also contribute to the coselection and persistence of the array of ARGs harbored by plasmids even in the absence of antibiotic selective pressure.

In this study, we identified plasmids harboring the fos operon for scFOS utilization. Plasmids carrying the fos operon also carried genes encoding resistance to antibiotics or virulence properties. We detected previously described IncHI1 (ST9) plasmids (7), but for the first time, we also identified plasmids of the IncF-type, IncK2, IncI1, and IncY families harboring the operon. These plasmids originated from genetically diverse bacteria of animal, human, and environmental origins. The fos operon was found mainly in animal isolates; nevertheless, there is a notable bias in the interpretation of the distribution of the fos operon due to the high number of animal isolates (66.21%) within the tested collection.

IncHI1 family plasmids were among the most common plasmids harboring the fos operon, and they all contained ARGs for resistance to multiple antimicrobials. However, all of the IncHI1 plasmids came from one study focusing on horse-related isolates from an equine clinic in the Czech Republic. IncHI1 plasmids harboring a fos operon in equine isolates were also recently detected in other studies (7–9). The connection of the fos operon to bacteria of the horse gut is probably related to the high levels of scFOSs in their diet, as discussed previously (11). Despite the undeniably beneficial effects of scFOSs (12), the spread of IncHI1 plasmids commonly harboring ARGs together with the fos operon might be promoted by scFOS supplementation.

The only identified exceptions for the origins of IncHI1 plasmids harboring a fos operon in this study are food-producing animals, namely, two pig isolates from the Czech Republic. Both isolates came from rectal swabs from healthy pigs collected in 2007 in one study (13). So far, no link has been established between the Czech pig farm and any horse environment. Therefore, the gain of the fos operon in food-producing animals remains to be elucidated. Interestingly, E. coli isolates harboring IncHI1 plasmids originating from pigs belonged to the same STs (ST224 and ST3858) and clades as those of the other isolates originating from the equine study. Recently, an IncHI1 plasmid of the same ST (ST9) harboring the fos operon was also detected in an E. coli isolate from 2016 originating from a domestic animal (dog from Canada) for the first time (14). An IncHI1/ST9 plasmid previously connected to solely equine isolates was found spreading in food-producing and domestic animals. Presumably, the fos operon is beginning to spread through bacterial populations and not only on IncHI1 plasmids.

The second most common plasmids carrying the fos operon were those of the IncF-type family. As recently described, ARGs and probably subsequently obtained metabolic genes stay behind a possible bacterial evolutionary advantage of numerous IncF-type plasmids (15). Palomino et al. suggested that IncF-type plasmids and probably other plasmid groups coharboring metabolic genes and ARGs may promote the spread of antibiotic resistance (15). They concluded that metabolic genes alone may mediate higher levels of bacterial growth and reduce the death rate in the presence of antibiotics. They also suggested that the selection of metabolic genes together with ARGs may be supported by antibiotic treatment. In our study, IncF-type plasmids did not carry ARGs, but they harbored the fos operon along with VAGs present in their variable regions. The detected VAGs, encoding siderophores, chelating agents for iron uptake, are associated with the survival of cells under the conditions of a low-iron environment (16). Our IncF-type plasmids carried genes encoding colicin V with bactericidal activity and its counterpart, the colicin V immunity protein, likely increasing their competitiveness with closely related bacterial cells (17). Despite the diverse origins and genetic backgrounds of the E. coli strains, the IncF-type plasmids shared the same genetic background of fos operon integration. Nevertheless, the acquisition process was not successfully verified by sequence analysis of flanking bases near ISs.

The IncI1 and IncK2 plasmids contained an incomplete fos operon and were associated with antibiotic resistance (18). Although these plasmids lacked the fosK, fosY, and fosGH2 genes and the fosX gene was truncated, phenotypic testing verified that bacteria carrying these plasmids are able to utilize scFOSs as the only sources of carbon. The only exception was an isolate that lacked, by an IS91 insertion, complete fosT and fosR genes. These findings highlight the previously described importance of the fosT transporter for scFOS utilization (6). Nevertheless, the genes encoding saccharide metabolism are crucial for successful scFOS utilization, but they would not fulfill their purpose without the fosT transporter. These plasmids carried genes that might mediate the utilization of rhamnose instead. Rhamnose can be found in plants of the genus Rhamnus and Uncaria and is also produced by microalgae, and it can be used as the sole source of carbon for multiple microorganisms (19). Genes encoding the l-rhamnulose-1-phosphate aldolase RhaD, the l-Rha mutarotase RhaM, and the carbohydrate porin RafY found on IncI1 and IncK2 plasmids were linked to IS91 and were probably transferred by it. IS91 poses a new way for fos operon mobilization compared to the one described previously (7).

The IncY phage-like plasmids may be the missing links in fos operon mobilization due to the resemblance of their integration and genetic background to those of the original E. coli isolate BEN2908 (GenBank accession number AY857617). Compared to the original isolate, the IncY plasmids showed 99.7% identity in the fos operon, including the nearby IS1 element. Moreover, in these plasmids, this operon was also associated with IS5 (ISKpn26), a mobile genetic element that was identical to the one present in the IncHI1-type plasmids. Interestingly, the IncY plasmids have often been reported to be accompanied by IncF-type, IncI, or IncHI plasmids within the same cell (18). These allegations fit the profile of the plasmids detected and obtained in this study. Our results suggest that IncY plasmids might serve as the transitional links for the fos operon between the chromosome and other plasmids. Nevertheless, this hypothesis still needs to be studied thoroughly.

The issue of the importance of metabolic genes for the spread of antibiotic resistance remains. Is the fos operon advantageous to the cell or merely cooccurring on plasmids harboring ARGs? It was proven, in vitro, that the fos operon together with scFOS supplementation may promote cell fitness and give a competitive advantage to E. coli BEN2908 (6). Future research should demonstrate if the connection between the fos operon and ARGs is advantageous as a way of promoting the spread of antibiotic resistance. Here, we show that the spread of the fos operon among plasmids harboring ARGs should not be overlooked, especially since the fos operon is not strictly bound to one group of plasmids.

MATERIALS AND METHODS

Antibiotic-resistant isolate collection.

A collection of over 11,538 AMR isolates consisting mostly of E. coli isolates was obtained between 2005 and 2019 from multiple studies using selective cultivation on MacConkey agar supplemented with antibiotics (cefotaxime [2 μg/mL], ciprofloxacin [0.05 μg/mL], colistin [3.5 μg/mL], or meropenem [0.125 μg/mL]). The isolates originated from animals (66.21%), the environment (18.92%), and humans (14.87%) from Europe, North and South America, Africa, and Australia, with a high prevalence of isolates from the Czech Republic (85.15%). The collection was made up of mainly animal isolates, and out of these, 43.31% originated from wildlife and 36.46% originated from food-producing animals. Genomic DNA was extracted by a boiling method and used for PCR screening for the fosT gene encoding an scFOS transporter using primers fosT-F (5′-ATGCCAACTGAACTTCGACC-3′) and fosT-R (5′-CGAATGGCATGACGAAGGTA-3′). A representative collection of 307 fosT-positive isolates was created. scFOS metabolic function was demonstrated for all of the fosT-positive isolates by a carbohydrate utilization experiment with the addition of an scFOS mixture (Baileys digest plus) and 1% bromothymol sulfone phthalein as a pH indicator. The medium for the metabolic test was prepared with 100 mL of sterile distilled water, 1 g of peptone, 0.5 g of NaCl, 1 g of the scFOS mixture, and 0.6 mL of 1% bromothymol sulfone phthalein to a final pH of 7.2. The overnight-grown cultures of the isolates inoculated into the above-mentioned medium showing yellow coloring demonstrated the fermentation process of these bacteria compared to the negative control (E. coli TOP10), which showed blue coloring. The ability to utilize scFOSs as the only sources of carbon was also tested by inoculating the isolates into minimal medium with the addition of the scFOS mixture. Minimal medium with the addition of scFOSs was prepared, and the test was conducted according to a protocol described previously (7). The experiment was also performed using minimal medium with the addition of nystose, an inulin that is a member of scFOSs with a tetrasaccharide structure.

Short-read sequencing.

A total of 237 fosT-positive isolates, selected based on their geographical origin and source (to exclude duplicates), were subjected to short-read whole-genome sequencing (WGS). Genomic DNA was extracted using the NucleoSpin tissue kit (Macherey-Nagel GmbH & Co. KG, Germany) and was used for DNA library preparation with a Nextera XT library preparation kit (Illumina, San Diego, CA, USA). Sequencing was performed on a NovaSeq 6000 platform (Illumina).

Long-read sequencing.

Long-read sequencing was performed to successfully assign the fos operon location and detect representative plasmids carrying the operon. Thirteen representatives (Table 1) were selected based on metadata and preliminary data analysis (isolate origin, source, representation of ARGs and VAGs, and plasmid replicons). The genomic DNA of the selected representatives was extracted by using a Circulomics Nanobind HMW (high-molecular-weight) DNA extraction kit (Pacific Biosciences [PacBio], CA, USA), and the plasmid DNA was extracted by using a Genopure plasmid midi kit (Roche Diagnostics GmbH). The sequence library was prepared by using an SQK-RBK004 rapid barcoding kit (Oxford Nanopore Technologies [ONT] Ltd., Oxford, United Kingdom) according to the manufacturer’s protocol, subsequently loaded into a FLO-MIN106D flow cell, and sequenced on a MinION Mk1B platform (ONT).

TABLE 1.

Plasmid sequences obtained by long-read sequencinga

Primer name Source Origin Inc group ST or RST ARGs Size (bp) GenBank accession no.
pH7_scFOS Rook CZ IncF type [F–:A–:B1] 147,156 OQ534111
pHP62_scFOS Rook CZ IncF type [F–:A1:B1] 139,264 OQ534112
pAP112_scFOS Poultry CZ IncF type [F–:A6:B1] 152,736 OQ534113
pB0009222_scFOS Human (infection) CZ IncF type [F77:A1:B1] 151,858 OQ534114
p29D_scFOS Human (healthy newborn) CZ IncK2 79,027 OQ534117
p1095_scFOS WWTP CZ IncK2 97,677 OQ534116
pDR19cip_scFOS Kite CZ IncK2 97,612 OQ534118
pOV-CH82_scFOS WWTP CZ IncK2 blaTEM-1A, sul2 110,793 OQ534119
pB0017981_scFOS Human (infection) CZ IncI1 ST103 94,321 OQ534115
pB-P_26_scFOS Broiler CZ IncY aadA5, dfrA17, sul2 97,015 OQ534120
p234cef_scFOS Human (infection) IC IncY aadA5, dfrA17, sul2 95,158 OQ534121
p1505h_scFOS WWTP CZ IncY aadA5, dfrA17, sul2, tet(A) 102,484 OQ534123
p5359h6_scFOS WWTP CZ IncY aadA5, dfrA17, sul2 88,952 OQ534122
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CZ, Czech Republic; IC, Ivory Coast; WWTP, wastewater treatment plant.

Data analysis and plasmid assembly.

The raw reads from the Illumina platform were low-quality trimmed (Q ≤ 20) and adaptor trimmed using Trimmomatic v0.39 (20) and assembled using SPAdes 3.13.1 (21) with the “–careful” remark. The fast5 data from the ONT MinION platform were base called, converted to fastq format using Guppy v6.0.1 (ONT), demultiplexed and adaptor trimmed using Porechop v0.2.2 (22), and quality trimmed (Q ≤ 9) using BBduk v38.68 (https://sourceforge.net/projects/bbmap/). The long reads were assembled using Unicycler v0.4.8 (23) or Flye v2.6 (24). The obtained assembled sequences were polished by long (Racon v1.4.20 [25] and medaka v1.2.3 [26]) and short (Pilon v1.23 [27]) reads. Partition of individual isolates based on their fos operon locations was performed using the BWA-MEM mapping algorithm (28) to link the obtained sequences to either the reference pEQ1 (IncHI1) plasmid sequence, our obtained long-read plasmid sequences, or the chromosomal reference.

In silico and phylogenetic analyses.

All isolates were analyzed using multilocus sequence typing (MLST) (https://github.com/tseemann/mlst) to assign them to a sequence type (ST). Plasmids were analyzed using ABRicate v0.9.8 (https://github.com/tseemann/abricate), namely, vfdb (29), to detect VAGs and CGE tools databases (PlasmidFinder v2.1 [30] to determine plasmid replicons, pMLST v2.0 [30] to distinguish plasmid RSTs [IncF-type plasmids], and pMLST and ResFinder v4.0 [31] to detect genes encoding antibiotic resistance). A Basic Local Alignment Search Tool (BLAST) search was performed to compare the obtained long-read sequences to publicly available genomes. The final assembled and closed plasmid sequences were manually verified and annotated using Geneious Prime v2022.2.1 (Biomatters, Auckland, New Zealand) with BLAST (BLASTn, BLASTp, and BLASTx), ISfinder (32), and the NCBI Conserved Domain Database (CDD) (33).

The phylogenetic analysis using short-read whole-genome sequences was based on a core-genome alignment of 2,740 genes created using Roary v3.12.0 (34) from precited open reading frames obtained using Prokka v1.14.6 (35). The phylogenetic tree was constructed using RAxML v8.2.11 (36) under the GTR+GAMMA model supported by 500 bootstraps. The phylogenetic tree was visualized and complemented by metadata using iTOL (37).

BRIG v0.95 (38) was used to compare and visualize all plasmid sequences with the long-read circular plasmid reference, clinker v0.0.25 (39) was used for the comparison of the linear visualizations of DNA sequences, and flourish.studio (https://flourish.studio/) was used to visualize the metadata.

Data availability.

The genome assemblies and the complete and annotated plasmid sequences have been deposited in GenBank under BioProject accession number PRJNA937289. The complete nucleotide sequences of the plasmids have been deposited in GenBank under accession numbers OQ534111 to OQ534123.

ACKNOWLEDGMENTS

We thank Dana Cervinkova, Martina Masarikova, Eva Suchanova, Iva Sukkar, Jana Siskova, and Ivana Jamborova for their excellent work in the laboratory. We thank Ivan Literak, Alois Cizek, and their teams for providing their collections of bacterial isolates, and we thank the terrain workers for collecting and providing the samples or bacterial isolates.

This study was funded by the Internal Grant Agency of the University of Veterinary Sciences Brno (203/2022/FVHE). The work of T.N. was supported by a Brno Ph.D. talent scholarship from the Brno City Municipality.

We have no potential conflicts of interest to declare.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Supplemental material. Download aem.00371-23-s0001.pdf, PDF file, 3.2 MB (3.2MB, pdf)

Contributor Information

Monika Dolejska, Email: dolejskam@vfu.cz.

Martha Vives, Unversidad de los Andes.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Supplemental material. Download aem.00371-23-s0001.pdf, PDF file, 3.2 MB (3.2MB, pdf)

Data Availability Statement

The genome assemblies and the complete and annotated plasmid sequences have been deposited in GenBank under BioProject accession number PRJNA937289. The complete nucleotide sequences of the plasmids have been deposited in GenBank under accession numbers OQ534111 to OQ534123.

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