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. 2023 Feb 17;11(2):515. doi: 10.3390/microorganisms11020515

Genomic Islands Identified in Highly Resistant Serratia sp. HRI: A Pathway to Discover New Disinfectant Resistance Elements

Samantha J McCarlie 1, Charlotte E Boucher 1, Robert R Bragg 1,*
Editors: Séamus Fanning1, Dirk P Bockmühl1
PMCID: PMC9964261  PMID: 36838480

Abstract

Molecular insights into the mechanisms of resistance to disinfectants are severely limited, together with the roles of various mobile genetic elements. Genomic islands are a well-characterised molecular resistance element in antibiotic resistance, but it is unknown whether genomic islands play a role in disinfectant resistance. Through whole-genome sequencing and the bioinformatic analysis of Serratia sp. HRI, an isolate with high disinfectant resistance capabilities, nine resistance islands were predicted and annotated within the genome. Resistance genes active against several antimicrobials were annotated in these islands, most of which are multidrug efflux pumps belonging to the MFS, ABC and DMT efflux families. Antibiotic resistance islands containing genes encoding for multidrug resistance proteins ErmB (macrolide and erythromycin resistance) and biclomycin were also found. A metal fitness island harbouring 13 resistance and response genes to copper, silver, lead, cadmium, zinc, and mercury was identified. In the search for disinfectant resistance islands, two genomic islands were identified to harbour smr genes, notorious for conferring disinfectant resistance. This suggests that genomic islands are capable of conferring disinfectant resistance, a phenomenon that has not yet been observed in the study of biocide resistance and tolerance.

Keywords: antimicrobial resistance, mobile genetic elements, multidrug efflux pumps, biocide resistance

1. Introduction

The COVID-19 pandemic has highlighted our need for effective disinfectants, antiseptics, and sanitisers (biocides). The antibiotic resistance crisis can be seen as a warning or foreshadowing of an equally alarming phenomenon of microbial resistance to disinfectants. This means it is troubling that, within the food and agricultural industries and medical environments, resistance to disinfectants amongst microorganisms is emerging at a startling rate [1,2,3,4].

Mobile genetic elements (MGEs) play a significant role in the transfer of genes which confer antimicrobial resistance [5,6,7,8]. Their mobility is brought about by horizontal gene transfer, resulting in populations with reduced susceptibility to various antimicrobials [5,8]. Resistance can develop against several antimicrobials simultaneously, without prior exposure [9]. Genomic island (GI) is an umbrella term for mobile genetic elements found on the bacterial chromosome that have been acquired through horizontal gene transfer, usually between 10 and 200 kb in length [6,10,11]. This overarching term also includes integrated plasmids, integrons, prophages, conjugative transposons, and integrative conjugative elements [6,10,11,12]. These MGEs are then given more specific identities based on their mechanism of transfer (conjugation, transduction, or transformation) and genes present (transposases, integrases etc.) [6,12].

Genomic islands can be further characterised based on the phenotype they confer. For example, pathogenicity islands encode genes that confer an advantage in pathogenicity [13], resistance islands encode antimicrobial resistance genes [14], and metabolic islands contain genes that confer an additive metabolic advantage [6,10].

The bioinformatic identification of genomic islands is achieved using two approaches. The first is via sequence composition, and the second is via comparative genomics [10,11]. Both techniques have respective advantages and limitations, and therefore, a combination of the two provides the most sensitive and precise output [10,12]. IslandViewer4 is the gold standard for genomic island prediction, as it incorporates four different genomic island prediction methods, IslandPick, IslandPath-DIMOB, SIGI-HMM, and Islander [15].

Genomic islands have been found to play a role in antibiotic resistance [8,16]. However, minimal research has been carried out on the role of genomic islands in disinfectant resistance. As this is an emerging issue, more insight into the molecular mechanisms of resistance to disinfectants and other biocides is needed. A genomic island in Listeria monocytogenes isolates was found to be responsible for food-borne outbreaks harbouring multiple resistance genes, including an efflux pump involved in benzalkonium chloride resistance (ErmE) [17,18]. Jiang and co-workers (2020) found that the sug operon on the bacterial chromosome encoding SMR efflux pumps conferred resistance to benzalkonium chloride. This research brings forth the idea that resistance islands may be the latest genetic element capable of conferring resistance to disinfectants.

Resistance islands are often harboured in multidrug-resistant bacteria as one of many mechanisms to increase survivability [19]. One of these bacteria, Serratia sp. HRI, has high disinfectant resistance capabilities and provides a unique opportunity to study resistance to disinfectants and other biocides [20]. Several mechanisms of resistance to disinfectants have been elucidated, with efflux pumps being the most common. However, molecular-based resistance has mostly been limited to the study of plasmids. Little is known about which other mobile genetic elements can play a significant role in the development and dissemination of the disinfectant resistance phenotype. In the search for novel mechanisms of disinfectant resistance, genomic islands and the hypothetical proteins they harbour are attractive targets in the search for novel, previously undescribed mechanisms of resistance. If the molecular basis of disinfectant resistance is better understood, this will help to safeguard our current disinfectants and ensure proper biosafety in the agricultural, food, and medical industries. The aim of this work is to use prediction software and bioinformatic analysis to determine whether genomic islands can contribute to disinfectant resistance. The finding of several resistance islands harbouring known disinfectant resistance genes within this highly resistant isolate suggests that genomic islands can be characterised as a molecular element capable of conferring disinfectant and biocide tolerance and resistance. This paper adds to the evidence that genomic islands are capable of conferring biocide tolerance and resistance.

2. Materials and Methods

Serratia sp. HRI was isolated from a bottle of Didecyldimethylammonium chloride (DDAC)-based disinfectant [20]. Upon analysis, high levels of resistance to Quaternary Ammonium Compound (QAC) disinfectants were found via Minimal Inhibitory Concentration (MIC) tests [20].

The unusually high level of resistance observed in this isolate, together with its isolation from a bottle of disinfectant, prompted research into this microorganism. The genome of Serratia sp. HRI was sequenced and previously published [20]. The raw reads from this sequencing run, described previously, were then assembled again using the PATRIC (v. July 2021) de novo Genome Assembly service with default parameters unless otherwise specified (available at https://www.bv-brc.org/app/Assembly2) [21].

This assembled genome is 5 533 130 bp long, with GC content of 59.1%, an N50 score of 348 770, an L50 of 5, 47 contigs, and 126 RNAs, deposited on NCBI under Genbank Accession No. CP083690.1. This genome was uploaded to IslandViewer4 [15] with Serratia marcescens strain N4-5 chromosome sequence as a reference. IslandViewer4 uses four genomic island prediction methods (IslandPick, IslandPath-DIMOB, SIGI-HMM, and Islander) to identify genomic islands [15]. Thereafter, resistance genes are identified by IslandViewer4 using the Resistance Gene Identifier (RGI) from the Comprehensive Antibiotic Resistance Database (CARD) [22], as well as virulence factors from the Virulence Factor Database (VFDB) [23], PATRIC [24], and Victor’s virulence factors (http://www.phidias.us/victors/ (accessed on 11 January 2022)), in addition to 18 919 pathogen-associated genes [25,26]. For further analysis and annotation, the sequence of each genomic island was uploaded to RAST and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) NCBI annotation tool for additional annotation [27,28].

In the GIs of interest (GI 11, 20, and 76), any gene annotated as a hypothetical or uncharacterised protein was finally run through the PSI-BLAST program [29] and annotated further if any significant hits were found.

3. Results

IslandViewer4 identified 92 genomic islands within the genome of Serratia sp. HRI, as depicted in Figure 1. Of the 92 genomic islands, 9 contained known antimicrobial resistance genes or genes implicated in antimicrobial resistance; these genomic islands were predicted via at least two prediction methods. Table 1, Table 2, Table 3 and Table 4 represent the structure of these genomic islands and annotated gene lists [27,30,31].

Figure 1.

Figure 1

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Circular map generated by IslandViewer4 depicting the location of genomic islands within the genome of Serratia sp. HRI. Orange bars represent GIs identified via the SIGI-HMM genomic island prediction software, blue bars are GIs identified via IslandPath-DIMOB program, and the integrated GIs identified via all programs used are represented by red bars. Adapted from IslandViewer4 [15].

Table 1.

Summary of the properties of resistance islands of Serratia sp. HRI, including a selection of genes within the resistance islands identified by IslandViewer4.

Genomic Island Antimicrobial Resistance Genes Hypothetical Proteins Toxin-Antitoxin Systems Mobility Genes Non-Resistance Efflux Genes Transcriptional Regulators
11 7 40 2 * 9 0 5
18 2 0 0 0 1 0
20 3 10 0 11 1 3
23 1 1 0 0 0 0
28 1 1 0 3 1 0
33 1 5 0 0 0 0
42 13 23 7 * 13 0 0
46 1 5 0 1 0 0
76 3 28 2 5 0 1
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* 1 partial toxin–antitoxin system.

Table 2.

Gene list of resistance island 11 of Serratia sp. HRI (1 370 193 bp–1 419 319 bp, GC content 49.2, size 49 126) identified by IslandViewer4. Gene function was annotated via RAST; any hypothetical or uncharacterised proteins were further analysed via NCBI PGAP and BLAST. Annotated drug resistance genes are highlighted in bold.

Function Start Stop Length (bp) Annotation
1 Periplasmic fimbrial chaperone StfD 3 764 762
2 Hypothetical protein 799 1455 657 Fimbrial protein (Serratia)
3 Hypothetical protein 1472 1966 495 Fimbrial protein (Serratia marcescens)
4 MrfF 1983 2474 492
5 Minor fimbrial subunit StfG 2484 3014 531
6 Hypothetical protein 3158 3697 540 LuxR C-terminal-related transcriptional regulator (Serratia marcescens)
7 Hypothetical protein 3715 3888 174
8 IS1 protein InsB 4211 3969 243
9 Inner-membrane proton/drug antiporter (MSF type) of tripartite multidrug efflux system 6496 4208 2289
10 Transcriptional regulator, LysR family 6637 7539 903
11 Colicin immunity protein PA0984 7645 8010 366
12 YpjF toxin protein 8619 8251 369
13 Uncharacterized protein YagB 9016 8678 339
14 UPF0758 family protein 9526 9047 480 DNA repair protein RadC (Serratia marcescens)
15 Hypothetical protein 9541 9765 225
16 Hypothetical protein 9887 10,069 183
17 FIG01222608: hypothetical protein 10,562 10,206 357
18 Hypothetical protein 11,008 10,697 312
19 Hypothetical protein 11,323 11,021 303
20 Hypothetical protein 11,845 11,342 504
21 Hypothetical protein 12,570 11,842 729 WYL-domain-containing protein (Serratia marcescens)
22 Hypothetical protein 13,008 12,772 237
23 Hypothetical protein 13,903 13,019 885
24 Hypothetical protein 14,462 15,091 630 Inovirus Gp2 family protein (Serratia marcescens)
25 Hypothetical protein 15,213 15,425 213 AlpA family phage regulatory protein (Serratia marcescens)
26 Hypothetical protein 15,474 15,632 159
27 Hypothetical protein 17,366 15,774 1593 DUF3987-domain-containing protein (Serratia marcescens)
28 Hypothetical protein 17,395 17,535 141
29 Hypothetical protein 17,784 17,963 180 ShlB/FhaC/HecB family hemolysin secretion/activation protein (unclassified Serratia)
30 Hypothetical protein 17,960 18,208 249
31 Phosphoglycerate mutase (EC 5.4.2.11) 18,243 18,860 618
32 Il-IS_2, transposase 19,280 18,843 438
33 Hypothetical protein 20,125 19,277 849 SMP-30/gluconolactonase/LRE family protein (Serratia marcescens)
34 Oxidoreductase, short-chain dehydrogenase/reductase family 20,988 20,122 867
35 Transcriptional regulator, LysR family 21,133 21,426 294
36 Mobile element protein 22,121 21,606 516
37 Insertion element IS401 (Burkholderia multivorans) transposase 22,400 22,173 228
38 Phage integrase 22,837 22,553 285
39 Phage-associated DNA N-6-adenine methyltransferase 23236 22,955 282
40 Hypothetical protein 23,677 23,531 147
41 Hypothetical protein 23,838 23,680 159
42 Hypothetical protein 23,837 23,971 135
43 Hypothetical protein 24,125 23,997 129
44 FIG01055438: hypothetical protein 24,208 24,387 180
45 Hypothetical protein 24,456 24,620 165
46 Hypothetical protein 24,617 24,712 96
47 Hypothetical protein 24,706 24,834 129
48 Hypothetical protein 25,094 24,936 159
49 Efflux transport system, outer membrane factor (OMF) lipoprotein 25,470 26,885 1416
50 ABC-type antimicrobial peptide transport system, permease component 26,885 28,021 1137
51 ABC-type antimicrobial peptide transport system, ATPase component 28,039 28,764 726
52 Probable Co/Zn/Cd efflux system membrane fusion protein 28,775 29,683 909
53 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate phosphatase related protein 29,715 30,416 702
54 Hydrolase, alpha/beta fold family 30,413 31,303 891
55 Permease of the drug/metabolite transporter (DMT) superfamily 31,300 31,659 360
56 Permease of the drug/metabolite transporter (DMT) superfamily 31,662 32,087 426
57 Hypothetical protein 33,118 32,228 891
58 FIG110192: hypothetical protein 34,184 33,120 1065 Peptidogalycan biosysnthesis protein (Serratia)
59 Aminotransferase, class III 35,560 34184 1377
60 Mobile element protein 35,743 35,856 114
61 Hypothetical protein 36,927 35,869 1059 ATP-binding protein (Serratia sp. HRI)
62 Two-component transcriptional response regulator, LuxR family 37,624 36,929 696
63 Hypothetical protein 37,940 38,161 222
64 Core lipopolysaccharide phosphoethanolamine transferase EptC 38,236 39,933 1698
65 Two-component response regulator 40,672 40,502 171
66 Two-component response regulator 40,948 40,685 264
67 Hypothetical protein 41,166 41,032 135
68 Hypothetical protein 42,468 41,395 1074 RelA/SpoT-domain-containing protein (Serratia)
69 Hypothetical protein 42,751 42,542 210
70 Hypothetical protein 42,965 42,822 144
71 Hydrolase, alpha/beta fold family 43,881 43,006 876
72 Monooxygenase, flavin-binding family 45,404 43,878 1527
73 Transcriptional regulator, AcrR family 46,310 45,717 594
74 Hypothetical protein 46,429 46,310 120
75 Hypothetical protein 46,428 46,628 201
76 MmcH 46,648 47,535 888
77 Hypothetical protein 47,657 47,857 201
78 Possible regulatory protein Trx 47,870 49,126 1257
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Table 3.

Gene lists of genomic island 20 of Serratia sp. HRI (1 822 085 bp-1 869 515 bp, GC content 52.4, size 47 430 bp) identified via IslandViewer4. Gene function was annotated via RAST; any hypothetical or uncharacterised proteins were further analysed via NCBI PGAP and BLAST.

Function Start Stop Length (bp) Annotation
1 Conjugative transfer protein TrbK 326 3 324
2 Conjugative transfer protein TrbJ 1082 339 744
3 Conjugative transfer protein TrbE 3529 1079 2451
4 Conjugative transfer protein TrbD 3811 3542 270
5 Conjugative transfer protein TrbC 4194 3808 387
6 Conjugative transfer protein TrbB 5261 4191 1071
7 CopG-domain-containing protein 5734 5258 477
8 Coupling protein VirD4, ATPase required for T-DNA transfer 7728 5731 1998
9 Transcriptional regulator, LysR family 8034 8939 906
10 Hypothetical protein 9221 9751 531
11 Transposase and inactivated derivatives 9796 10,032 237
12 Small multidrug resistance family (SMR) protein 10,578 10,261 318
13 Probable lipoprotein 10,900 10,637 264
14 Transcriptional regulator, LysR family 11,838 10,933 906
15 Hypothetical protein 13,335 11,932 1404 TolC family protein
16 Transcriptional regulator, TetR family 13,446 14,087 642
17 Probable Co/Zn/Cd efflux system membrane fusion protein 14,084 15,250 1167 MULTISPECIES: efflux RND transporter periplasmic adaptor subunit
18 Hypothetical protein 15,275 18,379 3105 MULTISPECIES: efflux RND transporter permease subunit
19 Hypothetical protein 18,460 18,807 348 MULTISPECIES: SMR family transporter
20 Hypothetical protein 18,823 19,443 621
21 ABC transporter, permease protein (cluster 9, phospholipid) 19,440 20,597 1158
22 Mobile element protein 21,909 21,205 705
23 Integron integrase IntI1 21,900 22,196 297
24 Mobile element protein 22,571 23,209 639
25 Transposase 23,176 26,100 2925
26 Beta-glucosidase (EC 3.2.1.21) 27,418 26,180 1239
27 Putative polysaccharide export protein YccZ precursor 27,383 28,471 1089
28 Tyrosine-protein kinase (EC 2.7.10.2) 28,730 30,892 2163
29 Hypothetical protein 30,933 32,171 1239
30 Hypothetical protein 32,197 33,204 1008
31 Hypothetical protein 33,223 33,972 750
32 Poly(glycerol-phosphate) alpha-glucosyltransferase (EC 2.4.1.52) 34,315 35,256 942
33 Hypothetical protein 35,283 36,419 1137
34 UDP-galactopyranose mutase (EC 5.4.99.9) 36,474 37,625 1152
35 Low-molecular-weight protein-tyrosine-phosphatase (EC 3.1.3.48) => Etp 38,004 38,438 435
36 Tyrosine-protein kinase (EC 2.7.10.2) 38,450 40,621 2172
37 Hypothetical protein 40,702 41,862 1161
38 Hypothetical protein 41,828 43,288 1461 MULTISPECIES: aldo/keto reductase
39 Glycosyltransferase 43,278 44,186 909
40 Glycosyl transferase, group 1 44,233 45,276 1044
41 Glycosyltransferase 45,351 47,300 1950
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Table 4.

Gene lists of genomic island 76 of Serratia sp. HRI (5 688 450 bp-5 725 416 bp, GC content: 44.0, Size: 36 966 bp) identified via IslandViewer4. Gene function was annotated via RAST; any hypothetical or uncharacterised proteins were further analysed via NCBI PGAP and BLAST.

Function Start Stop Length (bp) Annotation
1 Hypothetical protein 923 411 513 Hypothetical protein (Serratia sp. SSNIH1)
2 Polyketide synthase modules and related proteins 4124 1122 3003
3 Hypothetical protein 4338 4222 117
4 Autoinducer synthase 4424 5584 1161
5 Hypothetical protein 5859 6110 252
6 ABC-type multidrug transport system, permease component 6668 6546 123
7 Hypothetical protein 6969 6658 312 Multidrug efflux ABC transporter permease/ATP-binding subunit SmdA (Serratia marcescens) (WP_033641139.1)
8 Hypothetical protein 7032 8279 1248 MbeB family mobilization protein (Serratia marcescens)
9 MobA 8378 8599 222
10 Small multidrug resistance family (SMR) protein 8666 8998 333
11 Hypothetical protein 9165 8995 171 GNAT family N-acetyltransferase (Serratia marcescens)
12 Hypothetical protein 9377 9207 171
13 Hypothetical protein 9746 9531 216
14 Mobilization protein MobC 10,181 10,339 159
15 Hypothetical protein 11,258 10,875 384
16 Hypothetical protein 11,371 12,447 1077
17 Hypothetical protein 13,804 12,512 1293 Site-specific integrase (Serratia)
18 Probable site-specific recombinase 15,011 13,806 1206
19 Transcriptional regulator, AlpA-like 15,550 15,344 207
20 Hypothetical protein 16,511 15,651 861 DUF6387 family protein (Serratia)
21 Hypothetical protein 16,691 16,575 117
22 Hypothetical protein 17,617 16,709 909 DUF4760-domain-containing protein (Enterobacterales)
23 Hypothetical protein 17,972 17,856 117
24 Hypothetical protein 18,388 19,452 1065
25 Repeat region 19,395 19,521 127
26 Replication protein 20,789 19,809 981
27 Hypothetical protein 21,202 20,993 210
28 Hypothetical protein 21,229 21,357 129 Conjugal transfer protein TraD (Yersinia)
29 Hypothetical protein 21,836 21,384 453
30 Mobilization protein 21,871 23,106 1236
31 Hypothetical protein 23,121 23,711 591 tRNA modification GTPase (Yersinia enterocolitica)
32 Restriction enzyme BcgI alpha chain-like protein (EC:2.1.1.72) 23,769 25,805 2037
33 Hypothetical protein 25,847 26,941 1095
34 YoeB toxin protein 27,235 26,981 255
35 YefM protein (antitoxin to YoeB) 27,483 27,232 252
36 Hypothetical protein 27,667 28,959 1293
37 Repeat region 27,757 27,883 127
38 Phage integrase 28,952 29,149 198
39 Type I restriction-modification system, restriction subunit R (EC 3.1.21.3) 29,715 30,176 462
40 Hypothetical protein 30,943 30,173 771 MFS transporter (Serratia)
41 Hypothetical protein 31,191 31,382 192 GNAT family N-acetyltransferase (Paenibacillus xylanexedens)
42 Hypothetical protein 31,502 31,410 93 Phytanoyl-CoA dioxygenase family protein (Serratia)
43 Hypothetical protein 31,702 32,502 801
44 Nodulation protein nolO (EC 2.1.3.-) 32,512 34,344 1833
45 Hypothetical protein 34,355 34,492 138
46 Hypothetical protein 34,496 35,602 1107 G-D-S-L family lipolytic protein (Serratia)
47 Hypothetical protein 35,662 36,966 1305 ATP-grasp-domain-containing protein (Serratia)
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Three of the nine genomic islands are shown in more detail as they contain resistance genes of particular interest (Table 2, Table 3 and Table 4); the remaining six islands are depicted in more detail in the Supplementary section (Tables S1–S6). Resistance island 11 is studied closely due to the number of resistance genes and their combination with hypothetical proteins, transcriptional regulators, and toxin–antitoxin systems. Resistance islands 20 and 76 are of interest as they contain known disinfectant resistance genes and a number of hypothetical proteins.

Genomic island 11 is represented in Table 2. This resistance island contains 78 annotated genes, including 7 genes encoding various efflux pumps. Of the seven genes, these include two copies of permeases of the drug/metabolite transporter (DMT) superfamily and a probable Co/Zn/Cd efflux system membrane fusion protein. Various components of efflux systems, such as an inner-membrane proton/drug antiporter (MSF type) of a tripartite multidrug efflux system, an outer membrane factor (OMF) lipoprotein, and two ABC-type antimicrobial peptide transport system proteins, make up the permease component and ATPase component. There are about 40 hypothetical proteins and multiple transcriptional regulators within this genomic island, including those of the Trx, AcrR, LuxR, and LysR families.

Genes of interest in genomic island 20, represented in Table 3, include a small multidrug resistance efflux protein (SMR), an ABC transporter permease protein, and a probable Co/Zn/Cd efflux system membrane fusion protein. Several genes are associated with conjugative transfer, mobile element proteins, an integron-associated gene, and transposase-associated genes. Hypothetical protein 19 was further annotated by NCBI PGAP as an SMR family transporter, a well-known disinfectant resistance gene.

Genomic island 76, depicted in Table 4, contains 47 genes, including an smr gene and an ABC-type multidrug transport system gene, together with a complete toxin–antitoxin system (YoeB/YefM). This genomic island is also a mosaic of several mobile element associated genes, such as an integrase, repeat regions, recombinase, and multiple mobilisation proteins (MobA, MobC). Hypothetical protein 7 in GI 76 had a significant similarity hit in the BLAST program with a multidrug efflux ABC transporter permease/ATP-binding subunit SmdA (Max score: 25.0, Total score: 25.0, Query cover: 74%, E value: 1.9, Per. Ident: 26.51%). This protein is located next to a component of an ABC-type multidrug transport system and is likely part of an efflux system. Hypothetical protein 11, located adjacent to an SMR disinfectant resistance protein, had the highest similarity hit with GNAT family N-acetyltransferase (Serratia marcescens) when run through the BLAST program. This family of proteins is responsible for resistance to aminoglycoside antibiotics [32] and could play a role in the antimicrobial resistance of Serratia sp. HRI.

Although the following genomic islands were not highlighted, each has interesting characteristics and contains at least one antimicrobial resistance gene. Genomic island 18, depicted in Table S1 in the Supplementary section, contains heavy metal response genes to molybdenum and two ABC-type efflux pump permease components, YbhS and YbhR. These proteins, together with YbhF, form YbhFSR, which functions in tetracycline efflux and Na+(Li+)/H+ transport [33]. Adjacent to these genes is ybhL, a closely related gene whose function is unknown but is hypothesised to be involved in stress response and cell protection by unknown mechanisms [34].

Table S2 represents genomic island 23, which is one of the smallest GIs identified with only four genes. Some argue it should not be identified as a GI due to its small size [11]. However, as it contains a multidrug resistance gene from the DMT superfamily, it is noteworthy.

Genomic island 28, depicted in Table S3, contains genes encoding antibiotic multidrug resistance protein ErmB (macrolide and erythromycin resistance) and an adjacent ABC efflux gene [35,36]. This GI also contains multiple transposase genes and components from insertion sequence element IS911, suggesting this insertion sequence may have played a role in the evolution of this resistance island.

Genomic island 33 is a small island with only one annotated protein, shown in Table S4. The protein annotated is an HtpX protease, which, together with ClpA, is involved in aminoglycoside resistance in Stenotrophomonas maltophilia [37,38]. Although this island does not contain the ClpA gene, the HtpX protease has been co-selected with multiple hypothetical proteins, which may aid in its function and could be candidates for further study.

Genomic island 42 is a highly conserved metal response island, described in Table S5, harbouring 13 genes involved in metal response with three complete toxin–antitoxin systems. Multiple toxin–antitoxin systems and several MGE-associated genes suggest this genomic island is mobile and highly conserved within a population. The toxin–antitoxin system, HigA/HigB, has been found to play a regulatory role in virulence and biofilm formation in Pseudomonas aeruginosa [39,40]. The metal response genes include those for silver and copper, which are being promoted as used in some products an alternatives to current antimicrobials [41]. These characteristics threaten the efficacy of the potential of this alternative treatment.

A bicyclomycin resistance protein can be found on genomic island 46 in Table S6. This resistance protein, together with error-prone repair (UmuD) and error-prone DNA polymerase (UmuC), could introduce mutations and aid in the evolution of antimicrobial resistance.

4. Discussion

Resistance islands are a well-known molecular element capable of conferring antibiotic resistance [42], but little research has been carried out on whether these mobile elements play a role in disinfectant and biocide resistance. Improved sequencing technology and more accessible bioinformatic programs have opened the door to the study of these elements and their impact on the resistance profile. This work aims to use these advances in sequencing technology to identify regions likely characterised as resistance islands contributing to the high levels of disinfectant resistance observed in this isolate.

These results are integrated images and gene annotations generated by the IslandViewer4, RAST, PGAP, and PSI-BLAST programs. A total of 92 genomic islands were found within the genome of Serratia sp. HRI, and a few are highlighted here as they are of extrachromosomal origin, identified within a highly resistant microorganism, and harbour antimicrobial resistance genes. The vast amount of genomic islands identified within Serratia sp. HRI aligns with the predicted high level of plasticity within the Serratia genus [5]. High genomic plasticity can lead to a mosaic of MGEs and can be attributable to resultant antimicrobial resistance [8]. Iguchi and co-workers (2014) found high genome plasticity in a clinical Serratia marcescens isolate. Compared to a non-resistant isolate, a mosaic of mobile genetic elements and acquired resistance genes contributed to the high levels of antimicrobial resistance in the clinical isolate [5].

Genomic island 11 was the first presented here and can be described as an all-round resistance and fitness island, as it harbours several annotated resistance genes applicable to various antimicrobials. This genomic island includes partial efflux systems from the MFS, OMF, and ABC families and two copies of complete systems from the DMT efflux family. Efflux genes that are not labelled as resistance genes are also highlighted, as they are part of the genome of a highly resistant isolate, placed within a resistance island, and close to a resistance efflux system. Therefore, they are of interest for further study. This genomic island also carries genes involved in metal response, colicin immunity, transcriptional regulators, and multiple MGE components (insertion sequences, phage integrase, and mobility genes). All four transcriptional regulator families found within this GI have been shown to improve bacterial fitness and survivability. LysR-type transcriptional regulators have been reported to play a role in antibiotic resistance in Aeromonas sp. [43]. LuxR transcriptional regulators are involved in biofilm formation and stress response in Pseudomonas and Mycobacterium sp. [44,45]. AcrR transcriptional regulators and their mutations have been seen to contribute towards drug resistance in Salmonella sp. [46]. Finally, the possible regulatory protein thioredoxin (Trx) protects against oxidative stress, a well-established response after treatment by antimicrobials such as disinfectants [47]. Interestingly, more than half of all the genes present in this island are uncharacterised and are listed as hypothetical proteins. As this is a large genomic island and requires metabolic resources to maintain and transcribe these elements, it is intriguing that these genes have not been lost. This suggests that some of these hypothetical proteins which form the majority of this genomic island may have a function and are attractive candidates in the search for novel resistance genes and even novel mechanisms of resistance.

Genomic island 20 contains the first gene directly implicated in disinfectant resistance, the smr gene [19,48], as well as an ABC efflux permease protein. This island also contains a metal response gene and multiple conjugative transfer proteins alluding to the origin of this GI. Within this sequence, a mosaic of MGEs, including genes encoding transposases, an integrase, and mobile element proteins, were discovered. Multiple transcription regulators associated with antimicrobial resistance are again present in this GI, including regulators from the LysR family and Tetr families, linked to tetracycline resistance [49,50]. Within this resistance island, 11 out of 41 genes are uncharacterised and annotated as hypothetical proteins. This island contains multiple MGEs, suggesting high plasticity, and the probability of incorporating additional resistance determinants is high.

Genomic island 76 contains a complete toxin–antitoxin system (Yoe-B/YefM), an ABC multidrug efflux-encoding gene and, importantly an smr gene. This resistance island is conservable in a population due to the toxin–antitoxin system, and almost two-thirds of the genes in this island are uncharacterised. Out of the 47 genes making up this GI, 29 are hypothetical proteins that have been co-selected and maintained with the antimicrobial resistance genes in this island. These uncharacterised flanking sequences are potential targets in the search for new mechanisms of resistance.

When considered all together, these genomic islands contain multiple antimicrobial resistance genes harboured simultaneously within the genome of Serratia sp. HRI, which can confer a wide range of resistance within this single isolate. Although there were many incomplete efflux systems (GIs 11, 18, 19, 20, 28, and 76), bioinformatics and annotation software still have a way to go, and in the years to come, these systems may be annotated differently.

In a field such as disinfectant resistance, where knowledge of mechanisms is minimal, the vast numbers of hypothetical proteins within these resistance islands are attractive targets in searching for novel resistance genes and mechanisms of disinfectant resistance.

It is also interesting that very few genes identified in these islands were assigned to subsystems after annotation. This adds to the notion that bioinformatics and annotation programs need improvement, as more information is needed on where these genes fit into the bacterial metabolism and their function(s).

The plasticity and adaptability of the Serratia genome shows the capability of the this genus in acquiring MGEs that can contribute to the decreased susceptibility often observed in the Serratia genus [5]. The result is observed in isolates such as Serratia sp. HRI, whose genome is an assortment of fitness determinants gathered over time, increasing survivability to a wide range of antimicrobials. To confirm the phenotypic impact of these resistance islands and the extent of their impact, further work will be required.

5. Conclusions

There is limited information on whether genomic islands are capable of conferring resistance to disinfectants. Therefore, the genomic islands of Serratia sp. HRI will add to the knowledge of antimicrobial resistance and reinforce the idea that genomics islands can be described as the latest molecular element capable of conferring disinfectant resistance. This work also adds to the evidence for the cross-resistance and co-selection of antimicrobial resistance genes within a single organism. This work represents how predictive bioinformatic technology can lead targeted research into antimicrobial resistance. However, this is a starting point and only tells scientists where to look instead of providing a definitive answer. Phenotypic analysis needs to be coupled with predictive software to fully elucidate resistance mechanisms.

The increased use of disinfectants during the COVID-19 pandemic will inevitably give rise to less susceptible populations at an advanced rate. Amidst the pandemic, we are silently and unknowingly selecting disinfectant-resistant microorganisms. By getting ahead of disinfectant resistance, we will be able to safeguard our current disinfectants and ensure infection control in both the agricultural and medical industries.

Acknowledgments

The authors would like to acknowledge Jeffrey Newman for his advice in the conceptualisation of this research.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11020515/s1, Table S1: Gene lists of genomic island 18 of Serratia sp. HRI (1 655 571 bp–1 660 471 bp, GC content 62.3, Size 4 900 bp) identified via IslandViewer4 and annotated via RAST, Table S2: Gene lists of genomic island 23 of Serratia sp. HRI (1 875 362 bp–1 879 853 bp, GC content: 45.1, Size 4 491 bp) identified via IslandViewer4 and annotated via RAST, Table S3: Gene lists of genomic island 28 of Serratia sp. HRI (2 294 061 bp–2 309 315 bp, GC content: 48.1, Size: 15 254 bp) identified via IslandViewer4 and additional annotated via RAST, Table S4: Gene lists of genomic island 33 of Serratia sp. HRI (2 548 843 bp–2 553 244 bp, GC content 41.9, Size: 4 401 bp) identified via IslandViewer4 and annotated via RAST, Table S5: Gene lists of genomic island 42 of Serratia sp. HRI (3 188 478 bp–3 232 330 bp, GC content: 51.2, Size: 43 852 bp) identified via IslandViewer4 and annotated via RAST, Table S6: Gene lists of genomic island 46 of Serratia sp. HRI (3 571 957 bp–3 586 537 bp, GC content: 51.7, Size: 14 580) identified via IslandViewer4 and annotated via RAST.

Click here for additional data file. (126.9KB, zip)

Author Contributions

Conceptualisation (R.R.B.); data curation (S.J.M.); formal analysis (S.J.M.); funding acquisition (R.R.B., C.E.B.); investigation (S.J.M.); methodology (S.J.M., C.E.B., R.R.B.); project administration (R.R.B., C.E.B.); resources (R.R.B., C.E.B.); software (free, internet-based); supervision (R.R.B., C.E.B.); validation (S.J.M.); visualisation (S.J.M.); roles/writing—original draft (S.J.M.); writing—review and editing (R.R.B., C.E.B., S.J.M.). All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Sequence data used in this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Genbank Accession No. CP083690.1.

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research received no external funding.

Footnotes

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Associated Data

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

Supplementary Materials

Click here for additional data file. (126.9KB, zip)

Data Availability Statement

Sequence data used in this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Genbank Accession No. CP083690.1.

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