Prevalence of S. aureus and MRSA in seafood products
Multiplex PCR MRSA
Total of 498 samples collected were screened for the presence of coagulase-positive Staphylococcus aureus (CPS) and MRSA (Table 1). Out of the 498 samples, 68 were found positive for the CPS. The prevalence of CPS in the fish and fish products samples in the Veraval region was 13.65%. Similarly, 15 samples were contaminated with MRSA from the total tested samples. Hence, the prevalence of MRSA in fish and fishery products is around 3% in the Veraval region. Based on the result, it has been observed that the fresh and processed fish had a higher level of CPS and MRSA. Water and ice samples had a less number of CPS (15.0%) and MRSA (3.0%). While comparing the worldwide incidences of MRSA in seafood products, variation in MRSA percentage was observed from very low to high. Similarly, a higher incidence of 30.0% and 22.2% MRSA were found in raw fish and prepared fishery foods in public hospitals in Salvador, Bahia, and Brazil, respectively [12]. At the same time, Sivaraman et al., found a higher level of MRSA (50%) in the 173 market fish samples in Assam, India. Almost 60% of the shrimp aquaculture settings samples were positive for the MRSA in Kerala, India [47, 54]. However, Daniel Vazquez- Sanchez et al., did not find any MRSA in fish and fishery products [53]. The variation in MRSA level is based on the hygienic food status. The contaminations could either form the infected person, improper hygenic practices, or poor sanitary utensils. Here, it's essential to understand that most people are asymptomatic carriers and responsible for the continuous spread of MRSA in food. There is a high possibility of MRSA transmission from fish handlers to fish and vice versa. In addition, the infected processing utensils and unhygienic environment may act as a potential source for the transmission of MRSA. Several studies on MRSA found that the hygienic conditions of food handlers are generally undesirable in most situations, such as health conditions, personal hygienic conditions, and working habits, increasing cross-contamination in processed foods[16, 20, 23]. The current findings emphasize the necessity of Good Hygiene Practices (GHP) throughout multiple processing steps, beginning with transportation and retail outlets, to limit the risk of S. aureus and MRSA transmission from food products to humans [38].
Table 1
Incidence of S. aureus and Methicillin-resistant S. aureus MRSA contamination in seafood
Fish samples
|
No. of Samples
|
S. aureus
|
MRSA by multiplex PCR *
|
Fresh
|
108
|
20 (18.52%)
|
3 (2.7)
|
Chilled
|
79
|
14 (17.72%)
|
4 (5%)
|
Frozen
|
64
|
6 (9.38%)
|
2 (3.13%)
|
Processed fish
|
124
|
22 (17.74%
|
6 (4.84%)
|
Water
|
76
|
4 (5.26%)
|
0 (0%)
|
Ice
|
47
|
2 (4.26%)
|
0 (0%)
|
Total
|
498
|
68 (13.65%)
|
15 (3.01%)
|
Antimicrobial resistance patterns of MRSA isolates
Antibiogram was carried out for the 68 MRSA sample isolates. One isolate from each MRSA positive sample was taken and subjected to an antibiogram against 24 antibiotics. A total of 68 samples were subjected to an antibiogram as per the CLSI guidelines using Dodeca Staphylococci-1 and two strips (Himedia, Mumbai). The resistant pattern was varied between the type of samples; fresh and chilled sample isolates possess a higher level of resistance than the processed fish (Table 2). This might be due to the manual handling of the fish. The highest resistance was observed for the fresh and chilled fish products, and there were no MRSA isolates in the water and ice samples which revealed the chance from the workers. Linezolid is a recent antibiotic usually recommended for MRSA infection; in the present study, 69 to 75% of the isolates were resistant to it. Co-trimoxazole is a combination of Trimethoprim/Sulfamethoxazole; recommended for MRSA; whose resistance level varied from 69 to 87.5% in the collected samples.
Table 2
Percentage resistance level of S. aureus for different antibiotics of fish and fishery products
Samples
|
P (100U)
|
LZ
(30µg)
|
CoT 25µg)
|
CX
(30µg)
|
OF
(5µg)
|
GEN
(10µg)
|
RIF
(5µg)
|
A/S
(10/
10µg)
|
PIT
(100
/10µg)
|
Fresh (%)
|
18.2
|
72.7
|
81.8
|
60.9
|
18.2
|
72.7
|
100
|
36.4
|
72.7
|
Chilled (%)
|
37.5
|
75.0
|
87.5
|
46
|
12.5
|
62.5
|
87.5
|
37.5
|
75.0
|
Frozen (%)
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Processed (%)
|
23.1
|
69.2
|
69.2
|
16.9
|
23.1
|
53.9
|
76.9
|
23.1
|
53.8
|
Water (%)
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Ice (%)
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
100
|
P: Penicillin, LZ: Linezolid, COT; co-trimoxazole, CX: Cefoxitin, OF: ofloxacin, GEN; gentamicin, RIF: Rifampicin, A/S: ampicillin-Sulbactam, PIT: Piperacillin-Tazobactam |
Table 3
De novo CRISPR/Cas prediction with the help of CRISPRCasFinder
CRISPR_Id
|
CRISPR_Start
|
CRISPR_End
|
CRISPR_Length
|
Potential_ Orientation (AT%)
|
Consensus_Repeat
|
Repeat_
ID
(CRISPRdb)
|
Conservation_ Repeats (% identity)
|
Repeat_ Length
|
Evidence_ Level
|
SRR538_1
|
682402
|
682501
|
99
|
Unknown
|
AAGAGCCCCTAATTAATAAATTAAAAGGGG
|
R271
|
100
|
30
|
1
|
SRR538_2
|
768363
|
768447
|
84
|
Forward
|
CACCCCAACTTGCATTGTCTGTAGAA
|
R1692
|
96.1
|
26
|
1
|
SRR538_3
|
826136
|
826215
|
79
|
Reverse
|
CCGTCAGCTTCTGTGTTGGGGCCC
|
R2322
|
95.8
|
24
|
1
|
SRR538_4
|
875258
|
875343
|
85
|
Unknown
|
AAAGTCAGCTTACAATAATGTGCAAGTTGG
|
Unknown
|
96.6
|
30
|
1
|
SRR538_5
|
1169979
|
1170071
|
92
|
Reverse
|
TAAGAAACAGTAATCAATAAATTGATAACT
|
R7515
|
100
|
30
|
1
|
SRR538_6
|
1832483
|
1832610
|
127
|
Unknown
|
AATTATGGAGCGGAAGATAGGATTTACA CCTATACCTC
|
R441
|
97.3
|
38
|
1
|
SRR538_7
|
1859907
|
1859989
|
82
|
Forward
|
TCTGTGTTGGGGCCCCGCCAACCTGCA
|
Unknown
|
96.2
|
27
|
1
|
SRR538_8
|
2058028
|
2058117
|
89
|
Reverse
|
CAACTTTAGTTGTTAGGGGCTCTT
|
R1624
|
91.6
|
24
|
1
|
SRR538_9
|
2130159
|
2130284
|
125
|
Reverse
|
CCTCTTTACTCGAAAGCTCACAAAACTCT TGATATCA
|
Unknown
|
97.2
|
37
|
1
|
Skov et al recommended Cefoxitin as a marker for detecting methicillin resistance. Around 16–60% of isolates are resistant to these antibiotics, and Cefoxitin is used considered a surrogate marker for the detection of MRSA [50]. Ofloxacin is also a fluoroquinolone antibiotic, recommended for the treatment of multidrug-resistant, but the level of resistance was relatively low than the Ofloxacin; it varied from 12 to 23% of the isolates were resistant. Gentamicin is an Aminoglycoside antibiotic, around 53 to 72% of isolates were resistant. Rifampicin is an antibiotic recommended for the treatment of tuberculosis; resistance was observed in about 76 to 100% of isolates. Ampicillin/sulbactam combination is recommended for the beta-lactamase-producing bacteria; around 23 to 37% of isolates have exhibited the resistance. Piperacillin–tazobactam is an injectable form of antibiotic highly recommended for the beta-lactamase producing antibiotics, 53 to 75% of the isolates are resistant to these antibiotics and are used only in severe bacterial infections of endocarditis and osteomyelitis. Whereas susceptible to Azithromycin, Clarithromycin, Ciprofloxacin, Gatifloxacin, Clindamycin, Tigecycline, Moxifloxacin, Lomefloxacin, Norfloxacin, Novobiocin, Teicoplanin, Nitrofurantoin, and Pristinomycin.
Whole-genome sequence analysis of Novel MRSA ST243 strain by annotation, gene ontology and pathway analysis, rRNA genes
The Paired-end sequencing of the MRSA sample performed was later subjected to a quality check by Fastqc and trimmed to generate high-quality raw reads with phread score ≥ 30 excluding adaptor sequences (WGS information in supplementary table 1a). Appx. 6,157,589 raw reads with 92.87 average long reads with 164.27x of average coverage and 3,079,132 total nucleotides with 34% GC content. De novo contigs and scaffolds were generated using Spades, wherein 158 contigs and 136 scaffolds were identified, and a quality check was performed using the Quast against the reference sequence (CP000253.1) confirming the number of contigs to be 158 with GC% of 32.79%, 2397067 total aligned length and 64,451 bp N50 (the details furnished in the supplementary table 1b). Furthermore, annotation with prokka revealed that the MRSA-10 contains a 2,637,041 bases long genome, 2411 CDS, 2424 genes, 2424mRNA, 1 rRNA, 11 tRNA and 1 tmRNA (Fig. 1). On further analysis with the BLASTX, the first 15 organisms hit were in correspondence with Staphylococcus species, and appx. 62.8% of genes i.e. 1537 out of 2449 genes were annotated, providing information related to various function categories in the KEGG pathway (Fig. 2). The total number of Gene Ontology annotations identified for molecular functions was 870, with 586 annotations having to do with a biological process and 236 annotations having to do with cellular components. The phylogenetic relatedness was determined with the reference genome CPOOO253.1 and 3 other top blast searches with the E value 0.0 and visualized with the Figtree (Fig. 3). The variants were identified and filtered for Single Nucleotide Polymorphism (SNP) with the variant calling pipeline. In short, the variations identified were summarised in the form of two types of substitution mutation, i.e., 31,677 nt undergoing Transition mutation in which A to G and C to T transition was 16110 and 15567 respectively whereas, 18869 nt undergoing transversions out of which A to C: 3821, A to T: 9778, C to G: 1404, and G to T: 3866 variations was noticed. The sorting of the CRISPR sequence by the CRISPRcasFinder web server resulted in Nine CRISPR regions (Table no. 3). Using The WGS was submitted at NCBI GenBank with the accession number of NBZY00000000 [48]. Overall, 98.57% (2,424 CDs) were predicted with at least one hit in the NCBI database, and 100% of the predicted CDSs have a similarity of more than 60% at the protein level in the NCBI database.
Virulence genes analysis of Novel MRSA ST243 strain
The MRSA virulence gene profiles were analyzed by VirulenceFinder 2.0 from a whole-genome sequence to assess the extent of its pathogenicity/ toxins production nature. The presence of several virulence factors influences the capacity of S. aureus isolates to cause illness/ genes for adhesion, invasion, and host defence evasion. The ability of staphylococci to produce cytotoxins (hemolysins, leukotoxins, and leukocidins) and superantigenic toxins are linked to their virulence (enterotoxins, exfoliative toxins, and toxic shock syndrome toxin) (Table 4). This MRSA strain contains identical virulence factors for exoenzyme genes such as aureolysin (aur) and serine protease (spIE) with sizes of 1530 and 717bp, respectively accession numbers CP009554.1 and BX571856.1. MRSA virulence genes include gamma-hemolysin chain II precursor (hlgA), gamma-hemolysin component B precursor (hlgB), gamma-hemolysin component C (hlgC), Panton-Valentine leukocidin F component (lukF-PV), and Panton-Valentine leukocidin S component (lukF-PV), enterotoxin G (seg), enterotoxin I (sei), enterotoxin M (sem), enterotoxin N (sen), enterotoxin O (seo) and enterotoxin U (seu) with 100% identity. This MRSA strain has a diverse set of virulence factors/ toxin genes that have been identified as potential pathogen. These genes are predominantly expressed on mobile genetic elements and can be passed between strains via horizontal gene transfer. The presences of PVL (LukS and LukF proteins) are cytotoxic to different leukocytes and macrophages and other cells. PVL has been linked to community-acquired (CA)-MRSA infections and Sivaraman et al. found an increase in CA-MRSA infections in seafood [46]. Hemolysins, including hlgA, hlgB, and hlgC, are well-known toxins that cause cell lysis and death in blood cells whereas, Alpha hemolysin is the most potent membrane damaging toxin to blood cells, and is sensitive to platelets and monocytes lead to cell lysis and release of cytokines. Trigger the production of inflammatory mediators leads to septic shock symptoms during severe infections [31, 52]. Beta hemolysin/ b-toxin is sphingomyelinase that damages membrane rich in lipid.
Table 4
Detection of toxin genes in MRSA using VirulenceFinder 2.0
Virulence factor
|
Identity
|
Query/ Template length
|
Contig
|
Position in contig
|
Protein function
|
Accession number
|
hlgA
|
100
|
930/930
|
NBZY0100001.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_1.whole genome shotgun sequence
|
99768.100697
|
gamma-hemolysin chain II precursor
|
CP009554.1
|
hlgA
|
100
|
930/930
|
NBZY0100001.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_1.whole genome shotgun sequence
|
99768.100697
|
gamma-hemolysin chain II precursor
|
LN626917.1
|
hlgB
|
100
|
978/978
|
NBZY0100001.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_1.whole genome shotgun sequence
|
97292.98269
|
gamma-hemolysin component B precursor
|
BX571856.1
|
hlgC
|
100
|
948/948
|
NBZY0100001.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_1.whole genome shotgun sequence
|
98271.99218
|
gamma-hemolysin component C
|
CP009554.1
|
lukF-PV
|
100
|
978/978
|
NBZY01000018.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_18.whole genome shotgun sequence
|
52183.53160
|
Panton Valentine leukocidin F component
|
AB678716.1
|
lukF-PV
|
100
|
978/978
|
NBZY01000018.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_18.whole genome shotgun sequence
|
52183.53160
|
Panton Valentine leukocidin F component
|
HM584704.1
|
lukS-PV
|
100
|
939/939
|
NBZY01000018.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_18.whole genome shotgun sequence
|
53162.54100
|
Panton Valentine leukocidin S component
|
AB045978.2
|
lukS-PV
|
100
|
939/939
|
NBZY01000018.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_18.whole genome shotgun sequence
|
53162.54100
|
Panton Valentine leukocidin S component
|
AB256039.1
|
Seg
|
99.87
|
778/778
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
4972.5748
|
enterotoxin G
|
CP002388.1
|
Sei
|
100
|
729/729
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
2227.2599
|
enterotoxin I
|
CP002388.1
|
Sem
|
99.86
|
720/720
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
1473.2192
|
enterotoxin M
|
CP002388.1
|
Sen
|
97.43
|
777/777
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
3912.4688
|
enterotoxin N
|
AP014653.1
|
Seo
|
100
|
765/765
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
427.1191
|
enterotoxin O
|
CP002388.1
|
Seu
|
100
|
786/786
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
3109.3894
|
enterotoxin U
|
CP002388.1
|
Aur
|
100
|
1530/1530
|
NBZY01000017.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_17.whole genome shotgun sequence
|
44999..46528
|
aureolysin
|
CP009554.1
|
Sp1E
|
100
|
717/717
|
NBZY01000033.1 Staphylococcus aureus strain MRSA-10 MRSA10_Scaffold_33.whole genome shotgun sequence
|
13636..14352
|
serine protease splE
|
BX571856.1
|
Virulence factors for secreted exoenzyme genes, including aureolysin (aur) and serine protease (spIE) destroy host compounds or disrupt host metabolic and signalling pathways. The protease aureolysin (neutral proteinase of S. aureus) degrades numerous proteins, including insulin B, and inactivates PSMs, resulting in osteomyelitis pathogenesis [26]. It also causes the maturation of glutamyl endopeptidase SspA by cleaving glutamate residues. As a result, the aureolysin, glutamyl endopeptidase, and cysteine proteases staphopain A and B interfere with complement factors, causing bacterial death to be evaded [40]. Exfoliative toxins cleave desmosomal cadherins in the superficial skin layers, causing staphylococcal scalded skin syndrome (SSSS), a severe skin illness characterized by a rash, blisters, and severe lesional damage to the skin[42].
Spa typing of MRSA isolates by on Sanger sequencing
The standardized nomenclature and availability of spa types on the central spa server (http://spaserver.ridom.de) allow researchers to study clonal diversity and MRSA transmission in hospitals and community settings. T021 and its repetitions were the most prevalent spa Type in the strain 15-12-16-02-16-02-25-17-24, contig position (NBZY0100001601, 29990–30243) and plus orientation. There are 4568 strains on the Ridom spa server https://spa.ridom.de/spa-t021.shtml (repeat succession 15-12-16-02-16-02-25-17-24) In India; there is a frequency of 1.06% with the comment on cMRSA (lukS-lukF) with the CC30 and ST-30 and 3 spa types. The presence of leukocidin- Panton-Valentine leukocidin (PVL) (LukS and LukF proteins) has been linked to community-acquired (CA)-MRSA infections, according to this study. Sivaraman et al., reported on the prevalence of CA- MRSA in seafood [46]. However, an MLST study of the complete genome sequenced (NBZY00000000.1) found that MRSA isolates in fish and fisheries products in India were typed to a new ST 243 (Table 5) with arcC, aroE, glpF, gmk, pta, tpi, ygiL genes with 100% identity and coverage with the alleles of 2, 2, 5, 2,6, 3, and 2, respectively. Type t021 represents the so-called new ST 243 MRSA, often detected in Gujarat State, India, in fish and fisheries items.
Table 5
Multi Locus Sequence Typing (MLST) analysis of the Whole Genome Sequence (NBZY00000000.1) in MRSA Novel Sequence Type 243
Locus
|
Identity
|
Coverage
|
Alignment Length
|
Allele Length
|
Gaps
|
Allele
|
arcC
|
100
|
100
|
456
|
456
|
0
|
arcC_2
|
aroE
|
100
|
100
|
456
|
456
|
0
|
aroE_2
|
glpF
|
100
|
100
|
465
|
465
|
0
|
glpF_5
|
gmk
|
100
|
100
|
417
|
417
|
0
|
gmk_2
|
pta
|
100
|
100
|
474
|
474
|
0
|
pta_6
|
tpi
|
100
|
100
|
402
|
402
|
0
|
tpi_3
|
yqiL
|
100
|
100
|
516
|
516
|
0
|
yqiL_2
|
The present study shows that MRSA contamination occurred in the retail fish market and fish processing industrial samples; these isolates were resistant to rifampicin, cefoxitin Cefoxitin, and co-trimoxazole gentamicin, linezolid, penicillin, ofloxacin, piperacillin-tazobactam and ampicillin-sulbactam, i.e. multidrug-resistant. Thus, the presence of MDR S. aureus and beta-lactam generating MRSA may be the source of infected fish handlers and the handlers' poor hygienic profile, processing, and unsanitary environment of the fish source. To ensure the delivery of safe, wholesome seafood delivery, all fish handlers should be made aware of the need for personal hygiene and sanitary handling techniques at all stages of processing, preserving the cold chain, adequate cleaning and disinfection of equipment, and preventing cross-contamination. This study emphasizes the importance of ongoing antibiotic susceptibility testing for S. aureus and MRSA in seafood to identify the source of contamination.
The present study revealed that 13.65% of samples were contaminated with S. aureus, and 3% were with MRSA. Most of the S. aureus and MRSA isolates were multidrug-resistant (MDR) and to recent antimicrobial agents. MRSA carries virulence factors such as aureolysin (aur) & serine and toxin genes such as hlgA, hlgB, hlgC, lukF-PV, lukS-PV, seg, sei, sem, sen, seo and seu. The MRSA strain has spa type t021 with a novel MLST ST 243 in fish and fishery products. So, the presence of highly pathogenic, MDR and virulent MRSA strains in the fish and fishery products could pose a severe threat to the consumers, which will guide us to design a better surveillance protocol and control measures. It further suggested that Good Hygienic Practices, as recommended by WHO, need to be followed strictly during various stages of handling and processing of fish and fishery products to provide wholesome fish to the consumers [56, 57].