Research Article | Open Access
Naba Kumar Deka1,2 , Pratap Jyoti Handique1 , Probodh Borah2,3, Pranita Konwar4, Geetanjali Deka5, Rijumani Das6 and Mridusmita Choudhury1,2
1Department of Biotechnology, Gauhati University, Guwahati, Assam, India.
2Advanced Level State Biotech Hub, CVSc, AAU, Khanapara, Assam, India.
3Department of Animal Biotechnology, CVSc, AAU, Khanapara, Assam, India.
4Regional AI Centre, Bokakhat, Golaghat, Assam, India.
5Department of Obstetrics & Gynaecology, FAA Medical College & Hospital, Barpeta, Assam, India.
6Department of Surgery, Gauhati Medical College & Hospital, Guwahati, Assam, India.
Article Number: 8367 | © The Author(s). 2023
J Pure Appl Microbiol. 2023;17(2):951-965. https://doi.org/10.22207/JPAM.17.2.25
Received: 21 December 2022 | Accepted: 04 April 2023 | Published online: 10 May 2023
Issue online: June 2023
Abstract

Methicillin resistant Staphylococcus aureus (MRSA) is highly divergent antibiotic resistant bacteria earmarked as “High” in global pathogens’ priority list varying the severity and resistance geographically. Here, MRSA were screened using mecA gene with Cefoxitin and other 27 antibiotics of 19 classes using disc diffusion method from a highly humid climate of India. Multiple Antibiotic Resistance (MAR) index was calculated. Minimum Inhibitory Concentration (MIC) was determined against 11 classes of antibiotics. Detection of major virulence genes tst-1 and lukPV were done. A total of 95.24% Hospital Associated (HA)-MRSA, 56.14% Community Associated (CA)-MRSA and 82.53 % Livestock Associated (LA)-MRSA were detected. Cefoxitin, Oxacillin, Ciprofloxacin, Fusidic acid and Ticarcillin-Clavulinic acid resistance was observed in more than 60% of HA-MRSA, CA-MRSA and LA-MRSA. Across the hosts, Mupirocin, Gentamicin, Linezolid, Co-trimoxazole, and Rifampicin were found effective. Vancomycin Intermediate Staphylococcus aureus (VISA) detected in CA-MRSA & LA-MRSA. Multidrug Resistant (MDR) was found very high but extensively drug-resistant (XDR) was detected moderately. No pan drug-resistant (PDR) was detected. Virulence gene tst-1 and lukPV were detected in 7.69% and 32.69% MRSA isolates. The gene tst-1 is reported for the first time in pre and post-caesarian samples from Gynaecology department in this region with high MDR. This study showed S. aureus and subsequent prevalence of MRSA is higher in this region then national data. 2nd generation Cephalosporins were found effective which is very encouraging due to their limited uses. Detection of tst-1 in caesarian samples is a serious threat as neonatal transmission of MRSA from mother is reported.

Keywords

MRSA, OS-MRSA, BORSA, VISA, XDR, MDR, MAR Index

Introduction

Drug resistant disease causes almost 700,000 deaths every year globally.1 Staphylococcus aureus is classified as an important pathogen to be studied for Anti-Microbial Resistance (AMR). It is one of the 6 pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species) tagged as Critical and High importance by WHO and eponymously called as “ESKAPE”.2-3 MRSA is classified under the “serious threat” category by CDC in their “Antibiotic resistance threats in the united states-2019” report. The anterior nares are the most frequent site for S. aureus colonization and nasal carriage of S. aureus is an important risk factor for bacteremia and subsequent death. Carriers have higher rates of nosocomial S. aureus bacteraemia than non-carriers and a large proportion of nosocomial S. aureus infections originates from the patients’ own flora.4 Currently, Oxacillin-resistant staphylococci are resistant to all available β-lactam antimicrobial agents except some newer Cephalosporins. Vancomycin has been widely used in the treatment of MRSA infection for the past two decades.5 The majority of MRSA strains have remained susceptible to Vancomycin at the current Minimum Inhibitory Concentration (MIC) susceptibility breakpoint designated by the Clinical Laboratory Standards Institute (CLSI).

Antimicrobial resistance in Asia Pacific region is literary a global problem being the fact that more than 70% of world population lives here. Asian countries have shown very high rates (> 50%) of occurrence of MRSA.6 India has a large burden of infectious diseases and is one among the largest consumers of antibiotics in the world. Resistance was mostly found against Penicillin, Mupirocin, and Ciprofloxacin and greater susceptibility was observed towards Gentamicin and Co-trimoxazole. Vancomycin intermediate S. aureus (VISA) strains were also found to be very minimal.7 Studies have shown that geographically North India and East India have a greater burden of HA-MRSA then West and South India.8 In the same perspective, North East India needed to be studied separately being geo-climatically different from the rest of India. On the other hand, a few earlier studies in India detected higher frequency (5.4% –29.41%) of LA-MRSA in cattle and buffaloes with clinical mastitis9 and LA-MRSA is needed to be studied with a greater range of antibiotic classes.

Assam, a state of India, is characterized by alternate cool and warm periods with high humidity. As per Thornthwaite’s classification, Assam falls in “pre-humid” (moisture index Im 100 or more) and “humid” (moisture index Im between 20 to 100) climate zone.10 Assam has a temperature variation of 8°C-10°C in winter to up to 36°C in summer with a high humidity of 80%-85%, in average yearly. This region, as a whole, excluding the high mountain barriers, enjoys ‘Cwg’ or humid mesothermal Gangetic type of climate as per Koppen’s classification.11

High temperature and high relative humidity promotes sweat production and hydration of stratum corneum of the skin. These two factors provide suitable environmental conditions for maximized growth of S. aureus on skin, specifically during summer days.12 Increasing humidity was found to be associated with greater prevalence of MRSA and with Triclosan effectiveness found to be very less in humid and wet conditions against Staphylococcus aureus infections.13

India’s Antimicrobial Resistance Surveillance and Research Network by ICMR has already recognized six pathogenic groups and staphylococci (MRSA) is one among them. ICMR has emphasized on generating regional data focusing on One Health approach. Still, community data, surveillance on animals and data on antibiotics used in different regions of the country are missing so far. Hence, ICMR has asked researchers to gather antibiotic resistance data from livestock and poultry.14

Panton Valentine Leukocidin (Luk PV) is a multicomponent protein which severely damage the cell membrane by forming a hetero oligomeric transmembrane pore.15 Toxic Shock Syndrome (TSS) is a rare disease caused by a potentially lethal toxin characterized by high fever, hypotension and subsequent multiple organ dysfunction. This toxin is encoded by tst-1 (Toxic shock syndrome toxin) gene.16 Though earlier it was believed to be a disease of menstruating female, later it was also found in non-menstruating females and males.17 The toxin is hence divided into two distinct entities as “m-TSS” (menstruating Toxic Shock Syndrome) and “non-mTSS”( non-menstruating Toxic Shock Syndrome).

Prevalence of MRSA in human and animals and their antibiotic resistance patterns are poorly studied in Assam with variable data reported in limited number of studies. In fact, AMR studies involving various antibiotics classes with a view to identify multidrug-resistant (MDR), extensively-drug resistant (XDR) and pandrug-resistant (PDR) among Staphylococcus aureus field isolates have not been reported so far from the North Eastern region of India.18 Prevalence of tst-1 and lukPV are also reported less and due to the climatic and environmental differences, it is expected to be different from the national data.

In the present study, samples were collected from community, hospital wards (surgery and gynaecology) and livestock animals with a view to assess the MRSA prevalence as well as their antibiotic resistance patterns with respect to their virulence gene characterization.

Materials and Methods

Collection
In the present study, skin swabs from anterior nares and surgical wounds were collected from patients admitted in the clinical wards of Gynaecology and Surgery departments of two tertiary care hospitals viz. Silchar Medical College and Hospital and Gauhati Medical College and Hospital in Assam, India with preference to surgical wound samples, if available. Samples were collected during the summer season of 2019 and 2021. Wound swabs of under treatment animals from Veterinary Clinical Complex of College of Veterinary Sciences, Khanapara, Guwahati, Assam, domesticated animals, meat samples from poultry slaughterhouse and bovine milk samples from farm animals were selected for LA-MRSA isolation. Anterior nare swabs from communities and healthy human beings were collected as per CDC guidelines for CA-MRSA isolation. Except for slaughterhouse and milk samples, other samples were collected with sterile cotton swab (Hi-Media, PW009) in aseptic condition. Individual bovine milk samples were collected in sterile screw cap container (Hi-Media, PW126) directly from teats while milking and meat samples were aseptically collected from poultry slaughterhouses.

All swab samples were collected in sterile Cary Blair Transport Media w/o Charcoal (Sisco Research Lab, CM012) and processed within 48 hours of collection. All milk and meat samples were processed immediately after collection.

Isolation
The collected samples were inoculated in Peptone Water broth (Hi-Media, M614) followed by Mannitol Salt Agar (Hi-Media, MH118). Suspected isolates were confirmed on Baird Parker Agar (Hi-Media, M043) with concentrated egg yolk solution (Hi-Media, FD045) and Potassium Tellurite (Hi-Media, FD047) as per manufacturer’s instructions. Coagulase positivity were confirmed with Coagulase plasma (Hi-Media, FD248) as per manufacturer’s instructions.

DNA isolation
Genomic DNA was isolated from overnight grown pure cultures of S. aureus using PrestoTM Mini gDNA bacteria kit (Geneid, GBB300). Isolated DNA was quantified spectrophotometrically (Picodrop, Pico100). Average 300-500ng/µl DNA was obtained. Isolated DNA was immediately preserved at -20°C.

Molecular Confirmation
All suspected Staphylococcus aureus isolates were screened for aroA (Figure 1), nuc (Figure 2) and coa (Figure 3)  genes for confirmation.

Figure 1.  Amplification of aroA gene of Staphylococcus aureus by PCR
Lane 1-6 & 8-13: Isolates; Lane 7: 100 bp ladder; Lane 14: Negative control; Lane 15: Positive control (MTCC 96)

Figure 2.  Amplification of nuc gene of S. aureus
Lane 1-4, 6-8: Isolates; Lane 5: 50 bp ladder

Figure 3.  Amplification of coa gene of S. aureus by PCR
Lane 1-4, 6-8: Isolates; Lane 5: 50 bp ladder.

The primer set for aroA was designed in-house. A 50 µl PCR reaction mixture consisting of 1 µl each of 10 µM forward and reverse primers, 25µl of 2X Dream Taq PCR master mix (Thermo Scientific, K1071), 21 µl of nuclease free water (Thermo Scientific, AM9914G) and 2µl template DNA was prepared and PCR conditions were standardized at 95°C for 5 minutes followed by 35 cycles of 95°C for 1 minute, 52°C for 45 seconds and 72°C for 1 minute, and then 72°C for 5 minutes for final extension and 4°C for forever.

Primers and PCR conditions for nuc and coa gene (Table 1) were taken as reported.19 Annealing temperature for nuc was slightly modified using MTCC96 as control for standardization.

10 randomly selected isolates of S. aureus were subjected to 16s RNA amplification following reported protocol20 with slight modification of annealing temperature (Table 1). Gel products were purified with NucleoSpin® Gel and PCR Clean Up kit (Takara, 740609) and outsourced for Sanger Sequencing. Nucleotide BLAST (blastn) was performed with obtained sequences.

Table (1):
Primer pairs used for amplification of different genes of Staphylococcus aureus

No. Gene
name
Primer details Amplicon
size
Annealing
temp
Reference
1 aroA 5′-AAGGGCGAAATAGAAGTGCCG-3′ 872 bp 52 Self-designed
5′-ATTGGTTGAAGCATTGGTGT-3′
2 coa 5′-ACCACAAGGTACTGAATCAACG-3′ 211 bp 58 19
5′-TCTTGGTCTCGCTTCATATCC-3′
3 mecA 5′-AGAAGATGGTATGTGGAAGTTAG-3′ 584 bp 57 21
5′-ATGTATGTGCGATTGTATTGC-3′
4 nuc 5′-AAGCGATTGATGGTGATACGGTT-3′ 278 bp 50 19
5′-CAAGCCTTGACGAACTAAAGC-3′
5 16s RNA 5′-AGAGTTTGATCCTGGCTCAG-3′ 1513 bp 47 20
5′-GGTTACCTTGTTACGACTT-3′
6 tst-1 5′-TTATCGTAAGCCCTTTGTTG-3′ 398 bp 60 21
5′-TAAAGGTAGTTCTATTGGAGTAGG-3′
7 lukPV 5′-ATCATTAGGTAAAATGTCTGGA

CATGATCCA-3′

433 bp 55 24
5′-GCATCAASTGTATTGGATAG

CAAAAGC-3′

MRSA confirmation
Methicillin resistance was screened phenotypically with Oxacillin and Cefoxitin by disc diffusion method and genotypically with the screening of mecA gene (Figure 4). Bacterial turbidity of 0.5 McFarland constant was achieved with log phased growth of S. aureus isolates. The isolates were smeared on sterile Muller Hinton Agar plate using sterile swab (Hi-Media, PW003). Antibiotic discs were placed aseptically and first incubated at 35°C for 18 hours followed by another 6 hours at the same temperature to screen hetero-resistance. Zone of inhibition was measured for Cefoxitin and Oxacillin discs after 18 and 24 hours, respectively.

Figure 4.  Amplification of mecA gene of MRSA isolates
Lane 1: 100 bp ladder; Lane 2-7: Isolates; Lane 8: Positive control; Lane 9: Negative control

Reported primers and PCR conditions21 were used for mecA detection but it was found that MTCC96 is mecA negative. Hence, first three phenotypically Oxacillin and Cefoxitin resistant field samples were directly subjected to PCR amplification using the reported mecA primer and conditions. Amplified products were purified and sequenced to check the efficiency of the selected primer set to detect the presence of mecA gene. Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgiLINK_LOC=blasthome&PAGE_TYPE=BlastSearch&PROGRAM=blastn) was performed with the obtained sequences.

Details of Antibiotics used for disc diffusion and MIC test were provided in Supplementary Table 2 and Supplementary Table 3, respectively. Interpretation was done according to CLSI guideline.22

Table (2):
MIC distribution percentage details of HA-MRSA isolates

Antibiotics MIC (ug/ml) distribution percentage of 20 HA-MRSA isolates
<0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256
FC 100
RIF 100
TET 65 35
TMP 10 10 5 75
VAN 90 10
OXA 10 10 30 10 15 5 20
LNZ 20 70 10
TEI 60 40
ERY 20 45 5 30
PEN 10 15 15 10 50
CLI 5 65 30
FOX 15 15 40 30

Table (3):
MIC distribution percentage details of CA-MRSA isolates

Antibiotics MIC (ug/ml) distribution percentage of 32 CA-MRSA isolates
0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256
FC 3.1 50.0 6.3 6.3 3.1 3.1 6.3 15.6 6.3
RIF 56.3 43.8
TET 28.1 59.4 9.4 3.1
TMP 21.9 9.4 3.1 65.6
VAN 3.1 15.6 40.6 21.9 6.3 12.5
OXA 6.3 28.1 28.1 18.8 6.3 3.1 3.1 3.1 3.1
LNZ 3.1 9.4 40.6 34.4 12.5
TEI 34.4 37.5 15.6 9.4 3.1
ERY 9.4 9.4 18.8 18.8 9.4 15.6 9.4 9.4
PEN 3.1 3.1 9.4 9.4 28.1 12.5 15.6 18.8
CLI 9.4 6.3 21.9 46.9 15.6
FOX 28.1 43.8 18.8 6.3 3.1

Multi Drug Resistance (MDR), Extensive Drug Resistance (XDR) and Pan Drug Resistance (PDR) were interpreted as defined23 and reported accordingly.

Virulence gene characterization
All mecA positive isolates were screened for lukPV24 (Figure 5) and tst-121 (Figure 6) gene using reported primers. Details are provided in Table 1.

Figure 5. Amplification of lukPV gene of MRSA isolates
Lane 1,2,4,5: Isolates; Lane 3 : 100 bp ladder; Lane 6: Positive control (MTCC 96); Lane 7: Negative control

Figure 6. Amplification of tst-1 gene of MRSA isolates
Lane 1,2,4: Isolates; Lane 3: 100 bp ladder; Lane 5: Negative control; Lane 6: Positive control

Statistical analysis
Chi-Square Test (c2) was performed to find the significance level of Staphylococcus aureus isolation from different sources. A value of p< 0.01 was accepted as statistically significant. MAR (Multiple Antibiotic Resistance) index was calculated as described.25 A MAR index >0.2 signifies the high risk contamination where several antibiotics are used for treatment or growth promotion, A MAR index < 0.2 indicates the use of less antibiotics. MAR index =1 indicates that isolates were resistant against all antibiotics screened.

Figure 7. Resistance percentage of CA-MRSA, HA-MRSA and LA-MRSA isolates to different antimicrobial agents

RESULTS

A total of 611 samples were screened which included 154 hospital associated, 253 community associated and 204 livestock associated samples. Sequence similarity search by blastn with 16s RNA sequences of the isolates clearly identified the coagulase-positive isolates as Staphylococcus aureus with > 99 % similarity with identical isolates in the public database.

Details of sample isolation are provided in Supplementary Table 1. The Chi-square statistics of the three different types of sample source was 14.776. The p value was 0.000619. The result was found to be significant at p<0.01.

Blastn result with obtained mecA sequence showed > 99 % similarity with identical sequences of S. aureus in GenBank database. One of these sequences was used as positive control for further screening of other Methicillin resistant S. aureus (MRSA) isolates.

Depending upon phenotypic test and presence of mecA gene, the rate of occurrence of HA-MRSA was 95.24% (20/21) followed by 56.14% (32/57) CA-MRSA and 82.53% (52/63) LA-MRSA. The variation in the rate of occurrence was found to be significant (p < 0.01).

MRSA isolates resistant against different antibiotics are provided in Supplementary Table 4 & Figure 7.

All HA-MRSA isolates were Multi Drug Resistant (MDR) and 20% among them were Extensive Drug Resistant (XDR) with resistance against 10 to 11 antimicrobial classes.

MIC study revealed that except for Fusidic Acid and Rifampicin, other 10 antibiotics mostly fell within the MIC range of 0.5-16µg/ml. The Glycopeptides tested, viz. Vancomycin and Teicoplanin had MIC value between 1-2 µg/ml and 0.5-1 µg/ml, respectively. (Table 2).

It was found that 93.75% CA-MRSA were MDR and 10% among them were XDR with resistance shown against 10 to 17 antimicrobial classes.

MIC study revealed that except for Rifampicin, other 11 antibiotics mostly fell within the MIC range of 0.25-8 µg/ml. Vancomycin MIC was in the range of 0.25-8 µg/ml (Table 3).

It was found that all the LA-MRSA isolates were MDR and 5.77% among them were XDR with resistance shown against 10 to 14 antimicrobial classes.

MIC study revealed that all antibiotics mostly fell within the MIC range of 0.25-8 µg/ml.(Table 4).

No Pan Drug Resistance (PDR) was detected among the isolates in this study.

Out of 104 mecA positive Staphylococcus aureus isolates, tst-1 was detected in 8 (7.7%) isolates which comprises of 2 male, 5 female and 1 animal source. Detection of tst-1 in human samples were 6.73% (7/104) and all are from hospital-associated samples. All 5 female isolates were from the pre and post-caesarian ward of the Gynaecology department. No tst-1 was detected in CA-MRSA isolates.

Table (4):
MIC distribution percentage details of LA-MRSA isolates

Antibiotics MIC (ug/ml) distribution percentage of 52 LA-MRSA isolates
0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256
FC 17.3 51.9 9.6 11.5 9.6
RIF 92.3 1.9 3.8 1.9
TET 3.8 17.3 40.4 28.8 1.9 1.9 5.8
TMP 15.4 23.1 21.2 7.7 32.7
VAN 9.6 25.0 38.5 19.2 7.7
OXA 5.8 9.6 19.2 11.5 19.2 3.8 11.5 7.7 3.8 7.7
LNZ 13.5 50.0 30.8 5.8
TEI 7.7 48.1 26.9 17.3
ERY 3.8 23.1 21.2 25.0 3.8 5.8 9.6 7.7
PEN 3.8 1.9 11.5 19.2 7.7 15.4 7.7 19.2 13.5
CLI 3.8 46.2 32.7 7.7 5.8 3.8
FOX 1.9 23.1 23.1 21.2 19.2 5.8 5.8

A total of 34 (32.69%) isolates found harboring lukPV gene comprises of both human and animal isolates. Detection of lukPV gene in mecA positive samples were higher in Livestock (18/52) followed by hospital associated (9/ 20) and Community associated (7/32) samples. All mecA positive HA-MRSA and LA-MRSA carrying lukPV and/or tst-1 found mostly resistant against a diverse classes of antibiotics viz. Penicillinase stable Penicillin, Cephalosporins, Steroidal, Fluoroquinolones, Cephamycin and β lactam + β lactamase Inhibitor combination. CA-MRSA carrying lukPV were mostly found resistant against Fluoroquinolones and Folate pathway inhibitor classes whereas CA-MRSA carrying tst-1 were mostly resistant against Macrolides, Streptogramins and β lactam + β lactamase Inhibitor combination classes of antibiotics.

Details of MAR indexing results are provided in Table 5.

Table (5):
MAR indexing against antibiotics with respective no of isolates

No
No of
Antibiotics resistant (a)
No of
Sample
No of Antibiotics
screened (b)
MAR index
(a/b)
1
2
2
28
0.071
2
4
7
28
0.143
3
5
7
28
0.179
4
6
11
28
0.214
5
7
15
28
0.250
6
8
8
28
0.286
7
9
12
28
0.321
8
10
9
28
0.357
9
11
13
28
0.393
10
12
4
28
0.429
11
13
8
28
0.464
12
14
3
28
0.500
13
15
3
28
0.536
14
17
1
28
0.607
15
23
1
28
0.821
DISCUSSION

Asian Network for Surveillance of Resistant Pathogens (ANSORP) studies found that in Asian region, average MRSA prevalence was >50% with 25.5% and 67.4% being CA-MRSA and HA-MRSA, respectively. In India, they found 22.6% HA-MRSA and 4.3% CA-MRSA during the study period.26 Most of the samples received for ICMR-AMR surveillance network study were from ICU, OPD and ward admitted patients with superficial or blood stream infections. The study reported an increase of MRSA infections from 33.78% in 2015 to 42.6% in 2021.7,27-28 LA-MRSA occurrence varies worldwide (0.028%-9.3%) due to different geographical locations, samplings and methodologies.29 In India, it was reported to be higher (29.41%) in milk so as in Assam too. In some earlier studies in Assam, MRSA in animal meat was found to be 28.6%-48.57%.30-31 Our study showed that S. aureus as well as MRSA prevalence was very high in this region than the reported nationwide prevalence. This may be attributed to the high humidity in this region with substantial heat during summer.

It was also seen that S. aureus isolation was the lowest from hospitals but HA-MRSA was the highest from hospitals. ICMR-AMR surveillance network study have found deep wound infections by MRSA to be very low.27 In this study, however, we could not detect MRSA from wound infections, but a higher percentage (95.24 %) of MRSA were detected from anterior nares samples. This was perceptibly due to the use of effective regime of antibacterial for treatment of the wounds in the hospitalized patients. Isolation of 95.24% of MRSA from anterior nares of the hospitalized patients signified a high prevalence of MRSA in hospitals. However, sample volume may have influenced this result.

Worldwide, MRSA isolates are mostly resistant to Cefoxitin, Oxacillin, Penicillin, Methicillin, Tetracyclines, most of the 1st generation Cephalosporins, Macrolides, Fusidic Acid and Mupirocin.32 In Asian countries including India, it was observed that MRSA isolates were mostly resistant against both 1st and 2nd generation Cephalosporins along with both Penicillinase-labile and Penicillinase-stable β – lactam antibiotics, Cephamycin, Macrolides and Tetracyclines. In our study, the HA-MRSA isolates that were found to be resistant against 3rd and 4th generation Cephalosporins showed more than 60% resistance against β–lactam + β–lactam inhibitor combination also. Resistance was also recorded against Ciprofloxacin, which is otherwise mostly found effective outside India. It might be due to the indiscriminate and injudicious use of the Cephalosporins, β – lactam antibiotics and the common fluoroquinolone in this region. Resistance to the 2nd generation Cephalosporins was found to be very less in CA-MRSA, almost equivalent to 3rd and 4th generation Cephalosporins. Among LA-MRSA, resistance to the 2nd generation Cephalosporins was found to be very low compared to the 3rd and the 4th generation Cephalosporins. It was a very encouraging finding as currently, the 2nd generation Cephalosporins are rarely used in the treatment regime.

Isolates showing an MIC breakpoint value falling between 4– 8 µg/ml which was earlier 8-16 µg/ml prior to 2006,33 are considered as Vancomycin Intermediate S.aureus (VISA). After the first report of VISA,34 it is being an emerging concern in the MRSA treatment regime. It is believed that presence of MIC values towards the higher end of 2 µg/ml in Vancomycin susceptible isolates would make it slowly a less favourable antibiotic.35 ICMR AMRSN study8 found only 2.3% VISA strains among human associated MRSA. In our study, we didn’t find any VISA among HA-MRSA, but 18.75% and 26.92% of CA-MRSA and LA-MRSA, respectively, were found to be VISA isolates, clearly indicating the different patterns of MRSA prevalence in this region.

Oxacillin resistant S. aureus whose MIC value falls between 0.5– 4 µg/ml are characterized as BORSA36 and they are predominantly mecA negative. But, Yu-Tsung Huang37 reported mecA positive, Oxacillin resistant BORSA strains which also had an MIC of 0.5– 4 µg/ml. We found 4 no of CA-MRSA isolates which were mecA positive but Cefoxitin and Oxacillin susceptible falling under the range of BORSA. Marilyn Chung38 had mentioned such isolates as Oxacillin susceptible MRSA (OS-MRSA). Out of the four isolates, two were resistant to 6 groups of antibiotics (Aminoglycosides, Fluoroquinolones, Macrolides, Tetracyclin, Streptogramins and Folate pathway inhibitors) and other two were resistant to Fluoroquinolones, Folate pathway inhibitor, Macrolides, and Streptogramins, respectively. All the four isolates were susceptible to β–lactam + β–lactam inhibitor combination drug. Details study of these four isolates are further needed.

MDR strains of CA-MRSA were mostly resistant to 4-17 antibiotics classes which were mainly Fluoroquinolones, Steroidal, Streptogramins, β-lactam antibiotics with β-lactam inhibitors, Folate pathway inhibitor (Trimethoprim), Macrolides, 3rd generation Cephalosporins (Cefdinir), Aminoglycosides (Kanamycin), Penicillinase resistant penicillin and Cephamycin. High prevalence of MDR strains of CA-MRSA was also observed in China.39 Healthy individuals are a major asymptomatic reservoir of MRSA that may lead to its spread within the community.40-41 Interestingly, susceptibility of 2nd generation Cephalosporin (Cefaclor) and low resistance of Methicillin indicated restricted or obsolete use of these two drugs in this region.

MDR strains of HA-MRSA were mostly resistant to 5 -11 antibiotics classes consisting of mainly Cephamycin, Penicillinase resistant penicillin, Aminoglycoside (Kanamycin), Fluoroquinolones, Steroidal, 2nd/3rd/4th generation Cephalosporins, Folate pathway inhibitor (Trimethoprim) and β–lactam + β–lactam inhibitor combination drugs. All three generation Cephalosporins resistance indicated the drug abuse in human treatment regime and left with only choice of Glycopeptides.

MDR strains of LA-MRSA were mostly resistant to 4 -14 antibiotic classes mainly consisting of Cephamycin, Penicillinase resistant penicillin, Fluoroquinolones, Steroidal, 3rd generation Cephalosporin and β -lactam antibiotic with β -lactam inhibitor. Interestingly, above all these, MRSA from dogs showed resistance to Pseudomonic acid, Lincosamide, Ansamycin, Imipenem, Cephalosporins (all generation) and Folate pathway inhibitor. Small animals like dogs are mostly treated at the Veterinary Clinical Complex rather than other animals and they are more often treated with antibiotics. That’s why the higher level of antimicrobial resistance was observed.

Macrolide-Lincosamide-Streptogramin B (MLSB) regime are the most commonly used antibiotics against MRSA infections to spare the high-end antibiotics of Glycopeptides (Vancomycin) and Oxazolidinones (Linezolid) groups. Clindamycin resistance was found to be very high among HA-MRSA (100%) and LA-MRSA (69.23%), and moderate among CA-MRSA (43.75%) isolates. These findings are comparable to two earlier findings in other part of India42 and abroad.43 Though Clindamycin resistance was found to be higher in Hospital and Livestock samples, Erythromycin resistance was very less. This definitely indicates the possibility of using Clindamycin as an alternative therapy for Staphylococcus infection in India, especifically in Assam, both in human and animals.

Prevalence of Toxic shock syndrome toxin varies in Asian countries with detection of as high as 75%44 in Japan to as low as 2.9%45 in China. A recent study in Assam,16 tst-1 was detected in 50.79% MRSA consisting samples from hospital, community and environment, out of which only 7.9% were detected in hospital samples. In this study we have detected 35% hospital isolates harboring the tst-1 gene in MRSA strain which is corroborating with the earlier findings. Presence of tst-1 was thought to be connected with female menstrual hygiene with the use of tampons and menstrual cups, but later it was also detected in non-menstrual females and men. Most of the studies are concerned with mTSS and subsequent detection of tst-1. In this study, we have detected 5 no of MRSA strain from pregnant women in pre and post caesarian ward of the Gynecology department, which was first from this region. Mere detection of tst-1 doesn’t necessarily lead to the manifestation of disease as the level of gene expression controls the disease outcome. But the detection of the same in pregnant women is crucial as it has been reported that maternal carriages of MRSA plays a vital role in the neonatal carriages. No significance difference of antibiotic resistance was noticed in MRSA strain from mother and their neonates45 and the birth with highly resistant MRSA strain is a matter of concern. We found these five MRSA strains were highly resistant (Mean- 11.5 antibiotics, Mean MAR Index- 0.421). They were mostly resistant to Cephamycin, Penicillinase stable Penicillin, Fluoroqiunolones, Nitrofurans, Steroidal, Cephalosporins, Folate pathway inhibitor (only Trimethoprim) and β lactam+β lactamase inhibitor combination classes suggesting highly MDR strain.

Staphylococcus aureus isolates harboring lukPV in HA-MRSA and CA-MRSA were found to have higher antibiotic resistance with mean MAR Index 0.409 and 0.342 respectively than a lesser mean MAR Index of 0.289 for LA-MRSA. It definitely implies the use of higher antibiotics in human to cure severe Staphylococcus aureus infection than animals. Though lukPV was mostly found associated with CA-MRSA, we detected highest no in LA-MRSA with maximum in raw milk from bovine (14/18) and lowest in dog (1/18). In earlier studies, 41.6%- 47.6% Staphylococcus aureus isolates from bovine found harboring lukPV gene in India.46-47 It is probably due the practice of milking with hand in this region.

Strength and Limitations
In this study, we could not isolate MRSA from wound samples in human. Anterior nares work as a reservoir of MRSA, and isolation of MRSA from patients in hospital settings indicates the risk of nosocomial infection. MLST and SCCmec typing of these strains are underway in the lab for an even better understanding of the MRSA distribution.

CONCLUSION

As higher number of MDR and a few XDR isolates were reported in this region, Vancomycin resistance may be lurking around the corner. Uses of Glycopeptides to treat staphylococcal infection has to be very carefully monitored to prevent the growing Vancomycin resistance. Mupirocin, Gentamicin, Co-trimoxazole, 2nd generation Cephalosporins, Linezolid and Rifampicin may act as good alternatives to Glycopeptides to treat MRSA infections. In fact, restricted use of 2nd generation Cephalosporins in treatment may act as a much needed relief in the MRSA treatment. Antibiotic resistance to different classes in HA-MRSA and LA-MRSA are higher than CA-MRSA in this region shows the increased and abundant use of antibiotics in treatment regime. Increase of higher generation Cephalosporins resistance is a matter of concern. The clinical set up needs to look seriously on the uses of higher classes of antibiotics indiscriminately during treatment regime without assessing the susceptibility patterns. A detailed study of SCCmec typing and MLST will shade more light into the distribution of clonal complexes in livestock and human in this region. Uses of menstrual cups and tampons are very limited in this region. As we have detected tst-1 in pregnant woman with high MDR, it is further necessary to go for their expression study at protein level along with neonatal carriage study.

SUPPLEMENTARY INFORMATION

Additional file: Additional Table S1-S4.

Declarations

ACKNOWLEDGMENTS
The authors would like to thank Advanced Level State Biotech Hub, CVSc, AAU, Khanapara for providing with necessary help to carry out the research. Also thankful to Remya Parameswar Iyer, Kendriya Vidyalaya, IIT, Guwahati for her support.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
NKD, PB, PJH conceptualized the study and performed mthodology. PB, PJH performed formal analysis.NKD, MC, PK performed Investigation. NKD, PK, GD, RD, PB collected resources.NKD, PK wrote original draft. PJH, PB wrote, reviewed and edited the manuscript. PJH supervised the study. All authors read and approved the final manuscript for publication.

FUNDING
None.

DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript and/or in the supplementary files.

ETHICS STATEMENT
This study was approved by Institutional Ethics Committee, Gauhati Medical College & Hospital, Guwahati-781032 bearing approval no “MC/190/2007/Pt-11/MAR-2020/19” dated 04/06/2020 and Institutional Ethics Committee, Gauhati University, Guwahati- 781014 bearing approval no “GUIEC/2021/034” dated 29/09/2021.

References
  1. World Health Organisation. New report calls for urgent action to avert antimicrobial resistance crisis: WHO, New York. 2019. https://www.who.int/news/item/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis, Accessed on 22nd December, 2022.
  2. Tacconelli E, Carrara E, Savoldi A, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018;18(3):318-327.
    Crossref
  3. Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: No ESKAPE. J Infect Dis. 2008;197(8):1079-1081.
    Crossref
  4. Wertheim HFL, Vos MC, Ott A, et al. Risk and outcome of nosocomial Staphylococcus aureus bacteraemia in nasal carriers versus non-carriers. Lancet. 2004;364(9435):703-705.
    Crossref
  5. Kirst HA, Thompson DG, Nicas TI. Historical yearly usage of vancomycin. Antimicrob Agents Chemother. 1998;42(5):1303-1304.
    Crossref
  6. Kang CI, Song JH. Antimicrobial resistance in Asia: Current epidemiology and clinical implications. Infect Chemother. 2013;45(1):22-31.
    Crossref
  7. Walia K, Madhumathi J, Veeraraghavan B, et al. Establishing Antimicrobial Resistance Surveillance & Research Network in India: Journey so far. Indian J Med Res. 2019:149(2):164-179
    Crossref
  8. AMR ICMR Data, Annual report 2020. https://iamrsn.icmr.org.in/index.php/resources/amr-icmr-data, Accessed 28th March, 2023.
  9. Sunagar R, Hegde NR, Archana GJ, Sinha AY, Nagamani K, Isloor S. Prevalence and genotype distribution of methicillin-resistant Staphylococcus aureus (MRSA) in India. J Glob Antimicrob Resist. 2016;7:46-52.
    Crossref
  10. Thorpe E, Thorpe S. Geography and Environmental Ecology, Pearson India Education Services Pvt. Ltd,  Uttar Pradesh, India. 2020.  ISBN 978-93-325-4550-2
  11. Singh VP, Sharma N, Ojha CSP. The Brahmaputra basin water resources, 1st Ed. Springer Science+Business Media Dordrecht, Berlin, Germany. 2004.
    Crossref
  12. Mermel LA, Machan JT, Parenteau S. Seasonality of MRSA infections. PLoS One. 2011;6(3):e17925.
    Crossref
  13. Rios-Castillo AG, Ripolles-Avila C, Rodriguez-Jerez JJ. The effects of dry, humid and wear conditions on the antimicrobial efficiency of triclosan-containing surfaces. Appl Sci. 2019;9(8).
    Crossref
  14. Advancing Antimicrobial Stewardship. https://iamrsn.icmr.org.in/index.php/amsp/icmr-amsp. Accessed on 28th March, 2023.
  15. Amin DHM, Guler E, Baddal B. Prevalence of Panton-Valentine leukocidin in methicillin-resistant Staphylococcus aureus clinical isolates at a university hospital in Northern Cyprus: a pilot study. BMC Res Notes. 2020;13(1):490.
    Crossref
  16. Bhowmik D, Chetri S, Das BJ, Dhar Chanda D, Bhattacharjee A. Distribution of virulence genes and SCCmec types among methicillin-resistant Staphylococcus aureus of clinical and environmental origin: a study from community of Assam, India. BMC Res Notes. 2021;14(1):1-7.
    Crossref
  17. Tofte RW, Williams DN. Clinical and laboratory manifestations of toxic shock syndrome. Ann Intern Med. 1982;96(6p2):843-847.
    Crossref
  18. Borah P, Dutta R, Das L, et al. Prevalence, antimicrobial resistance and virulence genes of Salmonella serovars isolated from humans and animals. Vet Res Commun. 2022;46(3):799-810.
    Crossref
  19. Mitra SD, Velu D, Bhuvana M, et al. Staphylococcus aureus spa type t267, clonal ancestor of bovine subclinical mastitis in India. J Appl Microbiol. 2013;114(6):1604-1615.
    Crossref
  20. Eden PA, Schimdt TM, Blakemore RP, Pace NR. Phylogenetic analysis of Aquaspirillum magnetotacticum using Polymerase Chain Reaction-amplified 16s rRNA-specific DNA. Inter J System Bacteriol. 1991:41(2):324-325.
    Crossref
  21. Azimian A, Havaei SA, Fazeli H, et al. Genetic characterization of a vancomycin-resistant Staphylococcus aureus isolate from the respiratory tract of a patient in a University Hospital in Northeastern Iran. J Clin Microbiol. 2012;50(11):3581-3585.
    Crossref
  22. CLSI M100-ED29: 2021 Performance Standards for Antimicrobial Susceptibility Testing, 30th Edition. 2020;40.
  23. Magiorakos AP, Srinivasan A, Carey RB, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268-281.
    Crossref
  24. Lina G, Piemont Y, Godail-Gamot F, et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999;29(5):1128-1132.
    Crossref
  25. Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of faecal contamination of foods. Appl Environ Microbiol. 1983;46(1):165-170.
    Crossref
  26. Song JH, Hsueh PR, Chung DR, et al. Spread of methicillin-resistant Staphylococcus aureus between the community and the hospitals in Asian countries: An ANSORP study. J Antimicrob Chemother. 2011;66(5):1061-1069.
    Crossref
  27. AMR ICMR Data, Annual report 2021. https://iamrsn.icmr.org.in/index.php/resources/amr-icmr-data, Accessed on 28th March, 2023.
  28. Kaur J, Dhama AS, Buttolia H, et al. ICMR’s Antimicrobial Resistance Surveillance system (i-AMRSS): A promising tool for global antimicrobial resistance surveillance. JAC-Antimicrobial Resist. 2021;3(1):1-6.
    Crossref
  29. Mahanti A, Joardar SN, Bandyopadhyay S, et al. Characterization of methicillin-resistant and enterotoxins producing Staphylococcus aureus in bovine milk in India. J Agric Food Res. 2020;2:100017.
    Crossref
  30. Das P, Mazumder PB. Prevalence of Staphylococcus in raw meat samples in Southern Assam, India. J Agric Vet Sci. 2016;9(1):23-29.
    Crossref
  31. Phukan C, Anjum N, Ahmed G. In Vitro Activities of Linezolid, Daptomycin, Mupirocin and Tigecycline in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Skin and Soft Tissue Infections From North East India. Eur J Pharm Med Res. 2016;3(4):388-393
  32. Cuny C, Wieler LH, Witte W. Livestock-Associated MRSA: The impact on humans. Antibiotics. 2015;4(4):521-543.
    Crossref
  33. Maor Y, Rahav G, Belausov N, Ben-David D, Smollan G, Keller N. Prevalence and characteristics of heteroresistant vancomycin-intermediate Staphylococcus aureus bacteremia in a tertiary care center. J Clin Microbiol. 2007;45(5):1511-1514.
    Crossref
  34. Hiramatsu K, Hanaki H, Ino T, et al. Methicillin-resistant Staphylococcus aureus clinical strain with reduced Vancomycin susceptibility. J Antimicrob Chemother. 1997:40(1):135-146.
    Crossref
  35. Van Hal SJ, Lodise TP, Paterson DL. The clinical significance of vancomycin minimum inhibitory concentration in Staphylococcus aureus infections: A systematic review and meta-analysis. Clin Infect Dis. 2012;54(6):755-771.
    Crossref
  36. Hryniewicz MM, Garbacz K. Borderline oxacillin-resistant Staphylococcus aureus (BORSA) – a more common problem than expected? J Med Microbiol. 2017;66(10):1367-1373.
    Crossref
  37. Huang YT, Liao CH, Chen SY, Hsu HS, Teng LJ, Hsueh PR. Emergence of multidrug-resistant sequence type 45 strains among mecA-positive borderline oxacillin-resistant Staphylococcus aureus causing bacteraemia in a medical centre in Taiwan. Int J Antimicrob Agents. 2018;52(1):70-75.
    Crossref
  38. Chung M, Kim CK, Conceicao T, Aires-De-Sousa M, De Lencastre H, Tomasz A. Heterogeneous oxacillin-resistant phenotypes and production of PBP2A by oxacillin-susceptible/mecA-positive MRSA strains from Africa. J Antimicrob Chemother. 2016;71(10):2804-2809.
    Crossref
  39. Li Y, Tang Y, Qiao Z, et al. Prevalence and molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus in the respiratory tracts of Chinese adults with community-acquired pneumonia. J Infect Public Health. 2023;16(5):713-718.
    Crossref
  40. Tigabu A, Tiruneh M, Mekonnen F. Nasal Carriage Rate, Antimicrobial Susceptibility Pattern, and Associated Factors of Staphylococcus aureus with special emphasis on MRSA among urban and rural elementary school children in Gondar, Northwest Ethiopia: A Comparative Cross-Sectional Study. Adv Prev Med. 2018;9364757.
    Crossref
  41. Zajmi A, Shiranee F, Tang SGH, Alhoot MAM, Karim SA. Multidrug-Resistant Staphylococcus aureus as Coloniser in Healthy Individuals. In Bustos-Martínez J, Valdez-Alarcon JJ, eds. Staphylococcal Infections-Recent Advances and Perspective. IntechOpen Limited, London, UK. 2023.
  42. Bala R, Kaur N, Gupta N, et al. Detection of inducible resistance to clindamycin among methicillin resistant and sensitive strains of Staphylococcus aureus from India. J Pure Appl Microbiol. 2021;15(4):1957-1962.
    Crossref
  43. Shebl heba R, Zaki WK, Saleh AN, Salam shimaa AA. Prevalence of mecC gene among methicillin resistant Staphylococcus aureus isolated from patients in Ainshams University Hospital. J Pure Appl Microbiol. 2020;14(4):2807-2813.
    Crossref
  44. Nagao M, Okamoto A, Yamada K, Hasegawa T, Hasegawa Y, Ohta M. Variations in amount of TSST-1 produced by clinical methicillin resistant Staphylococcus aureus (MRSA) isolates and allelic variation in accessory gene regulator (agr) locus. BMC Microbiol. 2009;9:52.
    Crossref
  45. Lin J, Wu C, Yan C, et al. A prospective cohort study of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage in neonates: The role of maternal carriage and phenotypic and molecular characteristics. Infect Drug Resist. 2018;11:555-565.
    Crossref
  46. Annamanedi M, Sheela P, Sundareshan S, et al. Molecular fingerprinting of bovine mastitis-associated Staphylococcus aureus isolates from India. Sci Rep. 2021;11(1):1-15.
    Crossref
  47. Shrivastava N, Sharma V, Shrivastav A, Nayak A, Rai AK. Prevalence and characterization of Panton-Valentine leukocidin-positive Staphylococcus aureus in bovine milk in Jabalpur district of Madhya Pradesh, India. Vet World. 2018;11(3):316-320.
    Crossref

Article Metrics

Article View: 933

Share This Article

© The Author(s) 2023. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.