High prevalence of Panton-Valentine leukocidin positive, multidrug resistant, Methicillin-resistant Staphylococcus aureus strains circulating among clinical setups in Adamawa and Far North regions of Cameroon.

Staphylococcus aureus (S. aureus) is one of the earliest pathogens involved in human infections, responsible for a large variety of pathologies. Methicillin was the first antibiotic used to treat infections due to S. aureus but infections due to Methicillin resistant Staphylococcus aureus (MRSA) originated from hospital settings. Later, severe infections due to MRSA without any contact with the hospital environment or health care workers arose. Prevalence of MRSA has shown an alarming increase worldover including Cameroon. This Cross-sectional study was designed to evaluate the occurrence of MRSA infections in five different, most frequented Hospitals in northern Cameroon. Socio demographic data was recorded through questionnaire and different clinical specimens were collected for bacterial isolation. Identification of S. aureus was confirmed via 16s rRNA amplification using S. aureus specific primers. Molecular characterisation was performed through mecA gene, Luk PV gene screening and SCCmec typing. A total of 380 S. aureus clinical isolates were obtained of which 202 (53.2%) were nonduplicate multidrug resistant isolates containing, 45.5% MRSA. Higher number of MRSA was isolated from pus (30.4%) followed by blood culture (18.5%), and urine (17.4%). Patients aged 15 to 30 years presented high prevalence of MRSA (30.4%). Majority isolates (97.8%) carried the mecA gene, PVL toxin screening indicated 53.3% isolates carried the lukPV gene. Based on PVL detection and clinical history, CA-MRSA represented 53.3% of isolates. SCCmec typing showed that the Type IV was most prevalent (29.3%), followed by type I (23.9%). Amongst MRSA isolates high resistance to penicillin (91.1%), cotrimoxazole (86.7%), tetracycline (72.2%), and ofloxacin (70.0%) was detected. Meanwhile, rifampicin, fusidic acid, lincomycin and minocycline presented high efficacy in bacterial control. This study revealed a high prevalence of MRSA among infections due to S. aureus in Northern Cameroon. All MRSA recorded were multidrug resistant and the prevalence of CA MRSA are subsequently increasing, among population.

Introduction

Staphylococcus aureus (S. aureus) is recognized as one of the earliest pathogens involved in human infections and was first time isolated from pus by Louis Pasteur [1]. This pathogen is responsible for a large variety of pathologies worldwide and is still one of the most common infections in humans affecting all social groups [2, 3]. Infections caused by this bacteria include skin and soft tissue infections, bacteremia, endocarditis, central nervous system infections, pulmonary infections, muscle and skeletal infections, genitourinary tract infections, as well as toxin-induced diseases such as gastroenteritis [4-6].

Methicillin was the first antibiotic used to treat infections due to S. aureus but in 1961 resistant strains were discovered in the UK [7, 8]. Since this discovery, mortality, and morbidity of patients suffering from Stapholoccocal infection is increasing worldwide [9, 10]. Infections related to Methicillin resistant Staphylococcus aureus (MRSA) include pyogenic endocarditis, suppurative pneumonia, osteomyelitis and pyogenic infections of the skin, soft tissues [11]. MRSA originated from hospital settings, termed Hospital Acquired MRSA (HA-MRSA) [9] but since 1990, patients, particularly the young started to develope infections due to MRSA without any contact with the hospital environment or health care workers, and strains isolated were hence termed Community-Acquired MRSA (CA-MRSA) [9, 12]. This advent of CA-MRSA has drastically changed the epidemiology of past century MRSA isolates [13]. MRSA strains were favored by the carriage of certain genes such as mecA, which has a genetic element called staphylococcal chromosome cassette (SCCmec) [14, 15]. SCCmec consists of two essential elements, the mec complex, consisting of mecA and its regulators, and the cassette chromosome recombinase (ccr) genes that ensure the mobility of the cassette important for bacterial survival in stress conditions [16, 17]. SCCmec also encodes including a cytolysin psm-mec [17] which has an important role in virulence. Moreover, the comparison of mecA homologues and their neighboring genes carried by SCCmec are useful in determining phylogeny of the species in an evolutionary perspective [18]. Many SCCmec sequence types have been registered [17]. For genotypic differentiation of CA and HA MRSA, data indicate that CA-MRSA belong to SCCmec types IV and V [12]. In addition, the severity of S. aureus infection is related to its ability to adapt to human immune system through the production of diverse virulence factors that counteract the innate immune response and delay the adaptive immune response, promoting bacterial spread to deep tissues and organs. One such virulence factor that mainly targets leukocytes, is the pore forming Panton Valentine Leukocidin (PVL) [18, 19]. PVL toxin has two components called LukS-PV and LukF-PV and exhibits both toxic and immunomodulatory properties and is associated with severe infection outcomes including necrotizing pneumonia, pyomyositis, brain abscess, necrosis, and apoptosis [20-22]. CA-MRSA carry SCCmec types IV and V, and secrete the PVL toxin [12, 13]. While the HA MRSA strains which cause nosocomial infections carry SCCmec types I, II, and III [23, 24]. These strains do not secrete PVL toxin in general [25]. In addition, HA MRSA is resistant to non-Beta lactam antibiotics including aminoglycopeptides, fluoroquinolones, and macrolides [26, 27], while CA MRSA demonstrate resistance to Beta lactams, fluoroquinolones, and tetracyclines [28-30]. In Cameroon, the differentiation of these two types of strains is set up following the CDC definition based on surgical intervention, hospital environment contact, and hospital stay [31].

Multidrug resistance has became a major public health concern all over the world. Recently, many authors have reported the emergence of multidrug resistant bacterial pathogens, from various origins including veterinary and humans health systems [31-33]. Prevalence of MRSA shows an alarming increase in Cameroon with an increase from 20–30% in 2003 [34], to 34.6% in 2013 [35], and 78.6% from 2017 to 2019 [36]. The aim of this study was to evaluate distribution of CA and HA MRSA in Cameroon and assess their genetic diversity through MecA gene, Luk PV screening as well as SCCmec typing. Furthermore, antimicrobial resistance profiling is performed as a step towards better regulation of MRSA strains in circulation among hospitals and communities of Cameroon in sub-Saharan Africa.

Material and methods

A Cross-sectional study was conducted from April 2019 to December 2020 in five different, most frequented hospitals of Adamaoua and Far North Regions of Cameroon. Approval to conduct the study was obtained from the National Ethics Committee of Cameroon (No2017/12/958/CE/CNERSH/SP). Written authorization from each regional delegate of health and the various directors of hospitals were also obtained. Data were collected by questionnaire after the oral and written consent of patients or guardians. A literature review was conducted to design the questionnaire [37, 38]. Data acquired through questionnaire included sociodemographic information, Out or in- patients (according to the clinical definition of out or in patient care related to the CDC reference [31].

Bacterial isolation and identification

Bacterial trains were isolated from eight different types of clinical samples including pus, urine, blood culture, surgery wound, vaginal swab, urethral swab, semen culture, stool culture. Mannitol salt Agar (Bio-Rad, France) was used for culture.

Preliminary identification was carried out using a combination of colony morphology observation, Gram staining, catalase test to differenciate between catalase positive staphylococcal vs catalase negative streptococcus isolates [39]. Furthermore, mannitol fermentation, coagulase test (to differentiate Staphylococcus aureus isolates from others species of Staphylococcus) [40], and Dnase test (for identification of S. aureus) were performed as described earlier [41]. Isolate details are included in S2 Table.

Antimicrobial susceptibility

Antimicrobial susceptibility profiling was performed using the Kirby Bauer disc diffusion method on Muller Hinton Agar (Oxoid Ltd, Basingstoke, UK) according to the instruction of the European Committee on Antimicrobial Susceptibility Testing [42]. The turbidity of bacterial suspension was standardized by using 0.5 McFarland. A sterile cotton swab was dipped into the bacterial suspension and spread over the entire surface of Muller Hinton agar (Oxoid Ltd, Basingstoke, UK). Plates were left at room temperature for 3 to 5 min. Then, antimicrobial drug discs were placed by using a disc manual dispenser on to the Muller Hinton agar and incubated at 37°C for 18–24 h. Sixteen antibiotics (Oxoid Ltd, Basingstoke, UK) from eight classes of antibiotics were tested, namely: Penicillin (Oxacillin (5μg), Amoxicillin and Clavulamic acid (30μg),); Cephalosporin (Cefoxitin (30μg)), Novobiocin (5μg), Fluoroquinolon (Ofloxacin (15μg), Ciprofloxacin (5μg),): Aminoglycoside (Gentanicin (10 μg),): Macrolide/Lincosamide (Pristinamicin (15μg), Erythromycin (15μg), Lincomycin (15μg)); Tetracycline (Tetracyclin (30μg) and Minocyclin (30μg)); Others (Cotrimoxazol (75μg), Rifampicin (30μg), Fusidic Acid (10μg)); and Glycopeptide (Vanconicin (30μg)). At the end of the incubation period, the diameter of the growth inhibition area was measured by a digital caliper. The corresponding measured diameters were interpreted as susceptible (S), intermediate (I), or resistant (R) according to the European Committee on Antimicrobial Susceptibility Testing guidelines. Bacteria resistant to at least one antibiotic from three or more different antimicrobial classes were considered as MDR bacteria [43]. Methicillin resistance was characterized by Oxacillin (5μg) and Cefoxitin (30μg) double disc diffusion test. Isolates showing inhibition zone < 26 mm for oxacillin and < 22 mm for cefoxitin were considered methicillin-resistant Staphylococcus aureus (MRSA) according to the European Committee on Antimicrobial Susceptibility Testing and Antibiotic Committee of France Society of Microbiology 2019 [42].

Molecular identification of MRSA

DNA extraction

Methicillin-resistant Staphylococcus aureus (MRSA) isolated in this study were inoculated using Mannitol Salt Agar (Oxoid Ltd, UK) and incubated at 37°C for 24 hours. DNA extraction and purification of S. aureus was performed using the Solis BioDyne DNA extraction kit (Solis BioDyne, Estonia) according to manufacturers’ instructions [44, 45]. A couple of checks were realized to confirm DNA extraction: Evaluation of quality and quantity of DNA using Nanodrop machine and Gel electrophoresis using 1% agarose gel.

PCR confirmation of Staphylococcus aureus

Molecular identification of S. aureus strains was performed via amplification of the conserved 16s rRNA region in S. aureus amplifying a 409 kb product using 5’-ATTAGATACCCTGGTAGTCCACGCC- 3’and 5’-CGTCATCCCCACCTTCCTCC-3’ primers [46]. A total volume of 20 μL of PCR reaction mixture was constituted by adding 09 μL of PCR water, 1μL of forward, and reverse primers each, 04 μL of Master Mix (5x) (Solis BioDyne) and 05 μL of template DNA. Following conditions were established for this PCR: Initial denaturation 94°C for 5 min; denaturation 94°C for 45 seconds; annealing 60°C for 45 seconds; amplification 72°C for 30 seconds for 30 cycles; Final extension was performed at 72°C for 10 minutes. PCR products were resolved on 1% agarose gel at 90V for 40 min and visualized on UV transilluminator. Amplicon size of 409bp indicated the presence of S. aureus.

Detection of mecA and PVL

mecA and PVL were detected using a multiplex PCR screening method. MecA gene was amplified using mecA1 (5′ -GTAGAAATGACTGAACGTCCGATAA-3′’) and mecA2 (5′-CCAATTCCACATTGTTTCGGTCTAA-3′’) primers. Panton-Valentine Leucocidin was identified using primers sequence detecting lukS/F-PV genes. S/F primers use were Luk PV1 (5’-ATCATTAGGTAAAATGTCTGGACATGATCCA-3′’) and Luk PV2 (5’-GCATCAASTGTATTGGATAGCAAAAGC-3). The mixture was prepared by adding 1.5 μL of each of the four primers, 10 μL of PCR water, Master Mix 4 μL (Solis bioDyne), and template DNA 05 μL. While the thermocycling conditions were: Initial denaturation 95°C for 5 min; denaturation 95°C for 1 min; annealing 55°C for 1 min; amplification 72°C for 1 min 30 seconds; Final extension 72°C for 10 minutes for 30 cycles then resting at 4°C [38]. PCR products were resolved on 1% agarose gel at 90V. The gels were visualized under UV light to detect the expected band sizes (433 bp for Luk S/F and 310 for mec A) [47, 48].

Detection of Sccmec types I, II, III, IV, and V

In general, Community-acquired MRSA (CA-MRSA) carry PVL encoding genes LukS-PV and LukF-PV which are associated with increased virulence and HA-MRSA lack this gene. However, recent evidence shows that this difference has gradually become indistinct in recent CA and HA MRSA strains [13, 49]. For clear stratification of HA-MRSA and Community CA-MRSA, identification of Sccmec (Staphylococcal Chromosome Cassette mec) types is useful. HA-MRSA strains carry SCCmec types I, II, or III and do not have PVL encoding genes [25], while CA-MRSA carry SCCmec IV and V [50, 51]. The protocols used by Boyle et al and McClure-Warnier were followed for the molecular identification of Sccmec types I to V [44]. The reaction mixture was prepared as earlier but for the addition of oligomers mentioned in S1 Table.

Thermocycling parameters followed were: 95°C for 4 min, followed by 34 cycles of 95°C for 1 min, 53°C for 1 min, 72°C for 1 min 30 sec and final extension at 72°C for 4 min at 4°C. The PCR products were electrophoresed on 2% agarose gel containing ethidium bromide and visualized under UV transilluminator.

Statistical analysis

Data were maintained in Excel sofware, and exported to SPSS version 25.0 where statistical analysis like percentages, proportion and association were performed. Graphs were prepared using originPro 9.0 64 bit sofware. Descriptive statistics like mean, frequencies were performed on different variables. Chi-square and Fisher exact test were used to test categorical variables. A significant difference was considered at a P-value < 0.05.

Results

Phenotypic characteristics of the recovered isolates

During the nineteen (19) months of collection, 630 samples of Gram positive cocci were recorded from five clinical laboratory hospitals in northern Cameroon. Based on colony morphology observation, Gram staining, catalase, mannitol fermentation, coagulase, and Dnase test, 380/630 (60.3%) samples were identified as S. aureus, among these 201 (52.9%) originated from Adamaoua region and 179 (47.1%) from far north. Results of antimicrobial susceptibility showed that 202/380 (53.2%) of S.aureus were resistant to at least three antibiotics (multi drug resistant) from three different classes. Subsequent analysis was focused on these 202 nonduplicate multidrug resistant samples of Staphylococcus aureus. High prevalence of S. aureus was identified from pus 25.7% followed by urine 20.8%, urethral swab 12.5% and blood culture 11.2%. Among these MDR strains, 92 (45.5%) were MRSA (Table 1).

Table 1

Prevalence of Staphylococcus aureus among clinical samples collected between April 2019 to December 2020.

	Regions
	Adamawa (%)	Far north (%)	Total of samples
Gram positive cocci	332 (52.7)	298 (47.7)	630
S. aureus	201 (52.9)	179 (47.1)	380
Multi drug resistant (MDR) S. aureus	120 (59.4)	82 (40.6)	202
Methicillin resistant S. aureus (MRSA)	60 (57.7)	44 (42.3)	104
Methicillin resistant S. aureus after molecular confirmation	52 (56.5)	40 (43.5)	92

Socio demographical data of study participants showed that men were most represented (68.5%) compared to women (31.5%). The age of the patients ranged from 21 days to 85 years. Patients aged 15 to 30 presented the highest prevalence of MRSA (30.4%), followed by the 0 to 15 years age group. Most samples belonged to the Adamawa region (56.5%) while 43.5% were isolated from the Far North region of Cameroon. A higher number of MRSA were isolated from pus (30.4%) followed by blood culture (18.5%), and urine (17.4%). Urethral and vaginal isolates were equally represented with 9.8% isolates from these biological samples (Table 2).

Table 2

Repartition of MRSA based on age group and clinical sources.

	HA-MRSA (%)	CA MRSA (%)
Age group
0–15	18 (66.7)	9 (33.3)
15–30	4 (14.3)	24 (85.7)
30–45	7 (35)	13 (65)
45–60	2 (50)	2 (50)
60–75	6 (60)	4 (40)
≥75	3 (100)	0 (00)
Clinical source
Urine	4 (25)	12 (75)
Pus	18 (64.3)	10 (35.7)
Vaginal swab	1 (11.1)	8 (88.9)
Blood culture	12 (70.6)	5 (29.4)
Stool culture	0 (00)	4 (100)
Semen culture	0 (00)	4 (100)
Uretral collection	0 (00)	9 (100)
Sugery wound	5 (100)	0 (00)

Based on the CDC definition of Community-Acquired Methicillin-Resistant Staphylococcus aureus (CA-MRSA) which relies on surgical intervention, hospital environment contact, and hospital stay (32), 56.5% of the clinical isolates were categorized as CA-MRSA versus 43.5% Hospital-Acquired MRSA (HA-MRSA) isolates. The difference between CA-MRSA and HA-MRSA based on gender was not statistically significant (p ˃ 0.05), while a significant difference was observed between CA-MRSA, HA-MRSA concerning age group and clinical samples (p<0.05) (Table 2).

Distribution of LukS/F PV genes and SCCmec typing

Multiplex PCR detection of mecA and PVL genes revealed that 97.8% (n = 90) of our samples carried the mecA gene. While PVL toxin screening indicated that 53.3% (n = 49) carried the lukPV gene, 44.6% (n = 41) were Luk S/F negative. SCCmec typing showed that the Type IV was most prevalent (29.3%), followed by type I (23.9%), type V (22.8%), type III (14,1%), and type II (7.6%). Meanwhile, 2.2% isolates were not typable. Classification of Hospital Acquired MRSA (HA-MRSA) and Community-Acquired MRSA based on PVL production and SCCmec types showed that the CA MRSA (SCCmec types IV and V producing PVL toxin) was most prevalent 52.1% (n = 48) while 45.6% (n = 42) were HA MRSA (SCCmec types I, II and III and non producers of PVL). Similar distribution (CA-MRSA 56.5% and HA-MRSA 43.5%) was observed following the CDC definition as mentioned before, and there is no statistical difference between both methods with p = 0.577.

The most prevalent SCCmec type in all clinical samples was SCCmec Type IV (n = 27) and type I (n = 22). SCCmec type I was most prevalent in pus (n = 12) samples followed by type V (n = 7) which was most commonly observed in urine, and blood culture (n = 5). SCCmec type II was the least common and found in only 3 different isolates. Meanwhile type III was present in all the clinical samples, from pus (23.1%), urine (15.4%), and stool (7.7%) sources. Similarly, SCCmec type IV was common in isolates from pus (25.9%) and blood (33.5%) (Table 3).

Table 3

SCCmec typing of MRSA isolates.

		Urine (%)	Pus (%)	Vaginal cult (%)	Blood cult (%)	Stool (%)	Semen (%)	Urethral (%)	Surgery wound(%)	Total
SCCMec types	I	00 (00)	12(54,6)	2(9.1)	2(9.1)	2(9.1)	1(4.5)	2(9.1)	1(4.5)	22
	II	2(28.7)	1(14.2)	0(00)	2(28.7)	1(14.2)	0(00)	1(14.2)	0(00)	7
	III	02(15.4)	3(23)	2(15.4)	2(15.4)	1(7.7)	1(7.7)	1(7.7)	1(7.7)	13
	IV	03(11.1)	7(26)	2(7.4)	9(33.3)	0(00)	1(3.7)	3(11.1)	2(7.4)	27
	V	07(33.3)	5(23.8)	3(14.3)	2(9.5)	0(00)	1(4.8)	2(9.5)	1(4.8)	21
	Not typable	2(100)	-	-	-	-	-	-	-	2
Total		16	25	9	15	4	4	8	5	92

The geographical distribution of data showed that patients from Adamaoua region were most represented 52 (56.5%) than those from the far north region 40 (43.5%). While CA and HA MRSA repartition showed that HA MRSA were the most prevalent 21 (52.5%) in the Far north meanwhile CA MRSA were mostly present from out patient in the Adamaoua region 57.7% (n = 30) (Table 2). For SCCmec distribution, the type IV and V (n = 15 for each) were mostly represented in the Adamawa region followed by type I and III (n = 9 for each) while in the far north region the type I (n = 14) was most prevalent followed by type IV (n = 13) (Table 4).

Table 4

Region wise distribution of SCCmec types among clinical samples collected from the Adamawa and Far north regions of Cameroon.

Types of Sccmec	Adamawa region (%)	Far north region (%)
I	9 (40.9)	13 (59.1)
II	3 (42.9)	4 (57.1)
III	9 (69.2)	4 (30.8)
IV	15 (55.6)	12 (44.4)
V	15 (71.4)	6 (28.6)
Not typable	2 (100)	0

Antimicrobial susceptibility testing of isolates

All clinical S. aureus isolates were processed for antimicrobial susceptibility and classified as Multi drug-resistance (MDR), Extensively drug-resistance (XDR) and pandrug resistance (PDR) based on definition given by Magiorakos et al. [43].

MRSA isolates presented high resistance to penicillin (91.1%) followed by cotrimoxazol (86.7%), tetracycline (72.2%), and ofloxacin (70.0%). All (100%) MRSA isolates were multi-drug resistant. While rifampicin, fusidic acid, and minocycline presented high susceptibility respectively 90.0%, 75.6%, and 64.4% (Table 5).

Table 5

Antimicrobial susceptibility profile of MRSA isolates.

Group of ATB	ATB tested	Antibiotic susceptibility profile
		R (%)	S (%)	I (%)
Penicillin	AMC	43 (47.8)	11 (12.2)	36 (40.0)
	OX	92 (100)	00 (00)	00(00)
	P	82 (91.1)	05 (05.6)	03 (03.3)
Cephalosporin	FOX	92 (100)	00 (00)	00 (00)
Fluouroquinolon	CIP	39 (43.3)	34 (37.8)	17 (18,9)
	OFX	63 (70.0)	21 (23,3)	06 (06,7)
Aminoside	GEN	51 (56.7)	28 (31.1)	11 (12,2)
Macrolide /Lincosamide	E	40 (44.4)	43 (47.8)	07 (7.6)
	L	17 (18.9)	58 (64.4)	15 (16.7)
	PI	03 (3.3)	35 (38.9)	52 (57.8)
Tetracycline	TET	65 (72.2)	21 (23.3)	04 (04.4)
	MIN	08 (08.9)	54 (60.0)	28 (31.1)
Others	SXT	78 (86.7)	08 (08.9)	04 (04.4)
	RD	07 (07.8)	81 (90.0)	02 (02.2)
	FA	07 (07.8)	68 (75.6)	15 (16.7)
Glycopeptide	VA	12 (13.3)	34 (37.8)	44 (48.9)

Difference between, CA and HA MRSA is made on the basis of SCCmec and PVL secretion, antimicrobials comparison of these two types showed that the CA-MRSA were mostly resistant to beta-lactams, fluoroquinolones, tetracyclines, such as penicillin (95.6%), cotrimoxazole (87.4%), ofloxacin (79.2%) and tetracycline (75.0), meanwhile, HA-MRSA were most resistant to penicillin (85.7%), cotrimoxazole (85.7%), tetracycline (69.0%), ofloxacin (59.5%), gentamicin (47.6%). While most isolates were susceptible for rifampicin (88.1%), fusidic acid (76.2%), and lincomycin (66.7%) (Fig 1). HA MRSA are known to be resistant to non-Beta lactam antibiotics including aminoglycopeptides, fluoroquinolones, and macrolides [25] while CA MRSA demonstrate resistance to Beta lactams, fluoroquinolones, and tetracyclines [27]. Our study shows extensive drug resistance among CA MRSA that is steadily increasing in prevalence compared to previous data. It is alarming to note that while all MRSA were MDR, certain PDR isolates among both HA MRSA and CA MRSA were also identified (Table 6).

Fig 1

Antimicrobial susceptibility profile of CA and HA MRSA.

Kirby-bauer disc diffusion method was used to test Antimicrobial susceptibility of CA and HA MRSA against Amoxicilline+Clavulanic acid (AMC), Oxacillin (OX), Cefoxitin (FOX), Ciprofloxacin (CIP), Ofloxacin (OFX), Gentamicin (GEN), Erythromycin (E), Lincomycin (L), Tétrecyclin (TET) Cotrimoxazole (SXT), Rifampicin (R), FusidicD Acid (FA), Vancomycin (VA), Penicillin (P), Minociclin (MI). Zones of inhibition were interpreted according to EUCAST guidelines, R = Resistance; S = Sensible; I = Intermediaire.

Table 6

Antimicrobial susceptibility profile of S. aureus.

	Resistance profile	Most common antibiotic resistance patterns
Hospital acquired–MRSA	6 MDR	Aminoside (GEN); Fluoroquinolone (OFX, CIP); Macrolides (E, L)
	28 XDR
	2 PDR
Community acquired-MRSA	29 MDR	Betalactamines (P, OX, FOX, VA), Fluoroquinolone (CIP, OFX), Tetracycline (TET, MIN)
	41 XDR
	2 PDR

Distribution of SCCmec types according to antibiotic resistance phenotypes for MRSA

Distribution of SCCmec types according to antibiotic resistance phenotypes for MRSA isolates shows that sccmec type IV and V, which cause infections in community, were mostly resistant to penicillin, ofloxacine, cotrimoxazole and tetracycline. Meanwhile type I, II and III, responsible for hospital acquire infections were resistant to penicillin, cotromoxazole, tetracycline and erythromycine (Table 7).

Table 7

Distribution of SCCmec types according to antibiotic resistance phenotypes for MRSA.

		Types of SCCmec
Antibiotic classes	Antibiotic Tested	HA MRSA			CA MRSA
		I	II	III		IV	V
		R (%)	R (%)	R (%)		R (%)	R (%)
Penicillin	Amoxicillin	13 (59.1)	3 (42.8)	8 (61.5)		8 (29.6)	11(52.4)
	Oxacillin	22 (100)	7 (100)	13 (100)		27 (100)	21(100)
	Penicillin	20 (90.9)	6 (85.7)	10 (76.9)		26 (96.3)	20 (95.2)
Cephalosporin	Cefoxitin	22 (100)	7 (100)	13 (100)		27 (100)	21 (100)
Fluoroquinolone	Ciprofloxacin	7 (31.8)	3 (42.3)	5 (38.5)		11 (40.7)	13 (61.9)
	Ofloxacin	13 (59.1)	3 (42.3)	9 (69.3)		20 (74.1)	18 (85.7)
Aminoside	Gentamicin	9 (40.9)	4 (57.1)	7 (53.8)		19 (70.4)	12 (57.1)
Macrolide /Lincosamide	Erythromycin	10 (45.5)	3 (42.9)	7 (53.8)		10 (37.4)	10 (47.6)
	Lincomycin	4 (18.2)	2 (28.6)	2 (15.4)		6 (22.2)	3 (14.3)
	Pristynamicin	2 (8.9)	0 (0)	0 (0)		0 (0)	1 (4.8)
Tetracyclin	Tetracycline	13 (59.1)	5 (71.4)	11 (84.6)		22 (81.5)	14 (66.7)
	Minocyclin	1 (4.5)	1 (14.3)	1 (7.7)		4 (14.8)	1 (4.8)
Others	Cotrimoxazol	18 (81.8)	7 (100)	11 (84.6)		24 (88.9)	18 (85.7)
	Rifampicin	1 (4.5)	1 (14.3)	1 (7.7)		2 (7.4)	2 (9.1)
	Fusidic acid	0 (0)	0 (0)	1 (7.7)		3 (11.1)	3 (14.3)
Glycopeptide	Vancomycin	4 (18.2)	2 (28.6)	0 (0)		3 (11.1)	3 (14.3)

Correlation of clinical sources, and antimicrobial resistance

S. aureus isolated from all clinical samples were multi drug resistant. Samples from urine, blood culture and surgery wound were comming mostly from community sources, while S. aureus isolated from pus, stool culture were predominantly nosocomial. Our results showed that samples from CA MRSA, are commontly resistant to penicillin, cotrimoxazole, tetracycline and ofloxacine while rifampicin, lincomycin, erythromycin and minocycline presented high sensitivity rates. On the otherhand HA MRSA, were commontly resistant to penicillin, cotrimoxazole, tetracycline and gentamicine while showing sensitivity to rifampicin, lincomycin and fusidic acid (Table 8).

Table 8

Correlation of clinical sources, and antimicrobial resistance profiles.

Clinical sample source	Sccmec Type	Common resistant antibiotics	Common susceptible antibiotics
Urine	10/14 (71.4%) type IV to V	P, SXT, TET, OX, FOX	GEN, E, L, MI, RD
Pus	16/28 (57.1%) type I to III	P, SXT, TET, AMX, OX, FOX, GEN	L, MI, RD, FA
Vaginal collection	5/9 (55.6%) type IV to V	P, SXT, TET	GEN, E, L, MI, RD
Blood culture	11/17 (64.7%) type IV to V	P, SXT, TET, OFX, GEN,	E, L, PI, MI, RD, FA
Stool culture	4/4 (100%) type I to III	P, SXT, TET, GEN	RD
Semen culture	2/4 (50%) type IV to V	P, SXT, TET	L, PI, RD
Urethral collection	5/9 (55.6%) type IV to V	P, SXT, TET, OFX,	GEN, E, L, PI, RD, FA
Sugery wound	3/5 (60%) type IV to V	P, SXT, TET, OFX, L	RD

Amoxicilline+Clavulanic acid (AMC), Oxacillin (OX), Cefoxitin (FOX), Ciprofloxacin (CIP), Ofloxacin (OFX), Gentamicin (GEN), Erythromycin (E), Lincomycin (L), Tétrecyclin (TET) Cotrimoxazole (SXT), Rifampicin (RD), Fusidic Acid (FA), Vancomycin (VA), Penicillin (P), Minociclin (MI). MDR: Multi drugs resistance

Discussion

S. aureus is responsible for a large variety of pathologies worldwide [2].This pathogen has developed several resistance mechanismsagainst antimicrobial treatment. They include enzymatic inactivation of the antibiotic, alteration of the target with decreased affinity for the antibiotic, and also spontaneous mutations [44]. Methicillin-resistant Staphylococcus aureus remains an important issue associated with high mortality and morbidity of patients. MRSA are still a major public health concern worldwide, due to treatment challenges [52]. In the last century, infections related to MRSA only originated from hospitals known as Hospital-acquired Staphylococcus aureus [9]. However since 1990, infections due to MRSA without any contact with hospital or health care workers arose, thereafter termed Community-Acquired Staphylococcus aureus (CA-MRSA) [12]. In Cameroon Prevalence of MRSA has been increasing steadily with a rise from 20–30% prevalence since 2003 [34] to 80% in 2019 [53]. In this study, 202 nonduplicate multi drug resistant samples of S. aureus were isolated from the five most frequented hospitals of Adamawa and Far North regions of Cameroon. Among these clinical isolates 45.5% were identified as MRSA. Repartition of our clinical samples indicates that MRSA were most frequently detected in pus followed by blood culture and urine with respectively 30.4%, 18.5%, and 17.4% prevalence. Pus samples were mostly obtained from hospitalised patients while urine and blood culture were from outpatient wards. It is important to indicate that we have mostly focused our attention on the urinary tract infections associated S. aureus isolated from pregnant women, young children aged less than 5 years, and people living with comorbidities such as HIV and diabetes because they have compromised or immature immune systems. Antimicrobial susceptibility testing showed high resistance to penicillin, cotrimoxazole, tetracycline, ofloxacine and gentamicine. A similar report from Nepal also showed inefficacy of these antibiotics for treatment of local isolates [54]. Meanwhile rifampicin, fusidic acid, lincomycin and minocycline showed efficacy against our isolates. Similar efficacy of rifampicin was also observed in isolates from Creech [55]. Sociodemographic distribution showed that men were most susceptible with 68.5% (n = 63) prevalence. Similar prevalence among men was also observed in Ethiopia [56] and Barbados [57]. Samples from urine, blood culture and sugery wound were acquired mostly from community sources and showed resistance to penicillin, cotrimoxazole, tetracycline and ofloxacine while rifampicine, lincomycin, erythromycin and minocycline presented high sensitity rate. Meanwhile S. aureus isolated from pus, stool culture were from inpatient source. These isolates were resistant to penicillin, cotrimoxazole, tetracycline and gentamicine, while sensitivity to rifampicine, lincomycin and fusidic acid were observed. Similar results were reported in India recently [12]. In Cameroon betalactams, cephalosporin, tetracycline are mostly used to treat infections related to S. aureus [35, 58]. However, emergence of multiresistant strains carrying the PVL toxin worsen the situation. Therefore rifampicine, lincomycin and minocycline are recommended for effective control. Our study also highlights the fact that the rate of multidrug resistant MRSA among population is increasing in Cameroon as all our MRSA isolates were MDR. Mainstay antimicrobials including beta-lactams, and aminoglycoside tetracyclin, frequently use in treatment presented high resistance rate amongst our isolates SCCmec typing, depicted that the SCCmec type IV (29.3%) was most dominant followed by type I (23.9%), type V (22.8%) and type III (13.0%). This distribution was also observed in the study conducted in Uganda [52, 59]. On the basis of SCCmec molecular typing and PVL secretion, our study revealed that CA MRSA (which have SCCmec types IV and V) were more prevalent 52.1% than HA MRSA (Types I to III), as reported earlier among Dutch [60], American [61], and Arab populations [62]. The predominance of CA MRSA in our study can be explained by the fact that people aged 0 to 30 years were most represented (59.7%). Younger people mostly carry CA MRSA, due to infrequent hospitalization [63]. In addition, factors like poor hygiene, socio-cultural habits in these regions of study may contribute to increased prevalence of S. aureus as it is easily transmitted by hand contact. The increasing MDR MRSA strains in circulation in the youth is especially troubling as it comprises the majority of Cameroons population with major economic contribution. Moreover, our study describes a high prevalence of XDR and émergence of PDR among CA MRSA and HA MRSA. Alarming rate of XDR and PDR was also described by Haji et al. [64], Important meseares must be taken in Cameroon and over the world for limiting spread of these emerging resistant strains. Emergence of MDR MRSA harboring the PVL toxin worsen the situation in terms of infection control and therapy. Previously, the lukSF-PV genes were a frequent genetic marker of CA MRSA isolates with SCCmecIV or SCCmecV [65]. However, subsequent studies have demonstrated lukSF-PV+ HA MRSA strains [66]. In this study both HA MRSA and CA MRSA strains harbored the lukSF-PV genes. CA MRSA infection and carriage of PVL toxin is thought to be responsible for rapidly progressive and lethal infections including necrotizing pneumonia, severe sepsis, and necrotizing fasciitis [67]. Therefore it has been speculated that lukSF-PV+ HA MRSA might lead to severe clinical infections similar to that caused by lukSF-PV+ CA MRSA [68]. This is consistent with the fact that the mechanism of PVL toxicity involves cell lysis in human myeloid cells, promoting inflammation and possesses immunomodulatory properties, blocking immune activation therefore promoting tissue damage [69]. Further studies need to be undertaken with indepth genotypic analysis of resistance mechanisms and correlation with phenotypic data which is a limitation in this study. Moreover, detailed virulence profiling of isolates may indicate better control measures.

Conclusion

This study was designed to evaluate the prevalence of CA and HA MRSA circulating in major hospitals in Cameroon and assess their genetic diversity through MecA gene and PVL toxin screening. We observed a high prevalence of MDR, XDR and PDR MRSA strains as the etiological agent responsible for a wide variety of infections due to Staphylococcus aureus, in patients from Cameroon. While lukSF-PV genes were detected in both CA and HA MRSA, prevalence of PVL toxin secreting CA MRSA was alarmingly high, especially among patients below 30 years of age. Isolates depicted high resistance to betalactams, tetracyclin, fluouroquinolone meanwhile rifampicine, fusidic acid, lincomicine and minocycline presented high rate of sensitivity, and should subsequently be prescribed for S. aureus infection control. Rapid evolution of multidrugresistant CA MRSA strains observed in our society needs an action plan with a good diagnostic protocol before any treatment to reduce therapeutic failures. Also, implementation of strict aseptic measures for healthcare professionals and more education in hygiene measures are required to limit the spread of CA MRSA.

Oligonucleotide primers used for Sccmec types I to V identification [44].

(DOCX)

Click here for additional data file.

Metadata for bacterial isolates from patients with MRSA infections.

(XLSX)

Click here for additional data file.

(DOCX)

Click here for additional data file.

3 Jan 2022

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to authors:

-The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

Introduction: (it needs to be more informative)

-Give a hint about the virulence factors, different infections caused by MRSA, and the mechanism of disease occurrence.

- The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You should cite the following valuable studies:

1.PMID: 33177849

2. PMID: 32397408

3.PMID: 32994450

4. PMID: 32497922

5.PMID: 33061472

6.PMID: 33947875

7.PMID: 34445951

8.PMID: 33188216

9.https://doi.org/10.1016/j.aquaculture.2021.737643

10.PMID: 30150182

-Rephrase the aim of the work to be clear and better sound.

Material and methods: Illustrate your methods with subtitles:

-Add this subtitle: Bacterial Isolation and identification:

•Discuss in detail the methods of isolation and identification of S.aureus and MRSA. Besides, specific references should be added.

•Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

- Antimicrobial susceptibility testing:

•Illustrate the antimicrobial classes of the tested antimicrobial agents within the text.

•The authors are advised to classify the tested isolates to MDR , XDR, and PDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, 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; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

- Why did you ignore the detection of antibiotic resistance genes in the recovered isolates??

•Please use PCR to detect antibiotic resistance genes, followed by gene sequencing if possible. Afterward, the correlation between phenotypic and genotypic multidrug resistance should be performed.

-Add more details about the software used in the statistical analyses.

-Results:

-Add this subtitle: Phenotypic characteristics of the recovered isolates.

•Illustrate in detail the phenotypic characteristics of the recovered isolates.

-Antimicrobial susceptibility testing:

•-Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R, MDR, and XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).The antibiotic -resistance genes

- Increase the resolution of all figures (it should be 600 dpi).

-Discussion:

- The authors are advised to illustrate the real impact of their findings without repetition of results.

-Illustrate the different mechanisms of antimicrobial resistance in S.aureus.

-Conclusion

- Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

Reviewer #2: Comments to authors:

- The current study has a significant impact, but it needs a major revision:

- The manuscript should be revised for grammar mistakes.

- Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and in the References section (should be italic).

-The title is broad, please modify the title.

- Add more details about the used methods and most prevalent results in the abstract.

-In the introduction: discuss the public health importance of MRSA and its virulence determinants.

-Improve the aim of work.

Methods:

-Explain the methods of isolation and identification in detail??

-Specific references should be added to all the used methods and techniques.

- Antimicrobial susceptibility testing: Add the manufacturing company, city, and country for the used reagents and antimicrobial discs.

-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

--Results:

- Discuss in detail the phenotypic characters of the recovered isolates.

-increase the resolution of different Figures: Please improve.

-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

-The correlation between the phenotypic and genotypic MDR should be performed.

-Discussion:

- Please improve.

-Please improve the main conclusion of the manuscript.

**********

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18 Feb 2022

Responses to the reviewers comments

Dear Sir/Madam thank you for this careful review of our manuscript. We have responded to each question and comment below :

(NB : Line number refers to data from manuscript with track changes)

Academic Editor

2- Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.

- Financial disclosure statement has been incorporated in the meta data as suggested and in manuscript financial disclosure statement has also been incorporated.

2- Please also include the statement “There was no additional external funding received for this study.”

- Financial disclosure statement has been incorporated in the meta data as suggested and in manuscript financial disclosure statement has also been incorporated.

4- Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

- Thank you for indicating, we have included supporting Information file information at Line 166, 239 and 747.

Reviewers 1

Title

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

- Title has been modified according to suggestion as follows:

«High prevalence of Panton-Valentine leukocidin positive, multidrug resistant, Methicillin-resistant Staphylococcus aureus strains circulating among clinical setups in Adamaoua and Far North regions of Cameroon»

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

More details were included in the abstract highlighting the methods in Line 36 and major results stated in Line 37-58. The conclusion has also been rephrased, Line 59.

Introduction (it needs to be more informative). Give a hint about the virulence factors, different infections caused by MRSA, and the mechanism of disease occurrence.

Response : Further details have been incorporated in introduction Line 79 to 105

The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents.

Details are included in Line 118-123

Rephrase the aim of the work to be clear and better sound.

- The aim of the study has been rephrased. Lines 123-133

Material and methods: Illustrate your methods with subtitles:

Add this subtitle: Bacterial Isolation and identification :

Subtitle has been added at Line 147

Discuss in detail the methods of isolation and identification of S. aureus and MRSA. Besides, specific references should be added.

Details of methods for isolation and identification of S.aureus are provided on line 152 – 165. Moreover suggested references relevant to study are included, reference number 34, 11 and 35.

Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

Details of reagents including company and country have been incorporated and given in parenthesis after each Line. Biochemical tests used for preliminary bacterial identification are mentioned in line 155-165.

Antimicrobial susceptibility testing:

Illustrate the antimicrobial classes of the tested antimicrobial agents within the text

Name of each class of antibiotics tested was added and antibiotics were grouped per class

Line 175 - 185

The authors are advised to classify the tested isolates to MDR , XDR, and PDR as described by Magiorakos et al.

Isolates have been classified as suggested and mentioned in line 323 – 325 and presented in Table 6.

Please use PCR to detect antibiotic resistance genes, followed by gene sequencing if possible. Afterward, the correlation between phenotypic and genotypic multidrug resistance should be performed

Resistance gene mec A and virulence factors Luk S/F pvl, sccmec were screening by PCR in this study. It is certainly interesting to perform these additional analysis. However, due to funding limitations selected analysis could be performed. Moreover lab closure as a result of COVID 19 positive status of majority individuals (including 2 of our authors) rendered it impossible to perform additional screenings. Therefore this has been listed as a study limitation in discussion Line 470-473.

Add more details about the software used in the statistical analyses.

Software used in the statistical analyses are mentioned in line 245-247

Results:

-Add this subtitle: Phenotypic characteristics of the recovered isolates.

The subtitle was added line 254 and more details were provided for the MDR from line 263.

Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates (illustrate the names of the antimicrobial classes and different antibiotics): No. of strains%Type of resistance, MDR, and XDR Phenotypic multidrug resistance (Antimicrobial classes and different antibiotics).The antibiotic -resistance genes

Specific details have been incorporated in new tables 5 and 6 with classes of antibiotics line 322-327 page 12-13

Increase the resolution of all figures (it should be 600 dpi)

Figure 1 has been reformatted to improve resolution of image.

Discussion:

- The authors are advised to illustrate the real impact of their findings without repetition of results.

The discussion part has been restructured to avoid repetition of results.

-Illustrate the different mechanisms of antimicrobial resistance in S.aureus.

Appropriate response is provided in line 414-445, 442-449

Conclusion

Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

The conclusion has been revised, as suggested Line 476 - 489.

RESPONSE TO THE COMMENTS OF REVIEWER 2

Thank you so much sir/madam for your comments and appreciation to our manuscript. We are going to give responses to your comments in the following paragraphs.

- The manuscript should be revised for grammar mistakes.

Response : The manuscript has been thoroughly revised to remove grammatical mistakes.

- Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and in the References section (should be italic).

Response : Well received, correction have been made throughout the manuscript.

-The title is broad, please modify the title.

Response : New title has been proposed.

- Add more details about the used methods and most prevalent results in the abstract.

Response : Details have been included in the abstract.

-In the introduction: discuss the public health importance of MRSA and its virulence determinants.

Response : Details have been included in introduction Line 79 to 105

-Improve the aim of work.

Response : The aim of the study has been rephrased. Lines 123-133

Methods:

-Explain the methods of isolation and identification in detail??

Response : Details of methods for isolation and identification of S.aureus are provided on line 152 – 165.

-Specific references should be added to all the used methods and techniques.

Response : Thank you for pointing this oversight, we have inserted references in relevant sections.

- Antimicrobial susceptibility testing: Add the manufacturing company, city, and country for the used reagents and antimicrobial discs.

Response : We have inserted required information in methods section.

-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

Response : Resistance gene mec A and virulence factors Luk S/F pvl, sccmec were screening by PCR in this study. It is certainly interesting to perform these additional analysis. However, due to funding limitations selected analysis could be performed. Moreover lab closure as a result of COVID 19 positive status of majority individuals (including 2 of our authors) rendered it impossible to perform additional screenings. Therefore this has been listed as a study limitation in discussion Line 470-473.

Results:

- Discuss in detail the phenotypic characters of the recovered isolates.

Response : Specific details are provided in the text Line 254-277

-Increase the resolution of different Figures: Please improve.

Response : Figure resolution has been improved.

-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

Response :. This has been listed as a study limitation in discussion Line 470-473.

The correlation between the phenotypic and genotypic MDR should be performed.

Response : Phenotypic data is incorporated in new table (table 6) and details are listed in line 322-355.

-Discussion:

- Please improve.

Response : The discussion section has been revised completely.

-Please improve the main conclusion of the manuscript.

Response : The conclusion has been revised.

Submitted filename: Responses to the reviewers comments-FINAL.docx

Click here for additional data file.

24 May 2022

High prevalence of Panton-Valentine leukocidin positive, multidrug resistant, Methicillin-resistant Staphylococcus aureus strains circulating among clinical setups in Adamawa and Far North regions of Cameroon.

PONE-D-21-37074R1

Dear Dr. Javed,

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have carried out significant changes to the manuscript. They have addressed all the suggested corrections and comments. Really, it's an interesting study that has a significant impact. Now, the manuscript could be accepted.

Congratulations.

Reviewer #2: Most of my previous comments have been addressed. No further comments are needed. Now the manuscript could be accepted.

**********

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26 May 2022

PONE-D-21-37074R1

High prevalence of Panton-Valentine leukocidin positive, multidrug resistant, Methicillin-resistant Staphylococcus aureus strains circulating among clinical setups in Adamawa and Far North regions of Cameroon.

Dear Dr. Javed:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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