https://orcid.org/0000-0001-8352-8078
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Abstract
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.
Citation: Mohamadou M, Essama SR, Ngonde Essome MC, Akwah L, Nadeem N, Gonsu Kamga H, et al. (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. PLoS ONE 17(7): e0265118. https://doi.org/10.1371/journal.pone.0265118
Editor: Carla Pegoraro, PLOS, UNITED KINGDOM
Received: November 22, 2021; Accepted: May 13, 2022; Published: July 8, 2022
Copyright: © 2022 Mohamadou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Mohamadou Mansour was financially supported during his stay in Pakistan by Comsats University Islamabad and The World Academy of Sciences (TWAS) sandwich postgraduate fellowship award (FR number : 3240315420). No : The funders had no role in study design, collection and interpretation of data, decision to publish, no fund was received for publication. There was no additional external funding received for this study and we have not received any funds to cover APC fees.
Competing interests: The authors have declared no competing interest exist
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).
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).
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).
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).
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).
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).
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.
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).
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).
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.
Supporting information
S1 Table. Oligonucleotide primers used for Sccmec types I to V identification [44].
https://doi.org/10.1371/journal.pone.0265118.s001
(DOCX)
S2 Table. Metadata for bacterial isolates from patients with MRSA infections.
https://doi.org/10.1371/journal.pone.0265118.s002
(XLSX)
Acknowledgments
We thank The World Academy of Sciences (TWAS) and COMSATS University Islamabad (CUI) for the sandwich postgraduate fellowship award offer to Mohamadou Mansour.
References
- 1. Lyell A., Alexander Ogston. Micrococci and Joseph Lister. Journal of American Academy of Dermatology 1989;20:302–10. pmid:2644319.
- 2. Lakhundi S, Zhang K. Methicillin-Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology. Clinical Microbiology Reviews. 2018;31(4): pmid:30209034.
- 3.
Taylor TA, Unakal CG. Staphylococcus aureus. StatPearls Publishing, 2021; pmid:28722898 Bookshelf ID: NBK441868.
- 4. Foster TJ. Immune evasion by staphylococci. Nature Reviews Microbioly. 2005;3(12): P 948–58. pmid:16322743.
- 5. Fridkin SK, Hageman J, McDougal LK, Mohammed J, Jarvis WR, Perl TM, et al. Epidemiological and Microbiological Characterization of Infections Caused by Staphylococcus aureus with Reduced Susceptibility to Vancomycin, United States, 1997–2001. Clinical Infectious Diseases. 2003;36(4):429–39. pmid:12567300.
- 6. Köck R, Schaumburg F, Mellmann A, Köksal M, Jurke A, Becker K, et al. Livestock-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) as Causes of Human Infection and Colonization in Germany. de Lencastre H, PLoS ONE. 2013;8(2). pmid:23418434.
- 7. Brown DFJ, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ, et al. Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). Journal of Antimicrobial Chemotherapy. 2005;56(6):1000–18. pmid:16293678.
- 8. Jevons P. " Celbenin " resistant Staphylococci. British Medical Journal. 1961;1(5219): 124–125. PMC1952888.
- 9. Chambers HF, DeLeo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews of Microbiology. 2009;7(9):629–41. pmid:19680247.
- 10. Peng H, Liu D, Ma Y, Gao W. Comparison of community- and healthcare-associated methicillin-resistant Staphylococcus aureus isolates at a Chinese tertiary hospital, 2012–2017. Scientific Reports. 2018;8(1):17916. pmid:30559468
- 11. Algammal AM, Hetta HF, Elkelish A, Alkhalifah DHH, Hozzein WN, Batiha GE-S, et al. Methicillin-Resistant Staphylococcus aureus (MRSA): One Health Perspective Approach to the Bacterium Epidemiology, Virulence Factors, Antibiotic-Resistance, and Zoonotic Impact. 2020;13:3255–65. pmid:33061472.
- 12. Preeja PP, Kumar SH, Shetty V. Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus from Community- and Hospital-Associated Infections: A Tertiary Care Center Study. Antibiotics. 2021;10(2):197. pmid:33670648.
- 13. David MZ, Daum RS. Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic. Clinical Microbiology Reviews. 2010;23(3):616–87. pmid:20610826.
- 14. Aires de Sousa M, Lencastre H. Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunology & Medical Microbiology. 2004;40(2):101–11. https://doi.org/10.1016/S0928-8244(03)00370-5
- 15. Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus. Infection, Genetics and Evolution. 2008;8(6):747–63. pmid:18718557.
- 16. Katayama Y, Ito T, Hiramatsu K. A New Class of Genetic Element, Staphylococcus Cassette Chromosome mec, Encodes Methicillin Resistance in Staphylococcus aureus. Antimicrobial Agents Chemother. 2000;44(6):1549–55. pmid:10817707.
- 17. Queck SY, Khan BA, Wang R, Bach T-HL, Kretschmer D, Chen L, et al. Mobile Genetic Element-Encoded Cytolysin Connects Virulence to Methicillin Resistance in MRSA. Cheung A,. PLoS Pathog. 31 juill 2009;5(7):e1000533. pmid:19649313
- 18. Rolo J, Worning P, Nielsen JB, Bowden R, Bouchami O, Damborg P, et al. Evolutionary Origin of the Staphylococcal Cassette Chromosome mec (SCC mec). Antimicrob Agents Chemother. 2017;61(6). pmid:28373201.
- 19. Andrade MM, Luiz WB, da Silva Oliveira Souza R, Amorim JH. The History of Methicillin-Resistant Staphylococcus aureus in Brazil. Weina PJ, Canadian Journal of Infectious Diseases and Medical Microbiology. 2020;1–18. pmid:33082892.
- 20. Barcelo M, Chauvet E, Boukhari R, Mbieleu B. Pneumopathie nécrosante staphylococcique productrice de leucocidine de Panton-Valentine d’évolution favorable. Archives de Pédiatrie. 2009;16(1):32–6. pmid:19036566
- 21. Chaouch C, Kacem S, Tilouche L, Ketata S, Bouallegue O, Boujaafar N. Panton-Valentine leukocidin-positive osteoarticular infections. Médecine et Santé Tropicales. 2015;25(2):184–8. pmid:26039246
- 22. Young BC, Earle SG, Soeng S, Sar P, Kumar V, Hor S, et al. Panton–Valentine leucocidin is the key determinant of Staphylococcus aureus pyomyositis in a bacterial GWAS. ELife. 2019;8. pmid:30794157.
- 23. Okolie CE, Cockayne A, Penfold C, James R. Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for Diagnostic and Therapeutic Applications. BMC Biotechnology. 2013;13(1):103. pmid:24252611.
- 24. Archana GJ, Sinha AY, Annamanedi M, Asrith KP, Kale SB, Kurkure NV, et al. Molecular Characterisation of Methicillin-Resistant Staphylococcus aureus Isolated from Patients at a Tertiary Care Hospital in Hyderabad, South India. Indian Journal of Medical Microbiology. 2020;38(2):183–92. pmid:32883932.
- 25. Firoozeh F, Omidi M, Saffari M, Sedaghat H, Zibaei M. Molecular analysis of methicillin-resistant Staphylococcus aureus isolates from four teaching hospitals in Iran: the emergence of novel MRSA clones. Antimicrobial Resistance Infection Control. 2020;9(1):112. pmid:32680563
- 26. Mitevska E, Wong B, Surewaard BGJ, Jenne CN. The Prevalence, Risk, and Management of Methicillin-Resistant Staphylococcus aureus Infection in Diverse Populations across Canada: A Systematic Review. Pathogens. 2021;10(4):393. pmid:33805913.
- 27. Da Silva LSC, Andrade YMFS, Oliveira AC, Cunha BC, Oliveira EG, Cunha TS, et al. Prevalence of methicillin-resistant Staphylococcus aureus colonization among healthcare workers at a tertiary care hospital in northeastern Brazil. Infection Prevention in Practice. 2020;2(4):100084. pmid:34368723.
- 28. Lee Y-C, Chen P-Y, Wang J-T, Chang S-C. Prevalence of fosfomycin resistance and gene mutations in clinical isolates of methicillin-resistant Staphylococcus aureus. Antimicroial Resistance Infection Control. 2020;9(1):135. pmid:32807239.
- 29. Abdulgader SM, van Rijswijk A, Whitelaw A, Newton-Foot M. The association between pathogen factors and clinical outcomes in patients with Staphylococcus aureus bacteraemia in a tertiary hospital, Cape Town. International Journal of Infectious Diseases. 2020; 91:111–8. pmid:31790814.
- 30. McManus BA, Aloba BK, Earls MR, Brennan GI, O’Connell B, Monecke S, et al. Multiple distinct outbreaks of Panton–Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus in Ireland investigated by whole-genome sequencing. Journal of Hospital Infection. 2021;108:72–80. pmid:33259881.
- 31.
CDC. Antibiotic resistance threats in the United States. US Department of health and human services. USA; 2013. P. 114 http://dx.doi.org/10.15620/cdc:82532.
- 32. Makharita RR, El-kholy I, Hetta HF, Abdelaziz M, Hagagy F, Ahmed A, et al. Antibiogram and Genetic Characterization of Carbapenem-Resistant Gram-Negative Pathogens Incriminated in Healthcare-Associated Infections. 2020;13:3991–4002. pmid:33177849.
- 33. Abolghait SK, Fathi AG, Youssef FM, Algammal AM. Methicillin-resistant Staphylococcus aureus (MRSA) isolated from chicken meat and giblets often produces staphylococcal enterotoxin B (SEB) in non-refrigerated raw chicken livers. International Journal of Food Microbiology. 2020;328: pmid:32497922.
- 34. Kesah C, Ben Redjeb S, Odugbemi TO, Boye CS-B, Dosso M, Ndinya Achola JO, et al. Prevalence of methicillin resistant Staphylococcus aureus in eight African hospitals and Malta. Clinical Microbiology and Infection. 2003;9(2):153–6. pmid:12588338.
- 35. Gonsu K, Kouemo S. L., Toukam M, Ndze V. N., Koulla S. S. Nasal carriage of methicillin resistant Staphylococcus aureus and its antibiotic susceptibility pattern in adult hospitalized patients and medical staff in some hospitals in Cameroon. Journal of Microbiology and Antimicrobials. 2013;5(3):29–33. https://doi.org/10.5897/JMA2012.0232.
- 36. Foloum AK, Founou LL, Karang S, Maled Y, Tsayem C, Kuete M, et al. Methicillin resistant staphylococci isolated in clinical samples: a 3-year retrospective study analysis. Future Science OA. 2021;7(4): pmid:33815826
- 37. Artino AR, La Rochelle JS, Dezee KJ, Gehlbach H. Developing questionnaires for educational research: AMEE Guide No. 87. Medical Teacher. 2014;36(6):463–74. pmid:24661014.
- 38. Sansoni JE. Questionnaire design and systematic literature reviews. University of Wollongong. 2011;120:67.
- 39. England Deparment of Health. Antimicrobial Resistance Empirical and Statistical Evidence-Base. 2016;63.
- 40. Sperber WH, Tatini SR. Interpretation of the Tube Coagulase Test for Identification of Staphylococcus aureus. 1975;29:4. pmid:164821.
- 41. Kateete DP, Kimani CN, Katabazi FA, Okeng A, Okee MS, Nanteza A, et al. Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Annual Clinical Microbiology Antimicrob. 2010;9(1):23. pmid:20707914
- 42.
EUCAST/SFM. EUCAST / Comité de l’antibiogramme de la Société Française de Microbiologie (CA-SFM). 2019. 144 p. www.sfm-microbiologie.org
- 43. Magiorakos A-P, 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. Clinical Microbiology and Infection. 2012;18(3):268–81. pmid:21793988.
- 44. Boye K, Bartels MD, Andersen IS, Møller JA, Westh H. A new multiplex PCR for easy screening of methicillin-resistant Staphylococcus aureus SCCmec types I–V. Clinical Microbiology and Infection. 2007;13(7):725–7. pmid:17403127.
- 45. Oliveira DC, Lencastre H de. Multiplex PCR Strategy for Rapid Identification of Structural Types and Variants of the mec Element in Methicillin-Resistant Staphylococcus aureus. Antimicrobials Agents Chemother. 2002;46(7):2155–61. pmid:12069968
- 46. McClure-Warnier J-A, Conly JM, Zhang K. Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus. JoVE. 2013;(79):50779. pmid:24056633.
- 47. Vysakh P, Jeya M. A Comparative Analysis of Community Acquired and Hospital Acquired Methicillin Resistant Staphylococcus aureus. Journal of Clinical and Diagnostic Research. 2013;7(7):1339–42. pmid:23998061
- 48. Zhang K, McClure J-A, Elsayed S, Louie T, Conly JM. Novel Multiplex PCR Assay for Characterization and Concomitant Subtyping of Staphylococcal Cassette Chromosome mec Types I to V in Methicillin-Resistant Staphylococcus aureus. Journal of Clinical Microbiolog. 2005;43(10):5026–33. pmid:16207957
- 49. Kong EF, Johnson JK, Jabra-Rizk MA. Community-Associated Methicillin-Resistant Staphylococcus aureus: An Enemy amidst Us. Leong JM, éditeur. PLoS Pathogens. 2016;12(10):e1005837. pmid:27711163.
- 50. Alvarez CA, Yomayusa N, Leal AL, Moreno J, Mendez-Alvarez S, Ibañez M, et al. Nosocomial infections caused by community-associated methicillin-resistant Staphylococcus aureus in Colombia. American Journal of Infection Control. mai 2010;38(4):315–8. pmid:20042253.
- 51. Sola C, Paganini H, Egea AL, Moyano AJ, Garnero A, Kevric I, et al. Spread of Epidemic MRSA-ST5-IV Clone Encoding PVL as a Major Cause of Community Onset Staphylococcal Infections in Argentinean Children. Van Melderen L, éditeur. PLoS ONE. 2012;7(1):e30487. pmid:22291965.
- 52. Pantosti A, Sanchini A, Monaco M. Mechanisms of antibiotic resistance in Staphylococcus aureus. Future Microbiology. 2007;2(3):323–34. pmid:17661706.
- 53. Rasmussen RV, Fowler VG Jr, Skov R, Bruun NE. Future challenges and treatment of Staphylococcus aureus bacteremia with emphasis on MRSA. Future Microbiology. 2011;6(1):43–56. pmid:21162635.
- 54. Kengne M, Fotsing O, Ndomgue T, Mbekem Nwobegahay J. Antibiotic susceptibility patterns of Staphylococcus aureus strains isolated at the Yaounde Central Hospital,Cameroon: a retro prospective study. Pan African Medical Journal. 2019;32. pmid:31223393.
- 55. Pomorska K, Jakubu V, Malisova L, Fridrichova M, Musilek M, Zemlickova H. Antibiotic Resistance, spa Typing and Clonal Analysis of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Blood of Patients Hospitalized in the Czech Republic. Antibiotics. 2021;10(4):395. pmid:33917471.
- 56. Tsige Y, Tadesse S, G/Eyesus T, Tefera MM, Amsalu A, Menberu MA, et al. Prevalence of Methicillin-Resistant Staphylococcus aureus and Associated Risk Factors among Patients with Wound Infection at Referral Hospital, Northeast Ethiopia. Journal of Pathogens 2020;2020:1–7. pmid:32566311
- 57. Gittens-St Hilaire MV, Chase E, Alleyne D. Prevalence, molecular characteristics and antimicrobial susceptibility patterns of MRSA in hospitalized and nonhospitalized patients in Barbados. New Microbes and New Infections. 2020;35: pmid:32257222
- 58. Mansour M, Aline B, Bouba G, Chantal NEM, Didier MC, Benoit K, et al. Epidemiology Survey of Antibiotics Use in Hospitals and Veterinarian Practices in Northern Regions of Cameroon. International Journal for Research in Applied Sciences and Biotechnology. 21 avr 2020;7(2):9–16. http://dx.doi.org/10.2139/ssrn.3583315.
- 59. Kateete DP, Bwanga F, Seni J, Mayanja R, Kigozi E, Mujuni B, et al. CA-MRSA and HA-MRSA coexist in community and hospital settings in Uganda. Antimicrobial Resistance Infections Control. 2019;8(1):94. pmid:31171965
- 60. Popovich KJ, Hota B, Aroutcheva A, Kurien L, Patel J, Lyles-Banks R, et al. Community-Associated Methicillin-Resistant Staphylococcus aureus Colonization Burden in HIV-Infected Patients. Clinical Infectious Diseases. 2013;56(8):1067–74. pmid:23325428.
- 61. Khanfar Husam, Senok Abiola, Anani Adnan, Zinkevich Vitaly. Methicillin-resistant Staphylococcus aureus transmission in a low-prevalence healthcare setting. Journal of infections and public health. 7e éd. 2012; pmid:23021654.
- 62. Udo EE, Boswihi SS. Antibiotic Resistance Trends in Methicillin-Resistant Staphylococcus aureus Isolated in Kuwait Hospitals: 2011–2015. Medical Principles and Practice. 2017;26(5):485–90. pmid:28977793.
- 63. Prista-Leão B, Abreu I, Duro R, Silva-Pinto A, Ceia F, Andrade P, et al. Panton-Valentine Leukocidin-Producing Staphylococcus aureus Infection: A Case Series. Infectious Disease Reports.2020;12(3):61–9. pmid:33153134.
- 64. Haji SH, Aka STH, Ali FA. Prevalence and characterisation of carbapenemase encoding genes in multidrug-resistant Gram-negative bacilli. PLOS ONE. 2021;16(11): pmid:34723978
- 65. Dufour P, Gillet Y, Bes M, Lina G, Vandenesch F, Floret D, et al. Community‐Acquired Methicillin‐Resistant Staphylococcus aureus Infections in France: Emergence of a Single Clone That Produces Panton‐Valentine Leukocidin. Clinical infectious diseases 2002; 35(7): 819–24. pmid:12228818.
- 66. Yao D, Yu F, Qin Z, Chen C, He S, Chen Z, et al. RMeseoarlceh acrtuicllear characterization of Staphylococcus aureus isolates causing skin and soft tissue infections (SSTIs). 2010;5.
- 67. Boyle-Vavra S, Daum RS. Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton–Valentine leukocidin. Lab Invest. 2007;87(1):3–9. pmid:17146447.
- 68. Zhang C, Guo Y, Chu X. In Vitro generation of Panton-Valentine leukocidin (PVL) in clinical Methicillin-Resistant Staphylococcus aureus (MRSA) and its correlation with PVL variant, clonal complex, infection type. Sci Rep. 2018;8(1): https://doi.org/10.1038/s41598-018-26034-y
- 69. Sina H, Ahoyo TA, Moussaoui W, Keller D, Bankolé HS, Barogui Y, et al. Variability of antibiotic susceptibility and toxin production of Staphylococcus aureus strains isolated from skin, soft tissue, and bone related infections. BMC Microbiol.2013;13(1): pmid:23924370.