Molecular Diversity of Staphylococcus aureus from the Nares of Hospital Personnel, HIV-Positive and Diabetes Mellitus Patients in Yaounde Cameroon

Nasal carriage of Staphylococcus aureus has been identified as a risk factor for the development of staphylococcal infections caused by endogenous colonizing strains. Information on the genotypic diversity of Staphylococcus aureus is relevant for managing epidemiological and clinical challenges resulting from the evolutionary differences of this bacterium. The objective of this study was to determine and compare the molecular diversity of Staphylococcus aureus isolates from three high-risk populations in Yaounde, Cameroon. Molecular analysis confirmed that 95% of 100 tested isolates were S. aureus. The mecA and Panton Valentine-Leukocidin (PVL) genes (lukS/F-PV) were detected in 37% (35/95) and 43% (41/95) of isolates respectively and 18% lation. Multilocus Sequence Typing (MLST) of 12 selected isolates was assigned to six pandemic clonal complexes (CC): CC5 (ST5), CC8 [ST8, (n = 3)], CC15 (ST 15), CC25 (ST 25), CC72 [ST72 (n = 2)] and CC121 [ST 121 (n = 2)] and three atypical sequence types ST 508, ST 699 (CC45) and ST 1289 (CC 88). The study population represents an important reservoir for MRSA, MRSA-PVL and MSSA-PVL which could serve as focal point for further dissemination bringing about significant clinical and epidemiological implica-tions. The predominance of SCCmec IV and agr types in this setting warrants further investigation. Isolates were genetically diverse with MLST indicating that pandemic ST8 was predominant. Detection of atypical STs has provided an insight into the necessity for constant monitoring.


Introduction
Staphylococcus aureus is a Gram positive bacterium that is part of the microbiota of human nares, skin, intestine, upper respiratory tract and vagina [1]. These bacteria can cause serious disease when there is a breach of the mucosal barrier allowing sterile body sites to be infected by tissue invasion or toxin production [2]. The host and bacterial factors that facilitate colonization have not been fully characterized. However, polymorphism of genes that encode the glucocorticoid receptor, interleukin-4, complement inhibitor proteins and the reduced expression of antimicrobial peptides in nasal secretions have been associated with persistent nasal carriage [3] [4].
Bacteria adhesion is facilitated by loricrin, a major component of the squamous epithelium, which is the primary ligand for the clumping factor B (ClfB) expressed on the surface of S. aureus. Other bacterial factors like cell wall teichoic acid, capsular polysaccharide and iron-regulator surface determinants are also involved in Staphylococcus aureus colonization [5] [6] [7] [8]. Invasiveness of the bacteria is enhanced by the secretion of enzymes (nucleases, proteases, lipases, hyaluronidase and collagenases) and cytokines that inhibit complement activation, cause neutrophil chemotaxis and neutralize antimicrobial defensin peptides [9] [10]. Some strains produce toxic proteins such as enterotoxins, exfoliative toxins and leucocidin, which are responsible for diseases that vary in severity from superficial skin lesions to more serious invasive and life-threatening infections such as pneumonia, endocarditis, septicaemia, acute staphylococcal toxemia syndromes and staphylococcal food poisoning [11].
In vulnerable persons, colonization increases the risk of subsequent infection usually caused by colonizing strains [12]. Several studies have illustrated a causal association between S. aureus nasal carriage and an estimated 4-to 10-fold increase in the odds of carriers developing staphylococcal disease [13] [14]. The Human Immunodeficiency Virus/acquired immune deficiency syndrome (HIV/AIDS) associated infections are generally linked to the progressive depletion of cluster of differentiation 4 T (CD4 T) cells. A count lower than 200 cells/mm 3 of blood, recent antibiotic use, duration of hospital stay are considered to be risk factors for nasal colonization by methicillin, resistant S. aureus in HIV-infected patients [15] [16]. Diabetes mellitus, a debilitating chronic disease with ensuing complications such as renal failure, dermatological disorders and peripheral neuropathy with risk of foot ulcers, is considered risk factors for MRSA colonization [17]. Colonization of hospital personnel has been identified as a reservoir of healthy carriers of S. aureus in the hospital and community. Previous studies in Ethiopia, recorded a prevalence of 28.8% (34/118) S. aureus colonization of hospital personnel, among which 44.1% (15/34) were resistant to methicillin [18]. A similar study conducted in Yaounde Cameroon, recorded a prevalence of 23.7% [19]. Transmission of resistant strains from personnel to patients is likely to occur during routine patient care in the absence of effective hand hygiene. Acquisition of MRSA on hands by healthcare workers, was common after examination of sites like the chest, abdomen, forearm and hands [20].
Management of staphylococcal infections has become a burden to the global health community leading to increased cost of healthcare, prolonged hospital stay and higher morbidity and mortality among infected individuals [21] [22].
The epidemiology of S. aureus especially MRSA has evolved considerably over the years. Managing the upsurge in prevalence of community associated methicillin resistant Staphylococcus aureus (CA-MRSA) is challenging for several healthcare institutions which are already battling with high levels of hospital-associated methicillin resistant Staphylococcus aureus (HA-MRSA) [23]. Disease severity is further compounded by the presence of mobile genetic elements that confer antibiotic resistance such as Staphylococcus cassette chromosome (SCC) harboring mec elements for methicillin resistance or the A operon for vancomycin resistance. The SCCmec type II strains initially associated to the hospital settings have now been identified among community and animal isolates [24] [25]. Methicillin sensitive S. aureus (MSSA) and MRSA strains harboring Panton-Valentine Leukocidin (PVL), a bi-component cytotoxin encoded by luk S-PV with diverse genetic characteristics, are on the rise in Africa [26]. Given that luk S-PV is phage mediated and can easily be transmitted between isolates, this may explain high prevalence rates of gene among strains in Africa where monitoring is limited. PVL-encoding operon has been detected in 55.9% of S. aureus strains from certain African populations such as the Babongo Pygmies. Data from across Europe, however, depicts lower rates: 1.8% in Ireland, 4% in Turkey, 30% in Germany and 20% in United Kingdom [27] [28]. The expression of PVL and other S. aureus virulence determinants is thought to be controlled by multiple regulatory pathways including the accessory gene regulator (agr) characterized by a polymorphism of its auto-inducing peptide (AIP).
Based on this polymorphism, S. aureus has been classified into four agr groups.
Furthermore, molecular typing techniques such as pulsed field gel electrophore- sis (PFGE) and multilocus sequence typing (MLST) are currently being used to generate genotype information to categorize isolates into sequence types (STs) and clonal lineages or complexes (CCs) for effective epidemiological surveillance and for monitoring evolutionary trends [29].
The clinical relevance of characterizing both MRSA and MSSA has been demonstrated though such data is limited in Cameroon. The objective of this study was therefore to investigate the genetic diversity of S. aureus isolates obtained from hospital personnel, HIV-positive and diabetes mellitus patients in Yaounde, Cameroon for disease surveillance and better patient management.

Culture and Identification of Staphylococcus aureus Isolates
Identification was based on mannitol fermentation after incubation of plates at 37˚C for 24 to 48 hours. Isolates that fermented mannitol were further analysed using API staph (BioMérieux, Marcy l'Etoile, France). Antimicrobial susceptibility testing was carried out according to the disk diffusion method by Kirby Bauer. Identified isolates were stored at −20˚C pending molecular analyses performed at the Department of Medical Microbiology, University of Pretoria, South Africa.  [20]. Extracted DNA was stored at −20˚C.

Molecular Identification of Staphylococcus. aureus Isolates
The isolates were subjected to a multiplex Polymerase Chain Reaction (M-PCR) assay using specific primers to confirm biochemical identification. Staphylococcus species and genus specific upstream sense and downstream antisense primers shown in Table 1 were used to amplify the 16SrRNA and the nuc fragments of isolates respectively [30]. The PCR reaction mixture, volume and cycling conditions were adapted as previously described [30] [31]. Generated amplicons were run on 1.5% agarose gel electrophoresis after staining with 5 µl ethidium bromide (10 mg/ml; Sigma Aldrich) and visualized under UV light (Transilluminator Ultra-violet products incorporated, USA). A 100 bp DNA ladder (Promega, Madison, USA) was used as DNA molecular weight standard.

Detection of Antibiotic Resistance and Virulence Genes by M-PCR Assays
Primers specific for mecA were used to amplify the genes responsible for methicillin resistance. The ability to produce Panton-Valentine leukocidin (PVL) was determined by the presence of lukS-PV-lukF-PV and virulence regulation by the different agr (I to IV) groups, according to previously described protocols [28] [32]. Details of primer sequences, expected sizes of the amplified products, reaction volume and specific annealing temperatures are listed in Table 1.

Identification of the SCCmec Types of MRSA Isolates
The diversity of MRSA strains was established by multiplex PCR assays of the chromosomal cassette recombinase (ccr) types I to V and the Staphylococcal cassette chromosome mec allotypes (classes A to C). SCCmec types were assigned based on the combination of ccr type and mecA classes detected [33].

Pulsed Field Gel Electrophoresis
Pulsed Field Gel Electrophoresis analysis of 95 molecularly confirmed iso-  (34)). Each pair of primers amplified an internal fragment of a housekeeping gene of approximately 500 bp. Sequence analysis was performed by Inqaba Biotech South Africa. The identified loci and corresponding alleles were then compared with those from the S. aureus database.

Data Analysis
Data collected were entered into a spreadsheet and analyzed using Stata (version 13.0 STATA corps, Texas, USA

Results
In total, 95 (95%) of the 100 isolates were confirmed as S. aureus based on 16SrRNA and nucA genes (Figure 1). The other five isolates were not subjected to any further analysis.

Pulsed Field Gel Electrophoresis
Of the 95 (35 MRSA and 60 MSSA) isolates that underwent restriction digestion with Sma1 and electrophoresis on agarose gel, 93 had PFGE detected patterns. Thus 2 MSSA isolates were untypable with Sma1. These two isolates were not analysed any further. Relatedness of strains was analyzed by constructing a dendrogram based on distance value and 80% homology. The dendrogram showed 18 distinct pulsotype designated here as CI to CVIII ( Figure 3) and several singletons. Twelve representative isolates based on generated pulsotypes were sequenced using MLST.  Table 3 and Table 4 respectively.

Discussion
The present study has demonstrated the importance of molecular identification by revealing that community acquired-MRSA-PVL and community acquired-MSSA-PLV are common amongst S. aureus isolates colonizing the nares of hospital personnel, HIV-positive and diabetes mellitus patients in Yaounde. Three atypical sequence types were detected while pandemic strains were found to cluster irrespectively of studied genetic characteristics analysed or study population. Molecular identification and sequence typing of bacterial isolates is essential to confirm speciation, to monitor evolutionary trends and to determine clonal relatedness within the study population. The prevalence of colonization with MRSA has been reported to vary according to the population studied as well as study site [34]. MRSA colonization rate (45%) amongst isolates from hospital personnel identified in this study was higher than the 11.4% registered for a similar group in 2013 by Rongpharpi and collaborators in Assam, Nigeria [34] [35]. The 33% prevalence recorded for HIV-positive patients was higher than 2.4% reported in Mekelle, Northern Ethiopia [36]. Likewise the prevalence of MRSA amongst isolates from diabetes mellitus patients (39%) was higher than 9.9% registered by Kutlu and collaborators in 2012 [36]. These MRSA rates however fall within the 0% -59% range docu-mented in a systematic review 104 studies describing MRSA nasal carriage in several countries in Africa [37]. Discrepancy in results has been attributed to the level of compliance to control measures such as hand washing and techniques used for MRSA detection. The highest prevalence was recorded at the Yaounde Central Hospital which was the most crowded hospital facility among the study sites. Overcrowding is considered a favourable condition for the spread of MRSA through hand contact with contaminated surfaces or otherwise through the air. MRSA are resistant to several classes of antibiotics, rendering treatment of simple infections complicated and costly. Antimicrobial resistance is enhanced by ease in drug acquisition, automedication and poor hygiene.
Molecular characterisation of MRSA isolates revealed community-associated SCCmec type IV as the predominant type among the study population and study sites. A similar result was reported by Abdul et al., 2015 who identified SCCmec type IV as the leading type between SCCmec type IV and type V [38]. SCCmec type IV has been identified as the most virulent type because it carries a mecA gene, it has a functional recombinase and it is most mobile thus can be transmitted easily to MSSA within the community. Contrary to these findings, SCCmec V was reported as being the major type among MRSA isolated from the nares of Egyptian and Saudi Arabian outpatients. This variation could be due to the different multiplex-polymerase chain reaction (PCR) methods used in the assignment of SCCmec types as highlighted by Antiabong et al., 2016 [33]. The detection of hospital associated SCCmec type II among isolates from HIV positive and diabetes mellitus patients has been previously reported. This implies that community acquired-MRSA and hospital acquired-MRSA isolates might no longer be differentiated based only on SCCmec genotypes [29]. The circulation of MRSA strains within communities and hospitals may be driven by routine visits of patients and their family members (patients' care givers) to the hospital.
The frequency of MSSA/PVL and MRSA/PVL isolates from the three study populations were higher than reported prevalences of less than 10% in European countries, but falls within the 17% to 70% range reported for Madagascar, Morocco, Niger and Senegal [29]. While the MRSA/PLV prevalence for hospital personnel (41%) was comparable to the rate in Algiers (44%), rates for HIV-positive patients (35%) and diabetes mellitus patients (24%) were higher than has been documented in other parts of Africa [39]. Given that luk S-PV is phage mediated and can easily be transmitted between isolates, this may explain high prevalence rates of gene among strains in Africa where monitoring is limited. The MRSA/PVL strains sequenced were categorized as CC8, CC121 and CC1289. Most MSSA/PVL isolates were obtained from HIV-positive patients and those sequenced were classified in clonal complexes CC8, CC15, CC72 and CC121. The CC121 clone is a known African pandemic clone common in Nigeria, Togo, South Africa and Cameroon [38]. The relatively high rates of both MSSA/PVL and MRSA/PVL strains among asymptomatic carriage isolates may serve as important endogenous reservoirs for subsequent transmission to sterile body sites and an important epidemiological focal point for possible dissemination within the community. This may worsen clinical outcomes in financially over burden individuals and healthcare facilities. Africa. ST15 was reported in Mali, Gabon and Germany as the most prevalent serotype in asymptomatic carriers. The PVL-positive ST121 (MRSA-IV) clone detected, is prevalent in Africa and was also reported in Asia as a paediatric epidemic clone. [31] In addition to typical STs, three atypical STs (508, 699, and 1289) were identified. ST1289 (SCCmecV/PLV) which is a single-locus variant of ST88, with characteristics of an epidemic clone, was detected in 2008 among clinical isolates [15]. Similarly, Schaumburg et al., 2011, found ST508 to be significantly associated with asymptomatic carriage (p-value = 0.024) amongst isolates from Gabon.

Conclusion
Hospital personnel, HIV-positive and diabetes mellitus patients in Yaounde represent an important reservoir for MRSA, MRSA-PVL and MSSA-PVL strains. The virulence factor qgr type I was common among study populations. Studying the possible role of agr I in staphylococcal infections may provide insight for controlling risk factors among high risk patients. Isolates were genetically diverse with MLST revealing pandemic ST88 as being predominant and three atypical STs (508, 699, and 1289). The detection of uncommon STs especially ST1289 with characteristic of an epidemic clone has provided an insight into the necessity for routine monitoring to prevent spread and disease outbreaks. The clonal diversity of S. aureus reported in this study and the detection of some epidemic strains serve as the basis for informed decisions on better patient management.
municable Diseases of the Faculty of Medicine and Biomedical Sciences, The University of Yaounde 1, Cameroon and the Department of Medical Microbiology, University of Pretoria, South Africa for allowing us to conduct analyses at their facilities.

Funding
This work was partially financed by HIV-Research Trust, United Kingdom. The grant was used for molecular analysis techniques and purchase of reagents.

Availability of Data and Materials
The data used and or analysed during the current study are available from the corresponding author on reasonable request.

Authors' Contributions
AE designed the study with input from ME and ELM SKS. EM, GI, MM. CH collected samples from participants and isolated Staphylococcus aureus. AE, ELM, MCOA, MT and HGK did the molecular analysis. AE and MK did the PFGE. AE, JA and CF performed the MLST. MH and CF did the statistical analysis. AE, ME, ELM and JA wrote the first draft of the manuscript and all authors contributed to and had final approval of the article.

Consent for Publication
Not applicable.

Ethics Approval
Ethical clearance (No. 2014/07/475/CEA/CNERSH/SP) was obtained from the Cameroon National Ethics Committee for Human Health Research. Authorizations were obtained from recruitment sites and only participants who signed the assent form were enrolled in the study.

Conflicts of Interest
The authors declare that they have no competing interests.