Diversity of SCCmec Elements in Methicillin-Resistant Staphylococcus Aureus (MRSA) Recovered from a Healthy Student Population in Kenya

doi:10.21203/rs.3.rs-76228/v1

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Diversity of SCCmec Elements in Methicillin-Resistant Staphylococcus Aureus (MRSA) Recovered from a Healthy Student Population in Kenya

https://doi.org/10.21203/rs.3.rs-76228/v1

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Background: Methicillin-resistant S. aureus continues to be a concern for public health systems, particularly due to infections emerging in non-hospital settings. Resistance to methicillin is presently classified as a serious phenomenon because the majority of methicillin-resistant strains are also multi-drug resistant. The genetic determinant of resistance to methicillin and other β-lactam antibiotics is the mec-A gene, which lies in the SCCmec resistance island. In Kenya, studies done previously have shown the existence of SCCmec types in clinical isolates, but similar information on isolates recovered from healthy populations is scanty.

Methods: A cross-sectional study was conducted on healthy university students residing within the university residence halls to determine the carriage of MRSA. MRSA was detected using Cefoxitin (30µg), and mec-A gene and Sccmec elements were detected using conventional PCR methods. A total of 237 students were recruited, and 657 swabs were collected using standards methods for recovering S. aureus.

Results: A total of 231 S. aureus isolates were recovered, out of which 26 (11.3%) were MRSA. Out of the 26 MRSA strains, 17 carried the mecA gene in their gene cassettes. SCCmecV was the most prevalent (61.5%), followed by SCCmecII (53.9%) among the MRSA strains. SCCmecIVa, SCCmecIVb, SCCmecIVc and SCCmecIVd were absent in all the isolates. SCCmecV was found to be highly prevalent (64.7%) followed by SCCmecII, 8 (47.1%) among the mecA-positive MRSA strains. On the other hand, small proportions of mecA-negative isolates harbored SCCmecI (0.9%), SCCmecII (3.3%), SCCmec III (0.5%) and SCCmecV (2.3%).

Conclusion: This study revealed that the strains recovered from the student population were highly diverse in terms of the SCCmec elements they carried in their gene cassettes.

General Microbiology

Health Policy

MRSA

MSSA

MDR

mecA

SCCmec

university

healthy students

Kenya

Methicillin-resistant S. aureus infections have become a global public health concern, especially due to infections acquired in non-hospital settings (Qiao et al., 2014). In Kenya, the emergence of MRSA strains accounts for the major skin and soft-tissue infections (Aiken et al., 2014). The development of resistance to methicillin occurred in the 1960s, and this marked the beginning of the second wave of antibiotic resistance after the first wave, which was marked with the development of penicillin resistance. Resistance to methicillin by S. aureus is now classified as a serious phenomenon because the majority of such strains are also resistant to other classes of β-lactam antibiotics such as oxacillin, penicillin, amoxicillin, cephalosporins and carbapenems (Chambers & Deleo, 2009). Methicillin resistance is attributed to an extra penicillin-binding protein (PBP2a), which is coded by the mecA gene and has been shown to have a significantly reduced affinity for many β-lactam antibiotics (Arêde et al., 2012).

The genetic determinant of resistance to β-lactam antibiotics is the mecA gene (Fuda et al., 2004). The gene lies in the SCCmec resistance island and is present in about 95% of S. aureus isolates, displaying the phenotype of methicillin resistance (Wielders et al., 2002). Resistance to β-lactam has been shown not to be native to S. aureus, but rather, it has been acquired via the mecA gene for more than 40 years. The mecA gene encodes a protein known as penicillin-binding protein (PBP), which is designated as PBP2a. S. aureus produces four PBPs, namely, PBP1, PBP2 PBP3 and PBP4 that are anchored on the cytoplasmic membrane (Navratna et al., 2010). Penicillin-binding proteins function in the assembly and regulation of stages of the synthesis of a bacterial cell wall. Whereas the four PBPs are susceptible to alteration by β-lactam antibiotics resulting in the death of bacterial cells, PBB2a is refractory to the action of all presently used β-lactam antibiotics. The PBP2a can take over the functions of the four staphylococcal PBPs during exposure to β-lactam antibiotics (Kondo et al., 2007).

The mecA gene is known to be carried on a peculiar type of mobile genetic element inserted into the staphylococcal chromosome, known as the staphylococcal cassette chromosome mec (SCCmec) element (Katayama et al., 2000). The SCCmec elements share four characteristics. First, they carry the mec gene complex, which consists of a methicillin-resistance determinant gene (mecA), its regulatory genes and insertion sequences; second, they carry the ccr gene complex responsible for the mobility of the element and its associated sequences; third, they have distinct directly repeated nucleotide sequences and inverted complementary sequences at each end; and fourth, they integrate into the 3′ end of an open reading frame (ORF), orfX. Despite the similarities identified above, structures of SCCmec elements are also divergent (Kondo et al., 2007).

In Kenya, the few studies which sort to examine the diversities of SCCmec elements have revealed the existence of different types of SCCmec elements. In a recent study that analyzed isolates obtained from a government hospital and a private referral hospital in Nairobi County, SCCmecIII was found to be more prevalent, followed by SCCmecIV. The predominance of these two SCCmec elements was attributed to their small sizes, which facilitated their transmission in both community and hospital settings (Omuse et al., 2016). Another study that focused on isolates recovered from a government hospital setting also revealed the existence of SCCmecIII among all the six MRSA isolates obtained from inpatient screens (Aiken et al., 2014). In a study done by Maina et al., (2013), out of 69 MRSA strains obtained from patients with skin and soft tissue infections, 52 (75.4%) harbored SCCmecII, 5 (7.2%) harbored SCCmecI, 2 (2.9%) harbored SCCmecI and II, while 1 (1.4%) harbored SCCmecII in their gene cassettes. Based on this information, it is clear that previously published studies done in Kenya on the diversity of SCCmec elements focused on isolates recovered from patients only. Thus MRSA circulating in healthy populations outside hospital settings remain under-investigated. The previous studies have revealed the existence of SCCmec types II, III and IV, making this information available specifically for clinical MRSA isolates. As such, there is a lack of information on the SCCmec types harbored by S. aureus isolates circulating in otherwise healthy persons outside hospital settings in Kenya, and this is what this study sort to determine.

Recruitment of participants

Participants were recruited from the hostels. Students who resided at the university and agreed to provide written consent for participation by providing samples and filling the questionnaire were included in the study. Students who were on antibiotics and those who consented, but did not provide a nasal swab were excluded from the study.

This study was conducted in accordance with the regulations of the scientific and ethics review unit (SERU) of the Kenya Medical Research Institute (KEMRI) (Protocol No. KEMRI/SERU/CMR/0024/3123).

Study design

This cross-sectional study was conducted in 2016 on healthy students residing in a university’s residence halls. A systematic random sampling procedure was used to select the students’ hostel rooms where all occupants were recruited. A total of 237 healthy students were included in the study.

Collection of swabs

Swabs were collected from the nostrils, mobile phones, and pens used by participants using sterile swab sticks pre-moistened with sterile normal saline 0.85% (NaCl). All swabs were put in Amies transport media for transportation to the laboratory within a maximum of 4 hours. For enrichment, the swabs were transferred to trypticase soy broth (TSB) and incubated for 18 to 24 hours (Coia et al., 2006).

Isolation and identification of S. aureus isolates

A loopful of inoculum from the TSB cultures was inoculated in on Mannitol Salt Agar (Oxoid) plates and incubated aerobically at 37°C for 24 hours. Standard methods of identifying S. aureus, as described by Baron (1996), were employed. The plates were assessed for colony morphology and yellow pigment formation associated with S. aureus. Suspect colonies were subjected to Gram staining, coagulase, and catalase tests. Spa typing was also done for confirmation of all S. aureus isolates.

Antimicrobial susceptibility testing

The antimicrobial susceptibility tests were done using a modified Kirby-Bauer disk diffusion method describe by Rongpharpi et al. (2013). Commercially available antibiotic discs for Gram positive bacteria were used (Oxoid, CB, UK). The discs included ampicillin (10µg), amoxicillin-clavulanic acid (30), ciprofloxacin (10µg), erythromycin (15µg), gentamicin (10µg), cefoxitin (30µg), linezolid (30µg), norfloxacin (10µg), nitrofurantoin (300µg), chloramphenicol (30µg) and trimethoprim-sulfamethoxazole (25µg) (CLSI, 2015). S. aureus (MRSA) ATCC® 33591 (Oxoid) was used as control. Zones of inhibition were measured after overnight incubation at 37°C (Rongpharpi et al., 2013). Methicillin-resistant S. aureus was detected phenotypically using cefoxitin (30µg). S. aureus isolates that displayed zone sizes of less or equal to 21mm were characterized as MRSA, while S. aureus isolates that displayed zone sizes of more than 21mm were characterized as MSSA (CLSI, 2015). S. aureus isolates resistant to β-lactams, and antibiotics from at least three non-β-lactam classes were classified as MDRs.

Extraction of DNA

The extraction of DNA was done using a 10% Chelex solution prepared using 1X TE buffer following a modification of the procedure used by (HwangBo et al., 2010). A 300µl volume of 10% Chelex solution was added to fresh sterile Eppendorf tubes. To each Eppendorf tube filled with 300µl volume of 10% Chelex solution, a loopful of 24 hours old bacterial colonies were emulsified. The Chelex/sample tubes were then vortexed for 5-10 seconds. The tubes were then transferred to a heating block set at 95 ºC for 20 minutes. After boiling for 20 minutes, the tubes were left to cool for 10 minutes before centrifugation at 14700 rpm for 10 minutes. After that, 50µl of the supernatant was transferred to fresh Eppendorf tubes prefilled with 450µl of RNase DNase free PCR water. All the tubes were labeled carefully using the respective isolate codes.

Spa typing

Amplification and detection of mecA gene were done on all S. aureus isolates using conventional PCR methods. The primers used are listed in Table 1. In every PCR tube, 4µl of the ready to mix 5x FIREPol^®Master Mix (Solis Biodyne, UK), 0.4µl of both forward and reverse primers at a concentration of 10pmol of each primer, an aliquot of 1µl of BSA, 12.2 µl of RNase DNase free PCR water and 2µl of sample DNA was added. A single control strain of S. aureus was incorporated in the DNA amplification process. PCR reactions were set for 5 minutes at 80°C; 35 cycles of 45 seconds at 94°C, 45 seconds at 60°C, and 90 seconds at 72°C; a single final extension at 72°C for 10 minutes and a holding temperature at 4°C. The excepted band sizes were to be variable (Strommenger et al., 2008).

Amplification and detection of mecA gene

Amplification and detection of mecA gene were done on all S. aureus isolates using conventional PCR methods. The primers used are listed in Table 1. In every PCR tube, 4µl of the ready to mix 5x FIREPol^®Master Mix (Solis Biodyne, UK), 0.4µl of both forward and reverse primers, 1µl of BSA, 12.2µl of RNase DNase free PCR water and 2µl of sample DNA was added. Amplification reactions were set at an initial denaturation temperature of 95 ºC for 5 minutes. After that, the preparation was subjected to 30 cycles of denaturation at 94 ºC for 60 seconds, annealing at 56 ºC for 60 seconds, extension at 72 ºC for 60 seconds and a final extension at 72 ºC for 7 minutes. A holding temperature was set at 4°C. One control strain was incorporated into the DNA amplification process. The excepted PCR product encoding for mecA gene was 533 bp in size (Kondo et al., 2013).

SCCmec typing

SCCmec typing was done for all isolates using PCR. Primers specific for SCCmecI, SCCmecII, SCCmecIII, SCCmecIVa, SCCmecIVb, SCCmecIVc, SCCmecIVd and SCCmecV elements were used as published previously (Zhang et al., 2005). The primer sequences are provided in Table 1. PCR procedures were similar to those for the mecA gene, but the annealing temperature was set at 65°C for 45 seconds. A control strain of S. aureus known to be positive for the gene was incorporated in all the PCR sets.

Table 1

Primers used for amplification of selected genes
Type of primer/ gene	Primer sequences		Annealing temperature	Amplicon size	Reference
Type of primer/ gene	Forward primer	Reverse primer	Annealing temperature	Amplicon size	Reference
*spa*	5' TAA AGA CGA TCC TTC GGT GAG C3'	5' CAG CAG TAG TGC CGT TTG CTT3'	60°C	Variable	(Strommenger et al., 2008)
*mecA*	5'AAA ATC GAT GGT AAA GGT TGG C3'	5'AGT TCT GCA GTA CCG GAT TTG C3'	56°C	533bp	(Kondo et al., 2007)
SCCmecI	5’GCT TTA AAG AGT GTC GTT ACA GG 3’	5’GTT CTC TCA TAG TAT GAC GTC C3’	65°C*, 55°C	613bp	(Zhang et al., 2005)
SCCmecII	5’ CGT TGA AGA TGA TGA AGC G 3’	5’-CGA AAT TGG TTA ATG GAC C3’	65°C*, 55°C	398bp	(Zhang et al., 2005)
SCCmecIII	5’CCA TAT TGT GTA CGA TGC G 3’	5’ CCT TAG TTG TCG TAA CAG ATC G 3’	65°C*, 55°C	280bp	(Zhang et al., 2005)
SCCmecIVa	5’GCC TTA TTC GAA GAA ACC G 3’	5’-CTA CTC TTC TGA AAA GCG TCG 3’	65°C*, 55°C	776bp	(Zhang et al., 2005)
SCCmecIVb	5'TCT GGA ATT ACT TCA GCT GC3'	5'AAA CAA TAT TGC TCT CCC TC 3'	65°C*, 55°C	493bp	(Zhang et al., 2005).
SCCmecIVc	5’ACA ATA TTT GTA TTA TCG GAG AGC 3’	5’TTG GTA TGA GGT ATT GCT GG 3’	65°C*, 55°C	200bp	(Zhang et al., 2005).
SCCmecIVd	5'CTC AAA ATA CGG ACC CCA ATA CA3'	5'TGC TCC AGT AAT TGC TAA AG 3'	65°C*, 55°C	881bp	(Zhang et al., 2005).
SCCmecV	5'GAA CAT TGT TAC TTA AAT GAG GG 3'	5' TGA AAG TTG TAC CCT TGA CAC C3'	65°C*, 55°C	325bp	(Zhang et al., 2005).

Frequency of recovery of S. aureus

A total of 237 students, 120 (50.6%) males and 117 (49.4%) females were recruited, and 657 swabs were collected. The recovered S. aureus isolates were 231 in number, out of which 26 (11.3%) were MRSA. Overall, a large proportion of the participants 87 (36.7%) harbored S. aureus at least on their mobile phones compared to the proportions that carried the bacteria at least on their nostrils, 78 (32.9%), and pens, 66 (27.9%). It was also noted that a large proportion of MRSA strains were harbored in the nostrils (20, 76.9%), followed by mobile phones (7, 26.9%) and pens (5, 19.2%). In several cases, MRSA was also found to be harbored in the nostrils, mobile phones and pens used by the same student at the same time, as shown in Table 2.

Table 2

Number of S. aureus and MRSA recovered from nostrils and fomites
Type of sample/s analyzed	*Number of S. aureus* per sample type**		Number of MRSA per sample type
Type of sample/s analyzed	*S. aureus (N=231)*	Frequency (%)	MRSA (N=26)	Frequency (%)
Nostril	78	32.9	20	76.9
Phone	87	36.7	7	26.9
Pen	66	27.8	5	19.2
Nostril + Phone	40	16.9	4	15.4
Nostril + Pen	29	12.2	1	3.8
Phone + Pen	31	13.1	1	3.8
Nostril + Phone + Pen	17	7.2	9	34.6

Antimicrobial resistance phenotypes

All 231 isolates of S. aureus were tested against a panel of 11 antimicrobials. Resistance to ampicillin was very high (84%). Lower resistances of less than 20% were recorded on other isolates. Precisely, resistances to amoxicillin, chloramphenicol, gentamicin, ciprofloxacin, nitrofurantoin and norfloxacin were below 10%. None of the isolates was resistant to linezolid, and only a single isolate showed intermediate resistance to nitrofurantoin, as shown in Figure 1.

Following the screening of the 231 isolates for antimicrobial resistance, 26 (11.3%) were found to be MRSA, while 16 (9.9%) were MDRs. Out of the 26 MRSA strains, 13 (50%) were also MDRs. All the MRSA and MDR strains showed total resistance to ampicillin. Resistances above 40% were observed for erythromycin, norfloxacin and trimethoprim-sulfamethoxazole among the MRSAs. Similar observations were made for the MDRs, which also showed over 40% resistance levels to ciprofloxacin (50%) and norfloxacin (75%). For both MRSA and MDRs, less than 20% of resistance levels were recorded for amoxicillin-clavulanic acid, chloramphenicol and gentamicin, as shown in Figure 2. Statistical analysis revealed that MRSA strains were more likely to display multi-drug resistance phenotypes compared to the MSSA strains (P-value = 0.001).

mecA and Diversity of SCCmec types

All 231 S. aureus isolates were screened for the presence of the mecA gene and SCCmec elements. Only 17 (7.4%) of all the S. aureus isolates harbored the mecA gene. mecA gene was found in MRSA 17 (65.4%) of the MRSA strains. SCCmecI was more prevalent among the MRSA strains, 4 (15.4%), compared to MDR strains, 1 (6.3%), and MSSA strains, 2 (1%). Small proportions (less than 5%) of isolates recovered from the nostrils and fomites were also found to harbor SCCmecI. The prevalence of SCCmecII was found to be higher among the MDRs, 11 (68.8%) than in MRSA strains, 14 (53.9%) and the MSSA, which did not harbor SCCmecII. A large proportion of the isolates from the nostrils, 10 (12.8%), also harbored SCCmecI compared to the proportion of isolates from fomites, 5 (3.3%). SCCmecIII was only present in 3 (11%) of the MRSA isolates, and only 2 (2.6% ), and 1 (0.7%) of the isolates recovered from the nostril and fomites respectively. Similar to SCCmecII, the prevalence of SCCmecV was found to be slightly higher among the MDRs, 10 (62.5%), compared to MRSAs, 16 (61.5%), as shown in Table 3. SCCmecIVa, SCCmecIVb, SCCmecIVc and SCCmecIVd were absent in the pool of isolates.

Table 3

Proportion of isolates carrying SCCmec elements
Type/source of isolate	Number of isolates tested (N)	Frequency of mecA presence n (%)	Frequency of SCCmec elements n (%)
Type/source of isolate	Number of isolates tested (N)	Frequency of mecA presence n (%)	I	II	III	V
MRSA	26	17 (65.4%)	4, 15.4%	14, 53.9%	3 (11.5%)	16 (61.5%)
MSSA	205	0	2 (1.0%)	0	0	0
MDR	16	9 (56.3%)	1 (6.3%)	11 (68.8%)	0	10 (62.5%)
Nostrils	78	8 (10.3%)	3 (3.9%)	10 (12.8%)	2 (2.6%)	10 (12.8%)
Fomites	153	9(5.9%)	4 (2.6%)	5 (3.3%)	1 (0.7%)	6 (3.9%)

SCCmec elements in mecA positive isolates

All the mecA-positive strains were screened for the presence of selected SCCmec elements in their gene cassettes. Accordingly, SCCmecV was found to be highly prevalent, 11 (64.7%), compared to SCCmecII, 8 (47.1%), SCCmecI, 5 (29.4%), and SCCmec III, 2 (11.8%). Contrary to the mecA-positive MRSA strains, the SCCmecII were harbored in the majority, 7 (77.8%), of the mecA-positive MDR strains. A majority of mecA-positive isolates recovered from the nostrils, 6 (75%), and fomites, 5 (55.6%), were also found to harbor SCCmecV. The unidentified SCCmec elements were found in small proportions of the mecA-positive MRSA strains, 3 (17.6%). See Table 4.

Table 4

Proportion of mecA positive isolates carrying SCCmec elements
Type/source of isolate	*Number of mecA-positive isolates (N)*	SCCmec type, n(%)				Unidentified SCCmec elements, n (%)
Type/source of isolate	*Number of mecA-positive isolates (N)*	I	II	III	V	Unidentified SCCmec elements, n (%)
MRSA	17	5 (29.4%)	8 (47.1%)	2 (11.8%)	11 (64.7%)	3 (17.6%)
MDR	9	1 (11.1%)	7 (77.8%)	0	3 (33.3%)	0
Nostril	8	3 (37.5%)	5 (62.5%)	1 (12.5%)	6 (75%)	0
Fomites	9	2 (22.2%)	3 (33.3%)	1 (11.1%)	5 (55.6%)	3 (33.3%)

SCCmec elements in mecA negative isolates

Generally, the majority of the mecA-negative MRSA isolates harbored SCCmecII, 6 (66.7%), and SCCmecV, 5 (55.6%), compared to SCCmecIII, 1 (11.1%). The SCCmecI was the only gene detected in 2 (1.0%) of the mecA-negative MSSA strains. The SCCmecII and SCCmecV were present in 7 (57.1%) and 2 (28.6%) of the mecA-negative MDR strains, respectively. Small proportions of mecA-negative isolates recovered from nostrils (less than 10%) were found to harbor SCCmecI, II, III and V while those recovered from fomites harbored SCCmecI, II and V as presented in Table 5.

Table 5

Proportion of mecA negative isolates carrying SCCmec elements
Type/source of isolate	*Number of mecA-negative isolates (N)*	SCCmec type, n (%)
Type/source of isolate	*Number of mecA-negative isolates (N)*	I	II	III	V
MRSA	9	0	6 (66.7%)	1 (11.1%)	5 (55.6%)
MSSA	202	2 (1.0%)	0	0	0
MDR	7	0	4 (57.1%)	0	2 (28.6%)
Nostril	70	5 (7.1%)	1 (1.4%)	1 (1.4%)	4 (5.7%)
Fomites	144	2 (1.4%)	2 (1.4%)	0	1 (0.7%)

Multiple SCCmec elements

This study revealed four different combinations, each consisting of two distinct SCCmec elements. These included; SCCmecI and SCCmecIII, SCCmecI and SCCmecV, SCCmecII and SCCmecV and SCCmecII and SCCmecV. Almost half of the MRSA strains, 11 (42.3%), carried SCCmecII and SCCmecV compared to those that harbored SCCmecI and SCCmecV, 3 (11.5%), SCCmecI and SCCmecIII, 2 (7.7%), and SCCmecIII and SCCmecV, 1 (3.9%). Only two combinations of the SCCmec elements were seen among the MDR strains, where 8 (50%) of these strains carried SCCmecII and SCCmecV, and only a single MDR varied SCCmecI and SCCmecV. See Table 6 below.

Table 6

Proportion of S. aureus isolates carrying multiple SCCmec elements
Type/ source of isolate	*Number of S. aureus* isolates tested (N)**	SCCmec elements combination, n (%)
Type/ source of isolate	*Number of S. aureus* isolates tested (N)**	SCCmecI + SCCmecIII	SCCmecI + SCCmecV	SCCmecII + SCCmecV	SCCmecIII + SCCmecV
MRSA	26	2 (7.7%)	3 (11.5%)	11 (42.3%)	1 (3.9%)
MDR	16	0	1 (6.3%)	8 (50%)	0
Nostril	78	1 (1.3%)	2 (2.6%)	7 (9.0%)	1 (1.3%)
Fomites	153	1 (0.7%)	1 (0.7%)	4 (2.6%)	0

Carriage of S. aureus in a population that is otherwise considered as healthy has shown global variation. Previous studies have revealed colonization prevalence ranging between 20%-40%, which is reported as the prevalence limit in literature, in different study populations (Chambers & Deleo, 2009; Sivaraman et al., 2009). In this study, the prevalence of nasal, phone and pen colonization fell within the range of 20-40%. Similar results have also been reported by other studies which focused on college students in Tanzania (Okamo et al., 2016), China (Du et al., 2011), Nepal (Ansari et al., 2016) as well as the general population in Nigeria (Onanuga & Temedie, 2011), Gabon (Schaumburg et al., 2011), Brazil (Pires et al., 2014), Northern Manhattan (Miller et al., 2009) and Mexico (Hamdan-Partida et al., 2010). On the contrary, other published reports have indicated prevalences outside the 20-40% limit. A study conducted on healthy volunteers in Tunisia reported a nasal colonization prevalence of 13% (Ben Slama et al., 2011) while another study focused on a remote population in Guiana reported a colonization prevalence of 57.8% (Ruimy et al., 2008). As reported by Onanuga & Temedie (2011), these variations in the prevalence of nasal colonization could be linked to the characteristics of study populations. Therefore, the high prevalence of colonization observed on nasal cavities and phones could be attributed to specific aspects of this study’s participants.

In the present study, a prevalence of MRSA (11.3%) was found among a healthy population of university students aged between 18-30 years in Central Kenya. The MRSA strains were detected using cefoxitin (30 μg) discs, which, according to Rasheed & Ahmed (2010), is an alternative method to PCR in the detection of MRSA strains. So far in Kenya, this is the first study to present results on the prevalence of MRSA among healthy university students residing within institution-based residence halls. Other recent studies like (Zakai, 2015) have reported a higher MRSA prevalence (18.7%) among student populations in Jeddah, Saudi Arabia while others, Okamo et al. (2016) in Tanzania and Kitti et al. (2011) in Thailand, have reported very low prevalences of 0.3% and 1% respectively. These reports indicate the varied prevalence of MRSA strains in student populations. Even though other reports reveal MRSA prevalence that is as low as 1%, Kitti et al. (2011), the prevalence remains higher in different populations in other areas, thus posing a serious public health issue due to frequent contact with colonized individuals (Zakai, 2015). Based on the results reported by Kitti et al. (2011) (an MRSA prevalence of 1% in a student population), it could be possible that non-exposure to MRSA may result in reduced risks of MRSA colonization. It can be speculated that continued exposure to MRSA colonized environments may result in an increased risk of MRSA colonization. In addition, considering the case of this study where significantly high chances of MRSA colonization were reported in particular residence halls, it could be possible that other students visiting these residence halls would be increasing their risk for MRSA colonization.

Antimicrobial resistance phenotypes

This study established that MRSA strains were more likely to display multi-drug resistance compared to MSSA strains, a finding which has also been reported by Gupta et al. (2013) in India. Among the published studies done in Kenya (all of which focused on clinical isolates), none reported this property among the MRSA strains. Therefore, this study highlights crucial findings regarding the circulation of multi-drug resistant MRSA and MSSA strains among a healthy student population. Given the fact that S. aureus is transmitted via direct or indirect person-to-person contact, there is the possibility of an increased prevalence of multi-drug resistant MRSA and MSSA strains among the student population, which is a worrisome scenario. It is also worth noting that the multi-drug resistance property renders multi-drug resistant strains as one of the most intractable pathogenic organisms in the history of antibiotic therapy. Therefore, the circulation of these multi-drug resistant S. aureus among students presents a challenge to the management of Staphylococcal infections that may develop, particularly in regards to acquiring effective antibiotics, which may be expensive.

Carriage of mecA gene

Molecular analysis in this study showed that the S. aureus isolates were genetically diverse. A majority of the MRSA strains carried the mecA gene, while none of the MSSA strains carried this gene (Table 4-6). Among the studies done in Kenya, carriage of the mecA gene has been reported among the MRSA strains. In their study, Maina et al. (2013) reported that all the MRSA strains harbored the mecA gene, and similar to this study, none of the MSSA strains were found to harbor this gene. In a recent study conducted in Zambia, all the MRSA were found to harbor the mecA gene (Samutela et al., 2017). Similar to this study also, a majority of MRSA (90.2%) strains carried mecA gene, while 9.8% were negative for the gene in a study conducted in Sudan (Elhassan etal., 2015). According to available literature, the presence of the mecA gene forms the basis of resistance to beta-lactam antibiotics (Murakami et al., 1991; Sharma et al., 1998; Wongwanich et al., 2000). Therefore, basing on suggestions by the majority of researchers in this field, the mecA gene was linked to cefoxitin resistance among the mecA-positive isolates recovered in this study. Other genetic elements may also be considered for the explanation of cefoxitin-resistance mechanisms (Elhassan et al., 2015). Hence, the basis of cefoxitin-resistance among the mecA-negative MRSA isolates characterized in this study could be attributed to two possible reasons. Firstly, the hyperproduction of β-lactamases, as described by Olanyika et al., (2009) and secondly, development of specific alterations in the variable amino acids found in the PBPs cascades as highlighted by (Ba et al., 2014).

Diversity of SCCmec elements

Screening of all the S. aureus isolates for the presence of SCCmec elements revealed the existence of SCCmecV as the most prevalent mec type, followed closely by SCCmecII, then SCCmecI and lastly, SCCmecIII, which was only present in three isolates. Contrary to the clinical isolates recovered in Kenya, the non-clinical isolates examined in this study did not harbor any of the SCCmecIV types. SCCmecV has been shown to be of small molecular size, and this could be the possible reason for its high prevalence among isolates recovered in this study (Omuse et al., 2016). No other published study done in Kenya with similar findings was found. Previously, SCCmecIV has been reported to circulate highly in community settings, possibly because of its small size, which renders it more mobile and thus can be inserted into multiple S. aureus lineages (Omuse et al., 2016; Tong et al., 2012). It was unclear why none of the isolates recovered in this study harbored SCCmecIV.

The prevalence of SCCmecI was high among the MRSA strains compared to MDR strains. This observation was not unique to this study since SCCmecI is known to encode solely for resistance to β-lactam antimicrobials (Deurenberg et al., 2007). The prevalence of SCCmecII was found to be high among the MDRs compared to MRSA strains. Also, this observation was not unique to the present study because the SCCmecII cassettes are determinants of antimicrobial multi-resistance. The SCCmecII cassette carries additional genes for drug resistance, which are integrated into plasmids (pI258, pUB110 and pT181) and a transposon (Tn554) (Deurenberg et al., 2007). Similar to SCCmecII, the prevalence of SCCmecV was found to be slightly higher among the MDRs than MRSAs by 1%. SCCmecII (as stated before) determines antimicrobial multi-resistance, while SCCmecV encodes for resistance to β-lactam antimicrobials. The high numbers of SCCmecV strains, which were also found to be MDRs, could be attributed possibly to the fact that half the population of MDRs carried both SCCmecII and V in their genomes.

Multiple SCCmec elements

In this study, carriage of multiple SCCmec elements was reported on several occasions. Maina et al. made a similar observation in Kenya, where a single isolate was found to carry both SCCmec I and II. Almost half of the MRSA strains analyzed in the present study carried SCCmecII and SCCmecV compared to those that carried SCCmecI and SCCmecV (11.5%), SCCmecI and SCCmecIII (7.7%) and SCCmecII and SCCmecV (3.9%). Half the proportion of MDRs carried SCCmecII and SCCmecV. Characteristically, this study established that the SCCmecII and SCCmecV combination was more prevalent among the MRSA and MDR strains, and therefore, a likely genotypic characteristic of MRSA strains that are also MDRs. More studies could also be done to ascertain the prevalence of the existence of this combination of SCCmec elements among MRSA strains that are also MDRs.

Variants of SCCmec elements

In the present study, two MSSA strains were also found to carry SCCmecI. Additionally, among the mecA-negative MRSA strains, six carried SCCmecII, five carried SCCmecV while only a single isolate carried SCCmecIII. None of the previously published studies done in Kenya reported a similar finding. This finding was unclear, though other research reports demonstrated the existence of similar observations (Chlebowicz et al., 2010; Donnio et al., 2005; International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC), 2009; Shore et al., 2014; Vandendriessche et al., 2014; Wong et al., 2010). Donnio et al. (2005) characterized MSSA strains, which had the IS431: pUB110: IS431::dcs structure (SCCmec-associated elements), but did not carry mecA and ccrAB. Wong et al. (2010) characterized seven isolates, which resembled USA100 and carried SCCmecII but not mecA. For the two MSSA isolates that carried SCCmecI, it could be possible that the isolates lost the methicillin-resistant phenotype due to partial excision of the SCCmec Stamper et al. (2011). On the other hand, the mecA-negative MRSA carrying the various SCCmec types retained their methicillin-resistant phenotype possibly due to other genetic factors, which have been described previously in this context.

In summary, the present study demonstrated the existence of varied SCCmec types among S. aureus strains circulating in the studied student population. The SCCmecV was the most prevalent. Some strains carried multiple SCCmec elements in the gene cassettes, while other strains lacked the mecA gene but still carried the SCCmec elements in their gene cassettes.

SCCmec Staphylococcal cassette chromosome mec

MRSA Methicillin-resistant S. aureus

MDR Multi-drug resistance

MSSA Methicillin-susceptible S. aureus

PBP penicillin-binding protein

ORF Open reading frame

KEMRI Kenya Medical Research Institute

SERU Scientific and Ethics Review Unit

TSB Tryptone Soya Broth

PCR Polymerase Chain Reaction

UK United Kingdom

CLSI Clinical & Laboratory Standards Institute

AMP Ampicillin

AMC Amoxicillin clavulanic acid

FOX Cefoxitin

C Chloramphenicol

E Erythromycin

CN Gentamicin

CIP Ciprofloxacin

F Nitrofurantoin

SXT Sulphamethoxazole-trimethoprim

NOR Norfloxacin

LZD Linezolid

Ethics approval and consent to participate

This study was conducted in accordance with the regulations of the scientific and ethics review unit (SERU) of the Kenya Medical Research Institute (KEMRI) (Protocol No. KEMRI/SERU/CMR/0024/3123).

Consent for publication

Authors have approved the review and publication of this manuscript.

Availability of data and materials

Data collected in this study is available for perusal

Competing interests

Authors have to competing interests to declare.

Funding

This work was funded partly by the Social Ecology of Human Infectious Diseases Initiative (ESEI), Grant Reference: G1100783/1.

Authors' contributions

Osborn Khasabuli performed the laboratory work and wrote the manuscript. Dr. Caroline Ngugi and Dr. John Kiiru provided expert advice in designing the study and manuscript writing process.

Acknowledgements

The authors would like to thank the team at the molecular laboratory, Center for Microbiology Research (CMR), Kenya Medical Research Institute (KEMRI), for their technical assistance.

Authors' information (optional)

Not applicable

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Diversity of SCCmec Elements in Methicillin-Resistant Staphylococcus Aureus (MRSA) Recovered from a Healthy Student Population in Kenya

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Abstract

Figures

Background

Methods

Recruitment of participants

Study design

Collection of swabs

Isolation and identification of S. aureus isolates

Antimicrobial susceptibility testing

Extraction of DNA

Spa typing

Amplification and detection of mecA gene

SCCmec typing

Results

Frequency of recovery of S. aureus

Antimicrobial resistance phenotypes

mecA and Diversity of SCCmec types

SCCmec elements in mecA positive isolates

SCCmec elements in mecA negative isolates

Multiple SCCmec elements

Discussion

Antimicrobial resistance phenotypes

Carriage of mecA gene

Diversity of SCCmec elements

Multiple SCCmec elements

Variants of SCCmec elements

Conclusions

List Of Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgements

Authors' information (optional)

References

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