Staphylococcus aureus Nasal Carriage Among Patients and Health Care Workers in São Tomé and Príncipe

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen worldwide. However, data on MRSA prevalence in the African continent are scarce and nonexistent for São Tomé and Príncipe. In November 2010 and April 2012, a total of 332 individuals (258 patients and 74 health care workers [HCW]) from Hospital Dr. Ayres Menezes in São Tomé and Príncipe, were screened for S. aureus and MRSA carriage. Fifty-two persons (15.7%) were S. aureus nasal carriers out of which 14 (26.9%) were colonized with MRSA. MRSA isolates belonged to three clonal complexes: CC8 (PFGE type B-ST8-t064/t451-IVg/V), CC88 (PFGE E-ST88-t186/t786-IVa), and CC5 (PFGE K-ST5-t105-IVa/PFGE K-ST105-t002-II). A higher genetic diversity was found among methicillin-susceptible S. aureus (MSSA) isolates where 58.5% (n=24) belonged to four major lineages: PFGE type A-ST15-t084; PFGE C-ST508-t861 or related; PFGE D-ST152-t355 or related; and PFGE G-ST121-t159/t2304. Despite the common nonmultiresistant profile, 98% of the isolates harbored two or more virulence factors. Panton-Valentine leukocidine was detected in 36% of the isolates, all MSSA. S. aureus cross-transmission between HCW and patients in the pediatric and medicine wards and the detection of identical MRSA strains among patients in two different wards evidenced the need of implementation of additional infection control measures in this hospital.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen and a serious public health problem worldwide.^16,19 Although efforts to prevent MRSA transmission and infection in the most developed countries receive major research funding, the extent of the MRSA problem remains largely unknown in resource-poor regions. Moreover, surveillance systems to guide interventions require expertise and resources, which are very limited in developing countries, where social and health care system deficiencies such as overcrowding and understaffing result in a lack of infection control practices.⁸ For an integrated worldwide control of MRSA, studies to appraise the prevalence and profile of S. aureus disease in developing nations are mandatory.

MRSA surveillance data from African countries are scarce, refer to individual hospitals and the different study designs and selection of clinical specimens may constrain the comparison of values that are very variable. In West-African countries, MRSA prevalence ranges from 3.7% in carriage and 11% in clinical isolates from Gabon⁵⁰ to 15% in infection isolates from five sub-Saharan African towns,¹⁰ 16% in Nigeria⁵⁴ and 33.6% in Ghana.³⁸ Higher variability was also reported in East-South Africa: 6.3% in infection and 14.8% in carriage in Madagascar island,^44,45 23% in colonization among Ethiopian children and prisoners,²⁷ 27% in clinical isolates in South Africa,⁵³ 37.5% in surgical site infections and 52% in carriage among health care workers (HCW) and patients in surgical units of Mulago hospital, Uganda,^26,52 and 84.1% in patients with skin and soft tissue infections in Kenya.³¹ Sporadic MRSA isolates have been reported in Mali⁴⁸ and Tanzania.⁵⁷ Information from Portuguese-speaking African countries (PALOP) are almost inexistent and limited to a study that reported an MRSA prevalence of 8% in a single hospital in Mozambique between 2001 and 2006³² and a study dated from 1997 in Cape Verde islands.¹ Although no MRSA were isolated among nasal swabs from patients and HCW in Cape Verde, an unusually high prevalence (35%) of the highly potent Panton-Valentine leukocidine (PVL) was found among methicillin-susceptible S. aureus (MSSA) isolates, sharing the same genetic background as MRSA pandemic clones.³

The close relationships between Portugal and PALOP nations may influence the MRSA clonal types circulating in this country. Portugal has been reporting the highest MRSA rates among European countries for the last decade and is currently one of the two single countries reporting an MRSA prevalence above 50%.¹⁹ Therefore, the aim of the present study was to evaluate MRSA nasal carriage among patients and HCW in São Tomé and Príncipe, characterize its population structure, and identify eventual reservoirs and transmission routes within a hospital.

Materials and Methods

Hospital setting

São Tomé and Príncipe is an archipelago constituted by two major islands, São Tomé and Príncipe, located in the Equator in the Gulf of Guinea—Africa's western coast. São Tomé and Príncipe is the second smallest African country (Seychelles being the smallest) with a population of 183.000 with a mean age of 17.6 years.¹¹ The referral hospital in the country, Hospital Dr. Ayres Menezes is a medium-sized hospital (441 beds) located in the island of São Tomé, and constructed in 1918. The hospital has nine hospital wards (medicine, pediatrics, orthopedics, maternity, psychiatry, surgery, otorhinolaryngology, infectious diseases, and operating room), intensive care units (ICU) for adults (five beds) and children (three beds), several ambulatory services (medicine, pediatrics, orthopedics, surgery, gynecology and obstetrics, urology, ophthalmology, otorhinolaryngology, dermatology, psychiatry, and dentistry), and a routine analysis laboratory. It serves the entire population of the archipelago and has an admission rate of 45 to 70 entrances per day to the emergency ward.

Nasal screening and bacterial isolates

The institutional ethics committee approved the protocol. Oral informed consent was obtained at the time of screening. Inpatients and HCW were nasal swabbed for S. aureus carriage. Exclusion criteria included patients admitted for less than 48h, individuals with inaccessible nares due to the presence of medical devices, children being breastfed at the time of screening, and individuals who refused to participate in the study.

Sampling was performed by trained nurses in two periods of 1 week each—in November 2010 and April 2012—in the main services where the risk for S. aureus infection is usually high (medicine, orthopedics, surgery, pediatrics, burn unit, and ICU) and in gynecology. Samples from HCW were additionally recovered from the emergency ward and routine analysis laboratory.

Samples were taken by swabbing both nares of each individual with a sterile dry cotton swab, which was stored in Stuart transport medium and transferred to the Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica in Portugal. Swabs were processed within 4 to 8 days after sampling. In brief, each swab was inoculated on a rich medium, Tryptic Soy Agar (Becton, Dickinson & Co) and on selective medium, Mannitol Salt Agar (Becton, Dickinson & Co) for the first screening or on the chromogenic medium Chromagar Staph aureus (ChromAgar) for the second screening. All presumptive S. aureus colonies were tested for coagulase by latex agglutination test Staphytect Plus (Oxoid) or by agglutination of rabbit plasma in tubes (Becton Dickinson & Co) in case of prior ambiguous results. S. aureus species was confirmed by PCR amplification of the nuc gene.⁴²

Antimicrobial susceptibility testing and mecA detection

Screening of the mecA gene was performed on all S. aureus isolates by PCR.³⁹

Concomitantly, antimicrobial susceptibility testing was performed on all isolates by the disk diffusion method for a panel of 16 antibiotics: penicillin, oxacillin, erythromycin, gentamicin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, rifampin, tetracycline, fusidic acid, mupirocin, teicoplanin, vancomycin, linezolid, and quinupristin-dalfopristin. Breakpoints were defined according to the current Clinical Laboratory Standards Institute (CLSI) guidelines¹⁴ except for fusidic acid and mupirocin that were defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST_ www.eucast.org/) and British Society for Antimicrobial Chemotherapy (BSAC_http://bsac.org.uk/susceptibility/) guidelines, respectively. Strain S. aureus ATCC25923 was used as quality control.

Oxacillin minimum inhibitory concentrations were determined using E-test strips according to the manufacturer's instructions (Biomerieux, Marcy l'Etoile, France) for oxacillin susceptible, mecA-positive isolates.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was performed for all isolates after SmaI digestion as described by Chung et al.¹² The resulting band profiles were analyzed by both visual inspection using the criteria of McDougal et al.,³³ followed by automated analysis with the BioNumerics software version 6.6 (Applied Maths) for relatedness evaluation to define clusters. Dendrograms were generated as previously described²⁰ with a lower tolerance value of 1% for band pattern comparisons. Dice coefficient similarity cutoff at 80% and 95% were used for PFGE type and subtype clusters definition, respectively.

spa typing and multilocus sequence typing

spa typing was performed as previously described on at least one representative of each PFGE subtype, and spa types were assigned through the Ridom web server (http://spaserver.ridom.de).² Clonal relatedness of different spa types was inferred using the Based Upon Repeat Pattern (BURP) algorithm.³⁴ Representatives of the different PFGE types were further characterized by multilocus sequence typing (MLST).^15,17 Allelic profiles and sequence types (ST) were defined using the MLST online database (www.mlst.net). MLST clonal complexes (CC) were defined using the eBURST algorithm and restricted to single and double locus variants from each group founder or sub founder, inside each clonal group.

SCCmec typing

Characterization of the staphylococcal cassette chromosome mec (SCCmec) was performed for all MRSA.³⁶ SCCmec type IV isolates were subtyped by multiplex PCR as described,³⁵ after concentration adjustment for primer pairs J IVh F/R and ccrB2 F/R.

Detection of virulence determinants and agr type

The presence of 11 specific staphylococcal virulence genes, including three leukocidins (lukS-lukF, lukE-lukD, and lukM), three hemolysins (hlb, hlg, and hlgv) and five super-antigenic toxins (eta, etb, etd, sel, and sep) were determined by PCR for the entire collection as previously described.^37,58 The detection of the accessory gene regulator (agr) allele group (I-IV) was performed on all isolates by multiplex PCR according to Gilot et al.²²

Results

Study population

A total of 332 individuals (258 inpatients and 74 HCW), distributed by the two screening periods, were nasal swabbed for S. aureus carriage (Table 1). Four out of the 74 HCW were sampled twice (2010 and 2012), resulting in a total of 336 nasal swabs.

Table 1.

Staphylococcus aureus and MRSA Nasal Carriage in São Tomé and Príncipe by Individual Category (HCW or patients) and Sampling Period (November 2010 and April 2012)

	2010–2012 (%) ^a			2010 (%) ^a			2012 (%) ^a
No. of individuals	Total	HCW	Patients	Total	HCW	Patients	Total	HCW	Patients
Total	332	74	258	135	16	119	201	62	139
S. aureus-positive	52 (15.7)	15 (20.2)	37 (14.3)	23 (17.0)	6 (37.5)	17 (14.3)	30 (14.9)	10 (16.1)	20 (14.4)
MRSA-positive	14 (4.2)	3 (4.0)	11 (4.3)	5 (3.7)	2 (2.5)	3 (2.5)	9 (4.5)	1 (1.6)	8 (5.8)

Percentages relative to the total number of individuals screened in each period.

HCW, health care worker; MRSA, methicillin-resistant Staphylococcus aureus.

Among the 332 screened individuals, 52 were S. aureus nasal carriers (15.7%) out of which 14 (11 patients and 3 HCW) were colonized with MRSA (26.9% of the total S. aureus population). Concerning both screening periods individually, S. aureus nasal carriage slightly decreased from 17% in 2010 to 14.9% in 2012. However, the MRSA prevalence in the screened population increased from 3.7% in 2010 to 4.5% in 2012, mainly due to the rise of carriage among patients (2.5–5.8%) (Table 1). A single HCW was colonized in both screening periods, but with a different strain, a MRSA in 2010 and a MSSA in 2012.

The percentage of individuals colonized with MRSA was globally similar in both groups (4.3% [11 out of 258 patients swabbed] vs. 4% [3 out of 74 HCW swabbed]), but HCW seems to be more frequently colonized with S. aureus than patients (20.5% vs. 14.3%), although the difference was not statistically significant (p=0.22). Moreover, regarding the S. aureus population, the percentage of methicillin resistance in patients (29.7% [11 out of 37 S. aureus-positive swabs] seems to be considerable, but not significant (p=0.73) when compared with HCW (18.7% [3 out of 16 S. aureus-positive swabs]).

S. aureus carriage among physicians (25%, n=1), nurses (22.8%, n=8), and nurse-aids (21%, n=4) was similar.

Samples were collected in different hospital wards: pediatrics (n=103), medicine (n=100), orthopedics (n=41), surgery (n=56), burn unit (n=9), gynecology (n=3), emergency ward (n=2), ICU (n=5), and routine analysis laboratory (n=17). The highest percentages of S. aureus nasal carriers were observed in the burn unit (66.7%), ICU (40%), and orthopedics (31.7%), wards with a double internment time compared with other units (mean of 31.9 days vs. 15.3 days). The 14 MRSA isolates were recovered from the burn unit (44.4%, n=4), orthopedics (4.9%, n=2), pediatrics (4.9%, n=5), ICU (n=1), surgery (n=1), and medicine (n=1).

The 55 S. aureus isolates (two patients were co-colonized with two different isolates and a HCW was colonized in both periods with different isolates) recovered were further characterized as described below.

Antimicrobial resistance

All 55 S. aureus isolates were susceptible to gentamicin, quinupristin-dalfopristin, chloramphenicol, linezolid, vancomycin, fusidic acid, and mupirocin.

Among the 41 MSSA isolates, 36.6% showed resistance to trimethoprim-sulfamethoxazole, 12.2% to ciprofloxacin, 9.7% to tetracycline, and 4.9% to erythromycin. Higher resistance rates were found among the 14 MRSA; the isolates showed resistance to cefoxitin (100%), trimethoprim-sulfamethoxazol (85.7%), erythromycin (64.3%), tetracycline (57.1%), ciprofloxacin (42.8%), and rifampin (14.3%). Four MRSA and a single MSSA isolate showed induced resistance to clindamycin.

Among the 14 isolates that amplified the mecA gene and were considered MRSA, seven showed an oxacillin minimum inhibitory concentration between 0.5 to 3 mg/L, indicating a highly heterogeneous resistance phenotype.

Clonal types distribution

The MSSA isolates were distributed into eleven CCs (CC1, 5, 8, 15, 45, 72, 80, 88, 121, 152, and 707) and one singleton. Most (n=24; 58.5%) belonged to four major clonal lineages (Table 2): PFGE type A, spa type t084, ST15, agr II (n=10); PFGE C, t086/t2771/t5602/t10763, ST508/ST2446, agr I (n=6); PFGE type D, t355/t9564, ST152, agr I (n=4), and PFGE type G, t159/t2304, ST121, agr IV (n=4).

Table 2.

Molecular Characterization of the S. aureus Isolates (14 MRSA and 41 MSSA) Recovered from 336 Nasal Swabs in São Tomé and Príncipe in November 2010 and April 2012

					spa typing^b		MLST				Virulence determinants ^c
Isolate ID	Individual ^a	Year	MR/MSSA	PFGE type	spa type	spa_CC	ST	CC	SCCmec type	agr type	lukF-lukS-PV	lukD-lukE	hlg	hlgv	hlb	sel	sep	etd
STP151	patient	2012	MRSA	B2	t064	451	8	8	IVnt	I	−	+	−	+	−	−	−	−
STP297	patient	2012	MRSA	B2				8	IVnt	I	−	+	−	+	−	−	−	−
STP152	patient	2012	MRSA	B2				8	IVnt	I	−	+	−	+	+	−	−	−
STP153A	patient	2012	MRSA	B2				8	IVnt	I	−	+	−	+	−	−	−	−
STP277	patient	2012	MRSA	B3	t451	451		8	V	I	−	+	−	+	−	−	−	−
STP280	patient	2012	MRSA	B3				8	V	I	−	+	−	+	−	−	−	−
STP292	patient	2012	MRSA	B4	t451	451	8	8	V	I	−	+	−	+	−	−	−	−
STP59	patient	2010	MRSA	B5	t451	451	8	8	V	I	−	+	−	+	−	+	−	−
STP74A	HCW6	2010	MRSA	E1	t186	186	88	88	IVa	III	−	+	−	+	+	+	−	−
STP33	HCW2	2010	MRSA	E2	t786	186	88	88	IVa	III	−	+	−	+	+	+	−	−
STP182	patient	2012	MRSA	E3	t186	186		88	IVa	III	−	+	−	+	−	+	+	−
STP254	HCW11	2012	MRSA	E4	t786	186		88	IVa	na	−	+	−	+	+	−	−	−
STP46A	patient	2010	MRSA	K1	t105	2	5	5	IVa	II	−	+	−	+	−	−	−	−
STP65A	patient	2010	MRSA	K2	t002	2	105	5	II	II	−	+	−	+	−	−	−	−
STP273	HCW13	2012	MSSA	A1				15		II	+	+	−	+	−	−	−	−
STP302A	patient	2012	MSSA	A1				15		II	+	+	−	+	−	−	−	−
STP136A	patient	2010	MSSA	A1				15		II	+	+	−	+	−	+	−	−
STP286	patient	2012	MSSA	A1				15		II	+	+	−	+	−	−	−	−
STP80	patient	2010	MSSA	A1	t084	sing		15		II	+	+	−	+	−	+	−	−
STP76	patient	2010	MSSA	A1	t084	sing		15		II	+	+	−	+	−	+	−	−
STP24A	patient	2010	MSSA	A2	t084	sing	15	15		II	+	+	−	+	−	+	−	−
STP25A	patient	2010	MSSA	A2				15		II	+	+	−	+	−	+	−	−
STP252A	patient	2012	MSSA	A2				15		II	+	+	−	+	−	−	−	−
STP126	patient	2010	MSSA	A3	t084	sing		15		II	−	+	−	+	−	+	−	−
STP30A	HCW1	2010	MSSA	C1	t2771	2771/10763	508	45		I	−	−	+	−	−	+	+	−
STP165A	HCW8	2012	MSSA	C2	t861	2771/10763	2446	45		I	−	−	+	−	−	+	+	−
STP249A	HCW10	2012	MSSA	C2				45		I	−	−	+	−	−	+	+	−
STP334	HCW15	2012	MSSA	C3				45		I	−	−	+	−	−	+	+	−
STP155	patient	2012	MSSA	C4	t861	2771/10763		45		I	−	−	+	−	+	+	+	−
STP306	patient	2012	MSSA	C5	t10763	2771/10763		45		I	−	−	+	−	+	+	+	−
STP115	patient	2010	MSSA	D1	t355	355/9564	152	152		I	+	−	−	−	+	−	−	−
STP12B	patient	2010	MSSA	D2	t9564	355/9564	152	152		I	+	−	−	−	−	−	−	−
STP26B	patient	2010	MSSA	D3	t355	355/9564	152	152		I	+	−	+	−	+	−	−	−
STP327	HCW14	2012	MSSA	D4	t355	355/9564		152		I	+	−	−	−	+	−	−	−
STP214	HCW9	2012	MSSA	G1	t159	sing	121	121		IV	+	+	−	+	−	−	−	−
STP336	HCW6	2012	MSSA	G1				121		IV	+	+	−	+	−	−	−	−
STP252B	patient	2012	MSSA	G1				121		IV	+	+	−	+	−	−	−	−
STP196	patient	2012	MSSA	G2	t2304	excluded		121		IV	+	+	−	+	−	−	−	−
STP301	patient	2012	MSSA	J1	t939	sing	2447	45		na	−	−	+	−	−	+	−	−
STP179	patient	2012	MSSA	J2	t939	sing		45		na	−	−	+	−	−	+	−	−
STP29A	patient	2010	MSSA	J3	t939	sing	45	45		na	−	−	+	−	−	+	−	−
STP34A	HCW3	2010	MSSA	B1	t064	451	8	8		I	−	+	−	+	−	−	−	−
STP60	patient	2010	MSSA	B1				8		I	−	+	−	+	−	−	−	−
STP141	HCW7	2012	MSSA	F1	t701	451	6	5		I	−	+	−	+	−	−	−	−
STP260	HCW12	2012	MSSA	F2	t701	451		5		I	−	+	−	+	−	−	−	−
STP28B	patient	2010	MSSA	H1	t1458	sing	2367	707		III	−	+	−	+	−	−	−	−
STP108B	patient	2010	MSSA	H2	t1458	sing		707		III	−	+	−	+	−	+	−	−
STP35	HCW4	2010	MSSA	L1	t148	148/11082	72	72		I	−	+	−	+	−	+	+	−
STP322	patient	2012	MSSA	L2	t11082	148/11082	2448	72		I	−	+	−	+	−	+	+	−
STP26A	patient	2010	MSSA	W1	t590	sing	1	1		III	+	+	−	+	+	+	+	−
STP38A	HCW5	2010	MSSA	W2	t1931	sing	1	1		III	−	+	−	+	+	+	−	−
STP62A	patient	2010	MSSA	E5	t4195	186	88	88		III	−	+	−	+	−	−	−	−
STP206	patient	2012	MSSA	K3	t311	2	5	5		II	+	+	−	+	−	−	−	−
STP183	patient	2012	MSSA	R	t11083	sing	718	sing		II	−	+	−	+	−	−	−	−
STP298	patient	2012	MSSA	U	t934	sing	80	80		III	+	+	−	+	−	−	−	+

Each number refers to a different individual.

spa_CC, spa clonal complexes defined by BURP analysis; clonal assignment was performed based on the following default parameters: “exclude spa types that are shorter than five repeats” and “spa types are clustered if cost is less than or equal to 6.”

All isolates were tested for 11 virulence genes. Only virulence determinants that showed variance in presence/absence are listed. All the isolates were negative for lukM, eta, and etb genes; encoding leukocidin M; and exfoliative toxins A and B, respectively. lukF-lukS-PV, Panton-Valentine leukocidin genes; lukD-lukE, leukocidins D and E genes; hlb, β-hemolysin gene; hlg and hlgv, γ-hemolysin and γ-hemolysin variant genes; sel and sep, staphylococcal enterotoxins L and P genes; and etd, exfoliative toxin D-codifying gene.

MLST, multilocus sequence typing; ST, sequence type; CC, clonal complex, defined by eBurst v3 assessed on July 9, 2013. The previously accepted classification of CC5 and CC8, where ST5 and ST8 were considered the predicted founders, was maintained in the present classification; sing, singleton; na, no amplification was obtained for the agr system; +, gene presence; −, gene absence.

The MRSA isolates were distributed into three lineages only, associated to three clonal complexes (Table 2): CC8 (PFGE type B, t064/t451, ST8-IVg/V); CC88 (PFGE type E, t186/t786, ST88-IVa-agrIII), and CC5 (PFGE type K - K1, t105, ST5-IVa-agrII and PFGE K2, t002, ST105-II-agrII).

Clonal distribution was similar in the two screening periods, except for PFGE type H, which was exclusively found in 2010, and types F and G recovered in 2012 only. PFGE A1 and A2, belonging to the major clone found in the present study, were the only subtypes detected in both screening periods.

Concerning the different wards, high clonal type variability was found in orthopedics (n=9 clonal types), medicine (n=8), and pediatrics (n=5). Moreover, PFGE types A, B, C, and E were found in more than three different wards. No ward-specific clonal type was established, but MRSA was found in all but one ward screened.

Virulence factors

Among the entire collection, 98% of the isolates harbored two or more virulence factors, namely leukocidins and hemolysins. PVL was detected in 36% of the isolates (n=20), all MSSA, belonging to three prevalent clonal lineages and three minor clones (Table 2). All isolates other than PFGE types C, D, and J were positive for LukD-LukE and the γ-hemolysin variant. None of the isolates showed LukM, ETA, or ETB. ETD was present in the unique PFGE U-ST80 isolate.

Except for LukD-LukE and γ-hemolysin variant that were present in all MRSA, the MSSA isolates showed a higher prevalence and variability of virulence factors (Table 2). Interestingly, PFGE W1-t590-ST1 was positive for six out of the 11 virulent factors tested, including PVL. This isolate showed a nonmultiresistant pattern and co-colonized a patient with another PVL-positive isolate (PFGE D3-t355-ST152).

The occurrence of virulence factors was similar in both screening periods with the exception of enterotoxin L that decreased from 62.5% in 2010 to 29% in 2012 mainly due to the decline of clonal type PFGE A-ST15.

Concerning the accessory gene regulator (agr), almost half of the isolates (n=24, 43.6%) belonged to agr type I including five PFGE types (Table 2). agr types II and III included 25.4% and 16.4% of the isolates, respectively. agr type IV was restricted to PFGE G, ST121 isolates. Four isolates showed no amplification for the agr system. Although a unique situation of two different agr types was observed among PFGE E isolates, agr type was homogeneous inside each clonal lineage.

MRSA cross-transmission and S. aureus reservoirs

Three patients colonized with the same MRSA strain (PFGE B2) were identified in the burn unit and in two patients sharing another MRSA strain (PFGE B3) in the pediatric ward. No HCW colonized with related MRSA were detected in these wards and therefore no transmission route could be traced.

However, possible transmission routes could be traced for MSSA isolates in different wards: (1) PFGE type A1 was shared by a nurse-aid and one child in the pediatric ward; (2) highly related isolates were recovered from two HCW (subtype G1) and a child (subtype G2) in this same ward; and (3) a nurse and one patient in the medicine ward carried PFGE C1 and C5 isolates, respectively. All these cases were described in 2012, suggesting HCW as a possible reservoir of S. aureus in these hospital wards.

Discussion

This study presents the first report on S. aureus and MRSA nasal carriage in patients and HCW in São Tomé and Príncipe. The overall S. aureus nasal carriage rate ranged from 17% in 2010 to 14.9% in 2012. This value is less than half of the prevalence reported in 1997 in Cape Verde islands (41%), another PALOP country,¹ and slightly lower than in Mali (19.6%) and Gabon (29%), Central African countries geographically close to São Tomé and Príncipe.^1,7,48

Although previous studies on the African continent reported sporadic or nonexistence of MRSA carriage in Mali,⁴⁸ Tanzania,⁵⁷ and Cape Verde islands,¹ in São Tomé and Príncipe the MRSA prevalence of 4.2% in the entire population was similar to the 3.7% observed in Gabonese population.^7,50 On the other hand, the 26.9% methicillin resistance among S. aureus carriers was significantly higher than the 14.8% observed in nasal carriage among Malagasy community^44,45 or even the 16% reported in Nigeria.⁵⁴ Considering the two sampling periods, no significant difference was observed in the global occurrence of MRSA nasal carriage among patients and HCW as reported in a Portuguese hospital.⁶ However, the percentage of methicillin resistance was higher among S. aureus isolates recovered from patients (29.7% vs. 18.7%), probably due to antibiotic selective pressure.

The MRSA isolates described in the present study belonged to three major clonal complexes, CC8, CC88, and CC5. The major MRSA clonal lineage, ST8-t064/t451-IVg/V, shared a common genetic background with the community-acquired MRSA (CA-MRSA) ST8-IV, widely spread in the United States and Europe in colonization and infection,^28,47 but so far only occasionally reported in Africa.^7,10,54 Interestingly, the single ST8 isolates reported from Cameroon and Gabon, countries geographically close to São Tomé and Príncipe carried SCCmec type IV cassette, whereas in Nigeria, ST8-spa type t064/t451 carried exclusively type V.⁵⁴

The second most prevalent MRSA clonal type (ST88-IVa, spa types t186/t786) was found in both screening periods mainly among HCW, suggesting this lineage was endemic in the hospital. ST88-IV was reported as a major clone, associated with infection isolates in Madagascar, Morocco, Nigeria, Senegal, and Cameroon, in Gabon and in a single MRSA isolate in Mali.^10,48,50 Moreover, ST88 was reported as a minor MSSA clonal lineage in Nigeria and in a remote population of Babongo Pygmies.^21,49 Therefore, Africa seems to be a main reservoir of ST88 S. aureus genetic background, as already suggested by others but sporadically found elsewhere.^7,10

Two CC5 MRSA were found in São Tomé and Príncipe, ST105 (SLV of ST5)-II-t002 and ST5-IVa-t105, which are highly related to the New York/Japan (ST5-II) and the pediatric (ST5-VI or IV) epidemic clones, respectively. Although almost inexistent in Africa, ST105-II was previously found in Portugal, in colonization and infection isolates from the community and as the second major clone in a central hospital in Lisbon.^18,56 São Tomé and Príncipe has a direct demographic relationship with Portugal, namely concerning exchange of critically ill patients, and is a tropical touristic hot spot, and therefore the spread of MRSA isolates between these countries could not be excluded. However, in the present study we did not find any isolate belonging to the prevalent MRSA clonal type currently found in Portugal, EMRSA-15 (ST22-IV).^4,18

High genetic diversity was found among MSSA isolates, as previously found in other African countries.^1,7,9 Nevertheless, four major lineages could be identified: ST15, ST508/2446, ST152, and ST121.

ST15, a successful MSSA lineage worldwide commonly found in both carriage and disease, was reported as a major clone in Africa in Mali and Gabon.^7,48 In a multicentric study across Europe, ST15-t084 was the second most prevalent clone among invasive MSSA isolates.²³ ST508 has been reported in some African countries, usually as a minor penicillin-susceptible clone,^7,21,48 which was not the case in the present collection of São Tomé and Príncipe, where all isolates were penicillin resistant. Although ST152 has been responsible for sporadic cases of PVL-positive CA-MRSA infections in Europe and Australia⁴⁰ and recently reported in nasal carriage in the Balkan area,¹³ among African countries it has been associated not only with MSSA isolates, but also harboring PVL.^7,48,54 ST121-PVL positive, another major MSSA lineage in the present collection, is one of the most prevalent PVL-positive MSSA in infection across Europe.⁴⁶ In Africa, it has been described as a minor clone associated to nasal carriage or infection cases of otitis media, wound infections, and patients with tropical pyomyositis.^3,30,54

In São Tomé and Príncipe, 98% of the S. aureus isolates harbored at least two genes codifying virulence factors, namely leukocidins and hemolysins. Of major concern was the high prevalence (36%) of isolates carrying PVL. Similar values were found in other African countries, such as Cape Verde islands (35%), Mali (23.9%), and in a remote population of Gabonese Babongo Pygmies (55.9%).^3,48,49 Although in these studies none of the isolates harbored SCCmec, the spread of MRSA PVL-positive clones has been broadly reported in African hospitals.^10,43,50 The wide distribution of PVL-positive MSSA isolates in both carriage and infection confirms the African continent as a major reservoir of PVL-positive S. aureus isolates. Spread of virulent S. aureus out of Africa, including MRSA was evidenced in infected travelers returning from the tropics and subtropics to Europe.^24,51,55,60

In Hospital Dr. Ayres Menezes, São Tomé and Príncipe, trimethoprim-sulfamethoxazol, penicillins, and erythromycin are the most used antimicrobial agents, which explains the high resistance to these antibiotics observed in both MRSA and MSSA isolates. In addition, tetracycline resistance was also relevant in both collections, as recently described in Nigeria, which may be due to its wide clinical application, ease of administration and availability at low prices and over the counter.⁵⁴ Interestingly, seven oxacillin-susceptible MRSA (OS-MRSA) isolates were detected in the present collection. This situation has been recently reported among clinical isolates in India, Japan, and Greece^25,29,41 bringing new challenges to the laboratory identification and empirical antibiotherapy. OS-MRSA are frequently misreported as MSSA, leading to empirical treatments with beta-lactam antibiotics promoting potentially life-threatening consequences for patients.

HCW are known to be major reservoirs of S. aureus.^5,59 In São Tomé and Príncipe, we described three cases of highly related MSSA isolates colonizing nurses or nurse-aids and patients in the same wards. In addition, although no transmission route could be traced, patients colonized with a same MRSA strain were found in different wards, suggesting cross-transmission. Nurses and nurse-aids are the HCW categories with closer contact with patients, and therefore representing higher risk of S. aureus cross-transmission.⁶ The World Health Organization claims that hand hygiene among HCW is the leading measure to prevent the spread of health care-associated infections. Moreover, the introduction of prevention programs based on hand hygiene in some African countries has been successful.⁸ However, the nonexistence of lavatories inside the rooms in the hospital in São Tomé and Príncipe, and the irregular running water availability combined with the scarce usage of alcohol solutions for hand rub, may compromise the adequacy of this prevention programs and explain cross-transmission between HCW and patients.

This study had some limitations, namely the small number of HCW included in the first screening (2010), which could explain the nondetection of S. aureus cross-transmission links in this period. The identification of risk factors for MRSA carriage in this population was not possible due to the scarce demographic data available. Moreover, future studies including infection isolates will be of major interest.

In conclusion, we described for the first time S. aureus and MRSA nasal carriage rates in the nosocomial setting in São Tomé and Príncipe, which were not negligible and showed an increasing trend among patients. S. aureus clones circulating in this hospital harbored a high prevalence of virulence factors, namely PVL. The evidence of cross-transmission between HCW and patients indicate the need of implementation of infection control measures adequate to this African reality.

Footnotes

Acknowledgments

This work was partially supported by project PTDC/SAU-SAP/118813/2010 and grant No. PEst-OE/EQB/LA0004/2011 from Fundação para a Ciência e a Tecnologia (FCT), Portugal. T. Conceição was supported by grant SFRH/BPD/72422/2010 from FCT, Portugal.

We are grateful to the health care workers at Hospital Dr. Ayres Menezes for the given assistance during the screenings.

Disclosure Statement

The authors have declared that no competing interests exist.

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