MICROBIAL DRUG RESISTANCE Volume 20, Number 5, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2014.0007
Epidemiology of Methicillin-Resistant and -Susceptible Staphylococcus aureus in Luanda, Angola: First Description of the Spread of the MRSA ST5-IVa Clone in the African Continent Teresa Conceic¸a˜o,1 Ce´line Coelho,1 Isabel Santos-Silva,2 Hermı´nia de Lencastre,1,3 and Marta Aires-de-Sousa 2
Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen worldwide, and although surveillance studies are available in the most developed countries, data from Angola are inexistent. In June 2012, 295 inpatients and 199 healthcare workers from three hospitals in Luanda, Angola were nasal swabbed for S. aureus and MRSA carriage. A total of 117 individuals (23.7%) were S. aureus nasal carriers, out of which 68 (58.1%) were colonized with MRSA. The majority of the MRSA isolates (74%) belonged to a single clonal lineage, pulsed-field gel electrophoresis (PFGE) A-ST5-IVa associated with three spa types (spa types t105/ t311/t11657), followed by PFGE C-ST88-IVa (spa types t186/t325/t786/t1951/t3869) (n = 9; 12%); the other 11 MRSA isolates were representatives of 4 additional lineages. Almost half (49%) of the methicillin-susceptible S. aureus (MSSA) isolates belonged to three major clones: PFGE B-ST508 (spa types t050/t861/t1346/t1574/ t2626/t12218), PFGE D-ST45 (spa types t939/t11656), and PFGE E-ST30 (spa types t1202/t9118). MSSA isolates presented a high variability of virulence factors, including Panton-Valentine leukocidine (7.9%). MRSA carriage in Luanda is considerably high, and the major clone corresponds to a worldwide epidemic lineage, so far scarcely reported in Africa. Additional infection control measures in this metropolis are mandatory for a global MRSA control.
Introduction
M
ethicillin-resistant Staphylococcus aureus (MRSA) is a major cause of infection worldwide in both hospitals and community, leading to increased morbidity, mortality, length of hospital stay, and costs.8 Nasal carriage is a major risk factor for infection, since colonizing strains may serve as endogenous reservoirs for subsequent clinical infections, or may spread in the nosocomial setting by means of indirect patient-to-patient transmission, by direct transmission from colonized healthcare workers (HCW), or through contaminated environmental surfaces.5,54 Surveillance systems are key elements to prevent MRSA transmission and infection that receive substantial funding in the most developed countries, but are scarce or almost inexistent in resource-poor regions. However, populations in African countries are at high risk for MRSA infections due to reduced
financial resources, poor hygienic conditions, overcrowding, and understaffing that result in a lack of infection control practices.7 MRSA surveillance data from Africa are sporadic, but a few recent studies highlight a considerable diversity among MRSA nasal carriage rates: sporadic isolates in Mali,46 1.3% in Ghana,21 3.7% in Gabon,48 4.2% in healthy children under 5 years in Tanzania,39 14.8% in Madagascar,44,45 12.7% in HCW, 23% among children and prisoners in Ethiopia,32,49 21% in tuberculosis patients in KwaZuluNatal, South Africa,30 or 52% among HCW and patients in surgical units of Mulago hospital, Uganda.31 In Portuguese-speaking African countries (PALOP), former colonies with close relationships with Portugal, the MRSA prevalence was reported as null in two hospitals in Cape Verde islands in a study dated from 19971 and 4.2% nasal carriage among patients and HCW in Sa˜o Tome´ and Prı´ncipe between
1 Laboratory of Molecular Genetics, Instituto de Tecnologia Quı´mica e Biolo´gica (ITQB), Universidade Nova de Lisboa (UNL), Oeiras, Portugal. 2 Escola Superior de Sau´de da Cruz Vermelha Portuguesa (ESSCVP), Lisboa, Portugal. 3 Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, New York.
441
˜ O ET AL. CONCEICxA
442
2010 and 2012.16 In Mozambique, between 2001 and 2006, 8% of the S. aureus recovered from blood of children < 15 years old in a single hospital were methicillin resistant.35 Moreover, a significant prevalence of the highly potent PantonValentine leukocidine (PVL) was found among methicillinsusceptible S. aureus (MSSA) isolates in Sa˜o Tome´ and Prı´ncipe (36%) and Cape Verde (35%).3,16 The description of MRSA clonal lineages in Sa˜o Tome´ and Prı´ncipe that had been previously reported in Portuguese hospitals and in the community (ST5, ST8, and ST88) suggests that the close relationships between Portugal and PALOP nations may influence the MRSA clonal types circulating in the European countries.4,15 Data from Angola are inexistent. The aim of the present study was to evaluate the MRSA and MSSA nasal carriage among patients and HCW in different hospitals in Luanda, the capital of Angola, and characterize its population structure to identify eventual reservoirs in a city that has important demographic and economic exchanges with Portugal. Materials and Methods Hospital setting
Angola is a former Portuguese colony, situated in the West Coast of Africa. It is the seventh largest African country with 1,246,620 km2 and a population of 19,183,590 ( July, 2013) individuals with a mean age of 17.7 years.11 Hospital Ame´rico Boavida (HAB) and Hospital Pedia´trico David Bernardino (HPDB) are the major public hospitals in the capital city, Luanda. HAB is a large hospital with 624 beds constructed in 1968, with different internment services, namely, medicine, pediatrics, orthopedics, surgery, infectious diseases, urology, cardiology, and a 17-bed intensive care unit (ICU). This hospital serves an outpatient population of *2,500,000 inhabitants. HPDB is a national reference medium-sized pediatric hospital (350 beds) constructed in 1958 with three major internment services, namely, pediatrics, surgery, infectious diseases, and a 4-bed ICU. The hospital has also various ambulatory services, namely, pediatrics, surgery, orthopedics, cardiology, pneumology, ophthalmology, otorhinolaryngology, and an emergency ward. Clı´nica Sagrada Esperanc¸a (SE) is a 150-bed private clinic created in 1991 that serves an outpatient population of 500,000 inhabitants. The clinic has several internment services, namely, medicine, pediatrics, orthopedics, surgery, infectious diseases, and a nine-bed ICU in addition to several isolation rooms, individual rooms, and some ambulatory services. The emergency ward receives 80 entrances per day. Nasal screening and bacterial isolates
The inpatients and HCW from the three hospitals were nasal swabbed for S. aureus carriage. Exclusion criteria included patients admitted for < 48 hr, 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. The institutional ethics committee approved the protocol and an oral informed consent was obtained at the time of screening for each participant. Sampling was performed by a trained nurse during a 5day period in June 2012, in the main services where the risk for S. aureus infection is usually high (medicine, surgery,
pediatrics, infectious diseases, orthopedics, isolation rooms, and intermediate and ICUs), and in the cardiology, pulmonology, urology, hemodynamics, suite rooms, and emergency wards. Samples from HCW were additionally recovered from the pharmacy and sterilization department. Samples were taken by swabbing both nares of each individual with a sterile cotton swab, which was stored in the Stuart transport medium. Swabs were processed within 10 days at the Laboratory of Molecular Genetics, Instituto de Tecnologia Quı´mica e Biolo´gica in Portugal. In brief, each swab was inoculated on a rich medium, Tryptic soy agar (Becton, Dickinson & Co, Franklin Lakes, NJ) and on a selective chromogenic medium Chromagar Staph aureus (ChromAgar, Paris, France). All presumptive S. aureus colonies were tested for coagulase by latex agglutination test Staphaurex (Remel, Lenexa, KS) 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.43 Antimicrobial susceptibility testing and mecA detection
Antimicrobial susceptibility testing was performed on all isolates by the disk diffusion method for a panel of 15 antibiotics: penicillin, oxacillin, erythromycin, gentamicin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, rifampin, tetracycline, fusidic acid, teicoplanin, vancomycin, linezolid, and quinupristin-dalfopristin. Breakpoints were defined according to the current Clinical Laboratory Standards Institute (CLSI) guidelines,13 except for fusidic acid that was defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.eucast .org/). Strain S. aureus ATCC25923 was used as quality control. The presence of the mecA gene was tested on all S. aureus isolates by PCR.41 Oxacillin and vancomycin minimum inhibitory concentrations were determined using E-test strips according to the manufacturer’s instructions (Biomerieux, Marcy l’Etoile, France) for oxacillin-susceptible but mecApositive isolates and for all MRSA, respectively. Molecular typing Pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) was performed on all S. aureus isolates as described by Chung et al.12 The resulting SmaI restriction band profiles were analyzed by visual inspection using the criteria of McDougal et al.,36 followed by automated analysis with the BioNumerics software version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) for relatedness evaluation to define clusters. Dendrograms were generated as previously described24 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 cluster definition, respectively. spa typing and multilocus sequence typing. spa typing was performed on at least one representative of each PFGE subtype, and spa types were assigned through the Ridom web server (http://spaserver.ridom.de).2 Multilocus sequence typing (MLST) was conducted as described17,22 for representative isolates of the different PFGE types, previously characterized by spa typing. Allelic profiles and sequence types (ST) were defined using the MLST online database (www.mlst.net).
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
443
MLST clonal complexes were defined using the eBURST algorithm and restricted to single locus variant (SLV) and double locus variant from each group founder or subfounder, inside each clonal group. SCCmec typing. SCCmec was characterized by multiplex PCR in all MRSA isolates.38 Additionally, SCCmec type IV isolates were further subtyped by multiplex PCR, as described,37 after primer concentration adjustment: 0.3 mM for primer ccrB1; 0.7 mM for primer ccrB2; 1 mM for primers J IVg F and J IVg R; and 1.9 mM for primers J IVh F and J IVh R. Detection of virulence determinants and agr type
The presence of 11 specific staphylococcal virulence genes, including 3 leukocidins (lukS-lukF, lukE-lukD, and lukM), 3 hemolysins (hlb, hlg, and hlgv), and 5 superantigenic toxins (eta, etb, etd, sel, and sep), was determined by PCR for all S. aureus isolates as previously described.53 All S. aureus isolates belonging to ST8 or PVL + were screened for the presence of the arginine catabolic mobile element (ACME) by PCR amplification of arcA and opp3.19,20 Subtypes of the accessory gene regulator (agr I–IV) were determined for the entire collection by multiplex PCR.27 Statistical analysis
S. aureus and MRSA carriage differences were verified by the Pearson’s Chi-square test. All statistics were calculated using the SPSS software package version 21 (SPSS, Inc., Chicago, IL), and p-values of £ 0.05 were considered as statistically significant. Results
The patients colonized with MRSA were predominantly male (57.8%), half were < 18 years old (50%) and 57.8% were hospitalized for < 15 days (internment period ranged between 3 and 98 days). The leading internment causes were related to bone fractures (13%), malaria (11%), liver diseases (6.7%), and neoplasms (6.7%). Moreover, 82.2% of the patients had been taking antibiotics at the time of screening or in the previous month, namely, penicillins (86.5%), imidazole (40.5%), and aminoglycosides (18.9%), and 24.2% underwent surgery within the month preceding the screening. Comparing the colonization rates in the three hospitals, the global S. aureus carriage was considerably lower in HPDB (18.4% vs. 26.2/26.9%), while the MRSA carriage was significantly higher in HAB (17.6% vs. 10.7/11.7%)— Table 1. Moreover, the percentage of methicillin-resistant isolates among S. aureus was considerably inferior in the private clinic compared with the two public hospitals. Despite some intrahospital variability, the global prevalence of MRSA was similar between HCW and patients (Table 1). However, patients in public hospitals showed higher MRSA carriage rates (HAB: 18.9%, HPDB: 16.6%), compared with patients in the private clinic (SE: 4.8%) ( p = 0.030). No major differences were observed in both S. aureus and MRSA carriage rates among the different HCW categories (Table 1). Among 19 different wards included in the study, regardless of the hospital, the highest MRSA frequency was observed in the pediatric special care unit (25%, n = 6), orthopedics (23.8%, n = 5), and cardiology (21.7%, n = 5). Among the 117 nasal carriers, 7 patients and 4 HCWs were cocolonized with two different isolates, resulting in a total of 128 S. aureus (77 MRSA and 51 MSSA) recovered that were further characterized.
Prevalence of S. aureus and MRSA nasal colonization
Among the 494 individuals screened, 117 were S. aureus nasal carriers (23.7%). Of these, 68 (45 inpatients and 23 HCW) were colonized with MRSA indicating an extremely high (58.1%) prevalence of methicillin resistance among the S. aureus carriers.
Genetic diversity
The molecular characterization of the 128 S. aureus clustered the isolates into 22 clonal types (defined by PFGE): MRSA were classified in 6 types and the MSSA in 17 with one type common to both MRSA and MSSA (Table 2).
Table 1. Staphylococcus aureus and MRSA Nasal Carriage in Different Hospitals in Angola Individual characteristics Hospital SE-private HAB-public HPDB-public Individual Patients HCW HCW Physicians Nurses Othersa
S. aureus carriers n (%)
p-Value
% of methicillin resistance among S. aureus
p-Value
13 (10.7) 34 (17.6) 21 (11.7)
0.251 0.047 0.323
40.6 65.4 63.6
0.019 0.154 0.449
0.373
45 (15.2) 23 (11.6)
0.242
60.8 53.5
0.439
0.405 0.579 0.552
6 (13.0) 12 (12.3) 5 (9.4)
0.741 0.804 0.552
75.0 52.2 41.7
0.176 0.853 0.334
p-Value
MRSA carriers n (%)
32 (26.2) 52 (26.9) 33 (18.4)
0.446 0.172 0.038
74 (25.0) 43 (21.6) 8 (17.4) 23 (23.5) 12 (22.6)
a Includes: nurse’s aides, healthcare technicians, students, administrative personnel, cleaning staff, cooks, social workers, and pharmacists. Significant p-values are in bold. MRSA, methicillin-resistant S. aureus; HCW, healthcare worker; HAB, Hospital Ame´rico Boavida; HPDB, Hospital Pedia´trico David Bernardino; SE, Sagrada Esperanc¸a clinic.
˜ O ET AL. CONCEICxA
444
Table 2. Molecular Characterization of the 77 MRSA and 51 MSSA Isolates Recovered in Angola in June 2012 MR/ MSSA
PFGE No. type (No. of subtypes) isolates
Hospital
Type of individual
spa type
MRSA
A C J M G T
(13) (7) (4) (3) (2) (1)
57 9 5 3 2 1
HAB, HPDB, SE HAB, HPDB HAB, HPDB HPDB, SE SE SE
P/HCW P/HCW P/HCW HCW HCW HCW
t105/t311/t11657 t186/t325/t786/t1951/t3869 t104/t1774 t3092 t6065 t091
MSSA
B D E F A L H N R P J K Q S V W Z
(8) (5) (5) (3) (3) (2) (2) (2) (2) (2) (1) (1) (1) (1) (1) (1) (1)
11 7 7 4 3 3 2 2 2 3 1 1 1 1 1 1 1
HAB, HPDB, HAB, HPDB, HAB, HPDB, HPDB, SE HAB, HPDB, HAB, SE SE HAB, SE HPDB SE HAB SE HPDB SE HAB HAB HAB
P/HCW P/HCW P/HCW P/HCW P/HCW P/HCW P P HCW P P P HCW P P P P
t050/t861/t1346/t1574/t2626/t12218 t939/t11656 t1202/t9118 t349/t1350/t6205 t002/t1215/t6071 t148/t1346 t008/t1476 t084/t346 t1458/t12259 t314/t1077 t064 t693 t1476 t335 t12219 t5187 t957
SE SE SE SE
ST 5 88 8 72 5/2629 789 508 45 30 25 5 72 8 15 2367 121 8 1 2229 152 672 2728 601
SCCmec agr type type IVa IVa IVd/V V V V
II III I I II I I IV III/na I II I I II III IV I III I I I III II
PVL +/+/+/+/+/+ -
HDB, Hospital David Bernardino; P, patient; na, no amplification; PVL, Panton-Valentine leukocidine; ST, sequence type; MSSA, methicillin-susceptible S. aureus; PFGE, pulsed-field gel electrophoresis.
The overwhelming majority of the MRSA isolates (74%, n = 57) belonged to PFGE type A, spa types t105/t311/t11657, ST5, SCCmec IVa, agrII, highly related to the pandemic pediatric clone. Clonal type PFGE C, spa types t186/t325/t786/ t1951/t3869, ST88-IVa-agrIII represented 12% of the isolates (n = 9), while the remaining MRSA belonged to three minor clones and one single isolate (Table 2). Regarding the MSSA collection, almost half of the isolates (49%, n = 25) were distributed into three major clones: PFGE B, spa types t050/t861/t1574/t2626/t1346/t12218, ST508 (SLV of ST45)-agrI (n = 11); PFGE D, spa types t939/t11656, ST45-agrIV (n = 7); and PFGE E, spa types t1202/t9118, ST30-agrIII (n = 7). The remaining MSSA belonged to seven minor clones; seven other MSSA were represented by single isolates only (Table 2). Interestingly, although PFGE A was a major MRSA clonal lineage, it was sporadically found among MSSA isolates and associated to distinct spa types (Table 2). Distribution of clonal types among hospitals and populations
The major MRSA clonal type PFGE A-ST5-IVa was the only MRSA lineage found in the three hospitals and was detected in all wards screened. Two MRSA lineages (PFGE C-ST88-IVa and J-ST8-IVd/V) were exclusively found in public hospitals (HAB and HPDB), while one minor clone (PFGE G-ST5/ST2629-V) and one singleton (T-ST789-V) were detected in the private clinic SE only (Table 2). Concerning the MRSA distribution among patients and HCW, the overwhelming majority of the MRSA carriers (patients: 82.2%; HCW: 60.9%) were colonized with PFGE
A-ST5-IVa isolates. PFGE types M, G, and T were found exclusively among HCW (Table 2), namely, nurses and physicians. All HCW other than physicians and nurses who were MRSA carriers were exclusively colonized with PFGE A isolates. Although MSSA isolates showed a high genetic variability, the three more prevalent lineages (PFGE B, D, and E) were found in all three hospitals widely spread among patients and HCW (Table 2). Nurses showed the highest MSSA variability, being colonized with six different lineages (PFGE B: 27.3% of all nurse carriers; PFGE A: 18.2%; PFGE D and L: 18.2%; PFGE E: 9.1%; and PFGE F: 9.1%). Antimicrobial susceptibility
All 128 S. aureus isolates were susceptible to fusidic acid, quinupristin-dalfopristin, linezolid, and vancomycin (MIC values between 1.5 and 4 mg/L for MRSA isolates). Among the MSSA isolates, 92% showed resistance to penicillin, 59% to rifampin, 25% to trimethoprim-sulfamethoxazole, 18% to tetracycline, 4% to erythromycin, and only one isolate was resistant to ciprofloxacin and gentamicin. A single MSSA isolate showed induced resistance to clindamycin. Higher resistant phenotypes were found among the 77 MRSA. All isolates were resistant to penicillin, cefoxitin, and trimethoprim-sulfamethoxazol. In addition, 87% of the MRSA isolates were resistant to rifampin, 30% to gentamicin, 14% to chloramphenicol, 12% to ciprofloxacin, 9% to tetracycline, and 8% to erythromycin. Interestingly, 8 of the 77 MRSA isolates (belonging to ST5-V, ST88-IVa, or ST2629-V) that previously amplified the mecA gene,
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
445
FIG. 1. Distribution of the 11 virulence determinants among methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) clonal lineages. Percentages (%) of positive isolates are relative to the total number of isolates regarding each clonal lineage. Pulsed-field gel electrophoresis (PFGE) types are ordered according to the number of isolates in each type, in a descending trend from left to right. showed a susceptible or intermediate phenotype to oxacillin by the disk diffusion method, but an oxacillin MIC ranging between 0.75 and 2 mg/L, and were therefore considered as MRSA with a heterogeneous resistance phenotype. Virulence factors
Among the entire S. aureus collection, 66.4% of the isolates harbored two or more virulence factors, namely, leukocidins and hemolysins (Fig. 1). Although the MSSA isolates showed a higher variability of virulence factors, LukD-LukE and g-hemolysin variant were the most prevalent virulence determinants detected in both MRSA and MSSA (68.4% and 43% of the isolates for both toxins, respectively) (Fig. 1). None of the isolates showed LukM or ETB. PVL was detected in 10 isolates (7.9%), belonging to six different clonal types, all MSSA (Table 2 and Fig. 1). Six of the PVL + isolates were isolated in the private clinic SE. ETA and ETD were restricted to MRSA PFGE C-ST88-IVa and MSSA PFGE F-ST25 isolates, respectively (Fig. 1). MRSA PFGE M-ST72-V clonal type was the only lineage negative to all toxin determinants tested. Interestingly,
MSSA PFGE L-ST72 clonal type, although belonging to the same ST72, included the isolates with the highest virulence content, harboring 5 of the 11 toxins tested, but with a low antimicrobial resistance profile (resistance to penicillin only, and a single isolate resistant to rifampin). Concerning the accessory gene regulator (agr), half of the isolates (n = 66, 51.6%) belonged to agr type II, including four clonal types (Table 2). agr type I was the second most prevalent type (n = 33, 25.8%) and included the greatest clonal variability, followed by agr type III (n = 19, 14.8%). agr type IV was found in nine MSSA isolates (7%) associated to ST45 and ST121. One isolate showed no amplification for the agr locus and was considered as agr negative. All S. aureus isolates PVL + or belonging to ST8 (PFGE types H, J, or T) were screened for the presence of ACME, but none of the isolates harbored this element. MRSA cross-transmission
MRSA cross-transmission was detected in public hospitals only (Fig. 2). In HAB, isolates with the same PFGE types were shared between patients and between patients and HCW in different wards. In HPDB, cross-transmission
FIG. 2. MRSA cross-transmission between patients and between patients and healthcare workers (HCW) in different wards of the two public hospitals.
446
was found between patients only, in the pediatric special care and in the intermediate care units. No HCW colonized with related MRSA isolates were detected in these wards and, therefore, no transmission routes could be traced. Discussion
In the present study, we provide for the first time, data on the carriage rates and population structure of S. aureus and MRSA among patients and HCW in Angola, a major African country with close demographic relationships with Europe, namely with Portugal. Although the overall S. aureus nasal carriage rate observed in Angola (23.7%) parallels the rates described for other African countries, we found an extremely high (58.1%) prevalence of methicillin resistance among S. aureus isolates. Among the few reports available focusing on MRSA nasal carriage in African countries, this is the highest value, only comparable to rates reported in Ethiopia (44.1%) and Morocco (44.4%).49,56 In Sa˜o Tome´ and Prı´ncipe, another PALOP country, methicillin resistance among S. aureus was 26.9%.16 The higher availability and consumption of antibiotics in the hospitals in Luanda compared with other PALOP nations may explain the higher methicillin resistance in Angola. Angola has close demographic relationships with Portugal, which is currently together with Romania, the only European country that shows an MRSA prevalence above 50%.23 No significant difference was observed in the global occurrence of MRSA nasal carriage regarding patients and HCW in Luanda as reported in Sa˜o Tome´ and Prı´ncipe.16 In contrast, in Ghana and Gabon, none of the HCW screened were MRSA carriers.6,21 However, public hospitals showed higher levels of methicillin resistance among S. aureus compared to the private clinic, which may indicate that infection control policies are more effective in private and smaller hospitals. High antimicrobial resistance rates were observed for penicillin and trimethoprim-sulfamethoxazole in both MRSA and MSSA isolated in Angola, which is in agreement with reports from Sa˜o Tome´ and Prı´ncipe, Gabon, and Nigeria.6,16,26 This is not surprising given the widespread use of trimethoprim-sulfamethoxazole in Africa recommended by the World Health Organization to prevent opportunistic bacterial infections in patients with HIV/ AIDS, malaria, and suffering from malnutrition. Furthermore, and in contrast to other African countries, we found an unexpectedly high resistance to rifampin and gentamicin,1,16,21,26 which may be explained by the fact that rifampin is a first line antituberculosis drug and gentamicin is one of the mostly used antibiotics in the pediatric HPDB hospital. In our study, 27% of the patients screened had taken recently at least one of the three antibiotics (gentamicin, trimethoprim-sulfamethoxazole, or rifampin), and all MRSA recovered from these patients showed resistance to the referred antibiotics. Moreover, the resistance to chloramphenicol and ciprofloxacin among MRSA may be explained by the fact that chloramphenicol is one of the principal drugs used in HPDB and ciprofloxacin is a first line drug to treat MRSA infections in HAB. Therefore, an antimicrobial management program to optimize antimicrobial use is recommended in the participating Angolan hospitals.
˜ O ET AL. CONCEICxA
In Angola, both HCW and patients were mainly colonized with isolates belonging to ST5 lineage (SCCmec IVa, spa types t105 and t311), related to the epidemic Pediatric MRSA clone. This clone was isolated for the first time in 1992 in a Portuguese Pediatric hospital,47 and although no longer present in that hospital in 2006, one year later it was reported as the second major clonal lineage in the Portuguese Azores islands as well as in sporadic isolates in the mainland.4,14,42 The Pediatric MRSA clone has also been detected in other European countries, Poland, and France,18 but was mainly spread in South American countries, namely in Argentina and Colombia, and in the United States.28,33,36,47 The dissemination of epidemic MRSA isolates ST5-IV-PVL + was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003–2008)51 and was also responsible for infection cases in two African cities, Casablanca and Dakar.10 In recent reports, the Pediatric MRSA clonal lineage has been restricted to infection isolates, but was reported for the first time in nasal carriage in PALOP countries, as a minor clone in Sa˜o Tome´ and Prı´ncipe,16 and now as the prevalent clonal lineage among children and adults in the three Angolan hospitals. Since infection isolates were not included in this study, we could not assess to what extent this MRSA clonal lineage was also responsible for infection in these hospitals. The ST5 lineage is known to frequently acquire mobile genetic elements, such as different SCCmec elements, antibiotic resistance and toxin genes.40 Various SCCmec acquisition events occurred within the ST5 lineage, on multiple and independent occasions, evidencing that MRSA emerged in numerous circumstances and in distinct locations rather than exclusively by geographic dispersal of isolates.40 Moreover, description of local emergence of ST5PVL + lineages, as well as the emergence of multiresistant isolates corroborate this idea.14,28,51 Although SCCmec IVa, highly prevalent in African countries, might have been introduced de novo in MSSA ST5 isolates found in Angolan hospitals, the spread of this lineage from abroad, namely from Portugal, could not be excluded, since Angolan children frequently travel to Portugal to meet pediatricians or even to be admitted to the hospitals for critical medical treatments. Moreover, although not currently prevalent in Portuguese hospitals,25 ST5-IVa isolates sharing the same spa types t002 and t105 with isolates from our collection were recently reported in the community in public buses in Lisbon and Oporto.15,50 On the other hand, the description of ST5-I isolates in South African hospitals40 suggests that a local exchange of SCCmec cassette type I to type IVa could also explain the emergence of ST5-IVa clonal lineage in Angola. However, genome sequencing is needed to corroborate this hypothesis. ST88-IVa, the second major clonal lineage in Luanda, was a major MRSA lineage in Sa˜o Tome´ and Prı´ncipe and in other African countries, confirming its endemicity in the African continent.6,10,16,26,46 Among the other MRSA, ST8 was already reported in other African countries, namely as a major clone in Sa˜o Tome´ and Prı´ncipe and Nigeria.6,10,16,21,26 Although the MSSA population showed a higher variability as previously found in other African countries,1,6,9 half of the isolates were distributed into three major clonal lineages related to ST508, ST45, and ST30. ST508 has been reported in some African countries, being the second major
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
MSSA clone in Gabon and Sa˜o Tome´ and Prı´ncipe.6,16,26,46 Although sporadically found in Africa, ST45 and ST30 MSSA were widely spread in Europe, namely among the MSSA population in Portugal, where ST30 is endemic since 1992 and currently the predominant clonal lineage.25,29,52 Since these clonal lineages share the same genetic background as the major international MRSA epidemic clones, the Berlin clone (ST45-IV) and the community-associated Southwest Pacific clone (ST30-IV), these MSSA could represent a suitable reservoir for the acquisition of the highly prevalent SCCmec type IV in Angola. The African continent has been reported as a major reservoir of PVL + MSSA isolates in both carriage and infection.3,16,34,46,48 However, in Angola, only 7.9% of the isolates (all MSSA) carried PVL, and except three ST30 isolates, all the other PVL + belong to minor MSSA clones, which suggests that the spread of PVL in African countries is MSSA clonal related. It is well known that HCW who are nasal carriers play an important role in S. aureus transmission.5,55 In Angola, MRSA cross-transmission between HCW and patients was observed in the public hospital HAB, and the patients colonized with a same MRSA strain were found in different wards, in both public hospitals. In the private SE clinic, no cross-transmission events were detected, probably due to a low occupation rate in each ward, which contrasts to public hospitals. In conclusion, the MRSA nasal carriage rate in the nosocomial setting in Luanda, Angola, is extremely high in contrast with the much lower rates recently reported in another PALOP country, Sa˜o Tome´ and Prı´ncipe. Moreover, we described a massive spread of the MRSA Pediatric clone (ST5-IVa), a worldwide epidemic lineage scarcely reported in Africa so far. Antimicrobial resistance levels and MRSA cross-transmission within the hospitals suggest the need for implementation of additional infection control measures for a global MRSA control.
447
3.
4.
5. 6.
7. 8. 9.
10.
11.
Acknowledgments
This work was supported by project PTDC/SAU-SAP/ 118813/2010 and grant no. PEst-OE/EQB/LA0004/2011 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal. T.C. and C.C. were supported by grants SFRH/BPD/ 72422/2010 and 036/BI-BI/2012 from FCT, Portugal. The authors are grateful to the healthcare workers from Hospital Ame´rico Boavida, Hospital Pedia´trico David Bernardino, and Clı´nica Sagrada Esperanc¸a for the assistance during the screenings and to Ana Delgado for the statistical analysis.
12.
Disclosure Statement
14.
The authors interests exist.
have
declared
that
no
13.
competing
References
1. Aires-de-Sousa, M., I. Santos Sanches, M.L. Ferro, and H. de Lencastre. 2000. Epidemiological study of staphylococcal colonization and cross-infection in two West African Hospitals. Microb. Drug Resist. 6:133–141. 2. Aires-de-Sousa, M., K. Boye, H. de Lencastre, A. Deplano, M.C. Enright, J. Etienne, et al. 2006. High inter-
15.
16.
laboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. J. Clin. Microbiol. 44:619–621. Aires-de-Sousa, M., T. Conceic¸a˜o, and H. de Lencastre. 2006. Unusually high prevalence of nosocomial PantonValentine leukocidin-positive Staphylococcus aureus isolates in Cape Verde Islands. J. Clin. Microbiol. 44: 3790–3793. Aires-de-Sousa, M., B. Correia, and H. de Lencastre. 2008. Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period. J. Clin. Microbiol. 46:2912–2917. Albrich, W.C., and S. Harbarth. 2008. Health-care workers: source, vector, or victim of MRSA? Lancet Infect. Dis. 8:289–301. Ateba Ngoa, U., F. Schaumburg, A.A. Adegnika, K. Kosters, T. Moller, J.F. Fernandes, et al. 2012. Epidemiology and population structure of Staphylococcus aureus in various population groups from a rural and semi urban area in Gabon, Central Africa. Acta Trop. 124:42–47. Bagheri Nejad, S., B. Allegranzi, S.B. Syed, B. Ellis, and D. Pittet. 2011. Health-care-associated infection in Africa: a systematic review. Bull. World Health Organ 89:757–765. Boucher, H.W., and G.R. Corey. 2008. Epidemiology of methicillin-resistant Staphylococcus aureus. Clin. Infect. Dis. 46 Suppl 5:S344–S349. Breurec, S., C. Fall, R. Pouillot, P. Boisier, S. Brisse, F. Diene-Sarr, et al. 2011. Epidemiology of methicillin-susceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes. Clin. Microbiol. Infect. 17:633–639. Breurec, S., S.B. Zriouil, C. Fall, P. Boisier, S. Brisse, S. Djibo, et al. 2010. Epidemiology of methicillin-resistant Staphylococcus aureus lineages in five major African towns: emergence and spread of atypical clones. Clin. Microbiol. Infect. 17:160–165. Central Intelligence Agency. 2013. The World Factbook. Available at www.cia.gov/library/publications/the-worldfactbook/geos/tp.html. (Accessed November 25, 2013). (Online.) Chung, M., H. de Lencastre, P. Matthews, A. Tomasz, I. Adamsson, M. Aires-de-Sousa, et al. 2000. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb. Drug Resist. 6:189–198. [CLSI] Clinical Laboratory Standards Institute. 2009. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement M100–S19. Wayne, Pennsylvania. Conceic¸a˜o, T., A. Tavares, M. Miragaia, K. Hyde, M. Aires-de-Sousa, and H. de Lencastre. 2010. Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI. Eur. J. Clin. Microbiol. Infect. Dis. 29:543–550. Conceic¸a˜o, T., F. Diamantino, C. Coelho, H. de Lencastre, and M. Aires-de-Sousa. 2013. Contamination of public buses with MRSA in Lisbon, Portugal: a possible transmission route of major MRSA clones within the community. PLoS One 8:e77812. Conceic¸a˜o, T., I.S. Silva, H. de Lencastre, and M. Airesde-Sousa. 2013. Staphylococcus aureus nasal carriage
˜ O ET AL. CONCEICxA
448
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
among patients and health care workers in Sa˜o Tome´ and Prı´ncipe. Microb. Drug Resist. 20:57–66. Criso´stomo, M.I., H. Westh, A. Tomasz, M. Chung, D.C. Oliveira, and H. de Lencastre. 2001. The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones. Proc. Natl. Acad. Sci. U.S.A. 98:9865–9870. Dauwalder, O., G. Lina, G. Durand, M. Bes, H. Meugnier, V. Jarlier, et al. 2008. Epidemiology of invasive methicillin-resistant Staphylococcus aureus clones collected in France in 2006 and 2007. J. Clin. Microbiol. 46:3454–3458. Diep, B.A., S.R. Gill, R.F. Chang, T.H. Phan, J.H. Chen, M.G. Davidson, et al. 2006. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet 367:731– 739. Diep, B.A., G.G. Stone, L. Basuino, C.J. Graber, A. Miller, S.A. des Etages, et al. 2008. The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus. J. Infect. Dis. 197:1523–1530. Egyir, B., L. Guardabassi, S.S. Nielsen, J. Larsen, K.K. Addo, M.J. Newman, et al. 2013. Prevalence of nasal carriage and diversity of Staphylococcus aureus among inpatients and hospital staff at Korle Bu Teaching Hospital, Ghana. J. Global Antimicrob. Resist. 1:189–193. Enright, M.C., N.P. Day, C.E. Davies, S.J. Peacock, and B.G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillinsusceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008–1015. European Antimicrobial Resistance Surveillance System. 2013. EARSS annual report 2012. Available at www.ecdc.europa.eu/. Stockholm, Sweden: European Centre for Disease Prevention and Control (ECDC). (Accessed December 10, 2013). (Online.) Faria, N.A., J.A. Carric¸o, D.C. Oliveira, M. Ramirez, and H. de Lencastre. 2008. Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. J. Clin. Microbiol. 46:136–144. Faria, N.A., M. Miragaia, H. de Lencastre, and Multi Laboratory Project Collaborators. 2013. Massive dissemination of methicillin resistant Staphylococcus aureus in bloodstream infections in a high MRSA prevalence country: establishment and diversification of EMRSA-15. Microb. Drug Resist. 19:483–490. Ghebremedhin, B., M.O. Olugbosi, A.M. Raji, F. Layer, R.A. Bakare, B. Konig, et al. 2009. Emergence of a community-associated methicillin-resistant Staphylococcus aureus strain with a unique resistance profile in Southwest Nigeria. J. Clin. Microbiol. 47:2975–2980. Gilot, P., G. Lina, T. Cochard, and B. Poutrel. 2002. Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis. J. Clin. Microbiol. 40:4060–4067. Gomes, A.R., I.S. Sanches, M. Aires-de-Sousa, E. Castaneda, and H. de Lencastre. 2001. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Colombian hospitals: dominance of a single unique multidrug-resistant clone. Microb. Drug Resist. 7:23–32. Grundmann, H., D.M. Aanensen, C.C. van den Wijngaard, B.G. Spratt, D. Harmsen, and A.W. Friedrich. 2010. Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis. PLoS Med. 7:e1000215. Heysell, S.K., S.V. Shenoi, K. Catterick, T.A. Thomas, and G. Friedland. 2011. Prevalence of methicillin-resistant Staphylococcus aureus nasal carriage among hospitalised patients with tuberculosis in rural KwaZulu-Natal. S. Afr. Med. J. 101:332–334. Kateete, D.P., S. Namazzi, M. Okee, A. Okeng, H. Baluku, N.L. Musisi, et al. 2011. High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago hospital in Kampala, Uganda. BMC Res. Notes 4:326. Kejela, T., and K. Bacha. 2013. Prevalence and antibiotic susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) among primary school children and prisoners in Jimma Town, Southwest Ethiopia. Ann. Clin. Microbiol. Antimicrob. 12:11. Machuca, M.A., L.M. Sosa, and C.I. Gonzalez. 2013. Molecular typing and virulence characteristic of methicillin-resistant Staphylococcus aureus isolates from pediatric patients in Bucaramanga, Colombia. PLoS One 8:e73434. Maina, E.K., C. Kiiyukia, C.N. Wamae, P.G. Waiyaki, and S. Kariuki. 2013. Characterization of methicillinresistant Staphylococcus aureus from skin and soft tissue infections in patients in Nairobi, Kenya. Int. J. Infect. Dis. 17:e115–e119. Mandomando, I., B. Sigauque, L. Morais, M. Espasa, X. Valles, J. Sacarlal, et al. 2010. Antimicrobial drug resistance trends of bacteremia isolates in a rural hospital in southern Mozambique. Am. J. Trop. Med. Hyg. 83:152–157. McDougal, L.K., C.D. Steward, G.E. Killgore, J.M. Chaitram, S.K. McAllister, and F.C. Tenover. 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol. 41: 5113–5120. Milheiric¸o, C., D.C. Oliveira, and H. de Lencastre. 2007. Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: ‘SCCmec IV multiplex’. J. Antimicrob. Chemother. 60:42–48. Milheiric¸o, C., D.C. Oliveira, and H. de Lencastre. 2007. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob. Agents Chemother. 51:3374–3377. Moyo, S.J., S. Aboud, B. Blomberg, N. Mkopi, M. Kasubi, K. Manji, et al. 2014. High nasal carriage of methicillin-resistant Staphylococcus aureus among healthy Tanzanian under-5 children. Microb. Drug Resist. 20:82– 88. Nubel, U., P. Roumagnac, M. Feldkamp, J.H. Song, K.S. Ko, Y.C. Huang, et al. 2008. Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 105: 14130–14135. Okuma, K., K. Iwakawa, J.D. Turnidge, W.B. Grubb, J.M. Bell, F.G. O’Brien, et al. 2002. Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community. J. Clin. Microbiol. 40:4289–4294.
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
42. Oliveira, D.C., C. Milheiric¸o, and H. de Lencastre. 2006. Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI. Antimicrob. Agents Chemother. 50:3457–3459. 43. Poulsen, A.B., R. Skov, and L.V. Pallesen. 2003. Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit. J. Antimicrob. Chemother. 51:419–421. 44. Randrianirina, F., J.L. Soares, E. Ratsima, J.F. Carod, P. Combe, P. Grosjean, et al. 2007. In vitro activities of 18 antimicrobial agents against Staphylococcus aureus isolates from the Institut Pasteur of Madagascar. Ann. Clin. Microbiol. Antimicrob. 6:5. 45. Rasamiravaka, T., S. Rasoanandrasana, N.J. Zafindraibe, A.O. Rakoto Alson, and A. Rasamindrakotroka. 2013. Evaluation of methicillin-resistant Staphylococcus aureus nasal carriage in Malagasy patients. J. Infect. Dev. Ctries 7:318–322. 46. Ruimy, R., A. Maiga, L. Armand-Lefevre, I. Maiga, A. Diallo, A.K. Koumare, et al. 2008. The carriage population of Staphylococcus aureus from Mali is composed of a combination of pandemic clones and the divergent PantonValentine leukocidin-positive genotype ST152. J. Bacteriol. 190:3962–3968. 47. Sa´-Lea˜o, R., I. Santos Sanches, D. Dias, I. Peres, R.M. Barros, and H. de Lencastre. 1999. Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain? J. Clin. Microbiol. 37:1913–1920. 48. Schaumburg, F., U.A. Ngoa, K. Kosters, R. Kock, A.A. Adegnika, P.G. Kremsner, et al. 2011. Virulence factors and genotypes of Staphylococcus aureus from infection and carriage in Gabon. Clin. Microbiol. Infect. 17:1507– 1513. 49. Shibabaw, A., T. Abebe, and A. Mihret. 2013. Nasal carriage rate of methicillin resistant Staphylococcus aureus among Dessie Referral Hospital Health Care Workers; Dessie, Northeast Ethiopia. Antimicrob. Resist. Infect. Control 2:25.
449
50. Simo˜es, R.R., M. Aires-de-Sousa, T. Conceic¸a˜o, F. Antunes, P.M. da Costa, and H. de Lencastre. 2011. High prevalence of EMRSA-15 in Portuguese public buses: a worrisome finding. PLoS One 6:e17630. 51. Sola, C., H. Paganini, A.L. Egea, A.J. Moyano, A. Garnero, I. Kevric, et al. 2012. Spread of epidemic MRSA-ST5-IV clone encoding PVL as a major cause of community onset staphylococcal infections in Argentinean children. PLoS One 7:e30487. 52. Tavares, A., N.A. Faria, H. de Lencastre, and M. Miragaia. 2013. Population structure of methicillin-susceptible Staphylococcus aureus (MSSA) in Portugal over a 19-year period (1992–2011). Eur. J. Clin. Microbiol. Infect. Dis. 33:423–432. 53. Vandenesch, F., T. Naimi, M.C. Enright, G. Lina, G.R. Nimmo, H. Heffernan, et al. 2003. Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence. Emerg. Infect. Dis. 9:978–984. 54. von Eiff, C., K. Becker, K. Machka, H. Stammer, and G. Peters. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. N. Engl. J. Med. 344:11–16. 55. Wertheim, H.F., D.C. Melles, M.C. Vos, W. van Leeuwen, A. van Belkum, H.A. Verbrugh, et al. 2005. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis. 5:751–762. 56. Zriouil, S.B., M. Bekkali, and K. Zerouali. 2012. Epidemiology of Staphylococcus aureus infections and nasal carriage at the Ibn Rochd University Hospital Center, Casablanca, Morocco. Braz. J. Infect. Dis. 16:279–283.
Address correspondence to: Marta Aires-de-Sousa, PhD Escola Superior de Sau´de da Cruz Vermelha Portuguesa (ESSCVP) Avenida de Ceuta n1, Edifı´cio UrbiCeuta Lisboa 1300-125 Portugal E-mail: [email protected]
Epidemiology of Methicillin-Resistant and -Susceptible Staphylococcus aureus in Luanda, Angola: First Description of the Spread of the MRSA ST5-IVa Clone in the African Continent Teresa Conceic¸a˜o,1 Ce´line Coelho,1 Isabel Santos-Silva,2 Hermı´nia de Lencastre,1,3 and Marta Aires-de-Sousa 2
Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen worldwide, and although surveillance studies are available in the most developed countries, data from Angola are inexistent. In June 2012, 295 inpatients and 199 healthcare workers from three hospitals in Luanda, Angola were nasal swabbed for S. aureus and MRSA carriage. A total of 117 individuals (23.7%) were S. aureus nasal carriers, out of which 68 (58.1%) were colonized with MRSA. The majority of the MRSA isolates (74%) belonged to a single clonal lineage, pulsed-field gel electrophoresis (PFGE) A-ST5-IVa associated with three spa types (spa types t105/ t311/t11657), followed by PFGE C-ST88-IVa (spa types t186/t325/t786/t1951/t3869) (n = 9; 12%); the other 11 MRSA isolates were representatives of 4 additional lineages. Almost half (49%) of the methicillin-susceptible S. aureus (MSSA) isolates belonged to three major clones: PFGE B-ST508 (spa types t050/t861/t1346/t1574/ t2626/t12218), PFGE D-ST45 (spa types t939/t11656), and PFGE E-ST30 (spa types t1202/t9118). MSSA isolates presented a high variability of virulence factors, including Panton-Valentine leukocidine (7.9%). MRSA carriage in Luanda is considerably high, and the major clone corresponds to a worldwide epidemic lineage, so far scarcely reported in Africa. Additional infection control measures in this metropolis are mandatory for a global MRSA control.
Introduction
M
ethicillin-resistant Staphylococcus aureus (MRSA) is a major cause of infection worldwide in both hospitals and community, leading to increased morbidity, mortality, length of hospital stay, and costs.8 Nasal carriage is a major risk factor for infection, since colonizing strains may serve as endogenous reservoirs for subsequent clinical infections, or may spread in the nosocomial setting by means of indirect patient-to-patient transmission, by direct transmission from colonized healthcare workers (HCW), or through contaminated environmental surfaces.5,54 Surveillance systems are key elements to prevent MRSA transmission and infection that receive substantial funding in the most developed countries, but are scarce or almost inexistent in resource-poor regions. However, populations in African countries are at high risk for MRSA infections due to reduced
financial resources, poor hygienic conditions, overcrowding, and understaffing that result in a lack of infection control practices.7 MRSA surveillance data from Africa are sporadic, but a few recent studies highlight a considerable diversity among MRSA nasal carriage rates: sporadic isolates in Mali,46 1.3% in Ghana,21 3.7% in Gabon,48 4.2% in healthy children under 5 years in Tanzania,39 14.8% in Madagascar,44,45 12.7% in HCW, 23% among children and prisoners in Ethiopia,32,49 21% in tuberculosis patients in KwaZuluNatal, South Africa,30 or 52% among HCW and patients in surgical units of Mulago hospital, Uganda.31 In Portuguese-speaking African countries (PALOP), former colonies with close relationships with Portugal, the MRSA prevalence was reported as null in two hospitals in Cape Verde islands in a study dated from 19971 and 4.2% nasal carriage among patients and HCW in Sa˜o Tome´ and Prı´ncipe between
1 Laboratory of Molecular Genetics, Instituto de Tecnologia Quı´mica e Biolo´gica (ITQB), Universidade Nova de Lisboa (UNL), Oeiras, Portugal. 2 Escola Superior de Sau´de da Cruz Vermelha Portuguesa (ESSCVP), Lisboa, Portugal. 3 Laboratory of Microbiology and Infectious Diseases, The Rockefeller University, New York, New York.
441
˜ O ET AL. CONCEICxA
442
2010 and 2012.16 In Mozambique, between 2001 and 2006, 8% of the S. aureus recovered from blood of children < 15 years old in a single hospital were methicillin resistant.35 Moreover, a significant prevalence of the highly potent PantonValentine leukocidine (PVL) was found among methicillinsusceptible S. aureus (MSSA) isolates in Sa˜o Tome´ and Prı´ncipe (36%) and Cape Verde (35%).3,16 The description of MRSA clonal lineages in Sa˜o Tome´ and Prı´ncipe that had been previously reported in Portuguese hospitals and in the community (ST5, ST8, and ST88) suggests that the close relationships between Portugal and PALOP nations may influence the MRSA clonal types circulating in the European countries.4,15 Data from Angola are inexistent. The aim of the present study was to evaluate the MRSA and MSSA nasal carriage among patients and HCW in different hospitals in Luanda, the capital of Angola, and characterize its population structure to identify eventual reservoirs in a city that has important demographic and economic exchanges with Portugal. Materials and Methods Hospital setting
Angola is a former Portuguese colony, situated in the West Coast of Africa. It is the seventh largest African country with 1,246,620 km2 and a population of 19,183,590 ( July, 2013) individuals with a mean age of 17.7 years.11 Hospital Ame´rico Boavida (HAB) and Hospital Pedia´trico David Bernardino (HPDB) are the major public hospitals in the capital city, Luanda. HAB is a large hospital with 624 beds constructed in 1968, with different internment services, namely, medicine, pediatrics, orthopedics, surgery, infectious diseases, urology, cardiology, and a 17-bed intensive care unit (ICU). This hospital serves an outpatient population of *2,500,000 inhabitants. HPDB is a national reference medium-sized pediatric hospital (350 beds) constructed in 1958 with three major internment services, namely, pediatrics, surgery, infectious diseases, and a 4-bed ICU. The hospital has also various ambulatory services, namely, pediatrics, surgery, orthopedics, cardiology, pneumology, ophthalmology, otorhinolaryngology, and an emergency ward. Clı´nica Sagrada Esperanc¸a (SE) is a 150-bed private clinic created in 1991 that serves an outpatient population of 500,000 inhabitants. The clinic has several internment services, namely, medicine, pediatrics, orthopedics, surgery, infectious diseases, and a nine-bed ICU in addition to several isolation rooms, individual rooms, and some ambulatory services. The emergency ward receives 80 entrances per day. Nasal screening and bacterial isolates
The inpatients and HCW from the three hospitals were nasal swabbed for S. aureus carriage. Exclusion criteria included patients admitted for < 48 hr, 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. The institutional ethics committee approved the protocol and an oral informed consent was obtained at the time of screening for each participant. Sampling was performed by a trained nurse during a 5day period in June 2012, in the main services where the risk for S. aureus infection is usually high (medicine, surgery,
pediatrics, infectious diseases, orthopedics, isolation rooms, and intermediate and ICUs), and in the cardiology, pulmonology, urology, hemodynamics, suite rooms, and emergency wards. Samples from HCW were additionally recovered from the pharmacy and sterilization department. Samples were taken by swabbing both nares of each individual with a sterile cotton swab, which was stored in the Stuart transport medium. Swabs were processed within 10 days at the Laboratory of Molecular Genetics, Instituto de Tecnologia Quı´mica e Biolo´gica in Portugal. In brief, each swab was inoculated on a rich medium, Tryptic soy agar (Becton, Dickinson & Co, Franklin Lakes, NJ) and on a selective chromogenic medium Chromagar Staph aureus (ChromAgar, Paris, France). All presumptive S. aureus colonies were tested for coagulase by latex agglutination test Staphaurex (Remel, Lenexa, KS) 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.43 Antimicrobial susceptibility testing and mecA detection
Antimicrobial susceptibility testing was performed on all isolates by the disk diffusion method for a panel of 15 antibiotics: penicillin, oxacillin, erythromycin, gentamicin, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, rifampin, tetracycline, fusidic acid, teicoplanin, vancomycin, linezolid, and quinupristin-dalfopristin. Breakpoints were defined according to the current Clinical Laboratory Standards Institute (CLSI) guidelines,13 except for fusidic acid that was defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.eucast .org/). Strain S. aureus ATCC25923 was used as quality control. The presence of the mecA gene was tested on all S. aureus isolates by PCR.41 Oxacillin and vancomycin minimum inhibitory concentrations were determined using E-test strips according to the manufacturer’s instructions (Biomerieux, Marcy l’Etoile, France) for oxacillin-susceptible but mecApositive isolates and for all MRSA, respectively. Molecular typing Pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) was performed on all S. aureus isolates as described by Chung et al.12 The resulting SmaI restriction band profiles were analyzed by visual inspection using the criteria of McDougal et al.,36 followed by automated analysis with the BioNumerics software version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) for relatedness evaluation to define clusters. Dendrograms were generated as previously described24 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 cluster definition, respectively. spa typing and multilocus sequence typing. spa typing was performed on at least one representative of each PFGE subtype, and spa types were assigned through the Ridom web server (http://spaserver.ridom.de).2 Multilocus sequence typing (MLST) was conducted as described17,22 for representative isolates of the different PFGE types, previously characterized by spa typing. Allelic profiles and sequence types (ST) were defined using the MLST online database (www.mlst.net).
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
443
MLST clonal complexes were defined using the eBURST algorithm and restricted to single locus variant (SLV) and double locus variant from each group founder or subfounder, inside each clonal group. SCCmec typing. SCCmec was characterized by multiplex PCR in all MRSA isolates.38 Additionally, SCCmec type IV isolates were further subtyped by multiplex PCR, as described,37 after primer concentration adjustment: 0.3 mM for primer ccrB1; 0.7 mM for primer ccrB2; 1 mM for primers J IVg F and J IVg R; and 1.9 mM for primers J IVh F and J IVh R. Detection of virulence determinants and agr type
The presence of 11 specific staphylococcal virulence genes, including 3 leukocidins (lukS-lukF, lukE-lukD, and lukM), 3 hemolysins (hlb, hlg, and hlgv), and 5 superantigenic toxins (eta, etb, etd, sel, and sep), was determined by PCR for all S. aureus isolates as previously described.53 All S. aureus isolates belonging to ST8 or PVL + were screened for the presence of the arginine catabolic mobile element (ACME) by PCR amplification of arcA and opp3.19,20 Subtypes of the accessory gene regulator (agr I–IV) were determined for the entire collection by multiplex PCR.27 Statistical analysis
S. aureus and MRSA carriage differences were verified by the Pearson’s Chi-square test. All statistics were calculated using the SPSS software package version 21 (SPSS, Inc., Chicago, IL), and p-values of £ 0.05 were considered as statistically significant. Results
The patients colonized with MRSA were predominantly male (57.8%), half were < 18 years old (50%) and 57.8% were hospitalized for < 15 days (internment period ranged between 3 and 98 days). The leading internment causes were related to bone fractures (13%), malaria (11%), liver diseases (6.7%), and neoplasms (6.7%). Moreover, 82.2% of the patients had been taking antibiotics at the time of screening or in the previous month, namely, penicillins (86.5%), imidazole (40.5%), and aminoglycosides (18.9%), and 24.2% underwent surgery within the month preceding the screening. Comparing the colonization rates in the three hospitals, the global S. aureus carriage was considerably lower in HPDB (18.4% vs. 26.2/26.9%), while the MRSA carriage was significantly higher in HAB (17.6% vs. 10.7/11.7%)— Table 1. Moreover, the percentage of methicillin-resistant isolates among S. aureus was considerably inferior in the private clinic compared with the two public hospitals. Despite some intrahospital variability, the global prevalence of MRSA was similar between HCW and patients (Table 1). However, patients in public hospitals showed higher MRSA carriage rates (HAB: 18.9%, HPDB: 16.6%), compared with patients in the private clinic (SE: 4.8%) ( p = 0.030). No major differences were observed in both S. aureus and MRSA carriage rates among the different HCW categories (Table 1). Among 19 different wards included in the study, regardless of the hospital, the highest MRSA frequency was observed in the pediatric special care unit (25%, n = 6), orthopedics (23.8%, n = 5), and cardiology (21.7%, n = 5). Among the 117 nasal carriers, 7 patients and 4 HCWs were cocolonized with two different isolates, resulting in a total of 128 S. aureus (77 MRSA and 51 MSSA) recovered that were further characterized.
Prevalence of S. aureus and MRSA nasal colonization
Among the 494 individuals screened, 117 were S. aureus nasal carriers (23.7%). Of these, 68 (45 inpatients and 23 HCW) were colonized with MRSA indicating an extremely high (58.1%) prevalence of methicillin resistance among the S. aureus carriers.
Genetic diversity
The molecular characterization of the 128 S. aureus clustered the isolates into 22 clonal types (defined by PFGE): MRSA were classified in 6 types and the MSSA in 17 with one type common to both MRSA and MSSA (Table 2).
Table 1. Staphylococcus aureus and MRSA Nasal Carriage in Different Hospitals in Angola Individual characteristics Hospital SE-private HAB-public HPDB-public Individual Patients HCW HCW Physicians Nurses Othersa
S. aureus carriers n (%)
p-Value
% of methicillin resistance among S. aureus
p-Value
13 (10.7) 34 (17.6) 21 (11.7)
0.251 0.047 0.323
40.6 65.4 63.6
0.019 0.154 0.449
0.373
45 (15.2) 23 (11.6)
0.242
60.8 53.5
0.439
0.405 0.579 0.552
6 (13.0) 12 (12.3) 5 (9.4)
0.741 0.804 0.552
75.0 52.2 41.7
0.176 0.853 0.334
p-Value
MRSA carriers n (%)
32 (26.2) 52 (26.9) 33 (18.4)
0.446 0.172 0.038
74 (25.0) 43 (21.6) 8 (17.4) 23 (23.5) 12 (22.6)
a Includes: nurse’s aides, healthcare technicians, students, administrative personnel, cleaning staff, cooks, social workers, and pharmacists. Significant p-values are in bold. MRSA, methicillin-resistant S. aureus; HCW, healthcare worker; HAB, Hospital Ame´rico Boavida; HPDB, Hospital Pedia´trico David Bernardino; SE, Sagrada Esperanc¸a clinic.
˜ O ET AL. CONCEICxA
444
Table 2. Molecular Characterization of the 77 MRSA and 51 MSSA Isolates Recovered in Angola in June 2012 MR/ MSSA
PFGE No. type (No. of subtypes) isolates
Hospital
Type of individual
spa type
MRSA
A C J M G T
(13) (7) (4) (3) (2) (1)
57 9 5 3 2 1
HAB, HPDB, SE HAB, HPDB HAB, HPDB HPDB, SE SE SE
P/HCW P/HCW P/HCW HCW HCW HCW
t105/t311/t11657 t186/t325/t786/t1951/t3869 t104/t1774 t3092 t6065 t091
MSSA
B D E F A L H N R P J K Q S V W Z
(8) (5) (5) (3) (3) (2) (2) (2) (2) (2) (1) (1) (1) (1) (1) (1) (1)
11 7 7 4 3 3 2 2 2 3 1 1 1 1 1 1 1
HAB, HPDB, HAB, HPDB, HAB, HPDB, HPDB, SE HAB, HPDB, HAB, SE SE HAB, SE HPDB SE HAB SE HPDB SE HAB HAB HAB
P/HCW P/HCW P/HCW P/HCW P/HCW P/HCW P P HCW P P P HCW P P P P
t050/t861/t1346/t1574/t2626/t12218 t939/t11656 t1202/t9118 t349/t1350/t6205 t002/t1215/t6071 t148/t1346 t008/t1476 t084/t346 t1458/t12259 t314/t1077 t064 t693 t1476 t335 t12219 t5187 t957
SE SE SE SE
ST 5 88 8 72 5/2629 789 508 45 30 25 5 72 8 15 2367 121 8 1 2229 152 672 2728 601
SCCmec agr type type IVa IVa IVd/V V V V
II III I I II I I IV III/na I II I I II III IV I III I I I III II
PVL +/+/+/+/+/+ -
HDB, Hospital David Bernardino; P, patient; na, no amplification; PVL, Panton-Valentine leukocidine; ST, sequence type; MSSA, methicillin-susceptible S. aureus; PFGE, pulsed-field gel electrophoresis.
The overwhelming majority of the MRSA isolates (74%, n = 57) belonged to PFGE type A, spa types t105/t311/t11657, ST5, SCCmec IVa, agrII, highly related to the pandemic pediatric clone. Clonal type PFGE C, spa types t186/t325/t786/ t1951/t3869, ST88-IVa-agrIII represented 12% of the isolates (n = 9), while the remaining MRSA belonged to three minor clones and one single isolate (Table 2). Regarding the MSSA collection, almost half of the isolates (49%, n = 25) were distributed into three major clones: PFGE B, spa types t050/t861/t1574/t2626/t1346/t12218, ST508 (SLV of ST45)-agrI (n = 11); PFGE D, spa types t939/t11656, ST45-agrIV (n = 7); and PFGE E, spa types t1202/t9118, ST30-agrIII (n = 7). The remaining MSSA belonged to seven minor clones; seven other MSSA were represented by single isolates only (Table 2). Interestingly, although PFGE A was a major MRSA clonal lineage, it was sporadically found among MSSA isolates and associated to distinct spa types (Table 2). Distribution of clonal types among hospitals and populations
The major MRSA clonal type PFGE A-ST5-IVa was the only MRSA lineage found in the three hospitals and was detected in all wards screened. Two MRSA lineages (PFGE C-ST88-IVa and J-ST8-IVd/V) were exclusively found in public hospitals (HAB and HPDB), while one minor clone (PFGE G-ST5/ST2629-V) and one singleton (T-ST789-V) were detected in the private clinic SE only (Table 2). Concerning the MRSA distribution among patients and HCW, the overwhelming majority of the MRSA carriers (patients: 82.2%; HCW: 60.9%) were colonized with PFGE
A-ST5-IVa isolates. PFGE types M, G, and T were found exclusively among HCW (Table 2), namely, nurses and physicians. All HCW other than physicians and nurses who were MRSA carriers were exclusively colonized with PFGE A isolates. Although MSSA isolates showed a high genetic variability, the three more prevalent lineages (PFGE B, D, and E) were found in all three hospitals widely spread among patients and HCW (Table 2). Nurses showed the highest MSSA variability, being colonized with six different lineages (PFGE B: 27.3% of all nurse carriers; PFGE A: 18.2%; PFGE D and L: 18.2%; PFGE E: 9.1%; and PFGE F: 9.1%). Antimicrobial susceptibility
All 128 S. aureus isolates were susceptible to fusidic acid, quinupristin-dalfopristin, linezolid, and vancomycin (MIC values between 1.5 and 4 mg/L for MRSA isolates). Among the MSSA isolates, 92% showed resistance to penicillin, 59% to rifampin, 25% to trimethoprim-sulfamethoxazole, 18% to tetracycline, 4% to erythromycin, and only one isolate was resistant to ciprofloxacin and gentamicin. A single MSSA isolate showed induced resistance to clindamycin. Higher resistant phenotypes were found among the 77 MRSA. All isolates were resistant to penicillin, cefoxitin, and trimethoprim-sulfamethoxazol. In addition, 87% of the MRSA isolates were resistant to rifampin, 30% to gentamicin, 14% to chloramphenicol, 12% to ciprofloxacin, 9% to tetracycline, and 8% to erythromycin. Interestingly, 8 of the 77 MRSA isolates (belonging to ST5-V, ST88-IVa, or ST2629-V) that previously amplified the mecA gene,
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
445
FIG. 1. Distribution of the 11 virulence determinants among methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) clonal lineages. Percentages (%) of positive isolates are relative to the total number of isolates regarding each clonal lineage. Pulsed-field gel electrophoresis (PFGE) types are ordered according to the number of isolates in each type, in a descending trend from left to right. showed a susceptible or intermediate phenotype to oxacillin by the disk diffusion method, but an oxacillin MIC ranging between 0.75 and 2 mg/L, and were therefore considered as MRSA with a heterogeneous resistance phenotype. Virulence factors
Among the entire S. aureus collection, 66.4% of the isolates harbored two or more virulence factors, namely, leukocidins and hemolysins (Fig. 1). Although the MSSA isolates showed a higher variability of virulence factors, LukD-LukE and g-hemolysin variant were the most prevalent virulence determinants detected in both MRSA and MSSA (68.4% and 43% of the isolates for both toxins, respectively) (Fig. 1). None of the isolates showed LukM or ETB. PVL was detected in 10 isolates (7.9%), belonging to six different clonal types, all MSSA (Table 2 and Fig. 1). Six of the PVL + isolates were isolated in the private clinic SE. ETA and ETD were restricted to MRSA PFGE C-ST88-IVa and MSSA PFGE F-ST25 isolates, respectively (Fig. 1). MRSA PFGE M-ST72-V clonal type was the only lineage negative to all toxin determinants tested. Interestingly,
MSSA PFGE L-ST72 clonal type, although belonging to the same ST72, included the isolates with the highest virulence content, harboring 5 of the 11 toxins tested, but with a low antimicrobial resistance profile (resistance to penicillin only, and a single isolate resistant to rifampin). Concerning the accessory gene regulator (agr), half of the isolates (n = 66, 51.6%) belonged to agr type II, including four clonal types (Table 2). agr type I was the second most prevalent type (n = 33, 25.8%) and included the greatest clonal variability, followed by agr type III (n = 19, 14.8%). agr type IV was found in nine MSSA isolates (7%) associated to ST45 and ST121. One isolate showed no amplification for the agr locus and was considered as agr negative. All S. aureus isolates PVL + or belonging to ST8 (PFGE types H, J, or T) were screened for the presence of ACME, but none of the isolates harbored this element. MRSA cross-transmission
MRSA cross-transmission was detected in public hospitals only (Fig. 2). In HAB, isolates with the same PFGE types were shared between patients and between patients and HCW in different wards. In HPDB, cross-transmission
FIG. 2. MRSA cross-transmission between patients and between patients and healthcare workers (HCW) in different wards of the two public hospitals.
446
was found between patients only, in the pediatric special care and in the intermediate care units. No HCW colonized with related MRSA isolates were detected in these wards and, therefore, no transmission routes could be traced. Discussion
In the present study, we provide for the first time, data on the carriage rates and population structure of S. aureus and MRSA among patients and HCW in Angola, a major African country with close demographic relationships with Europe, namely with Portugal. Although the overall S. aureus nasal carriage rate observed in Angola (23.7%) parallels the rates described for other African countries, we found an extremely high (58.1%) prevalence of methicillin resistance among S. aureus isolates. Among the few reports available focusing on MRSA nasal carriage in African countries, this is the highest value, only comparable to rates reported in Ethiopia (44.1%) and Morocco (44.4%).49,56 In Sa˜o Tome´ and Prı´ncipe, another PALOP country, methicillin resistance among S. aureus was 26.9%.16 The higher availability and consumption of antibiotics in the hospitals in Luanda compared with other PALOP nations may explain the higher methicillin resistance in Angola. Angola has close demographic relationships with Portugal, which is currently together with Romania, the only European country that shows an MRSA prevalence above 50%.23 No significant difference was observed in the global occurrence of MRSA nasal carriage regarding patients and HCW in Luanda as reported in Sa˜o Tome´ and Prı´ncipe.16 In contrast, in Ghana and Gabon, none of the HCW screened were MRSA carriers.6,21 However, public hospitals showed higher levels of methicillin resistance among S. aureus compared to the private clinic, which may indicate that infection control policies are more effective in private and smaller hospitals. High antimicrobial resistance rates were observed for penicillin and trimethoprim-sulfamethoxazole in both MRSA and MSSA isolated in Angola, which is in agreement with reports from Sa˜o Tome´ and Prı´ncipe, Gabon, and Nigeria.6,16,26 This is not surprising given the widespread use of trimethoprim-sulfamethoxazole in Africa recommended by the World Health Organization to prevent opportunistic bacterial infections in patients with HIV/ AIDS, malaria, and suffering from malnutrition. Furthermore, and in contrast to other African countries, we found an unexpectedly high resistance to rifampin and gentamicin,1,16,21,26 which may be explained by the fact that rifampin is a first line antituberculosis drug and gentamicin is one of the mostly used antibiotics in the pediatric HPDB hospital. In our study, 27% of the patients screened had taken recently at least one of the three antibiotics (gentamicin, trimethoprim-sulfamethoxazole, or rifampin), and all MRSA recovered from these patients showed resistance to the referred antibiotics. Moreover, the resistance to chloramphenicol and ciprofloxacin among MRSA may be explained by the fact that chloramphenicol is one of the principal drugs used in HPDB and ciprofloxacin is a first line drug to treat MRSA infections in HAB. Therefore, an antimicrobial management program to optimize antimicrobial use is recommended in the participating Angolan hospitals.
˜ O ET AL. CONCEICxA
In Angola, both HCW and patients were mainly colonized with isolates belonging to ST5 lineage (SCCmec IVa, spa types t105 and t311), related to the epidemic Pediatric MRSA clone. This clone was isolated for the first time in 1992 in a Portuguese Pediatric hospital,47 and although no longer present in that hospital in 2006, one year later it was reported as the second major clonal lineage in the Portuguese Azores islands as well as in sporadic isolates in the mainland.4,14,42 The Pediatric MRSA clone has also been detected in other European countries, Poland, and France,18 but was mainly spread in South American countries, namely in Argentina and Colombia, and in the United States.28,33,36,47 The dissemination of epidemic MRSA isolates ST5-IV-PVL + was the main cause of increasing staphylococcal community-onset infections in Argentinean children (2003–2008)51 and was also responsible for infection cases in two African cities, Casablanca and Dakar.10 In recent reports, the Pediatric MRSA clonal lineage has been restricted to infection isolates, but was reported for the first time in nasal carriage in PALOP countries, as a minor clone in Sa˜o Tome´ and Prı´ncipe,16 and now as the prevalent clonal lineage among children and adults in the three Angolan hospitals. Since infection isolates were not included in this study, we could not assess to what extent this MRSA clonal lineage was also responsible for infection in these hospitals. The ST5 lineage is known to frequently acquire mobile genetic elements, such as different SCCmec elements, antibiotic resistance and toxin genes.40 Various SCCmec acquisition events occurred within the ST5 lineage, on multiple and independent occasions, evidencing that MRSA emerged in numerous circumstances and in distinct locations rather than exclusively by geographic dispersal of isolates.40 Moreover, description of local emergence of ST5PVL + lineages, as well as the emergence of multiresistant isolates corroborate this idea.14,28,51 Although SCCmec IVa, highly prevalent in African countries, might have been introduced de novo in MSSA ST5 isolates found in Angolan hospitals, the spread of this lineage from abroad, namely from Portugal, could not be excluded, since Angolan children frequently travel to Portugal to meet pediatricians or even to be admitted to the hospitals for critical medical treatments. Moreover, although not currently prevalent in Portuguese hospitals,25 ST5-IVa isolates sharing the same spa types t002 and t105 with isolates from our collection were recently reported in the community in public buses in Lisbon and Oporto.15,50 On the other hand, the description of ST5-I isolates in South African hospitals40 suggests that a local exchange of SCCmec cassette type I to type IVa could also explain the emergence of ST5-IVa clonal lineage in Angola. However, genome sequencing is needed to corroborate this hypothesis. ST88-IVa, the second major clonal lineage in Luanda, was a major MRSA lineage in Sa˜o Tome´ and Prı´ncipe and in other African countries, confirming its endemicity in the African continent.6,10,16,26,46 Among the other MRSA, ST8 was already reported in other African countries, namely as a major clone in Sa˜o Tome´ and Prı´ncipe and Nigeria.6,10,16,21,26 Although the MSSA population showed a higher variability as previously found in other African countries,1,6,9 half of the isolates were distributed into three major clonal lineages related to ST508, ST45, and ST30. ST508 has been reported in some African countries, being the second major
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
MSSA clone in Gabon and Sa˜o Tome´ and Prı´ncipe.6,16,26,46 Although sporadically found in Africa, ST45 and ST30 MSSA were widely spread in Europe, namely among the MSSA population in Portugal, where ST30 is endemic since 1992 and currently the predominant clonal lineage.25,29,52 Since these clonal lineages share the same genetic background as the major international MRSA epidemic clones, the Berlin clone (ST45-IV) and the community-associated Southwest Pacific clone (ST30-IV), these MSSA could represent a suitable reservoir for the acquisition of the highly prevalent SCCmec type IV in Angola. The African continent has been reported as a major reservoir of PVL + MSSA isolates in both carriage and infection.3,16,34,46,48 However, in Angola, only 7.9% of the isolates (all MSSA) carried PVL, and except three ST30 isolates, all the other PVL + belong to minor MSSA clones, which suggests that the spread of PVL in African countries is MSSA clonal related. It is well known that HCW who are nasal carriers play an important role in S. aureus transmission.5,55 In Angola, MRSA cross-transmission between HCW and patients was observed in the public hospital HAB, and the patients colonized with a same MRSA strain were found in different wards, in both public hospitals. In the private SE clinic, no cross-transmission events were detected, probably due to a low occupation rate in each ward, which contrasts to public hospitals. In conclusion, the MRSA nasal carriage rate in the nosocomial setting in Luanda, Angola, is extremely high in contrast with the much lower rates recently reported in another PALOP country, Sa˜o Tome´ and Prı´ncipe. Moreover, we described a massive spread of the MRSA Pediatric clone (ST5-IVa), a worldwide epidemic lineage scarcely reported in Africa so far. Antimicrobial resistance levels and MRSA cross-transmission within the hospitals suggest the need for implementation of additional infection control measures for a global MRSA control.
447
3.
4.
5. 6.
7. 8. 9.
10.
11.
Acknowledgments
This work was supported by project PTDC/SAU-SAP/ 118813/2010 and grant no. PEst-OE/EQB/LA0004/2011 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal. T.C. and C.C. were supported by grants SFRH/BPD/ 72422/2010 and 036/BI-BI/2012 from FCT, Portugal. The authors are grateful to the healthcare workers from Hospital Ame´rico Boavida, Hospital Pedia´trico David Bernardino, and Clı´nica Sagrada Esperanc¸a for the assistance during the screenings and to Ana Delgado for the statistical analysis.
12.
Disclosure Statement
14.
The authors interests exist.
have
declared
that
no
13.
competing
References
1. Aires-de-Sousa, M., I. Santos Sanches, M.L. Ferro, and H. de Lencastre. 2000. Epidemiological study of staphylococcal colonization and cross-infection in two West African Hospitals. Microb. Drug Resist. 6:133–141. 2. Aires-de-Sousa, M., K. Boye, H. de Lencastre, A. Deplano, M.C. Enright, J. Etienne, et al. 2006. High inter-
15.
16.
laboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. J. Clin. Microbiol. 44:619–621. Aires-de-Sousa, M., T. Conceic¸a˜o, and H. de Lencastre. 2006. Unusually high prevalence of nosocomial PantonValentine leukocidin-positive Staphylococcus aureus isolates in Cape Verde Islands. J. Clin. Microbiol. 44: 3790–3793. Aires-de-Sousa, M., B. Correia, and H. de Lencastre. 2008. Changing patterns in frequency of recovery of five methicillin-resistant Staphylococcus aureus clones in Portuguese hospitals: surveillance over a 16-year period. J. Clin. Microbiol. 46:2912–2917. Albrich, W.C., and S. Harbarth. 2008. Health-care workers: source, vector, or victim of MRSA? Lancet Infect. Dis. 8:289–301. Ateba Ngoa, U., F. Schaumburg, A.A. Adegnika, K. Kosters, T. Moller, J.F. Fernandes, et al. 2012. Epidemiology and population structure of Staphylococcus aureus in various population groups from a rural and semi urban area in Gabon, Central Africa. Acta Trop. 124:42–47. Bagheri Nejad, S., B. Allegranzi, S.B. Syed, B. Ellis, and D. Pittet. 2011. Health-care-associated infection in Africa: a systematic review. Bull. World Health Organ 89:757–765. Boucher, H.W., and G.R. Corey. 2008. Epidemiology of methicillin-resistant Staphylococcus aureus. Clin. Infect. Dis. 46 Suppl 5:S344–S349. Breurec, S., C. Fall, R. Pouillot, P. Boisier, S. Brisse, F. Diene-Sarr, et al. 2011. Epidemiology of methicillin-susceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes. Clin. Microbiol. Infect. 17:633–639. Breurec, S., S.B. Zriouil, C. Fall, P. Boisier, S. Brisse, S. Djibo, et al. 2010. Epidemiology of methicillin-resistant Staphylococcus aureus lineages in five major African towns: emergence and spread of atypical clones. Clin. Microbiol. Infect. 17:160–165. Central Intelligence Agency. 2013. The World Factbook. Available at www.cia.gov/library/publications/the-worldfactbook/geos/tp.html. (Accessed November 25, 2013). (Online.) Chung, M., H. de Lencastre, P. Matthews, A. Tomasz, I. Adamsson, M. Aires-de-Sousa, et al. 2000. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb. Drug Resist. 6:189–198. [CLSI] Clinical Laboratory Standards Institute. 2009. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement M100–S19. Wayne, Pennsylvania. Conceic¸a˜o, T., A. Tavares, M. Miragaia, K. Hyde, M. Aires-de-Sousa, and H. de Lencastre. 2010. Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI. Eur. J. Clin. Microbiol. Infect. Dis. 29:543–550. Conceic¸a˜o, T., F. Diamantino, C. Coelho, H. de Lencastre, and M. Aires-de-Sousa. 2013. Contamination of public buses with MRSA in Lisbon, Portugal: a possible transmission route of major MRSA clones within the community. PLoS One 8:e77812. Conceic¸a˜o, T., I.S. Silva, H. de Lencastre, and M. Airesde-Sousa. 2013. Staphylococcus aureus nasal carriage
˜ O ET AL. CONCEICxA
448
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
among patients and health care workers in Sa˜o Tome´ and Prı´ncipe. Microb. Drug Resist. 20:57–66. Criso´stomo, M.I., H. Westh, A. Tomasz, M. Chung, D.C. Oliveira, and H. de Lencastre. 2001. The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones. Proc. Natl. Acad. Sci. U.S.A. 98:9865–9870. Dauwalder, O., G. Lina, G. Durand, M. Bes, H. Meugnier, V. Jarlier, et al. 2008. Epidemiology of invasive methicillin-resistant Staphylococcus aureus clones collected in France in 2006 and 2007. J. Clin. Microbiol. 46:3454–3458. Diep, B.A., S.R. Gill, R.F. Chang, T.H. Phan, J.H. Chen, M.G. Davidson, et al. 2006. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet 367:731– 739. Diep, B.A., G.G. Stone, L. Basuino, C.J. Graber, A. Miller, S.A. des Etages, et al. 2008. The arginine catabolic mobile element and staphylococcal chromosomal cassette mec linkage: convergence of virulence and resistance in the USA300 clone of methicillin-resistant Staphylococcus aureus. J. Infect. Dis. 197:1523–1530. Egyir, B., L. Guardabassi, S.S. Nielsen, J. Larsen, K.K. Addo, M.J. Newman, et al. 2013. Prevalence of nasal carriage and diversity of Staphylococcus aureus among inpatients and hospital staff at Korle Bu Teaching Hospital, Ghana. J. Global Antimicrob. Resist. 1:189–193. Enright, M.C., N.P. Day, C.E. Davies, S.J. Peacock, and B.G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillinsusceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008–1015. European Antimicrobial Resistance Surveillance System. 2013. EARSS annual report 2012. Available at www.ecdc.europa.eu/. Stockholm, Sweden: European Centre for Disease Prevention and Control (ECDC). (Accessed December 10, 2013). (Online.) Faria, N.A., J.A. Carric¸o, D.C. Oliveira, M. Ramirez, and H. de Lencastre. 2008. Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. J. Clin. Microbiol. 46:136–144. Faria, N.A., M. Miragaia, H. de Lencastre, and Multi Laboratory Project Collaborators. 2013. Massive dissemination of methicillin resistant Staphylococcus aureus in bloodstream infections in a high MRSA prevalence country: establishment and diversification of EMRSA-15. Microb. Drug Resist. 19:483–490. Ghebremedhin, B., M.O. Olugbosi, A.M. Raji, F. Layer, R.A. Bakare, B. Konig, et al. 2009. Emergence of a community-associated methicillin-resistant Staphylococcus aureus strain with a unique resistance profile in Southwest Nigeria. J. Clin. Microbiol. 47:2975–2980. Gilot, P., G. Lina, T. Cochard, and B. Poutrel. 2002. Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis. J. Clin. Microbiol. 40:4060–4067. Gomes, A.R., I.S. Sanches, M. Aires-de-Sousa, E. Castaneda, and H. de Lencastre. 2001. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Colombian hospitals: dominance of a single unique multidrug-resistant clone. Microb. Drug Resist. 7:23–32. Grundmann, H., D.M. Aanensen, C.C. van den Wijngaard, B.G. Spratt, D. Harmsen, and A.W. Friedrich. 2010. Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis. PLoS Med. 7:e1000215. Heysell, S.K., S.V. Shenoi, K. Catterick, T.A. Thomas, and G. Friedland. 2011. Prevalence of methicillin-resistant Staphylococcus aureus nasal carriage among hospitalised patients with tuberculosis in rural KwaZulu-Natal. S. Afr. Med. J. 101:332–334. Kateete, D.P., S. Namazzi, M. Okee, A. Okeng, H. Baluku, N.L. Musisi, et al. 2011. High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago hospital in Kampala, Uganda. BMC Res. Notes 4:326. Kejela, T., and K. Bacha. 2013. Prevalence and antibiotic susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) among primary school children and prisoners in Jimma Town, Southwest Ethiopia. Ann. Clin. Microbiol. Antimicrob. 12:11. Machuca, M.A., L.M. Sosa, and C.I. Gonzalez. 2013. Molecular typing and virulence characteristic of methicillin-resistant Staphylococcus aureus isolates from pediatric patients in Bucaramanga, Colombia. PLoS One 8:e73434. Maina, E.K., C. Kiiyukia, C.N. Wamae, P.G. Waiyaki, and S. Kariuki. 2013. Characterization of methicillinresistant Staphylococcus aureus from skin and soft tissue infections in patients in Nairobi, Kenya. Int. J. Infect. Dis. 17:e115–e119. Mandomando, I., B. Sigauque, L. Morais, M. Espasa, X. Valles, J. Sacarlal, et al. 2010. Antimicrobial drug resistance trends of bacteremia isolates in a rural hospital in southern Mozambique. Am. J. Trop. Med. Hyg. 83:152–157. McDougal, L.K., C.D. Steward, G.E. Killgore, J.M. Chaitram, S.K. McAllister, and F.C. Tenover. 2003. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol. 41: 5113–5120. Milheiric¸o, C., D.C. Oliveira, and H. de Lencastre. 2007. Multiplex PCR strategy for subtyping the staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus: ‘SCCmec IV multiplex’. J. Antimicrob. Chemother. 60:42–48. Milheiric¸o, C., D.C. Oliveira, and H. de Lencastre. 2007. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob. Agents Chemother. 51:3374–3377. Moyo, S.J., S. Aboud, B. Blomberg, N. Mkopi, M. Kasubi, K. Manji, et al. 2014. High nasal carriage of methicillin-resistant Staphylococcus aureus among healthy Tanzanian under-5 children. Microb. Drug Resist. 20:82– 88. Nubel, U., P. Roumagnac, M. Feldkamp, J.H. Song, K.S. Ko, Y.C. Huang, et al. 2008. Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A. 105: 14130–14135. Okuma, K., K. Iwakawa, J.D. Turnidge, W.B. Grubb, J.M. Bell, F.G. O’Brien, et al. 2002. Dissemination of new methicillin-resistant Staphylococcus aureus clones in the community. J. Clin. Microbiol. 40:4289–4294.
PREDOMINANCE OF MRSA ST5-IVA IN ANGOLA
42. Oliveira, D.C., C. Milheiric¸o, and H. de Lencastre. 2006. Redefining a structural variant of staphylococcal cassette chromosome mec, SCCmec type VI. Antimicrob. Agents Chemother. 50:3457–3459. 43. Poulsen, A.B., R. Skov, and L.V. Pallesen. 2003. Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit. J. Antimicrob. Chemother. 51:419–421. 44. Randrianirina, F., J.L. Soares, E. Ratsima, J.F. Carod, P. Combe, P. Grosjean, et al. 2007. In vitro activities of 18 antimicrobial agents against Staphylococcus aureus isolates from the Institut Pasteur of Madagascar. Ann. Clin. Microbiol. Antimicrob. 6:5. 45. Rasamiravaka, T., S. Rasoanandrasana, N.J. Zafindraibe, A.O. Rakoto Alson, and A. Rasamindrakotroka. 2013. Evaluation of methicillin-resistant Staphylococcus aureus nasal carriage in Malagasy patients. J. Infect. Dev. Ctries 7:318–322. 46. Ruimy, R., A. Maiga, L. Armand-Lefevre, I. Maiga, A. Diallo, A.K. Koumare, et al. 2008. The carriage population of Staphylococcus aureus from Mali is composed of a combination of pandemic clones and the divergent PantonValentine leukocidin-positive genotype ST152. J. Bacteriol. 190:3962–3968. 47. Sa´-Lea˜o, R., I. Santos Sanches, D. Dias, I. Peres, R.M. Barros, and H. de Lencastre. 1999. Detection of an archaic clone of Staphylococcus aureus with low-level resistance to methicillin in a pediatric hospital in Portugal and in international samples: relics of a formerly widely disseminated strain? J. Clin. Microbiol. 37:1913–1920. 48. Schaumburg, F., U.A. Ngoa, K. Kosters, R. Kock, A.A. Adegnika, P.G. Kremsner, et al. 2011. Virulence factors and genotypes of Staphylococcus aureus from infection and carriage in Gabon. Clin. Microbiol. Infect. 17:1507– 1513. 49. Shibabaw, A., T. Abebe, and A. Mihret. 2013. Nasal carriage rate of methicillin resistant Staphylococcus aureus among Dessie Referral Hospital Health Care Workers; Dessie, Northeast Ethiopia. Antimicrob. Resist. Infect. Control 2:25.
449
50. Simo˜es, R.R., M. Aires-de-Sousa, T. Conceic¸a˜o, F. Antunes, P.M. da Costa, and H. de Lencastre. 2011. High prevalence of EMRSA-15 in Portuguese public buses: a worrisome finding. PLoS One 6:e17630. 51. Sola, C., H. Paganini, A.L. Egea, A.J. Moyano, A. Garnero, I. Kevric, et al. 2012. Spread of epidemic MRSA-ST5-IV clone encoding PVL as a major cause of community onset staphylococcal infections in Argentinean children. PLoS One 7:e30487. 52. Tavares, A., N.A. Faria, H. de Lencastre, and M. Miragaia. 2013. Population structure of methicillin-susceptible Staphylococcus aureus (MSSA) in Portugal over a 19-year period (1992–2011). Eur. J. Clin. Microbiol. Infect. Dis. 33:423–432. 53. Vandenesch, F., T. Naimi, M.C. Enright, G. Lina, G.R. Nimmo, H. Heffernan, et al. 2003. Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence. Emerg. Infect. Dis. 9:978–984. 54. von Eiff, C., K. Becker, K. Machka, H. Stammer, and G. Peters. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. N. Engl. J. Med. 344:11–16. 55. Wertheim, H.F., D.C. Melles, M.C. Vos, W. van Leeuwen, A. van Belkum, H.A. Verbrugh, et al. 2005. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis. 5:751–762. 56. Zriouil, S.B., M. Bekkali, and K. Zerouali. 2012. Epidemiology of Staphylococcus aureus infections and nasal carriage at the Ibn Rochd University Hospital Center, Casablanca, Morocco. Braz. J. Infect. Dis. 16:279–283.
Address correspondence to: Marta Aires-de-Sousa, PhD Escola Superior de Sau´de da Cruz Vermelha Portuguesa (ESSCVP) Avenida de Ceuta n1, Edifı´cio UrbiCeuta Lisboa 1300-125 Portugal E-mail: [email protected]
