Characterization of methicillin-susceptible and -resistant staphylococci in the clinical setting: a multicentre study in Nigeria

The staphylococci are part of the normal cutaneous ecosystem, but they are also opportunistic and invasive pathogens that cause a variety of human infections. Staphylococcus aureus is the most important human pathogen in this genus accounting for a high proportion of severe infections in hospitals and outpatient medical care [1‐3]. Although the clinical relevance of coagulase negative staphylococci (CNS) is still controversial, patients at risk of CNS infections include neonates, those with intravascular catheters, prosthetic devices, postoperative sternal wound infections and immunocompromised hosts [4‐7]. The remarkable ability of S. aureus and CNS to acquire antibiotic resistance limits therapeutic options, and morbidity and mortality rates of staphylococcal infections have increased the financial burden on health care systems worldwide [8‐14].

Although there are reports on the characterization of staphylococci in Nigeria [15‐18], many of the studies were limited to antibiotic susceptibility profiles. Moreover, one geographical region or health care institution was investigated, and CNS were not identified to species level. Only a few studies have investigated the molecular epidemiology and diversity of this group of organisms [19‐23], however, they were not multi-centre surveys on S. aureus and CNS. This work describes a multi-centre study which was conducted in order to provide clinicians and infection control practitioners with information on antibiotic resistance profiles, molecular characteristics of coagulase positive and negative staphylococci, and to alert clinicians to the emergence of new clones in health care institutions (HCIs) in Nigeria.

The identification of bacterial isolates to species level is of great importance in the clinical microbiology laboratory to obtain information on the diversity and significance of each species in human infection. Moreover, species identification is a prerequisite for epidemiological studies. Many clinical microbiology laboratories in Nigeria do not identify staphylococci (in particular CNS) to species level, which is important in understanding their variety and clinical relevance. Two species, S. haemolyticus and S. sciuri, accounted for the majority of the 40 CNS isolates investigated in this study (Table 3). Most of the S. haemolyticus isolates were obtained from blood culture samples in HCIs in Lagos2 and Jos, and S. sciuri from samples in Ogbomosho (South-West Nigeria). S. haemolyticus is regarded as the second most frequently isolated staphylococci from blood cultures [32] and one of the most clinically relevant CNS, particularly in immunocompromised patients [33]. S. sciuri, principally found in animal species, and although not considered important from the clinical standpoint, has been associated with infections such as endocarditis [34], urinary tract infection [35] and wound infections [19, 36]. Multiresistance to antibiotics was detected in 37 of the 40 CNS isolates, and all the S. haemolyticus isolates were resistant to at least three classes of antibiotics. Apart from the possible clinical importance because of its serious impact on the course of infection, multiresistant CNS are a potential source of genes encoding resistance to antibiotics for other staphylococci pathogenic for man. The isolation of multiresistant mecA positive S. haemolyticus from blood samples (septicaemia) and S. sciuri from various clinical materials (Table 3) in some HCIs clearly indicate that they could be of clinical importance in Nigeria. A number of reports have indicated an increase in the resistance of staphylococci to cotrimoxazole in Nigeria [15, 18, 21, 23, 37]. In this study, 72.5% of CNS and 82.3% of S. aureus isolates were resistant to cotrimoxazole. The antibiotic has wide clinical application, inexpensive, orally administered and available over-the-counter where they are sold with or without prescription in Nigeria. This could possibly explain the high level of staphylococcal resistance observed in this study. Most of the cotrimoxazole-resistant S. aureus were dfrA-negative; hence more studies are needed to understand the molecular mechanism of resistance in Nigeria.

Methicillin resistance in staphylococci is due to the expression of the mecA gene, which mediates penicillin binding protein 2a (PBP2a), a transpeptidase with low affinity for beta-lactams [7, 38]. The mecA gene is carried by a mobile genetic element (MGE) termed the staphylococcal cassette chromosome (SCCmec), and though the mec origin remains unknown, it has been suggested that mecA CNS may act as potential SCCmec donor accounting for the rise of new MRSA clones [39]. Eleven SCCmec elements have been described to date: SCCmec I-IV [40‐42], and V-XI [43‐48], but few reports exist on the detection of mecA gene and characterization of SCCmec types in Nigeria [21‐23]. There was excellent correlation between results of antibiotic susceptibility testing (VITEK) and detection of the mecA gene for the confirmation of methicillin-resistant staphylococci. However, the MRCNS were non-typeable using the multiplex PCR protocol [26]. Based on the method of Kondo et al. [27], the SCCmec V element (ccrC/Class C2 mec) was identified in representative S. haemolyticus isolates, as well as untypeable SCCmec elements (ccr-negative/Class A mec and ccr type 4/Class C2 mec gene complex) in one representative isolate each of S. sciuri and S. haemolyticus (Table 3). Recent studies have indicated that SCCmec V is the predominant element among methicillin-resistant S. haemolyticus isolates [33, 49]. To the best of our knowledge, this is the first report on the characterization of SCCmec elements in CNS in Nigeria. It is likely that the mec gene complex and ccr genes in CNS are diverse and distinct, and as indicated in previous reports [7, 28], there is the need to develop new classification schemes for non-typeable SCCmec types in MRCNS.

Molecular typing of the MRSA isolates indicated that two clones (CC8-SCCmecnon-typeable and CC88-SCCmec IV) existed in Maiduguri, North-East Nigeria, and four clones in South-West Nigeria (CC8-SCCmecIII/IV/V; CC30-SCCmecII/III; CC88-SCCmecIV and ST152-SCCmecnon-typeable) (Table 1). Previous studies on the epidemiology of MRSA in Nigeria identified MRSA clones t064-ST8-CC8 and t037-ST241-CC8 in South-West and North-East Nigeria, respectively [22, 23]. A community-associated clone, CC88-MRSA-IV, previously reported in Ibadan, Nigeria [21] was identified from wound and blood samples in Maiduguri and Ile-Ife, however, we were unable to determine whether the isolates were community-associated. Untypeable SCCmec types (ccrC/Class A mec; ccr-negative/Class C2 mec) were detected in two representative MRSA isolates, of which one was a PVL-positive isolate (t4690-ST152) from a patient with osteomyelitis in Ile-Ife (South-West Nigeria).

The MSSA were assigned mainly to clonal complexes CC5, CC8, CC15, CC30, CC45, CC80 and ST152 (Table 2). A high proportion of MSSA from Maiduguri were grouped in ST15 or ST152 indicating that these clones were successful in North East Nigeria. PVL-encoding genes were detected in 16 MSSA (44.4%) isolates belonging to almost all clonal complexes identified in this study. Moreover, 56% of these strains were associated with ST152 and 10 of 16 (62.5%) PVL positive MSSA isolates were associated with SSI and septicaemia. These observations confirm previous studies that PVL-positive MSSA ST152 are widespread in African countries [50‐52] including Nigeria [22, 23]. Furthermore, MSSA isolates (ST121) from nasal samples of HCWs were PVL positive. Our data suggests that the sequence types for pandemic PVL-positive MSSA identified in this study did not function as a reservoir for PVL-positive MRSA supporting the argument of Schaumburg et al. [52] that the intimate inter-relation between PVL-positive MSSA and MRSA, which could lead to the emergence and spread of PVL-positive MRSA clones has not been established. However, the detection of an ST152-PVL-positive MRSA in this study suggests that surveillance for this clone is strongly advocated in Nigeria.

This is the first multi-centre study on the characterization of coagulase positive and negative staphylococci using phenotypic and molecular methods in Nigeria. However, there were a number of limitations in this study. The number of isolates from the HCIs was low and variable which did not allow for comparison of data among the participating centres. Moreover, the diversity of the staphylococci analysed in this study does not represent their distribution patterns in health care institutions in Nigeria. Lastly, we could not conduct full characterization of the mecA-positive CNS isolates, and only representative isolates were selected for SCCmec typing and MLST. However, this study has highlighted the importance of species identification for CNS, their clinical relevance, and the need for longitudinal multi-centre surveys to provide data on antibiotic resistance and epidemiology of the staphylococci in Nigeria. Characterization of multiresistant mecA positive S. haemolyticus and S. sciuri from clinical samples is important in understanding their epidemiology within hospitals in Nigeria. The identification of untypeable SCCmec types in the study will be further evaluated and characterized; however, there is the need to develop new SCCmec classification schemes for non-typeable methicillin-resistant staphylococci. There is an urgent need to curtail the spread and establishment of PVL-positive MSSA, in particular, the MSSA ST152 clone, in Nigeria.