S. aureus, the prominent gram-positive pathogen is responsible for a wide variety of diseases encompassing both hospital and community-acquired infections. While these bacteria deploy numerous virulence factors in the initiation, persistence and dissemination of infections, antibiotic resistance stands as a formidable attribute enhancing their survival and success. This study aimed at characterizing the S. aureus isolates by whole genome sequence and determining their quinolones resistance level.
The major finding from this study was a nearly equal proportion of MRSA and MSSA strains comprising of 49.3% and 50.7% respectively. The occurrence of this potential MRSA strain exhibited positive associations albeit not statistically significant, with factors such as the presence of septic wounds, patient age, department of admission, and admission type. Given the broad range of infections caused by MRSA, there is an urgent need for an effective and sustainable surveillance system to monitor MRSA-associated infections in hospitals and communities. The presence of the mecA gene in a mobile genetic element emphasizes the importance of such surveillance. This MRSA proportion aligns with previous studies conducted in Moshi, Tanzania (43.3%), and among patients and healthcare workers in Uganda (Kumburu et al., 2018; Busingye, Bazira, and Musinguzi, 2023). However, it contrasts with a study using whole-genome sequencing, which reported a lower MRSA proportion of 33.3%. Nonetheless, this proportion is consistent with MRSA prevalence in several African countries (Wangai et al., 2019) but higher than a study in Libya (16%) (Ahmad et al., 2018). The disparity can be attributed to sample size, demographics, infection control measures, and MRSA detection methods.
Nonetheless, majority of these MRSA 57(82.6%) demonstrated resistance to quinolones (ciprofloxacin/Nalidixic acid). However, none isolates from MSSA demonstrated resistance to quinolones. The observed resistance to quinolones in MRSA were found to be mediated by combination of single point mutations in two non-resistance genes i.e., grlA (p.S80Y) and gyrA (p.S84L). These genes encode for two replicating enzymes topoisomerase IV and DNA gyrase respectively. The point mutations in grlA gene took place at position 80 whereas amino acid Serine was replaced by Tyrosine and that from gyrA took place at position 84 whereas amino acid Serine was replaced by Leucine. The observed mutations were simultaneously found to occur in all MRSA isolates that exhibited resistance to quinolones. It is worth noting that, a high level of quinolone resistance results from the observed combination of point mutations. Nonetheless, no point mutation in either grlB or gyrB genes, whereby a sole or combining mutations in gyrB/gyrB would confer quinolone resistance of low level.
Nevertheless, since no point mutations were found in either grlB/gyrB which alone would confer low level of resistance, therefore no isolates exhibited quinolone resistance of low level. This finding is consistent with a study in India (Gade and Qazi, 2013) but contrasts with studies in Uganda and Egypt, which reported no quinolone resistance among MRSA strains and different mutation patterns (Hashem et al., 2021; Busingye, Bazira, and Musinguzi, 2023). This discrepancy can be explained by population differences and detection methods used. Since quinolone resistance was not observed in MSSA strains, suggesting a need to reconsider the use of quinolones for MRSA-related infections.
Multilocus sequence typing identified 14 unrelated sequence types among the 140 S. aureus isolates, with ST8 being dominant among MRSA and ST152 among MSSA. This diversity suggests epidemiological unrelatedness among the studied isolates, enhancing their adaptability and spread. The SCCmec elements harbored in MRSA were further typed and all isolates were observed to harbored mec elements type IV. Further genotyping was done regarding Staphylococcus protein A (Spa) and again, diverse numbers were found. However, spa type 1476 and 355 were predominantly found in MRSA isolates and MSSA respectively. Thus makes the overall common MRSA genotype of ST8-SCCmecIV-spa 1476, which is a typical genotype of Community Acquired Methicillin-resistant S. aureus (CA-MRSA). The predominance of these CA-MRSA isolates, commonly associated with community-acquired MRSA, raises concerns about its prevalence in hospital settings. This highlights the urgency of controlling its spread and suggests that Community-acquired MRSA may eventually replace Hospital-acquired MRSA. Therefore, hospital authorities should apply proper infection control measures to prevent nosocomial transmission. These results are in line with a similar study conducted in Dar-es-salaam Tanzania (Manyahi et al), but contrary to study conducted in Eastern Tanzania. The discrepancy can be explained by differences in study sites and nature of samples sequenced (Tutu Mzee 2023)
High genetic diversity among the isolates was observed among those isolated from Dodoma Regional Referral Hospital and likewise from Tabora Regional Referral Hospital. Nevertheless, despite the high genetic diversity observed, some isolates have been found to form clusters suggesting they are closely related and share a common ancestor (Fig. 5). Most of the MRSA isolates from Mnazi Mmoja Referral Hospital were observed to exhibit closer genetic relatedness, suggesting they share a common ancestor between them. In this instance, a standalone cluster made up of 7 isolates with SCCmecIV-spa-t498 genotype with minor SNPs difference of 0 to 9 between them (FFZB_126, FFZB_79, FFZB_189, FFZB_154, FFZB_64, FFZB_48, and FFZB_201). It is worth considering that, these 7 isolates were collected from patients enrolled from different wards with the Surgical ward being the predominant one. Moreover, the same scenario has been observed among isolates from Morogoro Hospital, with four clusters found to exist among the isolates. The isolates forming all of the above-mentioned clusters were observed to comprise MRSA with genotype ST8-SCCmecIV-spa-t1476. Notably, a subset of MRSA isolates all originating from Mnazi Mmoja Hospital exhibited the genotype of ST8- SCCmecIV-spa-t498.
Aside from mecA gene, several other resistance genes such as blaZ, dfrG, aac (6’)-aph (2’’), ermC tet(K), were found distributed among the isolates. However, majority of these resistance genes were significantly observed in MRSA compared to MSSA. Intriguingly, numerous combinations of resistance genes were observed within the isolates, with a significant proportion exhibiting co-occurrence of two or more genes. Only 12% of isolates carried blaZ as the sole resistance determinant. Prominent combinations included blaZ-dfrG, aac (6’)-aph (2’’)-blaZ-dfrG-ermC-mecA and aac (6’)-aph (2’’)-blaZ-dfrG-ermC-mecA-tet(K), accounting for 20.7%, 17% and 10% respectively. The latter two combinations were exclusively identified in MRSA isolates, while the first combination was solely observed in MSSA isolates. Of more concern, majority of these genes confer resistance to non β-lactams drugs which to an already β-lactams drugs resistant MRSA isolates, thus further narrowing the treatment options. It is therefore important that the surveillance, screening for drug resistance should be routinely done in our hospitals for earlier detection of resistant strains.