Methicillin resistant staphylococci isolated in clinical samples: a 3-year retrospective study analysis

Aim: To determine the prevalence and describe the antimicrobial resistance patterns of circulating methicillin-resistant staphylococci (MRS) isolated from clinical specimens during a 3-year period in Yaoundé, Cameroon. Materials & methods: From January 2017 to December 2019, 1683 clinical samples were plated onto Mannitol salt agar. Bacterial identification was performed followed by antibiotic susceptibility testing. Data were analyzed using R program. Results: Staphylococci were identified in 90 (5.35%) of the 1683 clinical samples. Among these, 83.33% were MRS with 78.67% being methicillin-resistant Staphylococcus aureus (MRSA). The prevalence of MRS infection increased significantly with age. Conclusion: The study offers a good baseline for surveillance intervention to contain antimicrobial resistance and highlights the need to strengthen antimicrobial stewardship and infection, prevention and control programs in the country.

sulfamethoxazole were the antibiotics being tested. The resulting breakpoints were interpreted according to Clinical Laboratory and Standards Institute guidelines [6].

Data collection & statistical analysis
Information regarding patient personal details, specimen collection, sampling and laboratory results is deposited on the CEDBCAM (www.cedbcam.com) laboratory database under a software named ToolPro Manager for Health. The access to and use of this database is password-protected and restricted to laboratory staff working within the CEDBCAM. Therefore, data collection was undertaken within the CEDBCAM with extraction of isolate information being sourced from this computerized laboratory database. Data received was deduplicated and all the patient information was codified to ensure the ethical confidentially at all times. Participant demographics information including age, gender, isolate information, specimen type and antimicrobial susceptibility testing results were extracted and tabulated in an MS Excel Spreadsheet (version 2016). Upon data extraction, trends in the total number of staphylococcal isolates and their antimicrobial resistance patterns were assessed. These trends were subsequently compared over a 3-year period. R and inZight R (version 3.5.3) were used for the statistical analyses. Discrete variables were expressed as percentages and proportions with trends or associations being assessed using the standard Pearson's Chi-square (χ 2 ) test. A p-value below 0.05 was considered statistically significant.
The mean age of patients infected by MRS was quite similar in female (35.17 years, SD ± 15.02) and males (34.70 years, SD ± 12.59) with 31 and 34 years being the median in females and males, respectively. The Chi-square analysis revealed that the number of patients with MRS infection increased significantly with age (χ 2 = 16.936; p = 0.005). More specifically, the Figure 2 shows that MRS was more frequently detected in patients of ages within 26-35 years (34/75; 45.33%) and over 45 years (16/75; 21.33%). This age correlation was further observed to the species level where, MRSA prevalence was associated with similar age groups of 26-35 years (24/59; 40.68%) and over 45 years (14/59; 23.73%; Figure 2).

Discussion
MRSA was recognized as pathogen of high priority for research and development of new antibiotics by the WHO [7] given its serious socio-economic repercussions globally. In this study, the prevalence and antimicrobial resistance patterns of MRS isolated from clinical samples in a private laboratory in Cameroon were retrospectively analyzed.
Of the 1683 samples analyzed over the 3-year period, 90 (5%) staphylococcal isolates were recorded. The results obtained in the present study demonstrated the high prevalence of MRS (83%) with 78.67% being MRSA. The   Table 4. Antimicrobial resistance profiles of methicillin resistant coagulase negative staphylococci isolated from clinical samples.

Staphylococcus spp. (n = isolates) Resistance patterns (n = isolates) Antibiotics (n) Classes (n)
S. xylosus ( MRS prevalence reported in our study is consistent with studies from Cameroon where 72 and 75% of MRS were detected in clinical [3] and carriage [1] samples in Yaounde, respectively. In contrast, the results are lower than that reported from a survey conducted in a tertiary hospital in Northern Thailand where a maximum prevalence of MRS (100%) was detected from clinical isolates [8]. These results are however, higher in comparison with that reported in studies from other part of the world. A report from Congo demonstrated a 63.5% prevalence of MRS among hospitalized surgical patients with 63.5 and 60% being MRSA and MR-CNS, respectively [9]. Likewise, a 4-year cross-sectional study conducted in India revealed a 34% prevalence of MRS from clinical specimens [10], while Ramsamy et al. showed a decreasing MRSA prevalence ranging from 28% to 18% over a 5-year period (2011)(2012)(2013)(2014)(2015) in KwaZulu-Natal, South Africa [11]. In contrast to low-and-middle-income countries, lower MRSA prevalence (<25%) has been reported in European countries [2]. These discrepancies could be explained by the lack of effective implementation of infection, prevention and control measures in hospitals as well as irrational use of antibiotics in resource-constrained settings [4]. Moreover, the high MRS prevalence, especially MRSA, observed in our study could be attributed to the high burden of infectious diseases coupled with suboptimal hygiene and sanitation. Additionally, given that antimicrobial resistance is a neglected concern and antibiotic use is not necessarily well regulated in Cameroon, lack of monitoring, prevention and control measures likely engender extensive antibiotic consumption and subsequent high resistance rates in the country. Comprehensively delineate the molecular nature and epidemiology of MRS circulating in communities and hospitals in Cameroon is required in order to inform evidence-based strategies for rapid monitoring, prevention and containment of antimicrobial resistance.
Our findings revealed that the prevalence of MRS varied across year. Although, these changes may be attributable to shifts in incidence between hospital and community-based patients, we postulated that the variation observed could also be related to several factors including difference in demographics (ethnicity, socio-economic status, geography, etc.), practice variability among clinicians and laboratorians, as well as seasonal variation. This suggests that population-based studies are required to establish appropriate distribution and determinants of MRS infectious diseases.
Overall, females were more positive to MRS than males. It is unclear, why females were more positive than males especially given that previous reports revealed that males are more prone to bacterial carriage and infection albeit females might have a poorer outcome [12,13]. Our result shows that further research investigating the relationship between gender and risk of infection, the reason for higher MRS infection rates in females as well as other factors of infection incidence such as age and comorbid conditions are required.
The analyses of resistance profiles revealed that MRS isolates exhibited high level of MDR (70%) with >90% resistance to β-lactams including penicillin and cefoxitin for both MRSA and MR-CNS. This MDR prevalence is lower than that reported in a previous Cameroonian study where 100% of MRSA were MDR [3]. Co-resistance to non-β-lactam antibiotics, including trimethoprim-sulfamethoxazole, ciprofloxacin, tetracyclin, doxycyclin, gentamicin and fosfomycin was also observed. Such co-resistance to β-lactams together with other antibiotic families is frequent in MRS and could likely result from the indiscriminate and/or extensive antibiotic use (misuse, overuse and inappropriate use) in the country [14]. The co-resistance could also be explicated by the presence of mobile genetic elements such as plasmids, integrons, transposons and insertion sequences that are responsible of further resistance. One MRSA isolate displayed the phenotype FOX.P.CN.VA.DA.E.LEV.TE.FF.FA.C.TMP/SXT showing resistance to 12 antibiotics including the last resort drug vancomycin while one MR-CONS isolate displayed the profile FOX.P.CN.NET.E.CIP.LEV.SPA.TE.DOX exhibited resistance to ten antibiotics.
It is noteworthy to mention that the heterogeneity of the samples that were collected from both clinically ill and asymptomatic patients preclude any conclusion it was not possible to distinguish between infection from colonization.

Conclusion
The high prevalence of MRS and MDR MRSA and MR-CONS observed in this study highlights the need to strengthen antimicrobial stewardship and infection, prevention and control programs in the country. More multidimensional molecular epidemiological studies are urgently needed if the country is to reach United Nations Sustainable Development Goal (UN SDG) three of ensuring healthy lives and promoting well-being for all.

Limitations
Heterogeneity of the samples preclude robust conclusion regarding the association of MRS with infections given that sample were collected from both clinically ill and apparently asymptomatic patients. Moreover, although being performed over a 3-year period, the study was conducted in a single geographic area, hence the findings might not represent the broad population and whole country. The absence of molecular tests and genotyping also hinder appropriate molecular epidemiological data that could be used not only to prevent outbreak situations but also to invigorate adequate antibiotic resistance containment measures.

Future perspective
This study underscores the importance of routine screening, and monitoring of MRS as with other resistant bacteria. It further reveals that molecular techniques despite being uneconomical at small scale should also be integrated in the diagnostic confirmation of infectious diseases and especially MDR in developing countries such as in Cameroon. Upcoming efforts for better understanding of MRS and MRSA should therefore focus on two main areas namely: host and pathogen interaction and evaluation of MRS genomics, proteomics, metabolomics and epigenetics in animal models. More imminently, high-quality clinical and molecular epidemiological studies are needed to inform appropriate public health strategies and interventions. The study revealed that females were more infected by MRS than males, thus future studies investigating MRS in neonates should further be implemented to evaluate the incidence of MRS on neonates born from a similar cohort.

Summary points
• Methicillin-resistant staphylococci (MRS) are major source of infections in hospital and community settings.
• Trends and antimicrobial resistance profiles of circulating MRS strains were ascertained in Yaoundé, Cameroon.
• MRS was detected in over 80% of positive specimen with 79% being methicillin-resistant Staphylococcus aureus (MRSA). • Altogether, 65 resistance patterns were observed in MRS with resistance to a minimum of two (n = 4) and a maximum of 12 antibiotics (n = 1). • MRS isolates displayed around 70% of multi-drug resistance with both MRSA and methicillin-resistant coagulase-negative staphylococci displaying similar multi-drug resistance prevalence (around 70%). • The prevalence of MRS infection increased significantly with age.
• The high prevalence of MRS especially of MRSA observed in this study highlights the need to strengthen antimicrobial stewardship and infection, prevention and control programs in the country.