Isolation and Identification of Methicillin Resistant Staphylococci at Bethzatha Advanced Laboratory, Addis Ababa, Ethiopia

Introduction

Staphylococci are gram positive bacteria most of which colonize human and animal skin as transitory or commensal flora [1]. Coagulase negative staphylococci (CoNS) have been frequently isolated as an agent of nosocomial infections in last twenty years although they are pathogens which have low virulence rate [2]. They are colonized in hospital environment and hospitalized patients’ skin. Immunosuppressive therapies, invasive procedures, common usage of broad-spectrum antibiotics tend to result in bacterial infections. CoNS are an important agent that may lead to hospital acquired bacteraemia [3]. However when any bacteria are in the wrong site or other than their normal niche can cause infections. Staphylococcus aureus can be pathogenic if introduced into normally sterile sites such as blood stream, urinary tract, cerebrospinal and other internal tissues.

Methicillin is a drug that was introduced to treat patients with infections that is caused by penicillin resistant staphylococcus aureus [4]. Methicillin, the first semisynthetic penicillinase resistant penicillin, was widely used initially until methicillin resistant S aureus (MRSA) was found in England in 1961 [3]. MRSA acquired drug resistance via incorporation of the mecA gene, which encodes an alternative penicillin-binding protein, PBP 2a or its ortholog into the chromosome. Shortly after the introduction of methicillin in clinical world to treat infections caused by penicillinase producing S. aureus, methicillin resistant S.aureus (MRSA) emerged and spread worldwide [4]. The high rate of methicillin resistance among Staphylococcus aureus has resulted into the increased interest for the use of other drugs such as clindamycin, vancomycin and oxacillin for treatment of infections caused by MRSA.

Methicillin resistance in S. aureus is mediated through an altered protein called low-affinity penicillin binding protein (PBP2a). PBP2a is encoded by mecA gene which is present in chromosomal mobile genetic element called Staphylococcal cassette chromosome mec (SCCmec) specific site, encoding an alternative penicillin-binding protein that has low affinity for almost the entire class of β-lactam drugs including methicillin, oxacillin, and most cephem agents [5, 6]. Thus, MRSA is defined as S. aureus isolates genetically containing mecA or mecC or phenotypically showing resistance to oxacillin conventionally or to cefoxitin [7]. Cefoxitin is a more potent inducer of mecA and disk diffusion tests using Cefoxitin reveal clearer endpoints that are easier to read than tests with oxacillin [8]. In the recent past, there have been multiple reports on the use of cefoxitin as a surrogate marker for detection of mecA. However, some clinical isolates are mecA-positive and oxacillin susceptible and defined as oxacillin-susceptible MRSA (OSMRSA). Due to possible association of MRSA with multiple antibiotic resistance and relatively difficult and higher cost of treatment, the accurate and rapid identification of MRSA is crucial in clinical world for timely management of the infections caused by this superbug. Thus the present work is to determine methicillin resistant S. aureus and coagulase negative staphylococci (CoNS) from different clinical specimens.

Result

A total of 114 staphylococci, 40(35%) S. aureus and 75(65.8%) coagulase negative staphylococci (CoNS) were isolated from different clinical specimens The most dominant coagulase negative isolates were S.haemolyticus, 21% (16/75), S.epidermidis, 20%(15/75), and S.auricularis,

19% (14/75) (Table 1). S. aureus was most frequently 54% (26/48) isolated from pus followed by 34% (15/44) from blood cultures. The most frequent CoNS isolate from blood culture was S. haemolyticus, 36% (13/36) followed by S.epidermidis, 19% (7/36) (Table 1).

S. auricularis was isolated most frequently, 28.6 % (6/21) followed by S.epidermidis, 19 % (4/21) from pus and body fluids. Out of 114 staphylococci isolates, 51(44.3%) were methicillin resistant (both cefoxitin screening positive and oxacillin resistant) (Table 2). Out of 40 S. aureus isolates,

30% (12/40) were methicillin resistant. Similarly, of 75 CoNS, 52% (39/75) were methicillin resistant. S.hemolyticus, S.homini and S. epidermidis 16/16(100%), 7/9 (78%) and 11/15(73%) were methicillin resistant respectively (Table 2).

Table 3 shows antimicrobial susceptibility patterns of methicillin resistant staphylococci isolated from different clinical samples. All methicillin resistant S. aureus isolates

12/12(100%) were resistant to amoxicillin clavulnate, azithromycin, ampicillin and 11/12(91.7%) to oxacillin and clindamycin. Among CoNS, S. haemolyticus, 16/16(100%)

was resistant to amoxicillin clavulnate, oxacillin and 15/16(93.7%) to ciprofloxacin, ampicillin and imipenem. Simillarly all 11/11(100%) methicillin resistant S. epidermidis were resistant to amoxicillin clavulnate, ampicillin, oxacillin and penicillin (Table 3). On the other hand 9/12(75%) of S. aureus isolates were susceptible to Daptomycin, vancomycin, Fusidic, linozolin and all, 16/16 (100%) S. haemolyticus were susceptible to teicoplanin and vancomycin.

Discussion

In the present study methicillin resistant S.aureus was frequently isolated from pus and discharges and blood culture. The rate of methicillin resistant S. aureus, 30% in the present study is comparable to 28.3% rate of MRSA isolation from Desie hospital, North West Ethiopia [10], but lower compared to the national pooled rate 32.5% [6], 68% reported from Awasa, southern Ethiopia [11] and 35.6% from Tikur Anbesa Hospital Addis Ababa, Ethiopia [12]. However 30% methicillin resistant rate of S.aureus in the present study is higher than 23% methicillin resistance reported from Jimma, South west Ethiopia [13], 13.2% from Debrmarkos referral hospital, north west Ethiopia [14]. A study from Arbaminch Southern Ethiopia Mekuriya E, et al. [15] reported only 7.4% methicillin resistant S. aureus from nasal colonization which is relatively lower rate than most of the above studies. The variation observed could be due to the source of bacteria, the nature of the study participants, the laboratory methods used, and the study methods applied. Most (92%- 100%) of methicillin resistant S.aureus in the present study were multiply resistant to commonly used antibiotics, amoxicillin clavulnate, azithromycin, ampicillin, penicillin, clindamycin, ciprofloxacin and erythromycin. Multi-drug resistance to penicillin, ampicillin, amoxicillin, and ciprofloxacillin of MRSA isolates has been reported by many other workers from Ethiopia and elsewhere [16]. On the other hand, most (75%, 9/12) methicillin resistant S.aureus in the present study were susceptible to daptomycin, vancomycin, linezolid and fusidic acid. Dhunggel S, et al. [17] studied the prevalence and antimicrobial susceptibility of methicillin resistant staphylococcus aureus from tertiary heart care centre, Nepal, and found that none of the isolates was resistant to vancomycin. A similar pattern of susceptibility was documented by Nystinga J, et al. [16] from Kenya. In the present study Coagulase negative staphylococci (CoNS) were predominantly isolated from blood culture and pus and discharges and were frequently methicillin resistant. Other workers have also reported that Coagulase-negative staphylococci are among the most frequently isolated microorganisms in blood cultures [18]. The commonest CoNS isolated from blood cultures were S.haemolyticus, S.homini and S.epidermidis. In the present study, these CoNS were most resistant to azithromycin, ampicillin, and penicillin. Researchers from elsewhere also reported that infection-associated with CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin and multidrug-resistant isolates [17]. Asante J, et al. [18] has documented a similar observation from South Africa. Coagulase negative staphylococci in the present study were highly susceptible to daptomycin, teicoplanin and vancomycin. The high susceptibility of CoNS to vancomycin in present study agrees with reports from Osaka city Yamada K, et al. [19] and south India [20]. Coagulase negative staphylococci are mostly opportunistic and are increasingly associated with nosocomial infections, especially among the immunocompromised and those with invasive medical devices, and raise a significant health problem [21].

Conclusion and Recommendation

Multidrug resistant bacterial infection has become global problem making treatment option restricted. The situation is more challenging to low and middle economy countries. In the present study isolation of methicillin resistant Staphylococcus aureus and coagulase negative staphylococci is considerably high, besides, these strains showed extreme resistance to commonly prescribed antimicrobials such as penicillin, ampicillin, erythromycin and azithromycin. In principle, appropriate use of antibiotics, applying safety precautions are the key to reduce the spread of multidrug resistant bacteria including methicillin resistant staphylococci and other bacteria. So Periodic surveillance and appropriate control measures and safety precautions of nosocomial and community acquired MRSA and CoNS must be given prior attention. Furthermore, strengthening microbiology laboratory with necessary reagents, equipment and skilled man power is critical if we are to control the spread of these pathogens.