Antibiogram of Bacterial Isolates from the Anterior Nares and Hands of Health Care Workers in University of Uyo Teaching Hospital (UUTH) Uyo, AkwaIbom State, Nigeria

Frequently, most of the nosocomial pathogens are multidrug resistant bacteria which pose serious therapeutic challenges. Hospital outbreaks of methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), extended-spectrum beta lactamases (ESBLs) producing Gram negative bacteria which are resistant to cephalosporins and monobactams, and multidrug resistant Pseudomonas have severally been reported [1-3].


Introduction
Hospital acquired infections are increasingly becoming a major concern in both developed and developing economies. In hospital admitted patients, surgical site infections, catheter associated urinary tract infections, intravenous devices infection and respiratory infections are frequent causes of prolonged hospital stay, morbidity and mortality.
Frequently, most of the nosocomial pathogens are multidrug resistant bacteria which pose serious therapeutic challenges. Hospital outbreaks of methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), extended-spectrum beta lactamases (ESBLs) producing Gram negative bacteria which are resistant to cephalosporins and monobactams, and multidrug resistant Pseudomonas have severally been reported [1][2][3].
The role of health care workers in the transmission of infections has been extensively described [2]. Most often, spread is from patient to patient on the hands of health care workers, person to person through direct contact and on medical devices. The importance of hands in the transmission of hospital infections is worldwide accepted [4,5].
However, it may not be the regular practice to include hand washing (HW) as routine behavior in health-care workers, since microorganisms are invisible. or there are no adequate elements to carry out this practice [6].
Health-workers hands by themselves, or after contact with patients, increase the risk of virus and bacteria transmission that are sometimes resistant to antimicrobial agents (AMA) [4]. This is a two-way hazard that could be noxious to both patients and health-care workers, and which depends on the nature and frequency of contact with infectious materials, inoculum and prevalence of susceptible patients [4]. Despite the knowledge of the importance of this fact by health-care workers (HCW), they do not practice hand washing [6][7][8], which is more neglected by physicians nurses [9,5].
The need for intervention is imperative with several reports still documenting poor hand washing among health-care workers [10,11]. A targeted surveillance culture to identify asymptomatic carriers of multidrug resistant bacteria and subsequent isolation and treatment had been recommended for minimizing their spread within hospitals [1]. The knowledge of sensitivity patterns of isolates will also be useful in predicting possible risk of infection with multi-drug resistant bacteria in health-care settings.
This study is therefore undertaken as part of a wide scale hospital infection control surveillance programme of the University of Uyo Teaching Hospital, (UUTH), Uyo, Nigeria. It seeks to ascertain the frequency of bacterial colonization of the anterior nares and hands of healthcare workers who are in regular contact with patients. It also seeks to determine multidrug resistant isolates for possible intervention.

Materials and Methods
This study was carried out at the University of Uyo Teaching Hospital (UUTH) located in Uyo, AkwaIbom State. The hospital is a tertiary center that receives referrals from primary and secondary health institutions in the State and beyond.
A total of 60 samples consisting of 30 nasal swabs and 30 hand swabs were collected from 30 healthcare workers in University of Uyo Teaching Hospital. Of all the healthcare workers, 15 (50%) were doctors and 15 (50%) were nurses, which consisted of 11 (36.7%) males and 19 (63.3%) females. The media used in this study were Mannitol Salt Agar, Blood Agar, MacConkey Agar and Mueller Hinton, which were provided by the tertiary hospital. Sterile cotton wool swabs, which were premoistened in sterile normal saline, were used to swab the anterior nares and another used to swab the interdigital spaces of both hands of the participants. All media were prepared according to the manufacturer's instructions. The nasal swabs were aseptically swabbed and cultured on Mannitol salt agar plates. Yellowish colonies from the Mannitol salt agar plates were sub-cultured on blood agar base supplemented with 5% sheep blood. Plates were incubated aerobically at 35°C for 24-48 hrs. Isolates were presumptively identified as S. aureus using coagulase test. The swabs from interdigital spaces of both hands were cultured on Mannitol salt agar, Blood agar, MacConkey agar and Mueller Hinton agar plates. The bacterial isolates were identified using bacteriological procedures, involving morphology, microscopy and biochemical tests [12,13]. Antimicrobial susceptibility testing was carried out using the modified Kirby-Bauer disc diffusion method, according to the Clinical Laboratory Standard Institute (CLSI) guidelines (CLSI, 2008).
Methicilin resistance was tested using 1 μg oxacillin disc (Oxoid Ltd, Cambridge, UK), according to CLSI guidelines. Mueller Hinton Agar was inoculated with 0.5 McFarland preparation of the inoculum using the spread plate technique. 1 μg Oxacillin disc was placed on the agar and incubated aerobically at 35°C for 24 hrs. Organisms with zones of inhibition ≤ 12 mm were interpreted as methicillin resistant, while those with zones of inhibition ≥ 13 mm were interpreted as methicillin susceptible.
Inducible Macrolide Lincomycin Streptogramin B (iMLS B ) was done for all Staphylococcus isolates which were resistant to Erythromycin, but susceptible to Clindamycin, as described by the CLSI (CLSI, 2008). Mueller Hinton Agar plates were inoculated with 0.5 McFarland preparation of the inoculum using the spread plate technique. Then a 15 µg Erythromycin (Oxoid ltd, Cambridge, UK) and 2 µg Clindamycin discs were placed at 15-26 mm apart. The plates were incubated for 18-24 hrs at 35°C aerobically. Isolates that showed flattening of the Clindamycin zone of inhibition adjacent to the erythromycin disc (referred to as a D-zone) was regarded as exhibiting inducible Clindamycin resistance.
Enterobacteriaceae isolates with cefotaxime, ceftazidime and cefpodoxime zones of inhibition less than 27 mm, 22 mm, 17 mm, respectively, were suspected to be ESBL producing. A confirmatory test using the Double Disc Synergy technique was carried out according to CLSI (2008) guidelines. Mueller Hinton Agar plates were inoculated with 0.5 McFarland preparation of the inoculum using the spread plate technique. Then amoxicillin-clavulanate (20 µg-10 µg) disc was sandwiched by 30 µg ceftazidime, 30 µg cefpodoxime and 30 µg cefotaxime discs placed 15 to 20 mm, edge to edge from the amoxicillin-clavulanate disc. The plates were incubated aerobically for 16-18 hrs at 35°C. Isolates which showed increase in the inhibition zone of the cephalosporin adjacent to the amoxicillin-clavulanate disc was considered to be ESBL-producing.
Staphylococcus aureus ATTC 25923 and Escherichia coli ATTC 25922, and locally isolated MRSA and ESBL producing E. coli designated ASU11 and AFU11, respectively, were used as controls during the study.

Results
A total 60 samples, consisting of 30 nasal swabs and 30 hand swabs, were collected from 30 healthcare workers in University of Uyo Teaching Hospital. Of all the healthcare workers, 15 (50%) were doctors and 15 (50%) were nurses, which consisted of 11 (36.7%) males and 19 (63.3%) females.
Of the 60 samples analyzed, 48 organisms were isolated. Fourty-six isolates were identified as Staphylococcus spp, of which 30 (65.2%) were Staphylococcus aureus and 16 (34.8%) were Staphylococcus epidermidis. The other two isolates were identified as E. coli and Proteus mirabilis, and were isolated from 2 hand swabs of doctors. Staphylococcus spp were isolated from 26 (56.5%) specimens obtained from doctors and 20 (43.5%) from nurses. Of the 26 isolates from doctors, 20 (76.9%) were Staphylococcus aureus and from the total 20 isolates from nurses, 10 (50%) were Staphylococcus aureus. Generally, 28 (60.9%) Staphylococcal isolates were obtained from the noses of the healthcare workers, while 18 (39.1%) Staphylococcal isolates were obtained from their hands (Table  2a and   (MRSA) isolates were obtained from 30 Staphylococcus aureus isolated from healthcare workers. Out of the 30 healthcare workers included in this study, 6 (20%) showed MRSA colonization in their noses, and 6 (20%) also showed MRSA colonization in their hands (Table 3).
The frequency of bacterial colonization was seen to be higher among doctors than the nurses, and a higher frequency of colonization was seen in the noses than the hands of healthcare workers (Figure 1).

Discussion
A total of 30 healthcare workers, which included 15 doctors and 15 nurses, were included in this study. Out of the 60 (30 nasal swabs and 30 hand swabs) obtained from these healthcare workers, 46 (76.7%) yielded staphylococci, of which 30 (65.2%) were coagulase positive and 16 (34.8%) were coagulase negative, 2 (3.3%) yielded Gram negative bacteria (E. coli and Proteus mirablis), while 12 (20%) showed no bacterial growth. This can be compared to a research carried out by Kumar et al. [14], which included a total of 84 healthcare workers, consisting 60 doctors and 24 laboratory technicians. Of the 84 samples collected from the healthcare workers, 66 (78.6%) yielded Staphyloccus spp, which included 40 (60.6%) Staphylococcus aureus and 26 Staphylococcus epidermidis, while 18 (21.4%) specimens showed no growth of Staphylococci colonies, but showed growth of Gram negative bacteria from the nasal swab collected from the anterior nares. The difference in the number of isolates could be attributed to the difference in sample size.
In this study, the prevalence of Staphylococcus aureus was 50%; out of these, 40% were MRSA, and the rate of Staphylococcus epidermidis was 26.7%. Farzana et al. [15] conducted a similar study and reported the carriage of S. aureus, 48%, out of these, 29% were MRSA. Another study by Akoua et al. [16] showed the carriage rate of S. aureus 45.4%, and out of these, 38.7% strains were methicillin resistant.
This study revealed that 93% of healthcare workers carry Staphylococcus in their nostrils and 66.6% on their hands; this goes to prove that one of the ecological niches for colonization of Staphylococcus is the anterior nares, as most of the nasal specimens yield Staphylococcal growth on culture.
The greater number of bacteremia cases recorded has been due to Staphylococcus aureus of endogenous origin, since they originated from colonies of nasal mucosa. Although nasal carriage of S. aureus is harmless in healthy individuals, they can pose the risk of spreading infections to the community at large, and since the section of individuals under this study were healthcare workers, their interaction and exposure to hospital environment could cause major risks in transmitting to hospital patients and spreading nosocomial infections. Kumar et al. [14] also carried out similar research and showed that almost 25% of healthcare workers are stable nasal carriers and 30%-50% of them possess the bacteria in their hands. Tammelin et al. [17] also showed that 50.7% of healthcare workers carry bacteria in their nose and 26.3% in their hands.  Majority of the Staphylococci were multidrug resistant. Clindanycin was found to be very effective, but the rate of resistance against Trimethoprim Sulphamethoxazole was highest in the study.
The carriage of Gram negative bacteria among healthcare workers was found in 6.6% of the healthcare workers. Fifty percent of the Gram negative bacteria isolated were ESBL producing. A research by Metri et al. [3] shows that out of 218 Enterobacteriaceae isolates, 200 (91.7%) were ESBL producing. Previous studies from India have reported the prevalence of ESBL producers to be 6.6 to 91% [3].
Several researchers have revealed that most of the bacteria that cause nosocomial infections are those that have developed resistance to antibiotics used in treating those [15].
This study indicated high incidence of nasal carriage of Staphylococci and a low incidence of Gram-negative bacteria carriage among healthcare workers. There was also the presence of MRSA, ESBL producing bacteria and also inducible Clindamycin resistance Staphylococcus. There was also the presence of Methicillin Resistant Staphylococcus aureus (MRSA), extended spectrum Beta lactamases (ESBLs) producing bacteria, and also inducible clindamycin resistant Staphylococcus. The antibiotic sensitivity of these isolates was seen to be higher for Clindamycin in Staphylococcus aureus and ciprofloxacin in Staphylococcus epidermidis. E.coli was 100% sensitive to five of the antibiotics, while Proteus mirabilis was 100% sensitive to four of the antibiotics used, and completely resistant to the rest. The least sensitivity was seen for Trimethoprim Sulphamethoxazole.
There is need for Healthcare workers to wash their hands regularly with antiseptic soap, or to disinfect the hand by rubbing with alcohol solution. Proper infection control measures should be adopted and further research on multidrug resistant organisms and surveillance of nosocomial infection should be carried out. Public enlightenment against the abuse of antibiotics should be carried out.