Profiling of the bacterial pathogens associated with hospital acquired infections in hospitals within makurdi metropolis , middle belt , nigeria

Background: Nosocomial bacteria are bacteria that cause diseases acquired from the hospital environments. Aim: This study looked into profile of bacterial pathogens associated with nosocomial infections in hospitals within Makurdi metropolis. Result: A total of 71 bacterial pathogens were encountered from 240 samples analysed from three hospitals in Makurdi metropolis. Of the 71 isolates, 46(64.8%) were Gram positive and 25(35.2%) were Gram negative. The Gram positive constituting S. aureus (50.70%) and staphylococcus spp (14.08%) while the Gram negative constituting E. coli (15.50%), klebsiella spp (7.04%) and P. aeruginosa (12.68%). Out of 21(29.58%) isolates cultured from hand swabs, the bacterial pathogens from hand swab at F M C was the highest 12(57.14%) followed by C H M 5(23.81%) while B M M C has the least profile of 4(19.05%). With regards to hospital air, the highest profile of nosocomial bacteria was also from F M C 8(61.54%), followed by B M M C 4(30.77%) while C H M had just only one isolate 1(7.69%). Of 21(29.58%) isolates from B M M C; 10(47.62%) S. aureus, 4(19.05%) Staphylococcus spp, 2(9.52%) P. aeruginosa and 5(23.81%) Escherichia coli were obtained. In C H M, out of 18(25.35%) bacterial pathogens evolved constituting 12(66.67%) S. aureus, 2(11.11%) Staphylococcus spp, 1(5.56%) P. aeruginosa and 3(16.67%) E. coli. Whereas, the profile of 32(45.07) isolates in Federal Medical Centre Makurdi constituting 14(43.75%) S. aureus, 4(12.50%) Staphylococcus spp, 6(18.75%) P. aeruginosa, 3(9.38%) E. coli and 5(15.63%) Klebsiella spp were obtained. The antibiotic susceptibility pattern to Staphylococcus spp reveals that the highest level of sensitivity were demonstrated by Cloxacillin and Ofloxacin with 50%, followed by Augumentin and Cefuroxime with 40%, Gentamicin (30%), Erythromycin (20%) and least sensitivity with Ceftriazone (10%) while Ceftazidime has the highest resistance recorded 0% sensitivity. E. coli has the highest sensitivity on Ceftazidime having 81.8% and Gentamicin 63.6%, Ofloxacin also displayed a high level of sensitivity to isolates tested with 63.6% sensitive, follow by Cefuroxime (45.5%). Conclusion: This study showed that nosocomial bacterial pathogens particularly, S. aureus, P. aeruginosa, Staphylococcus spp and enteric bacteria, E. coli and Klebsiella spp are the predominant pathogens associated with infections acquired in hospital environment.So, information on resistance patterns of isolates encountered in this study will assist the clinicians in making improvement in management of nosocomial infections.


Introduction
The fact that bacteria are ubiquitous in nature is now a common knowledge which makes it easier for them to survive in different environments of which the hospital environment is not an exception. These organisms are found on different surfaces in hospital and as well as hospital air. They serve as agents of an infection referred to as nosocomial infection 1,2,3 Nosocomial infection is any infection acquired in hospital environment, hence it is also known as hospital acquired infections (HAI). 4 Apart from the fact that nosocomial infections affect the general health of patients, it also have a negative effect on their finance due to increase in patients' illnesses as well as their emotional stress which invariably may lead to situation that shorten the life span of such an infected person 16 . Nonetheless, Awosika et al. (2012) 5 reported that nosocomial infections can be contracted from contact with a carrier directly or indirectly through inanimate objects or air. Ishida et al. (2006) 6 also showed that, airborne bacteria are major source of infection after operation and a serious illness in the Intensive Care Unit. In the United States, approximately 1.7 million hospital-associated infections from all types of bacteria are responsible for 99,000 deaths each year; so about 10%, or 2 million patients are being infected each year, 7,8 In France, According to Lepoutre et al. (2005) 9 Heshowed that prevalence of nosocomial infection was 5.9% in 2001. Worse still, around 2004-2005, about 9,000 people died each year with a nosocomial infection, of which about 4,200 would have survived without this infection 10 . In Italy a survey in Lombardy gave a rate of 4.9% of patients in 2000 11 . While in United Kingdom in 2012 the Health Protection Agency reported the prevalence rate of hospital acquired infection in England was 6.4% in 2011, against a rate of 8.2% in 2006 12 . With regards to Switzerland estimates range between 2 and 14% 13 reports revealed that a survey conducted nationally gave a rate of 7.2% in 2004, Lyytikainen et al. (2005) 14 shows that in Finland the rate of epidemiology of nosocomial infections were estimated at 8.5% of patients in 2005. Moreover according to a report release by Federaal Kenniscentrum voor de Gezondheidszorg (2009) 15 the prevalence of nosocomial infections is about 6.2% in Belgium; therefore about 125,500 patients contract the infection every year which usually leads to about 3000 deaths. In addition, the mainroutes of transmission of nosocomial infection can either be via airborne transmission,contact transmission, vector borne transmission ordroplet transmission. Microbes carried in this mode can spread in air and may become inhaled by a susceptible host in the same room or over a longer distance from an infected patient, depending on environmental factors; so adequate precautions should be observed in those ill individuals suspected to have airborne, contact or droplet infections. Clostridiumspp, an anaerobic Gram-positive rods, causes gangrene; while a wide variety of lung, bone, heart and bloodstream infections which are normally resistant to drugs as in the case of beta-haemolytic Streptococci.Gram-positive bacteria, Staphylococcus aureus causes cutaneous bacteria that colonize the skin and nose of both health care staff and patients. In people with compromise immunity, Gram-negative bacteria, family Enterobacteriacae like Escherichia coli, Serratia marcescens, Klebsiella, Proteus, Enterobacter may colonize catheter insertion, bladder catheter, cannula insertion or surgical site in the body and then cause severe infections 16,17 ; while Pseudomonas spp are often isolated in water and damp areas may localize in the digestive tract of sicked people in hospitals 18 . This study aimed at investigating the profile of bacterial pathogens associated with nosocomial infections in different hospital environment within Makurdi metropolis, Benue State, North Central, Nigeria.

Materials and methods Collection of samples
The samples were collected in the morning before commencement of work but hand swab of the staff were collected during working hours. Thus, the analyses of samples collected are as follows: (a) City Hospital Makurdi: 10 hand swabs, 30 surface swabs, and 10 air samples; so total of 50 samples were collected from the hospital. (b) Bishop Murray Medical Centre: 10 hand swabs, 32 surface swabs, and 15 air samples; so total of 57 samples were collected from the hospital. (c) Federal Medical Centre: 77 hand swabs, 42 surface swabs, and 14 air samples; so total of 133 samples were collected from the hospital. Method of isolation Culture method: The surface samples were cultured using sterile swab sticks to make an inoculum on the plates while air samples were cultured by plate exposure method before streaking is done using a sterile wire loop.

Isolation and characterization of isolates
The organisms cultured from the air and the swabs were directly inoculated on nutrient agar and later sub-cultured on blood agar, chocolate and MacConkey agar near benzene burner. The inoculated media were incubated at 37oC for 24 h and then examined for bacterial growth. Therefore, the organisms evolved were characterized using cultural characteristics, microscopy (Gram's staining), biochemical test (catalase test, coagulase test, indole test, citrate utilization test, oxidase test, mannitol salt agar test, and sugar fermentation test) as described by Harley and Prescott (2002) 19 ; Cheesbrough (2000) 21 ; Cheesbrough (2006) 20 . In-vitro determination of antibiotic susceptibility: Antibiotic susceptibility of isolates was performed by disk diffusion according to Clinical Laboratory Standards Institute 22 guidelines. The multidisc contained the following antibiotics; Augmentin (AUG) 30μg, Ceftazidime (CAZ) 30μg, Ceftriazone (CTR) 30μg, Cefuroxime (CRX) 10μg, Cloxacillin (CXC) 10μg, Erythromycin (ERY) 30μg, Gentamicin (GEN) 10μg and Ofloxacin (OFL) 5μg. Zones of inhibition were used to determine the level of susceptibility of the isolates to the test antibiotics. Statistical Analysis: Data were analysed by the use of descriptive statistics via percentages and charts to show the frequency distribution nosocomial bacterial pathogens. Table 1 displays the distribution of the isolates in relation to the sample size analysed from hospitals within Makurdi metropolis; thus a total of seventyone bacterial isolates including 21(29.58%), 37(52.11%) and 13(18.31%) were cultured from hand swabs, surfaces of hospital environments and hospital air respectively. Therefore, two-hundred and forty samples were examined for profile of bacterial pathogens responsible for nosocomial infections in hospitals within Makurdi metropolis; 97(40.4%) were collected from hand palm of nurses and some of the hospital staff, 104(43.3%) were from surfaces in hospital environments and 39(16.3%) of the sample collected were from the air.   Table Top,

Table 3: Comparison of nosocomial bacteria in hospitals within Makurdi metropolis Hospitals
Profle of The data in Table 4 shows the incidence of nosocomial bacterial pathogensin relation to the hospitals environments within Makurdi metropolis. With regards to hand swabs FMC has the highest profile of bacteria 12(57.14%), followed by CHM 5(23.81%), while BMMC 4(19.05%) has the least profile bacteria cultured from the hand swabs. Of 37 isolate evolved from the surface swabs highest profile 13(35.14%) emerged from BMMC while FMC and CHM has the same rate of bacterial pathogens constituting 12(32.43%) each. In the profile from hospital air, findings shows that these bacteria thrive more in the air of FMC with the prevalent rate of 13(18.32%) followed by BMMC 4(30.77) and the least profile emerged from the air of samples of CHM with prevalent of 1(7.69%). The profile of staphylococcus aureus was very high from hand swabs which constituting 16(44.4%) and followed by bed rails and toilet seats constituting 4(11.1%) each as shown in Bar chart in Figure 2 shows that profile of staphylococcus aureus was very high from hand swabs which constituting 16(44.4%) and followed by bed rails and toilet seats constituting 4(11.1%)    (Table 4). HS-Hand swab, NT-Nurse Table Top,

AUG -Augumetin ERY -Erythromycin CAZ-Ceftazidime CTR-Ceftriazone CRX -Cefuroxime CXC-Cloxacillin GEN -Gentamicin OFL -Ofloxacin MDR-Multidrug Resistance
The data in Table 5 is theprofile of multidrug resistance bacteria isolates in hospitals within Makurdi metropolis. Of the 36 Staphylococcus aureus isolates, 30 constituting 5, 7 and 18 isolates were multi-resistant to 3, 4 and 5 or more of the eight antibiotics employed respectively. 2, 3 and 5 isolates of 10 Staphylococcus spp were multi-resistant to three, four and five or more antibiotics of the eight antibiotics. Whereas, 9 Pseudomonas aeruginosa encountered were resistant to 5 or more antibiotics employed; while 3, 1 and 5 out of 11 isolates of Escherichia coli were multi-resistance to 3, 4 and 5 or more of the of the eight antibiotics. With regards to Klebsiella spp none of the isolates was resistant to 4 antibiotics but 1 was resistant to three and five or more antibiotics employed each.

AUG -Augumetin ERY -Erythromycin CAZ-Ceftazidime CTR-Ceftriazone CRX -Cefuroxime CXC-Cloxacillin GEN -Gentamicin OFL -Ofloxacin Values in brackets (%) are the susceptibility rates
The data in Table 6 is the antibiogram of bacteria isolates from Hospitals within Makurdi metropolis. Thus the susceptibility patterns of the 71 isolates cultured from hospitals within Makurdi metropolis revealed 50 and 33 isolates were sensitive to Ofloxacin (OFL) and Cloxacillin (CXC) respectively, while 30 isolates each were resistance to Augumetin (AUG) and Gentamicin (GEN) suggesting that the four antibiotics would be very useful for combating of nosocomial bacterial pathogens. The profile of nosocomial bacterial pathogens in hospitals within Makurdi metropolis reveals that; seventy-one isolates were encountered from two hundred and forty samples collected from the three selected hospitals in Makurdi. 21 isolates cultured from hand swabs of nurses and other hospital staff, 37 isolates from hospital surfaces and 13 isolates cultured from hospital air as shown in Table 1, suggesting a polymicrobic nature of bacterial pathogens in hospital premises as it had been previously reported by Hammuel, et al. (2015) 23 and Chikere et al. (2008) 24 . In addition, out of 71 bacterial isolates in Table 3 (Table 4). On the other hand, Table 4 also reveals the Staphylococcus spp obtained constituting 21.8% of the Gram positive cocci from this investigation corroborates the findings by Nwankwo and Azeez (2015) 18 that coagulase negative Staphylococcus spp is among organisms of clinical importance in transmission of nosocomial infections. The profile of Gram negative bacteria shows that P. aeruginosa constituting 9(36.0%) from the six 6(60%) source out of the ten sample sources examined suggesting that they are commonly involved in nosocomial infection in general senses as shown in Figure 4. This finding is similar and also in agreement to the findings executed in Zaria and Kaduna metropolis in Nigeria by Aloma et al. (2013) 29 and Chrinius et al.
(2014) 27 respectively that one of the most commonly used surfaces in the hospital environment was found contaminated by P. aeruginosa which could be as a result of inadequate decontamination of the surfaces and that P. aeruginosa can be isolated from toilet seat and other moist environment in hospitals. Other Gram negative rods include E. coli and Klebsiella spp constituting 11(44.0%)and 5(20.0%) of the isolates encountered respectively ( Figure 5). Therefore the microbiology of bacterial pathogens responsible for nosocomial infections cannot be overemphasized among other bacterial illnesses, it is clearly seen from the present study that a good number of bacterial species are associated with infections acquired in hospital environments; similar studies by Nwankwo and Azeez (2015) 18 shows that, the pattern of bacteria most frequently isolated bacterial pathogens from operating theatres could cause nosocomial infections in patients undergoing surgery abound on inanimate objects in the operating theatre which is one of the environments under consideration in this study.  Figure 4); this is result shows consistency to the reports of findings by Tambekar et al. (2007) 32 ; Saana and Amani (2010) 30 . The antibiotics testing on the nosocomial bacterial pathogens carried out to provide vital information for those whose study antibiotics to which these organisms were sensitive. Although resistance of nosocomial bacterial pathogens against antibiotics are regularly reported; so it is difficult to compare results since variation in methodology may contribute to some extent to these differences. The susceptibility pattern Staphylococcus spp shown in Table 6 29 ; so this present study is also in agreement with their reports 29 . The Klebsiella spp strains encountered from this study were highly sensitive to six of the eight antibiotics employed which include Augumetin, Ceftazidime, Ceftriazone, Cefuroxime, Cloxacillin, Gentamicin and Ofloxacin with 80% sensitivity while very high level of resistance was also recorded from the bacteria with 0% sensitive from Erythromycin and Ceftriazone ( Table 6). The resistant Klebsiella spp were encountered in the air sample of the public hospital environments in Makurdi, Benue State, Nigeria.
Further study in this research shows that in Bishop Murray Medical Centre, the cell wall inhibitor drug cloxacillin are highly sensitive to S. aureus;while ofloxacin a member of fluoroquinolones which is inhibitors of nucleic acid synthesis also shows a very high sensitivity to the same pathogens at Federal Medical Centre and City Hospital Makurdi. Third generation cephalosporins (Ceftriazone), are active against P. aeruginosa in City Hospital Makurdi while erythromycin (macrolides) a member of cell inhibitors of protein synthesis is also active against Klebsiella spp in Federal Medical Centrel Makurdi. Gentamicin (an aminoglycosides) and Cefuroxime (Second generation cephalosporins) are active towards Staphylococcus spp. Third generation cephalosporins, (Ceftazidime and Ceftriazone), amino glycosides (Gentamicin), fluoroquinolones the inhibitor of nucleic acid synthesis (Ofloxacin and Augmentin) are active against Escherichia coli. Therefore, there was a degree of constitency in the in-vitro antibiotics susceptibility patterns of the nosocomial bacteria against the commonly used antibiotics employed in this study 34 .

Conclusion
This present study reveals that the profile of Gram positive bacteria was more than the profile of Gram negative bacteria in hospitals environment. It also shows a higher profile of bacterial pathogens from the hand swab than air and other surfaces in hospitals. This study also reveals nosocomial bacterial pathogens particularly, Staphylococcus aureus,Pseudomonas aeruginosa, Staphylococcus spp Escherichia coli and Klebsiella spp as the predominant pathogens associated with infections acquired in hospital environments. Antibiotics susceptibility tests indicates the presence of highly resistant species of Pseudomonas aeruginosa which suggests the risk of reliance on specific susceptibility patterns of bacteria to antibiotics employed for treatment of nosocomial infections.