INTRODUCTION:

Hospital environment play an important role in different infections, due toit is contains diverse population of microorganisms such as bacteria, fungi and viruses[1]. Microorganisms are present in large numbers in moist hospital environments, but some can also survive under hard environmental conditions[2]. Gram-negative and Gram-positive bacteria are one of the most important microorganisms are found in hospitals environments and cause different infections in in-patients such as urinary tract infection, respiratory tract infection, burns infections and blood infection[3, 4].

Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli have been found to be the most commonly pathogenic bacteria associated with different infections worldwide[4, 5, 6].

Hospitals were considered as a reservoir for many types of pathogenic bacteria, usually many of which are multi-resistant to antimicrobials[7, 8].

In hospitals, morbidity, increased health care costs and longer stays caused by the emergence bacterial resistance to antimicrobial agents is a global public health problem[9]. Recently, in hospitals environments especially in developing countries the emergence of multidrug resistant bacteria is an increasing infection control problem lead to difficult to treat inpatients infected with different infections because of increased antibiotic resistance rate[3, 7, 10]. Recently, vertical and horizontal gene transfer is two of the most important methods of bacterial evolution and enabling these pathogens to resist different type of antimicrobials such as aminoglycosides, chloramphenicol and penicillins[11]. In developing countries such as Iraq, annually, there is high emergence of different infections such as, urinary tract infection, burns infections and respiratory tract infection caused by different multidrug resistant bacteria. Therefore, the main aim of this study was to identify the most prevalent pathogenic bacteria that cause different infections in Al-Kufa Central Hospital – Iraq and determine the antibiotic susceptibility patterns of these pathogens during the period from 2015 to 2017 to evaluate the changing trend of antimicrobial susceptibility in this hospital.

MATERIALS AND METHODS:

Patients, Specimens collection and bacterial identification:

A total number of 517of in patients infected with different infections were included in this study, age range (18-60) years old (males and females) attending to the Al-Kufa General Hospital in Al-Kufa City –Iraq, from 1-1-2015to 1-1-2017. A total number of 238 swabs were collected from skin of in patients infected with burns infections, 171 urine samples were collected from in patients with urinary tract infections, 55 sputum samples were collected from in patients with respiratory tract infection and 53 blood samples were collected from in patients infection with blood infection. All burns swabs were streaked by sterile swab (Bioanalyse – Turkey) on the surface of blood agar (Oxoid – UK) plates, Chocolate agar (Oxoid – UK) plates and MacConkey agar (Oxoid – UK) plates and incubated aerobically in 37°C for 24h.¹².All urine and sputum specimens were collected in sterile container (Hi-media – India) and streaked by sterile swab (Bioanalyse – Turkey)on the surface of blood agar (Oxoid – UK) plates, Chocolate agar (Oxoid – UK) plates and MacConkey agar (Oxoid – UK) plates and incubated aerobically in 37°C overnight¹². Five ml of blood specimen was taken from in patients suspected infected with blood infection by sterile disposable syringe (Himedia – India) and added to 45 ml of brain heart infusion broth (Oxoid – UK) and incubated aerobically in 37°C for 7 days with continuous shaking every day, then streaked (by sterile loop, (Himedia – India) on the surface of blood agar (Oxoid – UK) plates, Chocolate agar (Oxoid – UK) plates and MacConkey agar (Oxoid – UK) plates and incubated aerobically in 37^oC overnight[12]. All growing pathogenic bacteria were identified by using standard bacteriological procedures according to Collee et al., (1996)¹², MacFadden (2000)[13] and CLSI (2015)[14].

Antimicrobial susceptibility testing:

This test was done by using disc diffusion method according to Bauer et al., (1966)[15]. Clinical and Laboratory Standards Institute (CLSI)[14] guidelines were used to interpret results (CLSI, 2015)[14] and the susceptibility patterns were determined following CLSI guidelines (2015)[14].

Five pure and fresh colonies from each type of bacterial growth was adjusted according to MacFarland nephelometer scale (1.5 × 10⁸ CFU/ml) and streaked on Mueller Hinton agar (Oxoid, UK) surface by sterile swab (Bioanalyse – Turkey). The antibiotics disks were placed aseptically on it and incubated at 37°C overnight. Eleven antimicrobials were used: Amoxicillin(AX, 25 µg), Ticarcillin (TIC, 75 µg), Amoxicillin + Clavulanic acid (AMC, 30 µg) Gentamicin (CN, 10 µg), Amikacin (AK, 30 µg), Tobramycin (TM, 10 µg), Tetracycline (TE, 30 µg), Doxycycline (DO, 30 µg), Ciprofloxacin (CIP, 5 µg), Chloramphenicol (C, 30 µg) and Nitrofurantoin (F, 300 µg).All antimicrobials were providing from Bioanalyse – Turkey.

RESULTS:

Total specimens:

Out of the 517 total specimens there were 290 specimens (56.092 %) were gram negative bacteria, 195 specimens (37.719 %) were gram positive bacteria and 16 specimens (3.094 %) with no any growth(Table 1).

Table 1: Total specimens in this study (N=517).

Bacterial growth	No.	%
Gram negative	226	43.713
Gram positive	275	53.191
No growth	16	3.094
Total	517	100

From the 517 total specimens there were 171 bacterial isolates (33.075 %) have been isolated from urine, 238 bacterial isolates (46.034 %) have been isolated from burns, 55 bacterial isolates (10.638 %)have been isolated from sputum, 53 bacterial isolates (10.253 %) have been isolated from blood and 16 specimens (3.094 %) with no bacterial growth(Table 2).

Table 3. Numbers and percentages of gram positive and gram negative bacterial isolates according to sites of infection (N= 517).

Bacteria	Urine (100%)	Burns (100%)	Sputum (100%)	Blood (100%)	Total (100%)
P.aeruginosa	45 (8.704)	44 (8.51)	20 (3.86)	13 (2.514)	122 (23.597)
K.Pneumoniae	76 (14.700%)	15 (2.901%)	5 (0.967%)	0 (0.0%)	96 (18.568)
E.coli	10 (1.934%)	41 (7.930%)	13 (2.514%)	0 (0.0%)	64 (12.379)
Proteus.spp.	33 (6.382%)	14 (2.707%)	0 (0.0%)	0 (0.0%)	47 (9.090)
Salmonellaspp.	0 (0.0%)	0 (0.0%)	0 (0.0%)	14 (2.707%)	14 (2.707)
S. aureus	0 (0.0%)	46 (8.897%)	0 (0.0%)	5 (0.967%)	51 (9.864)
S.pyogenes	0 (0.0%)	44 (8.510%)	9 (1.740%)	6 (1.160%)	59 (11.412)
S.pneumoniae	0 (0.0%)	34 (0.065%)	8 (1.547%)	6 (1.160%)	48 (9.288)
No growth	7 (1.353%)	0 (0.0%)	0 (0.0%)	9 (1.740%)	16 (3.094)
Total	171 (33.075)	238 (46.034)	55 (10.638)	53 (10.253)	517 (100)

Figure 9: Total percentage of antimicrobials resistance of bacterial isolates from different infections.

Amoxicillin (AX, 25 µg), Ticarcillin (TIC, 75 µg), Amoxicillin + Clavulanic acid (AMC, 30 µg) Gentamicin (CN, 10 µg), Amikacin (AK, 30 µg), Tobramycin (TM, 10 µg), Tetracycline (TE, 30 µg), Doxycycline (DO, 30 µg), Ciprofloxacin (CIP, 5 µg), Chloramphenicol (C, 30 µg) and Nitrofurantoin (F, 300 µg).

Table 4. Prevalence of multidrug resistant bacteria isolated from different infections (N= 501).

Bacteria	Total isolates	Urine (%)	Burns (%)	Sputum (%)	Blood (%)	Total (%)
P.aeruginosa	122	45 (100)	44 (100)	20 (100)	13 (100)	122 (100)
K.Pneumoniae	96	76 (100)	15 (100)	5 (100)	0 (0.0%)	96 (100)
E.coli	64	10 (100)	41 (100)	8 (61.53)	0 (0.0%)	59 (92.18)
Proteus.spp.	47	30 (90.90)	14 (100)	0 (0.0%)	0 (0.0%)	44 (93.61)
Salmonellaspp.	14	0 (0.0%)	0 (0.0%)	0 (0.0%)	9 (64.25)	9 (64.25)
S. aureus	51	0 (0.0%)	46 (100)	0 (0.0%)	5 (100)	51 (100)
S.pyogenes	59	0 (0.0%)	30 (68.18)	9 (100)	3 (50)	42 (71.18)
S.pneumoniae	48	0 (0.0%)	24 (70.58)	8 (100)	4 (66.66)	36 (75)
Total	501 (100)	161	214	50	34	459 (91.61)

DISCUSSION:

Pseudomonas aeruginosa, K. pneumoniae and S.aureus are associated with many infections such as urinary tract infection, burns infections and blood infection and they are recognized public health threat often acquired from the hospital environment and contaminated medical devices[16]. They are not only an important cause of morbidity but also increase the stay of the patient in the hospital and increase the cost of treatment[17].In this study, Pseudomonas aeruginosa and K. pneumoniae were the most prevalent bacteria at rate 23.597%, 18.568%and 9.864% respectively, and both of them were multidrug resistant with percentage 100%. This result is in agreement with many studies such as Apondi et al. (2016)16, Mitiku et al. (2015)[18], Kossow et al. (2017)[19], Dinh et al. (2017)[20] and Saba et al. (2017)[21] when they found that these bacteria were the most predominant pathogens and were multidrug resistance to most antimicrobials. Escherichia coli and Proteus spp. are the most important pathogenic bacteria cause urinary tract infection and became more virulent and more resistant to antimicrobials (multidrug resistant) in last five years[22, 23]. In this study, there was high prevalence of multidrug resistant E.coli and Proteus spp. with percentage 92.18% and 93.61%respectively, this result are in agreement with Abduzaimovic et al. (2016)[24], Pal et al. (2016)[25] and Ma et al. (2017)[26]. Other pathogenic bacteria were prevalent in this study in different percentages (Salmonella spp.2.707%), (S.pyogenes 11.412%) and (S. pneumoniae 9.288%). But, these bacteria were multidrug resistant with high rates 64.25%, 71.18% and 75% respectively. This result is in agreement with Forkus et al., (2017)[27], Babbar et al., (2017)[28] and Thapa et al., (2017)[29]. Salmonella typhi is the main causative agent of enteric fever, is a majorpublic health concern in Iraq and other developing countries. Several studies from developing countries have already identified this pathogen as multidrug resistant bacteria and a common cause of blood infection in hospitals of this regions[30, 31]. Streptococcus pyogenes and S.pneumoniae are the most important pathogenic bacteria causing different infections such as respiratory tract infection and the most pathogenic bacteria can resist different types of antimicrobials and therefore they are called multi-drug resistant bacteria[32].

The antibiotic susceptibility analysis of the bacterial isolates showed that about 20% were wild type, while 91.61% were multidrug resistant, particularly to cephalosporins and aminoglycosides. These resistance profiles can be due to expression of different enzymes such as extended-spectrum beta-lactamases and to over expression of efflux systems. Multi antibiotic resistance concerned all virulence profiles. Studies showed that resistance mechanisms such as efflux pump can contribute to virulence of bacteria[33]and a high proportion of multidrug bacterial isolates were found with virulence factors[34, 35]. The coevolution or coexistence of resistance and virulence may be inherent to their genetic linkage in the same genetic determinants and accessory elements (transposons, integrons and plasmids)[36].The use of antimicrobials is the single most important factor leading to antimicrobial resistance around the world[34]. Antibiotics are among the most commonly prescribed drugs used in human medicine. However, up to 55% of all the antibiotics prescribed for people are not needed or are not optimally effective as prescribed. Antibiotics are also commonly used in food animals to prevent, control, and treat disease, and to promote the growth of food-producing animals. The use of antibiotics for promoting growth is not necessary, and the practice should be phased out [37]. In the present study, most bacterial strains were resistant to Amoxicillin, Ticarcillin and Amoxicillin + Clavulanic acid, on the other hand Ciprofloxacin, Tetracycline and Tobramycin were provided good antibacterial effect. When a microbe acquires a genetic mutation making it resistant to the effect of one or more antimicrobial agents that were once effective. This genetic mutation may be acquired by gene transfer or spontaneously. Pathogenic bacteria that are resistant to different antimicrobials (more than three classes) are called multidrug resistant[38]. In conclusions: During past of the two years, P. aeruginosa and K. pneumoniae were in the first place that cause infections in Al-kufa General hospital. Most pathogenic bacteria became high resistant to most antimicrobials.

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Received on 10.08.2017 Modified on 24.09.2017

Research J. Pharm. and Tech 2017; 10(10):3264-3270.

DOI: 10.5958/0974-360X.2017.00579.0

Site of isolate	G+ve Bacteria (100%)	G-ve Bacteria (100%)	No growth (100%)	Total (100%)
Urine	96 (18.568 %)	68 (13.152 %)	7 (1.353 %)	171 (33.075 %)
Burns	114 (22.051 %)	124 (23.985%)	0 (0.0 %)	238 (46.034 %)
Sputum	38 (7.350 %)	17 (3.289 %)	0 (0.0 %)	55 (10.638 %)
Blood	27 (5.223 %)	17 (3.289 %)	9 (1.740 %)	53 (10.253 %)
Total	275 (53.191 %)	226 (43.713 %)	16 (3.096%)	517 (100%)