Antibacterial Susceptibility Of Bacteria Isolated

Published Date: 02 Nov 2017

from burns and wounds of cancer patients

Abstract In this study 540 burns and wound swabs were collected from cancer patients of some

Egyptian hospitals. The single infection was detected from 210, and 70 cases among wounded

and burned patients, while mixed infection was 30 and 45, respectively. We recovered where 60 isolates

of Pseudomonas aeruginosa, 60 isolates of Staphylococcus aureus, 7 isolates of Staphylococcus

epidermidis, 4 isolates of Streptococcus pyogenes, 25 isolates of Escherichia coli, 23 isolates of

Klebsiella pneumoniae and 27 isolates of Proteus vulgaris from 355 burn and surgical wound infections

. All bacterial isolates showed high resistance to the commonly used b-lactams (amoxycillin,

cefaclor, ampicillin, vancomycin, amoxicillin/clavulonic), and low resistance to imepenim and

ciprofloxacin. Plasmid analysis of six multidrug resistant and two susceptible bacterial isolates

revealed the same plasmid pattern. This indicated that R-factor is not responsible for the resistance

phenomenon among the isolated opportunistic bacteria. The effect of ultraviolet radiation on the

isolated bacteria was studied.

1. Introduction

Hospital-acquired infections remain a cause of morbidity,

extended hospital stay and death for patients (Holzheimer

et al., 1990; Pruitt et al., 1998; Naeem et al., 2006). The burn

and wound represent a susceptible site for opportunistic colonization

by organisms of endogenous and exogenous origin

(Pruitt et al., 1998). Bacterial infections in burn and wound

patients are common and are difficult to control. Sepsis consequently

is common and sepsis is often fatal (Lee et al., 1990;

Armour et al., 2007). In Egypt, nosocomial infection constitutes

a major problem. It requires more interest and attention

than it currently receives as it is responsible for a great deal of

morbidity and mortality among hospitalized patients in addition

to unavailability of records, statistics or enough information

about the problem as well as lack of universal program or

approach to control it (Abdel Rahman et al., 2010). Burns,

wounds, trauma, multiorgan failure and use of invasive devices

for surgery, and exposure of microorganisms in the environment

of hospital to a number of antimicrobial agents leading

to selective resistance are all some of the factors facilitating

colonization, transmission and susceptibility to infection

(Poh and Yeo, 1993).

The infection of burn wounds with multiple organisms, with

superadded problem of drug resistance, illustrates the need for a

drug policy by the hospitals for burn patients. The isolated bacteria

exhibited multiple resistance to antibiotics (Roberts et al.,

2008). Burns provide a suitable site for bacterial multiplication

and are more persistent richer sources of infection than surgical

wounds, mainly because of the larger area involved and longer

duration of patient stay in the hospital (Agnihotri et al., 2004).

Bacterial infections in burn and wound patients are common

and are difficult to control. Sepsis consequently is common

and sepsis is often fatal (Lee et al., 1990; Armour et al., 2007).

Plasmids are extrachromosomal self-replicating genetic

materials found in a variety of bacterial species and not essential

for growth of bacteria. Plasmids could carry genes that

code for drug resistance, virulence, production of antimicrobial

agents and metabolic activities (Ibrahim, 2002).

The aims of this paper are to isolate and identify bacterial

species causing burn and surgical wound post infections from

some Egyptian hospitals as well as determination of the antimicrobial

susceptibility of the isolated microorganisms and

plasmid profile analysis of the most frequent isolated organisms

which acquired multiple drug resistance.

2. Materials and methods

2.1. Sample collection

Swabs were collected from 540 cancer inpatients (radiotherapy

treated) of burn and surgical units and transported aseptically

to bacteriological labs for analysis. The patients’ samples comprise

365 post operative wounds and 175 burns from three hospitals

namely: El-Hussein University, Ain-Shams University

and Mansoura University.

2.2. Media

The following media were prepared according to the instructions

of the manufacturer, which include MacConkeys agar,

nutrient agar, nutrient broth, mannitol salt agar, trypticase

soya agar and urea agar base. On the other hand, blood agar

medium, indole test medium, sugar fermentation medium, gelatin

liquefaction medium and motility test medium were prepared

according to Collee et al. (1996).

Swabs were taken from all septic wounds, one week after

radiotherapy treatment. Swabs were transported into 2 ml trypticase

soya bean broth and incubated aerobically at 37 _C for

18 h. Then, one loopful from each sample was streaked on

MacConkey’s agar, mannitol salt agar, blood agar; the plates

were incubated at 37 _C for 24–48 h. Bacterial growth was

identified by colony characteristics, blood hemolysis, microscopic

examination of Gram stained preparations and motility

techniques. Biochemical activities including oxidase test; glucose,

lactose and mannitol fermentation, indole production,

gelatin liquefaction, catalase activity, nitrate reduction, urease

production, H2S production, coagulase and pigment production

were performed to confirm the identification of each

isolate according to the methods of Manual of Methods for

General Bacteriology (1981).

2.3. Antimicrobial susceptibility test

The identified isolates were tested using some antibiotics, such

as amoxicillin (25 lg), Cefaclor (25 lg), ampicillin (30 lg),

amoxicillin/clavulonic acid (25 lg), Ciprofloxacin (10 lg),

Imepenim (10 lg) and Vancomycin (35 lg) (Oxoid, UK). The

test was performed according to the Kirby–Bauer technique

(Bauer et al., 1966) and results interpreted using chart of

NCCLS (1994).

2.4. Plasmid profile

Plasmids of multi-drug resistant isolates were analyzed by rapid

screening procedure for plasmid DNA (Kado and Lui,

1981). Plasmid DNA of susceptible antibiotic was used as a

control for comparative studies.

2.5. Effect of ultraviolet (UV) irradiation on viability of

bacterial growth

Nearly 2 · 108 cells/ml, for each isolate, were exposed to UV

Lamp at 2600 nm (famed 1, Poland) at a distance of 30 cm

for 0, 30, 60, 90, 120 and 150 s, respectively. 0.05 ml was

spread (homogeneously) over nutrient agar plates after each

exposure in order to obtain the viable cell count. Assay plates

were also inoculated, after being diluted to a factor of 2 · 105

as 0.03 and 0.07 ml per plate, respectively, prior to exposure,

to confirm the viable cell count in the original culture. One

percent (1%) survival level of each isolate was calculated

from the given results to show the killing effect of UV light.

Results were expressed as viable cell count after each exposure

as well as the 1% survival level of each isolate under

investigation.

3. Results

Out of 540 cancer patients of burn and surgical wounds, 355

cases were infected with bacteria and represent 65.74% of total

patients included in this study. From the 260 cases of El-Hussein

University Hospital, 180 cases developed infections with an

incidence rate of 69.23%. These comprised of 125 wounds

infections out of 185 (67.57%) and 55 infections out of 75

(73.33%) burn cases. Out of 150 cases of surgical operations

of Ain-Shams University Hospital, 90 (60%) developed wound

infections. While out of 85 burn cases from Mansoura University

Hospital, 45 (52.94%) developed infections (Table 1).

Table 2 shows single and mixed bacterial infections of cancer

patients with wounds and/or burns. The prevalence of single

bacterial infections among wounded patients was high (280

cases) and only 75 cases showed mixed bacterial infections.

However, the prevalence of mixed bacterial infection in the

case of burned patients was significantly higher than surgically

wounded patients (42.8%), while single bacterial infections

were (57.2%). At El-Hussein University Hospital out of 125

wounded patients only 15 (12%) were having mixed bacterial

infections and only 20 (57.14) out of 55 burned patients were

having mixed bacterial infection. At Ain-Shams University

Hospital, 10 (11.11%) out of 90 wounded patients were infected

with mixed infection. However, at Mansoura University

Hospital, there were 20 (44.44%) out of 45 burned patients

having mixed bacterial infection.

The prevalence of bacterial species isolated from wounded

and burned patients: Pseudomonas aeruginosa was the most

frequent microorganism isolated from burned patients (30

isolates, 36.14%), followed by Staphylococcus aureus (20 isolates,

30.12%), Proteus vulgaris (15 isolates, 18.07%), Klebsiella

pneumonea (8 isolates, 9.64%) and Escherichia coli (5 isolates,

6.02%). While S. aureus was the most frequent microorganism

isolated from wounds, it was isolated from 35 wounded patients

(28.23%) followed by P. aeruginosa 30 isolates (24.19%), E. coli

20 isolates (16.13%), K. pneumonea 15 isolates (12.10%),

P. vulgaris 12 isolates (9.68%), Staphylococcus epidermidis 7

isolates (5.65) and Streptococcus pyogenes 4 isolates (3.23%)

(Table 3).

The prevalence of bacteria from three hospital universities

(Ain-Shams, El-Hussein and Mansoura) is illustrated in Figs.

1–3, respectively.

3.1. Antibacterial susceptibility pattern

K. pneumonia isolates were resistant to amoxicillin and amoxicillin/

clavulonic acid in a ratio of 90% while 27.2% of the isolates

were resistant to ciprofloxacin, 67% to cefaclor, 76% to

ampicillin, and 63% to imipenim (Fig. 4). Fig. 5 showed that

isolates of P. vulgaris were resistant to cefaclor (75%), imipenim

and vancomycin, each (72.1%), and ciprofloxacin

(25%). The resistance of the S. aureus isolates showed that

29% of the isolates were resistant to ciprofloxacin, 64% to

amoxicillin, 66.9% to cefaclor and 47% to imipenim (Fig. 6).

Ciprofloxacin was found to be the most powerful antibiotic

and only 17% of E. coli isolates were resistant. However, E. coli

isolates were 52%, 23%, and 18% resistant to amoxicillin/

clavulonic acid, imipenim and vancomycin, respectively (Fig. 7).

P. aeruginosa is known to be naturally resistant to amoxicillin

and ampicillin. These antibiotics were tested against P.

aeruginosa to make a comparison with other organisms possible.

All isolates of P. aeruginosa were resistant to amoxicillin,

cefaclor and amoxicillin/clavulonic acid. Data showed 93.4%,

90% and 80% of the isolates were resistant to vancomycin,

ampicillin and Imipenim, respectively (Fig. 8).

3.2. Mutagenic effect of UV irradiation

The experiment of UV mutant was conducted after preliminary

tests on seven types of bacteria to determine the suitable initiation time of irradiation by which the plates were countable.

It can be concluded from (Table 4) that the number of

mutant was increased as the UV doses increased. E. coli and

S. pyogenes currently not tolerated UV irradiation over 90 s.

The most tolerant organism to UV was S. epidermidis

(3.7 · 102) followed by S. aureus (2 · 102) and P. vulgaris

(1 · 102) CFU/ml.

Eight isolates of P. aeruginosa were tested against different

antimicrobial agents; two of them were different antibiotic sensitivity

patterns. Plasmids of these eight strains were analyzed

and results revealed that all the six P. aeruginosa isolates

contained the same plasmid pattern. On the other hand,

P. aeruginosa isolates (two isolates) that were sensitive to the

above mentioned antibiotic was used as a control and gave

the same plasmid pattern.

Fig. 9 shows photograph of plasmid analysis as follows:

Pattern 1: (two isolates) which were resistant to ciprofloxacin

and amoxicillin; lanes 1 and 2. Pattern II: (one isolate) shows

resistance to amoxycillin; lane 3. Pattern III: (two isolates),

shows resistance to amoxicillin and cefaclor; lanes 4 and 5.

Pattern 4: (one isolate), which was resistant to impenim, ciprofloxacin,

and amoxicillin; lane 6. The other two lanes: 7 and 8

represented two sensitive isolates.

4. Discussion

Nosocomial infections play a role in quality and control in

health care. Surveillance of these infections is the only way

to gain more insight into their frequency and cause (Beaujean

et al., 2002). Surgical site infections are a problem in all fields

of surgery (Steinbrecher et al., 1992). In addition, burned patients

are at a high risk for nosocomial infections by multiresistant

bacteria, a large proportion of which are gram negative

(Mokaddas et al., 1998). Within 24 h, burned patients can start

suffering from opportunistic bacterial attacks that can vary

from simple infections, such as those easily treatable by antibiotics

to more complicated bacteria, which may have natural or

acquired resistance to drugs. Infection by multiple drug resistant

bacteria could create additional complexity to the problem

(Ahmad, 2002).

Hussein et al. (1989) stated that the infection at the burn

unit was 84.9%. The same result obtained by Mago (2009)

who made a burn sepsis revealed that bacterial colonizaion

reached 80.6%; also, Cremer et al. (1996) found the infection

in burn unit was 94%. The incidence of bacterial infection of

burned patients obtained from El-Hussein University Hospital

and Mansoura University Hospital was 73.33% and 52.94%,

respectively. Our results obtained agreed with other workers

including Hussein et al. (1989), Cremer et al. (1996) and Mago

(2009) who stated that the infection at the burn unit was

84.9%, 94% and 80.6%, respectively. Naeem et al. (2006) at

the burns centre, totally agreed with the result obtained from

Mansoura University Hospital where the incidence of burn

infection was 10.1%. The marked reduction in the percentage

of infection in Mansoura University Hospital may be attributed

to the advanced surgical techniques, instruments and

the precise application of aseptic technique.

P. aeruginosa was the most common organism encountered

in burn infection (n= 30, 36.14%) as indicated in (Table 3).

The obtained results agreed with Cremer et al. (1996) and

Branski et al. (2009) who found P. aeruginosa in burn infection

in a percentage of 49% and 53.9%, respectively. P. aeruginosa

remains a significant pathogen in burn infection, its pathogenicity

being associated with the production of a cocktail of

virulence determinants, which is regulated by a population–

density-dependant mechanism and diffusion of signaling

molecules in the burn-wound environment (Koeber et al.,

2002). The high predominance of P. aeruginosa among the

burned patients must reflect a proper attention to the wounds

of the burned patients.

On other hand, the isolated P. aeruginosa were at a lower

frequency rate as in Ain-Shams Hospital where P. aeruginosa

Incidence of 20% of burn exudates (Saleh, 2000). Also,

Kluyatmans (2007) isolated P. aeruginosa from burn exudates

at frequencies of 19.7% and 21%, respectively.

The second most important microorganism isolated from

burned patients was S. aureus (n = 25, 30.12%). Hussein

et al. (1989) and Mohamed et al. (2000) also isolated S. aureus

as the second most important microorganism encountered in

burned patients following P. aeruginosa, S. aureus and

S. pyogenes that were found to tolerate sunlight up to

120 min of exposure.

These results agreed with Mohamed et al. (2000) who isolated

K. pneumonia and E. coli from burn infections at frequencies

of 2% and 7%, respectively in addition to Prot. mirabilis.

On the other hand, this result was not in agreement with

Khashaba (1981) who found that the most predominant isolated

organism was S. aureus (53.8%), followed by Klebsiella

sp. (26.3%), P. aeruginosa (18.8%), Proteus sp. (6.3) and

E. coli (3.8%). On the other hand, this result was not in agreement

with Khashaba (1981) who found that the most predominant

isolated organism was S. aureus (53.8%), followed by

Klebsiella sp. (26.3%), P. aeruginosa (18.8%), Proteus sp.

(6.3) and E. coli (3.8%).

This variation in the frequency rates may be due to variations

in both environmental conditions and attitudes toward

management of the burn wound. The number of wound infections

involved was 365 patients (150 patients from Ain-Shams

University Hospital, 30 patients from Mansoura University

Hospital, and 185 patients from El-Hussein University Hospital).

Out of 30 patients from Mansoura University Hospital, 25

cases (83.33%) developed bacterial infection and out of 185

patients from surgery department at El-Hussein Hospital,

125 patients (67.57%) developed bacterial infections. This result

was in accordance with Aganovic et al. (1994) whose rate

of nosocomial infection of post-operative wounds was 69.45%.

On the other hand, post-operative infection was at a lower

rate. Mostafa (2006) found that surgical wound infection

was the commonest nosocomial infection (40%). Mohamed

et al. (2000) found that the infection rate in postoperative

wounds was 37.3% Our results disagreed with the results

obtained by Holzheimer et al. (1990), Marroni et al. (2003),

Lee et al. (1990) and El-Daghestany (1992). They found that

the overall nosocomial infection of post-operative was 13%,

2.1%, 11.4%, and 11.1%, respectively, while Nageb (1990),

Eltahawy et al. (1992) and Abussaud (1996) found that the

overall incidence of surgical infections was 8.7%, 9%, and

8%, respectively.

The most common microorganisms isolated from wounded

patients were S. aureus (n= 35, 28.23%). The obtained result

more or less agreed with the result of Mostafa (2006) who

found S. aureus in significantly high prevalence in wound

infection (36.2%). Also Mohamed et al. (2000) reported that

S. aureus was the most common pathogen responsible for

the post operative wound infection (33.7%).

Different results were obtained by Nageb (1990) and Zaghloul

(1993), they isolated S. aureus at a prevalence of 19.2%

and 13.2% respectively. Other isolated S. aureus at higher frequency

rates: Khozam (1987) (59%) and Master et al. (2010)

(88%).

The second most important microorganism isolated from

the wounds was P. aeruginosa (n = 30, 24.19%). Mohamed

et al. (2000) reported that P. aeruginosa was one of the most

common pathogens responsible for the post-operative wound

infection (25.3%), Saleh (2000) and Cestari et al. (1999) isolated

P. aeruginosa from 20% and 31.2% of surgical wound

exudates samples, respectively. Low frequency rate was presented

by Nageb (1990) and Kluyatmans (2007) they isolated

P. aeruginosa from 11.5% and 8% of surgical wound exudates,

respectively.

Mohamed et al. (2000) isolated K. pneumonia (18%) from

the post-operative wound infection. Khozam (1987) found

Klebsiella sp. (9.9%). Abussaud (1996) isolated Klebsiella sp.

at (10%).

The policy of antibiotic treatment was always based on

in vitro susceptibility test. P. aeruginosa was always among

the bacteria most readily acquiring resistance toward antimicrobial

drugs. It could cause septicemia in burned patients.

So careful attention should be paid to inpatients whose

wounds are colonized by this organism (Ashour, 2000).

Seven antimicrobial agents were used which are Amoxycillin,

Cefaclor, Imipenim, Amoxycillin/clavulonic acid, Ciprofloxacin,

Vancomycin, and Ampicillin.

Amoxycillin, Cefaclor, were of low activity, and this may be

attributed to the extensive use of these drugs. The resistance

pattern toward Amoxicillin was as follows: P. aeruginosa

(100%), E. coli (89.5%), K. pneumonia (90.0%), P. vulgaris

(80.1%) and S. aureus (59%). The resistance pattern to Cefaclor

was as follows: P. aeruginosa (100%), E. coli (79.9%),

K. pneumonia 67%), Prot. mirabilis (69%), and S. aureus

(61%). The resistance pattern to Cefaclor was as follows: P.

aeruginosa (100%), E. coli (48%), K. pneumonia (90%),

P. vulgaris (75%) and S. aureus (44%). Imipenim was the most

potent antimicrobial agents against the isolated gram-negative

bacilli where 89% of P. aeruginosa, 82% of E. coli, 100% of K.

pneumonia and 97.5% of P. vulgaris isolates were resistant to

imipenim. Ciprofloxacin comes after imipenim in activity

against the isolated Gram-negative bacilli.

Imipenim was the most active antibiotic against

P. aeruginosa, El-Naggar (1984), Mohamed et al. (2000) and

Saleh (2000), they demonstrated the resistance of P. aeruginosa

isolates to imipenim were 98%, 85% and 100%, respectively.

Survey at the North of Portugal was performed to assess the

level of susceptibility to the most common antibiotics with anti-pseudomonal activity against P. aeruginosa. It revealed

that out of 525, 10% of the isolates were resistant to imipenim

(Gardoso et al., 2002). Saleh (2000) found that 64% of

P. aeruginosa strains were resistant to ciprofloxacin El-Daker

(2002) found that imipenim and ciprofloxacin were the most

effective antibiotics toward multidrug resistant Gram-negative

bacilli at Ain-Shams University Hospitals. Hussein et al.

(2001) investigated that the sensitivity rate for imipenim was

71% and for ciprofloxacin 52%, in contrast most of the tested

isolates showed resistance to the third generation Amoxycillin/

clavulonic acid (90%).

The prevalence of Cefaclor-resistant P. aeruginosa in

Thailand was 24%, which is higher than the value reported

for P. aeruginosa isolated of North America and Europe

(Girlich et al., 2002). Vancomycin was the effective drug in

surgical and medical patients infected with methicillin-resistant

S. aureus isolated from Ain-Shams University Hospitals. Susceptibility

testing of isolates S. aureus collected from different

hospitals in Riyadh, Saudi Arabia to vancomycin showed that

all isolates were sensitive (Fouda et al., 2005).

The effect of UV radiation on isolated bacteria was carried

out to figure out the role of UV to control the growth of the

bacteria contaminating the burns and wounds. The lethal effect

of UV on the isolated bacteria varied considerably among

the species examined, e.g., it is as low as 90 s in the case of

E. coli and S. pyogenes, whereas it is as high as 150 s in case

of S. aureus, P. vulgaris and S. epidermidis. Bacteria that were

resistant to UV rays and endured their effect for up to 150 s

may be characterized by a DNA with a high GC mol.% value

and consequently, low thymine content, thus reducing the

probability for thymine dimerization. Bacteria may also exhibit

high activity and complete repair systems for photo reactivation,

excision repair and post-replication repair.

In the present work six multi-drug resistant and two sensitive

strains of P. aeruginosa were selected for study concerning

their plasmid profile. They were tested against seven antimicrobial

agents. The P. aeruginosa isolates revealed five different

antibiotic patterns. The result revealed that all the eight P.

aeruginosa isolates contained the same plasmid pattern, which

means that there was no correlation between plasmid pattern

and their antimicrobial activity. This result was in agreement

with that of Fouda et al. (2005).