Mohammed Jasim Al-Shamarti*, Ahmed A. Hussein
and Adil Ibadi AL-Luhaiby

University of Kufa, Faculty of Science, Department of Laboratory Investigations, Najaf, Iraq.

Abstract

Ninety-one Bacterial samples were collected from Al-Sader Medical City in Al-Najaf province during a period of 11 weeks from 2-7-2016 to 15-9-2016. The isolates were identified according to cultural characteristics and biochemical activities. The results have revealed that 25 (27%) samples were E.coli, 22 (24%) samples were Staphylococcus spp, 27 (30%) samples were Proteus spp, and 17 (22%) samples were Enterococcus spp. The susceptibility of bacterial isolates to 22 antibiotics was tested using disc diffusion method. The results have revealed that in E.coli the most resistance was seen to Cotrimoxaz antibiotic with percentage of 67% . In Staphylococcus spp, most resistance was seen to Azactam antibiotic with percentage of 83% while susceptibility to Vancomycin was the most. In Enterococcus spp, the most resistance was seen to Vancomycin. Proteus spp, showed the most resistance against Ampicillin/cloxacillin. The distribution of bacterial infection among different genders and their effect on bacterial resistance were also tested.  The results showed that infections with E.coli were 12 (48%) in males while in females 13 (52%). Most of Staphylococcus spp 13 (59%), has been isolated from males, while in females 9 (41%). Males infected with Proteus spp constitute  13 (48%), on the other hand, 14 (52%) were isolated from females. Infections with Enterococcus spp were distributed as 8 (47%) and 9 (53%) in males and females respectively. The different bacterial sites where also correlated and tested against the susceptibility results. All isolates displays different resistance to different antibiotics that varies with infection site and patient gender. In E. coli the resistance to antibiotics in UTI and intestinal disease was approximately the same. In Staphylococcus spp. and Proteus spp. wounds infections exhibit more resistant than other types of infections. In Enterococcus spp. UTI infections exhibit more resistant than wounds infections.

Keywords: Enterococcus spp., Ampicillin/Cloxacin.

Introduction

Antibiotics are produced naturally by some fungi and bacteria as a secondary metabolites to kill other microorganisms that live in the same habitat for the sake of nutritional competition especially in the environments of limited nutritional resources (Bennett and Feibelman, 2001). The antibiotics used to treat people nowadays are usually derived from these natural products (Clardy et al., 2009). Antibiotics are used to treat many illnesses caused by bacterial infections, from ear and skin infections to pneumonia, food poisoning, meningitis, and other life-threatening infections so that antibiotics are essential tools for physicians at any given time.

Between 25% and 40% of hospital patients are receiving antibiotics intravenously (Equi et al., 2002; Wilson, 2006). As antibiotic became widely used, resistant bacterial strains having the ability to inactivate the drug became prevalent, therefore, chemical and structural studies that deals with the antibiotic synthesis were undertaken to modify penicillin chemistry in order to prevent its enzymatic destruction by penicillinases (β-lactamases) which are produced by drug resistant bacteria (Gold and Moellering Jr, 1996; Kardos and Demain, 2011). Interestingly, the ability of bacteria to produce β-lactamases became possible by using the gene based molecular techniques (Al-Shamarti, 2010). This, however, is an appreciated prospect to provide resistance profile of pathogenic bacteria in order to determine the effective antibiotic to be used in treatment (D’costa et al., 2006). Antibiotic resistance could arise as a result of spontaneous or induced genetic alteration in bacteria in addition to the horizontal gene transfer by means of conjugation among different bacterial cells. Thus, antibiotic resistance genes which had evolved as a result of natural selection could be shared (Mazodier and Davies, 1991). According to the concept of natural selection, random exposure to antibiotic puts evolutionary stress on bacteria to develop antibiotic resistance traits. Many antibiotic resistance genes are found on plasmids, enabling their transfer (Bennett, 2008). When a bacterium has the ability to resist several types of different antibiotic as a result of having several resistance genes, it is formally termed as multidrug resistant (MDR) or, informally, super bacterium or superbug (D’Costa et al., 2011).

Aim of the study
The aim of this study is to correlate between the type of infection and antibiotic resistance. This aim is reached via surveying many infections caused by many bacteria, then performing antibiotic susceptibility test.

Methodology
Preparation of Culture media
Preparation of culture media was according to the instruction manual supplied with each medium.

Collection and Identification of Bacterial Samples
The samples were collected from the hospital and bridged directly to the laboratory for identification. The identification of bacterial samples was carried out depending on the cultural characteristics and biochemical tests according to (Mac Faddin, 2000).

Procedure for Performing the Disc Diffusion Test
Inoculum Preparation
Direct Colony Suspension Method
As a convenient alternative to the growth method, the inoculums were prepared by making a direct broth or saline suspension of isolated colonies selected from a 18- to 24-hour agar plate (a nonselective medium, such as blood agar or nutrient agar, has been used).  The suspension is adjusted to match the 0.5 McFarland turbidity standard, using saline and a vortex mixer.

Inoculation of Test Plates
Optimally, within 15 minutes after adjusting the turbidity of the inoculums suspensions, a sterile cotton swab is dipped into the adjusted suspension.  The swab was rotated several times and pressed firmly on the inside wall of the tube above the fluid level. This is necessary to remove excess inoculum from the swab.

The dried surface of a Mueller-Hinton agar plates was inoculated by streaking the swab over the entire sterile agar surface. This procedure is repeated by streaking two more times, rotating the plate approximately 60° each time to ensure an equal distribution of inoculum.  As a final step, the rim of the agar is swabbed. The plate lids were left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for any excess surface moisture to be absorbed before applying the drug impregnated disks.

Application of discs to inoculated agar plates
Antibiotics discs produced by bioanalysa company were used. The predetermined battery of antimicrobial discs is dispensed onto the surface of the inoculated agar plate. Each disc was pressed down to ensure complete contact with the agar surface. Then, the plates were inverted and placed in an incubator set to 37°C within 15 minutes after the discs were applied.

Results and Discussion

The correlation of infection with the type of bacteria
The 91 Bacterial isolates from different infections obtained from Al-Sader medical city in Al-Najaf province over the period from 2-7-2016 to 15-9-2016, were identified and correlated with the site of infection as shown below in table 1.

Table 1. Distribution of bacteria and infections

Infection
Bacteria
Wound UTI Otitis
media
Intestinal
Disease
Total Percentage
E.coli 0 11 0 14 25 27%
Staphylococcus 10 7 5 0 22 24%
Proteus 9 10 8 0 27 30%
Enterococcus 8 9 0 0 17 19%
total 27 37 13 14 91 100%

The identification of bacterial samples showed that most of the isolates (30%) were  Proteus folled by E. coli (27%), Staphylococcus (24%) , and  Enterococcus (19%).

The results showed that UTI is mainly caused by E. coli. This goes in agreement with (Nicolle, 2008) who showed that E. coli is the cause of 80–85% of urinary tract infections, with Staphylococcus saprophyticus as a causative agent in 5–10% of UTI. Most of wound infections has been shown to be caused by Staphylococcus in the present study. This agrees with (Toshkova et al., 2001; Flora, 2002; Baggett et al., 2004) in explaining that Staphylococcus aureus, is the common cause of wound infections.

Correlation between bacterial infections and gender
The type of infection has been correlated with the gender of the patient and with the type of bacteria as shown in table 2.

Table 2. Correlation between bacterial infections and patient gender

Bacteria
Infection
E.coli Staphylococci Proteus Enterococci Total
male female male female male female male female male female
Wound 8 2 8 1 6 2 22 5
UTI 4 7 2 5 3 7 2 7 11 26
Otitis media 3 2 2 6 5 8
Intestinal Disease 8 6 8 6
Total & percentage 12

48%

13

52%

13

59%

9

41%

13

48%

14

52%

8

47%

9

53%

   46

50.5%

45

49.5%

The results showed that the ratio of male to female patients is tend to be equal regardless the type of infections, but, when we consider the type of infection it is clear that males are significantly more affected by wound infections than females as 81.5% of wound infections appeared in mlase. This could be due to the outdoor working environment of the males which makes more exposed to have injuries than females and, consequently, having wound infections more frequently than females. On the other hand, the results showed that UTI is more frequent in females than in males as 70% of UTI appeared in urine samples taken from females. This is most likely due to the anatomy of the female urethra and genital area which makes female more vulnerable to have UTI than males. This agrees with the findings that has been shown previously that UTI is more common in females (Stamm, 1991; Foxman et al., 2000; Harrington and Hooton, 2000; Chittagong, 2011).

Correlation between the type of infection and antibiotic resistance in E.coli
coli was isolated only from urine and stool as a causative agent of UTI and intestinal infection. The antibiotic resistance against 13 different antibiotics was evaluated and correlated to the type of infection which are UTI and intestinal infections. The resistance percentage for each antibiotic was calculated, then the mean of resistance percentages was calculated for both types of the previously mentioned infections (Table 3). The results showed approximate similarity of antibiotic resistance in both UTI and intestinal infections (Figure1).

The most resistance was seen to ampicillin with a percentage of 64% in UTI. This agrees  with (Chittagong, 2011) who showed that the most resistance rates for E. coli detected from urine culture were Ampicillin, Doxycycline, Cephalexin, Cephradine, Cotrimoxazole, Cefixime, Ceftroxone and Ciprofloxacin except for Cefuraoxime to which E. coli was significantly sensitive. The antibiotic resistance pattern by E.coli has been shown to be 40% for Amoxicillin, 23% for Cephradine, 21% for Cotrimoxazole, 6% for Doxycycline, 4% for Cefixime, 2% for Cephalexin, 2% for Ceftroxone and 2% for Ciprofloxacin (Bhowmick and Rashid, 2004; Mohammadi et al., 2010; Manikandan et al., 2011). (Omoregie et al., 2011) showed that 60% of E. coli isolates were susceptible to Gentamycin.

Table 3. Correlation between the type of infection and antibiotic resistance in E. coli

 

Fig. 1. The type of infection and antibiotic resistance in E. coli

Correlation between the type of infection and antibiotic resistance in Staphylococcus spp.
Staphylococcus isolated from wound infections has been shown to be more resistant against 12 antibiotics if compared with isolates from otitis media and UTI (figure 2). The most resistance was seen to Aztreonam antibiotic with a percentage of 83%, while the most effective antibiotic was Vancomycin with a resistance percentage of (8%) (table 4).

(Diekema et al., 2001) who shwed that more than 80% of coagulase-negative staphylococcal isolates were resistant to methicillin and semisynthetic penicillins. (Liu et al., 2011) showed that Community-acquired MRSA (CA-MRSA) isolates often maintain susceptibility to tetracyclines (tetracycline, doxycycline, minocycline, tigecycline). The results by (Omoregie et al., 2011) revealed that Staphylococcus aureus have great suseptibility to Amoxicillin (96.2%). Schwalbe et al,. (1987) showed that Staphylococcus spp. are resistance to Vancomycin.

Table 4. Correlation between the type of infection and antibiotic resistance in Staphylococcus spp.

Fig. 2. The type of infection and antibiotic resistance in Staphylococcus spp.

Correlation between the type of infection and antibiotic resistance in Enterococcus spp.
E
nterococcus was isolated from UTI and wound infections. The results revealed that isolates of UTI were more resistant than those isolated from wounds when tested against 14 antibiotics (figure 3) (table 5). (Karmarkar et al., 2004) showed that 77.23% of enterococcal isolates were resistant to more than six drugs. (Herman and Gerding, 1991) studied serious enterococcal infections (e.g., bacteraemia and endocarditis) and showed that such infections require treatment with a bactericidal combination of antibiotics that should include a penicillin (ampicillin or penicillin G) to which the isolates were susceptible and an aminoglycoside (gentamicin or streptomycin) to which they were resistant.

Table 5. Correlation between the type of infection and antibiotic resistance in Enterococcus spp.

(Courvalin, 2006) showed that more than 55% of enterococcal isolates in ICUs of more than 300 hospitals were vancomycin-resistant.

(Choudhury et al., 2015) revealed that there were rare isolates that lack resistance aginst gentamicin and streptomycin, while high level of resistance were seen against amikacin and kanamycin. And Results obtained by (Sapkota et al., 2007) revealed significant resistance by Enterococcus against erythromycin and tetracycline.

Fig. 3. The type of infection and antibiotic resistance in Enterococcus spp.

Table 6. Correlation between the type of infection and antibiotic resistance in Proteus spp.:

Correlation between the type of infection and antibiotic resistance in Proteus spp.
The isolates of Proteus spp. were obtained from wounds, UTI and otitis media. The most resistant isolates against 11 antibiotics were those isolated from wound infections (figure 4) The most resistance where against Ampicillin/cloxacillin (table 6).

(Feglo et al., 2010) showed that all proteus species were resistant to chloramphenicol, ampicillin and co-trimoxazole. However, 70 – 90 % of P. mirabilis and P. vulgaris isolates exhibited resistance to ampicillin, cotrimoxazole, tetracycline and chloramphenicol. (Newman et al., 2006) revealed that Proteus spp. isolates exhibited high antimicrobial resistance against tetracycline (85 %), chloramphenicol (82.5 %), co-trimoxazole (81 %) and ampicillin (77 %).

Fig. 4. The type of infection and antibiotic resistance in Proteus spp.

Conclusion

  1. coli was the main cause of intestinal diseases.
  2. coli was the most frequent bacteria casing UTI, Proteus spp. otitis media, and Staphylococcus spp. was the main cause of wound infections.
  3. In males the infections with Staphylococcus were more than in females, while infections with E. coli, Proteus spp. and Enterococcus spp. were more in females than in males.
  4. In coli the resistance to antibiotics in UTI and intestinal disease was approximately the same.
  5. In Staphylococcus and Proteus spp. wounds infections exhibit more resistant than other types of infections.
  6. In Enterococcus UTI infections exhibit more resistant than wounds infections.

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