Colistin Resistant Rate among Gram's Negative Bacteria Isolated from Sharg Alnile and Yastabsheroon Hospitals from 2016 to 2017

  

    
Burkholderia    mallei
    
Sensitive
    
Resist
    
Intermediate
  
  

    
CAZ(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
GM(10μg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
CXM(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
AX(10mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
CRO(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  

Table 15: Results of Antimicrobial Susceptibility testing of Burkholderia mallei. (100%) sensitive to Ceftazidime, (100%) sensitive to Gentamicin, (100%) sensitive to cefuroxime, (100%) resist to Amoxicillin and (100%) sensitive to Ceftrixon.

Increase in the prevalence of Gram’s negative pathogens that are resistant to Fluoroquinolones and Aminoglycosides as well as all β-lactams, including Carbapenems, Monobactam, Cephalosporins and broad-spectrum Penicillin, has prompted the reconsideration of Colistin as a valid therapeutic option [8]. Gram’s negative bacteria can develop resistance to Colistin through mutation or adaptation mechanisms. Mutation is inherited, low-level, and independent of the continuous presence of the antibiotic, whereas adaptation is the opposite. Studies of polymyxin-resistant Pseudomonas aeruginosa strains have suggested that alterations of the outer membrane of the bacterial cell (reduction in LPS, reduced levels of specific outer membrane proteins, reduction in cell envelope Mg⁺² and Ca⁺² contents, and lipid alterations) are related to the development of resistance. In addition, a recent study in Yersinia species demonstrated that an efflux pump/potassium system may be associated with resistance to polymyxin B. Although enzymatic resistance of bacteria to Colistin has not been reported, it is interesting that Bacillus polymyxa subspecies colistinus produces colistinase that inactivates Colistin [9].

Colistin is an old-generation antimicrobial agent; however, because it is one of the few agents that remain effective against multidrug-resistant Gram’s negative bacteria (e.g., carbapenem-resistant Pseudomonas aeruginosa and Enterobacteriaceae), its clinical usefulness is being increasingly recognized [10], among the most clinically significant multidrug-resistant bacteria are carbapenemase-producing Enterobacteriaceae. Because these bacteria usually remain susceptible to polymyxins, because of their potential toxicity, interest in polymyxins (colistin and polymyxin B) has been renewed worldwide. However, the increasing use of colistin explains why acquired Colistin resistance may now be added to the carbapenem resistance trait in Enterobacteriaceae [11]. Previous reports have described the mechanisms of Colistin resistance as being chromosomally mediated and not associated with horizontal gene transfer. However, from 2011 through 2014, a plasmid-encoded Colistin-resistance gene was identified in Colistin-resistant Escherichia coli isolated in China, particularly from animals, and 1% of hospitalized human patients [11,12].

For identification of polymyxin resistance in Enterobacteriaceae, they used many tests either by Antibiotic susceptibility testing that performed per CLSI guidelines (dick diffusion methods, dilution methods and E test) or by genotypes (PCR Amplification and Sequencing) [13].

Prevention of antibiotic resistance

Antimicrobial agents have been greatly important cornerstones of clinical medicine since the second half of the 20^th century and have saved a great number of people from life threatening bacterial infections. However, the last decade of the 20^th century and the first decade of the 21^th century have witnessed the emergence and spread of antibiotic resistance in pathogenic bacteria around the World, and the consequent failure of antibiotic therapy, especially in intensive care units (ICUs), which has led to hundreds of thousands of deaths annually [14].

Antibiotic resistance occurs when bacteria outsmart drugs [15]. Everyone has a role in helping to prevent antibiotic resistance. Canadians and healthcare professionals must work together to reduce its impacts on our health and healthcare system [16].

The doctors, nurses, veterinarians and other health workers must be aware that do not prescribe or dispense antibiotics unless they are truly necessary and you have made all efforts to test and confirm which antibiotic your human patient or the animal you are treating should have [17].

So if you are sick and doctor give you antibiotics you should take antibiotics exactly as directed by your health care professional; make sure you know how much to take (the right dosage),when to take your antibiotics, and how many days you should take them, Even if you feel better, finish your antibiotics as directed to make sure that all of the bacteria are destroyed, Do not share your antibiotics with anyone, use leftover antibiotics or use antibiotics prescribed for someone other than yourself [18].

The development of quick, effective molecular techniques for identifying resistance genes and the search of diagnostic biomarkers such as procalcitonin for using as a guide to cessation of antibiotics treatment are useful for reducing the use of antibiotics. Ultimately, if all members of society take on responsibility for maintaining the effectiveness of antibiotics and perform their role, minimization of antibiotic resistance can be successful [19].

Materials and Methods

Study design

Cross sectional hospital based study.

Study area

This study was carried out in Sharg Alnile and yastabsheroon hospitals.

Study population

Isolated Gram’s negative bacteria from different samples.

Sample size

According to duration between November 2016 and March 2017.

Included criteria

Isolated Gram's negative bacteria.

Excluded criteria

 Gram's positive bacteria.

Table 16: Results of Antimicrobial Susceptibility testing of Citrobacter koseri. (100%) resist to Norfloxacin (NOR), (100%) resist to Ciprofloxacin (CIP), (100%) resist to Nitrofurantoin (F), (100%) resist to Piperacillin (PRL), (100) resist to Cefixime (CFM), (100%) sensitive to Meropenem (MEM) and to Amikacin (AK).

  

    
Citrobacter    koseri
    
Sensitive
    
Resist
    
Intermediate
  
  

    
NOR(10mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
CIP(5mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
F(300mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
PRL(100mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
CFM(30mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
MEM(10mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
AK(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  

Table 16: Results of Antimicrobial Susceptibility testing of Citrobacter koseri. (100%) resist to Norfloxacin (NOR), (100%) resist to Ciprofloxacin (CIP), (100%) resist to Nitrofurantoin (F), (100%) resist to Piperacillin (PRL), (100) resist to Cefixime (CFM), (100%) sensitive to Meropenem (MEM) and to Amikacin (AK).

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Table 17: Results of Antimicrobial Susceptibility testing of Edwardsiella tarda. (100%) Norfloxacin (NOR), (100%) Ciprofloxacin (CIP), (100%) Nitrofurantoin (F), (100%) Piperacillin (PRL), (100) Cefixime (CFM), (100%) Meropenem (MEM) are resistance and (100%) sensitive to Amikacin (AK).

  

    
Edwardsiella    tarda
    
Sensitive
    
Resist
    
Intermediate
  
  

    
NOR(10mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
CIP(5mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
F(300mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
PRL(100mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
CFM(30mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
MEM(10mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
AK(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  

Table 17: Results of Antimicrobial Susceptibility testing of Edwardsiella tarda. (100%) Norfloxacin (NOR), (100%) Ciprofloxacin (CIP), (100%) Nitrofurantoin (F), (100%) Piperacillin (PRL), (100) Cefixime (CFM), (100%) Meropenem (MEM) are resistance and (100%) sensitive to Amikacin (AK).

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Methods

The isolated Gram's negative organisms were collected from different clinical samples, and sub cultured on MacConkey Agar for purification, incubate aerobically at 37 ⁰C for 24 hours [20-27].

MacConkey (Appendix 1) media which was used for cultivation of enterobacteria, contain a bile salt to inhibit non-intestinal bacteria and lactose with neutral red to distinguish the lactose-fermenting (pink) from non lactose-fermenting (yellow) [28].

Culture method

 The organisms were isolated and subculture on MacConkey, a colony was taken by using a wire loop sterilized by holding it in Bunsen flame so that the whole length becomes red-hot and waits until cooled. The inoculums were streaked thoroughly over area A to give a well-inoculums. The loop was re-sterilized and then drawn from the well in two or three parallel lines on to the fresh surface of the medium this process was repeated [27].

Preparation of smear

On sterile microscopic slide by sterile wire loop few drops of sterile normal saline was putted and re-sterile the loop and cooled, touch the colony from microorganisms grown in culture. And emulsify on the normal saline and the smear dry by air and fixed by passing three times through the flame [27].

Gram's stain

The Gram’s stain and microscopic evaluation of cultured bacteria were used with colony morphology to decide which identification steps are needed.

The dry and fixed smear was flood by crystal violet (Appendix 2) for few second and washed with tap water, and flood by iodine (Appendix 3) for few second and washed with tap water, the slide was flood on decolorize (Appendix 4) for second and washed with tap water, the slide was flood by counter stain (safranine) (Appendix 5) for 30 second and washed with tap water, then the slide was examined under the microscope [27].

Table 18: Results of Antimicrobial Susceptibility testing of Proteus mirablis. (100%) Norfloxacin (NOR), (100%) Ciprofloxacin (CIP) are sensitive, (100%) Nitrofurantoin (F) is resist, (100%) sensitive to Piperacillin (PRL), (100%) resist to Cefixime (CFM).

  

    
Proteus    mirablis
    
Sensitive
    
Resist
    
Intermedate
  
  

    
NOR(10mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
CIP(5mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
F(300mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
PRL(100mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
CFM(30mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  

Table 18: Results of Antimicrobial Susceptibility testing of Proteus mirablis. (100%) Norfloxacin (NOR), (100%) Ciprofloxacin (CIP) are sensitive, (100%) Nitrofurantoin (F) is resist, (100%) sensitive to Piperacillin (PRL), (100%) resist to Cefixime (CFM).

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Table 19: Results of Antimicrobial Susceptibility testing of Providencia retegerii. (100%) Ceftazidime (CAZ), (100%) Gentamicin (GM), (100%) Cefuroxime (CXM) are sensitive, (100%) resist to Amoxicillin (AX), (100%) is sensitive to Ceftrixon (CRO).

  

    
Providencia    retegerii
    
Sensitive
    
Resist
    
Intermediate
  
  

    
CAZ(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
GM(10μg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
CXM(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  
  

    
AX(10mcg)
    
0(0%)
    
1(100%)
    
0(0%)
  
  

    
CRO(30mcg)
    
1(100%)
    
0(0%)
    
0(0%)
  

Table 19: Results of Antimicrobial Susceptibility testing of Providencia retegerii. (100%) Ceftazidime (CAZ), (100%) Gentamicin (GM), (100%) Cefuroxime (CXM) are sensitive, (100%) resist to Amoxicillin (AX), (100%) is sensitive to Ceftrixon (CRO).

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Results

Antimicrobial susceptibility test

Standard antimicrobial susceptibility testing was done for isolated organism, and we used polymxin family (Colistin), for Multi-Drug Resistance (MDR) organism and was performed for all Gram’s negative bacteria isolated by Kirby –Bauer disk diffusion method using Muller-Hinton agar media according to the clinical laboratory standards institute (CLSI) guidelines [28].

Method

Inoculation preparation: Used of pure cultured Gram’s negative bacteria, by inoculated 4-5 colonies have same morphology in broth media and the suspension was standardized by Macfarland (Appendix 12) turbidity (concentration equaled to 1.5*10⁸) [27].

Sterile cotton swab was dipped into broth media and the excess was removed by rotation of the swab against the side of the tube and streaked on the Muller-Hinton agar (Appendix 13), the disc of the antibiotic was applied with sterile forceps into the surface of the media and incubated aerobically at 3⁰C overnight [27].

Antibiotic used

After incubation the zone of inhibition was measured and compared to the sheet provided by manufacture [27] (Table 1 & Figure 2).

Table 20: Frequency and the percentage of the Merobenem and Amickacin resistance organisms.

  

    
Type    of organism
    
Frequency
    
%Percentage
  
  

    
MEM&AK    sensitive
    
117
    
98%
  
  

    
MEM&AK    resistance
    
2
    
2%
  
  

    
Total
    
119
    
100%
  

Table 20: Frequency and the percentage of the Merobenem and Amickacin resistance organisms.

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Table 21: Frequency and the Percentage of gram negative according to susceptibility to colistin.

  

    
COLISTIN
    
Frequency
    
%Percentage
  
  

    
Resistance
    
0
    
0%
  
  

    
Sensitive
    
2
    
100%
  
  

    

    
2
    

  

Table 21: Frequency and the Percentage of gram negative according to susceptibility to colistin.

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Control organisms

The control organisms were applied to checked Gram’s stain, culture media, biochemical test and sensitivity test by standard organisms of Staphylococcus aureus American type culture collection (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), obtained from department of Microbiology in central lab (Tables 2-21).

Discussion

Development of antimicrobial resistance is a phenomenon inevitably related to microbial evolution and antibiotic use. This study to isolate and identify the Colistin resistant Gram’s negative bacteria.

The resistance to colistin antibiotic in this study was 0%, which is not agree with study that conducted in Central Greece (2015) by Oikonomou O, and et al, Sassera D, and et al in Nigeria (2011), and Matthaiou DK et al in Athens, Greece (2008). Which showed that resistance to colistin antibiotic was 21.1%, 17.6% and 41% respectively, this may be due to difference in the sample size, study area and method that applied [21,24,26].

In this research the isolated MDR E.coli is sensitive to colistin was inconsistence with study done by Hua Yu et al. which show that there is detection of colistin resistant Eschriachia coli: it may be due to different in sample size, study area [27].

A research done by Goli HR et al, in Iran (2016), and Sassera D, et al. in Italy (2014) which the colistin resistance rate is 2% from 100 sample, this result Differs from the presented study result there is no colistin resistance detected from 119 sample. May be due to the different area [22,23].

A study conducted by Antoniadou A et al, they found that the isolated K. pneumoniae were resistant to colistin. Their results inconsistence with this research result, since the isolated K. pneumoniae was resistant to Meropenem and Amickacin and sensitive to Colistin [1].

References

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Citation: Ahmed NAA. Colistin Resistant Rate among Gram's Negative Bacteria Isolated from Sharg Alnile and Yastabsheroon Hospitals from 2016 to 2017. J Blood Disord. 2018; 5(1): 1050. ISSN 2379-8009

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