Pathogenicity Study of a number of Gram- negative bacilli isolated from clinical sample

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Pathogenicity Study of a number of Gram- negative bacilli isolated from clinical sample

Abstract

The knowledge of pathoginicity of different clinical isolates has been the global necessity for control pathogenic bacteria.Fifty isolates form different clinical samples (urine and wounds) were collected from Al-Nu'man General Hospital in Baghdad city.The Gram-negative isolates were subjected to antibiotic susceptibility, β- lactamase production, biofilm formation, hemolysin production, and plasmid profile analysis. It is fond E. coli(54%) was the most common followed by Proteus vulgaris (18%), Enterobacter aerogenes (8%), Proteus mirabilis (6%), Enterobacter cloacae (4%), Acinetobacter baumannii (4%), and (2%) for each Klebsiella oxytoca, Enterobacter gergaviae and Pseudomonas mendocina. All isolates were observed resistance 100% for Ampicillin , Carbenicillin, Cefalotin, Cefotaxi, and Meropenem. Proteus vulgaris isolates showed only one large plasmid.

Key words: Gram negative, multidrug resistance, β- lactamase, biofilm , hemolysin, plasmid

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1.Introduction

Gram-negative bacteria cause infections including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis(1).The most important Gram-negative bacilli are members of the family Enterobacteriaceae such as Escherichia, Klebsiella, Enterobacter, Serratia, Citrobacter,and Proteus, (2) normal intestinal flora of humans and animals and may be isolated from a variety of environmental sources.Previous study showed 87.8% from clinical isolates was gram negative bacilli (3).E.coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Klebsiella spp., Haemophilus influenza, Proteus spp., Enterobacter spp. most common gram negative bacteria isolated from clinical samples (4). Multidrug-resistant Gram-negative organisms (MDRGNs) have emerged as a major threat to hospitalized patients and have been associated with mortality rates ranging from 30 to 70% (5).

Several virulence factors may be responsible for the pathogenicity of the bacteria. Extended spectrum β-lactamases (ESBLs) were found in 63.6 % E. coli and 66.7% K. pneumonia isolates(6), and also found in Proteus species and Acinetobacter species (7,8).The bacterial biofilms in the human body is a major cause of recurrent or chronic infections (9). There arefour basic criteria to define biofilm-associated infections: (i) Bacterial cell adherence to or association with a surface, (ii) In vivo observation of bacterial cell clusters, (iii) a localized infection pattern, and (iv) increased resistance to antibiotic treatment in the host compared to resistance of genetically equivalent planktonic bacteria (10). Previous study showed Escherichia coli isolates were assessed for their ability to produce biofilm In vitro by slime production (11).Hemolysin is a cytolytic protein capable of lysing human, horse, and rabbit erythrocytes(1), the haemolysin production analysis showed that 96.6% of Enterobacteriaceae produced cytolytic protein toxins (α-haemolysin) (12),and β-hemolysin production by Proteus spp.and E.coli(13,14).

Plasmid profile analysis is useful in determining the epidemical strain in outbreaks caused by multiple species: Escherichia, Klebsiella, Pseudomonas, Serratia, Streptococcus, and so on (15).Various diverse phenotypic characteristics are encoded by plasmids; these include antibiotic and metal resistance, degradation of complex organic compounds, pro­duction of enterotoxins and colicins, and the production of restriction enzymes (16).

This study was undertaken to identify the various Gram-negative bacilli, isolated from patients admitted in Al-Nu'man General Hospital in Baghdad city and to estimate the virulence factor.

2. Materials and Methods

2.1 Specimens

In this fifty sample analyzed, 30 wound samples and 20 urine samples were collected from Al-Nu'man General Hospital in Baghdad City. Sampling activates were carried out from 23April to 23June 2014. Age range between 40- 70 Years.

2.2 Isolation and identification of gram negative bacteria

2.2.1 Biochemical test

Gram negative bacteria isolated on respective selective and differential media were identified on the basis of colonial, morphological, Gram stain and biochemical tests, IMViC, Urea, Kligler Iron Agar (17) and also used automatically identification system Vitek 2 with GN card (Gram –negative fermenting and non- fermenting bacilli).

2.3 Biofilm formation

The Gram-negative isolates ability to colonize abiotic surface was investigated by using Christensen et al method (18). The E. coli isolates were cultivated in tubes with Trypton soy broth and incubated aerobically at 370C for 48 hours and thereafter the culture tubes were emptied carefully and stained with crystal violet solution 1% for 30 minutes, then tubes rinsed with distilled water and left to dry at room temperature. Results were compared with negative control and notice biofilm formation as a layer at the internal wall of tubes by naked eye indicate appositive result.

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2.4 Hemolysin production

The tested Gram-negative isolates for blood hemolysis were streaked on blood agar plates containing 5% (v /v) human blood and incubated aerobically at 370C for 24 hours. The clear zones around the growth colonies indicate a positive reaction (19).

2.5 β- lactamase production

Iodometric tests was used to detect β- lactamase activity (20).

2.6 Antibiotic sensitivity test

Antibiotic susceptibility profiles of Gram-negative isolates was determined by the standard Kirby-Bauer disk diffusion method (21). These antibiotics with their respective disk concentrations are Amoxacillin-clavulanic (30μg), Carbenicillin (100μg), Cefodizime (30μg), Chloramphenicol (30μg), Gentamycin (10μg), Imipenem (10μg), Norfloxacin (10μg), Piperacillin (100μ g), Rifampcin (5μ g) and Pefloxacin (5μg). Bacterial cultures suspension equivalent of 0.5 tube McFarland turbidity standards were spread on Muller-Hinton agar plates using sterile swabs and incubated aerobically at 370C for 24 hours, then inhibition zones diameter around antibiotic disks were measured. Results were expressed susceptible or resistant according to the criteria recommended by the CLSI (22).

2.7 Plasmid DNA isolated procedure

All Gram-negative isolates were screened for plasmid content by the alkaline method of Brinboim and Doly (23). Separated on a 1% agarose, at 50 vol. for 1hr. and 1.30 hr. The DNA bands were visualized and photographed under UV light after the gel had been stained with ethidium bromide.

3. Results and Discussion

The results showed that the 50 Gram negative isolates obtained from wound and urine samples were classified into six genera and 9 species (table1). 35/50 isolates (70%) were lactose fermenter and 15/50 isolates (30%) were non lactose fermenter (fig. 1 & 2). Recent study form Nigeria appeared 80.4% were lactose-fermenting members of the Enterobacteriaceae, and 19.6% were non-lactose fermenting gram-negative bacteria (24). The most common bacterial isolate was E.coli (54%), followed by Proteus vulgaris (18%), Enterobacteraerogenes(8%), Proteus mirabilis (6%), Enterobacter cloacae (4%), Acinetobacter baumannii (4%), and (2%) for each Klebsiella oxytoca, Enterobacter gergaviae and Pseudomonas mendocina. Study by Panta etal in 2013, showed E.coli the major isolates in the different clinical samples (urine and pus) followed by Salmonella typhi, Klebsiella spp., Salmonella paratyphi, Acinetobacter, Proteus spp.(3).Other study showed the most prevalent Gram-negative bacteria were E.coli (31.6%), Pseudomonas aeroginosa (31.2%), Acinetobacter baumannii (10.8%), Klebsiella pneumonia (8.3%), Klebsiella spp. (6.2%), Haemophilus influenze (3.7%), Proteus spp. (3.3%), and Enterobacter spp. (1.9%) (4).

Table 1: Distribution of bacterial species in different clinical samples

No.

Bacteria species

Urine samples

Wound samples

No. & % of Isolates

1

Escherichia coli

16

11

27 (54%)

2

Enterobacter cloacae

2

0

2 (4%)

3

Enterobactergergaviae

1

0

1 (2%)

4

Enterobacteraerogenes

0

4

4 (8%)

5

Klebsiella oxytoca

1

0

1 (2%)

6

Proteus vulgaris

0

9

9 (18%)

7

Proteusmirabilis

0

3

3 (6%)

8

Pseudomonas mendocina

0

1

1 (2%)

9

Acinetobacterbaumannii

0

2

2 (4%)

Total

20 (40%)

30 (60%)

50 (100%)

*wound W, ** urine U *wound W

Figure 1. Number of lactose ferment bacteria Figure 2. Number of non- lactose ferment bacteria

Antibiotic resistance is a worldwide problem (25).Figure 3 shows the sensitivity pattern of the gram negative isolates. All isolates was observed resistance in 100% for Ampicillin , Carbenicillin, Cefalotin, Cefotaxim, and Meropenem. This results related with previous global and local research (3,4,14, 26).

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*urine sample, **wound sample

Figure 3. Resistance percentage of bacterial species isolated from urine and wound

samples.

β-lactamase, β –hemolysis and Biofilm are a factor related with pathoginicity of infection according to many previous study. Our study appear β-lactamase recorded 86% positive result, β –hemolysis 48% and Biofilm 84%.β-lactamase mainly found in Escherichia coli, Klebsiellaspecies, Proteus species but can also occur in other members of Enteroacteriaceae family and in some nonenteric organisms such as Acinetobacter species (27, 28). Proteus spp. isolates shoed high number of β –hemolysis and then E. coli. Hemolysin is an extracellular protein toxin which is produced by some strains of E. coli, particularly those which cause extraintestinal infections in man (29,30) and also produce by many strain of Proteus (31). Most isolates revaled high activety of biofilm formation. 84% of isolates showed biofilm production except Klebsiella oxytoca. The ability of bacteria to form biofilms helps them to survive hostile conditions within host and is considered to be responsible for chronic or persistent infections (32).

Table 2.Number of bacterial species produce β-lactamase, β –hemolysis and Biofilm

No.

Bacteria Species

No. of Isolates

β-lactamase

β -hemolysis

Biofilm

1

Escherichia coli (U)

16

10

4

10

2

Escherichia coli (W)

11

11

4

11

3

Enterobacter cloacae(U)

2

2

1

2

4

Enterobacter gergaviae(U)

1

1

0

1

5

Enterobacter aerogenes(W)

4

4

2

4

6

Klebsiella oxytoca (U)

1

1

1

0

7

Proteus vulgaris (W)

9

9

7

9

8

Proteus mirabilis (W)

3

3

3

2

9

Pseudomonas mendocina(W)

1

1

1

1

10

Acinetobacter baumannii (W)

2

1

1

2

Total number (%)

50

43 (86%)

24 (48%)

42 (84%)

There are no evidence of plasmid copy in selected 36 clinical isolates (Figure 4,5) except one isolates of Proteus vulgaris (Figure 4/sample number 29) shwod large plasmid (10000 bp).Genetic information can be passed horizontally by transposons, plasmids and bacteriphage: for example when antibiotic resistance genes are carriedon plasmids they can be passed between unrelatedtypes of bacteria. Since genes carried on plasmidsare sometimes incorporated into the chromosome, agene can easily move from one organism to anunrelated one (33). In many cases, virulence genes are foundin large contiguous blocks known as chromosomal inserts or pathogenicity islands (34, 35).

C:\Documents and Settings\y a s s e n\Local Settings\Temporary Internet Files\Content.MSO\28DB78F2.jpgC:\Documents and Settings\y a s s e n\Local Settings\Temporary Internet Files\Content.MSO\A206E176.jpg 1 2 3 4 5 6 7 8 9 10 11 12 s 13 14 15 16 17 18 19 20 21 22 23 24 25

A b

Figure 4: Agarose gel electrophoresis of plasmids extracted from various clinical sample: a- sample 1-12 b-sample 13- 24. S. 1kb(250-10000bp) DNA ladder (Promega); (1% agarose, 50 vol. 1.30 hours).

C:\Documents and Settings\y a s s e n\Local Settings\Temporary Internet Files\Content.MSO\73D3F58A.jpgC:\Documents and Settings\y a s s e n\Local Settings\Temporary Internet Files\Content.MSO\C4630A6D.jpg

S 26 27 28 29 30 31 32 33 34 35 36

Figure 5: Agarose gel electrophoresis of plasmids extracted from various clinical sample: a- sample 25-36b-negatve picture. S. 1kb(250-10000bp) DNA ladder (Promega); (1% agarose, 50 vol. 1.30 hours).

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