Isolation of Proteus mirabilis and Proteus vulgaris from Different

Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(21): 2013

Isolation of Proteus mirabilis and Proteus vulgaris from Different Clinical Sources and Study of some Virulence Factors* Mays Hadi Jabur

Elham Abbas AL- Saedi Jawad Kadhum Trad Babylon university /College of Medicine

Abstract A total of 15 isolates of proteus out of 100 samples comprising of proteus mirabilis (7) (7%), proteus vulgris (8) (8%) isolated from various clinical sources (urin, otitis media, stool, burn, wound and peri anus infection). Maximum isolates were obtained from urine (33.33%), stool with otitis media (20%), burn (13.33%), wound and perianus infection. (6.66%). All isolates were tested for their resistance to fifteen different antibiotics and the results showed that all isolates were entirely resistant to cephalothin, cloxacyclin, tetracycline, doxycycline, lincomycin, erythromycin, azithromycin and rifampicin (100%), but lesser to penicillin and streptomycin (93.3%) and much lesser to amoxicillin (80%), ciprofloxacin (53.3%), gentamycin (46.6%). All isolates were sensitive to amikacin. Also all isolates were examined for colonization factor antigen, haemolysin production and protease.

‫اﻟﺧﻼﺻﺔ‬ ‫ ( ﻋـزﻻت ﻣـن‬8 ) ‫ و‬Proteus mirabilis ‫ ( ﻋزﻻت ﻣن‬7 ) ‫ ﺗﺿﻣﻧت‬Proteus ‫ ﻋزﻟﺔ ﻋﺎﺋدة ﻟﺑﻛﺗرﯾﺎ‬١٥ ‫ﺗم ﻋزل‬ ‫ اﻟﺟــروح واﺻــﺎﺑﺎت ﻓﺗﺣــﺔ‬، ‫ اﻟﺣــروق‬، ‫ اﻟﺧــروج‬، ‫ اﻻذن اﻟوﺳــطﻰ‬، ‫ﻋﯾﻧــﺔ ﺷــﻣﻠت ﻋﯾﻧــﺎت ) اﻻدرار‬100 ‫ ﻣــن ﺑــﯾن‬Proteus vulgaris ‫ﻣﺧﺗﻠﻔ ــﺔ اذ ﺗ ــم اﻟﺣ ــﺻول ﻋﻠ ــﻰ اﻋﻠ ــﻰ ﻧ ــﺳﺑﺔ ﻣ ــن ﻋﯾﻧ ــﺔ اﻻدرار واﻟﺗ ــﻲ‬

Proteus

‫اﻟ ــﺷرج ( وﻗ ــد ﻛﺎﻧ ــت ﻣﻌ ــدﻻت اﻻﺻ ــﺎﺑﺔ ﺑﺑﻛﺗرﯾ ــﺎ‬

‫وﺗـم اﻟﺣـﺻول ﻋﻠـﻰ‬13.33% ‫ وﻧـﺳﺑﺔ ﻋﯾﻧـﺎت اﻟﺣـروق ﻓﻘـد ﺑﻠﻐـت‬،20% ‫ اﻣـﺎ ﻋﯾﻧـﺎت اﻟﺧـروج واﻻذن اﻟوﺳـطﻰ ﻓﻘـد ﺑﻠﻐـت‬33.33% ‫ﺑﻠﻐـت‬ . 6.66% ‫اﻗل ﻧﺳﺑﺔ ﻣن اﻟﺟروح وﻓﺗﺣﻪ اﻟﺷرج واﻟﺗﻲ ﻛﺎﻧت ﺑﻧﺳﺑﺔ‬ ‫ ﻧوع ﻣن اﻟﻣﺿﺎدات وﻗد ﺑﯾﻧت اﻟﻧﺗﺎﺋﺞ ﺑﺎن ﻛل اﻟﻌـزﻻت ﻛﺎﻧـت ﻣﻘﺎوﻣـﺔ‬15 ‫ﺗم اﻟﺗﺣري ﻋن ﻗﺎﺑﻠﯾﺔ ﺟﻣﯾﻊ اﻟﻌزﻻت ﻟﻣﻘﺎوﻣﺗﻬﺎ اﻟﻣﺧﺗﻠﻔﺔ اﻟﻰ‬ rifampicin , , lincomycin , doxycycline, tetracycline ,cloxacyclin ,cephalothin

‫ﻛﻠﯾـﺎ اﻟـﻰ‬

streptomycin ‫ و‬penicillin ‫ ﻟﻣﻘﺎوﻣﺗﻬـﺎ ﻟﻛــل ﻣــن‬93.3% ‫ وﺑﻧــﺳﺑﺔ اﻗــل ﺑﻠﻐــت‬100 %‫ﺑﻧـﺳﺑﺔ‬azithromycin ,erythromycin ‫ ﻛﻣـﺎ ان‬، ‫ ﻋﻠـﻰ اﻟﺗـواﻟﻲ‬%(80, 53.3, 46.6 )‫ ﻓﻘد ﺑﻠﻐت‬amoxicillin , ciprofloxacin , gentamycin ‫اﻣﺎ ﻧﺳﺑﺔ اﻟﻣﻘﺎوﻣﺔ اﻟﻰ‬ .amikacin ‫ﺟﻣﯾﻊ اﻟﻌزﻻت ﻛﺎﻧت ﺣﺳﺎﺳﺔ اﻟﻰ‬ .‫ اﻟﻬﯾﻣوﻻﯾﺳﯾن واﻟﺑروﺗﯾﯾز‬،‫ﻛذﻟك ﺗم اﺧﺗﺑﺎرﻗﺎﺑﻠﯾﺔ اﻟﻌزﻻت ﻋﻠﻰ اﻧﺗﺎج ﻋﺎﻣل اﻻﺳﺗﻌﻣﺎر‬

Introduction Proteus species is known uropathogen especially causing urine tract infections in catheterized patients (Mobely and Warn, 1987, Warren et al., 1992) and those with urinary tract abnormalities. It may lead to pyelonephritis, stones, fever and bacteraemia. (Mishara et al., 2001). Proteus can cause a variety of community or hospital acquired infections including those of urinary tract, respiratory tract, wound, otitismedia, and burns, bacteraemia, neonatal meningoencephalitis, empyema, and osteo- mylitis (O'Hara et al., 2000). The routine use of antimicrobial agents in human has resulted in wide spread antibiotic resistance and the development of antibiotic resistance gene especially within and between the gram-negative bacteria (Enabulele el al., 2006) with the *

This paper is apart of M.SC thesis, college of medicine, university of Babylon . The comprehensive data are included in the thesis (in press). ٤٣

presence of antibiotics sensitive pressure, these resistant Proteus species tend to persist, enabiling the organism to cause extrainfection such as septicemia. Mechanism of pathogenesis of these organisms include several virulence factors, these include adhesion to uroepithelium (Old and Adegbola, 1992), haemolysin production (Welch, 1997), urease production (Jones and Mobley, 1997), swarming, protease and lipase (Allison et al., 1992). It has been suggested that heavily fimbriated organisms were better able to intiate the infection (Mobley and Chippendale, 1990). Similarly production of haemolysin has been correlated with cytotoxicity and with increased virulence of strains isolated from UTI, also the ability of isolates to produce protease considered as important virulence factor between Proteus species (Welch, 2004).

Aim of the study 1 – Isolation and identification of Proteus spp. from different clinical speciemens. 2 – Study of the effect of some antibiotics on Proteus isolates. 3 – Study of some factors associated with pathogenecity of Proteus such as colonization factor antigen, haemolysin, and protease production.

Material and methods:1 – Antibiotics sensitivity test A Kirby – Baur method was used for testing the antibiotics sensitivity (Baron and Fingold, 1990). 2 – Detection of colorization factor antigen: C.F.A. was achieved according to method of (Symth, 1982). 3- Detection of haemolysin production haemolysin production was achieved according to (Collee et al., 1996). 4 –Detection of protease production protease production was achieved according to (Macfaddin, 2000).

Results and discussion:This study deals with (100) samples isolated from different clinical sources (urine, otitis media, stool, burn, wounds, and perianus infection). Isolated and identification of microorganism comprising of proteus mirabilis 7 (7%), p. vulgaris 8 (8%). (Table 1, Table 2). Table (1): Frequency of positive and negative culture: Patients No. Positive culture Negative culture p. mirabilis p. vulgaris 100 7 8 85 % 7% 8% 85% Table (2): Distribution of Proteus species among various clinical sources: Samples Number % Urine 5 33.34% Otitis media 3 20% Stool 3 20% Burn 2 13.34% Wound 1 6.67% Perianus 1 6.67% Total 15 100% ٤٤


This results obtained in this study are identical with those obtained by ( ALBaghdadi et al ., 2009 ) who has succeed to isolate P. mirabilis and P. vulgaris at rate reaching to ( 10 %), ( 14 %) respectively but (Mishra et al., 2001) have isolated P.mirabilis from different sources of infection at rate reaching to (78.3%), (16.2%) respectively. The antibiotics profile of all isolates were investigated (Table 3). It showed no difference of antibiotic sensitivity among isolates. It was found that p.mirabilis and p. vulgaris are sensitive to amikacin (100%) and resistant (100%) to cephalothin, tetracycline, lincomycin, erythromycin, doxycycline rifampcin, azithromycin, and cloxacline. Some isolates shown resistance in lesser degree to penicillin and streptomycin (93.3%), amoxicillin (80%), ciprofloxacin (53.3%) and gentamycin (46.6%). The isolates are also resistant to cefotaxime (26.6%). Table (3): effect of antibiotics on the growth of proteus Isolate no. KF P AX CX CTX E AZM TE DO 1. + + + + + + + + + 2. + + + + + + + + 3. + + + + + + + + + 4. + + + + + + + + 5. + + + + + + + + + 6. + + + + + + + + 7. + + + + + + 8. + + + + + + + + 9. + + + + + + + + 10. + + + + + + + 11. + + + + + + + + 12. + + + + + + + + 13. + + + + + + + + 14. + + + + + + + + 15. + + + + + + + + Rute of 100 93.3 80 100 26.6 100 100 100 100 resistant% + resistant, - sensitive

CIP + + + + + + + + 53.3

CN + + + + + + + 46.6

S + + + + + + + + + + + + + + 93.3

Ak 0

RA + + + + + + + + + + + + + + + 100

KF= cephalothin , P = penicillin , AX = amoxicillin , CX= cloxacyclin , CTX= cefotaxime , E= erythromycin , Azm = azithromycin , TE= tetracycline , DO= doxycycline , CIP= ciprofloxacin , CN= gentamycin S= streptomycin , AK= amikacin , RA = rifampcin, L= lincomycin . All the isolates have shown resistance to beta-lactam groups include Cefotaxime, it is a synthetic broad spectrum cephalosporin antibiotic. The bacteriocidal activity of cefotaxime results from the inhibition of cell wall synthesis. (Mary et al., 2000). This results is identical with those obtained by (Pagani et al., 2002) and (Fluit et al., 2000) whom reported, most strains of P. mirabilis and P. vulgaris one increase of beta-lactam resistance mediated by the production of beta-lactamases .beta-lactam resistance in p. mirabilis is mainly due to the plasmid-mediated pencillinases TEM-1 and TEM-2. (Chanal et al., 2000). Furthermore, all the isolates have shown resistance to erythromycin and azithromycin this result agreement with (Mishra et al., 2001) ٤٥

L + + + + + + + + + + + + + + + 100

resistance to erythromycin is most commonly due to the acquisition of one of 21 genes these code for rRNA methylases that bring about methylation of adinine residues in 23 s rRNA preventing the binding of macrolides to 50s ribosomal subunit. Azithromycin is abacteriostatic agent which inhibit the growth of microorganisms by binding to the 50s subunit of the prokaryotic ribosome blocking protein synthesis at the peptidyl transferase step. The results also show that all isolates were resistante to tetracycline and this result agreed with Ahmad et al., (2007). Tetracycline is bacteriostatic especially in large doses and it is short acting. The results of the present study show high resistance of aminoglycosides (streptomycin and gentamycin). Aminoglycosides resistance is mediated by drug-modifying enzyme (Ahmed et al., 2007). There is no difference between P. mirabilis and P. vulgaris in response to antibiotic sensitivity test. The correct identification and antibiotic sensitivity testing of such isolates are essential for the proper management of infected individuals. In this study three virulence factors, colonization factor antigen, haemolysin and protease were examined The results showed that all isolates (100(% agglutinate human red blood cells type A and O in the presence of mannose or tannic acid which indicates that all Proteus isolates have CFA I and CFA III . Also the results of the present study show that all tested isolates (100%) have the colonization factor antigen II . The first necessary step for any pathogenic bacterium to initiate infection is the attachment to suitable receptor .This result agreement with (Mishara et al., 2000). Heavily fimbriated organism were show to initiate infection better than lightly fimbriated organisim , two fimbrial types mannose – resistance / protease like (MR/P) and mannose – resistance Klebsiella like ( MR/K) have been associated with the ability to hemagglutinate untreated or tannic acid – treated erythrocyte . (Bahrani et al., 1991). Haemolysin has been considered to be an important virulence factor (Zunino et al., 1999). Suggested that haemolysin is not essential during early infection but this factor is important at late stages of infection. As many as 100% isolates in the present study , produced beta- haemolysin , there was no significant differences in the haemolysin among isolates from different clinical source together . This result agree with results of (Bahraini et al ., 1991) who indicate that all isolates (100%) isolated from different clinical sources exhibit B – haemolysin on blood agar plates . In the ability of Proteus mirabilis and P. vulgaris to produce extracellular protease by using M9 media (supported by 2% glucose and 1% gelatin ) is investigated and it is found that all isolates (100%) able to produce extracellular protease after 48hrs. of incubation there is transparent area around the colony after the addition of 3 ml (5%) of trichloro acetic acid ( TCA) Table (4) .

٤٦


Table (4): Detection of Virulence factors on the Proteus species Isolation NO.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

CFA

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Hemolysin

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Protease

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Virulence factors

The results of the present study agree with the results of (Bahrani et al., 1991). Who indicate that Proteus species have been reported to produce extracellular protease. Proteases which have the capacity to degrade host proteins releasing amino acid as nutrients and may degrade proteins such as IgA which are involved in host defence , and may also be involved in host tissue damage ( Jost and Billington , 2005 ) .

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