Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 3 (September) 2008; 176 - 190 176 Referred by Referred by Prof. Dr. Jakeen K. A. El-Jakee Professor of Microbiology , Fac. Vet. Med., Cairo University Prof. Dr. Mona El-Enbeawy Professor of Microbiology , Fac. Vet. Med., Cairo University Minimum inhibitory concentrations for selected antimicrobial agents against Fusobacterium necrophorum isolated from bovine hepatic abscesses By El- Shorbagy M. M., Eman M. Nasr and Mona A. Maghawry Bacteriology Department. Animal Health Research Institute, Dokki, Giza SUMMARY T his study was carried out to determine bacterial agents causing liver abscesses in cattle slaughtered in El-Basateen abattoir in 2007 and to determine the resistance and susceptibility of Fusobacterium necropho- rum isolated from bovine hepatic abscesses to antimicrobial compounds. The following bacteria were isolated either alone or currently mixed to- gether, Fusobacterium necrophorum in the percentage of (47.4%), Clos- tridium perfringens (28.9%), Arcanobacterium pyogenes (44.7%), Staphylococcus aureus (23.7%), Escherichia coli (7.9%) and Peptostrep- tococcus anaerobius (5.3%). C. perfringens isolates were subjected to toxin-typing by using dermonecrotic test in guinea pig, C. perfringens types A and D were identified and type D was the most prevalent. Antim- icrobial susceptibility testing to different chemotherapeutic agents indi- cated that F. necrophorum, A. pyogenes and C. perfringens isolates were sensitive to penicillin G, clindamycin, lincomycin and tylosin. The same isolates were resisting to erythromycin, gentamycin, ampcillin, strepto- mycin, doxycillin HCl and nalidixic acid. MICs of penicillin G against F. necrophorum were as follows; 0.29 IU while it was 2.5 μg for lincomycin and MBCs were 1.17 for IU for penicillin G/ml, while it was 2.5 μg of lincomycin/ml. INTRODUCTION H epatic abscesses constitutes a serious economic problem. Due to the nature of its pathogene- sis and development. The disease is generally not diagnosed until the animals are slaughtered in the ab- attoir. Clinical symptoms are ap- parent only in cases of acute he- patic necrobacillosis (Mateos et
Referred byReferred by Prof. Dr. Jakeen K. A. El-Jakee Professor of Microbiology , Fac. Vet. Med.,
Cairo University Prof. Dr. Mona El-Enbeawy Professor of Microbiology , Fac. Vet. Med.,
Cairo University
Minimum inhibitory concentrations for selected antimicrobial agents
against Fusobacterium necrophorum isolated from bovine hepatic
abscesses
By El- Shorbagy M. M., Eman M. Nasr and Mona A. Maghawry
Bacteriology Department. Animal Health Research Institute, Dokki, Giza
SUMMARY
T his study was carried out to determine bacterial agents causing liver abscesses in cattle slaughtered in El-Basateen abattoir in 2007 and to
determine the resistance and susceptibility of Fusobacterium necropho-rum isolated from bovine hepatic abscesses to antimicrobial compounds. The following bacteria were isolated either alone or currently mixed to-gether, Fusobacterium necrophorum in the percentage of (47.4%), Clos-tridium perfringens (28.9%), Arcanobacterium pyogenes (44.7%), Staphylococcus aureus (23.7%), Escherichia coli (7.9%) and Peptostrep-tococcus anaerobius (5.3%). C. perfringens isolates were subjected to toxin-typing by using dermonecrotic test in guinea pig, C. perfringens types A and D were identified and type D was the most prevalent. Antim-icrobial susceptibility testing to different chemotherapeutic agents indi-cated that F. necrophorum, A. pyogenes and C. perfringens isolates were sensitive to penicillin G, clindamycin, lincomycin and tylosin. The same isolates were resisting to erythromycin, gentamycin, ampcillin, strepto-mycin, doxycillin HCl and nalidixic acid. MICs of penicillin G against F. necrophorum were as follows; 0.29 IU while it was 2.5 µg for lincomycin and MBCs were 1.17 for IU for penicillin G/ml, while it was 2.5 µg of lincomycin/ml.
INTRODUCTION
H epatic abscesses constitutes a serious economic problem.
Due to the nature of its pathogene-sis and development. The disease
is generally not diagnosed until the animals are slaughtered in the ab-attoir. Clinical symptoms are ap-parent only in cases of acute he-patic necrobacillosis (Mateos et
al., 1997). Liver abscesses repre-sent a major economic liability to producers and ultimately consum-ers. Besides liver condemnation, economic impacts include reduced feed intake, reduced weight gain, decreased feed efficiency and de-creased carcass yield. F. necrophorum is a normal inhabitant of the bovine rumen. Hepatic abscesses in cattle are re-lated to feeding of high-grain diets. Rapid fermentation of grain in the rumen and the consequent accu-mulation of organic acids, particu-larly lactate, result in ruminal aci-dosis and ruminitis Fusobacterium necrophorum penetrates the dam-aged rumen epithelial wall, reaches the liver via the portal circulation and causes abscesses (Nagaraja and Chengapa, 1998). F. necrophorum is non motile, non- spore- forming pleomorphic, Gram negative, strictly anaerobic, and is considered the primary etio-logic agent at hepatic abscesses in cattle (Scanlan and Hathcock, 1983; Kanoe et al., 1984; Jang and Hirsh, 1994 and Mateos et al., 1997). The bacterium has been iso-lated from 81 to 100 % of liver ab-scesses where systematic studies have been conducted (Tan et al., 1994). In some instances, the or-ganism has been involved as a sin-gle pathogen (Berg and Scanlan,
1982 and Lechtenberg et al., 1988), but often it was associated with a variety of other anaerobic or facultative anaerobic bacteria (Scanlan and Hathcock 1983). Such as; Arcanobacterium pyo-genes, Bacteroides, Clostridium, Pasteurella, Peptostreptococcus, Staphylococcus, Streptococcus (Simon and Stovell, 1971; Lechtenberg et al., 1988). Due to the importance of Fusobacterium necrophorum as an animal patho-gen, antimicrobial susceptibilities of the organism, particularly to clinically relevant antimicrobials have been reported (Lechtenberg et al., 1988). As control of liver abscesses in feed lot cattle gener-ally has depended on the use of an-timicrobial compounds. (Nagaraja and Chengappa, 1998). The pur-poses of the present study were to determine by cultural examination, the aerobic and anaerobic bacterial flora from bovine hepatic ab-scesses, and also to determine the resistance and susceptibility of F. necrophorum of bovine hepatic ab-scesses origin to some antimicro-bial compounds. MATERIALS AND METHODS 1- Samples: Thirty eight hepatic abscesses were obtained at slaughter from liver of slaughtered cattle at El-Basateen abattoir. Only abscesses with a purulent pocket were used. Portion of the liver contain the ab-
scesses on ice were transported to the laboratory. The specimens were processed the same day they were collected or kept at – 20 ºC until use.
2- Bacteriological examination: 38 abscesses from 38 livers
were examined, the abscesses were seared with a hot spatula and then opened with a sterile surgical blade. Samples were divided into two portions and were submitted to aerobic and anaerobic examina-tions from the first portion a loop-ful of the purulent material was streaked onto the surface of nutri-ent agar, MacConkey agar and blood agar media, all inoculated plates were incubated for 24 hour at 37ºC. Then all different colonies were selected and purified by sub-culturing on its selective medium include eosin methylene blue agar medium for the isolation of E. coli, mannitol salt agar for the isolation of S. aureus, Downes medium for pure culture colonies of coryne-bacterium group according to Cru-ickshank et al. (1975). Bacterial isolates were examined morpho-logically, culturally and biochemi-cally according to Burrows (1985) and Bailey and Scott (1990). A loopful from the second portion of purulent material was inoculated into brain heart infusion broth sup-plement with hemin, yeast extract, vitamin K and cysteine hydrochlo-ride incubated anaerobically at
37ºC for 48 hours (Lechtenberg and Nagaraja, 1998) followed by subculture onto brain heart infu-sion agar, blood agar plate and 200 μg/ ml neomycine sulphate sheep blood agar plates, incubated an-aerobically at 37ºC for 48 hours. Each colony type was subcultured for identification. The anaerobic bacteria were identified using the techniques and criteria of identification described by Koneman et al. (1992). Typing of C. perfringens isolates was made using dermonecrotic test in guinea- pigs (Stern and Batty, 1975) using C. perfringens diag-nostic antisera (Welcome, Eng-land). 3. Antibiogram study:
Antibiogram susceptibility were determined for (13) antibiot-ics, (pencillin G, Cephaloridine, chloramphenicol, clindomycin, erythromycin, gentamycin, oxytet-racycline, ampicillin, lincomycin, streptomycin, doxycillin, tylosin and nalidixic acid) against 5 iso-lates of F. necrophorum, 6 isolates of A. pyogenes and 13 isolates of C. perfringens. The disc diffusion method for anaerobic bacteria as described by Koneman et al. (1992) and Quinn et al. (1994) was used, except that it was neces-sary to add cysteine (0.59/L) to Muller Hinton medium to support the growth of isolates of F. necro-
phorum S, R, I (Intermediate) measured according to diameter of the zone of inhibition (mm), as re-corded in tables of interpretation (NCCLS, 1994).
The MIC for five strains rep-resented of F. necrophorum were subjected against penicillin G and lincomycin where most of isolates were sensitive (S) to them.
Different dilutions were pre-pared as follows:
Serial 2- fold dilutions of penicillin G and lincomycin were prepared in brain- heart infusion broth according to NCCLS (1979) was used, starting from concentra-tion 400 IU/ ml followed by 200, 100, 50, 25, 12.5, 6.25, 3.215, 1.562, 0.781, 0.39 and 0.195 IU/ml for penicillin G. While, lincomycin dilutions starting from 100 μg/ml followed by 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.39 then 0.195 μg/ml.
0.25 ml of standard bacterial suspension prepared from the tested organism was added into each tube where the final dilution for penicillin G were 300, 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17, 0.59, 0.29 and 0.15 IU/ml. While final dilutions for lincomycin were 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 μg/ml.
Control tube was made by adding 0.25 ml of the tested organ-ism into a tube containing 1 ml
broth alone without antibiotic. The tube were incubated anaerobically at 37ºC for 24 hours. The lowest concentration showing no visible bacterial growth (No turbidity) is the end point (MIC). The tube of end point and all the tubes in which there was no growth were centrifuged and the supernatants were discarded. The sediments were subcultured onto the surfaces of 10% sheep blood agar without antibiotic to determine MBC (Quinn et al, 1994). The MBC is the lowest concentration of antim-icrobial showing no growth (kill bacterial) on the solid media.
RESULTS
A ll collected liver abscesses samples were subjected for
aerobic and anaerobic bacteriologi-cal examination and the results ob-tained showed that 14 samples were harbored mixed cultures of facultative and obligatory anaero-bic bacteria only with the percent-age of 36.8% while 12 samples harbored obligatory anaerobic bac-teria only with the percentage of 31.6% and 12 samples harbored facultative anaerobic organisms only with percentage of 31.6% (Tables, 1 & 2). Table (1) also showed that 29 out of 38 samples were positive to aerobic examination with the per-centage of 76.3% and the most predominant isolates were A. pyo-
genes with a percentage of 44.7% followed by S. aureus and E. coli with the percentage of 23.7% and 7.9%, respectively, while, the an-aerobic isolation were 31 out of 38 with a percentage of 81.6% and the most predominant isolates were F. necrophorum with a percentage of 47.4% followed by C. perfringens and peptostreptococcus with per-centage of 28.9% and 5.3%, re-spectively. As shown in Table (2) it was noticed that F. necrophorum iso-lated either in single or mixed cul-ture with an incidence of 18.4% and 29% respectively, while, the mixed cultures include F. necro-phorum with A. pyogenes or C. perfringens in a percentage of 15.8 and 13.2 respectively. Similarly A. pyogenes was recovered either in single or mixed infections with an incidence of 13.2% and 31.6%, respectively. The mixed cultures include A. pyo-genes with F. necrophorum or C. perfringens in a percentage of 15.8% for both. Also, S. aureus was isolated either in pure culture or in mixed one with an incidence of 10.5% and 13.2% respectively. As, the mixed cultures include S. aureus with E. coli or Peptostrep-tococcus with a percentage of 7.9% and 5.3%, respectively. Table (3) showed that all C.
perfringens isolates (11) were tested for toxin production and showed that type “A” and “D” rep-resented 54.5% and 36.4% respec-tively while non toxigenic isolates represented 9.1%. Table (4) reveals that F. ne-crophorum, A pyogenes and C. perfringens isolates were sensitive to penicillin G, clindomycin, linco-mycin and tylosin and the same isolates were resistant to erythro-mycin, gentamicin, ampcillin, streptomycin, doxycillin HCl and nalidixic acid. As shown in Tables (5, 6) MIC of penicillin G to F. necro-phorum was 0.29 IU, MBC was 1.17 IU, while MIC and MBC of lincomycin was 2.5 μg/ ml for both.
Table (3): Typing of C. perfringens isolated from liver abscesses.
No of C. per-
fringens iso-
lates
Toxigenic isolates Non toxigenic
isolates Type A Type D
No % No % No %
11 6 54.5 4 36.4 1 9.1
Table (4): Susceptibility of obtained isolates of F. necrophorum, A. pyo-genes and C. perfringens to different chemotherapeutic agents.
Antimicrobial
discs Conc.
F.necrophorum
(5)
A. pyogenes
(6)
C. perfringens
(11) No. % A.A No. % A.A No. % A.A
Ampicillin 10 μg 1 20.0 R 2 33.3 R 4 36.3 R Cephaloridine 30 μg 3 60.0 S 3 50.0 S 5 45.5 R Chlorampheni-
col 30 μg 1 20.0 R 5 83.3 S 6 54.5 R
Clindamycin 2 μg 4 80.0 S 4 66.7 S 11 100 S Doxycillin HCl 30 μg 2 40.0 R 1 16.7 R 4 36.3 R Erythromycin 15 μg 2 40.0 R 1 16.7 R 4 36.3 R Gentamicin 10 μg 1 20.0 R 2 33.3 R 3 27.2 R Lincomycin 15 μg 4 80.0 S 4 16.7 S 9 81.8 S Nalidixic acid 30 μg 1 20.0 R 2 33.3 R 6 54.5 R Oxytetracycline 30 μg 3 60.0 S 1 16.7 R 2 18.1 R Pencillin G 10 μ 5 100 S 5 83.3 S 10 90.9 S Streptomycin 10 μg 2 40.0 R 2 33.3 R 5 45.5 R Tylosin 16 μg 3 60.0 S 4 66.7 S 7 63.6 S
No = Number of sensitive isolates. % = percents of sensitive isolates in relation total isolates species. A.A = Antibiogram activity.
Table (5): Count of F. necrophorum post- exposure to different dilution of
penicillin G. and lincomycin in comparison to bacterial growth
in the form turbidity in liquid media.
Dilutions (IU)
Penicillin G Dilutions (µg)
Lincomycin
T C T C
300 - - 80 - -
150 - - 40 - -
75 - - 20 - -
37.5 - - 10 - -
18.75 - - 5 - -
9.38 - - 2.5 - -
4.69 - - 1.25 + 15
2.34 - - 0.625 ++ 50
1.17 - - 0.313 +++ 145
0.59 - 2 0.156 +++ UHG
0.29 - 6 0.15 + 45
- = No bacterial growth and no bacterial count + = Weak bacterial growth. ++ = Moderate bacterial growth. +++ = Heavy bacterial growth. UHG = uncountable heavy growth. T = Turbidity C = Count
Table (6): MIC or MBC of penicillin G and lincomycin to F. necrophorum.
T he liver is particularly suscep-tible to abscesses because it
receives blood from several sources, including the hepatic ar-tery, the portal system and the um-bilical vein in fetus and neonate. Entry via portal vein is most com-mon route (Nagaraja et al., 1996 a). In cattle (and probably in the other ruminants) erosion of the ru-minal epithelium secondary to grain overload, lactic acidosis and ruminitis is thought to be the most common mechanism allowing bac-teria like F. necrophorum coloniza-tion of the liver (Nagaraja and Chengappa, 1998). Hepatic ab-scesses do not cause clinical signs of hepatic dysfunction unless they are particularly massive or exten-sively metastatic (Radostits et al., 2000). The greatest economic im-pact of liver abscesses is from re-duced animal performance. A num-ber of studies involving compari-sons of cattle with and without ab-scesses have documented that cat-tle with abscessed livers have re-duced feed intake, reduced weight gain, decreased feed efficiency and decreased carcass yield (Nagaraja et al., 1996). In addition hepatic abscess leads to the rejection of the affected livers at the abattoir (Radostits et al., 2000). Liver ab-scesses occur in any species, but the abscesses of significant eco-nomic impact occur in feed lot cat-tle (Nagaraja et al., 1996b).
Bacteriological examination of liver samples revealed that the incidence of facultative and obligatory anaerobic organisms were 71.3% and 81.6% respec-tively similar results were obtained by Lechtenberg et al. (1988) and Zaki et al. (2000). Also, results of this study in-dicates that many facultative an-aerobic and / or anaerobic microor-ganisms in single or mixed forms were identified and found to play a role in this infection as F. necro-phorum, A. pyogenes, C. perfrin-gens , S. aureus, E. coli and Pepto-streptococcus.
Similar findings were de-scribed by El-Sayed et al. (1991); Zaki et al. (2000) and Mashhadi et al. (2006). Table (2) showed also that F. necrophorum was isolated in sin-gle from 7 cases of liver abscesses in an incidence of 18.4% while 11 isolates were mixed with A. pyo-genes, C. perfringens in a percent-age of 15.8 and 13.2 respectively. This results confirm the finding of Lechtenberg et al. (1988); Mak-hareta (1988) and Ciftci et al. (1993) who isolated F. necropho-rum either in pure or mixed culture from hepatic abscesses which may give an idea about its role in liver abscesses.
factors of F. necrophorum play a critical role in the penetration and colonization of the ruminal epithe-lium and entry and establishment of infection in the liver. The prote-ase activity, dermonecrotic activity and cytotoxic effect of leukotxin on ruminal cells may aid in pene-tration and colonization of the ru-minal wall (Nagaraya and Chen-gappa, 1998).
A. pyogenes isolates were re-covered from liver abscesses in a single and mixed form, this results coincides with that observation of Darwish (1996); Zaki et al., (2000) and Mashhadi et al., (2006). It is believed that A. pyo-genes is the second most frequent pathogen isolated from liver ab-scesses (Nagaraja and Chen-gappa, 1998). The origin of A. pyogenes and its pathogenic mechanism are not well under-stood, but evidence exists for pathogenic synergy between A. pyogenes and F. necrophorum (Takeuchi, 1983).
C. perfringens was isolated in mixed culture with either F. necro-phorum or A. pyogenes and it is believed that C. perfringens played a secondary role in initiating he-patic lesion in animals (Tydon et al., 1980 and Itman et al., 1989).
C. perfringens type A was re-covered in an incidence of 54.5% while C. perfringens type D was isolated in an incidence of 36.3%.
Nearly similar results were ob-tained by Darwish (1996) and Zaki et al. (2000).
The susceptibility patterns of F. necrophorum, A. pyogenes and C. perfringens isolates to antim-icrobial agents were similar to pat-terns reported previously 100% of the F. necrophorum strains em-ployed in the study proved sensi-tive to the action of penicillin G., 80% of the strains studied were susceptible to lincomycin and clin-damycin and 60% were susceptible for cephaloridine, tylosin and oxyteteracycline. While, gen-tamicin, streptomycin, ampicillin, nalidix acid and chloramphenicol had low effect. The results is in agreement with the results ob-tained previously by Lechtenberg and Nagaraja (1989) who stated that F. necrophorum isolates were generally susceptible to penicillins, tetracyclines (chlortetracycline and oxytetracycline), lincosamides (clindamycin and lincomycin), and were resistant to aminoglycosides (Kanamycin, neomycin, gen-tamicin and streptomycin). Pres-cott and Baggot (1993) and Mateos et al. (1997) and explained the resistance to aminoglycosides may be because the action of them depend on their penetrating with the bacteria, in part by active trans-port and in part by passive diffu-sion such active transport doesn’t usually occur under anaerobic con-ditions.
Meanwhile the isolated Gram +ve bacteria as A. pyogenes and C. perfringens were sensitive to peni-cillin G, clindamycin, lincomycin and tylosin while both A. pyogenes and C. perfringens isolates were resistant to erythromycin, gen-tamicin, streptomycin, oxytetracy-clin, ampicillin, doxycillin HCl and nalidixic acid. Similar results obtained by Tyrrell et al. (2006). The suscepti-bility pattern of A. pyogenes (the second most common organism in liver abscesses) to an antimicrobial compounds is typical of Gram- positive bacteria (Specht et al., 1988). Similar results obtained by Vogel and Laydert (1994) and Rogers et al. (1995). Minimal In-hibitory concentrations of penicil-lin G and lincomycin for isolates of F. necrophorum were as follows 0.29 IU and 2.5 μg/ ml respec-tively while the MBC were 1.17 IU and 2.5 μg/ ml for penicillin G and lincomycin, respectively. The MIC of 12 isolates of F. necropho-rum examined by Berg and Scanlan (1982) were as follows: ≤ 0.06 IU of penicillin G/ ml; ≤ 0.5 μg of cephaloridine/ ml; ≤ 8 μg of erythromycin/ ml; ≤ 128 μg of gentamicin/ ml; ≤ 0.25 μg of oxytetracycline/ ml, and ≤ 4 μg of tylosin/ ml. The FDA- approved antibi-otic vary in their effectiveness in
preventing liver abscesses (Lecht-enberg and Nagaraja, 1989). It is important to emphasize the good activity of the penicillin G or ty-losin which is used as treatment and tylosin used as additive in ani-mal feed for the prophylaxis of he-patic abscessation, these results in vitro correspond to those obtained in animals by various authors (Galyean et al., 1992; Clary et al., 1993; Tan et al., 1994) in which a significant decrease of he-patic abscesses incidence was ob-served when tylosin was in corpo-rate together with the feed.
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