Osjetljivost bakterija Escherichia coli i Staphylococcus aureus izdvojenih iz pilića u Maiduguri (Nigerija) prema betalaktamskim antibioticima

283ISSN 0372-5480Printed in Croatia

VETERINARSKI ARHIV 80 (2), 283-297, 2010

Antibiotic susceptibility patterns of beta-lactamase-producingAntibiotic susceptibility patterns of beta-lactamase-producing Escherichia coliEscherichia coli and and StaphylococcusStaphylococcus aureusaureus isolated from chickens in isolated from chickens in

Maiduguri (Arid zone), NigeriaMaiduguri (Arid zone), Nigeria

Sunday A. MamzaSunday A. Mamza11*, Godwin O. Egwu*, Godwin O. Egwu22, and Gideon D. Mshelia, and Gideon D. Mshelia33

11Senator Ali Sheriff (SAS) Veterinary Hospital Maiduguri,Senator Ali Sheriff (SAS) Veterinary Hospital Maiduguri, NigeriaNigeria22Department of Veterinary Medicine, University of Maiduguri, Maiduguri, Nigeria Department of Veterinary Medicine, University of Maiduguri, Maiduguri, Nigeria

33Department of Veterinary Surgery and Theriogenology, University of Maiduguri, Maiduguri, NigeriaDepartment of Veterinary Surgery and Theriogenology, University of Maiduguri, Maiduguri, Nigeria

MAMZA, S. A., G. O. EGWU, G. D. MSHELIA: Antibiotic susceptibility patterns Antibiotic susceptibility patterns of beta-lactamase-producingof beta-lactamase-producing Escherichia coli Escherichia coli and and StaphylococcusStaphylococcus aureusaureus isolated isolated from chickens in Maiduguri (Arid zone), Nigeriafrom chickens in Maiduguri (Arid zone), Nigeria. Vet. arhiv 80, 283-297, 2010.

ABSTRACTEscherichia coliEscherichia coli and and Staphylococcus aureusStaphylococcus aureus species though opportunist pathogens, are becoming a global species though opportunist pathogens, are becoming a global

clinical problem in both human and veterinary medicine. This study was designed to determine the antibiotic clinical problem in both human and veterinary medicine. This study was designed to determine the antibiotic susceptibility patterns of β-lactamase-producing susceptibility patterns of β-lactamase-producing E. coli E. coli serotypes and serotypes and S. aureus strains S. aureus strains isolated from chickens isolated from chickens in the Maiduguri Arid zone, Nigeria. Various tissue samples from apparently healthy and diseased chickens were in the Maiduguri Arid zone, Nigeria. Various tissue samples from apparently healthy and diseased chickens were collected and examined for the presence of collected and examined for the presence of E. coliE. coli and and S. aureus.S. aureus. Isolates were identifi ed by relevant biochemical Isolates were identifi ed by relevant biochemical tests. β-lactamase-producing strains of the isolates were determined by the chromogenic cephalosporin method, tests. β-lactamase-producing strains of the isolates were determined by the chromogenic cephalosporin method, using nitrocefi n-impregnated sticks and cephalosporin (nitrocefi n) solution. The antibiotics susceptibility using nitrocefi n-impregnated sticks and cephalosporin (nitrocefi n) solution. The antibiotics susceptibility patterns of the isolates were determined for ten antibiotics (ampicillin, chloramphenicol, cephalexin, patterns of the isolates were determined for ten antibiotics (ampicillin, chloramphenicol, cephalexin, ciprofl oxacin, lincomycin, doxycycline, tetracycline, tylosin, tylosin tartrate and penicillin) by the micro-broth ciprofl oxacin, lincomycin, doxycycline, tetracycline, tylosin, tylosin tartrate and penicillin) by the micro-broth dilution method. dilution method. E. coliE. coli was isolated in 805 and was isolated in 805 and S. aureusS. aureus in 660 of 1300 tissue samples examined; from in 660 of 1300 tissue samples examined; from which 89 (11.1%) and 58 (8.8%) were β-lactamase-positive isolates respectively. Out of 540 which 89 (11.1%) and 58 (8.8%) were β-lactamase-positive isolates respectively. Out of 540 E. coliE. coli isolates isolates serotyped, 57 (10.6%) serogroups were identifi ed from which 17 (29.8%) were serogroups O1, 5 (8.8%) were serotyped, 57 (10.6%) serogroups were identifi ed from which 17 (29.8%) were serogroups O1, 5 (8.8%) were O2, and 2 (3.5%), 9 (15.8%), 6 (10.5%) and 18 (31.6%) were serogroups O26, O78, O86 and O141 respectively, O2, and 2 (3.5%), 9 (15.8%), 6 (10.5%) and 18 (31.6%) were serogroups O26, O78, O86 and O141 respectively, whilst, 483 (89.4%) isolates were not typable with the available sera. Serogroups O141, O1 and O78 were more whilst, 483 (89.4%) isolates were not typable with the available sera. Serogroups O141, O1 and O78 were more frequently isolated and serogroups O1 and O78 were more prevalent in sick chickens than in healthy chickens. frequently isolated and serogroups O1 and O78 were more prevalent in sick chickens than in healthy chickens. E. coliE. coli exhibited high resistance to ampicillin, chloramphenicol, tetracycline, lincomycin, penicillin and tylosin exhibited high resistance to ampicillin, chloramphenicol, tetracycline, lincomycin, penicillin and tylosin with MIC values >8.0 μg/μL, as did with MIC values >8.0 μg/μL, as did S. aureusS. aureus to all the antibiotics tested with MIC values >8.0 μg/μL. In to all the antibiotics tested with MIC values >8.0 μg/μL. In conclusion, the study has demonstrated the presence of conclusion, the study has demonstrated the presence of E. coliE. coli serotypes and serotypes and S. aureusS. aureus in various tissues of in various tissues of chickens and their antibiotic susceptibility patterns, clearly demonstrating multiple drug resistance.chickens and their antibiotic susceptibility patterns, clearly demonstrating multiple drug resistance.

Key wordsKey words: beta-lactamase, : beta-lactamase, Escherichia coliEscherichia coli, , Staphylococcus aureusStaphylococcus aureus, antibiotics susceptibility, chickens, , antibiotics susceptibility, chickens, Maiduguri Maiduguri

*Corresponding author:Dr. Sunday Akidarju Mamza, DVM, MVSc, SAS Veterinary Hospital, P.O.Box, 819, Maiduguri, Borno State, Nigeria, Phone: Dr. Sunday Akidarju Mamza, DVM, MVSc, SAS Veterinary Hospital, P.O.Box, 819, Maiduguri, Borno State, Nigeria, Phone: +234 0802 799 3611; +234 0809 487 5441; E-mail: [email protected]+234 0802 799 3611; +234 0809 487 5441; E-mail: [email protected]

284 Vet. arhiv 80 (2), 283-297, 2010

IntroductionIntroductionEscherichia coli (E. coli) Escherichia coli (E. coli) and and Staphylococcus aureusStaphylococcus aureus (S. aureus(S. aureus) infections constitute ) infections constitute

one of the most important bacterial diseases affecting the poultry industry (one of the most important bacterial diseases affecting the poultry industry (RAJI et al.RAJI et al., , 20032003) in Nigeria. ) in Nigeria. E. coliE. coli is is the predominant facultative anaerobic normal bacterial fl ora the predominant facultative anaerobic normal bacterial fl ora in the avian intestine and plays an important role in maintaining intestinal homeostasis. in the avian intestine and plays an important role in maintaining intestinal homeostasis. The organism is a major pathogen of worldwide importance in commercially raised The organism is a major pathogen of worldwide importance in commercially raised poultry, contributing signifi cantly to economic losses in both chickens and turkeys (poultry, contributing signifi cantly to economic losses in both chickens and turkeys (RAJI RAJI et al., 2003et al., 2003). ). S. aureus S. aureus occurs among the normal fl ora of live poultry, and broiler chicks occurs among the normal fl ora of live poultry, and broiler chicks become contaminated with the organism within the fi rst few days after being hatchedbecome contaminated with the organism within the fi rst few days after being hatched ((NORTTERMANS et al., 1982NORTTERMANS et al., 1982). ). S. aureusS. aureus is important in relation to poultry and meat is important in relation to poultry and meat hygiene because of its ability to produce entero-toxins, which may cause food poisoning hygiene because of its ability to produce entero-toxins, which may cause food poisoning in humans. About 30% of all outbreaks of food borne diseases have been reported to in humans. About 30% of all outbreaks of food borne diseases have been reported to be associated with poultry, and more than 25% of poultry disease outbreaks have been be associated with poultry, and more than 25% of poultry disease outbreaks have been attributed to attributed to S. aureusS. aureus globally ( globally (NORTTERMANS et al., 1982NORTTERMANS et al., 1982). Some of the poultry diseases ). Some of the poultry diseases ascribed to these agents such as coli septicaemia, coligranuloma or Hjarre’s disease and ascribed to these agents such as coli septicaemia, coligranuloma or Hjarre’s disease and omphalitis are incriminated with omphalitis are incriminated with E. coliE. coli ( (RAJI et al., 2003RAJI et al., 2003); whilst joint infections (swollen ); whilst joint infections (swollen hocks, arthritis) and plantar abscess (bumble foot) are associated with hocks, arthritis) and plantar abscess (bumble foot) are associated with S. aureusS. aureus ( (SAEED SAEED et al., 2000; JOHN, 2006et al., 2000; JOHN, 2006).).

The elaboration of entero-and invasive-toxins, cytotoxic necrotizing factor (CNF), The elaboration of entero-and invasive-toxins, cytotoxic necrotizing factor (CNF), attaching and effacing (eae) genes, fi mbrial adhesins and production of haemolysins, attaching and effacing (eae) genes, fi mbrial adhesins and production of haemolysins, siderophores, plasmids, integrons and haemolysins (siderophores, plasmids, integrons and haemolysins (GONZALEZ and BLANCO, 1996; PASS GONZALEZ and BLANCO, 1996; PASS et al., 2000et al., 2000) by ) by E. coliE. coli and the specifi c production of coagulase, DNase, hyaluronidase, and the specifi c production of coagulase, DNase, hyaluronidase, staphylokinase, nuclease and penicillinase by staphylokinase, nuclease and penicillinase by S. aureusS. aureus ( (JOHN, 2006JOHN, 2006) constitute some of ) constitute some of the pathogenic-markers by which these agents produce diseases in their hosts. Some of the pathogenic-markers by which these agents produce diseases in their hosts. Some of these markers contribute to the emergence of multiple drug resistant strains (these markers contribute to the emergence of multiple drug resistant strains (LAURA et al., LAURA et al., 20022002) in the two organisms and consequently to therapeutic failure in humans and animals ) in the two organisms and consequently to therapeutic failure in humans and animals ((YOSHICHIKA et al., 2000; VAN DEN BOGAARD et al., 2001; HANCHUN et al., 2004YOSHICHIKA et al., 2000; VAN DEN BOGAARD et al., 2001; HANCHUN et al., 2004) which ) which is becoming a global health concern. Though opportunist pathogens, these organisms also is becoming a global health concern. Though opportunist pathogens, these organisms also provide a large reservoir of antibiotic resistance in tissues of the bodies of their hosts, and provide a large reservoir of antibiotic resistance in tissues of the bodies of their hosts, and might transfer this to other organisms if the resistance genes concerned are translocated might transfer this to other organisms if the resistance genes concerned are translocated ((SAEED et al., 2000; DEBORAH et al., 2005SAEED et al., 2000; DEBORAH et al., 2005).).

Both organisms are known to produce beta-lactamases, possibly contributing to the Both organisms are known to produce beta-lactamases, possibly contributing to the emergence of multiple drug resistant strains (emergence of multiple drug resistant strains (LAURA et al., 2002LAURA et al., 2002). ).

Several studies have demonstrated that patterns of antibiotic usage greatly affect the Several studies have demonstrated that patterns of antibiotic usage greatly affect the number of resistant organisms which develop. Although the extent and speed to which number of resistant organisms which develop. Although the extent and speed to which bacteria develop resistance to antimicrobial drugs vary, indiscriminate broad-spectrum bacteria develop resistance to antimicrobial drugs vary, indiscriminate broad-spectrum antibiotic therapy has been shown to result in increased antimicrobial resistance. So far, antibiotic therapy has been shown to result in increased antimicrobial resistance. So far, resistances by resistances by E. coliE. coli and and S. aureusS. aureus have been shown to occur to most antimicrobial have been shown to occur to most antimicrobial

S. A. Mamza et al.: Antibiotic susceptibility patterns of beta-lactamase E. coli and S. aureus

285Vet. arhiv 80 (2), 283-297, 2010


drugs (drugs (HOWARD et al., 1996HOWARD et al., 1996) making this a serious setback in the treatment of bacterial ) making this a serious setback in the treatment of bacterial infections (infections (CHAH and OBOEGBULEM, 2005CHAH and OBOEGBULEM, 2005) in both humans and animals. ) in both humans and animals.

The emergence of bacterial species with extended spectrum beta-lactamases and The emergence of bacterial species with extended spectrum beta-lactamases and acquired resistance to various broad spectrum beta-lactam antibiotics is becoming a acquired resistance to various broad spectrum beta-lactam antibiotics is becoming a clinical problem globally in both human and veterinary medicine (clinical problem globally in both human and veterinary medicine (YOSHICHIKA et al., YOSHICHIKA et al., 2000; CARL et al., 20022000; CARL et al., 2002). Field reports have shown evidence of emerging multiple drug ). Field reports have shown evidence of emerging multiple drug resistant strains of these organisms in Nigeria following treatment of some bacterial resistant strains of these organisms in Nigeria following treatment of some bacterial diseases in poultry, coupled with the frequency of complaints of respiratory, diarrhoea, diseases in poultry, coupled with the frequency of complaints of respiratory, diarrhoea, and joint problems.and joint problems.

There is paucity of information on the distribution of avian There is paucity of information on the distribution of avian E. coliE. coli serotypes and serotypes and S. S. aureusaureus in the tissues of chickens in Nigeria, as well as the role of these disease agents in in the tissues of chickens in Nigeria, as well as the role of these disease agents in the elaboration of beta-lactamases responsible for multiple antimicrobial drug resistance the elaboration of beta-lactamases responsible for multiple antimicrobial drug resistance and their likely public health implications in both animals and humans. This study was and their likely public health implications in both animals and humans. This study was therefore undertaken to determine the antibiotic susceptibility patterns of β-lactamase-therefore undertaken to determine the antibiotic susceptibility patterns of β-lactamase-producing strains of producing strains of E. coliE. coli and and S. aureusS. aureus isolated from chickens in Maiduguri, Nigeria. isolated from chickens in Maiduguri, Nigeria.

Materials and methodsMaterials and methodsSample collection and processing.Sample collection and processing. Various tissue samples (196 each of the trachea, Various tissue samples (196 each of the trachea,

lungs, small intestines and liver, 135 hock joints, 136 digital pads and 201 cloacal swabs), lungs, small intestines and liver, 135 hock joints, 136 digital pads and 201 cloacal swabs), totalling 1300, were collected aseptically from both apparently healthy and sick chickens totalling 1300, were collected aseptically from both apparently healthy and sick chickens (of which 892 samples were from 332 apparently healthy and 408 samples from 68 (of which 892 samples were from 332 apparently healthy and 408 samples from 68 sick chickens) distributed across one hundred randomly selected poultry farms within sick chickens) distributed across one hundred randomly selected poultry farms within Maiduguri Metropolis, Borno State, Nigeria between April 2005 and April 2007. Maiduguri Metropolis, Borno State, Nigeria between April 2005 and April 2007.

Samples were transported on ice packs within one hour of collection to the research Samples were transported on ice packs within one hour of collection to the research and diagnostic laboratory of the Department of Veterinary Medicine, University of and diagnostic laboratory of the Department of Veterinary Medicine, University of Maiduguri where they were processed. Maiduguri where they were processed.

Bacteriological isolation.Bacteriological isolation. Homogenized samples were inoculated onto MacConkey’s Homogenized samples were inoculated onto MacConkey’s agar (LAB 2, idgagar (LAB 2, idg®®, Lancashire), Lancashire) plates and incubated at 37 plates and incubated at 37 OOC for 24 hrs. Some presumptive C for 24 hrs. Some presumptive E. coliE. coli and and S. aureusS. aureus colonies were selected from each sample and sub-cultured onto colonies were selected from each sample and sub-cultured onto EMB agar (LAB 61, idgEMB agar (LAB 61, idg®®, Lancashire) for , Lancashire) for E. coliE. coli and modifi ed Baird Parker agar (CM and modifi ed Baird Parker agar (CM 0961, Oxoid0961, Oxoid®®) for ) for S. aureusS. aureus. .

Identifi cation of isolates.Identifi cation of isolates. Isolates were tested for Gram-staining, methyl red and Isolates were tested for Gram-staining, methyl red and Voges-Proskuaer (Voges-Proskuaer (BAKER et al., 2001BAKER et al., 2001), catalase and coagulase (), catalase and coagulase (MacFADDIN, 1980MacFADDIN, 1980) and ) and then stored on nutrient agar slants until used for β-Lactamase production tests, then stored on nutrient agar slants until used for β-Lactamase production tests, E. coliE. coli serotyping and antimicrobial susceptibility tests. Isolates were routinely sub-cultured serotyping and antimicrobial susceptibility tests. Isolates were routinely sub-cultured every 4 months to maintain purity and viability during the course of the work.every 4 months to maintain purity and viability during the course of the work.

286 Vet. arhiv 80 (2), 283-297, 2010

Beta-lactamase test.Beta-lactamase test. The production of β-lactamase enzymes was tested by The production of β-lactamase enzymes was tested by the chromogenic cephalosporin method using commercially prepared nitrocephin-the chromogenic cephalosporin method using commercially prepared nitrocephin-impregnated touch sticks (BR0066A, Oxoidimpregnated touch sticks (BR0066A, Oxoid®® UK) according to the manufacture’s UK) according to the manufacture’s instructions, and cephalosporin (500 mg/mL nitrocephin) solution as described by instructions, and cephalosporin (500 mg/mL nitrocephin) solution as described by MILES MILES and AMYES (1996)and AMYES (1996)..

The stick test was carried out according to the manufacturer’s instructions. A The stick test was carried out according to the manufacturer’s instructions. A representative pure colony from the growth medium was selected. This colony was representative pure colony from the growth medium was selected. This colony was touched with the impregnated end of the stick. The stick was then rotated to pick up touched with the impregnated end of the stick. The stick was then rotated to pick up a small mass of pure cells and was observed for 10 minutes for a small mass of pure cells and was observed for 10 minutes for E. coliE. coli and up to 1hr and up to 1hr for for S. aureus. S. aureus. AA colour change from yellow to pink-red indicated positive β-lactamase-colour change from yellow to pink-red indicated positive β-lactamase-producing organisms.producing organisms.

Cephalosporin C (22237, Sigma-AldrichCephalosporin C (22237, Sigma-Aldrich®®, South-Africa) was used for the , South-Africa) was used for the cephalosporin solution method, according to the method described by cephalosporin solution method, according to the method described by MILES and AMYES MILES and AMYES (1996)(1996). Cephalosporin C was said to exhibit resistance or stability to β-lactamases but was . Cephalosporin C was said to exhibit resistance or stability to β-lactamases but was not suffi ciently potent for clinical use. Therefore, its use as a β-lactamase test reagent is not suffi ciently potent for clinical use. Therefore, its use as a β-lactamase test reagent is suitable for the detection of β-lactamase production in bacteria that can hydrolyze the β-suitable for the detection of β-lactamase production in bacteria that can hydrolyze the β-lactam ring in its molecule. A colour change from yellow to red was recorded positive for lactam ring in its molecule. A colour change from yellow to red was recorded positive for β-lactamase-producing strains.β-lactamase-producing strains.

Serotyping of E. coli isolates.Serotyping of E. coli isolates. The kits for serotyping avian The kits for serotyping avian E. coliE. coli comprising comprising serogroups O1, O2, O26, O78, O86 and O141 antisera were obtained from Prof. J.A. serogroups O1, O2, O26, O78, O86 and O141 antisera were obtained from Prof. J.A. Blanco, Director of the Blanco, Director of the E. coliE. coli Reference laboratory (LREC) in Spain. The serotyping Reference laboratory (LREC) in Spain. The serotyping was carried out according to the protocol (was carried out according to the protocol (ANOANONYM.,NYM., 2006 2006). ).

The pure isolate of The pure isolate of E. coliE. coli was inoculated onto a Tryptone Soya agar (TSA, 22091, was inoculated onto a Tryptone Soya agar (TSA, 22091, Sigma-Aldrich, South Africa) plates and incubated for 48 hrs at 37 Sigma-Aldrich, South Africa) plates and incubated for 48 hrs at 37 OOC. Some of the C. Some of the growths from the agar were then suspended in 2 μL of 0.85% saline solution in sterile growths from the agar were then suspended in 2 μL of 0.85% saline solution in sterile vaccuutainer tubes. The bacterial concentrations were adjusted by comparing the tubes to vaccuutainer tubes. The bacterial concentrations were adjusted by comparing the tubes to correspond with 1.8 x 10correspond with 1.8 x 1099 MacFarland Barium sulfate turbidity standard. The tubes were MacFarland Barium sulfate turbidity standard. The tubes were heated in a boiling water bath for 1hr at 100 heated in a boiling water bath for 1hr at 100 OOC to inactivate K antigens. The suspensions C to inactivate K antigens. The suspensions were then allowed to cool and 2 μL of formalinized saline solution (0.5%, v/v) containing were then allowed to cool and 2 μL of formalinized saline solution (0.5%, v/v) containing gentian violet (0.005%, w/v) were added. gentian violet (0.005%, w/v) were added.

The antisera were diluted (1x) to 1/80 using saline solution with sodium azide The antisera were diluted (1x) to 1/80 using saline solution with sodium azide according to the protocol (according to the protocol (ANONANONYMYM., 2006., 2006). The prepared antigen suspensions were ). The prepared antigen suspensions were tested with each of the diluted O antisera: 50 μL of each of the diluted antisera (1/80) were tested with each of the diluted O antisera: 50 μL of each of the diluted antisera (1/80) were added to each corresponding well of a sterile V-shaped polystyrene micro-titre tray and added to each corresponding well of a sterile V-shaped polystyrene micro-titre tray and equal volumes of the O antigen suspensions were then added. The trays were covered and equal volumes of the O antigen suspensions were then added. The trays were covered and incubated at 37 incubated at 37 OOC overnight and were then examined for agglutination. Negative reactions C overnight and were then examined for agglutination. Negative reactions were indicated by a sharp point, whereas positive reactions by a carpet-like appearance. were indicated by a sharp point, whereas positive reactions by a carpet-like appearance. Positive and negative controls were also prepared alongside the test samples.Positive and negative controls were also prepared alongside the test samples.


287Vet. arhiv 80 (2), 283-297, 2010

Serial dilutions of the O antisera were prepared in micro-titre dilution trays to give Serial dilutions of the O antisera were prepared in micro-titre dilution trays to give dilutions of 1/80, 1/160, 1/320 to 1/40960. Then 100 μL of each of the diluted O antisera dilutions of 1/80, 1/160, 1/320 to 1/40960. Then 100 μL of each of the diluted O antisera (1/80) were added to the fi rst well of the corresponding micro-titre tray and 50 μL of (1/80) were added to the fi rst well of the corresponding micro-titre tray and 50 μL of saline solution to the remaining 11 wells. Serial two-fold dilutions in 50 μL amounts saline solution to the remaining 11 wells. Serial two-fold dilutions in 50 μL amounts were then carried out from well 1 up to well 10, while wells 11 and 12 served as negative were then carried out from well 1 up to well 10, while wells 11 and 12 served as negative controls. Then 50 μL of the corresponding O antigen suspension was added to each of the controls. Then 50 μL of the corresponding O antigen suspension was added to each of the 12 wells of corresponding trays, starting from well 12 through to well 1, making the fi nal 12 wells of corresponding trays, starting from well 12 through to well 1, making the fi nal dilution of the antiserum to 1/81920 in the last well (well 12). Then the trays were covered dilution of the antiserum to 1/81920 in the last well (well 12). Then the trays were covered and incubated at 37 and incubated at 37 OOC overnight and were examined for agglutination. Strains showing C overnight and were examined for agglutination. Strains showing agglutination in dilutions of 1/160 or greater were considered to contain the same O agglutination in dilutions of 1/160 or greater were considered to contain the same O antigen as the corresponding antiserum and this constituted the fi nal identifi cation of the antigen as the corresponding antiserum and this constituted the fi nal identifi cation of the somatic antigen or O antigen of the isolate.somatic antigen or O antigen of the isolate.

Antibiotics susceptibility (MIC) test.Antibiotics susceptibility (MIC) test. The following ten antimicrobial agents The following ten antimicrobial agents (ampicillin (10044), cephalexin (22238), chloramphenicol (46109), ciprofl oxacin (ampicillin (10044), cephalexin (22238), chloramphenicol (46109), ciprofl oxacin (62143), doxycycline hydrochloride (44577), penicillin G benzathine (PEN-B), (62143), doxycycline hydrochloride (44577), penicillin G benzathine (PEN-B), tetracycline hydrochloride (T3383), lincomycin hydrochloride (62143), tylosin tartrate tetracycline hydrochloride (T3383), lincomycin hydrochloride (62143), tylosin tartrate (93806) and tylosin (T3397) obtained from Sigma Aldrich(93806) and tylosin (T3397) obtained from Sigma Aldrich®®, South-Africa, were tested. , South-Africa, were tested. With the exception of ciprofl oxacin, these antibiotics were found to be commonly used in With the exception of ciprofl oxacin, these antibiotics were found to be commonly used in the treatment of poultry infections in Maiduguri, Nigeria. the treatment of poultry infections in Maiduguri, Nigeria.

The minimum inhibitory concentrations (MICs) were determined by micro broth The minimum inhibitory concentrations (MICs) were determined by micro broth dilution according to standard method (dilution according to standard method (GAIL and JOHN, 1995; MILES and AMYES, 1996GAIL and JOHN, 1995; MILES and AMYES, 1996), ), using inoculums of 5 x 10using inoculums of 5 x 104 4 CFU/well in aCFU/well in a nutrient agar broth (No 1; 70122, Sigma-nutrient agar broth (No 1; 70122, Sigma-AldrichAldrich®®, South-Africa) with the ten antibiotics at concentrations ranging from 0.25 μg/, South-Africa) with the ten antibiotics at concentrations ranging from 0.25 μg/μL to 8.0 μg/μL. The MICs of the test solutions were read as the lowest concentrations of μL to 8.0 μg/μL. The MICs of the test solutions were read as the lowest concentrations of the antibiotics that inhibited a colour change (the antibiotics that inhibited a colour change (EGWU et al., 1994EGWU et al., 1994) or inhibited growth in the ) or inhibited growth in the wells of the micro-dilution trays after incubation for 18-24 hrs. Results were interpreted wells of the micro-dilution trays after incubation for 18-24 hrs. Results were interpreted as susceptible and resistant based on the interpretive standards for dilution susceptibility as susceptible and resistant based on the interpretive standards for dilution susceptibility testing (testing (GAIL and JOHN, 1995GAIL and JOHN, 1995). ).

Statistical analysis.Statistical analysis. Results were compared by Results were compared by XX2 2 test with Yates correction for test with Yates correction for continuity.continuity.

Results ResultsPrevalence of E. coli and S. aureus in chickens in Maiduguri, Nigeria. Prevalence of E. coli and S. aureus in chickens in Maiduguri, Nigeria. Table 1 Table 1

shows the prevalence of shows the prevalence of E. coli E. coli and and S. aureusS. aureus isolates from chickens in Maiduguri. Out isolates from chickens in Maiduguri. Out of a total of 1300 tissue samples collected, 1,256 (96. 6%) yielded growth, of which of a total of 1300 tissue samples collected, 1,256 (96. 6%) yielded growth, of which 805 (64.1%) were 805 (64.1%) were E. coli E. coli and 660 (52.5%) were and 660 (52.5%) were S. aureusS. aureus isolates. 313, 263 and 229 isolates. 313, 263 and 229 representing 38.9%, 32.7% and 28.4% of the representing 38.9%, 32.7% and 28.4% of the E. coli E. coli isolatesisolates were from broilers, layers were from broilers, layers and local chickens respectively. Similarly, 211, 231 and 218 representing 32%, 35% and local chickens respectively. Similarly, 211, 231 and 218 representing 32%, 35%


288


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and 33% of the and 33% of the S. aureusS. aureus isolates were also from broilers, layers and local chickens isolates were also from broilers, layers and local chickens respectively.respectively. Furthermore, 33 (10.5%), 27 (10.3%) and 29 (12.7%) of the Furthermore, 33 (10.5%), 27 (10.3%) and 29 (12.7%) of the E. coli E. coli and 9 and 9 (4.3%), 41(17.7%) and 8 (3.7%) of the (4.3%), 41(17.7%) and 8 (3.7%) of the S. aureusS. aureus isolates were β-lactamase producing isolates were β-lactamase producing strains isolated from broilers, layers and local chickens, respectively. Of the isolates from strains isolated from broilers, layers and local chickens, respectively. Of the isolates from healthy chickens, 25 (4.3%) healthy chickens, 25 (4.3%) E. coliE. coli and 17 (4.5%) and 17 (4.5%) S. aureusS. aureus were β-lactamase positive were β-lactamase positive strains, whilst 64 (28.7%) strains, whilst 64 (28.7%) E. coliE. coli and 41 (14.6%) and 41 (14.6%) S. aureusS. aureus were β-lactamase producing were β-lactamase producing strains isolated from diseased chickens. strains isolated from diseased chickens.

Table 1. Prevalence of Table 1. Prevalence of E. coliE. coli and and S. aureusS. aureus strains in apparently healthy and diseased chickens in strains in apparently healthy and diseased chickens in Maiduguri, NigeriaMaiduguri, Nigeria

Number (%) of β-lactamaseNumber (%) of β-lactamase positive isolatespositive isolatesType of Type of chickens

Healthychickens Sick chickens TotalE. coli S. aureus E. coli S. aureus E. coli S. aureus

Broilers 9/226 (4.0)n 3/121 (2.5)l 24/87 (27.6)f 6/90 (6.7)j 33/313 (10.5)g 9/211 (4.3)h

Layers 11/190 (5.8)m 12/133 (9.0)4 16/73 (21.9e 29/98 (29.6)k 27/263 (10.3)g

41/231 (17.7)i

Local/chickens 5/165 (3.0)l 2/126 (3.0)l 24/64 (38.0)d 6/92 (6.5)j 29/229

(12.7)g 8/218 (3.7)h

Total 25/582 (4.3)a 17/380 (4.5)a 64/223 (28.7)b

41/280 (14.6)c

89/805 (11.1)k 58/660 (8.8l

Numerator = number of β-lactamase isolates; denominator = number of isolates screened; Means with different Numerator = number of β-lactamase isolates; denominator = number of isolates screened; Means with different superscripts differ signifi cantly (P<0.05)superscripts differ signifi cantly (P<0.05)

Table 2. Number (%) of Table 2. Number (%) of ββ-lactamase-positive-lactamase-positive E. coli E. coli and S. aureusand S. aureus isolates in tissues of chickens isolates in tissues of chickens in Maiduguri in Maiduguri

Tissue E. coli S. aureus Trachea 17/111 (15.3) a 9/93 (9.7) c Lung 19/123 (15.5) a 10/103 (9.7) c

Liver 15/89 (16.9) a 8/59 (13.6) d

Small/intestine 14/144 (9.7) c 9/56 (16.1) a Hock joint 3/71 (4.2) h 6/74 (8.1) e

Digital pad 12/84 (14.3) i 13/121(10.7) f

Cloacal swab 9/183 (4.9) j 3/154 (1.9) g Total 89/805 (11.1) 1 58/660(8.8)2

Means with different superscripts differ signifi cantly (P<0.05); Numerator = number of Means with different superscripts differ signifi cantly (P<0.05); Numerator = number of ββ-lactamase positive -lactamase positive isolates; Denominator = number of isolates screenedisolates; Denominator = number of isolates screened

289


Vet. arhiv 80 (2), 283-297, 2010

Table 3Table 3. . Distribution ofDistribution of E. coli E. coli serotypes isolates in tissues of chickens in Maiduguri, Nigeria serotypes isolates in tissues of chickens in Maiduguri, Nigeria Serogroups (%)Serogroups (%)

Number of samples O1 O2 O26 O78 O86 O141 NTTrachea (82) 1 - - 3 2 1 75Lung (88) 2 1 - 1 - 3 81Liver (72) 1 1 - 1 1 1 67Small intestine (80) 1 - 1 - - 1 77Hoch joint (61) 2 - - - - - 59Digital pad (61) 2 - - - - 2 57Cloacal swab (94) 8 3 1 4 3 1 65Total (540) 17 5 2 9 6 18 483

(29.8)1 (8.8)2 (3.5)3 (15.8)4 (10.5)2 (31.6)1 (89.4)5

Means with differentMeans with different superscripts differsuperscripts differ signifi cantly (P<0.05); NT = not typable with available antiserasignifi cantly (P<0.05); NT = not typable with available antisera

Table 4. O serogroups identifi ed in Table 4. O serogroups identifi ed in E. coliE. coli strains isolated from healthy and sick chickens in strains isolated from healthy and sick chickens in MaiduguriMaiduguri

Number of Number of E. coliE. coli serogroups (%) serogroups (%) O serogroup Healthy chickens Sick chickens Total (n=392) (n=148) (n = 540)O1 6 (1.5) 11 (7.4) 17 (29.8)O2 2 (0.5) 3 (2.0) 5 (8.8)O26 2 (0.5) 0 (0) 2 (3.5)O78 3 (0.8) 6 (4.1) 9 (15.8)O86 5 (1.3) 1 (0.7) 6 (10.6)O141 16 (4.1) 2 (1.4) 18 (31.6)NT 358 (91.3) 125 (84.5) 483 (89.4)Totals 34 (8.8) 23 (15.5) 57 (10.6)

NT = not typable with available antisera NT = not typable with available antisera

Distribution of E. coli and S. aureus isolates in tissues of chickens.Distribution of E. coli and S. aureus isolates in tissues of chickens. Table 2 shows the Table 2 shows the tissue distribution of β-lactamase-producing strains of tissue distribution of β-lactamase-producing strains of E. coliE. coli and and S. aureusS. aureus isolates from isolates from chickens in Maiduguri.chickens in Maiduguri. Most strains of Most strains of E. coliE. coli were isolated from the liver (16.9%), lung were isolated from the liver (16.9%), lung (15.5%), trachea (15.3%), digital pad (14.3%) and small intestine (9.7%), whilst most (15.5%), trachea (15.3%), digital pad (14.3%) and small intestine (9.7%), whilst most strains of strains of S. aureusS. aureus were similarly isolated from small intestines (16.1%), liver (13.6%) were similarly isolated from small intestines (16.1%), liver (13.6%) and digital pads (10.7%). and digital pads (10.7%).

290


Vet. arhiv 80 (2), 283-297, 2010

Tabl

e 5.

Min

imum

inhi

bito

ry c

once

ntra

tion

valu

es fo

r som

e re

pres

enta

tive β-

lact

amas

e st

rain

s of

Tabl

e 5.

Min

imum

inhi

bito

ry c

once

ntra

tion

valu

es fo

r som

e re

pres

enta

tive β-

lact

amas

e st

rain

s of E

. col

i E

. col

i (n

= 12

) and

(n =

12)

and

S. a

ureu

s S

. aur

eus

(n =

14)

(n =

14)

isol

ated

from

tiss

ues o

f chi

cken

s in

Mai

dugu

ri, N

iger

iais

olat

ed fr

om ti

ssue

s of c

hick

ens i

n M

aidu

guri,

Nig

eria

Ant

ibio

ticA

ntib

iotic

isol

ate

isol

ate

Num

ber o

f iso

late

s with

thei

r MIC

(μg/μL

) val

ues

Num

ber o

f iso

late

s with

thei

r MIC

(μg/μL

) val

ues

Susc

eptib

le

Susc

eptib

le

valu

e va

lue μμg

/g/μμLL

0. 2

50.

25

0. 5

0. 5

1. 0

1. 0

2. 0

2. 0

4. 0

4. 0

8. 0

8. 0

>8. 0

>8. 0

Susc

eptib

le

Susc

eptib

le

(%)

(%)

Res

ista

nt

Res

ista

nt

(%)

(%)

Am

pici

llin

Am

pici

llin

E. c

oli

E. c

oli

<0.2

5<0

.25

22--

--22

----

882

(16.

7)2

(16.

7)8

(66.

7)8

(66.

7)S.

aur

eus

S. a

ureu

s--

----

2244

2266

--14

(100

)14

(100

)C

epha

lexi

nC

epha

lexi

nE.

col

iE.

col

i<8

.0<8

.022

--22

22--

--66

6 (5

0)6

(50)

6 (5

0)6

(50)

S. a

ureu

sS.

aur

eus

----

----

----

1414--

14 (1

00)

14 (1

00)

Chl

oram

phen

icol

Chl

oram

phen

icol

E. c

oli

E. c

oli

≤8.0

≤8.0

22--

----

22--

884

(33.

3)4

(33.

3)8

(66.

7)8

(66.

7)S.

aur

eus

S. a

ureu

s--

----

----

--44

--14

(100

)14

(100

)C

iprofl o

xaci

nC

iprofl o

xaci

nE.

col

iE.

col

i≤1

.0≤1

.066

----

44--

22--

6 (5

0)6

(50)

2 (1

6.7)

2 (1

6.7)

S. a

ureu

sS.

aur

eus

----

--88

--66

----

14 (1

00)

14 (1

00)

Dox

ycyc

line

Dox

ycyc

line

E. c

oli

E. c

oli

≤2.0

≤2.0

----

--22

--44

662

(16.

7)2

(16.

7)6

(50)

6 (5

0)S.

aur

eus

S. a

ureu

s--

----

----

--1414

--14

(100

)14

(100

)Li

ncom

ycin

Linc

omyc

inE.

col

iE.

col

i≤0

.5≤0

.5--

----

----

--1212

--12

(100

)12

(100

)S.

aur

eus

S. a

ureu

s--

----

----

--1414

--14

(100

)14

(100

)Pe

nici

llinG

Peni

cilli

nGE.

col

iE.

col

i≤0

.12

≤0.1

2--

----

----

--1212

--12

(100

)12

(100

)S.

aur

eus

S. a

ureu

s--

----

----

--1414

--14

(100

)14

(100

)Te

tracy

clin

eTe

tracy

clin

eE.

col

iE.

col

i≤4

.0≤4

.0--

22--

----

--1010

2 (1

6.7)

2 (1

6.7)

10 (8

3.3)

10 (8

3.3)

S. a

ureu

sS.

aur

eus

----

----

----

1414--

14 (1

00)

14 (1

00)

Tylo

sint

artra

teTy

losi

ntar

trate

E. c

oli

E. c

oli

≤0.7

≤0.7

----

----

----

1212--

12 (1

00)

12 (1

00)

S. a

ureu

sS.

aur

eus

----

----

----

1414--

14 (1

00)

14 (1

00)

Tylo

sin

Tylo

sin

E. c

oli

E. c

oli

≤0.7

≤0.7

44--

----

----

884

(33.

3)4

(33.

3)8

(66.

7)8

(66.

7)S.

aur

eus

S. a

ureu

s--

----

----

--1414

14 (1

00)

14 (1

00)

Susc

eptib

le v

alue

s wer

e ad

apte

d fr

om in

terp

retiv

e st

anda

rds f

or d

ilutio

n su

scep

tibili

ty te

st (

Susc

eptib

le v

alue

s wer

e ad

apte

d fr

om in

terp

retiv

e st

anda

rds f

or d

ilutio

n su

scep

tibili

ty te

st (G

AIL

G

AIL

andan

d JO

HN

, JO

HN

, 199

5).

199

5).

291


Vet. arhiv 80 (2), 283-297, 2010

E. coli serotyping.E. coli serotyping. Out of the total of 540 isolates serotyped 57 (10.6%) strains were Out of the total of 540 isolates serotyped 57 (10.6%) strains were identifi ed to be associated to 6 serogroups from which 17 (29.8%) were of serogroup O1, identifi ed to be associated to 6 serogroups from which 17 (29.8%) were of serogroup O1, 5 (8.8%) were O2, whilst, 2 (3.5%), 9 (15.8%), 6 (10.5%) and 18 (31.6%) were serogroups 5 (8.8%) were O2, whilst, 2 (3.5%), 9 (15.8%), 6 (10.5%) and 18 (31.6%) were serogroups O26, O78 O86 and O141, respectively, whilst 483 (89.4%) strains were not typable with O26, O78 O86 and O141, respectively, whilst 483 (89.4%) strains were not typable with the available sera. With the exception of serogroup O26, each of the other serotypes the available sera. With the exception of serogroup O26, each of the other serotypes occurred in the liver; whilst all serotypes occurred in the cloacal swab. Only serotypes occurred in the liver; whilst all serotypes occurred in the cloacal swab. Only serotypes O1 and O141 were isolated from the digital pad. Serogroup O141 was more frequently O1 and O141 were isolated from the digital pad. Serogroup O141 was more frequently isolated from healthy, 16 (4.1%) than from sick, 2 (1.4%) chickens, whereas serogroups isolated from healthy, 16 (4.1%) than from sick, 2 (1.4%) chickens, whereas serogroups O1, 11 (7.4%), O2, 3 (2.0%), and O78, 6 (4.1%) were more frequently isolated from sick O1, 11 (7.4%), O2, 3 (2.0%), and O78, 6 (4.1%) were more frequently isolated from sick chickens than from healthy chickens, where 6 (1.5%), 2 (0.5%) and 3 (0.8%) were their chickens than from healthy chickens, where 6 (1.5%), 2 (0.5%) and 3 (0.8%) were their respective ratios (Table 4). Serogroups O1 and O141 differed signifi cantly (P<0.05) in respective ratios (Table 4). Serogroups O1 and O141 differed signifi cantly (P<0.05) in prevalence from O78, whereas serogroups O1 and O141 did not show any signifi cant prevalence from O78, whereas serogroups O1 and O141 did not show any signifi cant difference (P>0.05). difference (P>0.05).

Susceptibiliy to antibiotics. Susceptibiliy to antibiotics. The minimum inhibitory concentrations of 12 The minimum inhibitory concentrations of 12 E. coliE. coli and and 14 14 S. aureusS. aureus isolates to ten antibiotics are shown in Table 5. Ampicillin and cephalexin had isolates to ten antibiotics are shown in Table 5. Ampicillin and cephalexin had a range of 0.25 μg/μL-2.0 μg/μL, chloramphenicol (0.25 μg/μL - 2.0 μg/μL), ciprofl oxacin a range of 0.25 μg/μL-2.0 μg/μL, chloramphenicol (0.25 μg/μL - 2.0 μg/μL), ciprofl oxacin (0.25 μg/μL - 8.0 μg/μL), doxycycline (2.0 μg/μL - 8.0 μg/μL), tetracycline (0.5 μg/μL - (0.25 μg/μL - 8.0 μg/μL), doxycycline (2.0 μg/μL - 8.0 μg/μL), tetracycline (0.5 μg/μL - >8.0 μg/μL), and tylosin (0.25 μg/μL - >8.0 μg/μL) respectively to>8.0 μg/μL), and tylosin (0.25 μg/μL - >8.0 μg/μL) respectively to E. coli E. coli. All the strains . All the strains of of E. coli E. coli tested were resistant to lincomycin, penicillin and tylosin tartrate, indicating a tested were resistant to lincomycin, penicillin and tylosin tartrate, indicating a unimodal distribution. Two strains (16.7%) were susceptible to ampicillin, doxycycline unimodal distribution. Two strains (16.7%) were susceptible to ampicillin, doxycycline and tetracycline, 4 (33.3%) to chloramphenicol, whilst resistance of 66.7% was observed and tetracycline, 4 (33.3%) to chloramphenicol, whilst resistance of 66.7% was observed in 8 strains to tylosin, chloramphenicol and ampicillin, respectively. Furthermore, variable in 8 strains to tylosin, chloramphenicol and ampicillin, respectively. Furthermore, variable resistances of 50% and 83.3% respectively were also observed against cephalexin and resistances of 50% and 83.3% respectively were also observed against cephalexin and tetracycline. Ciprofl oxacin and doxycycline showed variation in susceptibility to tetracycline. Ciprofl oxacin and doxycycline showed variation in susceptibility to E. coliE. coli isolates. The tested strains of isolates. The tested strains of S. aureusS. aureus demonstrated high levels of resistance to nine demonstrated high levels of resistance to nine of the ten antibiotics, ampicillin, cephalexin, chloramphenicol, doxycycline, lincomycin, of the ten antibiotics, ampicillin, cephalexin, chloramphenicol, doxycycline, lincomycin, penicillin, tetracycline, and tylosin), considering the susceptible MIC values adapted. penicillin, tetracycline, and tylosin), considering the susceptible MIC values adapted. However, at higher concentrations between 2.0-8.0 μg/μL (i.e. above the adapted However, at higher concentrations between 2.0-8.0 μg/μL (i.e. above the adapted susceptible values) 57.1% of the susceptible values) 57.1% of the S. aureusS. aureus strains showed intermediate susceptibility to strains showed intermediate susceptibility to ciprofl oxacin. Ampicillin had MIC values of 2.0 μg/μL to 2 (14.3%) isolates, 4.0 μg/μL ciprofl oxacin. Ampicillin had MIC values of 2.0 μg/μL to 2 (14.3%) isolates, 4.0 μg/μL to 4 (28.6%) and 2.0 μg/μL to 8 (57.1) of the to 4 (28.6%) and 2.0 μg/μL to 8 (57.1) of the S. aureus S. aureus strains. strains.

DiscussionDiscussionIn Nigeria, as in other countries in the world, epizootics of bacterial diseases occur In Nigeria, as in other countries in the world, epizootics of bacterial diseases occur

frequently in poultry farms. These diseases occur in all age groups of chickens at any frequently in poultry farms. These diseases occur in all age groups of chickens at any period of time, especially the early stages of life. The economic implications range from period of time, especially the early stages of life. The economic implications range from weight loss, high mortality rates, carcass downgrade and reduced production (weight loss, high mortality rates, carcass downgrade and reduced production (BLANCO et BLANCO et al., 1994al., 1994). Some of these diseases are associated with resident or ingested proliferation of ). Some of these diseases are associated with resident or ingested proliferation of

292 Vet. arhiv 80 (2), 283-297, 2010


pathogenic pathogenic E. coliE. coli and/or and/or S. aureusS. aureus in different organs or tissues of chickens, manifesting in different organs or tissues of chickens, manifesting different signs. different signs.

In the present study the In the present study the E. coliE. coli serogroup O141 strain was identifi ed as the most serogroup O141 strain was identifi ed as the most widespread isolate amongst chickens in Maiduguri, Nigeria. The frequency of isolation widespread isolate amongst chickens in Maiduguri, Nigeria. The frequency of isolation showed that serogroups O1 and O78 were the most common serotypes isolated from sick showed that serogroups O1 and O78 were the most common serotypes isolated from sick chickens. This fi nding supports the reports of HANCHUN et al., (2004) and MISHRA chickens. This fi nding supports the reports of HANCHUN et al., (2004) and MISHRA (1994) in which they independently reported (1994) in which they independently reported E. coliE. coli serogroups O1 and O78 as the most serogroups O1 and O78 as the most prevalent serotypes in chickens with colibacillosis. However, in a fi eld study of clinical prevalent serotypes in chickens with colibacillosis. However, in a fi eld study of clinical cases and dead-in-shell embryos in Zaria, Nigeria, RAJI et al., (2003) examined 86 cases and dead-in-shell embryos in Zaria, Nigeria, RAJI et al., (2003) examined 86 E. E. colicoli strains isolated from colibacillosis and found that 17% of the strains belonged to strains isolated from colibacillosis and found that 17% of the strains belonged to only 2 serogroups, O8 and O9, whilst only 1.2% belonged to serogroups O26 and O78 only 2 serogroups, O8 and O9, whilst only 1.2% belonged to serogroups O26 and O78 respectively, and reported that serogroup O8 was the most frequently encountered strain. respectively, and reported that serogroup O8 was the most frequently encountered strain. The fi ndings in the present study did not agree with this report. This disagreement may The fi ndings in the present study did not agree with this report. This disagreement may likely arise from the use of specifi c antisera in the present study and /or probably from likely arise from the use of specifi c antisera in the present study and /or probably from differences between the geo-climatic zones of Zaria and Maiduguri where the two studies differences between the geo-climatic zones of Zaria and Maiduguri where the two studies were independently carried out. The occurrence of the were independently carried out. The occurrence of the E. coliE. coli serogroups O1, O2 and O78 serogroups O1, O2 and O78 in Nigeria as demonstrated by this study agrees with previous studies (in Nigeria as demonstrated by this study agrees with previous studies (ABDELKADER et ABDELKADER et al., 1995; HANCHUN et al., 2004al., 1995; HANCHUN et al., 2004). ).

Serogroup O26 was isolated in dairy products (raw cow milk) and water bodies Serogroup O26 was isolated in dairy products (raw cow milk) and water bodies (rivers) in Adamawa and Borno States of Nigeria ((rivers) in Adamawa and Borno States of Nigeria (MOSES et al., 2005MOSES et al., 2005). It was also reported ). It was also reported in chickens byin chickens by RAJI et al. (2003) RAJI et al. (2003) in Zaria, Nigeria. This serogroup was isolated in the in Zaria, Nigeria. This serogroup was isolated in the present study from the small intestine and cloaca of local chickens. The isolation of this present study from the small intestine and cloaca of local chickens. The isolation of this serogroup from local chickens in this study may suggest ingestion by the chickens of serogroup from local chickens in this study may suggest ingestion by the chickens of cattle faeces, since the serogroup was more frequently isolated from cattle (MOSES et cattle faeces, since the serogroup was more frequently isolated from cattle (MOSES et al., 2005), and local chickens, being free range scavengers, may have had easy access to al., 2005), and local chickens, being free range scavengers, may have had easy access to bovine faeces. There is also the likelihood that this serotype may cause food poisoning in bovine faeces. There is also the likelihood that this serotype may cause food poisoning in humans from consuming chickens harbouring this strain, since the serotype belongs to the humans from consuming chickens harbouring this strain, since the serotype belongs to the enterotoxigenic group of enterotoxigenic group of E. coli.E. coli.

The main factor compromising the effi cacy of drugs such as penicillins, cephalosporin The main factor compromising the effi cacy of drugs such as penicillins, cephalosporin and related compounds, is the production of β-lactamases (and related compounds, is the production of β-lactamases (MILES and AMYES, 1996MILES and AMYES, 1996) by ) by the affected bacteria. The present study shows that the affected bacteria. The present study shows that E. coliE. coli isolates from local chickens isolates from local chickens had higher (12.7%) β-Lactamase-producing strains, whilst β-lactamase-producing strains had higher (12.7%) β-Lactamase-producing strains, whilst β-lactamase-producing strains of of S. aureusS. aureus were higher (17.7%; P<0.05) in layers. The indiscriminate use of antibiotics were higher (17.7%; P<0.05) in layers. The indiscriminate use of antibiotics by poultry farmers has also been linked to the emergence of bacteria with multiple-drug by poultry farmers has also been linked to the emergence of bacteria with multiple-drug resistant strains (resistant strains (ABDELKADER et al., 1995ABDELKADER et al., 1995) in poultry. Evidence of multiple resistant ) in poultry. Evidence of multiple resistant strains was most commonly encountered in this study. The frequency of resistance to strains was most commonly encountered in this study. The frequency of resistance to antibiotics among enteric bacteria from domestic animals has increased markedly, antibiotics among enteric bacteria from domestic animals has increased markedly,

293Vet. arhiv 80 (2), 283-297, 2010


probably due to the excessive haphazard use of antibiotics by farmers (probably due to the excessive haphazard use of antibiotics by farmers (SAEED et al., 2000SAEED et al., 2000), ), which is a common phenomenon in Nigeria. which is a common phenomenon in Nigeria.

Previous studies (Previous studies (SAENZ et al., 2001; CARL et alSAENZ et al., 2001; CARL et al.., 2002; HANCHUN et al., 2004, 2002; HANCHUN et al., 2004), reported ), reported resistances of clinical isolates of resistances of clinical isolates of E. coliE. coli from chickens to tetracycline, chloramphenicol, from chickens to tetracycline, chloramphenicol, ampicillin and amino glycosides. Similar observations were seen in this study. Such ampicillin and amino glycosides. Similar observations were seen in this study. Such resistances could be ameliorated by the use of ethylene diamine tetra acetate (EDTA)-resistances could be ameliorated by the use of ethylene diamine tetra acetate (EDTA)-potentiated antibiotics potentiated antibiotics in vitroin vitro, as this compound was reported to have signifi cantly , as this compound was reported to have signifi cantly potentiated the activities of antibiotics such as tetracycline and ampicillin. It has also been potentiated the activities of antibiotics such as tetracycline and ampicillin. It has also been reported to produce reversal of antibacterial resistant strains of Gram-negative bacteria reported to produce reversal of antibacterial resistant strains of Gram-negative bacteria ((CHAH and OBOEGBULEM, 2005CHAH and OBOEGBULEM, 2005). This however, needs to be investigated ). This however, needs to be investigated in vivoin vivo during during infections due to these organisms. The present study however, did not agree with the infections due to these organisms. The present study however, did not agree with the fi ndings of fi ndings of WANG et al. (2001)WANG et al. (2001), , SAENZ et al. (2001)SAENZ et al. (2001) and and HANCHUN et al. (2004)HANCHUN et al. (2004) in which in which they reported they reported E. coliE. coli resistance of 79% and 90% to ciprofl oxacin, since in the present resistance of 79% and 90% to ciprofl oxacin, since in the present study study E. coliE. coli isolates from chickens were moderately sensitive to this antibiotic. This isolates from chickens were moderately sensitive to this antibiotic. This difference may arise from the lack of or low usage of this antibiotic in chickens in this difference may arise from the lack of or low usage of this antibiotic in chickens in this part of Nigeria. Nevertheless, ciprofl oxacin and other quinolones have been reported to part of Nigeria. Nevertheless, ciprofl oxacin and other quinolones have been reported to be ineffective in the treatment of animal diseases in China (be ineffective in the treatment of animal diseases in China (HANCHUN et al., 2004HANCHUN et al., 2004) and ) and developed countries like Spain, where previous studies were conducted. In the developing developed countries like Spain, where previous studies were conducted. In the developing countries like Nigeria, quinolones (enrofl oxacins, ciprofl oxacin, ofl oxacin etc) are less countries like Nigeria, quinolones (enrofl oxacins, ciprofl oxacin, ofl oxacin etc) are less commonly used in veterinary medicine, especially in the north-eastern part of the country commonly used in veterinary medicine, especially in the north-eastern part of the country and they account for 1.7% of all antibiotics used in poultry (and they account for 1.7% of all antibiotics used in poultry (MAMZA, 2008MAMZA, 2008). However, ). However, the level of resistance to ciprofl oxacin of poultry the level of resistance to ciprofl oxacin of poultry E-coliE-coli isolates observed in this study isolates observed in this study was similar to that found in The Netherlands, where approximately 10% resistance was was similar to that found in The Netherlands, where approximately 10% resistance was reported (reported (VAN DEN BOGAARD et al., 2001VAN DEN BOGAARD et al., 2001). This low resistance rate may also be associated ). This low resistance rate may also be associated with the low usage of this drug by poultry farmers in that country. with the low usage of this drug by poultry farmers in that country.

A most striking observation in this study was the wide-spread resistance of A most striking observation in this study was the wide-spread resistance of S. aureusS. aureus strains to all the ten antibiotics. These strains may probably possess other resistant or strains to all the ten antibiotics. These strains may probably possess other resistant or genetic markers in addition to production of β-lactamases; or perhaps the isolates may be genetic markers in addition to production of β-lactamases; or perhaps the isolates may be methicillin-resistant strains (MRSA): as methicillin-resistant methicillin-resistant strains (MRSA): as methicillin-resistant Staphylococcus aureus Staphylococcus aureus has has been reported to show resistance to almost all antibiotics, due to the presence of a novel been reported to show resistance to almost all antibiotics, due to the presence of a novel penicillin-binding protein (PBP2a) which showed a decreased binding affi nity for β-penicillin-binding protein (PBP2a) which showed a decreased binding affi nity for β-lactams (lactams (JOHN, 2006JOHN, 2006); hence, the popular reference to it as a “superbug”. This fi nding may ); hence, the popular reference to it as a “superbug”. This fi nding may likely portend further complication in treatment of likely portend further complication in treatment of E. coliE. coli and and S. aureusS. aureus infections in both infections in both humans and animals from this region, as these organisms are becoming a major threat in humans and animals from this region, as these organisms are becoming a major threat in both human and veterinary medicine. In a study in the US, both human and veterinary medicine. In a study in the US, SAEED et al. (2000)SAEED et al. (2000) isolated isolated S. aureusS. aureus from the hock joints and internal organs of diseased chickens and reported that from the hock joints and internal organs of diseased chickens and reported that the isolates were sensitive to Ampicillin, Penicillin and ciprofl oxacin. The fi nding in the the isolates were sensitive to Ampicillin, Penicillin and ciprofl oxacin. The fi nding in the

294

present study is in contrast with the above report. In this study present study is in contrast with the above report. In this study S. aureus S. aureus isolated from isolated from hock joints and internal organs of chickens were resistant to these drugs. The difference in hock joints and internal organs of chickens were resistant to these drugs. The difference in the observations as presently reported could be linked to the production of β-Lactamases, the observations as presently reported could be linked to the production of β-Lactamases, which are capable of producing penicillinases and cephalosporinases that hydrolyze which are capable of producing penicillinases and cephalosporinases that hydrolyze the β-lactam rings in these antibiotics (the β-lactam rings in these antibiotics (LAURA et al., 2002LAURA et al., 2002). The widespread resistance ). The widespread resistance to antibiotics by to antibiotics by S. aureus S. aureus could be due to acquisition of plasmids and/or transposons could be due to acquisition of plasmids and/or transposons ((SAEED et al., 2000SAEED et al., 2000) by the bacteria. Some transposons (plasmids) have genetic elements ) by the bacteria. Some transposons (plasmids) have genetic elements called integrons that enable them to capture exogenous genes (called integrons that enable them to capture exogenous genes (HOWARD et al., 1996HOWARD et al., 1996). ). A number of such genes may therefore be inserted into a given integron, resulting in A number of such genes may therefore be inserted into a given integron, resulting in multiple antimicrobial drug resistance (multiple antimicrobial drug resistance (HOWARD et al., 1996HOWARD et al., 1996), a typical case observed in ), a typical case observed in the present study.the present study.

In conclusion, we have demonstrated the existence of various pathogenic strains of In conclusion, we have demonstrated the existence of various pathogenic strains of E. coliE. coli serogroups from various tissues of chickens; Phenotypically, serogroups from various tissues of chickens; Phenotypically, E. coliE. coli and and S. aureus S. aureus have demonstrated multi-resistance patterns (based on the defi nition of multi-resistance have demonstrated multi-resistance patterns (based on the defi nition of multi-resistance by by DUIJKEREN et al. (2004) DUIJKEREN et al. (2004) to antibiotics such as ampicillin, doxycycline, tetracycline, to antibiotics such as ampicillin, doxycycline, tetracycline, penicillin, lincomycin, chloramphenicol and Tylosin that are commonly used in poultry penicillin, lincomycin, chloramphenicol and Tylosin that are commonly used in poultry in Nigeria. The resistance problem demands that a renewed effort be made to seek newer in Nigeria. The resistance problem demands that a renewed effort be made to seek newer antibacterial agents effective against such pathogenic bacteria (antibacterial agents effective against such pathogenic bacteria (E. coliE. coli and and S. aureus S. aureus) ) that are resistant to antibiotics, and the need to search for other compounds that could that are resistant to antibiotics, and the need to search for other compounds that could effectively inhibit the growth of these common members of organisms.effectively inhibit the growth of these common members of organisms.

_______AcknowledgementsThe authors are grateful to Prof. George Blanco, Director of the The authors are grateful to Prof. George Blanco, Director of the E. coliE. coli reference centre Spain for supplying reference centre Spain for supplying the antisera used in this study, Mr Isa Gulani, for his technical assistance, the Head of department Veterinary the antisera used in this study, Mr Isa Gulani, for his technical assistance, the Head of department Veterinary Medicine, University of Maiduguri, Dr A.U Mani, for his great assistance towards seeing the success of this Medicine, University of Maiduguri, Dr A.U Mani, for his great assistance towards seeing the success of this work and Mrs Patience wakirwa for the secretariat work. work and Mrs Patience wakirwa for the secretariat work.

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Received: 18 August 2008Accepted: 22 December 2009

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MAMZA, S.A., G. O. EGWU, G. D. MSHELIA: Osjetljivost bakterija MAMZA, S.A., G. O. EGWU, G. D. MSHELIA: Osjetljivost bakterija Escherichia Escherichia colicoli i i Staphylococcus aureus Staphylococcus aureus izdvojenih iz pilića u Maiduguri (Nigerija) prema beta-izdvojenih iz pilića u Maiduguri (Nigerija) prema beta-laktamskim antibioticima. laktamskim antibioticima. Vet. arhiv 80, 283-297, 2010

SAŽETAKSAŽETAKPremda uvjetno patogene vrste Premda uvjetno patogene vrste Escherichia coliEscherichia coli i i Staphylococcus aureusStaphylococcus aureus predstavljaju globalni klinički predstavljaju globalni klinički

problem u humanoj i veterinarskoj medicini. Ovo istraživanje provedeno je sa svrhom određivanja osjetljivosti problem u humanoj i veterinarskoj medicini. Ovo istraživanje provedeno je sa svrhom određivanja osjetljivosti prema antibioticima serovarova prema antibioticima serovarova E. coliE. coli i izolata i izolata S. aureusS. aureus izdvojenih iz pilića u sušnom području Maiduguri izdvojenih iz pilića u sušnom području Maiduguri u Nigeriji. Različiti uzorci tkiva od naizgled zdravih te od bolesnih pilića bili su pretraženi na prisutnost u Nigeriji. Različiti uzorci tkiva od naizgled zdravih te od bolesnih pilića bili su pretraženi na prisutnost bakterija bakterija E. coliE. coli i i S. aureusS. aureus. Izolati su bili identifi cirani različitim biokemijskim testovima. Sojevi što su . Izolati su bili identifi cirani različitim biokemijskim testovima. Sojevi što su proizvodili β-laktamazu bili su identifi cirani kromogen cefalosporinskom metodom uz uporabu nitrocefi nom proizvodili β-laktamazu bili su identifi cirani kromogen cefalosporinskom metodom uz uporabu nitrocefi nom impregniranih stikova i otopine cefalosporina (nitrocefi na). Osjetljivost spomenutih bakterija bila je određivana impregniranih stikova i otopine cefalosporina (nitrocefi na). Osjetljivost spomenutih bakterija bila je određivana mikrodilucijskom metodom na 10 antibiotika (ampicilin, koramfenikol, cefaleksin, ciprofl oksacin, linkomicin, mikrodilucijskom metodom na 10 antibiotika (ampicilin, koramfenikol, cefaleksin, ciprofl oksacin, linkomicin, doksiciklin, tetraciklin, tilozin, tilozin tartrat i penicilin). doksiciklin, tetraciklin, tilozin, tilozin tartrat i penicilin). E. coliE. coli je bila izdvojena iz 805, a je bila izdvojena iz 805, a S. aureusS. aureus iz 660 od iz 660 od 1300 pretraženih uzoraka tkiva od čega je 89 (11,1%) izolata 1300 pretraženih uzoraka tkiva od čega je 89 (11,1%) izolata E. coliE. coli i 58 (8,8%) izolata i 58 (8,8%) izolata S. aureusS. aureus bilo pozitivno bilo pozitivno na β-laktamazu. Od 540 serološki tipiziranih izolata na β-laktamazu. Od 540 serološki tipiziranih izolata E. coliE. coli identifi cirano je bilo 57 (10,6%) seroloških skupina identifi cirano je bilo 57 (10,6%) seroloških skupina od čega je 17 (29,8%) pripadalo serološkoj skupini O1, 5 (8,8%) serološkoj skupini O2, 2 (3,5%) skupini O26, 9 od čega je 17 (29,8%) pripadalo serološkoj skupini O1, 5 (8,8%) serološkoj skupini O2, 2 (3,5%) skupini O26, 9 (15,8%) skupini O78, 6 (10,5%) skupini O86 i 18 (31,6%) serološkoj skupini O141, dok se 483 (89,4%) izolata (15,8%) skupini O78, 6 (10,5%) skupini O86 i 18 (31,6%) serološkoj skupini O141, dok se 483 (89,4%) izolata nisu mogla tipizirati raspoloživim antiserumima. Najčešće su bile izdvojene serološke skupine O141, O1 i O78 nisu mogla tipizirati raspoloživim antiserumima. Najčešće su bile izdvojene serološke skupine O141, O1 i O78 s time da su serološke skupine O1 i O78 prevladavale u bolesnih pilića. s time da su serološke skupine O1 i O78 prevladavale u bolesnih pilića. E. coliE. coli je bila vrlo otporna na ampicilin, je bila vrlo otporna na ampicilin, kloramfenikol, tetraciklin, linkomicin, penicilin i tilozin s vrijednostima minimalne inhibitorne koncentracije kloramfenikol, tetraciklin, linkomicin, penicilin i tilozin s vrijednostima minimalne inhibitorne koncentracije >8,0 μg/μL. I >8,0 μg/μL. I S. aureusS. aureus je bio otporan prema svim pretraživanim antibioticima s minimalnim inhibitornim je bio otporan prema svim pretraživanim antibioticima s minimalnim inhibitornim vrijednostima >8,0 μg/μL. U istraživanju je dokazana prisutnost serovarova vrijednostima >8,0 μg/μL. U istraživanju je dokazana prisutnost serovarova E. coliE. coli i vrste i vrste S. aureusS. aureus u različitim u različitim tkivima pilića te njihova osjetljivost na antibiotike s jasno dokazanom multiplom rezistencijom.tkivima pilića te njihova osjetljivost na antibiotike s jasno dokazanom multiplom rezistencijom.

Ključne riječi: Ključne riječi: beta-laktamaza, beta-laktamaza, Escherichia coliEscherichia coli, , Staphylococcus aureusStaphylococcus aureus, antibiotici, osjetljivost, pilići, , antibiotici, osjetljivost, pilići, MaiduguriMaiduguri


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Osjetljivost bakterija Escherichia coli i Staphylococcus aureus izdvojenih iz pilića u Maiduguri (Nigerija) prema betalaktamskim antibioticima