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CHANGES IN ANTIBIOTIC RESISTANCE INDICES OF ANIMAL ESCHERICHIA COLI STRAINS WITH NUMBER OF ISOLATES Marina Spînu, L. Köbölkúti, D. Cadar, Mihaela Niculae, G. Bianu, Silvana Popescu, L. Lukács UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE, CLUJ-NAPOCA, ROMANIA, STR. MĂNĂŞTUR NO.3-5 Summary E. coli infections are probably the most frequent infectious diseases of young, both in intensive and extensive raising systems, with serious negative economic consequences. The research aimed to investigate the dependence of antimicrobial resistance of E.coli strains isolated from dairy cows with mastitis (n=8), newborn calves with diarrhea (n=12), piglets with edema disease (n=11) and hens with granuloma lesions (n=12). Microbial isolates were cultured on broth, agar and then identified as E. coli by use of API 20E biochemical tests. Serotypes were identified by classical rapid agglutination with use of specific anti-O, H and K sera. A Kirby-Bauer type antibiogram was performed for each strain with a range of eighteen antibiotics and antimicrobial agents: amoxicillin, ampicillin, ceftazidime, cephalexin, cephalotin, colistin sulphate, erythromicin, furazolidone, nitrofurantoin, novobiocin, oxacillin, penicillin G, polimixin B, rifampicin, spectinomicin, tetracycline, streptomicin, sulphametaxazole. The results indicated the presence of different serotypes of E. coli, such as O9:K103:H- in mastitic cows, O8:K25 (75%) and, O9:K35, O8:K28, O101:K28 (8.33% each) in calves, O141:(H4)(18.18%), O139:K82:H1 (36.36%) and O138:H- (45.46%) in swine oedema disease cases, while chicken E. coli strains belonged to serotypes O1:K1 (n=3, 25%), O2:K1 (n=6, 50%) and O78:K80 (n=3, 25%). Antibiotic resistence indices (ARI) were of 0.145 in cows, 0.44 in calves and hens and 0.49 in piglets. The results indicated an increased ARI with the increase of isolated strain numbers and with species. Key words: E.coli, bovine, swine, chickens, antibiotic resistance index
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Introduction piglets (Oanh et al., 2010) caused by Shigatoxigenic E. coli (STEC) strains (Konstantinova et al., 2008). Colibacillosis, caused by avian pathogenic Escherichia coli (APEC) is one of the main causes of economic losses in the poultry industry worldwide. Most studies on colibacillosis refer to broilers, even though laying hens can also be severely affected (Vandekerchove et al., 2012). E. coli infections have also been described in turkeys, geese, and ducks and are thought to be the cause of significant economic losses (Landman and Cornelissen, 2006). A progressive increase in antibacterial-drug resistance was demonstrated among E. coli isolates from the feces of calves and piglets reared on farms (Hinton et al., 1984).
E. coli infections are probably the most frequent infectious diseases of young, both in intensive and extensive raising systems, with serious negative economic consequences. Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection (Dogan et al., 2006). Nevertheless, dairy cattle with clinical mastitis caused by Escherichia coli exhibit a wide range of disease severity, from mild, with only local inflammatory changes of the mammary gland, to severe, with significant systemic derangement (Weny et al., 2006). Diarrhea due to E. coli is one of the most frequent diseases in young piglets and calves (Hariharan et al., 2004), while edema disease (ED) is a common fatal disease in newly weaned 361
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Methods. All fourty-three isolates were subjected to isolation and serotyping of E. coli strains. The samples were cultivated on simple broth then agar, and 24 hour cultures were identified by the use of API 20 E biochemical tests and the specific software. To establish the incidence of various serotypes within the samples, the antigenic formula of each strain was identified by a clasical rapid agglutination test, using specific anti-O, K and H sera. A droplet of the 24 hour culture on MuellerHinton agar was mixed in turn, with a droplet of each anti-serum and the reaction was considered positive in the case of serum where the agglutination was present. The resistance to antibiotics of all isolates was tested by use of the Kirbz-Bauer radial diffusion technique (Barry et al., 1985). All E. coli strains were inseminated on Mueller Hinton agar plates, using 1 ml of a 24 h culture in simple broth, adjusted to McFarland scale density 0.5 (tube 1). After removing the exceeding culture, the plates were allowed to dry and antibiotic discs were placed on the surface of the agar. Eighteen antibiotics and chemotherapeutic agents, most currently involved in veterinary therapy, were used: amoxicillin (AMC), ampicillin (AX), ceftazidime (CAZ), cephalexin (CL), cephalotin (KF), colistin sulphate (CT), erythromicin (E), furazolidone (FR), nitrofurantoin (F), novobiocin (NV), oxacillin (OX), penicillin G (P), polimixin B (Pb), rifampicin (RD), spectinomicin (SH), streptomicin (S), sulphametaxazole (SXT), tetracycline (TE). Subsequently, the plates were incubated for 24 h at 37ºC. Growth inhibition diameters were expressed in mm and compared to estimate the efficacy of each antimicrobial agent. Resistant colonies and total resistence were indicated as „CR” and „R” respectively. Antibiotic resistance index was calculated according Hinton and Linton (1983), by a formula y/nx, where y was the number of total resistance (R) determinants in a population n and x=18, that is the number of drugs included in the sensitivity test in this study.
The ease with which bacteria become resistant to currently used antimicrobial agents has been of concern to clinicians, public health officials, and researchers. Transferable drug resistance represents a major threat to the treatment of infectious diseases in both humans and animals. The use of antimicrobial agents in both human and veterinary medicine exerts a strong selective pressure inducing resistance to antimicrobial agents, but most of these experiments have been performed in laboratories under standardized conditions (Kruse and Sørum, 1994). Although antimicrobial suscep-tibility testing is recommended, information on drug resistance trends in a geographic area is helpful to veterinarians in drug selection for empirical therapy (Hariharan et al., 2004). The research aimed to investigate the dependence of antimicrobial resistance of E.coli strains isolated from infections of different species (mastitis, edema disease, diarrhea, poliserositis and granulomas) in connection to previous treatments and serotype.
Material and methods Clinical cases.The studies were carried out on dairy cows showing E. coli mastitis (n=8), calves with neonatal dierrhoea (n=12), piglets with oedema diseases (n=11) and hens showing E. coli induced granulomas (n=12) Samples. Cows were sampled at the morning milking, while feces from calves were taken from the rectum, with sterile tampons. Pathological samples from piglets and hens were taken during the necropsy, consisting of liver, mesenteric lymph nodes, spleen, an unopend bone and fragments of the intestine, as well as ascitic fluid in piglets and liver, serous membranes and bones in hens, respectively. All samples were sampled and transported under sterile conditions. When needed, the intestine was separatly packed from other anatomical parts. 362
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Results and discussion O8:K25 (75%) and, O9:K35, O8:K28, O101:K28 (8.33% each). O141:(H4)(18.18%), O139:K82:H1 (36.36%) and O138:H- (45.46%) were the serotypes identified in swine oedema disease cases. Chicken Escherichia coli strains belonged to serotypes O1:K1 (n=3, 25%), O2:K1 (n=6, 50%) and O78:K80 (n=3, 25%). The serotypes affecting verious animals depend on the species, age, raising system and geographical area, as samples coming from various regions of Transylvania indicated in this study. Resistence to antibiotics of the isolated E. coli strains was presented in tables 1 and 2.
Facing the problem of development and spreading of bacterial resistance, preventive strategies are considered the most appropriate means to counteract. The establishment of corresponding manage-ment options relies on scientifically defensible efforts to obtain objective data on the prevalence of bacterial resistance in healthy and diseased livestock (Wallmann, 2006). In mastitic cows, coming from the same farm, only one serotype of E. coli (O9:K103:H- ) was isolated, considered to be present in the Transylvania (Perianu et al., 2011). In calves, the isolated serotypes were
Table 1. Antibiotic resistance and E.coli serotypes in cows and calves
Calves
Cows Antibiotic AMC
O9:K103:H-
9X O8:K25
O9:K35
O101:K28
O8:K28
R
15mm
16mm
R
14mm
AX
3mm
13mm
13mm
R
16mm
CAZ
5mm
10mm
10mm
15mm
11mm
CL
R
R
R
R
R
KF
R
R
R
R
R
CT E FR
9mm 6mm
9mm R
10mm CR
10mm R
8mm CR
16mm
12mm
13mm
16mm
19mm
F
17mm
20mm
15mm
17mm
18mm
NV
7mm
7mm
CR
CR
R
OX
R
R
R
R
CR
P
8mm
R
R
R
R
Pb
12mm
11mm
11mm
10mm
13mm
RD
13mm
13mm
10mm
15mm
12mm
SH
R
R
R
R
CR
S SXT
R R
R R
R R
7mm R
R R
TE
CR
CR
R
R
CR
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Table 2. Antibiotic resistance and E.coli serotypes in hens and piglets
Hens Antibiotic AMC AX CAZ CL KF CT E FR F NV OX P Pb RD SH S SXT TE
3x O1:K1 10mm 8mm CR R 10mm 11mm R
6xO2:K1 15mm 13mm 20mm R 16mm 10mm R
3x O78:K80 R R CR R R 11mm R
2x O141:(H4) 17mm 18mm 17cm R 15mm 9mm R
Piglets 4x O139:K82:H1 15mm R 16mm R R 9mm R
CR CR R R R 14mm 14mm R R R 16mm
20mm 20mm R R R 14mm 16mm R 10mm R 16mm
9mm 10mm R R R 10mm 16mm R 11mm R 14mm
20mm 20mm 10mm R 9mm 10mm 10mm R R R R
13mm 17mm R R R 11mm 11mm R R R R
Older results suggest that ETEC strains of E. coli found in calves with diarrhea belong to serogroups O9:K35, O101:K30, O8:K85, O20:K? O8:K25, and O101:K28 (Braaten and Myers, 1977). Serotype O8:K25 and O101:K(A)28 were considered K99+ (Blanco et al., 1993). Serotype O8:K28 was also found in the present study in calves. In a more recent research (Sting and Stermann, 2008), E. coli O-types O139 and O141, present in piglets with edema disease of this study, accounted for 43.8% of all porcine strains and for 55.4% of the strains exhibiting haemolytic activity. Escherichia coli serogroups O1, O2, and O78 were considered the main causative agents of avian colibacillosis (Jamalludeen, 2009) and these correspond to the findings of the present research. Phenotypic resistance of veterinary pathogens to more than one antimicrobial
5x O138:H17mm 17mm R R 16mm 9mm R 20mm 20mm R 9mm R 11mm 13mm R 13mm R R
agent (multi-resistance) may be caused by intrinsic resistance to the antimicrobial agents, acquired cross-resistance, or acquired co-resistance (Werckenthin, 2005). E.coli isolates from mastitic milk were found by some researchers, sensitive to cefoperazone, polymyxin B, colistin and gentamycin (Stephan and Rüsch, 1997). However, in the treatment of mastitis the resistance of a particular agent is only one among several contributing factors. All bovine isolates proved to be resistant in a high proportion to antiobiotics. Although cefoperazone was not used in this experiment, the serotype isolated from mastitic cows proved to be resistant to other two cephalosporins, cephalexin and cephalotin. Such cases were mentioned in infant medicine as well (Thaver et al., 2009). Nevertheless, the very low ARI (0.145) could be the result of the single isolated serotype. In calves with diarrhea, 364
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Blanco M., Blanco J.E., Blanco J. Enterotoxigenic Escherichia coli K99+, serotype 08:K25, produce cytotoxic necrotizing factor CNF1 and alphahemolysin. Microbiologia.; 9(1):72-6, 1993. Braaten B.A., Myers L.L. Biochemical characteristics of enterotoxigenic and nonenterotoxigenic Escherichia coli isolated from calves with diarrhea. Am. J. Vet. Res., 38(12):1989-91, 1977. Dogan B., Klaessig S., Rishniw M., Almeida R.A., Oliver S.P., Simpson K., Schukken Y.H. Adherent and invasive Escherichia coli are associated with persistent bovine mastitis. Vet. Microbiol. ;116 (4):270-82, 2006 . Hariharan H., Coles M., Poole D., Page R. Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves from piglets and calves with diarrhea, Can. Vet. J..;45:605–606, 2004. Hinton M., Rixson P. D., Allen V. The persistance of drug resistant Escherichia coli strains in the majority faecal flora of calves, J. Hyg., Camb., 93, 547-557 547, 1984. Hinton, M., Linton, A. H. Antibacterial drug resistance among Escherichia coli isolated from calves fed on a milk substitute diet. Veterinary Record 112, 567-568, 1983. Jamalludeen N., She Y.M., Lingohr E.J., Griffiths M. Isolation and characterization of virulent bacteriophages against Escherichia coli serogroups O1, O2, and O78. Poult Sci.;88(8):1694-702, 2009. Konstantinova L., Hamrik J., Kulich P., Kummer V., Maskova J., Alexa P. The effect of intramuscular administration of colistin on the development and course of experimentally induced oedema disease in weaned piglets. Vet. Microbiol..;128(12):160-6, 2008. Kruse H., Sørum H. Transfer of Multiple Drug Resistance Plasmids between Bacteria of Diverse Origins in Natural Microenvironments, Applied And Environmental Microbiology, Vol. 60, No. 11, p. 4015-4021, 1994. Landman W.J., Cornelissen R.A. Escherichia coli salpingitis and peritonitis in layer chickens: an overview. Tijdschr Diergeneeskd.;131(22):814-22, 2006. Oanh T.K., Nguyen V.K., Do T.N., Goddeeris B.M., De Greve H. Escherichia coli strains causing edema disease in northern Vietnam share an identical verotoxin 2e. op Anim. Health Prod.; 42(8):1797-804, 2010.
the ARI increased to 0.44, due to a higher number of strains/serotypes and resistance to a higher number of antibiotics in each strain, ranging from 6 of 18 (33.33%) to 10 of 18 (55.55%). This was probably a consequence of non-discriminating antibiotic treatments, without previous tests on sensitivity, in both mastites and cases of neonatal diarrhea of calves. E.coli strains isolated from piglets differed as resistance to antibiotics. The highest ARI was calculated (0.49) and the highest number of inefficient antibiotics was observed for these animals (61.11%). Serotypes O139:K82:H1 (61.11%) and O138:H- (44.44%) were the most resistant. In general, the inhibition diameters were higher in hens and piglets than in bovine. The results indicated an emerging resistance in all isolated E. coli serotypes which is of concern not only for veterinary medicine, but also for consumers. Limited data pose a challenge in devising simple community-based management strategies. Further studies are needed to determine prevalence of resistant strains, as well as assess regional trends.
Conclusions These was no overlap between the E. coli serotypes isolated from different species, but all shared the increased resistance to antibiotics, suggesting an inneficinet use of antibacterial treatments on farm. ARI increased with the species and number of isolated strains. Newer generation antibiotics seemed least efficient, trigerring the compulsory caracter of pre-treatment antibacterial tests. In enedemic areas, vaccination could represent an alternative.
References Barry A.L., Thornsberry C. Susceptibility tests: Diffusion test procedures. In: Lennette E.H., Balows A., Hausler Jr W.J., Shadomy H.J., eds. Manual of Clinical Microbiology. 4th ed, Washington, D.C.: Am. Soc. Microbiol, p.978–987, 1985.
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Perianu T. (ed), 2011. Infectious disease of animals, Bacterial diseases, Millenium Publishing House, Iasi Stephan R., Rüsch P. Current resistance status of Escherichia coli strains from bovine mastitis milk samples. Schweiz Arch. Tierheilkd.;139(11):495-9, 1997. Sting R, Stermann M. Duplex real-time PCR assays for rapid detection of virulence genes in E. coli isolated from post-weaning pigs and calves with diarrhoea. Dtsch. Tierarztl Wochenschr. ;115(6):231-8, 2008. Thaver D., Ali S.A., Zaidi A.K. Antimicrobial resistance among neonatal pathogens in developing countries. Pediatr. Infect. Dis. J.; 28 (1 Suppl):S19-21, 2009.
Vandekerchove D., De Herdt P., Laevens H., Pasmans F. Colibacillosis in caged layer hens: characteristics of the disease and the aetiological agent. Avian Pathol.;33(2):11725, 2004. Wallmann J. Monitoring of antimicrobial resistance in pathogenic bacteria from livestock animals. Int. J. Med. Microbiol.; 296 Suppl 41:81-6, 2006. Wenz J.R., Barrington G.M., Garry F.B., Ellis R.P., Magnuson R.J. Escherichia coli isolates' serotypes, genotypes, and virulence genes and clinical coliform mastitis severity. J. Dairy Sci.; 89(9):3408-12, 2006.
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