Abstract
We investigated the antibiotic resistance of a collection of 94 strains (55.6%) of haemolytic Escherichia coli (E. coli) isolated in 169 diarrhoeic faecal samples from buffalo (Bubalus bubalis) calves. Bacterial colonies on McConkey and EHLY agar that showed the morphology of E. coli were biochemically tested and then, furtherly classified as haemolytic, using PCR-based assays for enterohemorrhagic E. coli hly (hlyEHEC) virulence gene. When the pathogenic isolates were tested for their susceptibility to 13 different antibiotics, each tested isolate was found to be highly resistant to more than three antibiotics. In fact, absolute resistance (100% of resistance) to penicillin G, lincomycin, neomycin, was detected. Amoxicillin/clavulonic acid and ampicillin were found to be moderately effective against the majority of isolates (46.8% of resistance). Thirty-two (34%) of the haemolytic E. coli strains were phenotypically resistant to tetracycline. None of the isolated strains of E. coli was resistant to colistin sulfate. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in food producing animals is highly recommended.
Introduction
In Italy, buffalo (Bubalus bubalis) population consists of approximately 200,000 animals (CitationZicarelli, 2004). Buffalo herds are mainly located in the southern regions, where they represent an important economic resource for the industry of milk-derived products. A key role in the aetiology of bacterial infectious diarrhoea in buffalo calves has been attributed to enteropathogenic Escherichia coli mainly during the first three weeks of life (CitationZaman et al., 2006). The pathogenic E. coli adhere to the mucosa and proliferate in the lumen of intestine, producing a potent enterotoxin, which stimulate excessive secretion of fluid from intestinal mucosa. This loss of fluid causes the principal sign (diarrhoea) and often leads to dehydration and high rate of death in the buffalo calves, and consequently causes heavy economical losses (CitationRadostits et al., 1994).
E. coli can produce several types of haemolysin, including an extracellular protein (α-haemolysin), a cell-bound protein (β-haemolysin) and a haemolysin expressed to be nalidixic acid-resistant mutants (γ-haemolysin) (CitationCavalieri et al., 1984; CitationBeutin, 1991; CitationLin et al., 2008). α-haemolysin (HlyA) is a 107-kDa protein toxin secreted by pathogenic strains of E coli, that induces osmotic lysis of erythrocytes because of its pore-forming activity (CitationGoñi and Ostolaza, 1998). It is a member of the so-called “RTX family”, a group of proteins characterized by the presence of a Gly and Asp-rich nonapeptide sequence repeated in tandem near the protein C-terminus (CitationYoung and Holland, 1999; CitationWelch, 2001). Several studies provide evidences that slaughtered animals like buffalo, cows and goats are reservoirs for enterohemorrhagic (EHEC) E coli, including the potentially virulent strain designated E. coli O157:H7 (CitationIslam et al., 2008). Previous studies have documented that prevalence (ranges from 2–3% to as high as 80%), and shedding patterns of E. coli O157:H7 in the faeces of beef cattle are highly variable (CitationElder et al., 2000; CitationCallaway et al., 2003).
Evaluation of a strain’s sensitivity to antibiotics is another useful way to differentiate enterohemorrhagic E. coli, given that strains with a greater pathogenic potential present resistance to a wide range of antibiotics (CitationKrumperman, 1983; CitationHarwood et al., 2000). In this study haemolytic E coli strains isolated from diarrhoeic buffalo calves samples living in the Campania Region (southern Italy) were biotyped and tested for their antibiotic susceptibility to evaluate the pathogenic potential of the strains that buffaloes carry and shed into the environment and the related public health risks implied. The primary objective of the present study was to determine the prevalence of EHEC in diarrhoeic faecal samples collected from buffalo calves and establish their antibiotic resistance.
Materials and methods
Laboratory isolation and identification of haemolytic E. coli
A total of 169 diarrhoeic faecal specimens from buffalo calves with diarrhoea (30-days-old and younger), were collected between October 2006 and October 2008. The samples were presented for screening at the Infectious Diseases Section, Faculty of Veterinary Medicine of Naples, and the microbiological examination started within 5 h after the collection time. The samples were cultivated after running an enrichment broth Brain heart Infusion (BHI) incubated at 37°C for 24 h and the results were evaluated according to the turbidity of the broth. Loopful from these broth cultures were then streaked onto MacConkey’s Agar (MAC 3) (from Oxoid), differential and selective medium to grow gram-negative bacteria, and Enterohaemolysin Agar with blood (EHLY) (from Oxoid), differential and selective medium to grow enterohemorrhagic E. coli strains (EHEC) enterohaemolysin productors. Indole and oxidase tests were performed for lactose-positive colonies. Suspected colonies were identified morphologically and then biochemically by the API 20E Kit system (bioMérieux, France). As positive control, E. coli O157:H7 strain ATCC 43895 was used as quality control strains in each experiment.
E. coli 0157 latex test and serotyping
A latex agglutination test for the identification of E. coli serogroup 0157 (Oxoid) was used. This test demonstrated by slide agglutination E. coli strains possessing the O157 serogroup antigen. Furthermore, the determination of O:111, O:26, O:157, O:128 and O:103 antigens was carried out, utilizing antisera obtained from Biolife Italiana srl.
PCR
For confirmation of haemolytic activity of E. coli isolates PCR amplification of the ehxA gene was performed, as described previously (CitationFeng and Monday, 2000).
The following primers were used (sense: 5′-GTTTATTCTGGGGCAGGCTC-3′ and antisense: 5′- CTTCACGTCACCATACATAT- 3′) to amplify a fragment of 158 bp. Bacteria were harvested from EHLY agar, suspended in 250 µL of sterile water, incubated at 100°C for 5 min to release the DNA and centrifuged. The supernatant was used in the PCR reaction as described below. Amplification of bacterial DNA was performed using 30 µL volumes containing 7 µL of the prepared sample supernatant. Each reaction mixture also contained 1× AmpliTaq Gold DNA polymerase reaction buffer, 2.5 mM MgCl2, 1 mM deoxynucleoside triphosphates, 1 pmol of each primer per µL, and 1 U AmpliTaq Gold DNA polymerase.
The conditions for the PCR were 95°C for 5 min for initial denaturation of DNA within the sample followed by 30 cycles of 94°C for 45 s (denaturing), 52°C for 45 s (annealing), and 72°C for 1 min, with a final extension step at 72°C for 5 min, performed with a thermal cycler (Thermocycler Eppendorf). The amplified products were analyzed by gel electrophoresis in 2% agarose containing ethidium bromide (0.5 µg/mL). Negative controls contain all reagents except template DNA. As positive control, genomic DNA isolated from a suspension of E. coli O157:H7 strain ATCC 43895 was used as quality control strain. All PCR assays were carried out in duplicate. The products were visualized under UV transillumination of apparatus BioRad Gel Doc 1000 (BioRad, Italy).
Antibiotic susceptibility testing
Laboratory trials were performed in accordance with the principles described in the standard method of the National Committee for Clinical Laboratory Standards (NCCLS, 2002), using the following antimicrobial agents: nalidixic acid (NA 30 µg), amoxicillin/clavulonic acid (AMC 30 µg), ampicillin (AMP 10 µg), apramycin (APR 15 µg), colistin sulfate (CS 10 µg), enrofloxacin (ENR 5 µg), gentamicin (CN 10 µg), tetracycline (TE 30 µg), trimethoprim/sulphametoxazole (SXT 25 µg), ceftiofur (EFT 30 µg), neomycin (N 30 µg), lincomycin (MY 2 µg), penicillin G (P 10 µg). The susceptibility of each isolate to the panel of antibiotics was revealed by the diameter size of the clear zones around the dish, as directed by the manufacturer.
Statistical analysis
Statistical analysis was performed by Fisher’s exact test using GraphPad InStat Version 3.00 for Windows 95 (GraphPad Software, San Diego, CA, USA).
Results
From a total of 169 faecal samples from buffalo (Bubalus bubalis) calves affected with diarrhoea have been isolated 94 E. coli strains (55.6%) showing haemolytic activities. E. coli strains were considered haemolytic when on the blood agar plates a clear halo was observed around isolated colonies after overnight incubation. Of the 94 strains for which serotyping was attempted, 40 samples were positive in latex agglutination test for the identification of E. coli serogroup 0157 and were confirmed employing another antisera test, as showed in . We found by serotyping an extremely significant prevalence of 0157 antigen (42.5%, P<0.0001) compared with all other antigens tested. The isolated O26, O111 and O128 showed a prevalence ranging between 4.3% and 12.8%, while no presence of O103 antigen was detected ().
Table 1 Serogroups of Escherichia coli isolated from diarrhoeic faecal samples from buffalo (Bubalus bubalis) calves.
PCR amplification of ehxA gene virulence marker gave the confirmation of haemolytic activity of all E. coli isolates (data not shown).
The results of in vitro resistance of the isolates from diarrhoeic faecal buffalo samples against 13 different antibacterial agents (all commonly used by clinicians and veterinarians for the treatment of infections with Gram-negative bacteria), are presented in . Haemolytic E. coli were highly resistant to penicillin G, lincomycin and neomycin. Amoxicillin/clavulonic acid and ampicillin were found to be moderately effective against the majority of isolates (46.8% of resistance). Thirty-two (34%) of the haemolytic E. coli strains were phenotypically resistant to tetracycline, whereas none of the isolated strains of E. coli was resistant to colistin sulfate.
Table 2 Resistance to thirteen antimicrobial agents among 94 haemolytic isolates of E. coli.
Most strains belonged to serotypes O157, O26, O111 and O128 showed resistance to three or more antimicrobial agents (). When the prevalence of the ampicillin resistance was compared with different serotypes it showed significantly higher occurrence in O157 than in O128 (62.5% vs 25%, P<0.05), whereas tetracycline resistance appeared to be very significantly higher in O26 than in O157 (100% vs 22.5%, P<0.005).
Table 3 Antibiotic resistance of different serotypes of E. coli isolates from diarrhoeic faecal samples in buffalo (Bubalus bubalis) calves.
Discussion
Enterohaemorrhagic Escherichia coli (EHEC) is a causative agent for haemorrhagic colitis and haemolytic uremic syndrome, both in humans (CitationFitzpatrick, 1999) and animals (CitationWang, 2006). The gastrointestinal tract of domestic ruminants is considered as the major reservoir for EHEC (CitationGriffin and Tauxe, 1991), which may be transmitted to humans via contaminated food and water. E.coli infection in calves continues to be a major problem worldwide. They cause substantial economic loss, both directly by mortality and poor growth after clinical disease, and indirectly from animal carriage leading to cases of human E. coli infection.
Several serotypes of EHEC, such as O157, O26 and O111 have been known to be associated with diarrhoea and dysentery in calves (CitationChina et al., 1997; CitationPaton and Paton, 1999; CitationSandhu and Gyles, 2002). Recently E. coli O157:H7 was also isolated from water buffaloes (Bubalus bubalis) in southern Italy (CitationGaliero et al., 2005).
In the present study the occurrence of EHEC O157 is resulted to be significantly higher with respect to other serotypes. This implies that the serotype O157 is certainly the major cause of disease in naturally infected buffalo calves of southern Italy. Moreover our results show that resistance levels of haemolytic E. coli isolates are high when measured against a panel of thirteen antibiotics (all commonly used by clinicians and veterinarians for the treatment of infections with Gram-negative bacteria), and that these strains carry overall high levels of resistance to penicillin G, lincomycin and neomycin (100% of resistance). A substantial number of haemolytic E. coli isolates were amoxicillin/clavulonic acid (46.8%) and ampicillin (46.8%) resistant. In contrast to penicillin G, the aminopenicillins, as ampicillin and amoxicillin, are active against several aerobic gram-negative enteric bacilli, such as E. coli, and this justifies our results. The aminopenicillins, the more recent parenteral penicillins, by the addition of an amino group to the side chain of penicillin G, show a better ability to penetrate through the pores in the outer membrane of gram-negative bacteria. However, over time, they may increased resistance to the β-lactamases produced by many gram-negative bacteria and surely patterns of drug resistance may be geographically based depending on the degree of local use.
Furthermore we observed, among 94 haemolytic isolates of E. coli, phenotypically resistance to tetracycline (34%). Tetracycline is not used to treat E. coli infections in humans, but resistance to tetracycline is still common in E. coli (CitationCalva et al., 1996; CitationDominguez et al., 2002), which suggests that resistance has been selected by a bystander effect on commensal E. coli, during treatment of other pathogens in humans or animals. The fate of antibiotic-resistant commensal strains, therefore, deserves more attention.
In our study E. coli O157 and E. coli O128 have shown a resistance of 25% to nalidixic acid (NA). NA belongs to a group of broad spectrum antibiotics called quinolones and the mechanism of NA resistance of bacteria is through the inhibition of DNA-gyrase, which is required for DNA synthesis. NA-resistant isolates have been reported in clinic (CitationZhang et al., 2008). It has already been suggested that Escherichia coli O157:H7 from cattle may become more frequently pathogenic to humans as a side effect of the increasing use of veterinary fluoroquinolones (CitationMaurer et al., 2009). However, the increasing incidence of antibiotic-resistant bacteria came forth because of too frequent and inappropriate use of antibiotics. CitationPiddock (2006) described that a growing number of bacterial species are simultaneously becoming resistant to more than one antibiotic. Furthermore, for certain strains fluctuations are possible in the percentage of resistance from year to year, suggesting that a continuous monitoring on the antibiotic resistance is necessary. A rapid diagnosis is important to prevent an inappropriate administration of antibiotic therapy, which may support antimicrobial resistance diffusion. The reason is that antibiotics kill off not only “bad” bacteria, but can also kill the “good” bacteria in the gut. This leads to “digestive imbalance” where there are too few remaining “good” bacteria in the digestive system. Of course, the treatment with “probiotics”, employed successfully in vivo, promotes immune responses against the E. coli cytotoxin and enhances the elimination of O157 from the intestinal tract (CitationOgawa et al., 2001). Probiotics play an important role in attenuating host epithelial responses to pathogenic E. coli infections and are also effective at reducing O157:H7 gut colonization in ruminants (CitationSherman et al., 2005), which serve as the environmental reservoir for enterohemorrhagic E. coli.
Conclusions
In conclusion, since buffalo calves may house in multidrug-resistant E. coli, they could be an important source of infection spread, with impact on public health. For this reason a careful selection of appropriate antibiotics is critically important and a continued surveillance of the emerging antimicrobial resistance in rural environment is recommended.
References
- BeutinL. 1991 The different hemolysins of Escherichia coli Med. Microbiol. Immun 180 167–182
- CallawayT.R. ElderR.O. KeenJ.E. AndersonR.C. NisbetD.J. 2003 Forage feeding to reduce preharvest Escherichia coli populations in cattle, a review J. Dairy Sci 86 852–860
- CalvaJ.J. Sifuentes-OsornioJ. CeronC. 1996 Antimicrobial resistance in fecal flora: longitudinal community-based surveillance of children from urban Mexico Antimicrob. Agents Chem 40 1699–1702
- CavalieriS.J. BohachG.A. SnyderI.S. 1984 Escherichia coli alpha-hemolysin: characteristics and probable role in pathogenicity Microbiol. Rev 48 326–343
- ChinaB. PirsonV. MainilJ. 1997 Typing of bovine attaching and effacing Escherichia coli by multiplex in vitro amplification of virulence-associated genes Appl. Environ. Microbiol 62 3462–3465
- DominguezE. ZarazagaM. SaenzY. BrinasL. TorresC. 2002 Mechanisms of antibiotic resistance in Escherichia coli isolates obtained from healthy children in Spain Microb. Drug Resist 8 321–327
- ElderR.O. KeenJ.E. SiragusaG.R. Barkocy-GallagherG.A. KoohmaraieM. LaegreidW.W. 2000 Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing Proc. Natl. Acad. Sci. USA 97 2999–3003
- FitzpatrickM. 1999 Haemolytic uraemic syndrome and E coli 0157 BMJ 318 684–685
- Feng P Monday SR. 2000 Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes Mol Cell Probes 14 333–7
- GalieroG. ConederaG. AlfanoD. CaprioliA. 2005 Isolation of verocytotoxin-producing Escherichia coli O157 from water buffaloes (Bubalus bubalis) in southern Italy Vet. Rec 156 382–383
- GoñiF.M. OstolazaH. 1998 E. coli α-hemolysin: a membrane-active protein toxin Braz. J. Med. Biol. Res 31 1019–1034
- GriffinP.M. TauxeR.V. 1991 The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E.coli, and the associated hemolytic uremic syndrome Epidemiol. Rev 13 60–98
- HarwoodV.J. WhitlockJ. WithingtonV. 2000 Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis: use in predicting the source of fecal contamination in subtropical waters Appl. Environ. Microbiol 66 3698–3704
- IslamMA MondolAS de BoerE BeumerRR ZwieteringMH TalukderKA HeuvelinkAE 2008 Prevalence and genetic characterization of shiga toxinproducing Escherichia coli isolates from slaughtered animals in Bangladesh Appl Environ Microbiol 74 5414–5421
- KrumpermanP.H. 1983 Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foods Appl. Environ. Microbiol 46 165–170
- LinX.M. LiH. WangC. PengX.X. 2008 Proteomic analysis of nalidixic acid resistance in Escherichia coli: identification and functional characterization of OM proteins J. Proteome Res 7 2399–2405
- MaurerC. MeunierD. MadecJ.Y. 2009 Shiga toxin Stx2 production is promoted by enrofloxacin in experimental in vitroselected mutants of Escherichia coli O157:H7 resistant to fluoroquinolones Foodborne Pathog. Dis 6 257–259
- OgawaM. ShimizuK. NomotoK. TanakaR. HamabataT. YamasakiS. TakedaT. TakedaY. 2001 Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid Int. J. Food Microbiol 68 135–140
- PatonA.W. PatonJ.C. 1999 Direct detection of Shiga toxigenic Escherichia coli strains belonging to serogroups O111, O157, and O113 by multiplex PCR J. Clin. Microbiol 37 3362–3365
- PiddockL.J. 2006 Multidrug-resistance efflux pumps - not just for resistance Nat. Rev. Microbiol 4 629–636
- RadostitsO.M. BloodD.C. GayC.C. 1994 Diseases Caused by Inorganic and Farm Chemicals, Veterinary Medicine 8th ed. Bailliere Tindall London, UK
- SandhuK.S. GylesC.L. 2002 Pathogenic Shiga toxin-producing Escherichia coli in the intestine of calves Can. J. Vet. Res 66 65–72
- ShermanP.M. Johnson-HenryK.C. YeungH.P. NgoP.S.C. GouletJ. TompkinsT.A. 2005 Probiotics reduce enterohemorrhagic Escherichia coli O157:H7- and enteropathogenic E. coli O127:H6-induced changes in polarized T84 epithelial cell monolayers by reducing bacterial adhesion and cytoskeletal rearrangements Infect Immun 73 5183–5188
- WangJ.Y. WangS.S. Pin-Zhang YinP.Z. 2006 Haemolytic–uraemic syndrome caused by a non-O157:H7 Escherichia coli strain in experimentally inoculated dogs J. Med. Microbiol 55 23–29
- WelchR.A. 2001 RTX toxin structure and function: a story of numerous anomalies and few analogies in toxin biology Curr. Top. Microbiol 257 85–111
- YoungJ. HollandI.B. 1999 ABC transporters: bacterial exportersrevisited five years on Biochim. Biophys. Acta 1461 177–200
- ZamanT. KhanA. AkhtarM.Z. 2006 Some of the risk factors of niliravi buffalo (Bubalus bubalis) neonatal calf mortality in Pakistan Pak. Vet. J 26 121–125
- ZhangD.F. JiangB. XiangZ.M. WangS.Y. 2008 Functional characterization of altered outer membrane proteins for tetracycline resistance in Escherichia coli Int. J. Antimicrob. Ag 32 315–319
- ZicarelliL. 2004 Buffalo milk: its properties, dairy yield and mozzarella production Vet. Res. Commun 28 127–135