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Veterinary Microbiology 134 (2009) 305–310 j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v e t m i c In vitro antimicrobial activity against 10 North American and EuropeanLawsonia intracellularis isolates Suphot Wattanaphansak Randall S. Singer Connie J. Gebhart a Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, 205 Veterinary Science Building,1971 Commonwealth Ave, St. Paul, MN 55108, United Statesb Instituto de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile The objective of this study was to determine the in vitro minimum inhibitory concentration (MIC) of antimicrobials against 10 isolates of Lawsonia intracellularis, the etiological agent of proliferative enteropathy (PE). Antimicrobials tested included carbadox, chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin. The MIC ofeach antimicrobial against L. intracellularis was determined using a tissue culture system and was identified as the lowest concentration that inhibited 99% of L. intracellularis growth, as compared to the antimicrobial-free control. Each antimicrobial concentration was evaluated for both intracellular and extracellular activity against L. intracellularis, an obligately intracellular bacterium. When tested for intracellular activity, carbadox,tiamulin, and valnemulin were the most active antimicrobials with MICs of 0.5 mg/ml.
Tylosin (MICs ranging from 0.25 to 32 mg/ml) and chlortetracycline (MICs ranging from0.125 to 64 mg/ml) showed intermediate activities and lincomycin (MICs ranging from 8 to>128 mg/ml) showed the least activity. When tested for extracellular activity, valnemulin(MICs ranging from 0.125 to 4 mg/ml) was the most active against most L. intracellularisisolates. Chlortetracycline (MICs ranging from 16 to 64 mg/ml), tylosin (MICs ranging from1 to >128 mg/ml), and tiamulin (MICs ranging from 1 to 32 mg/ml) showed intermediateactivities. Lincomycin (MICs ranging from 32 to >128 mg/ml) showed the least activity.
Our in vitro results showed that each L. intracellularis isolate had a different antimicrobialsensitivity pattern and these data can be utilized as an in vitro guideline for the furtherantimicrobial evaluation of field L. intracellularis isolates.
ß 2008 Elsevier B.V. All rights reserved.
ities against L. intracellularis infection, the selection of anappropriate antimicrobial is difficult. The paucity of Proliferative enteropathy (PE) is one of the most information is due to the fact that standard antimicrobial prevalent enteric bacterial diseases in grower and finisher assays are not applicable to evaluate the antimicrobial pigs. The etiological agent of this disease is an obligate activities of most intracellular organisms since these intracellular, Gram-negative bacterium named Lawsonia bacteria only propagate themselves inside the host cell.
intracellularis. The treatment of a PE outbreak on a pig farm Therefore, most in vitro studies of antimicrobial activities often involves antimicrobial therapy. However, since little against obligate intracellular bacteria are undertaken information is available on in vitro antimicrobial sensitiv- through a complicated cell culture system Furthermore, few strains of L. intracellularis * Corresponding author. Tel.: +1 612 624 3444; fax: +1 612 625 5203.
have been successfully isolated and maintained in vitro. Of these, only three European isolates have been tested in 0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
S. Wattanaphansak et al. / Veterinary Microbiology 134 (2009) 305–310 vitro for antimicrobial susceptibilities using a tissue inhibitory concentration (MIC) of each antimicrobial against L. intracellularis. Briefly, the frozen bacteria were thawed and grown in cell culture for at least 3 continuous It has been a decade since these antimicrobial activity passages to achieve 100% infection of the McCoy cell studies of L. intracellularis have been reported, and no monolayer. All L. intracellularis isolates were tested twice further in vitro studies have been published to update or and each replicate was prepared independently. Each expand upon the limited data existing for the antibiotic strain of L. intracellularis was harvested from the mono- sensitivity of L. intracellularis. Therefore, the objective of this study was to determine the in vitro antimicrobial sensitivities of 10 isolates of L. intracellularis obtained from medium, and 100 ml of this bacterial suspension was both North America and Europe against six antimicrobial inoculated onto one-day-old McCoy cells in 96-well tissue compounds that have been used for the treatment and culture plates (Nalge Nunc International, New York, United In this study, the MICs were expressed for both intracellular and extracellular activities. Intracellular MIC testing was conducted in order to measure the effect 2.1. Source and preparation of antimicrobials of antimicrobials on L. intracellularis after the bacteria hadinfected the enterocytes. For intracellular testing, a The following antimicrobial agents were purchased as pure chemicals: carbadox, chlortetracycline hydrochlor- used with minor changes to the cell line and the bacterial ide, lincomycin hydrochloride and tylosin tartrate concentration. Briefly, 100 ml of bacterial suspension (Sigma–Aldrich, Missouri, United States). Tiamulin hydro- containing approximately 106–107 L. intracellularis organ- gen fumarate and valnemulin hydrochloride were sup- isms/ml, a quantification method described by plied as pure chemicals from Novartis Animal Health (Basel, Switzerland). The stock solutions of all antimicro- cells 24 h before exposure to the antimicrobials. This bial compounds were prepared to a final concentration of permitted sufficient time for L. intracellularis to penetrate 2560 mg/ml. Each antimicrobial solution was sterilized by the host cells prior to antimicrobial treatment. After filtration using 0.2 mm-pore size filters. The stock solution incubation, the bacterial suspension was removed and of carbadox was first dissolved with 0.1N NaOH and then replaced with 100 ml of fresh culture medium containing was diluted in sterile distilled de-ionized water. The stock various concentrations of antimicrobials at 1, 2, and 3 days solutions of the other compounds were dissolved directly post inoculation, followed by fresh culture media on day 4 in sterile distilled de-ionized water and all were kept at with no antimicrobial as previously described ( À20 8C until use. Once the antimicrobials were thawed, they were used and kept refrigerated for up to 3 days. A The extracellular MIC testing was designed to mimic series of two-fold dilutions were made from the stock the effect of antimicrobial on L. intracellularis when the solutions, and these were then diluted 1:10 with culture bacterium is free in the gut lumen before infecting the medium to resultant final concentrations of 0.125, 0.25, intestinal cells. For extracellular testing, we followed a 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/ml. Each concentra- tion of antimicrobial was tested in triplicate.
minor changes. Briefly, a series of two-fold dilutions ofstock antimicrobials were added to culture medium containing L. intracellularis. The suspension was incubatedat 37 8C in 8.0% oxygen, 8.8% carbon dioxide, and 83.2% A total of 10 L. intracellularis field strains collected nitrogen atmosphere for 2 h without mixing, allowing between 1983 and 2006 from infected pigs from North direct exposure of the bacteria to the antimicrobials. After America and Europe were tested. Six L. intracellularis incubation, 100 ml of the bacterial suspension was strains used were from North America: PHE/MN1-00, transferred to infect one-day-old McCoy cells. The medium VPB4, KKumn04, NWumn05, DBumn06 and 47216-06.
was removed after 24 h incubation and replaced with Three L. intracellularis strains used were from the United 100 ml of new culture medium without any antimicrobials Kingdom: LR189/5/83, 963/93 and 916/91; and one L.
for 3 consecutive days. Following the media removal each intracellularis strain used was from Denmark: D15540. All day, the infected plates were exposed to hydrogen gas and strains were stored at À72 8C until use.
the plates were then kept at 37 8C for 5 days in an incubator All strains of L. intracellularis were grown in murine with 8.0% oxygen, 8.8% carbon dioxide and 83.2% nitrogen fibroblast-like McCoy cells (CRL 1696, American Type Culture Collection, Virginia, United States) and were After 5 days incubation, supernatant from the infected maintained in a cell culture system as described previously plates was removed and the cell culture monolayer was fixed with 100 ml of cold 50% acetone and 50% methanolfor 1 min. To assess the inhibitory effect of each antimicrobial on L. intracellularis proliferation, the infectedplates were stained using a modified immunoperoxidase A tissue culture system was modified from a previous with primary antibody from a rabbit hyperimmunized Table 1Summary of intracellular and extracellular MIC endpoints for six antimicrobial agents against 10 L. intracellularis isolates, six obtained from North America and four from Europe, measured by using tissue culturesystem with 5 days of incubation Each strain of L. intracellularis was tested twice and the bacteria were prepared independently for each replicate. USA: the United States of America; Den: Denmark; UK: United Kingdom.
a The intracellular MIC.
b Extracellular MIC was defined as the lowest antimicrobial concentration that inhibited 99% of L. intracellularis proliferation, compared to antimicrobial-free control.
S. Wattanaphansak et al. / Veterinary Microbiology 134 (2009) 305–310 with L. intracellularis antigen. The L. intracellularis pro- included chlortetracycline with an MIC range of 0.25– liferation was evaluated by counting the number of heavily 16 mg/ml, lincomycin with an MIC range of 8–64 mg/ml, and tylosin with an MIC range of 0.5–2 mg/ml. The using an inverted microscope (Olympus, Tokyo, Japan) extracellular activity results showed that valnemulin with a 20Â objective lens. Cells were considered to be had the highest activity against L. intracellularis; all isolates HIC if the number of intracellular L. intracellularis had had MICs of 0.25 mg/ml. The antimicrobials that had proliferated to greater than 30 bacteria per cell. The moderate activity were carbadox with an MIC range of 1– number of HICs in each well was then expressed as a 4 mg/ml, chlortetracycline with an MIC range of 16–64 mg/ percentage compared to the average HIC of the control ml, tiamulin with an MIC range of 1–4 mg/ml, and tylosin wells. The intracellular and extracellular MIC endpoints for with an MIC range of 2–16 mg/ml. The antimicrobial that each antimicrobial in this study were defined as the lowest showed the least activity against L. intracellularis was antimicrobial concentration that inhibited 99% of L.
lincomycin with MICs of 32–>128 mg/ml.
intracellularis proliferation in the McCoy cells after 5 daysof incubation These inhibitions were indicated by the percentage of HIC of each anti-microbial concentration as compared to the antimicrobial- Although methodologies for determining antimicrobial sensitivity of intracellular organisms have been developed,the methods and interpretations of their results have not been standardized or uniformly accepted. In this study, thepracticality of assessing the in vitro antimicrobial activity The MIC endpoints of each antimicrobial were deter- against L. intracellularis was demonstrated using a tissue mined using the median value from a set of triplicate wells.
culture system, which was modified from a previous study MIC assays were performed in duplicate from independent bacterial preparations, and the duplicate MIC endpoints The MIC results in the earlier studies testing various were expressed for each antimicrobial for each isolate.
antimicrobials against L. intracellularis strains used only When the percentage of HIC of L. intracellularis in the three strains from the United Kingdom. This was due to the antimicrobial-free control was less than 50%, the MIC tests limited number of strains available and the difficulty of the for that L. intracellularis strain were repeated.
laboratory techniques for maintaining and culturing L.
intracellularis ( One European isolate (916/91 or NCTC 12657) thatwas tested in the previous study ( The intracellular and extracellular MIC values for was also retested for antimicrobial activity in this study.
antimicrobials used against the 10 L. intracellularis iso- Unfortunately, individual MIC endpoints were not shown lates in the present study are shown in The for individual isolates in the previous study. Therefore, a concentrations of L. intracellularis inocula were between direct comparison of the MIC endpoints of the current 1.2 Â 106 and 3.4 Â 107 L. intracellularis organisms/ml, study to the earlier study is difficult to perform. With the and each isolate had a range of less than one log between exception of tylosin and tiamulin (intracellular MIC), the the two replicates. For L. intracellularis isolates from North MIC results of the tested antimicrobials from the earlier America (n = 6), the intracellular activity results showed that carbadox, tiamulin, and valnemulin displayed the of a two-fold dilution compared to our results. In those highest activity with MICs from 0.5 mg/ml. Chlortetra- studies and this current study, the extracellular and cycline and tylosin showed moderate activity against L.
intracellular MICs for L. intracellularis were determined intracellularis with MIC ranges from 0.125 to 64 mg/ml and in an effort to mimic L. intracellularis infections in which 0.25 to 32 mg/ml, respectively. Lincomycin showed the the bacteria would be exposed to antimicrobials before and lowest activity against most L. intracellularis isolates with after invasion into intestinal cells. The results of the an MIC range from 16 to >128 mg/ml. The extracellular intracellular and extracellular MICs obtained from two activity results showed that only valnemulin had high different batches of each L. intracellularis strain demon- activity against L. intracellularis with MICs ranging from strated that the assay was reproducible. The median MIC 0.125 to 4 mg/ml. Antibiotics with moderate activities from the two replicates was always within a two-fold against L. intracellularis included carbadox with an MIC dilution, determined by assessing whether any duplicate range from 4 to 32 mg/ml, chlortetracycline with an MIC was more than a two-fold dilution away from the log 2 range from 32 to 64 mg/ml, tiamulin with an MIC range from 1 to 32 mg/ml, and tylosin with an MIC range from 1 Currently there are no antimicrobial MIC breakpoints to >128 mg/ml. All L. intracellularis isolates from North for intracellular organisms using a tissue culture system; America had the lowest extracellular activity to lincomy- therefore, interpretations of the sensitivity data are complicated. For various reasons, these data should only MIC results for the European isolates (n = 4) were be used as a guide to determine which antimicrobials similar to the North American isolates in that, for the could be effective in treating L. intracellularis infections in intracellular MICs, carbadox, tiamulin, and valnemulin vivo. First, it is unknown how the in vitro assay compares had the highest activity against L. intracellularis with MICs to in vivo L. intracellularis infections. Second, it is unknown of 0.125 mg/ml. Antimicrobials with moderate activities what concentration of each antimicrobial can be attained S. Wattanaphansak et al. / Veterinary Microbiology 134 (2009) 305–310 at the site of L. intracellularis infection. This concentration intracellular bacteria maintained under a tissue culture is critical in determining whether a specific isolate is system for an extended period of time have the potential susceptible or resistant. Finally, it is unknown whether to develop genomic mutations that could impact their the antimicrobial would have the greatest effect on L.
intracellularis while the bacteria are extracellular or Therefore, these results represent the most comprehen- intracellular. The data can be used, however, to pre- sive L. intracellularis MIC study to date. Further in vivo dict the utility of the antibiotic. If there is no diversity in studies should be conducted to confirm the antimicrobial activity levels and yet the MIC is very low, such as the observed variation in carbadox intracellular MICvalues, then this antibiotic might function very well. If there is a large range of MICs, such as was observed withchlortetracycline intracellular MIC levels, then some L.
Our in vitro data greatly expand the antimicrobial MIC intracellularis isolates may have less sensitivity to that information available for L. intracellularis. Based on our in vitro results, it is clear that L. intracellularis isolates have a According to our results, extracellular MICs were diversity of antimicrobial sensitivity patterns. Because it is higher than the intracellular MICs for all antimicrobials, unlikely that L. intracellularis will be isolated and tested while the previous report showed that both of them were during a PE outbreak, our data can serve as an in vitro guideline for the range of antimicrobial responses of L.
difference may be the effect of contact time with the intracellularis. Based on this guideline, we predict carba- antimicrobials. The extracellular activity assay was dox, tiamulin and valnemulin to be the most active designed to have less contact time than the intracellular antimicrobials, chlortetracycline and tylosin to be inter- activity assay (i.e., one day compared to three consecutive mediately active, and lincomycin to be the least active days). These differing contact times were performed due antimicrobial against L. intracellularis.
to the fact that L. intracellularis can penetrate the cellswithin 24 h (). By definition, an extracellular MIC is the effect of theantimicrobial on L. intracellularis before the bacteria enter This study was supported in part by a grant from the cells and, therefore, only one day of exposure to the Novartis Animal Health Inc. The authors would like to antimicrobial can be used to determine the extracellular thank Dr. Dean Dau for critical reading and comments on MIC. A fraction of the L. intracellularis could have survived the manuscript, Dana Beckler and Keith Kinsley for the single extracellular treatment and subsequently providing two L. intracellularis isolates and Benjawan returned to active phase upon the disappearance of the Wijarn, Molly Freese, and Beth Thompson for excellent antimicrobials. This could suggest that a single-dose antimicrobial treatment is insufficient to inhibit thegrowth of L. intracellularis. A potential explanation for the lower intracellular MIC is the accumulation ofantimicrobial inside the cells, making the intracellular Carryn, S., Bambeke, F.V., Mingeot-Leclercq, M.P., Tulkens, P.M., 2002.
Comparative intracellular (THP-1 Macrophage) and extracellular concentration much higher than the extracellular con- activities of b-lactams, azithromycin, gentamicin, and fluoroquino- centration. The increased intracellular concentrations lones against Listeria monocytogenese at clinically relevant concen- could enhance the chemotherapeutic activity of anti- trations. Antimicrob. Agents Chemother. 46, 2095–2103.
Carryn, S., Chanteux, H., Seral, C., Mingeot-Leclercq, M.P., Bambeke, F.V., microbials against intracellular bacteria. In contrast to the Tulkens, P.M., 2003. Intracellular pharmacodynamics of antibiotics.
macrolides, b-lactams, aminoglycosides and lincomycin Infect. Dis. Clin. North Am. 17, 615–634.
Drancourt, M., Raoult, D., 1993. Methodology of antibiotics testing for intracellular pathogens. In: Raoult, D. (Ed.), Antimicrobial Agents and ). Although this study did not determine the Intracellular Pathogens. CRC Press, Boca Raton, pp. 71–85.
antimicrobial concentration inside the cells, the intracel- Gnarpe, J., Eriksson, K., Gnarpe, H., 1996. In vitro activities of azithromycin lular MIC results seemed to show that most of the and doxycycline against 15 isolates of Chlamydia pneumoniae. Anti-microb. Agents Chemother. 40, 1843–1845.
antimicrobials tested can penetrate and bind to inter- Guedes, R.M.C., Gebhart, C.J., Deen, J., Winkelman, N.L., 2002. Validation of nalized L. intracellularis to exert their chemotherapeutic an immunoperoxidase monolayer assay as a serologic test for porcine proliferative enteropathy. J. Vet. Diag. Invest. 14, 528–530.
Guedes, R.M.C., Gebhart, C.J., 2003. Onset and duration of fecal shedding, Interestingly, one observation from this study was that cell-mediated and humoral immune responses in pigs after challenge the North American L. intracellularis isolates tended to with a pathogenic isolate or attenuated vaccine strain of Lawsonia have higher intracellular and extracellular MICs than the intracellularis. Vet. Microbiol. 91, 135–145.
European L. intracellularis isolates. Additional L. intracel- Horowitz, H.W., Hsieh, T.C., Aguero-rosenfeld, M.E., Kalantarpour, F., Chowdhury, I., Wormser, G.P., Wu, J.M., 2001. Antimicrobial suscept- lularis isolates from North America and Europe will need ibility of Ehrlichia phagocytophila. Antimicrob. Agents Chemother. 45, to be tested before drawing any conclusions about geographic differences and antimicrobial sensitivities Ives, T.J., Marston, E.L., Regnery, R.L., Butts, J.D., Majerus, T.C., 2000. In vitro susceptibilities of Rickettsia and Bartonella spp. to 14-hydroxy-clar- changing over time. This study evaluated most of the L.
ithromycin as determined by immunofluorescent antibody analysis intracellularis isolates currently available globally and of infected Vero cell monolayers. J. Antimicrob. Chemother. 45, 305– obtained from 2 different continents, including new Lawson, G.H.K., McOrist, S., Jasni, S., Mackie, R.A., 1993. Intracellular clinical (low passage) and well-established (high passage) bacteria of porcine proliferative enteropathy: cultivation and main- L. intracellularis isolates. This was important because tenance in vitro. J. Clin. Microbiol. 31, 1136–1142.
S. Wattanaphansak et al. / Veterinary Microbiology 134 (2009) 305–310 McOrist, S., Gebhart, C.J., 1995. In vitro testing of antimicrobial agents for McOrist, S., Mackie, R.A., Lawson, G.H.K., 1995b. Antimicrobial suscept- proliferative enteropathy (ileitis). Swine Health Prod. 3, 146–149.
ibility of Ileal symbiont intracellularis isolated from pigs with porcine McOrist, S., Jasni, S., Mackie, R.A., Berschneider, H.M., Rowland, A.L., proliferative enteropathy. J. Clin. Microbiol. 33, 1314–1317.
Lawson, G.H.K., 1995a. Entry of the bacterium Ileal symbiont intra- Wattanaphansak, S., Gebhart, C.J., Olin, M., Deen, J., 2005. Measurement cellularis into cultured enterocytes and its subsequent release. Res.
of the viability of Lawsonia intracellularis. Can. J. Vet. Res. 69,


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