1 Antimicrobial Susceptibility of Multidrug-Resistant

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1 AAC Accepts, published online ahead of print on December 00 Antimicrob. Agents Chemother. doi:./aac.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Antimicrobial Susceptibility of Multidrug-Resistant Campylobacter jejuni and C. coli strains: in vitro Activities of 0 Antimicrobial Agents 0 Mirva Lehtopolku,,* Ulla-Maija Nakari, Pirkko Kotilainen,, Pentti Huovinen, Anja Siitonen, and Antti J. Hakanen, Antimicrobial Resistance Unit and Gastrointestinal Infections Unit, National Institute for Health and Welfare, Turku and Helsinki, Finland; Department of Medicine, Turku University Hospital and University of Turku, Turku, Finland Running title: Susceptibility of Multiresistant Campylobacter spp. Keywords: Campylobacter spp., carbapenem, drug resistance, glycylcycline, macrolides, quinolones, tigecycline Word count of the abstract: 0 Word count of the text: *Corresponding author. Mailing address: Antimicrobial Resistance Unit, National Institute for Health and Welfare (THL), Kiinamyllynkatu, 00 Turku, Finland. Phone: Fax: mirva.lehtopolku@utu.fi

2 0 ABSTRACT There is a paucity of information regarding antimicrobial agents suitable to treat severe infections caused by multidrug-resistant Campylobacter spp. Our aim was to identify agents potentially effective towards multiresistant Campylobacter strains. The in vitro activities of 0 antimicrobial agents against Campylobacter spp. strains were analyzed by determining the MICs by the agar plate dilution method or the Etest. These strains were selected from 0 Campylobacter spp. isolates collected from Finnish patients between 00 and 00 and screened for macrolide susceptibility using the disk diffusion test. The strains consisted of strains with erythromycin inhibition zone diameters mm and strains with inhibition zone diameters > mm. Of the Campylobacter strains, were resistant to erythromycin by MIC determination (MIC µg/ml). Given that the resistant strains were identified among the collection of 0 isolates, the frequency of erythromycin resistance was.%. All erythromycin-resistant strains were multidrug-resistant, (.%) of them being resistant to ciprofloxacin (MIC µg/ml). The percentages of resistance to tetracycline and co-amoxiclav were.% and.%, respectively. All macrolide-resistant strains were susceptible to imipenem, meropenem and tigecycline. Ten (.%) multiresistant strains were identified as C. jejuni and (.%) as C. coli. These data demonstrate that the incidence of macrolide resistance was low, but the macrolide-resistant Campylobacter spp. strains were uniformly multidrug-resistant. In addition to the carbapenems, also tigecycline was in vitro highly effective against these multidrug-resistant Campylobacter strains. Its efficacy in the treatment of human campylobacteriosis should be evaluated in clinical trials.

3 0 INTRODUCTION Campylobacteriosis is usually a mild and self-limiting disease requiring no antimicrobial treatment (, ). Only rarely is it associated with extraintestinal manifestations, e.g. septicemia, skin and soft tissue infection, infective endocarditis or meningitis (). These infections usually require treatment with intravenous antimicrobial agents (). Infections in immunocompromised patients and in pregnant women as well as in very young and very old patients or those with persisting symptoms (> week) also require antimicrobial treatment (). Guillain-Barré syndrome and reactive arthritis are major postinfectious complications of campylobacteriosis (,, ). For many years, macrolides and fluoroquinolones have been the first and second choice alternatives in the antimicrobial treatment of campylobacter enteritis. Since the late 0s, however, the emergence of resistance against these antimicrobial groups has complicated the treatment of this disease (). For example in Thailand, the resistance against the fluoroquinolones has been up to 0% (, ). In Finland, fluoroquinone resistance increased between and 000 from 0% to 0% among all travellers isolates, and from % to % among those from Asia alone (). So far, the resistance against the macrolides has remained on a quite low level and stable. According to our previous study (), the frequency of macrolide resistance was % among Campylobacter jejuni strains collected from Finnish patients during However, there are a few studies showing that in some countries macrolide resistance may be slowly increasing (). The emergence of macrolide resistance is of serious concern since the macrolide-resistant strains are usually resistant to fluoroquinolones and other antimicrobial groups (, ). Very few alternatives exist with which to treat campylobacteriosis caused by these multidrug-resistant strains. Also, previous studies have shown that patients infected with a quinolone- or erythromycin-resistant Campylobacter strain have an increased risk of an adverse event, compared with patients infected with a quinolone- and erythromycin-susceptible Campylobacter strain (,, ). The aim of this study was to identify antimicrobial agents that, based on in vitro activities, could be useful in infections caused by multidrug-resistant Campylobacter spp. strains. To do so, we evaluated antimicrobial susceptibilities of 0 antimicrobial agents against Campylobacter strains collected in different laboratories in Finland between 00 and 00.

4 0 MATERIALS AND METHODS Study collection. Between May 00 and July 00, a total of 0 Campylobacter spp. strains were isolated from human stool specimens of Finnish patients in clinical microbiology laboratories in different parts of Finland and sent to the Gastrointestinal Infections Unit, National Institute for Health and Welfare, Helsinki, Finland. The cultivation of the stool samples and the preliminary identification of the isolates were carried out by standard microbiological methods. At the Gastrointestinal Infections Unit, hippurate hydrolysis test was used to differentiate C. jejuni and C. coli. All hippurate-negative isolates were confirmed by PCR as either C. jejuni or C. coli (0). All of the isolates were also screened for macrolide susceptibility using an erythromycin disk (content µg, BBL, Beckton Dickinson, Sparks, MD), which was added on the pure culture sheep blood plates. The screening was aimed at distinguishing the evidently macrolide-susceptible population from the population that contained the macrolide-resistant strains. An inhibition zone diameter > mm around the erythromycin disk was chosen to indicate macrolide susceptibility. This decision was supported e.g. by the French Society for Microbiology ( proposing the zone diameter mm as a breakpoint for susceptibility. Moreover, the recent results of Gaudreau et al. () have suggested that zone diameters of 0 mm and mm around the erythromycin disk in the disk diffusion testing could be considered as breakpoints of susceptibility and resistance, respectively, for the C. jejuni isolates. All isolates exhibiting inhibition zone diameters mm were sent to the Antimicrobial Resistance Unit, National Institute for Health and Welfare, Turku, Finland, for analyses of species and antimicrobial susceptibilities. In addition, a number of 0 randomly selected Campylobacter spp. isolates exhibiting zone diameters > mm were sent for further analyses to be included as macrolide-susceptible control strains. The isolates from patients travelling abroad within two weeks preceding their symptoms were classified as of foreign origin and all other isolates were classified as of domestic origin. Antimicrobial susceptibility testing. The MICs of the Campylobacter spp. isolates were determined by the agar plate dilution method as described previously () or by the Etest. The MIC

5 0 breakpoints used for resistance were those recommended by the Clinical and Laboratory Standards Institute (CLSI) for non-enterobacteriaceae to those antimicrobials for which such recommendations are available (). The antimicrobials evaluated using the agar plate dilution method were as follows: ampicillin, chloramphenicol, clarithromycin, clindamycin, co-amoxiclav, erythromycin, gentamicin, nalidixid acid, norfloxacin, ofloxacin, and tetracycline, Sigma (Steinheim, Germany); azithromycin, ciprofloxacin and levofloxacin, Fluka (Buchs, Switzerland); imipenem, MSD (UK); meropenem, AstraZeneca (Espoo, Finland); moxifloxacin, Bayer (Wuppertal, Germany); sitafloxacin, Daiichi Pharmaceutical (Tokyo, Japan); and telithromycin, Aventis Pharma (France). The reagent powder for each of these agents was obtained from its manufacturer. The MICs of tigecycline were determined by the Etest (Biodisk AB, Solna, Sweden) according to the manufacturer s instructions. In brief, after culturing the isolates by standard microbiological methods, inocula, prepared in NaCl at a density adjusted to a.0 McFarland turbidity standard, were delivered onto % sheep blood Mueller-Hinton agar plates. An Etest strip with a tigecycline concentration range from 0.0 to µg/ml was applied onto each plate. The plates were incubated at C for h in a microaerobic atmosphere (CampyPak, BBL). The MIC value was read at the point of the intersection between the growth zone edge and the Etest strip. C. jejuni DSM was used as a control in susceptibility testing and also as a growth control strain. In addition, Staphylococcus aureus ATCC and Escherichia coli ATCC were used as controls in susceptibility testing. The results were interpreted using the non-species related EUCAST ( breakpoints for susceptibility (MIC 0. µg/ml) and resistance (MIC >0. µg/ml). Multidrug resistance was defined as resistance to three or more antimicrobial groups. The antimicrobial groups were as follows: (i) quinolones; (ii) macrolides, clindamycin and telithromycin; (iii) tetracycline and tigecyclin; (iv) β-lactams; (v) gentamicin; and (vi) chloramphenicol.

6 Data analysis. The susceptibility data were analyzed using the WHONET. computer program (available from Comparisons of the susceptibility data between the erythromycin-resistant and susceptible Campylobacter spp. strains as well as between the C. jejuni and C. coli strains were performed by using the Fisher s exact test. P values less than 0.0 were considered significant. 0

7 0 RESULTS Strain collection. Of the 0 Campylobacter spp. isolates, all 0 isolates exhibiting inhibition zone diameters mm in erythromycin screening were included in this study. In addition, we randomly selected 0 isolates exhibiting erythromycin zone diameters > mm to serve as erythromycin-susceptible control strains. From those isolates, did not grow after freezing and storage. Thus, our strain collection consisted of Campylobacter spp. strains, of which exhibited erythromycin inhibition zone diameters mm and exhibited zone diameters > mm. Of these strains, were of foreign origin and were of domestic origin. The origin was unknown for strains. There were 0 (.%) strains identified as C. jejuni and (.%) strains identified as C. coli. Only four (.%) C. coli strains were domestic, while (0%) C. jejuni strains were domestic. Erythromycin susceptibility. Of the Campylobacter spp. strains with inhibition zone diameters mm in the erythromycin disk screening, were classified by the agar plate dilution method as erythromycin-resistant (MIC µg/ml) and as erythromycin-susceptible (MIC < µg/ml). All of the strains with inhibition zone diameters > mm were classified by the agar plate dilution method as erythromycin-susceptible. Thus, the final study collection consisted of erythromycin-susceptible and erythromycin-resistant Campylobacter spp. strains. Given that the erythromycin-resistant strains were identified among the initial study collection of 0 isolates, it can be estimated that the frequency of erythromycin resistance in Campylobacter spp. was.%. To confirm the adequacy of our screening approach, the results of the erythromycin disk screening were compared with the erythromycin MIC values. All strains classified as erythromycinresistant based on MIC determination had erythromycin inhibition zone diameters 0 mm, while all strains exhibiting zone diameters mm were classified by MIC determination as erythromycin-susceptible. Among the erythromycin-resistant strains, (.%) were foreign, one was domestic and for one strain, the origin was unknown. Ten (.%) resistant strains were identified as C. jejuni and

8 0 nine (.%) strains as C. coli. Erythromycin resistance was significantly more common among C. coli than among C. jejuni (P < 0.00). As compared to the C. jejuni strains, the C. coli strains were significantly more commonly resistant also to several other antimicrobial agents (Table ). Susceptibility of the macrolide-resistant and susceptible strains to other antimicrobials. The susceptibilities of the erythromycin-resistant and susceptible Campylobacter spp. strains to additional antimicrobial agents were different (Table ). Determined by the agar plate dilution method, the MIC 0 and MIC 0 of erythromycin for the erythromycin-resistant strains were > µg/ml. The MIC 0 and MIC 0 values were and > µg/ml for telithromycin, and and µg/ml for clindamycin, these being lower than those for azithromycin and clarithromycin. The respective MIC 0 and MIC 0 values were and > µg/ml for ciprofloxacin, and µg/ml for co-amoxiclav, and and µg/ml for ampicillin. Imipenem, meropenem, gentamicin, and sitafloxacin exhibited the lowest MIC 0 and MIC 0 values. Determined by the Etest, the MIC 0 and MIC 0 values of tigecycline were 0.00 and 0.0 µg/ml for the erythromycin-resistant strains, and 0.00 and 0.0 µg/ml for the erythromycin-susceptible strains, respectively. As compared to the erythromycin-susceptible Campylobacter spp. strains, the erythromycin-resistant strains were significantly more commonly resistant to several antimicrobial agents presented in Table. Of the erythromycin-resistant strains, (.%) were highly-resistant (MIC µg/ml), and also resistant to azithromycin (MIC µg/ml) and clindamycin (MIC µg/ml). Eighteen (.%) strains had telithromycin MICs µg/ml including strains with MICs > µg/ml. All erythromycin-resistant strains were multidrug-resistant, (.%) of them being resistant to ciprofloxacin (MIC µg/ml). Six strains had MICs of co-amoxiclav between and µg/ml, and seven strains had MICs of ampicillin between and > µg/ml. There were (.%) strains resistant to tetracycline (MIC µg/ml).

9 Of all Campylobacter spp. strains, (.0%) were ciprofloxacin-resistant; of them, (.%) were multidrug-resistant. The proportion of tetracycline resistance was 0.% (/) in the whole study population. 0

10 0 DISCUSSION In the present study, the macrolide-resistant Campylobacter spp. strains were uniformly multidrug-resistant. There is a paucity of information regarding antimicrobial agents that could be used in serious infections, if caused by these multidrug-resistant Campylobacter strains. Since extraintestinal manifestations are uncommon in campylobacteriosis, data on their antimicrobial therapy is based only on anecdotal case reports (,, ) and small retrospective case series (, ). Thus, the most appropriate protocols of antimicrobial treatment for bacteremic infections, whether caused by susceptible or resistant strains, have not been established. Successful outcomes have been reported especially with the carbapenems (,,, ). In previous studies, this antimicrobial group has also shown excellent in vitro activities against the Campylobacter spp. (, ). Correspondingly in the present study, all macrolide-resistant strains were susceptible to meropenem and imipenem. Among the older antimicrobial agents, gentamicin was effective, but it is not suitable for use e.g. in meningitis or during pregnancy. However, gentamicin may be effective in septicemia and other systemic infections in conjunction with carbapenem antibiotics. It is of note that also tigecycline was here highly active against all Campylobacter spp. strains including the macrolide- and multidrug-resistant strains. We consider this an important finding due to the limited number of other agents potentially effective in the treatment of multidrug-resistant campylobacteriosis. In campylobacter enteritis, antimicrobial treatment is required at least in immunocompromised patients and during pregnancy (). The treatment is also required, if the patient has severe or persisting symptoms, and in case of bloody diarrhea (). In enteric infections, administration of antimicrobials by peroral route is usually preferable to intravenous route. Based on the results of present study, no currently available peroral antimicrobial agent reliably covers the macrolideresistant Campylobacter spp. strains. All but one erythromycin-resistant strain were here resistant to ciprofloxacin, the majority of them being resistant also to telithromycin and tetracycline. Co-

11 0 amoxiclav may offer the best alternative, with only one third of our strains resistant. Among the fluoroquinolones, sitafloxacin exhibited low MIC values against the macrolide-resistant isolates, these results being consistent with our previous findings (). However, sitafloxacin is not presently on market being, thus, of no clinical consequence. In addition, all macrolide-resistant strains were susceptible to chloramphenicol, which is no more available in Finland for systemic use. The present study provides information on the frequency of macrolide resistance in campylobacters in Finland between 00 and 00. The finding that of the 0 Campylobacter spp. isolates initially included proved erythromycin-resistant by MIC determination indicates that the frequency of macrolide resistance was.%. We trust this material to be representative of the macrolide resistance situation in our country during that period, as these isolates comprised about one fifth of all campylobacters recovered from Finnish patients throughout the study. We also trust that our screening approach was appropriate to identify at least a great majority, if not all, of the macrolide-resistant strains present in the initial study population. The adequacy of the methodology was verified by comparing the results of the screening tests to the MIC determinations, with a finding that all isolates with inhibition zone diameters mm were determined by the MICs as susceptible. Among the strains undergoing species determination,.% were identified as C. coli, and yet, almost half (.%) of the macrolide-resistant strains were identified as C. coli. This is consistent with earlier studies showing that the C. coli strains are more often macrolide-resistant than C. jejuni (, ). Because of the increasing trend of resistance among the Campylobacter spp., routine susceptibility testing is an important tool to choose an appropriate antimicrobial treatment for the patient. Also species identification may have therapeutic implications. The in vitro activity of tigecycline against the Campylobacter spp. has been analyzed in one previous study (), in which it exhibited the lowest MIC values against the resistant Campylobacter strains. Clinically, the drug has shown excellent activity e.g. in the treatment of complicated skin and soft tissue infection (), which is known to be one manifestation of extraintestinal campylobacterioris (, ). Tigecycline circulates primarily as unchanged drug and

12 0 its major route of elimination is through feces, likely via biliary excretion (, ). On this basis, it seems reasonable to assume that tigecycline might be effective even in patients with gastroenteritis. In conclusion, the incidence of macrolide resistance among the Campylobacter spp. was low, but the macrolide-resistant strains were uniformly multidrug-resistant. C. coli was significantly more frequently macrolide-resistant as compared to C. jejuni. Thus, rapid species identification may have therapeutic implications. Based on our results, no perorally administered antimicrobial agent reliably covers the macrolide-resistant Campylobacter strains. Co-amoxiclav appears to offer the best treatment alternative, with only one third of our isolates resistant. In addition to imipenem and meropenem, tigecycline was in vitro highly effective also against the multidrug-resistant Campylobacter strains. The efficacy of tigecycline in the treatment of human campylobacteriosis should be evaluated in clinical trials.

13 0 ACKNOWLEDGMENTS We are indebted to Minna Lamppu, Erkki Nieminen and all the staff members of the laboratories of the study for expert technical assistance. This study was financially supported by grants from the Maud Kuistila Memorial Foundation, Suomen Kulttuurisäätiö foundation and the Turku University Central Hospital Research Fund. The postgraduate studies of Ulla-Maija Nakari were funded by the Finnish Graduate School on Applied Bioscience: Bioengineering, Food & Nutrition, Environment. This work was presented in part at the th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Helsinki, Finland, 00 (P0)

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18 TABLE. The comparison of the antimicrobial susceptibility between the Campylobacter jejuni and C. coli strains. Resistance C. jejuni (n=0) a C. coli (n=) Antimicrobial breakpoint b Number (%) of Number (%) of agent (µg/ml) resistant strains resistant strains P value Erythromycin (.%) (0%) <0.00 Azithromycin (%) (.%) <0.00 Clindamycin (%) (0%) <0.00 Nalidixic acid (.%) (.%) <0.00 Ciprofloxacin (.%) (.%) <0.00 Norfloxacin 0 (0.%) (.%) <0.00 Ofloxacin 0 (0.%) (.%) <0.00 Levofloxacin (.%) (.%) 0.00 Ampicillin 0 (.%) (.%) 0. Co-amoxiclav (.%) (.%) 0. Chloramphenicol (0.%) Imipenem 0 0 not applicable Meropenem 0 0 not applicable Gentamicin (0.%) Tetracycline (.%) (.%) 0.00 Tigecycline not applicable a For ciprofloxacin and tetracyclin data available for strains. For tigecycline data available for strains. For meropenem data available for strains. For imipenem data available for strains. b Resistance breakpoints available by the CLSI: ciprofloxacin µg/ml, norfloxacin µg/ml, ofloxacin µg/ml, levofloxacin µg/ml, imipenem µg/ml, meropenem µg/ml, tetracyclin µg/ml, gentamicin µg/ml and chloramphenicol µg/ml. Resistance breakpoints available by our previous work: erythromycin µg/ml, azithromycin µg/ml, clindamycin µg/ml, nalidixic acid µg/ml, ampisillin µg/ml, co-amoxiclav µg/ml. Resistance breakpoint available by EUCAST for tigecycline >0. µg/ml.

19 TABLE. The MICs (µg/ml) of 0 antimicrobial agents for Campylobacter spp. strains and the comparison of the erythromycin-resistant and -susceptible Campylobacter spp. strains. Erythromycin-resistant Erythromycin-susceptible strains (MIC µg/ml) strains (MIC < µg/ml) n= n= a Antimicrobial % of % of agent MIC0 MIC0 resistance b MIC0 MIC0 resistance b P value Erythromycin > > 0 0 Azithromycin > > <0.00 Clarithromycin > > Telithromycin > Clindamycin. 0.. <0.00 Nalidixic acid. 0. <0.00 Ciprofloxacin > <0.00 Norfloxacin >. >. <0.00 Ofloxacin <0.00 Levofloxacin >. 0.. <0.00 Moxifloxacin 0. Sitafloxacin Ampicillin Co-amoxiclav Chloramphenicol Imipenem not applicable Meropenem < < not applicable Gentamicin Tetracycline >. 0.. <0.00 Tigecycline not applicable a For clarithromycin, ciprofloxacin and tetracyclin data available for strains. For tigecycline data available for strains. For meropenem data available for strains. For imipenem data available for strains. b Resistance breakpoints available by the CLSI: ciprofloxacin µg/ml, norfloxacin µg/ml, ofloxacin µg/ml, levofloxacin µg/ml, imipenem µg/ml, meropenem µg/ml, tetracycline µg/ml, gentamicin µg/ml, and chloramphenicol µg/ml. Resistance breakpoints available by our previous work: erythromycin µg/ml, azithromycin µg/ml, clindamycin µg/ml, nalidixic acid µg/ml, ampicillin µg/ml, co-amoxiclav µg/ml. Resistance breakpoint available by EUCAST for tigecycline >0. µg/ml.