Prevalence and antimicrobial susceptibility of Arcobacter species in human stool samples: the prospective German Arcobacter prevalence study Arcopath

Background Arcobacter species, particularly A. butzleri, but also A. cryaerophilus constitute emerging pathogens causing gastroenteritis in humans. However, isolation of Arcobacter may often fail during routine diagnostic procedures due to the lack of standard protocols. Furthermore, defined breakpoints for the interpretation of antimicrobial susceptibilities of Arcobacter are missing. Hence, reliable epidemiological data of human Arcobacter infections are scarce and lacking for Germany. We therefore performed a 13-month prospective Arcobacter prevalence study in German patients. Results A total of 4646 human stool samples was included and Arcobacter spp. were detected in 0.85% of specimens from 3884 outpatients and in 0.40% of samples from 752 hospitalized patients. Overall, A. butzleri was the most prevalent species (n = 24; 67%), followed by A. cryaerophilus (n = 10; 28%) and A. lanthieri (n = 2; 6%). Whereas A. butzleri, A. cryaerophilus and A. lanthieri were identified in outpatients, only A. butzleri could be isolated from samples of hospitalized patients. Antimicrobial susceptibility testing of Arcobacter isolates revealed high susceptibilities to ciprofloxacin, whereas bimodal distributions of MICs were observed for azithromycin and ampicillin. Conclusions In summary, Arcobacter could be isolated in 0.85% of German outpatients and ciprofloxacin rather than other antibiotics might be appropriate for Further epidemiological studies needed, however, to provide a sufficient risk assessment of in humans.

patient populations, geographical aspects, examined sample sizes, and in particular, to the different microbiological methods applied. The impact of the detection method has been demonstrated in several studies (25-28). The authors each compared different cultural isolation strategies with varying incubation and medium conditions revealing differences in Arcobacter isolation frequency ranging from 7% to 36%. Notably, our study revealed a higher Arcobacter prevalence in an analyzed subgroup by using Arcobacter-specific enrichment (0.97%) than determined by nonspecific methods used in the three routine laboratories (0.49%). Future studies should address whether patients with Arcobacter spp. at low quantities that can only be detected by applying specific enrichment methods differ clinically from those patients in whom the pathogen is easily detected within the routine culture based procedures.
Furthermore, we determined a higher Arcobacter prevalence in stool samples of outpatients than of hospitalized patients (i.e., 0.85% (33/3884) and 0.40% (3/752), respectively). Thus, in most patients, Arcobacter spp. most likely do not cause serious infections requiring hospitalization. Likewise, in a previous German study, patients who were hospitalized for severe gastroenteritis (n = 104) were found to be positive mainly for norovirus or Campylobacter spp.; in contrast, no Arcobacter was isolated by using routine diagnostics (29).
Among the 36 Arcobacter isolates obtained in our study, A. butzleri was the most prevalent species (n = 24) followed by A. cryaerophilus (n = 10), which is in line with other studies (10, 21, 24). In addition, to best of our knowledge, this is the first report of A. lanthieri isolation from human specimens (n = 2) which might point towards its role as gastrointestinal pathogen. However, the applied selective enrichment media as well as the multiplex PCR are validated for the detection of the three species A. butzleri, A. cryaerophilus and A. skirrowii only, and could therefore bias the result according to species diversity (16,18).
Overall, in the analyzed subgroup Arcobacter spp. were the second most frequently isolated pathogens (0.97%) after Campylobacter spp. (4.39%), followed by azithromycin, and whole genome sequencing analysis revealed an amino acid substitution in ribosomal protein L22 (leading to azithromycin resistance), but no mutations in the 23S rRNA gene, which explains the susceptibility to erythromycin (38). Further analyses are needed to determine the genomic background being responsible for the divergent MIC distributions observed by us for Arcobacter spp.
As mentioned before, 86% of the investigated Arcobacter isolates showed low MICs for ciprofloxacin ranging from 0.032-0.50 µg/ml, which is comparable to other studies (36). In contrast, clinical Campylobacter isolates displayed high resistance rates (MICs ≥ 4 µg/ml) ranging from 45 to 71.4% (39,40). Notably, we found elevated MICs for ciprofloxacin predominantly in A. cryaerophilus strains similar to a Belgian study (34). Thus, ciprofloxacin might be the drug of choice, if antibiotic treatment of A. butzleri-infection is required.
In accordance with our data, only low resistance rates from 0-4% of Arcobacter spp.
to gentamicin have been reported before (36). Similarly, susceptibility to tetracycline might be common, although one recent study from retail food in Portugal demonstrated high resistance (95%) in A. butzleri (5, 41). Furthermore, 42% of our A. butzleri isolates displayed high MICs for ampicillin (24-64 µg/ml), which is similar to previous studies where 50 to 100% isolates with high ampicillin MICs have been shown (20,22,31,34).

Conclusions
In summary, Arcobacter spp. was not rare in our study and could be isolated more often from outpatients than from hospitalized patients. Furthermore, A. lanthieri was identified in fecal samples from human patients for the first time. Results from antimicrobial susceptibility testing indicate that Arcobacter spp. might be more susceptible to fluoroquinolones than to macrolides, particularly azithromycin. Future studies should provide reliable risk assessments of Arcobacter infections in humans.    Figure 1 Prevalence of bacterial pathogens in a subgroup of the study population (n=2257). Black bar Figure 2 MIC distribution of Arcobacter spp. originating from human stool samples for six antimicrobia