Comparison of Arcobacter Isolation Methods, and Diversity of Arcobacter spp. in Cheshire, United Kingdom

Abstract

The aims of this study were, firstly, to compare five published methods for the isolation of Arcobacter spp. from animal feces in order to determine the most sensitive and specific method. Second, we analyzed the resulting isolates by multilocus sequence typing (MLST) in order to investigate the diversity of the isolates recovered. Third, we investigated the ability to recover Arcobacter spp. from frozen fecal samples. Seventy-seven fecal samples from cattle, sheep, and badgers were subjected to five isolation methods, based on published methods for the isolation of Arcobacter and Campylobacter spp. Thirty-nine Arcobacter butzleri isolates were analyzed using a multilocus sequence typing scheme. The survival of Arcobacter spp. in frozen samples was investigated by freezing the fecal samples at ؊80°C for 7 days and then applying the same five isolation methods. The most sensitive and specific method used an Arcobacter-specific broth in conjunction with modified charcoal cefoperazone deoxycholate agar (mCCDA) with added antibiotics. Freezing of fecal samples led to a reduction in the recovery of Arcobacter spp. by approximately 50%. The 39 allelic profiles obtained by MLST could be divided into 11 sequence types (STs). We have identified the most sensitive and specific method for the isolation of Arcobacter spp. from animal feces and demonstrated that the freezing of fecal samples prior to isolation reduces arcobacter recovery. MLST analysis of the isolates revealed a high level of diversity.

Arcobacter

“…Further work is required to better understand the relationship between these putative virulence markers and human clinical outcomes, though some tantalizing evidence suggests that Arcobacter has the potential to induce tight junction dysfunction (Bücker et al, 2009;Karadas et al, 2016), which may lead to diarrhea . This is similar to the effect observed with enteroaggregative E. coli (EAEC) infection (Strauman et al, 2010 (Wesley et al, 2000;van Driessche et al, 2005;Merga et al, 2011;Shah et al, 2013;Grove-White et al, 2014). Isolation rates vary between studies, but young cattle/calves were consistently shown to have higher Arcobacter carriage rates.…”

“…It is therefore likely that temperature acts as the most important selective pressure in Arcobacter vs. Campylobacter culture. On this basis, recovery of Arcobacter from traditional Campylobacter media (Bolton and Preston broths) is not surprising and has been reported (Diergaardt et al, 2004;Merga et al, 2011;Figueras et al, 2014;Banting et al, 2016). Arcobacter is routinely cultured at 20 to 37°C under aerobic or microaerophilic conditions, with the most frequent temperature being 30°C, while Campylobacter spp.…”

“…Since that time various methods have been developed to isolate Arcobacter. All are relatively similar to Campylobacter isolation methods, with the exception of temperature (Merga et al, 2011). Media bases such as Mueller-Hinton broth, Brucella broth and Bolton broth have been used.…”

“…Antibiotics used in Arcobacter broths are often overlapping with those used in Campylobacter isolation. Freezing of fecal specimens is not recommended as it may produce a reduction of ~50% in the recovery of Arcobacter species (Merga et al, 2011) Sequencing of the A. butzleri genome revealed the presence of a large number of antibiotic resistance genes (Miller et al, 2007). In fact, A. butzleri RM4018 displayed resistance to 42 of 65 antibiotics tested, more than Campylobacter jejuni, C. coli or C. lari.…”

“…Historically, arcobacters were isolated from human feces as noncampylobacters via Campylobacter isolation protocols. However, the majority of Campylobacter isolation methods utilize a 42°C incubation temperature that is unlikely to be optimal for the majority of Arcobacter species/strains (Merga et al, 2011). Several large studies from the last decade have begun to shed light on Arcobacter in humans, but methods are not consistent between studies so caution must be used when interpreting results (Table 1).…”