Arcobacter spp. prevalence in various types of samples
Research progress on prevalence and pathogenicity has led A. butzleri and A. cryaerophilus to be ranked as serious hazards to human health by the International Commission on Microbiological Specifications for Foods (ICMSF, 2002) [32]. However, due to missing standardized isolation and identification methods, Arcobacter spp. prevalence data in various countries remain undetermined. This is the first study analyzing the prevalence of Arcobacter spp. in different sources in Lithuania by using Arcobacter-specific detection methods combined with molecular identification and verification.
Within the present study, Arcobacter spp. were isolated from 20 out of a total of 1200 (1.7%) human stool samples tested. There are no epidemiological data provided by other Baltic countries that could be used for comparative analysis. However, this finding is consistent with studies from Belgium and Portugal, where Arcobacter spp. were detected in 1.3% (89/6774) and 1.7% (5/298) of clinical stool samples respectively [14, 33]. Other studies, conducted in Turkey, Germany, New Zealand, India, Chile and Belgium, reported different prevalence rates ranging from 0.3 to 4% [17–19, 34–36]. After identification using multiplex PCR and verification by rpoB sequencing all isolates were classified as A. butzleri. This result is in agreement with previous studies in Turkey and Chile, where A. butzleri was the only species recovered from human feces [17, 35]. According to other authors, A. cryaerophilus and A. skirrowii can also be isolated from human stool samples. However, the latter is only rarely detected due to slow growth on culture media and overgrowth by other bacteria, while the prevalence of A. cryaerophilus is up to 6.7-fold lower compared to A. butzleri [14, 17, 19, 36, 37]. These are probably the main factors that caused lower species diversity in this study.
Improper hygienic practices at different stages of food supply chain may result in food contamination with Arcobacter spp. Handling and consumption of contaminated food products is considered as one of the main risk factors for human infection [7, 38]. The reported prevalence of Arcobacter species in foods varies greatly among different studies. However, most studies agree that the contamination rates of poultry meat are higher in comparison to red meat, raw cow milk and vegetables [24, 39–41]. In this study, Arcobacter spp. were isolated from all food products, with an overall prevalence of 28.5% (152 of 534 samples). As expected, chicken meat showed the highest contamination levels (36%, 119/331), followed by raw cow milk (25%, 26/104) and RTE salads (7.1%, 7/99). Part of these results are in agreement with studies from Malaysia and Italy where Arcobacter was detected in 39% (48/123) of chicken meat and in 21.6% (8/37) of raw cow milk samples [42, 43]. According to other authors, the reported prevalence of Arcobacter spp. in chicken meat and raw cow milk ranged from 12 to 85.7% and from 4.1 to 46%, respectively [18, 44–46]. The isolation rate of Arcobacter in RTE salad mixes was lower in comparison with studies conducted in Italy and Portugal (i.e. ranging from 27.5 to 47.6%), but higher than the reported contamination of leafy green vegetables (4.4%, 4/90) from a study in South Korea [24, 47, 48]. Regarding the distribution of species based on sample type, A. butzleri was the only species detected in raw cow milk and the most commonly isolated species in chicken meat (114 out of 119 isolates), whereas in RTE packaged vegetables the most common was A. cryaerophilus (5 out of 7 isolates). A. skirrowii was not recovered from tested food samples. These results are in concordance with previous studies that reported A. butzleri as the predominant or the only species (75.4-100% of isolates) detected in chicken meat and raw cow milk. A. cryaerophilus was the second most commonly isolated species (0-21.5% of isolates), while A. skirrowii was rarely found (0-3.1% of isolates) [43–46]. The ability of A. butzleri to grow in low temperatures (4-10 ºC), attach to various pipe surfaces (i.e. stainless steel, copper and plastic), form biofilms and survive sanitizing procedures explains its persistence in the food processing environment and high isolation rates [49–53]. In case of RTE salads, the higher prevalence of A. cryaerophilus was not reported by previous studies. During our survey, pre-washed RTE salad mix samples were tested; therefore, higher A. cryaerophilus occurrence in vegetables might be associated with a higher capacity to adhere and survive on plant surfaces.
Contaminated water is considered as another important risk factor for public health, and it has been estimated that 63% of A. butzleri infections in humans are related to the consumption of or contact with contaminated water [6]. Arcobacter spp. were isolated from 36 out of 128 (28.1%) examined environmental water samples. This finding is consistent with a study from Canada, where Arcobacter was detected in 25.6% (173/676) of surface water samples [54]. However, the prevalence in environmental waters varies greatly across studies, with rates ranging from 20.8 to 58.6% [55, 56]. Out of 36 Arcobacter isolates, A. butzleri was the most prevalent species (n = 30) followed by A. cryaerophilus (n = 6), which is in accordance with other studies [56, 57].
Differences between reported Arcobacter prevalence rates in various sources may be due to numerous factors, such as examined sample sizes, geographic and seasonal variation, implemented hygiene protocols and sanitation procedures on farms and food processing facilities, patient populations, sensitivity and specificity of used detection methods. Due to the lack of standard isolation and cultural identification protocols, the latter aspect is of particular importance. According to previous studies, factors like including a pre-enrichment step, media composition and incubation conditions may cause differences in recovery rates ranging from 7.1 to 38% [48, 58–60]. Furthermore, it should be taken into consideration that only stool samples of inpatients were included in this study. Arcobacter infections are generally mild and do not require hospitalization, hence the overall prevalence might be higher than the one reported here. Nonetheless, Arcobacter was frequently isolated from chicken meat, environmental water, raw cow milk and RTE salads, which is consistent with previous reports.
Antimicrobial susceptibility of isolated bacteria
At the European Union (EU) level, protocols that were developed by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) are mainly focused on the harmonized monitoring of antimicrobial resistance in Campylobacter and Salmonella from various sources (i.e. food, food-producing animals and humans) [61]. In contrast to these zoonotic pathogens, the AST of Arcobacter is not standardized (i.e. there are no reference protocols or defined standard interpretive criteria). Therefore, data on antimicrobial susceptibility of Arcobacter spp. are scarce. Furthermore, the use of different testing methods and breakpoints hinder harmonized monitoring or comparative analysis and can result in therapeutic misguidance. Nevertheless, recent reports have indicated resistance of Arcobacter spp., isolated from food products, environment and human clinical samples, to several classes of antibiotics (i.e. macrolides, fluoroquinolones, lincosamides, tetracyclines and penicillins) [25, 62–64]. In these studies, resistance was determined by applying European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for Campylobacter, Enterobacteriaceae and non-species related breakpoints, or Clinical and Laboratory Standards Institute (CLSI) breakpoints for Campylobacter, Enterobacteriaceae and Staphylococcus spp.
In our study, two different methods were used for the isolation of Arcobacter spp. from food products, environmental water and human stool samples. However, strains from different sources showed similar MIC distribution patterns (data not shown). Therefore, MIC data were aggregated and compared with EUCAST ECOFFs for C. jejuni [31]. Regardless of species, none of the tested Arcobacter isolates showed elevated MICs for gentamicin and tetracycline. These results are in concordance with previous studies from Belgium, Spain and Iran, where the determined resistance rates for gentamicin and tetracycline were between 0-3.6% and 0-11%, respectively [62, 63, 65]. In general, aminoglycosides (i.e. gentamicin, kanamycin and streptomycin) are highly effective against Arcobacter spp. and, therefore, are recommended for the treatment of severe infections [7]. However, in case of tetracycline, higher resistance rates (up to 90.5%) were recently reported [24].
Azithromycin is more effective than erythromycin against Campylobacter, which is reflected in 16-fold lower ECOFF value. Both of these antibiotics belong to the class of macrolides; thus, the changes (i.e. methylation or mutations) in ribosomal target sites and drug efflux usually cause cross-resistance in Campylobacter spp. [66]. Surprisingly, Arcobacter spp. AST revealed equal or up to 16 times higher azithromycin MIC values in comparison with those of erythromycin for 145 (69.7%; data not shown) isolates. Furthermore, MIC data for azithromycin were distributed bimodally, while an unimodal distribution for erythromycin was found. After applying C. coli EUCAST breakpoints, Van den Abeele et al. [63] found that 21.7% (23/106) of the Arcobacter strains were resistant to erythromycin. This finding is in agreement with our results, as 42 isolates (20.2%) had MICs > 8 µg/ml. According to other authors, from 2.8% to 100% of tested Arcobacter strains were resistant toward this antibiotic [65, 67]. High resistance rates pose a serious risk to public health as erythromycin is critically important for treatment of campylobacteriosis and it was suggested to be used in Arcobacter infections [68]. As described in previous studies, we also found that the majority of azithromycin MICs (96.2%) were equal to or above the C. jejuni ECOFF (0.25 µg/ml) [19, 63]. MIC data for azithromycin indicated the presence of a subpopulation with reduced susceptibility. Therefore, elevated MICs (≥ 8 µg/ml) were determined for 130 (67.7%) A. butzleri and 2 (12.5%) A. cryaerophilus strains. Similarly, Brückner et al. [19] observed elevated MIC values (> 8 µg/ml) for 54.2% A. butzleri and 10% A. cryaerophilus strains. Divergent MIC distribution patterns for macrolides are consistent with the results of a recent study from Germany that tested the in vitro susceptibility of clinical Arcobacter strains using the same methodology [19]. Additionally, erythromycin MIC values peaked at 4 µg/ml, while azithromycin MIC distribution was characterized by two peaks at 1 µg/ml and 16 µg/ml, which is also in agreement with our results. The authors have hypothesized that an amino acid substitution (A86E) in ribosomal protein L22 and the absence of mutations (A2074T or A2075G) in 23S rRNA gene may result in resistance to azithromycin and susceptibility to erythromycin, which is seen in Campylobacter [19, 69]. AST of clinical Legionella pneumophila strains showed that there is a correlation between reduced susceptibility to azithromycin and the presence of the lpeAB genes encoding a macrolide efflux pump [70]. Although Arcobacter spp. do not possess these genes, the presence of lpeAB functional homologs (encoding MacAB-TolC) was already reported [71]. However, whole genome sequence-based analysis of Arcobacter is needed in order to determine the genetic mechanisms affecting the MICs of different macrolides.
According to EFSA and ECDC [61], ciprofloxacin resistance increased during the period from 2015 to 2019 in C. jejuni strains isolated from humans. In 2019, the reported resistance at EU level for C. jejuni and C. coli from various sources (i.e. humans, poultry, broiler meat) was between 61.5-90% and 61.2-89.4%, respectively. In comparison with Campylobacter, the resistance rates in Arcobacter are lower (ranging from 0 to 27.4%) [25, 63, 64]. Results of this study are in accordance with previous reports as only 18 (8.7%) Arcobacter isolates had elevated MICs (≥ 8 µg/ml), while the rest displayed low values that ranged between 0.032-1 µg/ml. The majority of strains (16/18) that had elevated MIC values were isolated from chicken meat. This result can be explained by the use of fluoroquinolones in poultry rearing [24]. A slightly higher percentage of A. butzleri isolates (8.9%) showed reduced susceptibility in comparison to A. cryaerophilus (6.3%), which is in line with a study by Rahimi et al. [65]. In case of ampicillin, high MICs (≥ 24 µg/ml) were determined for 46.9% of A. butzleri isolates, while only one A. cryaerophilus strain (6.3%) showed a MIC of 24 µg/ml. This result is in agreement with previous studies reporting high MICs for A. butzleri and A. cryaerophilus strains with rates of 42-100% and 0-23.3%, respectively [17, 19, 63]. Furthermore, a majority (23/30, 76.7%) of A. butzleri strains isolated from environmental water showed MICs that ranged from 24 µg/ml to > 256 µg/ml. High rates of resistance (94.4-100%) were observed in previous studies involving A. butzleri isolates from aquatic environment [25, 72].
According to our results, in case of ciprofloxacin, the C. jejuni ECOFF (0.5 µg/ml) could be applied for Arcobacter as isolates with MICs ranging from 0.032 to 0.5 µg/ml formed a wild-type subpopulation (i.e. bacteria without acquired resistance mechanisms). This result is in agreement with previous reports [19, 73]. However, for gentamicin, tetracycline, erythromycin, azithromycin and ampicillin, various rates of presumptive wild-type isolates (i.e. 35.6%, 70.2%, 39.6%, 89.5% and 66%, respectively; Fig. 1) had MICs that were above the ECOFF values for C. jejuni. Therefore, Arcobacter ECOFFs for these antimicrobials may be higher and should be reassessed.
Prevalence of putative virulence genes
Although A. butzleri and A. cryaerophilus are considered as emerging zoonotic pathogens, data on virulence and pathogenic mechanisms is still limited [7]. The prevalence rates of putative virulence genes among Arcobacter spp. isolated from human, animal and food samples were previously reported by several authors [26, 48, 74–76]. However, this is the first study reporting the occurrence of virulence genes in Arcobacter strains isolated from different sources in Lithuania.
We examined A. butzleri and A. cryaerophilus isolates for the presence of ten genes that are homologous to virulence factors in C. jejuni and other pathogens. Regardless of isolation source, six genes, namely ciaB, mviN, pldA, tlyA, cj1349 and cadF, were identified in most or even all A. butzleri isolates (100%, 97.9%, 95.8%, 99%, 99% and 100%, respectively). The high occurrence of these genes (ranging between 77.5-100%) was reported in previous studies after testing A. butzleri isolates from human stool, food products, animal feces, in-line milk filters of cow dairy farms, slaughterhouse processing water and processing line equipment [23, 26, 74–77]. The remaining four genes, i.e., hecA, hecB, irgA and iroE, were less prevalent. Higher cadF, ciaB, cj1349, mviN, pldA and tlyA detection rates in comparison with irgA, iroE, hecA, and hecB are consistent between most of published studies. In general, the irgA gene showed the lowest occurrence rate (12.5%) and was not detected in isolates from human stool, raw cow milk, and RTE salad mixes. Similar prevalence rates (ranging from 7.1 to 17.6%) were reported previously [23, 62, 77]. The presence of irgA gene in A. butzleri from raw cow milk and RTE vegetables was rarely investigated; however, Girbau et al. [75] and Mottola et al. [47] did not detect irgA in strains that were isolated from these sources, which is in line with our study. The occurrence of hecA (20.3%), hecB (38.5%) and iroE (18.8%) genes is similar to that reported by other authors (ranging between 10.8-31.3%, 29-48% and 12-30%, respectively) [23, 26, 77–79]. Surprisingly, the presence of hecA, irgA and iroE was considerably lower in human stool and food isolates compared with environmental water isolates. This is in agreement with Karadas et al. [76] who determined higher detection rates for irgA (44%), hecA (44%) and iroE (67%) in isolates from water in comparison to isolates originating from humans, pork, chicken meat, and minced meat. However, in contrast to Karadas et al., our results revealed that the gene encoding hemolysin activation protein (hecB) was significantly more prevalent in strains from water and human clinical samples compared with strains from food (P < 0.05). This difference might be associated with the lower number of isolates tested in previous study. Fourteen A. butzleri isolates (7.3%), obtained from chicken meat (n = 7; 6.1%) and environmental water (n = 7; 23.3%), were found to carry all ten putative virulence genes. Slightly different rates (ranging from 1.7 to 22.5%) were determined in studies from Spain and Germany [75, 80]. This disparity might be due to differences in the origin of tested isolates.
In accordance with other reports [26, 47, 55, 75, 79], we observed fewer virulence genes (n = 5) among A. cryaerophilus strains in comparison to A. butzleri. For A. cryaerophilus, irrespective of origin, two genes (ciaB and mviN) were detected in all isolates, whereas cj1349 and hecA were present in 12.5%, and tlyA in 25% of isolates. The predominance of ciaB and mviN in A. cryaerophilus was reported in previous studies involving isolates from chicken meat, water and other sources [26, 74, 78]. For bacteria originating from vegetables, the data on virulence gene distribution is limited to one study, which showed partial agreement with our results. In particular, the study from Italy reported the presence of cadF and mviN in all A. cryaerophilus isolates, while other seven genes (i.e. ciaB, cj1349, irgA, hecA, tlyA, hecB and pldA) were not detected [47]. According to other authors, the occurrence of cj1349, hecA and tlyA in A. cryaerophilus varies greatly with rates ranging between 0-76.9%, 0-30%, and 0-31.8% respectively [47, 75, 81]. Furthermore, cadF (0-100%), pldA (0-61.5%) and irgA (0-15.9%) were also identified in A. cryaerophilus [47, 48, 81, 82]; however, we did not detect these genes among tested isolates. The above-mentioned virulence profile differences within A. cryaerophilus species might be associated with higher genomic heterogeneity in primer target sequences [83].