Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Comparative pangenomic analysis of Campylobacter fetus isolated from Spanish bulls and other mammalian species.

Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). The latter causes bovine genital campylobacteriosis, a sexually-transmitted disease endemic in Spain that results in significant economic losses in the cattle industry. Here, 33 C. fetus Spanish isolates were whole-genome sequenced and compared with 62 publicly available C. fetus genomes from other countries. Genome-based taxonomic identification revealed high concordance with in silico PCR, confirming Spanish isolates as Cff (n = 4), Cfv (n = 9) and Cfv biovar intermedius (Cfvi, n = 20). MLST analysis assigned the Spanish isolates to 6 STs, including three novel: ST-76 and ST-77 for Cfv and ST-78 for Cff. Core genome SNP phylogenetic analysis of the 95 genomes identified multiple clusters, revealing associations at subspecies and biovar level between genomes with the same ST and separating the Cfvi genomes from Spain and other countries. A genome-wide association study identified pqqL as a Cfv-specific gene and a potential candidate for more accurate identification methods. Functionality analysis revealed variations in the accessory genome of C. fetus subspecies and biovars that deserve further studies. These results provide valuable information about the regional variants of C. fetus present in Spain and the genetic diversity and predicted functionality of the different subspecies.

Whole-genome long-read sequencing to unveil Enterococcus antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of Enterococcus lactis.

Enterococcus faecalis (Efs) and Enterococcus faecium (Efm) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis (Elc). However, identification techniques do not routinely differentiate Elc from Efm. As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs, 23 Efm, and 59 Elc. Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCEEnterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis (Elc) from Enterococcus faecium (Efm) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis (Efs) and Efm, it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (Efs, Efm, and Elc) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

Monitoring within-farm transmission dynamics of antimicrobial-resistant Campylobacter in dairy cattle using broth microdilution and long-read whole genome sequencing.

Campylobacter jejuni and Campylobacter coli are important foodborne zoonotic pathogens and cause for concern due to the increasing trend in antimicrobial resistance. A long-run surveillance study was conducted in animals from different age groups in five dairy cattle farms to investigate the within-farm diversity and transmission dynamics of resistant Campylobacter throughout time. The resistance phenotype of the circulating isolates (170 C. jejuni and 37 C. coli) was determined by broth microdilution and a selection of 56 isolates were whole genome sequenced using the Oxford-Nanopore long-fragment sequencing technology resulting in completely resolved and circularized genomes (both chromosomes and plasmids). C. jejuni was isolated from all farms while C. coli was isolated from only two farms, but resistance rates were higher in C. coli than in C. jejuni and in calves than in adult animals. Some genotypes (e.g. ST-48, gyrA_T86I/tet(O)/blaOXA-61 in farm F1; ST-12000, aadE-Cc/tet(O)/blaOXA-489 in F4) persisted throughout the study while others were only sporadically detected. Acquisition of extracellular genes from other isolates and intracellular mutational events were identified as the processes that led to the emergence of the resistant genotypes that spread within the herds. Monitoring with Oxford Nanopore Technologies sequencing helped to decipher the complex molecular epidemiology underlying the within-farm dissemination of resistant Campylobacter.

Within-farm dynamics of ESBL-producing Escherichia coli in dairy cattle: Resistance profiles and molecular characterization by long-read whole-genome sequencing.

A longitudinal study was designed in five dairy cattle farms to assess the within-farm dynamics of ESBL-/AmpC-/carbapenemase-producing E. coli and their resistance profiles, along with the genes conferring the resistance phenotypes. Twelve samplings were performed over a period of 16 months, collecting rectal feces from apparently healthy animals in three age groups (calves, heifers, and lactating cows) that were subjected to selective isolation in cefotaxime-containing media. Minimum inhibitory concentrations were determined by broth microdilution for 197 cefotaxime-resistant E. coli (1-3 isolates per age group and sampling date), and 41 of them were selected for long-read whole-genome sequencing. Cefotaxime-resistant E. coli were detected in the five farms, but isolation frequency and resistance profiles varied among farms and age groups. The genetic profiling of a selection of isolates recovered in two of the farms was described in full detail, showing the predominance of a few genomic subtypes of E. coli in one farm (F1) and great variability of strains in another one (F4). Two predominant distinct strains carrying the bla CTX-M-1 gene in IncX1 plasmids successively spread and persisted in F1 over a prolonged period. In F4, 13 different MLST types carrying a high diversity of ESBL-encoding genes in 6 different plasmid types were observed, probably as the result of multiple source contamination events. In both farms, the presence of certain plasmid types with the same repertoire of ARGs in different E. coli STs strongly suggested the occurrence of horizontal transfer of such plasmids among strains circulating within the farms. Considering the public health importance of ESBL-producing E. coli both as pathogens and as vectors for resistance mechanisms, the presence of ß-lactamase- and other AMR-encoding genes in plasmids that can be readily transferred between bacteria is a concern that highlights the need for One Health surveillance.

Whole genome-based characterisation of antimicrobial resistance and genetic diversity in Campylobacter jejuni and Campylobacter coli from ruminants.

Campylobacter, a leading cause of gastroenteritis in humans, asymptomatically colonises the intestinal tract of a wide range of animals.Although antimicrobial treatment is restricted to severe cases, the increase of antimicrobial resistance (AMR) is a concern. Considering the significant contribution of ruminants as reservoirs of resistant Campylobacter, Illumina whole-genome sequencing was used to characterise the mechanisms of AMR in Campylobacter jejuni and Campylobacter coli recovered from beef cattle, dairy cattle, and sheep in northern Spain. Genome analysis showed extensive genetic diversity that clearly separated both species. Resistance genotypes were identified by screening assembled sequences with BLASTn and ABRicate, and additional sequence alignments were performed to search for frameshift mutations and gene modifications. A high correlation was observed between phenotypic resistance to a given antimicrobial and the presence of the corresponding known resistance genes. Detailed sequence analysis allowed us to detect the recently described mosaic tet(O/M/O) gene in one C. coli, describe possible new alleles of blaOXA-61-like genes, and decipher the genetic context of aminoglycoside resistance genes, as well as the plasmid/chromosomal location of the different AMR genes and their implication for resistance spread. Updated resistance gene databases and detailed analysis of the matched open reading frames are needed to avoid errors when using WGS-based analysis pipelines for AMR detection in the absence of phenotypic data.

Draft Genome Sequence of Escherichia marmotae E690, Isolated from Beef Cattle.

We report here the draft genome sequence of an extended-spectrum ß-lactamase (ESBL)-producing Escherichia species isolated from rectal feces collected from beef cattle in northern Spain. Analysis of the draft genome identified the strain as a member of the newly described species Escherichia marmotae.

Prevalence of Cefotaxime-Resistant Escherichia coli Isolates from Healthy Cattle and Sheep in Northern Spain: Phenotypic and Genome-Based Characterization of Antimicrobial Susceptibility.

In order to estimate herd-level prevalence of extended-spectrum ß-lactamase/AmpC ß-lactamase (ESBL/AmpC)- and carbapenemase-producing commensal Escherichia coli in ruminants in the Basque Country (northern Spain), a cross-sectional survey was conducted in 2014 to 2016 in 300 herds using selective isolation. ESBL-/AmpC-producing E. coli was isolated in 32.9% of dairy cattle herds, 9.6% of beef cattle herds, and 7.0% of sheep flocks. No carbapenemase-producing E. coli was isolated. Phenotypic antimicrobial susceptibility determined by broth microdilution using EUCAST epidemiological cutoff values identified widespread coresistance to extended-spectrum cephalosporins and other antimicrobials (110/135 isolates), particularly tetracycline, sulfamethoxazole, trimethoprim, and ciprofloxacin. All isolates were susceptible to tigecycline, imipenem, meropenem, and colistin. The genomes of 66 isolates were sequenced using an Illumina NovaSeq 6000 and screened for antimicrobial resistance determinants against ResFinder and PointFinder. The plasmid/chromosomal locations of resistance genes were predicted with PlasFlow, and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to 11 antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes, blaCTX-M-14 being the most common, and 11 carried determinants of the AmpC phenotype, mostly the blaCMY-2 gene. Additionally, genes coding for ß-lactamases of the CTX-M group 9 were detected as well as the sporadic presence of blaSHV-12, blaCMY-4, and a point mutation in the ampC promoter. Only a bovine isolate coharbored more than one ESBL/AmpC genetic determinant (blaCTX-M-14 and a mutation in the ampC promoter), confirming its ESBL- and AmpC ß-lactamase-producing phenotype. Most ESBL/AmpC genes were located in IncI1 plasmids, which also carried a great variety of other antimicrobial resistance genes.IMPORTANCE Extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase (AmpC)-producing E. coli isolates have emerged in recent years as some of the fastest spreading antimicrobial resistance determinants in humans and food-producing animals, becoming a concern for animal and public health. This study provided insight into the prevalence of cefotaxime-resistant E. coli in cattle and sheep in the Basque Country and the associated genetic determinants of antimicrobial resistance. These constituted an important contribution to the limited repository of such data for cattle in the region and for sheep worldwide. Antimicrobial susceptibility testing by phenotypic and molecular methods is key in surveillance programs to enhance early detection of resistance development, monitor resistance trends, and provide guidance to clinicians in selecting the adequate therapy.

Occurrence of Campylobacter jejuni and Campylobacter coli in Cattle and Sheep in Northern Spain and Changes in Antimicrobial Resistance in Two Studies 10-years Apart.

A cross-sectional survey was conducted in 2014-2016 in 301 ruminant herds to estimate C. jejuni and C. coli prevalence, and investigate their susceptibility to antimicrobials. Risk of shedding C. jejuni was higher in cattle than sheep (81.2% vs. 45.2%; ORadj = 5.22, p < 0.001), whereas risk of shedding C. coli was higher in sheep than in cattle (19.1% vs. 11.3%; ORadj = 1.71, p = 0.128). Susceptibility to six antimicrobials was determined by broth microdilution using European Committee for Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off values. C. coli exhibited higher resistance (94.1%, 32/34) than C. jejuni (65.1%, 71/109), and resistance was more widespread in isolates from dairy cattle than beef cattle or sheep. Compared to results obtained 10-years earlier (2003-2005) in a similar survey, an increase in fluoroquinolone-resistance was observed in C. jejuni from beef cattle (32.0% to 61.9%; OR = 3.45, p = 0.020), and a decrease in tetracycline-resistance in C. jejuni from dairy cattle (75.0% to 43.2%; OR = 0.25, p = 0.026). Resistance to macrolides remained stable at low rates and restricted to C. coli from dairy cattle, with all macrolide-resistant C. coli showing a pattern of pan-resistance. Presence of the single nucleotide polymorphisms (SNPs) associated to quinolone and macrolide resistance was confirmed in all phenotypically resistant isolates. The increase in fluoroquinolone resistance is worrisome but susceptibility to macrolides is reassuring.

16S rRNA amplicon sequencing characterization of caecal microbiome composition of broilers and free-range slow-growing chickens throughout their productive lifespan.

Gut microbiota affects health, metabolism and immunity of the host, and in the case of livestock, also food-safety. Here, 16S rRNA gene high-throughput Illumina sequencing was used to describe the microbiome of chicken caeca in two different breeds and management systems throughout their whole productive lifespan. Broilers (Ross-308), as a fast-growing breed reared in an intensive system for 42-days, and a slow-growing breed of chicken (Sasso-T451A) reared in an extensive farming system with outdoor access for 86-days, were compared. The core microbiome and differentially abundant taxa, as well as taxa associated with age were identified. Age was identified as the strongest influencing factor in caecal microbiota composition, and, in general, each age-group showed an age-associated community profile, with a transition period at the middle of their lifespan. However, substantial differences were observed in the composition of caecal microbiota of both chicken breeds, microbiota being richer and more complex in free-range chicken than in broilers. Several taxa positively/negatively correlated with Campylobacter relative abundance were also identified. Especially noteworthy was the identification by microbial community comparison of microbiota profiles suggestive of dysbiosis in several free-range chickens, probably associated to the typhlitis observed in the lumen of their caeca.

Salmonella spp. and Listeria monocytogenes shedding in domestic ruminants and characterization of potentially pathogenic strains.

Salmonella and Listeria monocytogenes are important human pathogens that can be isolated from the faeces of ruminants, and constitute potential sources of human infection via contamination of food and water or contact (direct or indirect) between humans and animals. A cross-sectional survey including 301 herds was carried out to estimate the prevalence of cattle herds (beef and dairy) and sheep flocks positive for these zoonotic organisms in ruminants in the Basque Country (Northern Spain). The proportion of herds where Salmonella shedding was detected was 5.8% for beef cattle, 3.7% for dairy cattle and 6.1% for sheep flocks. Apart from a multi-drug resistant (ASuT) monophasic variant of S. Typhimurium (4,[5],12:i:-) isolated from a beef cattle herd, the remaining 11 serotypes of S. enterica identified were not among the common causes of non-typhoidal salmonellosis in humans. L. monocytogenes was isolated from 42.3% of beef cattle herds, 46.3% of dairy cattle herds, and a significantly lower proportion of sheep flocks (23.5%). Presence of other animal species in the farm and geographical location were associated with herd risk of L. monocytogenes shedding. A total of 65.8% of the isolates of L. monocytogenes harboured the virulence gene, llsX, as determined by real-time PCR. In conclusion, ruminant herds in the Basque Country did not seem to pose a high risk for Salmonella serotypes commonly associated to human clinical cases of salmonellosis entering the food chain. Conversely, potentially pathogenic L. monocytogenes strains were widespread in ruminant farms, mostly in cattle.

Effects of dry whey powder and calcium butyrate supplementation of corn/soybean-based diets on productive performance, duodenal histological integrity, and Campylobacter colonization in broilers.

BACKGROUND: Campylobacter is the main cause of gastroenteritis in humans in industrialized countries, and poultry is its principal reservoir and source of human infections. Dietary supplementation of broiler feed with additives could improve productive performance and elicit health benefits that might reduce Campylobacter contamination during primary production. The aim of this study was to assess the effect of dietary supplementation with whey (a prebiotic) and calcium butyrate (a salt of a short-chain fatty acid) on productive traits, duodenal histological integrity, and Campylobacter colonization and dissemination in broiler chickens during the 42-day rearing period. RESULTS: Six hundred one-day-old Ross-308 chickens were placed into 20 ground pens and assigned to one of 4 corn/soybean-based dietary treatments (5 replicates of 30 chicks per treatment) following a randomized complete block design: 1) basal diet with no supplementation as the control, 2) diet supplemented with 6% dry whey powder, 3) diet containing 0.1% coated calcium butyrate, and 4) diet containing 6% whey and 0.1% calcium butyrate. At age 15 days, 6 chickens per pen were experimentally inoculated with Campylobacter jejuni. The results showed that supplementation of the corn/soybean-based diet with 6% whey alone or, preferably, in combination with 0.1% coated calcium butyrate improved growth and feed efficiency, had a beneficial effect on duodenal villus integrity, and decreased mortality. These favourable effects were particularly significant during the starter period. Six days after oral challenge, Campylobacter was widespread in the flock, and the birds remained positive until the end of the rearing period. Although Campylobacter was not isolated from environmental samples, it was detected by real-time polymerase chain reaction (PCR) in dust, air filters, and drinkers while birds shed culturable C. jejuni cells. No differences (p > 0.050) in colonization or shedding levels that could be attributed to the diet were observed during the assay. CONCLUSIONS: Beneficial effects on performance and intestinal health were observed, particularly during the starter period, when chickens were fed a diet supplemented with both whey and coated calcium butyrate. However, none of the tested diets provided the chicks any differential degree of protection against Campylobacter infection.