Background The SOS response, a conserved bacterial DNA repair system, plays a critical role in adaptation under stress, by inducing spontaneous mutations. This study compared the evolutionary dynamics of Escherichia coli and a derivative of this strain with a constitutively activated SOS response (SOS*), focusing on growth phenotype, competitive fitness, genome evolution, and metabolism over 90 days of evolution. Results Laboratory evolution revealed that both wild-type and SOS* populations increased in growth rates, reduced average cell lengths, improved competitive fitness, and expanded their catabolic substrate utilization over time. However, SOS* strains, which revealed initial reduced growth rate, fitness and carbon-utilization, adapted at a faster rate than the wild-type, and obtained levels comparable to the wild-type. Whole-genome sequencing identified higher mutation rates and distinct mutation patterns in the SOS* populations compared to the wild-type populations, particularly an increase in non-synonymous substitutions, suggesting positive selection for adaptive traits. Mutational analyses revealed convergence in regulatory and metabolic pathways in both wild-type and SOS* groups. However, SOS* accumulated a higher number of mutations in regulatory and metabolic genes, consistent with increased phenotypic flexibility during adaptation. Despite this, their fitness and carbon source utilization abilities did not surpass those of the evolved wild-types. Conclusion Our results indicate that while SOS-mediated mutagenesis enhances genetic diversity and accelerates adaptation, its long-term impact on adaptive evolution may be limited under non-selective environments due to the sustained SOS activation.

Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum lead to pullorum disease and fowl typhoid in avian species and have been of substantial economic significance to the poultry industry. Serotyping and biochemical assays are complicated for use in the differentiation of these two biovars owing to their antigenic similarity. The aims of this study were to establish a new multiplex polymerase chain reaction (PCR) method to detect and differentiate the two Salmonella biovars, Gallinarum and Pullorum. The novel multiplex PCR was established on the basis of three genes, SG1445, I137_14465, and stn. In silico analysis indicated the SG1445 gene exists in all Salmonella serovar, apart from S. Pullorum, which allowed the use of this gene for S. Pullorum identification. The I137_14465 gene is absent in S. Gallinarum and present in all other Salmonella serovars, allowing this gene to be utilized for S. Gallinarum detection. stn was employed as a control gene for the Salmonella genus. The multiplex PCR could identify and distinguish the two pathogens accurately from 74 strains of Salmonella, including 29 different serovars and without false positive reactions from 44 non-Salmonella bacteria. The specificity was verified with a broad collection of Salmonella isolates. The detection limit of the multiplex PCR was as low as 22.5 pg/μL genomic DNA of the two pathogens and 100 CFU of bacterial cells. Detection of clinical isolates from a chicken farm was carried out using this method with good agreement with detection based on traditional Salmonella serotyping and biochemical identification. The findings of this study showed that the developed multiplex PCR technique could sensitively detect and specifically differentiate the two biotypes of S. Gallinarum in laboratory and clinical samples.

Post-weaning diarrhoea (PWD) is a major driver of antimicrobial use in pigs. Optimising treatment duration may reduce selection pressure while maintaining clinical efficacy, but under EU Regulation (EU) 2019/6 veterinarians must adhere to authorised treatment durations, creating a need for evidence to support label changes. This study aimed to determine whether a 3-day apramycin treatment is non-inferior to the standard 7-day course for clinical cure of PWD and to assess its effect on the abundance of the apramycin resistance gene aac(3)-IV. A cluster-randomised non-inferiority field trial was conducted in 518 diarrhoeic piglets across 28 pens in a commercial herd. Piglets received apramycin for 3 or 7 days. Clinical cure was assessed by faecal scoring at end of treatment (EOT) and at a common Day 8 timepoint after treatment initiation. A non-inferiority margin of − 10

Salmonella is a major foodborne pathogen associated with poultry production. Data comparing Salmonella occurrence and resistance patterns across poultry production systems in Uganda remain limited. This study assessed the prevalence, serovar diversity, antimicrobial susceptibility, and genetic diversity of Salmonella isolated from semi-intensive poultry farms in Wakiso district and predominantly backyard, free-range poultry farms in Soroti district, Uganda. A cross-sectional study was conducted between October 2021 and March 2022. A total of 402 boot sock samples were collected from 402 farms and cultured for Salmonella. Isolates were confirmed to the genus-level using MALDI-TOF MS. Antimicrobial susceptibility testing was performed against eight antibiotics using disc diffusion, and whole-genome sequencing was used for serovar determination, multi-locus sequence typing, and antimicrobial resistance gene detection. Overall, Salmonella prevalence was 7.7% (31/402) with no statistically significant difference between semi-intensive farms in Wakiso (8.0%; 16/200) and predominantly free-range farms in Soroti (7.4%; 15/202). Eighteen distinct serovars and multiple STs were identified, demonstrating substantial genetic diversity. Phenotypic resistance was uncommon in both sampled farm groups. Among isolates from free-range farms in Soroti only one isolate expressed resistant to tetracycline, and in semi-intensive farms, one isolate was resistant to tetracycline, and two isolates were resistant to both tetracycline and sulfamethoxazole-trimethoprim. Whole genome sequencing revealed a broader resistome among isolates from semi-intensive farms, including tet(A), sul3, qnrS13, cmlA1, dfrA12, and mef(B), while most isolates from free-range farms carried only the intrinsic aac(6′)-Iaa gene. Encouragingly, both Salmonella occurrence and AMR levels were low overall, although the detection of acquired AMR genes in some isolates supports the need for strengthened antimicrobial stewardship, biosecurity improvement and continued surveillance.

Trueperella pyogenes is an important pathogen of ruminants and occasionally other animals. In the current study, we isolated bacteria from uterus of Danish healthy dairy cows (n = 56) and cows judged by veterinarians to suffer from post partum uterine disease (n = 25). T. pyogenes was more frequently isolated from cows judged to be sick than from healthy cows (odds ratio 37.09), and the average quantity of T. pyogenes was significantly higher in these cows (p = 0.0002). Eleven Danish isolates from uterus of cows and 26 strains from bronchial lavage samples of calves with or without clinical signs of bovine respiratory syndrome were de novo sequenced, and the sequences were compared to each other and to all available genomes of sufficient quality in NCBI database (n = 126). The genome size of T. pyogenes varied between 2.19 and 2.46 megabases with GC content within 59.3 % and 59.9 %. Core-genome SNP analyses showed that Danish strains did not form a cluster of their own, and that strains from healty and sick animals clustered together. In analyses of all available genomes, two main clusters were found where strains from pigs and strains from cattle with a few exceptions clustered in separate branches, however, without indications of clustering depending on geographical location or anatomical site of the strain. This lack of host, geographic, and anatomic location-specific clustering was also evident by analyses of gene presence/absence matrixes and sequence alignments of the proteins encoded by the major virulence associated genes plo, fimA, fimE, fimC and cbpA. Danish strains of T. pyogenes from uterus and bronchial lavage had MIC50 and MIC90 values at or below the lowest dilutions tested for 11 out of 18 antimicrobials tested, indicating a high susceptibility. Indications of widespread low susceptibility was observed for tetracyclines (MIC50 >8 µg/ml) and sulfonamides (MIC50 >256 µg/ml). High MIC values to tetracyclines were assocatied to the presence of acquired resistance genes, most commonly the tet(W) gene, and high values to sulphonamides to the presence of the gene sul1.

Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SGa) and Pullorum (SPu) are avian-specific pathogens causing systemic disease, while S. Enteritidis (SEnt) is a broad host range serovar causing gastroenteritis. The genomic mechanisms underlying this difference in host range and pathogenicity remain incompletely understood. Here, we performed a large-scale pan-genome analysis of 5440 poultry-derived genomes (4927 SEnt, 106 SGa, 407 SPu) integrated with functional chicken and macrophage experiments. Compared with SEnt, avian-specific SGa and SPu exhibited extensive pseudogenization and shared 87 genes absent in SEnt, organized into four major genomic clusters (PG_1-PG_4) enriched in type VI secretion system genes and prophage-derived elements. Conserved SNPs distinguishing SGa/SPu from SEnt were enriched in carbohydrate and nitrogen metabolism pathways, suggesting potential metabolic divergences during infection. Infection experiments in chickens using deletion mutants revealed that deletions of genes in SPI-2 (ssaE, ssaT) and fimbrial genes (stfA, safA) were important for systemic infection of chicken with both SGa and SEnt, despite pseudogenization of fimbrial operons in SGa. Mutants in SPI-13 and SPI-14 were only significantly attenuated in SGa. The specific prophage region PG_3 was important for systemic infection in SGa, while a distinct prophage element (ENT_2) enhanced infection in SEnt. Together, these findings bridge comparative genomics with experimental validation, identifying genomic degradation, prophage acquisition, and serovar-specific pathogenicity islands as putative mechanisms underlying avian host specificity and systemic pathogenesis in Salmonella.

BACKGROUND:Calf diarrhoea causes substantial welfare and economic losses, and it is one of the major drivers of antimicrobial use. This study aimed to characterize the faecal microbiome of diarrhoeic calves, with a specific focus on Escherichia coli, and to assess whether microbial profiles vary with age, diarrhoea severity, and high E. coli abundance in the absence of other detectable enteric pathogens. METHODS:Stool samples from Danish diary calves (n = 32) below 4 weeks of age were collected from 11 herds and were analysed using direct long-read sequencing (mgt) as well as analyses of a subset of samples by swiping microbiota from faecal samples grown on McConkey agar plates (plate-swipe). Metagenomes were analysed to characterise community structure (Shannon α-diversity; Bray-Curtis PCoA with PERMANOVA) and to assess differential abundance at the species level while adjusting for sample type (mgt/plate swipe), herd, age, number of other pathogens detected by qPCR (rotavirus, coronavirus, Cryptosporidium parvum, Salmonella Dublin, Clostridium perfringens A, B, C, Eimeria and Escherichia coli F5) and recorded as presence/absence and summarised into infection classes (None/Mono/Co-2/Co-3 +). Binning was performed to build metagenome assembled genomes (MAGs) of E. coli. RESULTS:Microbiome structure was dominated by methodological and contextual factors: sample type (direct metagenomic vs plate swipe) and herd explained far more variation than clinical severity and age. Metagenomic species profiles from plate swabs were comparatively homogeneous and E. coli-rich, whereas direct metagenomes captured higher diversity. Differential abundance identified species enriched with increasing diarrhoea severity and with infection classes, while pathogen-specific contrasts (e.g., C. perfringens A-positive vs negative) revealed discrete sets of bacterial co-occurrences. Classical pathotype markers (virulence-genes) were uncommon among E. coli MAGs. CONCLUSIONS:Long-read metagenomics revealed insignificant influence of severity of diarrhoea, age below 4 weeks and number of pathogens detected in stool samples on diversity and microbial communities in diarrheic dairy calves. In contrast, large variation was observed between herds. On average, E. coli constituted about half of the microbiota. MAGs generated by binning indicated non-specific blooming of strains without particular virulence genes.

Agmatine, the product of the decarboxylation of arginine, catalysed by arginine decarboxylase (ADC), is a bioactive compound that functions as a neuromodulator and co-transmitter and has gained increasing attention in recent years due to its therapeutic potential, particularly for its neuroprotective properties. Members of the genus Hafnia are the main agmatine producers in dairy products. In this regard, Hafnia is considered a beneficial microorganism due to its ability to enhance cheese organoleptic properties and its emerging probiotic potential, making it relevant for functional food development, specially agmatine-enriched dairy products. This study aimed to identify the genetic basis for agmatine production in Hafnia paralvei and to assess its role in bacterial fitness. Genomic analysis of the strain H. paralvei IPLA15029 revealed the presence of two genes encoding putative ADC enzymes, adiA and speA, however, organized slightly different than those in other enterobacteria. In some bacteria, ADC exists in two forms: one involved in polyamine biosynthesis, encoded by the constitutive speA gene, and another involved in acid stress resistance, encoded by the adiA gene, which is inducible under acidic conditions. In vivo experiments under controlled pH conditions showed that agmatine accumulation occurs exclusively under acidic conditions, which also stabilize the compound by preventing its catabolism to putrescine. Gene expression analysis revealed that adiA was transcribed as a monocistronic unit, and that in these conditions, adiA is the gene responsible for agmatine production. This was confirmed by generating an adiA knockout mutant after the implementation of the CRISPR-Cas9 system, marking the first successful application of this technology in the genus Hafnia. Moreover, the adiA knockout demonstrated that the encoded arginine decarboxylase is essential for survival under severe acid stress.

Antimicrobial resistance (AMR) poses a serious threat to public health, with the emergence of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae, particularly Escherichia coli, raising significant concerns. This study aims to elucidate the drivers of antimicrobial resistance, and the global spread of cefotaxime-resistant E. coli (CREC) strains. Whole-genome sequencing (WGS) was performed to explore genome-level characteristics, and phylogenetic analysis was conducted to compare twenty CREC strains from this study, which were isolated from broiler chicken farms in Bangladesh, with a global collection (n = 456) of CREC strains from multiple countries and hosts. The MIC analysis showed over 70% of strains isolated from broiler chickens exhibiting MIC values ≥ 256 mg/L for cefotaxime. Notably, 85% of the studied farms (17/20) tested positive for CREC by the end of the production cycle, with CREC counts increasing from 0.83 ± 1.75 log10 CFU/g feces on day 1 to 5.24 ± 0.72 log10 CFU/g feces by day 28. WGS revealed the presence of multiple resistance genes, including blaCTX-M, which was found in 30% of the strains. Phylogenetic comparison showed that the Bangladeshi strains were closely related to strains from diverse geographical regions and host species. This study provides a comprehensive understanding of the molecular epidemiology of CREC. The close phylogenetic relationships between Bangladeshi and global strains demonstrate the widespread presence of cefotaxime-resistant bacteria and emphasize the importance of monitoring AMR in food-producing animals to mitigate the spread of resistant strains.

The aim of this study was to investigate the effect of avoiding flock treatment on resistance levels in the intestine of pigs. To investigate this, studies, each in two pig herds, quantified resistant coliforms by culture method in pigs treated as flock or as individual animal treatments orally with neomycin against post-weaning diarrhea (PWD) or intramuscularly with amoxicillin to prevent umbilical infection. Individual oral treatment against PWD did not lead to a lower number or proportion of resistant coliforms compared to flock treated pigs in any of the two herds under study, despite reduction of treatment incidences to 32% and 35% of pigs. After intramuscular treatment against umbilical infection, fewer resistant bacteria were seen in individually treated piglets in a herd with low treatment incidence (33%), while no reduction of resistant coliform bacteria compared to flock treatment was observed in the other herd with higher treatment incidence (86%). Thus, individual animal treatment reduced the amount of antimicrobial used, but concurrent reduction in resistant coliform bacteria was not always observed.

Investigation of clonal development of dominant persistent clones of avian pathogenic Escherichia coli (APEC) is important to understand their evolution and to gain knowledge to improve their control in poultry production. Whole-genomic sequencing, including hybrid assembled genomes of short and long reads, was used to analyse clonal persistence and evolution of APEC. Two vertically transferred E. coli clones, represented by ten isolates from sequence type (ST) 95-PFGE type 65 and eight isolates from ST131-PFGE type 47, were selected to identify genomic variations. The isolates had been sampled in broiler production during a period of 9 months in a previous study. The main differences among strains within each clone were related to plasmids, transposases, incomplete phage elements and amino acid substitutions which by far exceeded the genetic variation related to core-genome SNPs (cgSNPs). Fourier-transform infrared spectroscopy was, for the most part, only able to trace clones within the same ST. The genome-wide mutation rate was equivalent to 1.48 mutations per genome per year for ST95-PFGE65 and 2.86 for ST131-PFGE47, respectively. The most recent common ancestors were estimated back to 2009 for ST95-PFGE65 and to 2011 for ST131-PFGE47, with further divergence occurring in years until sampling in 2014–2015. The methodology introduced is able to trace the temporal origin of APEC clones. The conventional threshold of ten or fewer cgSNPs to include strains in the same clone did not consider any gain or loss of plasmids for the strains compared. On average, one plasmid transfer event was predicted every second year. For strains expected to be vertically transferred during the long production periods of great-grandparents over grandparents and parents to broilers, one to two plasmid transfers are therefore predicted, and several cgSNPs may be introduced, whereas up to one cgSNP is expected to be manifested during a broiler production cycle and rarely involving plasmid transfer.

Background Globally, the increase in antimicrobial resistance is of great concern. In Denmark, the pig sector is accountable for the majority of antimicrobial usage in animals. As new-born piglets are at risk of developing infectious omphalitis, and many pigs are treated by antimicrobials within the first days of life, an early and accurate diagnosis of the disease is imperative to maintain animal welfare and reduce the antimicrobial usage. The aim of the present study was to compare histopathological and microbiological findings in piglets clinically diagnosed with and without omphalitis during the first three days after birth. Results A total of 98 case piglets with omphalitis and 98 control piglets without omphalitis, based on clinical examinations during the first three days of life, were included. Of the 196 piglets, 79 (38 cases and 41 controls) presented histopathological omphalitis. Post mortem, the volume of the umbilicus was significantly enlarged in cases compared to controls (p = 0.01). Except for a group of bacteria unidentified by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) (p = 0.02), no apparent association was found between histopathologically diagnosed omphalitis and the prevalence of specific pathogens (p > 0.05). The frequency of histopathologically diagnosed omphalitis tended to increase with increasing age in both cases and controls. The frequency of piglets with both histopathological omphalitis and arthritis/synovitis was significantly different among cases and controls (p = 0.05). This was due to all controls, and none of the cases, with arthritis/synovitis presented histopathological omphalitis. Conclusion The clinical differentiation between omphalitis cases and controls did not correspond to the histological diagnosis of omphalitis in zero-to-three days old piglets. An inaccurate clinical diagnosis complicates prudent use of antimicrobials in pig herds. In addition, animal welfare may be hampered in infected piglets due to lack of treatment and in healthy piglets due to the antimicrobial effect on the gut microbiome.

Background Innovative antibiotic discovery strategies are urgently needed to successfully combat infections caused by multi-drug-resistant bacteria. Methods We employed a direct screening approach to identify compounds with antimicrobial and antimicrobial helper-drug activity against Gram-positive and Gram-negative bacteria. We used this platform in two different strains of methicillin-resistant Staphylococcus aureus (MRSA) and aminoglycoside-resistant strains of Escherichia coli to screen for antimicrobials compounds, which potentiate the activity of aminoglycoside antibiotics. Screening was performed with 75 known microbial products and 880 extracts from Actinomycetes from a collection at the company Naicons. Results The antibiotics rifamycin O and thermorubin inhibited the growth of neomycin-resistant E. coli in combination with 1/8 MIC of neomycin, suggesting a potential application as adjuvant drugs for neomycin. Additionally, in the Actinomycetes extract screen, one extract with antimicrobial activity and one extract with gentamicin adjuvant activity against gentamicin-resistant E. coli were identified, demonstrating the applicability of the screening approach. Against MRSA, the paramagnetoquinones, the lantibiotic NAI-107 and the spirotetronate NAI-414 showed the most pronounced antimicrobial activity. Difference is susceptibility towards antimicrobials and extracts were observed between the two MRSA strains used for screening. Conclusion Compounds with antibacterial and helper drug activity were identified using our screening approach. The results demonstrate the importance of strain selection in antimicrobial screening and highlight the potential of natural products as a source of potential new antibacterial and adjuvant therapies against both Gram-positive and Gram-negative bacteria.

Antibiotic persistence arises when a subset of cells in an otherwise susceptible bacterial population temporarily exhibits elevated tolerance to bactericidal antibiotics. While significant progress has been made in elucidating persister mechanisms, much of this research has focused on Gram-negative bacteria, and these findings are not always applicable to Gram-positive species. This study explores genetic factors influencing persister cell formation in the Gram-positive Streptococcus agalactiae (Group B Streptococcus, GBS). The central finding in this study is the identification of the previously uncharacterized UvrD-like helicase homolog, gbs1341, as a factor affecting multidrug persistence and biofilm formation. Alongside gbs1341, mutants in relA and trmB were also found to be implicated. Mutations in these genes resulted in a phenotype characterized by elevated basal levels of the stringent response alarmone (p)ppGpp, linked with increased persister frequencies and enhanced biofilm production. In contrast, the gbs1341 deletion mutant displayed a significantly higher persister fraction following exposure to antibiotics, along with altered and enhanced biofilm formation, despite unaltered (p)ppGpp levels compared to the wild-type. These findings characterize gbs1341 as a novel genetic factor in both persister cell formation and biofilm production, highlighting a genetic link between these phenotypes, advancing our understanding of persistence in Gram-positive bacteria.

The SOS response is a critical DNA damage repair mechanism in bacteria, designed to counteract genotoxic stress and ensure survival. This system can be activated by different classes of antimicrobial agents, each inducing the SOS response through different mechanisms. Moreover, it has been observed that certain antibiotics can enhance conjugative plasmid transfer frequencies. However, while previous studies have suggested that the SOS response contributes to horizontal transfer of certain genes, its role in plasmid conjugation remains unclear. In this study, we investigated the relationship between the SOS response and conjugation of IncI1 and IncFII plasmids harboring various blaCTX-M resistance genes. Results showed that cefotaxime and mitomycin C induced both the SOS response and conjugation, while ciprofloxacin induced the SOS response without affecting conjugation frequencies. Further analysis of SOS mutants, ranging from constitutively inactive to hyper-induced states, revealed no correlation between SOS levels and conjugation frequencies, despite upregulation of tra gene expression in a SOS hyper-induced strain. Proteomic analysis revealed that cefotaxime-induced conjugation was associated with increased transfer and pilus protein expression. In contrast, the SOS hyper-induced strain displayed limited upregulation of plasmid-encoded proteins, suggesting post-transcriptional regulation. Additionally, putative LexA binding sites on the IncI1 plasmid revealed potential SOS-mediated regulation of plasmid genes, highlighting the interaction between the SOS response and plasmid, although it did not significantly affect conjugation.IMPORTANCEPlasmids play a critical role in the dissemination of antibiotic resistance through conjugation. Recent research suggests that the use of antibiotics not only selects for already resistant variants but further increases the rate of plasmid-encoded conjugative transmission by increasing expression of the conjugative system. At the same time, these antibiotics are known to induce the stress-related SOS response in bacteria. To be able to counteract an antibiotic-induced increase in conjugative transfer of resistance plasmid, there is a need for a fundamental understanding of the regulation of transmission, including whether this happens through activation of the SOS response. In this research, we show that antibiotic-induced conjugation and induction of the SOS response happen through different mechanisms, and thus that future strategies to control the spread of antibiotics cannot interfere with the SOS response as its target.

Plasmids play a major role in the spread of antibiotic resistance genes in bacteria. Plasmid copy number (PCN) is often tightly regulated. In plasmids of the ColE1-type, this regulation happens by a negative feedback mechanism using an antisense RNA. Here, we employed a sequencing-based method for determining PCN to demonstrate that copy number of different ColE1-family plasmids harboring antibiotic resistance genes increases during antibiotic treatment. Further, we show that deletion of the gene pcnB reduces the copy number of ColE1-family plasmids in E. coli MG1655, which in turn results in a reduced resistance to antimicrobials of the classes aminoglycosides, β-lactams and tetracyclines. In the absence of antibiotic selection, the deletion of pcnB also decreased the number of ColE1-type plasmids in a bacterial population. Hence, PcnB, which polyadenylates RNA, marking it for decay, represents a potential drug and helper-drug target that could be used to reduce PCN to re-sensitize bacteria with multi-copy-number resistance-plasmids to treatment with different antimicrobials.

Extracts, fractions and the pure compound epifriedelanol of the medicinal plant Synadenium glaucescens have antibacterial properties. Herbal products are generally considered less prone to resistance development than conventional antimicrobials, as they contain multiple compounds, which makes bacteria less likely to develop resistance. However, data supporting this notion are lacking. This study evaluated the development of resistance in Staphylococcus aureus subjected to extract, fractions and epifriedelanol of S. glaucescens. It also identified S. aureus fitness genes contributing to intrinsic resistance to extract of S. glaucescens. Fluctuation and gradient concentration assays were used to determine mutation rates and growth adaptation, respectively, which were lower following exposure to growth in crude extract than the pure compound epifriedelanol. By subjecting 1920 single gene mutants from the Nebraska Transposon Mutant Library to growth in the presence of extract of S. glaucescens, 12 genes were identified as important for natural resistance in S. aureus JE2; however, only mutation in the hemB gene decreased the minimum inhibitory concentration by greater than 4-fold (64-fold). In conclusion, purifying active antimicrobial compounds from S. glaucescens and using them as antibacterial substances as an alternative to crude extract increased the risk of resistance development. Further, the gene hemBappears to have a significant role in the natural resistance to the extracts obtained from S. glaucescens in this study.

BACKGROUND:Calf diarrhoea is a common disease manifestation caused by a variety of pathogens, which are often concurrently detectable in faecal samples from affected calves. It is unknown if co-infections lead to more severe diarrhoea than single-pathogen infections. METHODS:A total of 170 faecal samples were obtained from diarrhoeic calves, and the faecal material was graded in diarrhoeic severity from I to III. The pathogens present in the samples were then identified using a multiplex qPCR assay. RESULTS:Clostridium perfringens type A and Cryptosporidium parvum were the most frequently detected pathogens and were present in 47% and 42% of the faecal samples, respectively. C. parvum was the most frequently detected pathogen in samples with diarrhoeic grade III, while Escherichia coli F5-positive samples had the highest relative frequency of samples with diarrhoeic grade III. Approximately one-third of the samples contained at least two different pathogens. For all diarrhoeic grades, the number of samples with one or more pathogens detected was greater than the number of samples with no pathogens detected, but this difference was only significant for diarrhoeic grade III. LIMITATIONS:Only pathogens detected by one specific multiplex qPCR assay were identified, meaning that other pathogens causing diarrhoea in these calves could have been overlooked. CONCLUSIONS:Co-infections were not associated with a higher diarrhoeic grade compared to single-pathogen infections in calves. In single-pathogen infections, C. parvum was significantly more prevalent in samples with diarrhoeic grade III, when compared to diarrhoeic grades I and II combined.

Antibiotic treatment of piglets after birth is commonly carried out due to concern for development of omphalitis leading to umbilical outpouchings and/or systemic infections. Among others, the portal of entry for bacterial infections includes the umbilical cord at birth. The aim was to characterise the histological and bacteriological pattern of manifestations in the umbilicus of piglets with omphalitis that died during the suckling period in a Danish herd. A total of 37 piglets found dead or euthanised due to sickness before weaning were included. Histopathological omphalitis was diagnosed in 13 of these piglets, and umbilical lesions and bacteria were most often observed in association with the umbilical blood vessels. Neutrophilic granulocyte infiltrations were observed in association with both umbilical arteries and the vein, occurring most frequently in the arteries. Escherichia coli and Trueperella pyogenes were the most commonly isolated bacteria from piglets with histopathological omphalitis. Omphalitis in piglets was characterized by inflammation and presence of bacteria in the umbilical arteries and to a lesser extent the umbilical vein. Inflammation in urachus was not present.

Methods for assembly and annotation of whole genomic sequences were compared for six strains of avian pathogenic Escherichia coli (APEC). Two vertically transferred E. coli clones, represented by three isolates all belonging to pulse field genome electrophoresis (PFGE) type 65- sequence type (ST)95 and three isolates belonging to PFGE type 47- ST131, were selected for Illumina short read sequencing. There was no significant difference between SPAdes and CLC Genomic Workbench for benchmark parameters to assemble the short reads. The six strains were also sequenced by long read sequencing (Nanopore) and these reads were hybrid assembled with the short reads. Unicycler provided a lower number of contigs and higher NG50 compared to Flye. No significant differences between total length of genomes were obtained from the four assemblers. At least 2.1 and 0.9 % of coding gene sequences (CDSs) annotated with Rapid annotations using subsystems technology (RAST) and PROKKA, respectively were wrongly annotated. The errors were most often associated to CDS of shorter length (<150 nt) with functions such as transposases, mobile genetic elements or being hypothetical. The investigation points out the importance of controlling automatic annotations and suggest further work to improve annotations in strains not belonging to the K12 or B lineages.