Molecular characterization of Glaesserella parasuis strains circulating in North American swine production systems.

BACKGROUND: Glaesserella parasuis is the causative agent of Glässer's disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted. RESULTS: In silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency;  blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%).   CONCLUSION: This study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control.

Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

Streptococcus gallolyticus and Bacterial Endocarditis in Swine, United States, 2015-2020.

To evaluate trends in bacterial causes of valvular endocarditis in swine, we retrospectively analyzed 321 cases diagnosed at Iowa State University Veterinary Diagnostic Laboratory (Ames, IA, USA) during May 2015--April 2020. Streptococcus gallolyticus was the causative agent for 7.59% of cases. This emerging infection in swine could aid study of endocarditis in humans.

An internally validated prediction model for critical COVID-19 infection and intensive care unit admission in symptomatic pregnant women.

BACKGROUND: Pregnant women are at an increased risk of mortality and morbidity owing to COVID-19. Many studies have reported on the association of COVID-19 with pregnancy-specific adverse outcomes, but prediction models utilizing large cohorts of pregnant women are still lacking for estimating the risk of maternal morbidity and other adverse events. OBJECTIVE: The main aim of this study was to develop a prediction model to quantify the risk of progression to critical COVID-19 and intensive care unit admission in pregnant women with symptomatic infection. STUDY DESIGN: This was a multicenter retrospective cohort study including 8 hospitals from 4 countries (the United Kingdom, Austria, Greece, and Turkey). The data extraction was from February 2020 until May 2021. Included were consecutive pregnant and early postpartum women (within 10 days of birth); reverse transcriptase polymerase chain reaction confirmed SARS-CoV-2 infection. The primary outcome was progression to critical illness requiring intensive care. The secondary outcomes included maternal death, preeclampsia, and stillbirth. The association between the primary outcome and 12 candidate predictors having a known association with severe COVID-19 in pregnancy was analyzed with log-binomial mixed-effects regression and reported as adjusted risk ratios. All the potential predictors were evaluated in 1 model and only the baseline factors in another. The predictive accuracy was assessed by the area under the receiver operating characteristic curves. RESULTS: Of the 793 pregnant women who were positive for SARS-CoV-2 and were symptomatic, 44 (5.5%) were admitted to intensive care, of whom 10 died (1.3%). The 'mini-COvid Maternal Intensive Therapy' model included the following demographic and clinical variables available at disease onset: maternal age (adjusted risk ratio, 1.45; 95% confidence interval, 1.07-1.95; P=.015); body mass index (adjusted risk ratio, 1.34; 95% confidence interval, 1.06-1.66; P=.010); and diagnosis in the third trimester of pregnancy (adjusted risk ratio, 3.64; 95% confidence interval, 1.78-8.46; P=.001). The optimism-adjusted area under the receiver operating characteristic curve was 0.73. The 'full-COvid Maternal Intensive Therapy' model included body mass index (adjusted risk ratio, 1.39; 95% confidence interval, 1.07-1.95; P=.015), lower respiratory symptoms (adjusted risk ratio, 5.11; 95% confidence interval, 1.81-21.4; P=.007), neutrophil to lymphocyte ratio (adjusted risk ratio, 1.62; 95% confidence interval, 1.36-1.89; P<.001); and serum C-reactive protein (adjusted risk ratio, 1.30; 95% confidence interval, 1.15-1.44; P<.001), with an optimism-adjusted area under the receiver operating characteristic curve of 0.85. Neither model showed signs of a poor fit. Categorization as high-risk by either model was associated with a shorter diagnosis to intensive care unit admission interval (log-rank test P<.001, both), higher maternal death (5.2% vs 0.2%; P<.001), and preeclampsia (5.7% vs 1.0%; P<.001). A spreadsheet calculator is available for risk estimation. CONCLUSION: At presentation with symptomatic COVID-19, pregnant and recently postpartum women can be stratified into high- and low-risk for progression to critical disease, even where resources are limited. This can support the nature and place of care. These models also highlight the independent risk for severe disease associated with obesity and should further emphasize that even in the absence of other comorbidities, vaccination is particularly important for these women. Finally, the model also provides useful information for policy makers when prioritizing national vaccination programs to quickly protect those at the highest risk of critical and fatal COVID-19.

The role of laboratory parameters in predicting severity of COVID-19 disease in pregnant patients.

We aimed to examine the relationship between laboratory markers and the severity of the disease in pregnant women diagnosed with coronavirus disease 2019 (COVID-19). Clinical records were retrospectively reviewed for 112 pregnant women. Patients diagnosed with COVID-19 were divided into two groups as mild/moderate and severe. The relationship between predicting the severity of the disease and laboratory parameters was investigated. Neutrophil lymphocyte ratio, C-reactive protein (CRP), ferritin and aspartate aminotransferase levels were significantly higher in severe COVID-19 cases than mild/moderate cases (p = .048, p = .003, p = .015 and p = .035, respectively). CRP was found to be the most useful marker in terms of diagnostic performance with a cut off value of 10.8 (sensitivity 80%, specificity 56.1%, NPV 88.5% and PPV 40.0%). The best diagnostic performance was obtained using CRP and ferritin combined with cut-offs of 10.8 mg/L for CRP and 26.5 µg/L for ferritin. Combined CRP and ferritin showed sensitivity, specificity, negative predictive value and positive predictive value of 94.7%, 52.8%, 96.6% and 41.9%, respectively, in predicting severe COVID-19. The combination of CRP and ferritin parameters may be useful in estimating the severity of the disease in pregnant patients who were initially diagnosed with COVID-19. Impact StatementWhat is already known about this subject? Coronavirus disease 2019 (COVID-19) can rapidly develop into acute respiratory distress syndrome (ARDS) and result in serious complications in some pregnant patients. Therefore, timely diagnosis of patients is crucial. Most previous reports of COVID-19 laboratory results are based on data from the general population and limited information is available regarding pregnancy status. Although laboratory medicine makes an important contribution to clinical decision making in many infectious diseases, including COVID-19, studies to predict the severity of the disease with laboratory markers are limited and the results are contradictory.What do the results of this study add? Our study shows that C-reactive protein (CRP), neutrophil lymphocyte ratio (NLR), ferritin and aspartate aminotransferase (AST) are associated with severe disease in pregnant women diagnosed with COVID-19. In addition, the use of combined CRP and ferritin appears to have higher sensitivity and negative predictive value than using other tests alone. Furthermore, this study shows that coagulation markers are not useful in predicting disease severity in pregnancy.What are the implications of these findings for clinical practice and/or further research? Predicting the severity of COVID-19 disease in pregnancy can prevent unnecessary hospitalisations and allow the implementation of the necessary clinical approach. Further studies can focus on the clinical usefulness of these parameters in predicting severe COVID-19 in pregnancy.

Short-term outcomes of pregnant women with convalescent COVID-19 and factors associated with false-negative polymerase chain reaction test: A prospective cohort study.

AIM: To evaluate the clinical factors associated with false-negative RT-PCR results and to report the outcome of a cohort of pregnant women with COVID-19. METHODS: This cohort study was conducted in a tertiary referral pandemic hospital and included 56 pregnant women. A study including pregnant women with either a laboratory or clinical diagnosis for COVID-19 were included in the study. The primary outcome was clinical factors associated with false-negative RT-PCR results defined as a positive immunoglobulin M assessed by rapid testing in clinically diagnosed patients. Clinical outcomes of laboratory diagnosed patients were also reported. RESULTS: In total, 56 women with either RT-PCR or clinical COVID-19 diagnosis were included in the study. Forty-three women either had RT-PCR positivity or IgM positivity. The clinical outcome of these pregnancies was as follows: mean maternal age 27.7, immunoglobulin M positive patients 76.7%, RT-PCR positive patients 55.8%, maternal comorbidities 11.5%, complications in patients below 20 weeks 34.8%, complications in patients above 20 weeks 65.1%, elevated CRP 83.7%, lymphopenia 30.2%, time from hospital admission to final follow-up days 37 and stillbirth 8.3%. The proportion of women who tested positive for SARS-CoV-2 immunoglobulin M was 100% in the RT-PCR positive group and 56.5% in the clinical diagnosis group (P = .002). The symptom onset to RT-PCR testing interval longer than a week (risk ratio: 2.72, 95% CI: 1.14-5.40, P = .003) and presence of dyspnoea (risk ratio: 0.38, 95% CI: 0.14-0.89, P = .035) were associated with false-negative RT-PCR tests. The area under the curve of these parameters predicting false-negative RT-PCR was 0.73 (95% CI: 0.57-0.89). CONCLUSIONS: Symptomatic women with a negative RT-PCR should not be dismissed as potential COVID-19 patients, especially in the presence of prolonged symptom onset-test interval and in women without dyspnoea.

Role of metAB in Methionine Metabolism and Optimal Chicken Colonization in Campylobacter jejuni.

Campylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain type NCTC 11168); however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to l-methionine. Comparative genomics identified differential distribution of the genes metA and metB, which function to convert homoserine for downstream production of l-methionine, between IA3902 and W7, which could enable a secondary pathway for l-methionine biosynthesis in a W7 ΔluxS but not in an IA3902 ΔluxS strain. To test the hypothesis that the genes metA and metB contribute to l-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison, the W7 ΔmetAB ΔluxS and IA3902 ΔluxS::metAB mutants. Quantitative reverse transcription-PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in the IA3902 ΔluxS::metAB mutant and absent in the W7 ΔmetAB ΔluxS mutant. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated that l-methionine and S-adenosyl methionine concentrations decreased in the W7 ΔmetAB ΔluxS mutant and increased in the IA3902 ΔluxS::metAB mutant. Assessment of chicken colonization revealed that the IA3902 ΔluxS::metAB strain partially rescued the colonization defect of the IA3902 ΔluxS strain, while the W7 ΔmetAB ΔluxS strain showed significantly decreased colonization compared to that of the wild-type and the W7 ΔluxS strain. These results indicate that the ability to maintain l-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

Role of enterotoxigenic Escherichia coli prophage in spreading antibiotic resistance in a porcine-derived environment.

Enterotoxigenic Escherichia coli (ETEC) cause acute secretory diarrhoea in pigs, posing a great economic loss to the swine industry. This study analysed the prevalence and genetic characteristics of prophages from 132 ETEC isolates from symptomatic pigs to determine their potential for spreading antibiotic resistance. A total of 1105 potential prophages were identified, and the distribution of the genome size showed three 'overlapping' trends. Similarity matrix comparison showed that prophages correlated with the ETEC lineage distribution, and further identification of these prophages corroborated the lineage specificity. In total, 1206 antibiotic resistance genes (ARGs) of 52 different categories were identified in 132 ETEC strains; among these, 2.65% (32/1206) of ARGs were found to be carried by prophages. Analysis of flanking sequences showed that almost all the ARGs could be grouped into two types: 'blaTEM-1B ' and 'classic class 1 integron (IntI1)'. They co-occurred with a strictly conserved recombinase and transposon Tn3 family but with a difference: the 'blaTEM-1B type' prophages exhibited a classic Tn2 transposon structure with 100% sequence identity, whereas the 'IntI1 type' co-occurred with the TnAs2 transposon with only 84% sequence identity. These results imply that ARGs might be pervasive in natural bacterial populations through transmission by transposable bacteriophages.

The Rho-Independent Transcription Terminator for the porA Gene Enhances Expression of the Major Outer Membrane Protein and Campylobacter jejuni Virulence in Abortion Induction.

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. Its porA gene encodes the major outer membrane protein (MOMP) that is abundantly expressed and has important physiological functions, including a key role in systemic infection and abortion induction in pregnant animals. Despite the importance of porA in C. jejuni pathogenesis, mechanisms modulating its expression levels remain elusive. At the 3' end of the porA transcript, there is a Rho-independent transcription terminator (named T porA in this study). Whether T porA affects the expression and function of MOMP remains unknown and is investigated in this study. Green fluorescent protein (GFP) fusion constructs with the porA promoter at the 5' end and an intact T porA or no T porA at the 3' end of the gfp coding sequence revealed that both the transcript level of gfp and its fluorescence signals were more than 2-fold higher in the construct with T porA than in the one without T porA Real-time quantitative PCR (qRT-PCR) analysis of the porA mRNA and immunoblot detection of MOMP in C. jejuni showed that disruption of T porA significantly reduced the porA transcript level and the expression of MOMP. An mRNA decay assay demonstrated that disruption of T porA resulted in a shortened transcript half-life of the upstream gfp or porA gene, indicating that T porA enhances mRNA stability. In the guinea pig model, the C. jejuni construct with an interrupted T porA was significantly attenuated in abortion induction. Together, these results indicate that T porA enhances the expression level of MOMP by stabilizing its mRNA and influences the virulence of C. jejuni.

High Prevalence of Fluoroquinolone-Resistant Campylobacter Bacteria in Sheep and Increased Campylobacter Counts in the Bile and Gallbladders of Sheep Medicated with Tetracycline in Feed.

Campylobacter is a major foodborne pathogen in humans and a significant cause of abortion in sheep. Although ruminants are increasingly recognized as important reservoirs for Campylobacter species, limited information is available about the molecular epidemiology and antimicrobial resistance (AMR) profiles of sheep Campylobacter Here, we describe a two-trial study that examined Campylobacter profiles in sheep and determined whether in-feed tetracycline (TET) influenced the distribution and AMR profiles of Campylobacter Each trial involved 80 commercial sheep naturally infected with Campylobacter: 40 of these sheep were medicated with tetracycline in feed, while the other 40 received feed without antibiotics. Fecal and bile samples were collected for the isolation of Campylobacter The bacterial isolates were analyzed for antimicrobial susceptibility and genotypes. The results revealed that 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter (Campylobacter jejuni and Campylobacter coli), with no significant differences between the medicated and nonmedicated groups. All but one of the tested Campylobacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resistance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant to FQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coli ST902 were the predominant genotypes in the sheep. Feed medication with tetracycline did not affect the overall prevalence, species distribution, and AMR profiles of Campylobacter, but it did increase the total Campylobacter counts in bile and gallbladder. These findings identify predominant Campylobacter clones, reveal the high prevalence of FQ-resistant C. coli, and provide new insights into the epidemiology of Campylobacter in sheep.IMPORTANCECampylobacter is a major cause of foodborne illness in humans, and antibiotic-resistant Campylobacter is considered a serious threat to public health in the United States and worldwide. As a foodborne pathogen, Campylobacter commonly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Results from this study reveal the predominant genotypes and high prevalence of tetracycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter The finding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as this class of antibiotics is not used for sheep in the United States, and it may suggest the transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep. Additionally, the results demonstrate that in-feed medication with tetracycline increases Campylobacter counts in gallbladders, suggesting that the antibiotic promotes Campylobacter colonization of the gallbladder. These findings provide new information on Campylobacter epidemiology in sheep, which may be useful for curbing the spread of antibiotic-resistant Campylobacter in animal reservoirs.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Investigating the Suitability of a Laboratory Mouse Model to Study the Pathogenesis of Abortifacient Campylobacter jejuni.

The aim of this study was to assess whether pregnant mice represent a useful model to study the reproductive pathology of Campylobacter jejuni IA3902 using the end point of positive microbial culture of the organism from the fetoplacental unit. Pregnant BALB/c and CD-1 mice (14 days' gestation) were inoculated orally and intraperitoneally (IP) with 1 × 109 colony-forming units/ml of C. jejuni IA3902. The organism was recovered by microbial culture from the fetoplacental unit in 10 of 10 BALB/c and 10 of 10 CD-1 IP-inoculated pregnant mice and in 29% (2/7) BALB/c and 38% (3/8) CD-1 orally inoculated pregnant mice. Gross reproductive lesions included necrosuppurative placentitis, fetal resorption, intrauterine fetal death, stillborn pups (dead neonates), and multifocal hepatitis. Histological changes consisted of locally extensive neutrophilic and necrotizing placentitis with intralesional bacterial colonies of C. jejuni, ulcerative endometritis, random multifocal hepatitis, and rare cholecystitis. Immunohistochemistry for the major outer membrane protein of C. jejuni revealed moderate to large numbers of the organism at the periphery of the placental discs, within trophoblasts and extracellularly, with invasion into the placental disc largely via the vascular network. The organism is trophic for neutral mucin, iron, and L-fucose within the murine placenta. C. jejuni IA3902 has affinity for the murine reproductive tract, specifically the fetoplacental unit, where it results in a necrotizing placentitis with positive microbial recovery after both IP and oral challenge in BALB/c and CD-1 pregnant mice.

Point mutations in the major outer membrane protein drive hypervirulence of a rapidly expanding clone of Campylobacter jejuni.

Infections due to clonal expansion of highly virulent bacterial strains are clear and present threats to human and animal health. Association of genetic changes with disease is now a routine, but identification of causative mutations that enable disease remains difficult. Campylobacter jejuni is an important zoonotic pathogen transmitted to humans mainly via the foodborne route. C. jejuni typically colonizes the gut, but a hypervirulent and rapidly expanding clone of C. jejuni recently emerged, which is able to translocate across the intestinal tract, causing systemic infection and abortion in pregnant animals. The genetic basis responsible for this hypervirulence is unknown. Here, we developed a strategy, termed "directed genome evolution," by using hybridization between abortifacient and nonabortifacient strains followed by selection in an animal disease model and whole-genome sequence analysis. This strategy successfully identified SNPs in porA, encoding the major outer membrane protein, are responsible for the hypervirulence. Defined mutagenesis verified that these mutations were both necessary and sufficient for causing abortion. Furthermore, sequence analysis identified porA as the gene with the top genome-wide signal of adaptive evolution using Fu's Fs, a population genetic metric for recent population size changes, which is consistent with the recent expansion of clone "sheep abortion." These results identify a key virulence factor in Campylobacter and a potential target for the control of this zoonotic pathogen. Furthermore, this study provides general, unbiased experimental and computational approaches that are broadly applicable for efficient elucidation of disease-causing mutations in bacterial pathogens.

Genotypes and Antimicrobial Susceptibility Profiles of Hemolytic Escherichia coli from Diarrheic Piglets.

Hemolytic Escherichia coli are important pathogens in neonatal and weaned pigs. In this study, we analyzed 95 hemolytic E. coli isolated from intestinal contents or fecal samples of diarrheic piglets in 15 states of the United States between November 2013 and December 2014. Phenotypic antimicrobial susceptibility was determined through Sensititre BOFO6F plates for all the strains. They were all resistant to clindamycin, penicillin, tiamulin, tilmicosin, and highly resistant to oxytetracycline (91.6%), chlortetracycline (78.9%), ampicillin (75.8%), and sulfadimethoxine (68.4%). 86.2% of them were multidrug resistant. Whole genome sequencing (WGS) showed that 55 strains were enterotoxigenic E. coli (ETEC) and 40 strains were non-ETEC, and the strains belonged to 22 known and 2 novel sequence types (STs). ST100 and ST10 were the main and predominant STs in ETEC strains, whereas the non-ETEC strains were diverse with ST23 and ST761 as the main STs. Antibiotic resistance gene/mutation profiling of the genomes confirmed the results of antimicrobial susceptibility test. Notably, significant differences were found in the susceptibility to enrofloxacin between ETEC and non-ETEC (58.2% vs. 5.0%) and gentamicin (32.7% vs. 7.5%). ampH, ampC2, and ampC1 were the most common beta-lactamase genes in all E. coli strains, and extended-spectrum beta-lactamase (ESBL) genes were rare in these isolates. This study provides new insights into antibiotic resistance and genotypes of intestinal pathogenic E. coli associated with swine disease in the United States, and support the utility of WGS in accurate prediction of resistance to most antibiotics.

Utilization of macrophage extracellular trap nucleotides by Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia in swine, an important disease worldwide. It has finite biosynthetic capabilities, including a deficit in de novo nucleotide synthesis. The source(s) for nucleotides in vivo are unknown, but mycoplasmas are known to carry membrane-bound nucleases thought to participate in the acquisition of nucleotides from host genomic DNA. Recent research has demonstrated that neutrophils can produce extracellular traps (NETs), chromatin NETs decorated with granular proteins to interact with and eliminate pathogens. We hypothesized that M. hyopneumoniae could utilize its membrane nuclease to obtain nucleotides from extracellular traps to construct its own DNA. Using the human monocytic cell line THP-1, we induced macrophage extracellular traps (METs), which are structurally similar to NETs. The thymidine analogue ethynyl deoxyuridine (EdU) was incorporated into THP-1 DNA and METs were induced. When incubated with M. hyopneumoniae, METs were degraded and the modified nucleotide label could be co-localized within M. hyopneumoniae DNA. When the nucleases were inhibited, MET degradation and nucleotide transfer were also inhibited. Controls confirmed that the EdU originated directly from the METs and not from free nucleotides arising from intracellular pools released during extrusion of the chromosomal DNA. M. hyopneumoniae incorporated labelled nucleotides more efficiently when 'fed' on METs than from free nucleotides in the medium, suggesting a tight linkage between nuclease degradation of DNA and nucleotide transport. These results strongly suggest that M. hyopneumoniae could degrade extracellular traps formed in vivo during infection and incorporate those host nucleotides into its own DNA.

Campylobacter Prevalence and Quinolone Susceptibility in Feces of Preharvest Feedlot Cattle Exposed to Enrofloxacin for the Treatment of Bovine Respiratory Disease.

Campylobacter spp. can be pathogenic to humans and often harbor antimicrobial resistance genes. Data on resistance in relation to fluoroquinolone use in beef cattle are scarce. This cross-sectional study of preharvest cattle evaluated Campylobacter prevalence and susceptibility to nalidixic acid and ciprofloxacin in feedlots that previously administered a fluoroquinolone as primary treatment for bovine respiratory disease. Twenty fresh fecal samples were collected from each of 10 pens, in each of five feedlots, 1-2 weeks before harvest. Feces were cultured for Campylobacter using selective enrichment and isolation methods. Genus and species were confirmed via PCR. Minimum inhibitory concentrations (MICs) of ciprofloxacin and nalidixic acid were determined using a micro-broth dilution method and human breakpoints. Antimicrobial use within each pen was recorded. Data were analyzed using generalized linear mixed-models (prevalence) and survival analysis (MICs). Overall, sample-level prevalence of Campylobacter was 27.2% (272/1000) and differed significantly among feedlots (p < 0.01). Campylobacter coli was the most common species (55.1%; 150/272), followed by Campylobacter hyointestinalis (42.6%; 116/272). Within-pen prevalence was not significantly associated with the number of fluoroquinolone treatments, sex, body weight, or metaphylaxis use, but was associated with the number of days cattle were in the feedlot (p = 0.03). The MICs for the majority of Campylobacter isolates were above the breakpoints for nalidixic acid (68.4%; 175/256) and for ciprofloxacin (65.6%; 168/256). Distributions of MICs for nalidixic acid (p ≤ 0.01) and ciprofloxacin (p ≤ 0.05) were significantly different among feedlots, and by Campylobacter species. However, fluoroquinolone treatments, sex, body weight, days on feed, and metaphylaxis were not significantly associated with MIC distributions within pens. We found no evidence that the number of fluoroquinolone treatments within feedlot pens significantly affected the within-pen fecal prevalence or quinolone susceptibilies of Campylobacter in feedlots that used a fluoroquinolone as primary treatment for bovine respiratory disease.

Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni.

Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni.

Identification and functional analysis of two toxin-antitoxin systems in Campylobacter jejuni.

Toxin-antitoxin (TA) systems are widely distributed in bacteria and play an important role in maintaining plasmid stability. The leading foodborne pathogen, Campylobacter jejuni, can carry multiple plasmids associated with antibiotic resistance or virulence. Previously a virulence plasmid named pVir was identified in C. jejuni 81-176 and IA3902, but determining the role of pVir in pathogenesis has been hampered because the plasmid cannot be cured. In this study, we report the identification of two TA systems that are located on the pVir plasmid in 81-176 and IA3902, respectively. The virA (proteic antitoxin)/virT (proteic toxin) pair in IA3902 belongs to a Type II TA system, while the cjrA (RNA antitoxin)/cjpT (proteic toxin) pair in 81-176 belongs to a Type I TA system. Notably, cjrA (antitoxin) represents the first noncoding small RNA demonstrated to play a functional role in Campylobacter physiology to date. By inactivating the TA systems, pVir was readily cured from Campylobacter, indicating their functionality in Campylobacter. Using pVir-cured IA3902, we demonstrated that pVir is not required for abortion induction in the guinea pig model. These findings establish the key role of the TA systems in maintaining plasmid stability and provide a means to evaluate the function of pVir in Campylobacter pathobiology.

Emergence of a plasmid-borne multidrug resistance gene cfr(C) in foodborne pathogen Campylobacter.

Objectives: To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen. Methods: WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST. Results: The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion. Conclusions: These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens.

A Mutator Phenotype Promoting the Emergence of Spontaneous Oxidative Stress-Resistant Mutants in Campylobacter jejuni.

Campylobacter jejuni is a leading cause of foodborne illnesses worldwide. As a microaerophilic organism, C. jejuni must be able to defend against oxidative stress encountered both in the host and in the environment. How Campylobacter utilizes a mutation-based mechanism for adaptation to oxidative stress is still unknown. Here we present a previously undescribed phenotypic and genetic mechanism that promotes the emergence of oxidative stress-resistant mutants. Specifically, we showed that a naturally occurring mutator phenotype, resulting from a loss of function mutation in the DNA repair enzyme MutY, increased oxidative stress resistance (OXR) in C. jejuni We further demonstrated that MutY malfunction did not directly contribute to the OXR phenotype but increased the spontaneous mutation rate in the peroxide regulator gene perR, which functions as a repressor for multiple genes involved in oxidative stress resistance. Mutations in PerR resulted in loss of its DNA binding function and derepression of PerR-controlled oxidative stress defense genes, thereby conferring an OXR phenotype and facilitating Campylobacter survival under oxidative stress. These findings reveal a new mechanism that promotes the emergence of spontaneous OXR mutants in bacterial organisms.IMPORTANCE Although a mutator phenotype has been shown to promote antibiotic resistance in many bacterial species, little is known about its contribution to the emergence of OXR mutants. This work describes the link between a mutator phenotype and the enhanced emergence of OXR mutants as well as its underlying mechanism involving DNA repair and mutations in PerR. Since DNA repair systems and PerR are well conserved in many bacterial species, especially in Gram positives, the same mechanism may operate in multiple bacterial species. Additionally, we developed a novel method that allows for rapid quantification of spontaneous OXR mutants in a bacterial population. This method represents a technical innovation and may also be applied to other bacterial species. These findings significantly advance our understanding of bacterial mechanisms for survival under oxidative stress.