Absence of bovine leukemia virus in the buffy coats of breast cancer cases from Alabama, USA.

Breast cancer is reported as one of the most common and deadly cancers among females. Recent findings have suggested that bovine leukemia virus (BLV), a highly prevalent bovine virus worldwide, might be linked to human breast cancer. However, the involvement of BLV as a risk factor for breast cancer remains controversial. In this study, BLV FRET-PCR was carried out on 238 blood-derived DNA samples from breast cancer patients from the Alabama Hereditary Cancer Cohort. In addition, randomly selected samples (n = 20) were evaluated by WGS for the presence of BLV genome. No BLV proviral DNA was detected in any of 238 samples assayed by FRET-qPCR in this study. Similarly, the WGS analysis did not detect the presence of the BLV genome in the DNA of the buffy coats from 20 randomly selected patients with breast cancer. This study did not support the findings of suggesting an association between BLV and breast cancer. Notably, nearly all the studies using in situ PCR and immunohistochemistry demonstrated positive associations while other studies using whole-genome sequencing and other methods failed to identify the BLV association with breast cancer. Further studies including all reported BLV detection techniques/methods on the same breast cancer sample sets would appear to be the most likely way of resolving the current contradictory evidence.

Efficient affinity-tagging of M13 phage capsid protein IX for immobilization of protein III-displayed oligopeptide probes on abiotic platforms.

We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011 pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.

Molecular Detection of Bartonella spp. in China and St. Kitts.

Bartonella are vector-borne hemotropic bacteria that infect a wide variety of hosts, including people. While there are PCR assays that can identify individual or groups of Bartonella, there is no reliable molecular method to simultaneously detect all species while maintaining genus specificity and sensitivity. By comparing highly conserved 16S rRNA sequences of the better-recognized Bartonella spp. on GenBank, we selected primers and probes for a genus-specific pan-Bartonella FRET-qPCR. Then, a gltA-based Bartonella PCR was established by selecting primers for a highly variable region of gltA, of which the sequenced amplicons could identify individual Bartonella spp. The pan-Bartonella FRET-qPCR did not detect negative controls (Brucella spp., Anaplasma spp., Rickettsia spp., Coxiella burnetii, and Wolbachia) but reliably detected as few as two copies of the positive control (Bartonella henselae) per reaction. There was complete agreement between the pan-Bartonella FRET-qPCR and the gltA-based Bartonella PCR in detecting Bartonella in convenience test samples from China and St. Kitts: cats (26%; 81/310), Ctenocephalides felis (20%; 12/60), cattle (24%; 23/98), and donkeys (4%; 1/20). Sequencing of the gltA-based Bartonella PCR products revealed B. henselae (70%; 57/81) and B. clarridgeiae (30%; 24/81) in cats and C. felis (67%; 8/12, and 33%; 4/12, respectively) and B. bovis in cattle (23.5%; 23/98) and donkeys (4.0%; 1/24). The pan-Bartonella FRET-qPCR and gltA-based Bartonella PCR we developed are highly sensitive and specific in detecting recognized Bartonella spp. in a single reaction. The pan-Bartonella FRET-qPCR is convenient requiring no gel electrophoresis and providing copy numbers, while the gltA-based Bartonella PCR reliably differentiates individual Bartonella species. The use of these PCRs should greatly facilitate large-scale surveillance studies and the diagnosis of infections in clinical samples.

Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the "Big Six." The "Big Six" strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain's individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this "Big Six" reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.

Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx- strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx-) evolutionary paths.

Population structure and genetic diversity of Streptococcus suis isolates obtained from the United States.

Diseases caused by the zoonotic pathogen Streptococcus suis are an extensive economic problem as well as an animal welfare concern for the global swine industry. Previous studies have evaluated the genomic diversity and population structure of S. suis isolates, however, the majority of these studies utilized isolates obtained from countries other than the U.S. This study applied whole genome sequencing and cgMLST-based typing to evaluate the population structure and genetic relatedness among S. suis isolates obtained within the U.S. The established high-resolution phylogenomic framework revealed extensive genomic variation and diversity among the sampled S. suis isolates, with isolates from the U.S. and from countries outside the U.S. found interspersed in the phylogeny. S. suis isolates obtained within the U.S. did not cluster by state or geographic location, however, isolates with similar serotypes, both obtained from within and outside the U.S., generally clustered together. Average nucleotide identity (ANI) values determined for the S. suis genomes were extensively broad, approaching the recommended species demarcation value, and correlated with the phylogenetic group distribution of the cgMLST-based tree. Numerous antimicrobial resistance (AMR) elements were identified among both U.S. and non-U.S. isolates with ble, tetO, and ermB genes identified as the most prevalent. The epf, mrp, and sly genes, historically used as markers for virulence potential, were also observed in the genomes of isolates that grouped together forming a subclade of clonal complex 1 (CC1) isolates. Collectively, the data in this report provides critical information needed to address potential biosurveillance needs and insights into the genetic diversity and population structure of S. suis isolates obtained within the U.S.

Detection of zoonotic vector-borne pathogens in domestic dogs in Giza, Egypt.

The public health implications of zoonotic vector-borne pathogens are numerous because domestic animals, such as dogs, live in close proximity to humans. Blood was collected from 116 domestic dogs in Cairo, Egypt from three different settings at the human-animal interface. The three settings the dogs came from were: privately owned animals seeking care at the Cairo University Faculty of Veterinary Medicine Clinic, non-laboratory reared research dogs maintained at the Cairo University Faculty of Veterinary Medicine, and an urban private animal rescue in Shabramont, Giza, Egypt. Enrolled animals were visually inspected for presence of flea or tick ectoparasites, Rhipicephalus sanguineus sensu lato ticks were recovered from 56 enrolled animals and a flea identified as Ctenocephalides felis was recovered from one animal. To test for past and/or current infection with vector-borne pathogens, conventional PCR and IDEXX SNAP® 4Dx® Plus were performed on whole blood. Pathogen targets included: Anaplasma spp., Ehrlichia spp., Babesia spp., Borrelia spp., Bartonella spp., Dirofilaria spp., and Rickettsia spp. Among dogs sampled across all locations, one dog was positive for Babesia sp. infection and one dog was positive for Anaplasma sp. infection as detected by PCR and confirmed by Sanger sequencing. Three additional dogs were positive for infection but had incomplete sequences obtained: two for Ehrlichia sp. and one for Borrelia sp. The SNAP® test results for all sampled dogs included: eight dogs positive for Anaplasma spp., 14 dogs positive for Ehrlichia spp., and five additional dogs positive for both Anaplasma spp. and Ehrlichia spp. SNAP® test results by sampling location showed that 66% of the dogs at the animal rescue were positive for Anaplasma spp. and/or Ehrlichia spp., 17% of the privately owned dogs at the Faculty of Veterinary medicine were positive for Anaplasma spp. and/or Ehrlichia spp., and none of the research dogs were positive for any of the targets on the SNAP® test. This high proportion of seropositivity in the animals sampled indicates a vector population which is not well controlled and a need for continued owner education and promotion of consistent use of preventive medications and the risk for zoonotic transmission.

Genome Sequence Analysis and Characterization of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains Associated With a Laboratory Infection.

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae-positive O157:H7 strains isolated from the patient's stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains' individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher's life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection.

Disseminated Toxoplasma gondii Infection in an Adult Osprey (Pandion haliaetus).

An adult female osprey (Pandion haliaetus) was found weak and unable to fly in Auburn, Alabama in August 2019. The bird was captured and submitted to the Southeastern Raptor Center of the Auburn University College of Veterinary Medicine for evaluation. On presentation, the bird was thin with a body condition score of approximately 1.5 out of 5. The bird died during the examination and was submitted for necropsy. At the necropsy, there was a severe loss of muscle mass over the body, and the keel was prominent. The liver and spleen were moderately enlarged with pale tan to red foci randomly scattered throughout the parenchyma. A histopathologic observation revealed multifocal to coalescing areas of necrosis and hemorrhage with intralesional protozoans in the liver, spleen, lungs, kidney, sciatic nerve, esophagus, cerebrum, heart, and proventriculus. Immunohistochemistry using anti-Toxoplasma gondii-specific antibodies showed a strong positive labeling of the parasite. Semi-nested PCR, specific for the B1 gene of T. gondii, successfully identified T. gondii. This is the first confirmed case of T. gondii infection in an osprey.

Concurrent Resistance to Carbapenem and Colistin Among Enterobacteriaceae Recovered From Human and Animal Sources in Nigeria Is Associated With Multiple Genetic Mechanisms.

Resistance to last resort drugs such as carbapenem and colistin is a serious global health threat. This study investigated carbapenem and colistin resistance in 583 non-duplicate Enterobacteriaceae isolates utilizing phenotypic methods and whole genome sequencing (WGS). Of the 583 isolates recovered from humans, animals and the environment in Nigeria, 18.9% (110/583) were resistant to at least one carbapenem (meropenem, ertapenem, and imipenem) and 9.1% (53/583) exhibited concurrent carbapenem-colistin resistance. The minimum inhibitory concentrations of carbapenem and colistin were 2-32 µg/mL and 8 to >64 µg/mL, respectively. No carbapenem resistant isolates produced carbapenemase nor harbored any known carbapenemase producing genes. WGS supported that concurrent carbapenem-colistin resistance was mediated by novel and previously described alterations in chromosomal efflux regulatory genes, particularly mgrB (M1V) ompC (M1_V24del) ompK37 (I70M, I128M) ramR (M1V), and marR (M1V). In addition, alterations/mutations were detected in the etpA, arnT, ccrB, pmrB in colistin resistant bacteria and ompK36 in carbapenem resistant bacteria. The bacterial isolates were distributed into 37 sequence types and characterized by the presence of internationally recognized high-risk clones. The results indicate that humans and animals in Nigeria may serve as reservoirs and vehicles for the global spread of the isolates. Further studies on antimicrobial resistance in African countries are warranted.

Complete Genome Sequence of a Colistin-Resistant Klebsiella pneumoniae Isolate from Houseflies (Musca domestica) in a Trash Disposal Truck in the United States.

A Klebsiella pneumoniae strain isolated from houseflies in a trash disposal truck in the United States was resistant to colistin, a last-resort drug for treating infections caused by multidrug-resistant Gram-negative bacteria. Complete genome sequencing resulted in a total genome size of 5,337,408 bp for this isolate with a plasmid of 224,442 bp.

DIVERSE BARTONELLA SPP. DETECTED IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) AND ASSOCIATED KEDS (LIPOPTENA MAZAMAE) IN THE SOUTHEASTERN USA.

There are many known species of Bartonella, Gram-negative bacteria that can cause febrile illness and fatality in humans and animals. These pathogens are often transmitted through hematophagous arthropod vectors such as fleas and lice. Despite increasing awareness about Bartonella spp. and their zoonotic potential, as well as existing literature on Bartonella spp. in cervids, little is known about the diversity of Bartonella spp. in white-tailed deer (Odocoileus virginianus) and their associated keds in the southeastern US. We examined the prevalence and diversity of Bartonella spp. in an enclosed herd of white-tailed deer and their ectoparasites, deer keds (Lipoptena mazamae), in Alabama. The overall prevalence of Bartonella infection in this population of deer was 16% (10/63) and 24% (23/96) in keds associated with deer that we sampled. Three species of Bartonella were identified in both deer and their keds: Bartonella bovis, Bartonella schoenbuchensis, and Bartonella sp. 1. Additionally, Bartonella melophagi was detected in white-tailed deer but not in the sampled keds. The detection of four Bartonella species in one population of white-tailed deer, three of which have known zoonotic potential, highlights the importance of Bartonella diversity within host species.

Molecular and Serological Prevalence of Leptospira spp. in Feral Pigs (Sus scrofa) and their Habitats in Alabama, USA.

Leptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and a variety of wild and domestic animal species. In order to understand the prevalence and diversity of Leptospira spp. in feral pig populations of Alabama, we trapped 315 feral pigs in Bullock County east-central Alabama, and collected 97 environmental samples from riparian areas in Bullock County and Macon County east-central Alabama. Two previously published PCRs followed by DNA sequencing and BLASTn were performed to identify pathogenic Leptospira species in the kidney of feral pigs (3.2%, 10/315) as well as environmental samples collected from the habitats of feral pigs (2.1%, 2/97), but not in the whole blood samples (n = 276) or spleen (n = 51). An ELISA determined that 44.2% of serum samples (122/276) were antibody-positive for Leptospira. The identification of two pathogenic Leptospira species from environmental samples and the high sero-positivity in feral pigs suggests potential pathogen shedding from feral pigs to environments, and to humans and domestic animals. In order to better understand the risk to human health associated with feral swine presence, further studies are warranted to explore the interrelationship between Leptospira spp. shedding in the urine of feral pigs and bacterial culture to explore pathogenicity. Multi-locus sequencing typing (MLST) and microscopic agglutination tests (MAT) should be performed in future studies to make a definite determination of pathogenic Leptospira in feral pigs in Alabama.

Comparison of microbiota, antimicrobial resistance genes and mobile genetic elements in flies and the feces of sympatric animals.

Flies are well-known vectors of bacterial pathogens, but there are little data on their role in spreading microbial community and antimicrobial resistance. In this study, we compared the bacterial community, antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in flies with those in the feces of sympatric animals. A 16S rRNA-based microbial analysis identified 23 bacterial phyla in fecal samples and 25 phyla in flies; all the phyla identified in the fecal samples were also found in the flies. Bray-Curtis dissimilarity analysis showed that the microbiota of the flies were more similar to the microbiota of the feces of their sympatric animals than those of the feces from the three other animal species studied. The qPCR array amplified 276 ARGs/MGEs in fecal samples, and 216 ARGs/MGEs in the flies, while 198 of these genes were identified in both flies and feces. Long-term studies with larger sample numbers from more geospatially distinct populations and infection trials are indicated to further evaluate the possibility of flies as sentinels for antimicrobial resistance.

Identification of mobile colistin resistance genes (mcr-1.1, mcr-5 and mcr-8.1) in Enterobacteriaceae and Alcaligenes faecalis of human and animal origin, Nigeria.

Colistin is a last-resort drug used to treat infections caused by multidrug-resistant Gram-negative bacteria that have developed carbapenem resistance. Emergence and rapid dissemination of the nine plasmid-mediated mobile colistin resistance genes (mcr-1 to mcr-9) has led to fear of pandrug-resistant infections worldwide. To date, there is only limited information on colistin resistance in African countries where the drug is widely used in agriculture. In this Nigerian study, 583 non-duplicate bacterial strains were isolated from 1119 samples from humans, camels, cattle, dogs, pigs and poultry using colistin-supplemented MacConkey agar, among which 17.0% (99/583) were colistin-resistant. PCR (mcr-1 to mcr-9) and whole-genome sequencing (WGS) identified mcr in 21.2% (21/99) of colistin-resistant isolates: mcr-1.1 (n = 13), mcr-8.1 (n = 5), mcr-1.1 and mcr-8.1 (n = 2), and mcr-1.1 and mcr-5 (n = 1). Of the 21 mcr-positive strains, 9 were isolated from human samples, with 8 being Klebsiella pneumoniae, and 6 of these human K. pneumoniae had a high colistin MIC (>64 µg/mL). In contrast, 9 of the 12 mcr-positive animal isolates were Escherichia coli, of which only 2 had a colistin MIC of >64 µg/mL. This study is the first to report mcr-1 in Alcaligenes faecalis and the emergence of mcr-5 and mcr-8 in Nigeria. WGS determined that mcr-1 was localised on an IncX4 plasmid and that 95.2% of mcr-1 harbouring isolates (20/21) transferred colistin resistance successfully by conjugation. These findings highlight the global spread of colistin resistance and emphasise the urgent need for co-ordinated global action to combat resistant bacteria.

Peptide ELISA and FRET-qPCR Identified a Significantly Higher Prevalence of Chlamydia suis in Domestic Pigs Than in Feral Swine from the State of Alabama, USA.

Chlamydia suis is an important, highly prevalent, and diverse obligate intracellular pathogen infecting pigs. In order to investigate the prevalence and diversity of C. suis in the U.S., 276 whole blood samples from feral swine were collected as well as 109 fecal swabs and 60 whole blood samples from domestic pigs. C. suis-specific peptide ELISA identified anti-C. suis antibodies in 13.0% of the blood of feral swine (26/276) and 80.0% of the domestic pigs (48/60). FRET-qPCR and DNA sequencing found C. suis DNA in 99.1% of the fecal swabs (108/109) and 21.7% of the whole blood (13/60) of the domestic pigs, but not in any of the assayed blood samples (0/267) in feral swine. Phylogenetic comparison of partial C. suis ompA gene sequences and C. suis-specific multilocus sequencing typing (MLST) revealed significant genetic diversity of the C. suis identified in this study. Highly genetically diverse C. suis strains are prevalent in domestic pigs in the USA. As crowding strongly enhances the frequency and intensity of highly prevalent Chlamydia infections in animals, less population density in feral swine than in domestic pigs may explain the significantly lower C. suis prevalence in feral swine. A future study is warranted to obtain C. suis DNA from feral swine to perform genetic diversity of C. suis between commercial and feral pigs.

Multidrug-Resistant Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp. in Houseflies and Blowflies from Farms and Their Environmental Settings.

Background: Antimicrobial resistance is rising globally at an alarming rate. While multiple active surveillance programs have been established to monitor the antimicrobial resistance, studies on the environmental link to antimicrobial spread are lacking. Methods: A total of 493 flies were trapped from a dairy unit, a dog kennel, a poultry farm, a beef cattle unit, an urban trash facility and an urban downtown area to isolate Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp. for antimicrobial susceptibility testing and molecular characterization. Results: E. coli, K. pneumoniae and coagulase-negative Staphylococcus were recovered from 43.9%, 15.5% and 66.2% of the houseflies, and 26.0%, 19.2%, 37.0% of the blowflies, respectively. In total, 35.3% of flies were found to harbor antimicrobial-resistant bacteria and 9.0% contained multidrug-resistant isolates. Three Staphylococcus aureus isolates were recovered from blowflies while three extended spectrum beta lactamase (ESBL)-carrying E. coli and one ESBL-carrying K. pneumoniae were isolated from houseflies. Whole genome sequencing identified the antimicrobial resistance genes blaCMY-2 and blaCTXM-1 as ESBLs. Conclusion: Taken together, our data indicate that flies can be used as indicators for environmental contamination of antimicrobial resistance. More extensive studies are warranted to explore the sentinel role of flies for antimicrobial resistance.