Search

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.

The recently described Rickettsia felis is an emerging human pathogen causing flea-borne spotted fever [1]. Although found in a wide range of arthropods including fleas, ticks, mites, and lice, the...

Chlamydia gallinacea is an endemic Chlamydia agent in poultry with a worldwide distribution. The aim of this study was to investigate whether C. gallinacea can be transmitted via fecal-oral, respiratory and vertical routes. After co-housing with C. gallinacea-inoculated broilers (n = 10) for 15 days, over 90.0% of SPF broilers (n = 10) became C. gallinacea-positive in their oropharyngeal and cloacal swabs. Connection of isolators with ventilation tubing resulted in transmission of infectious bronchitis virus, but not of C. gallinacea, from infected broilers in one isolator to uninfected ones in the other isolator. Chlamydia-qPCR determined that 97.6% of shells of embryonated eggs (287/294) from a breeding farm were positive for C. gallinacea. C. gallinacea positivity in egg albumen increased significantly from 7.6% (10/128) before incubating to 44.4% (8/18) of 7-day incubation, and from 5.5% (7/128) to 38.9% (7/18) in egg yolk. After incubating for 19 days, C. gallinacea DNA was detected in heart (5/55, 9.1%), liver (3/55, 5.5%), spleen (7/55, 12.7%), lung (6/55, 10.1%), kidney (8/55; 14.5%) and intestine (4/55, 7.3%) of chicken embryos. Taken together, our data indicate that C. gallinacea can be efficiently transmitted by the fecal-oral route, but not via aerosol. Additionally, vertical transmission can occur via penetration of C. gallinacea from eggshell to albumen, yolk, and the growing embryo. Our findings provide essential information for the control of C. gallinacea in poultry farms.

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.

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.

With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 µL reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.

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.

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.

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.

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.

Colistin is a last resort drug used to treat infections caused by multidrug resistant Gram-negative bacteria that have developed resistance to carbapenem. The emergence and rapid dissemination of the nine plasmid-mediated mobile colistin resistance genes (mcr-1 to mcr-9) has led to fear of pan-drug resistant infections occurring worldwide. To date, there is only limited information on colistin resistance in African countries where the drug is widely used in agriculture. In this study from Nigeria, 583 non-duplicate bacterial strains were isolated from 1,119 samples collected from humans, camels, cattle, dogs, pigs and poultry by the use of colistin-supplemented MacConkey agar, and 17.0% of the isolates (99/583) were colistin resistant. PCRs (mcr-1 to mcr-9) and whole genome sequencing (WGS) identified mcr in 21.2% of the colistin-resistant isolates: mcr-1.1 (n=13), mcr-8.1 (n=5), both mcr-1.1 and mcr-8.1 (n=2), and mcr-1.1 and mcr-5 (n=1). Nine of the 21 mcr-positive strains were isolated from human samples, with eight being K. pneumoniae, and six of these human K. pneumoniae had a high colistin MIC of >64 µg/ml. In contrast, nine of the 12 mcr-positive animal isolates were E. coli of which only two had a colistin MIC of >64 µg/ml. Our study is the first to report mcr-1 in Alcaligenes faecalis, and emergence of mcr-5 and mcr-8 in Nigeria. WGS determined that mcr-1 was localized on IncX4 plasmid, and 95.2% of mcr-1 harboring isolates (20/21) transferred colistin resistance successfully in conjugation assay. Our findings highlight the global spread of colistin resistance, and emphasize the urgent need for coordinated global action to combat resistant bacteria.

Wild birds inhabit in a wide variety of environments and can travel great distances. Thus, wild birds can possibly spread antimicrobial resistance along the way, and this may represent a potential public health concern. We characterized antimicrobial resistance in fecal Escherichia coli and Enterococcus faecalis in wild raptors in the southeastern US. Cloacal samples were collected from 118 wild raptors of 17 species from 18 counties in Alabama and 15 counties in Georgia. A total of 112 E. coli and 76 E. faecalis isolates were recovered, and we found significantly more antimicrobial-resistant E. coli (20/112, 18%) than E. faecalis (6/76, 8%; P=0.05). Five antimicrobialresistant genes: blaTEM-1, blaCTX-M-1, tet(M), cmlA, cat, and gyrA, were identified in antimicrobial-resistant E. coli isolates. Five of 13 (38%) ampicillin-resistant E. coli harbored both bla-TEM-1 and blaCTX-M-1 genes, indicating they are extended-spectrum β-lactamase-carrying strains. Both of the tetracycline resistance genes, tet(M) and tet(L), were identified in E. faecalis isolates. Wild raptors seem to be a reservoir host of antimicrobial-resistant E. coli and E. faecalis and may represent a hazard to animal and human health by transmission of these isolates.

Berberine hydrochloride (BBR) has been reported to improve insulin resistance. However, the underlying mechanism remains largely elusive. We therefore investigate the anti-inflammatory role of BBR and the cell signaling pathways that involved. C2C12 myoblasts were treated with IL-1β (10ng/mL) alone or in combination with TNF-α (50 ng/mL) in the presence or absence of BBR (10 µM) for 24 hours. Western blot analysis was used to assess the protein level. In vivo study is conducted using C57BL/6 mice fed with high fat high sucrose (HFHS) diet to induce insulin resistance. Skeletal muscle was collected after 4 weeks treatment with either 5 mg/kg BBR or vehicle. BBR reduced activity of iNOS, and stress-related kinases, including p38 MAPK, NF-kB, SAP/JNK, MMK4 and C-JUN that was induced by cytokines in C2C12 myoblast. Furthermore, BBR reversed cytokine mediated suppression of AMPK-α, augmented activity of SIRT-1 and PGC-1α. Antioxidant enzyme or transcription factor, nuclear factor like 2 (NRF-2) were also elevated after BBR treatment. In addition, BBR improved insulin signaling in C2C12 myoblast. HFHS diet induced iNOS expression was reversed by BBR in skeletal muscle. Augmentation of the activity of uncoupled protein-3 (UCP-3) and PPARα in HFHS diet skeletal muscle was observed. Our results demonstrate that BBR ameliorates cytokine induced inflammation and oxidative stress. BBR improves mitochondrial function, promotes insulin sensitivity. The anti-inflammatory effect of BBR in skeletal muscle may mediated through multiple pathways including activation of AMPKα-SIRT-1-PGC-1α pathway, and inhibition of MMK4- SAP/JUNK-C-JUN pathway. BBR is a potential medication in treating obesity induced insulin resistance. Disclosure A. Poudel: None. J. Zhou: None. S. Massey: None. L. Li: None.

Feline immunodeficiency virus (FIV) is among the most common infectious agents of cats. Five well-characterized FIV subtypes, A, B, C, D, and E, are recognized worldwide. As in HIV diagnosis, serum antibodies against FIV classically serve as an indicator of infection status. After the introduction of an inactivated FIV vaccine, this approach has become problematic, since antibodies generated by vaccination are indistinguishable from antibodies in response to infection. However, PCR detection of host-cell-integrated FIV DNA will differentiate infection-derived antibody from vaccination-derived positivity because presumably the RNA of inactivated vaccine virus will not integrate into the host genome. In this study, we established a gag gene-based dual-emission fluorescence resonance energy transfer (FRET) real-time PCR that amplifies single-target copies of all known FIV strains and differentiates five FIV subtypes. All blood samples from experimentally FIV-infected cats ( n = 5) were antibody positive and highly positive in the FIV PCR. In contrast, nine cats became antibody positive after FIV vaccination but remained negative in the FIV PCR. Of 101 FIV antibody-positive feline blood specimens submitted for FIV PCR diagnosis, 61 were positive (60%). A total of 23 of the positive PCRs identified subtype A, 11 identified subtype B1, 11 identified subtype B2/E, and 16 identified subtype C. FIV subtype D was not detected in any submitted specimens even though 13 blood specimens were from cats known to have received the FIV vaccine, which contains FIV subtype A and D inactivated virions. Therefore, this PCR quantitatively identifies FIV subtypes and unambiguously discriminates between FIV-vaccinated and FIV-infected cats.

Babesia gibsoni and Babesia canis are the etiological agents of canine babesiosis, a protozoal hemolytic disease with global significance. Canine babesiosis has been diagnosed by microscopic identification of intra-erythrocytic trophozoites in blood smear, and by serological testing. Here we developed a quantitative fluorescence resonance energy transfer (FRET)-PCR that amplifies a fragment of the Babesia spp. 18S rRNA gene with high sensitivity and specificity. Melting curve analysis differentiates B. gibsoni, B. canis canis/B. canis vogeli, and B. canis rossi by the disassociation temperature of the fluorescent probes. Babesia gibsoni infection was detected in 8 of 48 canine breeds (17%) and 24 of a total of 235 specimens (10.2%) submitted from 22 states of the continental United States of America. A potential blood donor was positive for B. canis vogeli infection. In Hong Kong (China), B. gibsoni infection was detected in 30 of 64 specimens (46.9%) from 15 of the 24 breeds (63%). While the frequency of canine babesiosis did not associate with seasonal change in Hong Kong, positivity in the USA for Babesia spp. infection was higher in Spring and Summer than in Autumn and Winter. The data suggest that environmental factors associated with tick vector exposure rather than genetic susceptibility determine the incidence of canine babesiosis. Babesia spp. burdens in blood declined significantly with increasing age of the infected dogs, and therapy with atovaquone and tilmicosin eliminated B. gibsoni while doxcycline and berenil did not. This demonstrates that high-resolution real-time PCR analysis may advance diagnosis and therapy monitoring of canine babesiosis.

Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies againstChlamydiaspp. have been elusive due to high cross-reactivity of chlamydial antigens. To identifyChlamydiaspecies-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among allChlamydiaspecies. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against eachChlamydiaspecies in chemiluminescent ELISAs. For each of nineChlamydiaspecies, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous,Chlamydiamonospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of sixC. pecorum-specific peptides from five proteins withC. pecorum-reactive sera from cattle, the natural host ofC. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at Chlamydiaspp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

Intracellular Chlamydia (C.) bacteria cause in cattle some acute but rare diseases such as abortion, sporadic bovine encephalomyelitis, kerato-conjunctivitis, pneumonia, enteritis and polyarthritis. More frequent, essentially ubiquitous worldwide, are low-level, asymptomatic chlamydial infections in cattle. We investigated the impact of these naturally acquired infections in a cohort of 51 female Holstein and Jersey calves from birth to 15 weeks of age. In biweekly sampling, we measured blood/plasma markers of health and infection and analyzed their association with clinical appearance and growth in dependence of chlamydial infection intensity as determined by mucosal chlamydial burden or contemporaneous anti-chlamydial plasma IgM. Chlamydia 23S rRNA gene PCR and ompA genotyping identified only C. pecorum (strains 1710S, Maeda, and novel strain Smith3v8) in conjunctival and vaginal swabs. All calves acquired the infection but remained clinically asymptomatic. High chlamydial infection associated with reduction of body weight gains by up to 48% and increased conjunctival reddening (P

Identification of highly immunogenic antigens is critical for construction of an efficacious subunit vaccine against C. pneumoniae infections. A previous project used a genome-wide screen to identify twelve protective C. pneumoniae candidate genes in an A/J mouse lung disease model (Li et al., Vaccine 24:2917, 2006). Due to insufficient induction of Th1 immunity, these genes elicited only modest protection. Here, we used the E. coli heat-labile enterotoxin as a Th1-enhancing genetic adjuvant, and re-tested these twelve genes, in parallel with six genes identified by other investigators. Vaccine candidate genes cutE and Cpn0420 conferred significant protection by all criteria evaluated (prevention of C. pneumoniae-induced death, reduction of lung disease, elimination of C. pneumoniae). Gene oppA_2 was protective by disease reduction and C. pneumoniae elimination. Four other genes were protective by a single criterion. None of the six genes reported elsewhere protected by reduction of lung disease or elimination of C. pneumoniae, but three protected by increasing survival.