Genome Sequence of AvianEscherichia coliStrain IHIT25637, an Extraintestinal PathogenicE. coliStrain of ST131 Encoding Colistin Resistance Determinant MCR-1

Sequence type 131 (ST131) is one of the predominant Escherichia coli lineages among extraintestinal pathogenic E. coli (ExPEC) that causes a variety of diseases in humans and animals and frequently shows multidrug resistance. Here, we report the first genome sequence of an ST131-ExPEC strain from poultry carrying the plasmid-encoded colistin resistance gene mcr-1

The inhibition of the apoptosis pathway by the Coxiella burnetii effector protein CaeA requires the EK repetition motif, but is independent of survivin

Coxiella burnetii is an obligate intracellular bacterium that causes Query (Q) fever, a zoonotic disease. It requires a functional type IV secretion system (T4SS) which translocate bacterial effector proteins into the host cell cytoplasm and thereby facilitates bacterial replication. To date, more than 130 effector proteins have been identified, but their functions remain largely unknown. Recently, we demonstrated that one of these proteins, CaeA (CBU1524) localized to the host cell nucleus and inhibited intrinsic apoptosis of HEK293 or CHO cells. In the present study we addressed the question whether CaeA also affects the extrinsic apoptosis pathway. Ectopic expression of CaeA reduced extrinsic apoptosis and prevented the cleavage of the executioner caspase 7, but did not impair the activation of initiator caspase 9. CaeA expression resulted in an up-regulation of survivin (an inhibitor of activated caspases), which, however, was not causal for the anti-apoptotic effect of CaeA. Comparing the sequence of CaeA from 25 different C. burnetii isolates we identified an EK (glutamic acid/ lysine) repetition motif as a site of high genetic variability. The EK motif of CaeA was essential for the anti-apoptotic activity of CaeA. From these data, we conclude that the C. burnetii effector protein CaeA interferes with the intrinsic and extrinsic apoptosis pathway. The process requires the EK repetition motif of CaeA, but is independent of the upregulated expression of survivin.This work was supported by the Deutsche Forschungsgemeinschaft (SFB796 project B8) to AL and by the ERA-NET PathoGenoMics 3rd call to AL and JPG

Phenotype of Coxiella burnetii Strains of Different Sources and Genotypes in Bovine Mammary Gland Epithelial Cells

Despite the high prevalence of C. burnetii in dairy herds and continuous shedding via milk by chronically infected cows, bovine milk is not recognized as a relevant source of human Q fever. We hypothesized that the bovine mammary gland epithelial cell line PS represents a suitable in vitro model for the identification of C. burnetii-strain-specific virulence properties that may account for this discrepancy. Fifteen C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes (I, II, III and IV). The replication efficiencies of all strains were similar, even though strains of the MLVA-genotype II replicated significantly better than genotype I strains, and bovine and ovine isolates replicated better than caprine ones. Bovine milk isolates replicated with similar efficiencies to isolates from other bovine organs. One sheep isolate (Cb30/14, MLVA type I, isolated from fetal membranes) induced a remarkable up-regulation of IL-1β and TNF-α, whereas prototypic strains and bovine milk isolates tended to suppress pro-inflammatory responses. While infection with strain Nine Mile I rendered the cells partially refractory to re-stimulation with E. coli lipopolysaccharide, Cb30/14 exerted a selective suppressive effect which was restricted to IL-6 and TNF-α and spared IL-1β. PS cells support the replication of different strains of C. burnetii and respond in a strain-specific manner, but isolates from bovine milk did not display a common pattern, which distinguishes them from strains identified as a public health concern

Interaction of Coxiella burnetii Strains of Different Sources and Genotypes with Bovine and Human Monocyte-Derived Macrophages

Most human Q fever infections originate from small ruminants. By contrast, highly prevalent shedding of Coxiella (C.) burnetii by bovine milk rarely results in human disease. We hypothesized that primary bovine and human monocyte-derived macrophages (MDM) represent a suitable in vitro model for the identification of strain-specific virulence properties at the cellular level. Twelve different C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes. Infection efficiency and replication of C. burnetii were monitored by cell culture re-titration and qPCR. Expression of immunoregulatory factors after MDM infection was measured by qRT-PCR and flow cytometry. Invasion, replication and MDM response differed between C. burnetii strains but not between MDMs of the two hosts. Strains isolated from ruminants were less well internalized than isolates from humans and rodents. Internalization of MLVA group I strains was lower compared to other genogroups. Replication efficacy of C. burnetii in MDM ranged from low (MLVA group III) to high (MLVA group IV). Infected human and bovine MDM responded with a principal up-regulation of pro-inflammatory cytokines such as IL-1β, IL-12, and TNF-α. However, MLVA group IV strains induced a pronounced host response whereas infection with group I strains resulted in a milder response. C. burnetii infection marginally affected polarization of MDM. Only one C. burnetii strain of MLVA group IV caused a substantial up-regulation of activation markers (CD40, CD80) on the surface of bovine and human MDM. The study showed that replication of C. burnetii in MDM and the subsequent host cell response is genotype-specific rather than being determined by the host species pointing to a clear distinction in C. burnetii virulence between the genetic groups

Interaction of different Chlamydiae species with bovine spermatozoa

Abstract Background Interaction of spermatozoa and Chlamydiae spp. might contribute to reduced fertility in cattle. To proof this hypothesis, bovine semen was incubated with viable or heat inactivated Chlamydia (C.) abortus or psittaci (Multiplicity of infection = 1) and sperm motility was monitored with a computer-assisted sperm analyzer over 24 h. Additionally, the interaction with the spermatozoa was further investigated by means of light and transmission electron microscopy (TEM). Results Only viable Chlamydiae of both species decreased sperm motility and this only after about 9 h. Taking binding rates into account, the loss of sperm motility after about 9 h could likely be a consequence of Chlamydiae attachment to the spermatozoa. About two thirds of the Chlamydiae elementary bodies were bound to the front third of the sperm, the acrosomal region. No inclusions of Chlamydiae in spermatozoa were observed in TEM after 2 h co-incubation. Conclusions As initial motility was not affected following co-incubation of viable Chlamydiae and bovine sperm, it seems likely that sperm could serve as a carrier/vehicle for Chlamydiae facilitating cervical passage of Chlamydiae spp. in cattle. Additionally, our results suggest that spermatozoa carrying Chlamydiae may have no initial disadvantage in reaching the oviduct, but are immotile at the time of ovulation what might have an impact on fertilization capacities of the individual sperm. Consequently, high concentrations of the investigated Chlamydiae in the seminal plasma or female genital tract might play a role in reduced fertility in cattle

Multispecies and Clonal Dissemination of OXA-48 Carbapenemase in Enterobacteriaceae From Companion Animals in Germany, 2009—2016

The increasing spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a serious threat to public health. Recent studies suggested animals as a putative source of such bacteria. We investigated 19,025 Escherichia coli, 1607 Klebsiella spp. and 570 Enterobacter spp. isolated from livestock, companion animal, horse, and pet samples between 2009 and 2016 in our routine diagnostic laboratory for reduced susceptibility to carbapenems (CP) by using meropenem-containing media. Actively screened CP non-susceptible strains as well as 367 archived ESBL/AmpC-β-lactamase-producing Enterobacteriaceae were then tested for the presence of CP genes by PCRs. Among 21,569 isolates, OXA-48 could be identified as the sole carbapenemase type in 137 (0.64%) strains. The blaOXA-48 gene was located on an ∼60-kb IncL plasmid and sequence analysis revealed high similarity to reference plasmid pOXA-48a, which has been involved in the global spread of the blaOXA-48 gene in humans for many years. Klebsiella pneumoniae was the predominant OXA-48 producer (n = 86; 6.6% of all K. pneumoniae isolates), followed by E. cloacae (n = 28; 5.0%), Klebsiella oxytoca (n = 1; 0.3%), and E. coli (n = 22, 0.1%). OXA-48 was not found in livestock, but in dogs (120/3182; 3.8%), cats (13/792; 1.6%), guinea pig (1/43; 2.3%), rat (1/23; 4.3%), mouse (1/180; 0.6%), and one rabbit (1/144; 0.7%). Genotyping identified few major clones among the different enterobacteria species, including sequence types ST11 and ST15 for K. pneumoniae, ST1196 for E. coli, and ST506 and ST78 for E. cloacae, most of which were previously involved in the dissemination of multidrug-resistant strains in humans. The majority of OXA-48 isolates (n = 112) originated from a university veterinary clinic (UVC), while animals from further 16 veterinary institutions were positive. Clonal analyses suggested nosocomial events related to different species and STs in two veterinary clinics and horizontal transfer of the pOXA-48-like plasmid between bacterial species and animals. A systematic monitoring is urgently needed to assess the dissemination of CPE not only in livestock but also in companion animals and veterinary clinics

Image_4_Multispecies and Clonal Dissemination of OXA-48 Carbapenemase in Enterobacteriaceae From Companion Animals in Germany, 2009—2016.PDF

<p>The increasing spread of carbapenemase-producing Enterobacteriaceae (CPE) poses a serious threat to public health. Recent studies suggested animals as a putative source of such bacteria. We investigated 19,025 Escherichia coli, 1607 Klebsiella spp. and 570 Enterobacter spp. isolated from livestock, companion animal, horse, and pet samples between 2009 and 2016 in our routine diagnostic laboratory for reduced susceptibility to carbapenems (CP) by using meropenem-containing media. Actively screened CP non-susceptible strains as well as 367 archived ESBL/AmpC-β-lactamase-producing Enterobacteriaceae were then tested for the presence of CP genes by PCRs. Among 21,569 isolates, OXA-48 could be identified as the sole carbapenemase type in 137 (0.64%) strains. The bla_OXA-48 gene was located on an ∼60-kb IncL plasmid and sequence analysis revealed high similarity to reference plasmid pOXA-48a, which has been involved in the global spread of the bla_OXA-48 gene in humans for many years. Klebsiella pneumoniae was the predominant OXA-48 producer (n = 86; 6.6% of all K. pneumoniae isolates), followed by E. cloacae (n = 28; 5.0%), Klebsiella oxytoca (n = 1; 0.3%), and E. coli (n = 22, 0.1%). OXA-48 was not found in livestock, but in dogs (120/3182; 3.8%), cats (13/792; 1.6%), guinea pig (1/43; 2.3%), rat (1/23; 4.3%), mouse (1/180; 0.6%), and one rabbit (1/144; 0.7%). Genotyping identified few major clones among the different enterobacteria species, including sequence types ST11 and ST15 for K. pneumoniae, ST1196 for E. coli, and ST506 and ST78 for E. cloacae, most of which were previously involved in the dissemination of multidrug-resistant strains in humans. The majority of OXA-48 isolates (n = 112) originated from a university veterinary clinic (UVC), while animals from further 16 veterinary institutions were positive. Clonal analyses suggested nosocomial events related to different species and STs in two veterinary clinics and horizontal transfer of the pOXA-48-like plasmid between bacterial species and animals. A systematic monitoring is urgently needed to assess the dissemination of CPE not only in livestock but also in companion animals and veterinary clinics.</p

The anti-apoptotic Coxiella burnetii effector protein AnkG is a strain specific virulence factor

The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Effector proteins injected into the host cell by the C. burnetii type IVB secretion system (T4BSS) are required for the inhibition of host cell apoptosis. AnkG is one of these anti-apoptotic effector proteins. The inhibitory effect of AnkG requires its nuclear localization, which depends on p32-dependent intracellular trafficking and importin-α1-mediated nuclear entry of AnkG. Here, we compared the sequences of ankG from 37 C. burnetii isolates and classified them in three groups based on the predicted protein size. The comparison of the three different groups allowed us to identify the first 28 amino acids as essential and sufficient for the anti-apoptotic activity of AnkG. Importantly, only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Finally, using the Galleria mellonella infection model, we observed that AnkG from the first group has the ability to attenuate pathology during in vivo infection, as it allows survival of the larvae despite bacterial replication.This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the grant LU1357/5-1 to AL. We thank Prof. Robert Heinzen, Dr. Alyssa Ingmundson, Prof. James Samuel and Prof. Thomas Stamminger for providing plasmids, and the Lührmann lab for discussion. Open Access funding provided by Projekt DEAL.info:eu-repo/semantics/publishedVersio