European Chlamydia abortus livestock isolate genomes reveal unusual stability and limited diversity, reflected in geographical signatures.

BACKGROUND: Chlamydia abortus (formerly Chlamydophila abortus) is an economically important livestock pathogen, causing ovine enzootic abortion (OEA), and can also cause zoonotic infections in humans affecting pregnancy outcome. Large-scale genomic studies on other chlamydial species are giving insights into the biology of these organisms but have not yet been performed on C. abortus. Our aim was to investigate a broad collection of European isolates of C. abortus, using next generation sequencing methods, looking at diversity, geographic distribution and genome dynamics. RESULTS: Whole genome sequencing was performed on our collection of 57 C. abortus isolates originating primarily from the UK, Germany, France and Greece, but also from Tunisia, Namibia and the USA. Phylogenetic analysis of a total of 64 genomes shows a deep structural division within the C. abortus species with a major clade displaying limited diversity, in addition to a branch carrying two more distantly related Greek isolates, LLG and POS. Within the major clade, seven further phylogenetic groups can be identified, demonstrating geographical associations. The number of variable nucleotide positions across the sampled isolates is significantly lower than those published for C. trachomatis and C. psittaci. No recombination was identified within C. abortus, and no plasmid was found. Analysis of pseudogenes showed lineage specific loss of some functions, notably with several Pmp and TMH/Inc proteins predicted to be inactivated in many of the isolates studied. CONCLUSIONS: The diversity within C. abortus appears to be much lower compared to other species within the genus. There are strong geographical signatures within the phylogeny, indicating clonal expansion within areas of limited livestock transport. No recombination has been identified within this species, showing that different species of Chlamydia may demonstrate different evolutionary dynamics, and that the genome of C. abortus is highly stable.

Assessment of Chlamydia psittaci Shedding and Environmental Contamination as Potential Sources of Worker Exposure throughout the Mule Duck Breeding Process.

Chlamydia psittaci is an obligate intracellular bacterium responsible for avian chlamydiosis, otherwise known as psittacosis, a zoonotic disease that may lead to severe atypical pneumonia. This study was conducted on seven mule duck flocks harboring asymptomatic birds to explore the circulation and persistence of C. psittaci during the entire breeding process and assess the potential sources of worker exposure. Cloacal swabs and air samples were taken on each occasion requiring humans to handle the birds. In parallel, environmental samples, including dust, water, and soil, were collected. Specific real-time PCR analyses revealed the presence of C. psittaci in all flocks but with three different shedding patterns involving ducks about the age of 4, 8, and 12 weeks with heavy, moderate, and low excretion levels, respectively. Air samples were only positive in flocks harboring heavy shedders. Dust in flocks with heavy or moderate shedders carried chlamydial loads strongly associated with the loads detected in avian and soil samples. Environmental contamination, significantly correlated with shedding dynamics, was considered to be the most probable source of exposure. The high prevalence of bacteriophage Chp1 in all flocks, mostly jointly present with chlamydia, suggests an important factor in C. psittaci persistence, thus creating a greater risk for humans. A survey conducted in these flocks regarding farming practices and activities showed that disinfection seems to be the most promising practice for reducing C. psittaci prevalence in ducks and that the place and the duration of action during operations seem to be potential risk factors. Strict adherence to good practices is strongly recommended.

Chlamydiaceae in North Atlantic Seabirds Admitted to a Wildlife Rescue Center in Western France.

Birds are the primary hosts of Chlamydia psittaci, a bacterium that can cause avian chlamydiosis in birds and psittacosis in humans. Wild seabirds are frequently admitted to wildlife rescue centers (WRC) at European Atlantic coasts, for example, in connection with oil spills. To investigate the extent of chlamydial shedding by these birds and the resulting risk for animals in care and the medical staff, seabirds from a French WRC were sampled from May 2011 to January 2014. By use of a quantitative PCR (qPCR), 195 seabirds belonging to 4 orders, 5 families and 13 species were examined, of which 18.5% proved to be Chlamydiaceae positive. The highest prevalence of shedders was found in northern gannets (Morus bassanus) (41%), followed by European herring gulls (Larus argentatus) (14%) and common murres (Uria aalge) (7%). Molecular characterization and phylogenetic analysis of qPCR-positive northern gannet samples revealed two variants of a strain closely related to C. psittaci. In European herring gulls and in one common murre, strains showing high sequence similarity to the atypical Chlamydiaceae-like C122 previously found in gulls were detected. Our study shows that seabirds from the northeastern Atlantic Ocean carry several chlamydial organisms, including C. psittaci-related strains. The staff in WRCs should take protective measures, particularly in the case of mass admissions of seabirds.

Emergence and maintenance of multidrug-resistant Escherichia coli of canine origin harbouring a blaCMY-2-IncI1/ST65 plasmid and topoisomerase mutations.

OBJECTIVES: To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. METHODS: MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. RESULTS: Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. CONCLUSIONS: The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

Diversity of vanA-type vancomycin-resistant Enterococcus faecium isolated from broilers, poultry slaughterers and hospitalized humans in Greece.

OBJECTIVES: This study investigated the prevalence of vancomycin-resistant enterococci (VRE) in the broiler production environment after the avoparcin ban and their epidemiological relationship with human clinical VRE from the same geographical regions in Greece. METHODS: Caecal contents from broilers (n = 500) from eight livestock farms and faecal samples from poultry slaughterers (n = 50), all collected in two slaughterhouses during 2005-08, were analysed for species and vancomycin resistance gene identification using multiplex PCR. Sixty-three human clinical vancomycin-resistant Enterococcus faecium (VREF) isolates, obtained during 2006-09, were also examined. Discriminant analysis (DA) was used to establish the relationship of antimicrobial resistance profiles (ARPs) among broiler, poultry slaughterer and human clinical VREF. PFGE was conducted to study the genetic relatedness among VREF from the different sources. RESULTS: A total of 120 VRE were recovered from 113 (22.6%) broiler samples. VREF carrying the vanA gene were predominant, being recovered from 72 (14.4%) samples from five (62.5%) broiler farms. Concerning poultry slaughterers, VREF were recovered from 10 (20%) samples. Susceptibility testing revealed that broiler VREF were consistently resistant to tetracycline, whereas 93.7% of clinical VREF were resistant to ampicillin. Furthermore, 92.1% of clinical VREF compared with 54.4% of broiler VREF were multiresistant (resistant to at least five antimicrobial classes). DA classified broiler and human clinical VREF into their corresponding source with high classification rates (100% and 85.7%, respectively), while the classification rate of poultry slaughterer VREF was relatively low (50%), with 40% of them classified closely to broiler VREF. PFGE patterns were clearly related to the source of the VREF, with broiler isolates being clustered distinctly from all human isolates. CONCLUSIONS: A remarkable persistence of VREF was observed in the broiler production environment even >10 years after the avoparcin ban. Human and broiler VREF belonged to clearly unrelated populations, strongly indicating no clonal spread of VREF among the different sources, even between broilers and poultry slaughterers, despite them sharing common ARPs, as also supported by DA.

Serum acute phase proteins as clinical phase indicators and outcome predictors in naturally occurring canine monocytic ehrlichiosis.

BACKGROUND: Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is an important tick-borne disease of global importance. Currently, limited information is available on the diagnostic and prognostic value of acute phase proteins (APPs) in dogs naturally infected with E. canis. HYPOTHESIS: APPs may be useful indicators of the clinical phase of CME and predictive of the clinical outcome (death or survival). ANIMALS: Fifty-six dogs naturally infected with E. canis and 7 clinically healthy control dogs. METHODS: C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and albumin concentrations determined on admission were retrospectively compared among 27 dogs with nonmyelosuppressive CME, 29 dogs with myelosuppressive CME and 7 healthy dogs. Diagnosis of CME was based on clinical and clinicopathological findings, seropositivity to E. canis, polymerase chain reaction amplification of E. canis-specific 16S rDNA, microscopic observation of Ehrlichia sp. morulae in blood monocytes or some combination of these. RESULTS: Mean concentrations of CRP, SAA, and Hp were significantly higher in the myelosuppressed dogs compared with the other groups, but no significant differences were found in the concentration of albumin. Survival analysis of the affected animals indicated that APP concentrations were not associated with clinical outcome; the latter was strongly associated with pancytopenia (odds ratio for death 22.7) and neutropenia (odds ratio for death 7.7). CONCLUSIONS AND CLINICAL IMPORTANCE: CRP, SAA, and Hp serum concentrations on admission are useful indicators of the clinical phase of CME, but are not useful predictors of clinical outcome.

Pharmacodynamics of amoxicillin against Mannheimia haemolytica and Pasteurella multocida and pharmacokinetic/pharmacodynamic (PK/PD) correlation in sheep.

Silicone-made tissue cages were implanted in sheep. Blood serum (SBS) and tissue cage fluid (TCF) samples were collected after amoxicillin intravenous and intramuscular administrations, at the dose of 15 mg/kg. Amoxicillin pharmacodynamics were studied in an artificial culture medium, SBS and TCF with use of a Mannheimia haemolytica and a Pasteurella multocida strain. A concentration-independent antimicrobial activity of amoxicillin was confirmed for levels higher than 0.79-1.75×MIC. This result favored the use of the percentage of the 24 h dosing interval during which drug levels remain above MIC as the appropriate pharmacokinetic/pharmacodynamic index. The subsequent correlation revealed that intravenous administration could be considered effective against "deep" infections caused by bacteria with MICs<1 µg/mL or "shallow" infections caused by bacteria with MICs<0.1 µg/mL. Intramuscular administration could be safely considered effective against both "deep" and "shallow" infections when the MICs of the targeted pathogens are lower than 1 µg/mL.