Egle Kudirkiene

Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS.

Streptococcus equi includes very important animal and human pathogens. S. equi subsp. equi (SEE) is a highly pathogenic equine specific subspecies, while S. equi subsp. zooepidemicus (SEZ) and S. equi subsp. ruminatorum are opportunistic pathogens of various animal species and humans. Due to great phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies in horses. Superspectra created in this study enabled correct identification of 86 strains belonging to different subspecies of S. equi, isolated from various hosts, infection sites and years. In general, higher average identification accuracy was achieved for SEE (99.0±3.0%) than for SEZ (93.3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method with spectra analyses using the SARAMIS database. Additionally, first results on subtyping of SEZ indicated that a more refined discrimination, for example for epidemiological surveys, may be possible.

Prevalence and genotypes of Campylobacter jejuni from urban environmental sources in comparison with clinical isolates from children.

This study aimed to investigate the prevalence of Campylobacter jejuni in potential contamination sources that are not regularly monitored such as free-living urban pigeons and crows, dogs, cats and urban environmental water and to assess the possible impact on the epidemiology of campylobacteriosis in children using multilocus sequence typing (MLST). Campylobacter spp. were detected in 36.2 % of faecal samples of free-living urban birds and in 40.4 % of environmental water samples. A low prevalence of Campylobacter spp. was detected in dogs and cats, with 7.9 and 9.1 %, respectively. Further identification of isolates revealed that environmental water and pet samples were mostly contaminated by other Campylobacter spp. than C. jejuni, whereas C. jejuni was the most prevalent species in faecal samples of free-living birds (35.4 %). This species was the dominant cause of campylobacteriosis in children (91.5 %). In addition, the diversity of C. jejuni MLST types in free-living birds and children was investigated. Clonal complex (CC) 179 was predominant among free-living urban birds; however, only two isolates from children were assigned to this CC. One dog and one child isolate were assigned to the same clonal complex (CC48) and sequence type (ST) 918. The dominant two clonal complexes among the child clinical isolates (CC353 and CC21) were not detected among C. jejuni strains isolated from environmental sources examined in this study. As only two CCs were shared by environmental and child C. jejuni isolates and a high number of novel alleles and STs were found in C. jejuni isolated from free-living urban birds and environmental water, there is probably only a limited link between urban environmental sources and campylobacteriosis in children, particularly in rather cold climatic conditions.

In silico prediction of Gallibacterium anatis pan-immunogens

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

Characterisation of Commensal Escherichia coli Isolated from Apparently Healthy Cattle and Their Attendants in Tanzania.

While pathogenic types of Escherichia coli are well characterized, relatively little is known about the commensal E. coli flora. In the current study, antimicrobial resistance in commensal E. coli and distribution of ERIC-PCR genotypes among isolates of such bacteria from cattle and cattle attendants on cattle farms in Tanzania were investigated. Seventeen E. coli genomes representing different ERIC-PCR types of commensal E. coli were sequenced in order to determine their possible importance as a reservoir for both antimicrobial resistance genes and virulence factors. Both human and cattle isolates were highly resistant to tetracycline (40.8% and 33.1%), sulphamethazole-trimethoprim (49.0% and 8.8%) and ampicillin (44.9% and 21.3%). However, higher proportion of resistant E. coli and higher frequency of resistance to more than two antimicrobials was found in isolates from cattle attendants than isolates from cattle. Sixteen out of 66 ERIC-PCR genotypes were shared between the two hosts, and among these ones, seven types contained isolates from cattle and cattle attendants from the same farm, suggesting transfer of strains between hosts. Genome-wide analysis showed that the majority of the sequenced cattle isolates were assigned to phylogroups B1, while human isolates represented phylogroups A, C, D and E. In general, in silico resistome and virulence factor identification did not reveal differences between hosts or phylogroups, except for lpfA and iss found to be cattle and B1 phylogroup specific. The most frequent plasmids replicon genes found in strains from both hosts were of IncF type, which are commonly associated with carriage of antimicrobial and virulence genes. Commensal E. coli from cattle and attendants were found to share same genotypes and to carry antimicrobial resistance and virulence genes associated with both intra and extraintestinal E. coli pathotypes.

Draft Genome Sequence of Gallibacterium anatis bv. haemolytica 12656-12 Liver, an Isolate Obtained from the Liver of a Septicemic Chicken.

ABSTRACT We report the draft genome sequence of Gallibacterium anatis bv. haemolytica strain 12656-12 Liver. This strain was isolated from the liver of a septicemic layer chicken in Denmark in 1981. The strain has been used extensively for experimental purposes.

The Use of a Combined Bioinformatics Approach to Locate Antibiotic Resistance Genes on Plasmids From Whole Genome Sequences of Salmonella enterica Serovars From Humans in Ghana

In the current study, we identified plasmids carrying antimicrobial resistance genes in draft whole genome sequences of 16 selected Salmonella enterica isolates representing six different serovars from humans in Ghana. The plasmids and the location of resistance genes in the genomes were predicted using a combination of PlasmidFinder, ResFinder, plasmidSPAdes and BLAST genomic analysis tools. Subsequently, S1-PFGE was employed for analysis of plasmid profiles. Whole genome sequencing confirmed the presence of antimicrobial resistance genes in Salmonella isolates showing multidrug resistance phenotypically. ESBL, either blaTEM52−B or blaCTX−M15 were present in two cephalosporin resistant isolates of S. Virchow and S. Poona, respectively. The systematic genome analysis revealed the presence of different plasmids in different serovars, with or without insertion of antimicrobial resistance genes. In S. Enteritidis, resistance genes were carried predominantly on plasmids of IncN type, in S. Typhimurium on plasmids of IncFII(S)/IncFIB(S)/IncQ1 type. In S. Virchow and in S. Poona, resistance genes were detected on plasmids of IncX1 and TrfA/IncHI2/IncHI2A type, respectively. The latter two plasmids were described for the first time in these serovars. The combination of genomic analytical tools allowed nearly full mapping of the resistance plasmids in all Salmonella strains analyzed. The results suggest that the improved analytical approach used in the current study may be used to identify plasmids that are specifically associated with resistance phenotypes in whole genome sequences. Such knowledge would allow the development of rapid multidrug resistance tracking tools in Salmonella populations using WGS.

Impact of egg disinfection of hatching eggs on the eggshell microbiome and bacterial load

ABSTRACT Disinfection of hatching eggs is essential to ensure high quality production of broilers. Different protocols are followed in different hatcheries; however, only limited scientific evidence on how the disinfection procedures impact the microbiome is available. The aim of the present study was to characterize the microbiome and aerobic bacterial load of hatching eggs before disinfection and during the subsequent disinfection steps. The study included a group of visibly clean and a group of visibly dirty eggs. For dirty eggs, an initial wash in chlorine was performed, hereafter all eggs were submitted to two times fumigation and finally spray disinfection. The eggshell microbiome was characterized by sequencing of the total amount of 16S rRNA extracted from each sample, consisting of shell surface swabs of five eggs from the same group. In addition, the number of colony forming units (cfu) under aerobic conditions was established for each disinfection step. The disinfection procedure reduced the bacterial load from more than 104 cfu (initially visibly clean eggs) and 105 cfu (initially visibly dirty eggs) to less than 10 cfu per sample after disinfection for both groups of eggs. The microbiome of both initially visibly clean and initially visibly dirty eggs had the highest abundances of the phyla Firmicutes, Proteobacteria and Bacteroidetes. Within the phyla Firmicutes the relative abundances of Clostridiales decreased while Lactobacillus increased from before to after final disinfection. In conclusion, the investigated disinfection procedure is effective in reducing the bacterial load, and by adding a chlorine wash for initially visibly dirty eggs, the microbiome of initially visibly clean and initially visibly dirty eggs had a highly similar microflora after the final disinfection step.

Treatment with high-dose antidepressants severely exacerbates the pathological outcome of experimental Escherichia coli infections in poultry

There is an urgent need for novel antibiotics as the current antibiotics are losing their value due to increased resistance among clinically important bacteria. Sertraline, an on-marked anti-depressive drug, has been shown to modify bacterial activity in vitro, including increasing the susceptibility of Escherichia coli to antibiotics. The aim of the present study was to investigate if the antimicrobial activity of sertraline could be documented under clinical settings, hereunder if sertraline could potentiate the effect of tetracycline in treatment of an experimentally induced ascending infection in poultry. A total of 40 chickens were divided in four groups of 10 chickens each. All chickens were challenged with 4x103 colony forming units (CFU) of a tetracycline resistant E. coli strain using a surgical infection model, and subsequently treated with either high-dose sertraline, tetracycline, a combination hereof or received no treatment. Seven days post challenge all birds were submitted to necropsy and scored pathologically for lesions. The average lesion scores were significantly higher (P<0.05) in the groups that were treated with high-dose sertraline or high-dose sertraline combined with tetracycline. In conclusion high-dose treatments (four times the maximum therapeutic dose for treating human depression) with sertraline as an adjuvant for treatment of antibiotic resistant E. coli infections exacerbate the pathological outcome of infection in chickens.

Draft Genome Sequence of Chelonobacter oris Strain 1662T, Associated with Respiratory Disease in Hermann's Tortoises

ABSTRACT Chelonobacter oris 1662T is a type strain of the recently described species of the Pasteurellaceae family. The strain was isolated from the choanae of a captive tortoise with signs of respiratory tract infection. The genome reported here is approximately 2.6 Mb in size and has a G+C content of 47.1%.