Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms.

BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Pyocin-mediated antagonistic interactions in Pseudomonas spp. isolated in James Ross Island, Antarctica.

Interactions within bacterial communities are frequently mediated by the production of antimicrobial agents. Despite the increasing interest in research of new antimicrobials, studies describing antagonistic interactions among cold-adapted microorganisms are still rare. Our study assessed the antimicrobial interactions of 36 Antarctic Pseudomonas spp. and described the genetic background of these interactions in selected strains. The overall bacteriocinogeny was greater compared to mesophilic Pseudomonas non-aeruginosa species. R-type tailocins were detected on transmission electron micrographs in 16 strains (44.4%); phylogenetic analysis of the corresponding gene clusters revealed that the P. prosekii CCM 8878 tailocin was related to the Rp3 group, whereas the tailocin in Pseudomonas sp. CCM 8880 to the Rp4 group. Soluble antimicrobials were produced by eight strains (22.-2%); gene mining found pyocin L homologues in the genomes of P. prosekii CCM 8881 and CCM 8879 and pyocin S9-like homologues in P. prosekii CCM 8881 and Pseudomonas sp. CCM 8880. Analysis of secretomes confirmed the production of all S- and L-type pyocin genes. Our results suggest that bacteriocin-based inhibition plays an important role in interactions among Antarctic soil bacteria, and these native, cold-adapted microorganisms could be a promising source of new antimicrobials.

Carbapenemase-Producing Gram-Negative Bacteria from American Crows in the United States.

Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.

Genome sequences of two Antarctic strains of Pseudomonas prosekii: insights into adaptation to extreme conditions.

Pseudomonas prosekii is a recently described species isolated exclusively from James Ross Island close to the Antarctic Peninsula at 64° south latitude. Here, we present two P. prosekii genome sequences and their analyses with respect to phylogeny, low temperature adaptation, and potential biotechnological applications. The genome of P. prosekii P2406 comprised 5,896,482 bp and 5324 genes (GC content of 59.71%); the genome of P. prosekii P2673 consisted of 6,087,670 bp and 5511 genes (GC content of 59.50%). Whole genome sequence comparisons confirmed a close relationship between both investigated strains and strain P. prosekii LMG 26867T. Gene mining revealed the presence of genes involved in stress response, genes encoding cold shock proteins, oxidative stress proteins, osmoregulation proteins, genes for the synthesis of protection molecules, and siderophores. Comparative genome analysis of P. prosekii and P. aeruginosa PAO1 highlighted differences in genome content between extremophile species and a mesophilic opportunistic pathogen.

Genomic analysis of Escherichia coli strains isolated from diseased chicken in the Czech Republic.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) can cause various extraintestinal infections in poultry, resulting in massive economic losses in poultry industry. In addition, some avian E. coli strains may have zoonotic potential, making poultry a possible source of infection for humans. Due to its extreme genetic diversity, this pathotype remains poorly defined. This study aimed to investigate the diversity of colibacillosis-associated E. coli isolates from Central European countries with a focus on the Czech Republic. RESULTS: Of 95 clinical isolates subjected to preliminary characterization, 32 were selected for whole-genome sequencing. A multi resistant phenotype was detected in a majority of the sequenced strains with the predominant resistance to ß-lactams and quinolones being associated with TEM-type beta-lactamase genes and chromosomal gyrA mutations respectively. The phylogenetic analysis confirmed a great diversity of isolates, that were derived from nearly all phylogenetic groups, with predominace of B2, B1 and C phylogroups. Clusters of closely related isolates within ST23 (phylogroup C) and ST429 (phylogroup B2) indicated a possible local spread of these clones. Besides, the ST429 cluster carried blaCMY-2, - 59 genes for AmpC beta-lactamase and isolates of both clusters were generally well-equipped with virulence-associated genes, with considerable differences in distribution of certain virulence-associated genes between phylogenetically distant lineages. Other important and potentially zoonotic APEC STs were detected, incl. ST117, ST354 and ST95, showing several molecular features typical for human ExPEC. CONCLUSIONS: The results support the concept of local spread of virulent APEC clones, as well as of zoonotic potential of specific poultry-associated lineages, and highlight the need to investigate the possible source of these pathogenic strains.

Whole genome sequencing and function prediction of 133 gut anaerobes isolated from chicken caecum in pure cultures.

BACKGROUND: In order to start to understand the function of individual members of gut microbiota, we cultured, sequenced and analysed bacterial anaerobes from chicken caecum. RESULTS: Altogether 204 isolates from chicken caecum were obtained in pure cultures using Wilkins-Chalgren anaerobe agar and anaerobic growth conditions. Genomes of all the isolates were determined using the NextSeq platform and subjected to bioinformatic analysis. Among 204 sequenced isolates we identified 133 different strains belonging to seven different phyla - Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia, Elusimicrobia and Synergistetes. Genome sizes ranged from 1.51 Mb in Elusimicrobium minutum to 6.70 Mb in Bacteroides ovatus. Clustering based on the presence of protein coding genes showed that isolates from phyla Proteobacteria, Verrucomicrobia, Elusimicrobia and Synergistetes did not cluster with the remaining isolates. Firmicutes split into families Lactobacillaceae, Enterococcaceae, Veillonellaceae and order Clostridiales from which the Clostridium perfringens isolates formed a distinct sub-cluster. All Bacteroidetes isolates formed a separate cluster showing similar genetic composition in all isolates but distinct from the rest of the gut anaerobes. The majority of Actinobacteria clustered closely together except for the representatives of genus Gordonibacter showing that the genome of this genus differs from the rest of Actinobacteria sequenced in this study. Representatives of Bacteroidetes commonly encoded proteins (collagenase, hemagglutinin, hemolysin, hyaluronidase, heparinases, chondroitinase, mucin-desulfating sulfatase or glutamate decarboxylase) that may enable them to interact with their host. Aerotolerance was recorded in Akkermansia and Cloacibacillus and was also common among representatives of Bacteroidetes. On the other hand, Elusimicrobium and the majority of Clostridiales were highly sensitive to air exposure despite their potential for spore formation. CONCLUSIONS: Major gut microbiota members utilise different strategies for gut colonisation. High oxygen sensitivity of Firmicutes may explain their commonly reported decrease after oxidative burst during gut inflammation.

Two highly divergent lineages of exfoliative toxin B-encoding plasmids revealed in impetigo strains of Staphylococcus aureus.

Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETBTY4. Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETBTY4-based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids.

Curcuma and Scutellaria plant extracts protect chickens against inflammation and Salmonella Enteritidis infection.

After a ban on the use of antibiotics as growth promoters in farm animals in the European Union in 2006, an interest in alternative products with antibacterial or anti-inflammatory properties has increased. In this study, we therefore tested the effects of extracts from Curcuma longa and Scutellaria baicalensis used as feed additives against cecal inflammation induced by heat stress or Salmonella Enteritidis (S. Enteritidis) infection in chickens. Curcuma extract alone was not enough to decrease gut inflammation induced by heat stress. However, a mixture of Curcuma and Scutellaria extracts used as feed additives decreased gut inflammation induced by heat or S. Enteritidis, decreased S. Enteritidis counts in the cecum but was of no negative effect on BW or humoral immune response. Using next-generation sequencing of 16S rRNA we found out that supplementation of feed with the 2 plant extracts had no effect on microbiota diversity. However, if the plant extract supplementation was provided to the chickens infected with S. Enteritidis, Faecalibacterium, and Lactobacillus, both bacterial genera with known positive effects on gut health were positively selected. The supplementation of chicken feed with extracts from Curcuma and Scutelleria thus may be used in poultry production to effectively decrease gut inflammation and increase chicken performance.

Detecting horizontal gene transfer among microbiota: an innovative pipeline for identifying co-shared genes within the mobilome through advanced comparative analysis.

Horizontal gene transfer (HGT) is a key driver in the evolution of bacterial genomes. The acquisition of genes mediated by HGT may enable bacteria to adapt to ever-changing environmental conditions. Long-term application of antibiotics in intensive agriculture is associated with the dissemination of antibiotic resistance genes among bacteria with the consequences causing public health concern. Commensal farm-animal-associated gut microbiota are considered the reservoir of the resistance genes. Therefore, in this study, we identified known and not-yet characterized mobilized genes originating from chicken and porcine fecal samples using our innovative pipeline followed by network analysis to provide appropriate visualization to support proper interpretation.

The hare syphilis agent is related to, but distinct from, the treponeme causing rabbit syphilis.

The treponemes infecting lagomorphs include Treponema paraluisleporidarum ecovar Cuniculus (TPeC) and ecovar Lepus (TPeL), infecting rabbits and hares, respectively. In this study, we described the first complete genome sequence of TPeL, isolate V3603-13, from an infected mountain hare (Lepus timidus) in Sweden. In addition, we determined 99.0% of the genome sequence of isolate V246-08 (also from an infected mountain hare, Sweden) and 31.7% of the genome sequence of isolate Z27 A77/78 (from a European hare, Lepus europeaus, The Netherlands). The TPeL V3603-13 genome had considerable gene synteny with the TPeC Cuniculi A genome and with the human pathogen T. pallidum, which causes syphilis (ssp. pallidum, TPA), yaws (ssp. pertenue, TPE) and endemic syphilis (ssp. endemicum, TEN). Compared to the TPeC Cuniculi A genome, TPeL V3603-13 contained four insertions and 11 deletions longer than three nucleotides (ranging between 6 and2,932 nts). In addition, there were 25 additional indels, from one to three nucleotides long, altogether spanning 36 nts. The number of single nucleotide variants (SNVs) between TPeC Cuniculi A and TPeL V3603-13 were represented by 309 nucleotide differences. Major proteome coding differences between TPeL and TPeC were found in the tpr gene family, and (predicted) genes coding for outer membrane proteins, suggesting that these components are essential for host adaptation in lagomorph syphilis. The phylogeny revealed that the TPeL sample from the European brown hare was more distantly related to TPeC Cuniculi A than V3603-13 and V246-08.

High prevalence and genetic diversity of Treponema paraluisleporidarum isolates in European lagomorphs.

IMPORTANCE: Syphilis is an ancient disease of humans and lagomorphs caused by two distinct but genetically closely related bacteria (>98% sequence identity based on the whole genome) of the genus Treponema. While human syphilis is well studied, little is known about the disease in the lagomorph host. Yet, comparative studies are needed to understand mechanisms in host-pathogen coevolution in treponematoses. Importantly, Treponema paraluisleporidarum-infected hare populations provide ample opportunity to study the syphilis-causing pathogen in a naturally infected model population without antibiotic treatment, data that cannot be obtained from syphilis infection in humans. We provide data on genetic diversity and are able to highlight various types of repetitions in one of the two hypervariable regions at the tp0548 locus that have not been described in the human syphilis-causing sister bacterium Treponema pallidum subsp. pallidum.

The genomes of the yaws bacterium, Treponema pallidum subsp. pertenue, of nonhuman primate and human origin are not genomically distinct.

BACKGROUND: Treponema pallidum subsp. pertenue (TPE) is the causative agent of human yaws. Yaws is currently reported in 13 endemic countries in Africa, southern Asia, and the Pacific region. During the mid-20th century, a first yaws eradication effort resulted in a global 95% drop in yaws prevalence. The lack of continued surveillance has led to the resurgence of yaws. The disease was believed to have no animal reservoirs, which supported the development of a currently ongoing second yaws eradication campaign. Concomitantly, genetic evidence started to show that TPE strains naturally infect nonhuman primates (NHPs) in sub-Saharan Africa. In our current study we tested hypothesis that NHP- and human-infecting TPE strains differ in the previously unknown parts of the genomes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we determined complete (finished) genomes of ten TPE isolates that originated from NHPs and compared them to TPE whole-genome sequences from human yaws patients. We performed an in-depth analysis of TPE genomes to determine if any consistent genomic differences are present between TPE genomes of human and NHP origin. We were able to resolve previously undetermined TPE chromosomal regions (sequencing gaps) that prevented us from making a conclusion regarding the sequence identity of TPE genomes from NHPs and humans. The comparison among finished genome sequences revealed no consistent differences between human and NHP TPE genomes. CONCLUSION/SIGNIFICANCE: Our data show that NHPs are infected with strains that are not only similar to the strains infecting humans but are genomically indistinguishable from them. Although interspecies transmission in NHPs is a rare event and evidence for current spillover events is missing, the existence of the yaws bacterium in NHPs is demonstrated. While the low risk of spillover supports the current yaws treatment campaign, it is of importance to continue yaws surveillance in areas where NHPs are naturally infected with TPE even if yaws is successfully eliminated in humans.

Genomic analysis of Paenibacillus larvae isolates from the Czech Republic and the neighbouring regions of Slovakia.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybee larvae. In the Czech Republic, two large infested regions were recognised. This study aimed to analyse P. larvae strains occurring in the Czech Republic in the years 2016-2017 and to characterise the genetic structure of their population with the use of Enterobacterial Repetitive Intergenic Consensus genotyping (ERIC), multilocus sequence typing (MLST) and whole genome sequence (WGS) analysis. The results were complemented by the analysis of isolates collected in the year 2018 in areas of Slovakia located near the Czechia-Slovakia border. ERIC genotyping revealed that 78.9% of tested isolates belonged to the ERIC II genotype and 21.1% to ERIC I genotype. MLST showed six sequence types with ST10 and ST11 being the most frequent among isolates. Within six isolates we found discrepancies in correlations between MLST and ERIC genotypes. The use of MLST and WGS analysis of isolates revealed that each of the large infested geographic regions had its own dominating P. larvae strains. We assume that these strains represented primary sources of infection in the affected areas. In addition, the sporadic presence of strains identified by core genome analysis as genetically related was unveiled in geographically distant regions suggesting possible human-mediated transmission of AFB.

The distribution of antibiotic resistance genes in chicken gut microbiota commensals.

Antibiotic resistance in bacterial pathogens or several indicator bacteria is commonly studied but the extent of antibiotic resistance in bacterial commensals colonising the intestinal tract is essentially unknown. In this study, we aimed to investigate the presence of horizontally acquired antibiotic resistance genes among chicken gut microbiota members in 259 isolates with known whole genomic sequences. Altogether 124 isolates contained at least one gene coding for antibiotic resistance. Genes coding for the resistance to tetracyclines (detected in 101 isolates), macrolide-lincosamide-streptogramin B antibiotics (28 isolates) and aminoglycosides (25 isolates) were the most common. The most frequent tetracycline resistance genes were tet(W), tet(32), tet(O) and tet(Q). Lachnospiraceae and Ruminococcaceae frequently encoded tet(W). Lachnospiraceae commonly coded also for tet(32) and tet(O). The tet(44) gene was associated with Erysipelotrichaceae and tet(Q) was detected in the genomes of Bacteroidaceae and Porphyromonadaceae. Without any bias we have shown that antibiotic resistance is quite common in gut commensals. However, a comparison of codon usage showed that the above-mentioned families represent the most common current reservoirs but probably not the original host of the detected resistances.

Clinical Characteristics of Patients with Tick-Borne Encephalitis (TBE): A European Multicentre Study from 2010 to 2017.

Tick-borne encephalitis (TBE) virus is a major cause of central nervous system infections in endemic countries. Here, we present clinical and laboratory characteristics of a large international cohort of patients with confirmed TBE using a uniform clinical protocol. Patients were recruited in eight centers from six European countries between 2010 and 2017. A detailed description of clinical signs and symptoms was recorded. The obtained information enabled a reliable classification in 553 of 555 patients: 207 (37.3%) had meningitis, 273 (49.2%) meningoencephalitis, 15 (2.7%) meningomyelitis, and 58 (10.5%) meningoencephalomyelitis; 41 (7.4%) patients had a peripheral paresis of extremities, 13 (2.3%) a central paresis of extremities, and 25 (4.5%) had single or multiple cranial nerve palsies. Five (0.9%) patients died during acute illness. Outcome at discharge was recorded in 298 patients. Of 176 (59.1%) patients with incomplete recovery, 80 (27%) displayed persisting symptoms or signs without recovery expectation. This study provides further evidence that TBE is a severe disease with a large proportion of patients with incomplete recovery. We suggest monitoring TBE in endemic European countries using a uniform protocol to record the full clinical spectrum of the disease.

Whole genome sequence of the Treponema pallidum subsp. endemicum strain Iraq B: A subpopulation of bejel treponemes contains full-length tprF and tprG genes similar to those present in T. p. subsp. pertenue strains.

Treponema pallidum subsp. endemicum (TEN) is the causative agent of endemic syphilis (bejel). Until now, only a single TEN strain, Bosnia A, has been completely sequenced. The only other laboratory TEN strain available, Iraq B, was isolated in Iraq in 1951 by researchers from the US Centers for Disease Control and Prevention. In this study, the complete genome of the Iraq B strain was amplified as overlapping PCR products and sequenced using the pooled segment genome sequencing method and Illumina sequencing. Total average genome sequencing coverage reached 3469×, with a total genome size of 1,137,653 bp. Compared to the genome sequence of Bosnia A, a set of 37 single nucleotide differences, 4 indels, 2 differences in the number of tandem repetitions, and 18 differences in the length of homopolymeric regions were found in the Iraq B genome. Moreover, the tprF and tprG genes that were previously found deleted in the genome of the TEN Bosnia A strain (spanning 2.3 kb in length) were present in a subpopulation of TEN Iraq B and Bosnia A microbes, and their sequence was highly similar to those found in T. p. subsp. pertenue strains, which cause the disease yaws. The genome sequence of TEN Iraq B revealed close genetic relatedness between both available bejel-causing laboratory strains (i.e., Iraq B and Bosnia A) and also genetic variability within the bejel treponemes comparable to that found within yaws- or syphilis-causing strains. In addition, genetic relatedness to TPE strains was demonstrated by the sequence of the tprF and tprG genes found in subpopulations of both TEN Iraq B and Bosnia A. The loss of the tprF and tprG genes in most TEN microbes suggest that TEN genomes have been evolving via the loss of genomic regions, a phenomenon previously found among the treponemes causing both syphilis and rabbit syphilis.

Use of 16S rRNA gene sequencing for prediction of new opportunistic pathogens in chicken ileal and cecal microbiota.

In this study, we addressed differences in the development of gut microbiota in 4 successive batches of commercially hatched broiler parent chickens. When planning this study, we expected to find a batch with compromised performance which would allow identification of microbiota of suboptimal composition. Microbiota composition was determined only by sequencing the V3/V4 region of 16S rRNA genes in samples collected from chickens 5 to 18 wk of age. In a total, 100 and 160 samples originating from the ileum or cecum were processed, respectively. In one of the flocks with suboptimal performance we identified an increased abundance of Helicobacter brantae forming over 80% of ileal microbiota in individual chickens. Moreover, we also tested samples of 53-wk-old hens from the same genetic line in which egg production decreased. In this case, cecal microbiota was enriched for Fusobacterium mortiferum forming over 30% of total cecal microbiota. Although none of the identified unusual microbiota members have been well recognized as pathogenic, they may represent new opportunistic pathogens of chickens worth of further investigation. Analysis of gut microbiota composition by next generation sequencing thus proved as a useful and unbiased alternative to bacterial culture, especially in the cases of unspecific symptoms like decrease in flock performance.

Contact with adult hen affects development of caecal microbiota in newly hatched chicks.

Chickens in commercial production are hatched in a clean hatchery environment in the absence of any contact with adult hens. However, Gallus gallus evolved to be hatched in a nest in contact with an adult hen which may act as a donor of gut microbiota. In this study, we therefore addressed the issue of microbiota development in newly hatched chickens with or without contact with an adult hen. We found that a mere 24-hour-long contact between a hen and newly hatched chickens was long enough for transfer of hen gut microbiota to chickens. Hens were efficient donors of Bacteroidetes and Actinobacteria. However, except for genus Faecalibacterium and bacterial species belonging to class Negativicutes, hens did not act as an important source of Gram-positive Firmicutes. Though common to the chicken intestinal tract, Lactobacilli and isolates from families Erysipelotrichaceae, Lachnospiraceae and Ruminococcaceae therefore originated from environmental sources instead of from the hens. These observation may have considerable consequences for the evidence-based design of the new generation of probiotics for poultry.

Gut Anaerobes Capable of Chicken Caecum Colonisation.

Chicks in commercial production are highly sensitive to enteric infections and their resistance can be increased by administration of complex adult microbiota. However, it is not known which adult microbiota members are capable of colonising the caecum of newly hatched chicks. In this study, we therefore orally inoculated chicks with pure cultures of 76 different bacterial isolates originating from chicken caecum on day 1 of life and determined their ability to colonise seven days later. The caecum of newly hatched chickens could be colonised by bacteria belonging to phyla Bacteroidetes, Proteobacteria, Synergistetes, or Verrucomicrobia, and isolates from class Negativicutes (phylum Firmicutes). On the other hand, we did not record colonisation with isolates from phyla Actinobacteria and Firmicutes (except for Negativicutes), including isolates from families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Lactobacillaceae. Representatives of genera commonly used in probiotics such as Lactobacillus, Enterococcus, or Bacillus therefore did not colonise the chicken intestinal tract after a single dose administration. Following challenge with Salmonella enterica serovar Enteritidis, the best protecting isolates increased the chicken's resistance to S. Enteritidis only tenfold, which, however, means that none of the tested individual bacterial isolates on their own efficiently protected chicks against S. Enteritidis.