To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. Two ...experiments were undertaken, in which 14-day-old chickens were colonized with 1 x 10⁷-1 x 10⁹ CFU g⁻¹Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 μg ml⁻¹ ciprofloxacin, MICs of 16 μg ml⁻¹ and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.
The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific ...PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.
Abstract
Objectives
To assess stability and contribution of a large ESBL-encoding IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts.
Methods
...Specific-pathogen-free 3–4-day-old New Zealand White rabbits and conventionally reared 6-week-old weaned lambs were orally infected with WT E. coli O104:H4 or the ESBL-plasmid-cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time.
Results
Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonization of the two experimental hosts. The plasmid-cured strain was recovered at significantly higher levels than WT during late-stage colonization of rabbits, but at lower levels than WT in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined.
Conclusions
These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.
Verotoxigenic Escherichia coli (VTEC) serotype O157:H7 strains from a Swedish cattle prevalence study (n=32), and livestock-derived strains linked to human disease (n=13), were characterized by ...microarray and PCR detection of virulence genes. The overall aim of the study was to investigate the distribution of known virulence determinants and determine which genes are linked to increased pathogenicity in humans. A core set of 18 genes or gene variants were found in all strains, while seven genes were variably present. This suggests that the majority of VTEC O157:H7 found in Swedish cattle carry a broad repertoire of virulence genes and should be considered potentially harmful to humans. A single virulence gene type was significantly associated with strains linked to human disease cases (P=0·012), but no genetic trait to explain the increased virulence of this genotype could be found.
Enteric bacteria with resistance to third and fourth generation cephalosporin antibiotics, especially Escherichia coli bearing the blaCTX-M gene, have been detected in a wide range of food producing ...animals. However, commercial vaccines for these organisms are not currently available.
An autogenous vaccine was prepared from E. coli bearing the blaCTX-M-14 gene and evaluated as a potential control measure to reduce shedding and dissemination of these organisms in cattle. Calves (n=30) received either an autogenous vaccine prepared from E. coli serotype O33 bearing the blaCTX-M-14 gene or a placebo by intramuscular injection on three separate occasions. Two weeks after the final vaccination, all calves were challenged by oral gavage with the O33 CTX-M-14 strain of E. coli (1×1010 CFU). Faeces, intestinal mucosa and blood samples were taken for enumeration of total and CTX-M-14 E. coli and for assessment of the humoral immune response.
The cumulative number of total E. coli excreted at 7days post-challenge was significantly (p=0.006) lower in the vaccinated group than the placebo group. However, there was no significant difference in the shedding of either CTX-M-14 E. coli or total E. coli between vaccinated and placebo calves throughout the study period. The systemic immune response to E. coli O33 antigen was tested by ELISA and was significantly higher (p<0.001) in vaccinated than placebo calves. However, there was no significant difference in the mucosal immune response. These findings do not support the use of autogenous vaccination for the control of CTX-M-14 E. coli in calves.
The current understanding of the pathogenesis of avian pathogenic
Escherichia coli (APEC) in colisepticaemia is limited. This review discusses putative virulence determinants per se, such as a number ...of surface organelles including fimbriae and flagella; together with other factors such as iron sequestering mechanisms, which are involved in the survival of
E. coli in the host rather than initiation of infection. It is concluded that avian colisepticaemia is a multi-factorial disease and that to date only a limited number of virulence factors of APEC have been thoroughly elucidated.
Summary
The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including ...acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper‐susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB–TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.
Escherichia coli isolates were recovered from faecal samples taken from cattle, sheep and pigs at slaughter in England and Wales. Isolates (
n = 1227) selected at random from this collection were ...each hybridised in colony dot-blot experiments with an
eae gene probe that presumptively identified attaching–effacing
E. coli (AEEC). Of the 99 (8.1%)
eae positive isolates 72 were of ovine origin, 24 were of bovine origin and three of porcine origin. None were typed as O157:H7 whereas 78 were assigned to 23 serogroups and 21 were untypable. The most frequently isolated
eae positive serogroups were O156 (10), O26 (8), O103 (8), O108 (7) O56 (6) and O168 (6) of which serogroups O103 and O156 only were recovered from all three animal species. In tissue culture adherence assays, 36 representatives of
eae positive isolates of all serogroups and host of origin tested induced intimate attachment with varying degrees of actin accumulation and pedestal formation in the HEp-2 cells. The identity of the
eae type for these 36 was determined by specific PCR and the most prevalent intimin types were
eaeβ (15),
eaeγ (12) and
eaeε (4). Isolates were examined by PCR for the presence of other virulence determinants and five possessed
stx1 but none possessed
stx2. One O115
eaeε isolate possessed
cnf1 and 2,
hlyA,
etpD and
katP genes which is a novel combination of virulence determinants.
Recently, it has been reported that
Salmonella secrete flagellin in response to host produced lysophospholipids. However, this monomer of the bacterial flagella activates Toll-like receptor 5 (TLR5) ...in the innate immune system. The objective of this study was to examine the role of flagellin expression during infection of species-specific macrophages (MΦ) which either expressed or lacked TLR5. Initially, TLR5-activity was confirmed in bovine MΦ using
Salmonella typhimurium derived-flagellin. Within these cells, recombinant FliC induced a potent CXCL8 response when compared to the heterogeneous (FliC/FljB) form of purified flagellin. Furthermore, neither form of flagellin induced nitrite secretion which was subsequently detected after exposing bovine MΦ to LPS in the presence of IFN-γ. Flagellin enhanced the accumulation of
Salmonella enteritidis in TLR5-positive bovine and human MΦ which was independent of adhesion in bovine MΦ. In contrast, murine MΦs which lacked TLR5 were equally susceptible to hosting
S. enteritidis, with or without flagellin. However, lack of flagellin in
S. typhimurium marginally inhibited bacterial accumulation in bovine MΦ, where FljB and FliC compensated for the lack of each other. This study suggests that flagellin may be inducing TLR5-dependent internalisation mechanisms in MФ which vary qualitatively between different species and
Salmonella serotypes.
Newly hatched specific pathogen-free chicks were dosed with a suspension of
Bacillus subtilis spores prior to challenge with
Escherichia coli O78:K80, a known virulent strain associated with avian ...colibacillosis, 24
h later. A single oral inoculum of 2.5×10
8 spores was sufficient to suppress all aspects of
E. coli O78:K80 infection. Colonisation of deep organs was reduced by a factor of over
2
log
10
whilst colonisation of the intestine, as measured by direct caecal count, was reduced over
3
log
10
. Shedding of
E. coli O78:K80 was measured by semi-quantitative cloacal swabbing and was reduced significantly for the duration of the experiment, 35 days.
B. subtilis persisted in the intestine although with decreasing numbers over the same period. Challenge with the same dose 5 days after pre-dosing with spores overcame any suppressive effect of the spores.
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