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Aims: To phytosynthesize silver nanoparticles (AgNPs) and determine their antibacterial and antibiofilm capacity against gram-positive and gram-negative bacterial strains., Methods and Results: AgNPs were synthesized using Bothriochloa laguroides aqueous extract as reducing and stabilizing agent. After characterization, a phytochemical screening to the extract and the AgNPs was performed. Antibacterial activity, inhibition and eradication of biofilms against Staphylococcus aureus and Yersinia enterocolitica strains were tested. Spherical AgNPs with an average size of 8 nm were obtained. Tannins, flavonoids, carbohydrates, proanthocyanidins, anthocyanins and saponins were identified in aqueous extract; meanwhile, only carbohydrates were identified in AgNPs. The MIC and MBC were determined at pmol L -1  levels for all tested strains. Furthermore, AgNPs inhibited more than 90% of biofilms formation and eradicated more than 80% of mature biofilms at concentrations higher than MIC., Conclusions: The AgNPs obtained in this study inhibited planktonic and sessile growth, and eradicated mature biofilms of pathogenic bacterial strains at very low concentrations., Significance and Impact of Study: The current study showed the promising potential of AgNPs as antibiofilm agents opening the way for the future development of a new class of antibacterial products., (© 2021 The Society for Applied Microbiology.)

Aims of the Study: The ability of Yersinia enterocolitica strains to form biofilms and the capacity of different alkaloids to inhibit biofilm formation were investigated., Methods and Results: The capacity to form biofilm on polystyrene of 31 Y. enterocolitica strains was evaluated. Biofilm and quorum sensing (QS) inhibition of 17 alkaloids were assayed; furthermore, minimum biofilm inhibitory concentration (MBIC) was determined. The capacity to form biofilms among the examined strains seemed to be a strain-related feature. The best biofilm inhibitors at 100 µmol l -1 were oliverine (1), guatterine (3), liriodenine (4), oliveridine (5) and pachypodanthine (6), which showed biofilm inhibition higher than 87%. Pachypodanthine (6) was the most effective compound with MBIC value of 12·5 µmol l -1 at subinhibitory concentration and also was able to inhibit QS system and reduce yenR expression at this concentration., Conclusion: This is the first study to demonstrate that oliverine, liriodenine, and pachypodanthine are able to inhibit biofilm formation of Y. enterocolitica without critically disturbing its growing capacity. At MBIC, pachypodanthine inhibited biofilm formation and QS., Significance and Impact of the Study: The use of aporphinoid alkaloids as biofilms inhibitory agents might potentially be useful to treat biofilm-associated infections in the future., (© 2020 The Society for Applied Microbiology.)

Foodborne diseases have become a health issue worldwide, mainly due to the consumption of contaminated foods that are either raw, improperly heat treated or cross-contaminated after adequate heat treatment foods. A group of alkaloids extracted from plants were tested to evaluate their antimicrobial effect against different strains of Yersinia enterocolitica and other foodborne bacteria. The results obtained reveal that oliveridine and pachypodanthine inhibited Y. enterocolitica growth, with MIC values of 25 μmol l -1 and 100 μmol l -1 respectively. The results indicated that both alkaloids are good growth inhibitors, but oliveridine showed greater inhibitory effect with lower MIC values. Inhibitory alkaloids can be developed as potential antimicrobials in food system to prevent or treat foodborne diseases, thus contributing to solve the global issue of contaminated food consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Alkaloids are abundant secondary metabolites in plants and represent one of the most widespread class of compounds endowed with multiple and varied pharmacological properties. In this work, we propose two aporphinoid alkaloids extracted from plants as new antimicrobial agents. Oliveridine and pachypodanthine inhibited Yersinia enterocolitica growth for up to 96 h of culture. This is the first reported study of the activity of these alkaloids as antimicrobial compounds., (© 2019 The Society for Applied Microbiology.)

In this study, our aim was to estimate the extent of Yersinia enterocolitica contamination in frozen foods in China and determine the bioserotype, virulotype, antimicrobial resistance, and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) genotyping profiles of recovered Y. enterocolitica isolates. Out of 455 samples collected between July 2011 and May 2014, 56 (12.3%) tested positive for Y. enterocolitica. The 70 isolated strains were grouped into five clusters and one singleton based on their ERIC-PCR fingerprint, at a similarity coefficient of 70%. All strains were of biotype 1A, and 35.7% were of bioserotype 1A/O:8. Most strains lacked the virulence genes ail, virF, ystA, and ystC, but harbored ystB, fepD, ymoA, fes and sat. All strains were sensitive to ticarcillin but resistant to two or more antibiotics, and 48.6% of the strains were resistant to four to nine antibiotics. High resistance rates were observed for ampicillin, cephalothin, trimethoprim/sulfamethoxazole, amoxicillin/clavulanic acid, nalidixic acid and chloramphenicol (98.6%, 95.7%, 74.3%, 28.6%, 18.6% and 12.9%, respectively). This study provides a systematic surveillance of Y. enterocolitica prevalence in frozen foods in China and indicates its high antibiotic resistance, which could serve as useful information for the government to control Y. enterocolitica contamination in frozen foods and the use of antibiotics., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: The objective of this study was to investigate the survival of Salmonella and Yersinia enterocolitica strains in pig slurry and evaluate urea and ammonia as disinfection strategies., Methods and Results: Salmonella Anatum, Salmonella Derby, Salmonella Typhimurium DT19 and Y. enterocolitica bioserotypes 4, O:3, 2, O:5,27 and 1A, O:6,30 were selectively marked by insertion of the plasmid, pGLO encoding for green fluorescent protein and for ampicillin resistance. Strain cocktails were inoculated into fresh pig slurry (control), slurry treated with urea [final concentration 2% w/w, (0.33 mol l(-1) )] and slurry treated with ammonia [final concentration 0.5% w/w, (0.3 mol l(-1) )] and stored at 4, 14 and 25°C. Bacterial counts were determined at regular intervals on xylose lysine deoxycholate agar (XLD), and XLD supplemented with ampicillin (0.1 mg ml(-1) ) and arabinose (0.6 mg ml(-1) ) for Salmonella and cefsulodin-irgasan-novobiocin agar (CIN) and CIN supplemented with ampicillin and arabinose for Y. enterocolitica. The pH of the control-, urea- and ammonia-treated samples ranged from 7.1 to 7.7, 8.8 to 8.9 and 8.0 to 8.3, respectively. Salmonella D(4) values ranged from 2.71 to 21.29 days, D(14) values from 2.72 to 11.62 days and D(25) values from 1.76 to 6.85 days. The equivalent D values ranges for the Y. enterocolitica strains were 3.7-19.23, 1.8-16.67 and 1.63-7.09 days, respectively. Treatment significantly (P < 0.01) affected D values with control > ammonia > urea, as did incubation temperature; 4 > 14 > 25°C., Conclusions: Urea and to a lesser extent ammonia may be used to disinfect Salmonella- and/or Y. enterocolitica-contaminated pig slurry, decreasing the storage time required while increasing its fertilizer value., Significance and Impact of the Study: This study presents data supporting the treatment of pig slurry to kill important zoonotic agents, thereby reducing environmental contamination, cross-infection of other animals and decreasing zoonotic disease in the food chain., (© 2012 The Society for Applied Microbiology.)

Aims: To determine the effectiveness of sucrose monolaurate (SML) and sucrose monocaprate (SMC), alone and in combination with ethylenediaminetetraacetic acid (EDTA), propionic acid (PA) or citric acid (CA) in reducing mesophilic aerobic bacteria (MAB) and Yersinia enterocolitica O:9 populations on eggshells and their damage potential on the microstructure of shell cuticle., Methods and Results: Uninoculated eggs and eggs submerged in a solution of Y. enterocolitica were immersed in solutions of the various treatments. MAB and Y. enterocolitica counts on the surface of the eggs were carried out before and after treatment. MAB counts decreased less than 2 logs on uninoculated eggshells irrespective of treatment and reductions of 3.2 and 3.0 logs of Y. enterocolitica were obtained with 1000 microg ml(-1) SML plus 0.1% CA or 1000 microg ml(-1) SML plus 600 microg ml(-1) EDTA solutions, respectively. Y. enterocolitica 2/O:9 was recovered from natural microflora. Use of blue lake staining revealed minimal damage to the shells from the washing treatments., Conclusions: SML and SMC at 1000 microg ml(-1) combined with CA or EDTA could be effective in reducing Y. enterocolitica on eggshells with a minimal risk of later bacterial recontamination., Significance and Impact of the Study: Eggs are a recognized vehicle for transmission of Y enterocolitica although a prevalence of only 2.7% was detected in this study. Washing eggs in solutions containing SML or SMC could eliminate Y. enterocolitica contamination of egg shells.

The Tol system is an interactive set of envelope proteins that includes both outer and cytoplasmic membrane proteins. Central to this system is TolA, which spans the periplasmic space to communicate with residents of each membrane. To identify motifs involved in the protein/protein interactions through which TolA acts, the ability of a phylogenetically distinct TolA protein from Yersinia enterocolitica to function in the Tol system of Escherichia coli was examined. Although at least 59 codons shorter and only c. 67% identical to its E. coli homologue, the Y. enterocolitica gene encoded a protein that supported the physiological function of the Tol system in E. coli, and conferred sensitivity to the TolA-dependent colicins A, K, and E1, but interestingly, not to colicin N. The correlation of conferred phenotype with sequence similarities and differences provides a first step in defining essential structural motifs and their specific contributions to function.

The present study demonstrates that catecholamine responsiveness in Yersinia enterocolitica, a bacterial pathogen whose infectious spectrum is principally limited to the gut, is limited to norepinephrine and dopamine, and not epinephrine; this behavior contrasts with observations for two pathogens with a wider extra-gastrointestinal spectrum, Escherichia coli O157:H7 and Salmonella enterica, which respond to all three catecholamines. Epinephrine showed lower potency than norepinephrine and dopamine in inducing growth of E. coli and S. enterica, and was a potent antagonist of norepinephrine and dopamine growth responsiveness in Y. enterocolitica. Given that only norepinephrine and dopamine and not epinephrine-containing neurons are found with the enteric nervous system, the results suggest that certain of the more exclusive enteric pathogens may have developed response systems preferentially for those neuroendocrine hormones that are produced by the enteric nervous system as host-derived signals by which to sense the environment and initiate pathogenic processes.

Objectives: To study at the molecular level the heterogeneity of expression of the two chromosomal beta-lactamases, BlaA and BlaB, in Yersinia enterocolitica strains isolated from clinical samples., Methods: MIC determination by the agar dilution method and beta-lactamase assays was performed to determine the resistance level conferred by these enzymes. DNA cloning, PCR and direct sequencing were used to detect the presence of mutations., Results: The blaA allele from strain IP97 (blaA97) was found to carry a deletion of 51 bp which entirely abolished its beta-lactamase activity. Both the ampR gene and the promoter region of strain Y56 were shown to be functional by a gene swapping experiment. The blaB allele from strain Y56 was found to carry two point mutations, only one of them resulting in a change in the amino acid sequence of the protein. This single amino acid change created a practically inactive BlaB or AmpC cephalosporinase in Y. enterocolitica Y56., Conclusions: The lack of activity observed in the beta-lactamases of some Y. enterocolitica isolates was due to the presence of point mutations or small deletions in the corresponding genes.

Aim: In this study, the growth characteristics of Yersinia enterocolitica biotype 4, GER O:3 plasmid bearing (P+) and plasmid cured (P-) strain types were evaluated in brain heart infusion broth supplemented with cefsulodin, irgasan, and novobiocin alone or in combination., Methods and Results: Growth curves were obtained for the two strain types in broth supplemented with selective agents at 25 or 37 degrees C for 32 h to obtain data on the lag phase durations and growth rates of the strains. Generally, the lag times and growth rates of the P+ and P- strains were similar for cultures incubated at 25 degrees C regardless of the selective agent added and where plasmid replication and expression were not under any significant burden. However, where the lag times and growth rates of the strains were examined at 37 degrees C, significant differences were observed in the lag phase durations of the plasmid bearing strain type compared the plasmid cured strain, an effect that was due to the burden of the plasmid and the influence of selective agents. Generally, when two or more agents were present, lag phase durations were longer for the plasmid bearing strain. Some exceptions noted where in the presence of irgasan or full selective agent (CIN) the opposite case was observed. When growth rates were compared, the plasmidless strain type was typically faster than the plasmid bearing strain in the presence of most selective agents at 37 degrees C and the growth rates of both strain types at 25 degrees C were similar where the temperature appeared to negate the effects of plasmid., Conclusions: The data obtained in these studies suggest that selective agents (in particular irgasan) and incubation temperature play a significant role in influencing the growth characteristics of plasmid bearing and plasmid cured strains of Y. enterocolitica., Significance and Impact of the Study: This data presented in this study has significant implications for enrichment methods used in the detection or recovery of plasmid bearing Y. enterocolitica strains from food, environmental or clinical samples.

In this study, three structurally distinct disintegrins (flavoridin, echistatin, kistrin) were used as molecular probes to further characterize the molecular mechanisms underlying Yersinia enterocolitica infection of host cells. The activity of the three disintegrins on Y. enterocolitica uptake into fibronectin-adherent HeLa cells was evaluated at disintegrin doses which were non-cytotoxic and unable to induce cell detachment. Flavoridin resulted to be the most effective in inhibiting bacterial entry into host cells; echistatin was almost 50% less effective than flavoridin, whereas kistrin was definitely inactive. Our results suggest that alpha(5)beta(1) integrin receptor, which binds flavoridin with higher affinity than the other two disintegrins, plays a major role in Y. enterocolitica uptake into HeLa cells. Furthermore, flavoridin binding to this integrin prevented the disruption of the functional complex FAK-Cas, which occurs in the Y. enterocolitica uptake process.

Objectives: To detect beta-lactamases (A & B) and extended-spectrum beta-lactamases (ESBLs) in clinical and non-clinical isolates of Yersinia enterocolitica biovar 1A, and to determine their activity in the presence of specific lactamase inhibitors., Methods: The presence of beta-lactamases and ESBLs was detected by disc diffusion in 219 (36 clinical, 183 non-clinical) isolates. beta-Lactamase activity was assayed spectrophotometrically in all 36 clinical and 10 representative non-clinical isolates using nitrocefin as the substrate. Inhibition of beta-lactamases was studied by clavulanic acid, aztreonam and cloxacillin., Results: Of the 219 isolates, all except two non-clinical isolates indicated the presence of beta-lactamase A (Bla-A) based on the smaller (2-8 mm) radius of the inhibition zone around the ticarcillin disc. Synergy between ticarcillin and co-amoxiclav discs was, however, observed in only 34% of isolates of non-clinical origin. beta-Lactamase B (Bla-B) was found to be consistently positive among all the clinical and non-clinical isolates, as indicated by its characteristic appearance of flattening of the zone of inhibition around the cefotaxime disc adjacent to an imipenem disc. Bla-B was induced more strongly in clinical than in non-clinical isolates. Inhibition of enzyme A by clavulanic acid, aztreonam and cloxacillin was found to be similar, whereas enzyme B was inhibited more strongly by aztreonam and cloxacillin. None of the isolates showed the unequivocal presence of ESBL., Conclusion: This is the first report on beta-lactamases of Yersinia enterocolitica biovar 1A from Asia. Y. enterocolitica biovar 1A expressed both Bla-A and Bla-B. Heterogeneity was, however, discerned in the expression of Bla-A and by induction of Bla-B among clinical and non-clinical isolates of Y. enterocolitica biovar 1A.

Objectives: The aim of this study was to determine the roles of mutations in the gyrA and parC genes and the overexpression of efflux pump(s) as mechanisms of resistance to quinolones. Forty-five Yersinia enterocolitica O:3 clinical isolates (41 nalidixic acid-resistant, three nalidixic acid-susceptible and one nalidixic acid-resistant strain obtained in vitro) were analysed., Results: All the nalidixic acid-resistant strains showed mutations in the gyrA gene and none in the parC gene. The presence of the inhibitor produced decreases in the MIC values of nalidixic acid by two to six serial dilution steps in 37 of the 41 nalidixic acid-resistant strains. Meanwhile, the MIC value of ciprofloxacin was affected in two strains whose values diminished three serial dilution steps. The nalidixic acid-resistant mutant obtained in vitro was also affected by the inhibitor decreasing the MIC value of nalidixic acid three serial dilutions steps whereas the MICs for the nalidixic acid-susceptible strains were not affected., Conclusions: Our results show that the high level of resistance to nalidixic acid is likely due to an overexpression of an efflux pump plus a mutation in the gyrA gene, whereas decreased susceptibility to ciprofloxacin is only associated with the presence of a mutation in the gyrA gene.

Aims: Yersinia enterocolitica causes several syndromes in humans. The most common presentation is enterocolitis in children, presenting as fever and diarrhoea. A Y. enterocolitica multiple strain infection in twin infants was investigated., Methods and Results: One isolate was recovered from one patient and two morphologically-different isolates were recovered from the other infant. Biochemically, all isolates were identified as Y. enterocolitica group. The genomic DNA from each strain was purified and DNA fingerprinting was performed. The banding patterns observed for Y. enterocolitica isolates 2 and 3, from patients 1 and 2, respectively, were identical when comparing the presence or absence of major bands. However, Y. enterocolitica isolate 1, from patient 1, showed a distinctive banding pattern from isolates 2 and 3., Conclusion: The findings indicate that one infant was colonized by more than one strain of Y. enterocolitica, demonstrating that multiple strains can colonize and invade a patient., Significance and Impact of the Study: Recognition of multiple strain infections can be important in diagnosis, treatment and prognosis of Y. enterocolitica infections, as well as in disease epidemiology. The technique described here offers a straightforward method for strain comparison.

One hundred and forty-five isolates of Yersinia enterocolitica of different serotypes and biotypes, including atypical biotypes, collected from various parts of the world, were examined for their susceptibility to beta-lactam antibiotics and expression of intracellular beta-lactamases. The reasons for the specificity of patterns of susceptibility to beta-lactams for each biotype or subtype of Y. enterocolitica were elucidated by examining their ss-lactamase activity. Whilst the biotypes and subtypes were uniformly susceptible to the newer beta-lactam antibiotics, the susceptibility pattern observed with other beta-lactams was specific to each biotype or subtype, because of the characteristics of beta-lactamase expression by strains within these groups. The susceptibility to these beta-lactam agents depended entirely on the extent of elaboration or the absence of one of the two beta-lactamases, enzyme A and enzyme B, found in the species. Detection of enzyme B by a disc diffusion test yielded inconsistent results, but detection of enzyme A by disc diffusion was highly reliable. This test clearly distinguished strains of biotype 2, serotype O:5,27 from those of biotype 2, serotype O:9 and biotype 3, serotypes O:1, 2a-3, O:3 and O:5.

This study was aimed at elucidating the role of virulence plasmid (pYV) in the susceptibility of Yersinia enterocolitica to bactericidal agents such as chlorine and heavy metals. Plasmid-bearing (pYV+) Y. enterocolitica was less susceptible to the antimicrobial action of chlorine and heavy metals compared with the isogenic plasmidless (pYV-) derivative. This difference was, however, observed only with bacteria cultured at 25 degrees C. pYV-associated susceptibility apart, cells cultured at 37 degrees C were also found to be less susceptible to the antimicrobial action of these agents. The results indicate that the susceptibility of Y. enterocolitica to these agents was influenced both by the presence of the virulence plasmid and the temperature at which the cells were cultured.

Quantification of sanitary-important bacteria (e.g. Enterobacteriaceae), as well as indicators of environmental contamination, was assessed in samples of cattle dung from 25 cattle farms in 15 north-eastern Slovakia districts. The inhibitory effect of crude bacteriocin extract CBE V24 from Enterococcus faecalis V24 against Listeria monocytogenes Ohio and Yersinia enterocolitica YE85 was examined in cattle dung water with the aim of finding a new way of eliminating the health risk of the animal slurry. The following bacterial groups were quantified: Salmonella spp., Shigella-like spp. , Proteus spp., Enterobacter spp., Citrobacter spp., Pseudomonas spp. , Escherichia coli, Listeria spp., staphylococci, streptococci and enterococci (the average count ranged from 102 up to 104 cfu ml-1). Antagonistic effect of the crude bacteriocin from Enterococcus faecalis V24 in the range of 100-600 Arbitrary units per ml (AU ml-1) was shown against the following bacteria: Enterobacter cloacae, Ent. asburiae, Proteus spp., Salmonella spp., Acinetobacter lwoffi, L. monocytogenes as well as Y. enterocolitica YE85. During tests performed to study the inhibitory effect of the crude bacteriocin CBE V24 (concentration 800, 1600 AU ml-1) against L. monocytogenes Ohio and Y. enterocolitica YE85 in experimentally contaminated cattle dung, a reduction of 2.03 and 1.44 log cfu ml-1, respectively, was already noted after 1 h after crude bacteriocin CBE V24 addition.

Previously we have identified and characterized three murine microbicidal proteins purified from the granule fraction of cells from the murine macrophage cell line RAW264.7. During these studies evidence was obtained for the presence of an additional antimicrobial protein in the cytosolic fraction of RAW264.7 cells that had been activated with interferon-gamma (IFN-gamma). In this study we have purified this protein, designated ubiquicidin, to apparent homogeneity and demonstrated that it is a cationic, small (Mr 6654) protein. Ubiquicidin displayed marked antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium. Using a gel overlay procedure evidence was obtained that the protein also displays activity against Escherichia coli, Staphylococcus aureus, and an avirulent strain of Yersinia enterocolitica. Aminoterminal amino acid sequencing and mass spectrometry analysis of purified ubiquicidin indicated that it is most likely identical to the ribosomal protein S30. This protein is produced by posttranslational processing of the Fau protein, a 133-amino-acid fusion protein consisting of S30 linked to an unusual peptide with significant homology to ubiquitin. The fau gene has been reported to be expressed in a variety of tissues in humans and various animal species. The presence of ubiquicidin in the cytosol of macrophages may serve to restrict the intracellular growth of microorganisms. In addition, because macrophage disintegration will likely lead to release of ubiquicidin into the extracellular environment, it may contribute to host defense after macrophage death.

The effect of sodium lactate (NaL) (0, 2.4 or 4.8%), in heating and recovery media, on Yersinia enterocolitica and Listeria monocytogenes numbers recovered from minced beef heated at 55 degrees C, was examined. Survivors were enumerated on selective media at pH 5.7/7.4 (Y. enterocolitica) or pH 5.7/7.2 (L. monocytogenes). Recovery of the organisms depended on the pH and NaL levels in the recovery medium. The heat resistance of Y. enterocolitica (P < 0.001) and L. monocytogenes (P < 0.01) decreased as the concentration of NaL in the minced beef increased from 0 to 2.4% or 4.8%. The thermal destruction of pathogens in foods processed using mild temperatures may be enhanced by the addition of 2.4% NaL.

The antibiotic susceptibilities of 151 Yersinia enterocolitica strains isolated from humans, animals and the environment, and belonging to biovars 1A (n = 22), 1B (n = 13), 2 (n = 12), 3 (n = 31), 4 (n = 63) and 5 (n = 10) to 71 antibiotics were examined. Biovars could be characterized phenotypically by their similar antibiograms. For the majority of biovars only minor differences in susceptibilities were detected. Ninety-nine percent of all Yersinia strains were resistant to amoxycillin, but biovar-related differences in MICs were demonstrated. Significant differences, characteristic of specific biovars, were detected for fosfomycin, some beta-lactams (in particular amoxycillin/clavulanate, and ticarcillin). Strains of biovar 1A were resistant or of intermediate susceptibility to ticarcillin and had fosfomycin MICs of 16-64 mg/L and amoxycillin/clavulanate MICs of 4-32 mg/L. Most strains of biovar 1B (n = 11) were sensitive to amoxycillin/clavulanate (MIC = 0.5-2 mg/L). Most strains of biovar 2 (n = 10) showed an antibiotic phenotype resembling that of strains of biovar 1A, but they were more susceptible to fosfomycin (MIC = 4-16 mg/L). Twenty-seven strains of biovar 3 had amoxycillin/clavulanate MICs of 4-32 mg/L; 19 of them were sensitive to ticarcillin and fosfomycin. Biovar 4 strains were uniformly sensitive or of intermediate susceptibility to amoxycillin/clavulanate (MIC = 0.5-4 mg/L), sensitive to fosfomycin (MIC = 1-4 mg/L) but resistant to ticarcillin. The prevailing antibiotic phenotype of biovar 5 (n = 7) was similiar to the phenotype of biovar 4, although these biovar 5 strains were slightly more susceptible to amoxycillin/clavulanate and showed a heterogeneous pattern of susceptibility to fosfomycin (MIC = 1-32 mg/L). The results of this study show that unambiguous statements cannot be made about the natural antibiotic susceptibility to certain beta-lactams and fosfomycin of Y. enterocolitica. The data indicate a complex regulation of beta-lactamases in Y. enterocolitica. Some beta-lactamases are found more frequently or will be expressed predominantly in specific biovars.

The survival of Yersinia enterocolitica serotype O:3 was examined in an in vitro model of the porcine ileum in the presence of normal ileal microflora using a medium supplemented with the growth-promoting antimicrobials avoparcin, spiramycin, tylosin, carbadox and olaquindox, or without any antimicrobials, at 39 degrees C for 5 d. Growth of Y. enterocolitica was inhibited in all trials in the following order: avoparcin < carbadox < spiramycin < no feed additives < tylosin < olaquindox. The media supplemented with avoparcin, carbadox and spiramycin supported the survival of Y. enterocolitica up to 5 d. When incubated with the normal ileal microflora without any growth-promoting antimicrobials, Y. enterocolitica could not be isolated after 4 d. With olaquindox and tylosin, Y. enterocolitica was not detected after 3 d.

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55 degrees C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at approximately equal to 1-1.5 pH units below that of water-treated controls. Growth permitting pH at 4.8-5.2 was reached after 1% LAD in less than 0.5 d (pH 4.8-5.0), 2% LAD within 1.5 d (pH 4.9-5.1) and after 5% LAD (pH 5.0-5.2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0.9 log10 cfu per cm2 and on 5% LAD the reduction was more than 1.4 log10 cfu per cm2. The reductions in L. monocytogenes were about a third of those in Y. enterocolitica. On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2-5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2-5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2-5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.

The MICs of 24 antimicrobial agents were determined for 335 strains of Yersinia enterocolitica isolated from faeces in the Netherlands during 1982-1991. The isolates belonged to biotypes 1A, 1B, 2, 3, 4 and to serotypes O3, O5.27, O6.3, O7.8, O8, O9. Almost all strains were susceptible to piperacillin, piperacillin/tazobactam, imipenem, all cephalosporins except cefazolin, the aminoglycosides, quinolones, co-trimoxazole, doxycycline and chloramphenicol but resistant to amoxycillin, co-amoxiclav and macrolides. No association was observed between susceptibility patterns, biotype or serotype nor were there marked changes in the susceptibility during the last decade. The agents traditionally used to treat human infection, including co-trimoxazole, doxycycline and chloramphenicol may remain drugs of first choice.

The iron chelator deferoxamine (DFO) B enhances virulence of Yersinia enterocolitica and modulates cellular immune responses. Since cytokines mediate effector mechanisms in resolution of yersiniae from infected tissues, the impact of DFO B and DFO G1 on cytokine production by murine bone marrow macrophages (BMM) was investigated. BMM were stimulated with lipopolysaccharide (LPS) of Salmonella typhimurium or infected with Y. enterocolitica. DFO B inhibited interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-alpha mRNA production 4-fold (shown by semiquantitative reverse transcription polymerase chain reaction). TNF-alpha and IL-6 protein production was reduced 50% by DFO B. In contrast, DFO G1 had no effect on cytokine production. Moreover, cytokine production by Yersinia-infected BMM was decreased by plasmid-encoded Yersinia proteins. Thus, plasmid-cured strains induced higher cytokine responses in BMM than did the wild type strain. These results suggest that DFO B acts in a bimodal fashion in yersiniosis: iron supply to the pathogen and immunosuppression of the host.

The impact of desferrioxamine B (DFOB) on intracellular calcium mobilization, chemoluminescence (CL) signaling, phagocytosis, and bacterial killing of Yersinia enterocolitica by polymorphonuclear leukocytes (PMNL) was investigated. DFOB did not alter calcium signaling in PMNL but transiently increased phagocytosis. Likewise, higher counts of viable bacteria in PMNL and increased CL signals after stimulation with Y. enterocolitica were observed in the presence of DFOB. However, DFOB reduced the CL signal elicited individually by zymosan, PMA, and FMLP. Comparison of plasmid-cured with plasmid-harboring Y. enterocolitica revealed that plasmid-encoded determinants modulate phagocytosis, CL signal, and bactericidal properties of PMNL. These data demonstrate that DFOB alters microbicidal properties of PMNL and modulates their interaction with Y. enterocolitica and provide further evidence for a putative dual role of DFOB in the pathogenesis of Yersinia infection, namely growth support of the pathogen and modulation of the antimicrobial host response.

The effects of desferrioxamine B (DFOB) and G (DFOG) on growth promotion and virulence of Yersinia enterocolitica as well as on T cell activation and proliferation were investigated. Both desferrioxamines promoted growth of Y. enterocolitica O:8 (WA-314) and O:3 (Y-108). DFOB had a greater immunosuppressive effect on T cells than did DFOG. These results suggest a dual role of DFOB in yersiniosis: growth promotion of the pathogen and immunosuppression of the host. The LD50 of both Yersinia strains for mice was reduced by DFOB but not by DFOG. However, the LD50 of yersiniae was reduced by DFOB to a greater extent in Yersinia-resistant C57BL/6 than in Yersinia-susceptible BALB/c mice. The different impact of DFOB on the LD50 of Y. enterocolitica in C57BL/6 and BALB/c mice might be due to an immunomodulating effect of DFOB.

Of 53 documented cases of Yersinia enterocolitica septicemia reported to the French national registry between 1985 and 1991, 43 files contained sufficient information on antibiotic treatment to be analyzed retrospectively. All patients had at least two positive cultures of blood collected before the initiation of treatment. All strains were susceptible in vitro to the antibiotics that are usually active against gram-negative rods except for older beta-lactam agents (i.e., aminopenicillins and first-generation cephalosporins). No multiresistant strain was isolated. Only four (7.5%) of the 53 patients died. Aminopenicillins, first-generation cephalosporins, and--when prescribed alone--amoxicillin/clavulanate were not effective. Third-generation cephalosporins, most often used in combination with other antibiotics, were successful in 85% of cases. Fluoroquinolones--alone or in combination--cured all of 15 infections, with patients improving rapidly and becoming apyretic within 1-4 days. These agents therefore seem to constitute the best treatment.

One hundred clinical isolates of Yersinia enterocolitica were biotyped, serotyped and tested for in-vitro sensitivity to seven beta-lactam antibiotics and six other antibiotics. Twenty-four of the isolates belonged to Niléhn's and Wauters' biotype 1A, 12 to biotype 3 and 64 to biotype 4. No biotypes 1B or 2 strains were found. All isolates were sensitive to chloramphenicol, ciprofloxacin, gentamicin, tetracycline, trimethoprim and all but one to sulphafurazole. All were resistant to ampicillin with MIC's ranging from 32 to 256 mg/L, but were susceptible to ticarcillin/clavulanic acid and imipenem. With other beta-lactam agents there were different patterns of sensitivity, specific to each of the three biotypes. All biotype 4 strains were resistant to carbenicillin and ticarcillin but sensitive to amoxycillin/clavulanic acid and cefoxitin. Biotype 3 strains, by contrast, were sensitive to carbenicillin and ticarcillin but resistant to amoxycillin/clavulanic acid and cefoxitin, whilst biotype 1A strains were resistant to each of these four beta-lactam agents.

The intracellular beta-lactamases of 100 distinct clinical isolates of Yersinia enterocolitica were examined, to investigate the effect of selected beta-lactam agents on the enzymes. The enzyme inhibition profiles obtained were specific to each of the three biotypes 1A, 3 and 4. Biotype 4 strains elaborated only one beta-lactamase with a pI of 8.3 corresponding to the carbenicillin-hydrolysing beta-lactamase enzyme A of Cornelis & Abraham. Biotype 3 strains also produced a single beta-lactamase with a pI of 5.4-5.6 corresponding to enzyme B, the chromosomally mediated inducible cephalosporinase. Two beta-lactamases were found in biotype 1A strains, one was identical to enzyme A and the other, although showing a slight difference in pI to enzyme B, was similar to this enzyme in other respects. The differences in the distribution of enzyme A and enzyme B in strains of Y. enterocolitica belonging to the biotypes 1A, 3 and 4 isolated in Australia, explain the different patterns of susceptibility to beta-lactam antibiotics.

Strain IP134 of Yersinia enterocolitica, which produces two chromosomal-mediated beta-lactamases, was used to detect the presence of beta-lactamase inhibiting agents in plants. Aqueous and alcoholic extracts were obtained from the aerial parts of 179 phanerogamous species, belonging to 39 botanical families. In an assay to detect synergy, eight plants representing a wide taxonomic distribution showed beta-lactamase inhibitory activity. An iodometric assay confirmed the inhibition of beta-lactamases in six of these plants, and revealed beta-lactamase inhibition to be masked by antibacterial activity in two additional plant extracts. Thus, beta-lactamase inhibitory activity was present in 4.5% of the tested species, 1.1% of which had simultaneous antibacterial activity detectable.

Seventeen strains of Yersinia enterocolitica and related species were examined for their susceptibility to a variety of antimicrobial agents. Twelve of the strains were clinical isolates which carried a virulence plasmid; five were food isolates which lacked plasmids. When grown on agar containing Congo red or haemin, each strain yielded pigmented (CR+) and non-pigmented (CR-) variants. CR+ bacteria of clinical origin were virulent, whereas CR+ food isolates and all CR- bacteria were avirulent. The susceptibility of CR+ and CR- bacteria to the following agents was compared: ampicillin, chloramphenicol, latamoxef, nalidixic acid, novobiocin, polymyxin B, rifampicin, streptomycin, tetracycline and tobramycin. The results showed that bacteria which bound Congo red were more susceptible to each drug examined than their CR- counterparts. This relationship held regardless of whether the CR+ bacteria possessed a virulence plasmid or not. The findings suggest that binding of antimicrobial agents by yersiniae may parallel binding of haemin and Congo red.

A prospective study was undertaken to evaluate the incidence, course, effects of treatment and outcome of patients with Yersinia enterocolitica infections. A total of 189 patients were followed: 62.5% had enteric forms of illness, 20.6% extramesenteric forms, 23.2% arthritis and erythema nodosum. Lymphadenopathy with high fever and weight loss, a septic syndrome and hepatitis were predominant manifestations of the extramesenteric form. Ten per cent of the isolates (135) were susceptible to amoxycillin (4 mg/l), 38% to cephradine (8 mg/l), 82% to doxycycline (4 mg/l), 83% to chloramphenicol (4 mg/l), 85% to trimethoprim (1 mg/l), 87% to cefuroxime (8 mg/l), 92% to piperacillin (16 mg/l), 99% to gentamicin (1 mg/l) and 100% to cefotaxime (4 mg/l), pefloxacin (0.12 mg/l), ofloxacin (0.06 mg/l) and ciprofloxacin (0.016 mg/l). The majority of the patients with enteritis recovered without antibiotic therapy. The duration of enteritis was not significantly influenced by antibiotic treatment. Eighty five patients, 46 with enteric and 39 with extramesenteric forms were treated. The clinical response to co-trimoxazole was 71%, and to doxycycline 75%. Cefuroxime, ceftazidime, cefoperazone, piperacillin and gentamicin failed in seven of eight courses. Three patients treated with ciprofloxacin responded well. The role of quinolones in yersiniosis needs further attention.

Iron-overloaded mice were infected with a virulent strain of Yersinia enterocolitica by the oral route to study the effect of antimicrobial treatments. The effects of therapy were assessed by enumeration of viable yersiniae in Peyer's patches and in ileal contents. Combinations of cephalothin and clavulanic acid showed therapeutic effects, which were interpreted as in-vivo synergism, since each component alone was ineffective. Ceftazidime, which is relatively beta-lactamase resistant, showed in-vivo activity similar to that of the combination of cephalothin and clavulanic acid. These results suggest that clavulanic acid is able to protect cephalothin against Y. enterocolitica beta-lactamases in vivo, as has been shown previously in vitro.