Citrobacter freundii Activation of NLRP3 Inflammasome via the Type VI Secretion System.

Citrobacter freundii is a significant cause of human infections, responsible for food poisoning, diarrhea, and urinary tract infections. We previously identified a highly cytotoxic and adhesive C. freundii strain CF74 expressing a type VI secretion system (T6SS). In this study, we showed that in mice-derived macrophages, C. freundii CF74 activated the Nucleotide Oligomerization Domain -Like Receptor Family, Pyrin Domain Containing 3(NLRP3) inflammasomes in a T6SS-dependent manner. The C. freundii T6SS activated the inflammasomes mainly through caspase 1 and mediated pyroptosis of macrophages by releasing the cleaved gasdermin-N domain. The CF74 T6SS was required for flagellin-induced interleukin 1ß release by macrophages. We further show that the T6SS tail component and effector, hemolysin co-regulation protein-2 (Hcp-2), was necessary and sufficient to trigger NLRP3 inflammasome activation. In vivo, the T6SS played a key role in mediating interleukin 1ß secretion and the survival of mice during C. freundii infection in mice. These findings provide novel insights into the role of T6SS in the pathogenesis of C. freundii.

Citrobacter freundii impairs the phosphoryl transfer network in the gills of Rhamdia quelen: Impairment of bioenergetics homeostasis.

The precise coupling of spatially separated intracellular adenosine triphosphate (ATP)-producing and ATP-consuming, catalyzed by creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), is a critical process in the bioenergetics of tissues with high energy demand, such as the branchial tissue. The effects of Citrobacter freundii infection on gills remain poorly understood, limited only to histopathological studies. Thus, the aim of this study was to evaluate whether experimental infection by C. freundii impairs the enzymes of the phosphoryl transfer network in gills of silver catfish (Rhamdia quelen). The CK (cytosolic and mitochondrial) and AK activities decreased in infected compared to uninfected animals, while the PK activity did not differ between groups. The gill histopathology of infected animals revealed extensive degeneration with fusion and necrosis of secondary lamellae, detachment of superficial epithelium, aneurysm, vessel congestion and inflammatory process. Based on these evidences, the inhibition and absence of an efficient communication between CK compartments caused the impairment of the branchial bioenergetics homeostasis, which was not compensated by the augmentation on branchial AK activity in an attempt to restore energy homeostasis. In summary, these alterations contribute to disease pathogenesis linked to branchial tissue in animals infected with C. freundii.

κ-Carrageenan Enhances Lipopolysaccharide-Induced Interleukin-8 Secretion by Stimulating the Bcl10-NF-κB Pathway in HT-29 Cells and Aggravates C. freundii-Induced Inflammation in Mice.

Background. The dietary usage of carrageenan as common food additive has increased observably over the last 50 years. But there is substantial controversy about its safety. Methods. We investigated whether the κ-carrageenan could enhance lipopolysaccharide-induced IL-8 expression by studying its actions on the TLR4-NF-κB pathway. The aggravating effect of κ-carrageenan on Citrobacter freundii DBS100-induced intestinal inflammation was also investigated in a mouse model. Results. Our data show that κ-carrageenan pretreatment promoted LPS-induced IL-8 expression in HT-29 cells. Although CD14, MD-2, and TLR4 were upregulated, the binding of LPS was not enhanced. However, the pathway of Bcl10-NF-κB was triggered. Interestingly, κ-carrageenan competitively blocked the binding of FITC-LPS. Furthermore, pretreatment with κ-carrageenan for one week previous to gavage with C. freundii DBS100 markedly aggravated weight loss, mortality, and colonic damage. The secretion of cytokines was unbalanced and the ratio of Tregs was decreased significantly. In addition, κ-carrageenan, together with C. freundii DBS100, enhanced the transcription and secretion of TLR4 and NF-κB. Conclusions. κ-Carrageenan can synergistically activate LPS-induced inflammatory through the Bcl10-NF-κB pathway, as indicated by its aggravation of C. freundii DBS100-induced colitis in mice. General Significance. Our results suggest that κ-carrageenan serves as a potential inflammatory agent that magnifies existing intestinal inflammation.

Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump.

OBJECTIVES: To characterize the pNDM-CIT plasmid identified in Citrobacter freundii carrying genes encoding the metallo-ß-lactamase NDM-1 and the 16S RNA methylase ArmA. METHODS: The complete DNA sequence of pNDM-CIT was obtained by using the 454-Genome Sequencer FLX procedure on a library obtained using plasmid DNA purified from the pNDM-CIT Escherichia coli J53 transconjugant. Contig assembly and predicted gaps were confirmed and filled by PCR-based gap closure. Comparative analysis with IncHI1 incompatibility group plasmids was performed using BLASTN and BLASTP algorithms. RESULTS: Plasmid pNDM-CIT was 288::920 bp and revealed an IncHI1 plasmid scaffold, showing novel resistance and potential virulence determinants. The bla(NDM-1) gene was identified within a novel genetic context, flanked by a duplication of the class 1 integron on both sides. The replicase gene repAciN, originating from Acinetobacter spp. plasmids, was identified in a close association with the Tn1548::armA transposon and the macrolide resistance mel-mph2 cluster. The same structure was identified in silico from a series of enterobacterial plasmids carrying the armA gene. The repAciN gene probably represents a remnant sign of the original occurrence of the armA gene in Acinetobacter plasmids. A CP4-like prophage sequence was identified in pNDM-CIT, containing a resistance-nodulation-cell division/multidrug resistance (RND/MDR) efflux pump cluster surrounded by two IS1-like elements. This resistance determinant, associated with such a prophage sequence, has never been reported on plasmids. CONCLUSIONS: Plasmid pNDM-CIT differed significantly from all known bla(NDM-1)-carrying plasmids identified in Enterobacteriaceae, since it combines the metallo-ß-lactamase NDM-1, the 16S RNA methylase ArmA and a cryptic prophage carrying the RND/MDR efflux pump.

Hydrolysis spectrum extension of CMY-2-like ß-lactamases resulting from structural alteration in the Y-X-N loop.

The Citrobacter freundii isolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) ß-lactamase. The chromosome-borne bla(AmpC-CHA) gene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded the Escherichia coli TOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resulting E. coli TOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 ß-lactamase, which is structurally related to the chromosome-borne cephalosporinase of C. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficient E. coli strain. Moreover, it exhibited increased catalytic efficiency (k(cat)/K(m)) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (k(cat)), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A ß-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

Outbreak of Klebsiella pneumoniae carbapenemase-2-producing K. pneumoniae sequence type 11 in Taiwan in 2011.

From June to September 2011, a total of 305 ertapenem-nonsusceptible Enterobacteriaceae isolates (MICs of ertapenem ≥ 1 µg/ml) were collected from 11 hospitals in different parts of Taiwan. The MICs of 12 antimicrobial agents against these isolates were determined using the broth microdilution method, and genes for carbapenemases were detected using PCR. Genotypes of isolates possessing carbapenemase genes were identified by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The ertapenem-nonsusceptible Enterobacteriaceae isolates included Klebsiella pneumoniae (n = 219), Escherichia coli (n = 64), Enterobacter cloacae (n = 15), and other species (n = 7). Seven (2.3%) of the ertapenem-nonsusceptible Enterobacteriaceae isolates exhibited colistin MICs of >4 µg/ml, and 24 (7.9%) were not susceptible to tigecycline (MICs > 2 µg/ml). A total of 29 (9.5%) isolates carried genes encoding carbapenemases, namely, K. pneumoniae carbapenemase-2 (KPC-2) in 16 (7.3%) isolates of K. pneumoniae (KPC-2-KP) and IMP-8 in 5 (2.3%) isolates of K. pneumoniae, 5 (33.3%) isolates of E. cloacae, 1 isolate of E. coli, 1 isolate of Klebsiella oxytoca, and one isolate of Citrobacter freundii. The 16 KPC-2-KP isolates were isolated from patients at four different hospitals in northern Taiwan. All 16 of the KPC-2-KP isolates were susceptible to amikacin and colistin and had a similar pulsotype (pulsotype 1) and the same sequence type (sequence type 11). Infections due to KPC-2-KP mainly occurred in severely ill patients in the intensive care unit (n = 14, 88%). Four patients with infections due to KPC-2-KP died within 14 days of hospitalization. The findings are the first to demonstrate intrahospital and interhospital dissemination of KPC-2-KP in northern Taiwan.

Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea. RESULTS: During an epidemiologic study focusing on infantile diarrhea, aggregative C. freundii (EACF) and EAEC strains were concomitantly recovered from a severe case of mucous diarrhea. Thereby, the occurrence of synergic events involving these strains was investigated. Coinfection of HeLa cells with EACF and EAEC strains showed an 8-fold increase in the overall bacterial adhesion compared with single infections (P < 0.001). The synergic effect was mediated by physical interactions among the bacteria and primed in the absence of chemical signaling and without the participation of host cells. Thus, significant increases (2.7-fold on average) in bacterial adhesion were also observed during the formation of mixed biofilms on abiotic surfaces. Bacterial settling assays showed that EAEC strains harboring F-pili genes (traA) were capable of forming bacterial aggregates only in the presence of EACF. Scanning electronic microscopy analyses revealed that bacterial aggregates as well as enhanced biofilms formed by EACF and traA-positive EAEC were mediated by non-bundle forming, flexible pili. Moreover, mixed biofilms formed by EACF and traA-positive EAEC strains were significantly reduced using nonlethal concentration of zinc, a specific inhibitor of F pili. In addition, EAEC strains isolated from diarrheic children frequently produced single biofilms sensitive to zinc. CONCLUSIONS: Putative F pili expressed by EAEC strains boosted mixed biofilm formation when in the presence of aggregative C. freundii.

[Ability of opportunistic enterobacteria to adapt to different temperatures].

AIM: To study variability of enzymatic apparatus of opportunistic enterobacteria. MATERIALS AND METHODS: Clinical strains of Morganella morganii, Citrobacter freundii, Proteus mirabilis isolated from patients treated in Irkutsk Regional Hospital for Infectious Diseases. Activity of cellulase and lipase as well as amount of auxins and gibberellins was studied in these bacteria at different cultivation temperatures. RESULTS: It was shown that studied species isolated from humans enterobacteria are able to produce plant growth regulators amount of which depends from cultivation temperature and type of microorganism. Activity of cellulase sharply rises if temperature falls. CONCLUSION: Obtained results show high adaptation potential of opportunistic bacteria from Enterobacteriaceae family. Switch on saprophytic mechanism after fall of temperature to environment-corresponding values allows them to survive in soil and arrange different interactions with soil biota including plants.

Induction of plasmid-mediated AmpC ß-lactamase DHA-1 by piperacillin/tazobactam and other ß-lactams in Enterobacteriaceae.

Chromosomal AmpC ß-lactamase induction by several types of ß-lactams has been reported, but not enough data are available on DHA-1 ß-lactamase, a plasmid-mediated AmpC ß-lactamase. Therefore, we evaluated the DHA-1 ß-lactamase induction by various antibiotics including piperacillin/tazobactam (PIP/TZB) in this study. Six strains (Enterobacter cloacae 2 strains, Citrobacter freundii 1 strain, Serratia marcescens 2 strain, and Morganella morganii 1 strain) possessing chromosomal inducible AmpC ß-lactamase were used as controls. Four strains (Escherichia coli 2 strains, Klebsiella pneumoniae 1 strain, and C. koseri 1 strain) possessing DHA-1 ß-lactamase were used. The ß-lactamase activities were determined by a spectrophotometer using nitrocefin. ß-lactamase induction by PIP, PIP/TZB was not observed in any strains and ß-lactamase induction by third- and fourth-generation cephems was not observed in most strains. The induction ratios of the chromosomal AmpC ß-lactamase in the reference group by PIP/TZB were <1.51, and those of the DHA-1 ß-lactamase were <1.36, except for K. pneumoniae Rkp2004 (2.22). The ß-lactamase induction by first- and second-generation cephems, flomoxef, and carbapenem differed in each strain. Cefmetazole (CMZ) strongly induced ß-lactamase. This study demonstrated that the induction of DHA-1 ß-lactamase was similar to that of chromosomal AmpC using various Enterobacteriaceae, although the induction of ß-lactamase in both groups by PIP/TZB was low. We also reported that the induction of PIP/TZB, a ß-lactamase inhibitor combination antibiotic, against various AmpC-producing Enterobacteriaceae, including DHA-1 producers, was low.

Comparative analysis of two attacin genes from Hyphantria cunea.

A full-length clone corresponding to attacin was isolated from a cDNA library made from fat body of immunized Hyphantria cunea larvae. This newly isolated attacin B shows characteristics different from those previously reported for attacin A. The two attacin cDNAs encode precursor proteins of 233 and 248 amino acid residues, respectively. The two attacins show 45.9% identity at the amino acid level, and 35.2% identity at the nucleotide level. Attacins A and B of H. cunea show significant identities with the attacins of Lepidoptera. Attacin B is a typical glycine-rich protein, while attacin A is leucine-rich. Attacin B is expressed from last instar larvae to adult, while attacin A showed stage-specific expression during the prepupal and pupal stages. Attacins A and B are predicted to have different secondary structure in that attacin A has no tendency to form helices but attacin B contains a substantial number of helices. Attacin A is induced at a trace level in infected larvae, while attacin B is strongly induced against Gram-positive and negative bacteria, fungi, and viruses. The attacin B transcripts were detected in fat body, epidermis and hemocytes after injection with Escherichia coli, Citrobacter freundii, or Candida albicans, but not in the midgut and Malpighian tubule. Recombinant attacin A showed no antibacterial activity, while recombinant attacin B showed strong antibacterial activity in proportion to the amount of the protein injected.

Genetic Diversity, Multidrug Resistance, and Virulence of Citrobacter freundii From Diarrheal Patients and Healthy Individuals.

Objectives:Citrobacter freundii is a frequent cause of nosocomial infections and a known cause of diarrheal infections, and has increasingly become multidrug resistant (MDR). In this study, we aimed to determine the genetic diversity, the antimicrobial resistance profiles and in vitro virulence properties of C. freundii from diarrheal patients and healthy individuals. Methods: 82 C. freundii isolates were obtained from human diarrheal outpatients and healthy individuals. Multilocus Sequence Typing (MLST) of seven housekeeping genes was performed. Antimicrobial susceptibility testing was carried out using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Adhesion and cytotoxicity to HEp-2 cells were assessed. PCR and sequencing were used to identify blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, and qepA genes. Results: The 82 C. freundii isolates were divided into 76 sequence types (STs) with 65 STs being novel, displaying high genetic diversity. Phylogenetic analysis divided the 82 isolates into 5 clusters. All 82 isolates were sensitive to imipenem (IPM), but resistant to one or more other 16 antibiotics tested. Twenty-six isolates (31.7%) were multidrug resistant to three or more antibiotic classes out of the 10 distinct antibiotic classes tested. Five MDR isolates, all of which were isolated from 2014, harbored one or more of the resistance genes, blaTEM-1, blaCTX-M-9, aac(6')-Ib-cr, qnrS1, qnrB9, and qnrB13. All 11 qnrB-carrying C. freundii isolates belonged to cluster 1, and one C. freundii isolate carried a new qnrB gene (qnrB92). Six isolates showed strong cytotoxicity to HEp-2 cells, one of which was multidrug resistant. Conclusions:C. freundii isolates from human diarrheal outpatients and healthy individuals were diverse with variation in sequence types, antibiotic resistance profiles and virulence properties.

Enteric bacteria, lipopolysaccharides and related cytokines in inflammatory bowel disease: biological and clinical significance.

Ulcerative colitis (UC) and Crohn's disease (CD) [inflammatory bowel disease (IBD)] are both characterized by an exaggerated immune response at the gut associated lymphoreticular tissue level. Such an abnormal and dysregulated immune response may be directed against luminal and/or enteric bacterial antigens, as also supported by murine models of inflammatory bowel disease (IBD) caused by organisms such as Citrobacter rodentium and Helicobacter hepaticus. Bacterial endotoxins or lipopolysaccharides (LPS) have been detected in the plasma of IBD patients and an abnormal microflora and/or an increased permeability of the intestinal mucosa have been invoked as cofactors responsible for endotoxemia. At the same time, the evidence that phagocytosis and killing exerted by polymorphonuclear cells and monocytes and the T-cell dependent antibacterial activity are decreased in IBD patients may also explain the origin of LPS in these diseases. In IBD, pro-inflammatory cytokines and chemokines have been detected in elevated amounts in mucosal tissue and/or in peripheral blood, thus suggesting a monocyte/macrophage stimulation by enteric bacteria and/or their constituents (e.g. LPS). On these grounds, in experimental models and in human IBD, anti-cytokine monoclonal antibodies and interleukin receptor antagonists are under investigation for their capacity to neutralize the noxious effects of immune mediators. Finally, the administration of lactobacilli is beneficial in human IBD and, in murine colitis, this treatment leads to a normalization of intestinal flora, reducing the number of colonic mucosal adherent and translocated bacteria.

Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia.

Here we review the history, clinical significance, pathology and molecular pathogenesis of Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia. C. rodentium serves as an important model pathogen for investigating the mechanisms controlling attaching and effacing pathology, epithelial hyperproliferation, and tumor promotion in the distal colon of the mouse.

Host/pathogen interactions at mucosal surfaces: immune consequences.

The mucosal immune system has evolved to protect the host against the establishment of infections at or through the mucosal surfaces of the body. Protective immunity must be activated to specific pathogenic agents or their products but inappropriate immune responses to food/environmental antigens must be avoided. Thus, the mucosal immune system is under tight regulation. Pathogenic bacteria and their products can be exploited as specific probes of mucosal immune responses. Bacterial enterotoxins such as cholera toxin are potent mucosal immunogens and adjuvants that activate both mucosal and systemic immune responses. Infection models involving microorganisms such as Citrobacter rodentium can also be used to investigate the consequences of mucosal colonisation that lead to immune disfunction.

Royal Society of Tropical Medicine and Hygiene meeting at Manson House, London, 14 December 1995. Enteropathogenic Escherichia coli--mucosal infection models.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC and C. rodentium. In this study we found similar ultrastructural damage in small intestinal biopsies from an EPEC-infected child and large bowel specimens from C. rodentium-infected mice. The C. rodentium-infected large bowel biopsies revealed massive hyperplastic reactions and the infected human small intestinal biopsies showed an increase in total crypt cell number and mitotic index. EPEC-infected small intestinal organ cultures revealed bacteria adhering in a localized pattern and evidence of A/E lesions. Covaspheres coated with a biologically active cell-binding domain of intimin also adhered to cells in a localized fashion but did not induce the characteristic A/E lesions.

Citrobacter freundii empyema in a patient with occult pulmonary histoplasmosis.

The genus Citrobacter includes three species of organisms that are uncommonly associated with human infection. When they are pathogenic, there are usually one or more associated respiratory, urinary, skin-soft tissue, and central nervous system infections and neonatal sepsis. These infections occur in the wake of significant systemic illness or complicate antibiotic usage. Rarely, infection has been associated with active tuberculosis. The authors report a case of Citrobacter freundii empyema in a patient with occult pulmonary histoplasmosis.

Prophylactic feeding of Lactobacillus acidophilus NCFM to mice attenuates overt colonic hyperplasia.

The objective of this project was to determine if the probiotic Lactobacillus acidophilus NCFM would protect mice from developing transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium. Our hypothesis was that the oral administration of L. acidophilus NCFM to mice would mitigate colonic hyperplasia and modulate the host immune response. A concurrent administration (CA) study was performed by feeding mice phosphate-buffered saline (PBS), C. rodentium only, L. acidophilus NCFM only, or C. rodentium and NCFM concurrently on the same day. The mice in the CA study were not protected by the probiotic, since their mean colon sample weights (0.109 g) were significantly higher than those of the PBS controls (0.0774 g), and the hematoxylin and eosin-stained samples showed histological changes typically associated with TMCH. A prophylactic feeding (PF) study was performed by orally feeding mice PBS or NCFM once daily for 20 consecutive days; in addition, on day 7, mice were challenged with either PBS or C. rodentium. Mice in the PF study were protected when they consumed the probiotic prior to the pathogen challenge, since their mean colon sample weights (0.0812 g) were not significantly higher than those of the controls (0.0753 g). The hematoxylin and eosin-stained samples appeared similar to the control samples, and the intestinal interleukin (IL)-15 and gamma interferon (IFN-gamma) mRNA levels were reduced. L. acidophilus NCFM did attenuate overt colonic hyperplasia when fed to mice prior to challenge with C. rodentium. The mouse model used in this study enabled us to investigate the efficacy of the L. acidophilus NCFM in preventing gastrointestinal disease and is a valid model for future probiotic research.

Comparison of bacteriological, genetic and pathological characters between Escherichia coli O115a,c:K(B) and Citrobacter rodentium.

Murine pathogenic Escherichia coli O115a,c:K(B) (MPEC) is the causative agent of mouse megaenteron, the pathology of which resembles that of transmissible murine colonic hyperplasia caused by Citrobacter rodentium. We compared their genetic and pathological features to reveal the relationship between these two bacteria. To evaluate the genetic distances, 16S rDNA genes were sequenced and biochemical reactions were tested. Mouse strain susceptibility tests, using CF1 MPEC-susceptible germfree mice and BALB/cA(Jic) resistant mice were performed. MPEC strains and C. rodentium showed more than 99.6% identity by comparison of 16S rDNA gene sequences. All results from biochemical reactions and the mouse strain susceptibility tests were identical. It is proposed that MPEC should be reclassified as C. rodentium.

Isolation and characterization of cytotoxic, aggregative Citrobacter freundii.

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 "cytotoxic and aggregative C. freundii." Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii.

In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584.

Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H(2)O(2) and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H(2)O(2) and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture.