It has long been known that probiotics can be used to maintain intestinal homeostasis and treat a number of gastrointestinal disorders, but the underlying mechanism has remained obscure. Recently, increasing evidence supports the notion that certain probiotic-derived components, such as bacteriocins, lipoteichoic acids, surface layer protein and secreted protein, have a similar protective role on intestinal barrier function as that of live probiotics. These bioactive components have been named ‘postbiotics’ in the most recent publications. We previously found that the Lactobacillus rhamnosus GG (LGG) culture supernatant is able to accelerate the maturation of neonatal intestinal defense and prevent neonatal rats from oral Escherichia coli K1 infection. However, the identity of the bioactive constituents has not yet been determined. In this study, using liquid chromatography-tandem mass spectrometry analysis, we identified a novel secreted protein (named HM0539 here) involved in the beneficial effect of LGG culture supernatant. HM0539 was recombinated, purified, and applied for exploring its potential bioactivity in vitro and in vivo. Our results showed that HM0539 exhibits a potent protective effect on the intestinal barrier, as reflected by enhancing intestinal mucin expression and preventing against lipopolysaccharide (LPS)- or tumor necrosis factor α (TNF-α)-induced intestinal barrier injury, including downregulation of intestinal mucin (MUC2), zonula occludens-1 (ZO-1) and disruption of the intestinal integrity. Using a neonatal rat model of E. coli K1 infection via the oral route, we verified that HM0539 is sufficient to promote development of neonatal intestinal defense and prevent against E. coli K1 pathogenesis. Moreover, we further extended the role of HM0539 and found it has potential to prevent dextran sulfate sodium (DSS)-induced colitis as well as LPS/D-galactosamine-induced bacterial translocation and liver injury. In conclusion, we identified a novel LGG postbiotic HM0539 which exerts a protective effect on intestinal barrier function. Our findings indicated that HM0539 has potential to become a useful agent for prevention and treatment of intestinal barrier dysfunction- related diseases.

Inflammatory bowel disease (IBD) is a chronic and relapsing intestinal inflammatory condition with no effective treatment. Probiotics have gained wide attention because of their outstanding advantages in intestinal health issues. In previous studies, a novel soluble protein, HM0539, which is derived from Lactobacillus rhamnosus GG (LGG), showed significant protective effects against murine colitis, but no clear precise mechanism for this effect was provided. In this study, we hypothesized that the protective function of HM0539 might be derived from its modulation of the TLR4/Myd88/NF-κB axis signaling pathway, which is a critical pathway widely involved in the modulation of inflammatory responses. To test this hypothesis, the underlying anti-inflammatory effects and associated mechanisms of HM0539 were determined both in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and in dextran sulfate sodium (DSS)-induced murine colitis. Our results showed that HM0539 inhibited the expression of cyclooxygenase-2 (COX-2) and the expression inducible nitric oxide synthase (iNOS) by down-regulating the activation of their respective promoter, and as a result this inhibited the production of prostaglandin E2 (PGE2) and nitric oxide (NO). Meanwhile, we demonstrated that HM0539 could ultimately modulate the activation of distal NF-κB by reducing the activation of TLR4 and suppressing the transduction of MyD88. However, even though the overexpression of TLR4 or MyD88 obviously reversed the effect of HM0539 on LPS-induced inflammation, HM0539 still retained some anti-inflammatory activity. Consistent with the in vitro findings, we found that HM0539 inhibited to a great extent the production of inflammatory mediators associated with the suppression of the TLR4/Myd88/NF-κB axis activation in colon tissue. In conclusion, HM0539 was shown to be a promising anti-inflammatory agent, at least in part through its down-regulation of the TLR4-MyD88 axis as well as of the downstream MyD88-dependent activated NF-κB signaling, and hence might be considered as a potential therapeutic option for IBD.

Proteus mirabilis is commonly considered to be an opportunistic pathogen causing urinary tract infections (UTIs) in humans. However, some strains of P. mirabilis were found to be associated with food poisoning outbreaks, with the pathogenic mechanism still unclear. In our study, we described a novel strain of P. mirabilis C02011 isolated from patients’ specimens in a food poisoning in China. In order to determine its gastrointestinal pathogenicity, experiments were performed to compare P. mirabilis B02005 strain (isolated from healthy people) and P. mirabilis American Type Culture Collection (ATCC) 29906 strain both in vitro [Caco-2 cells: bacterial adhesion and invasion assays, Giemsa staining, and transmission electron microscopy (TEM)] and in vivo [BALB/c mouse model: fecal character, colon injury, histological examination, immunochemistry, and western blotting (WB)]. According to the results, C02011 strain exhibited almost identical characteristics with B02005 strain in bacterial appearance and proliferation. In vitro, Caco-2 cells were infected with P. mirabilis C02011, B02005, and P. mirabilis ATCC 29906 strains. After that, Giemsa staining and TEM were used for observing the infection process of C02011 strain. Meanwhile, the adhesive abilities of different strains were rated as follows: P. mirabilis B02005 > P. mirabilis C02011 > P. mirabilis ATCC 29906 (P < 0.01). Invasive abilities of different strains were rated as follows: P. mirabilis C02011 > P. mirabilis B02005 > P. mirabilis ATCC 29906 (P < 0.01). In vivo, BALB/c mice were infected with P. mirabilis C02011 and B02005 strains. C02011 strain shows more virulence than B02005 strain in terms of the following indicators: (1) feces water content and fecal character; (2) colon length of mice; (3) histological examination on mouse intestine tissues; (4) ELISA for detecting TNF-α level in the colon; and (5) WB and immunohistochemistry (IHC) for detecting occludin protein expression in the colon. On the basis of these results, we firstly validated that the novel strain of P. mirabilis C02011 shows more gastrointestinal pathogenicity than the other strains isolated from a healthy individual. In addition, type IV secretion system (T4SS) was preliminarily confirmed to play an important role in the pathogenesis of diarrheal P. mirabilis isolated from the food poisoning incident.

Purpose: Acinetobacter baumannii is an important pathogen in the nosocomial infections worldwide. Combining with carbapenemases, efflux pumps and outer membrane proteins (OMPs) have been thought to affect the development of carbapenem resistance in A. baumannii. This study aimed to investigate the contributions of different efflux pumps and OMPs in developing carbapenem resistance in a clinical isolate of A. baumannii and reveal the possible mechanism of overproduction of main efflux pumps. Patients and methods: In this study, an imipenem-susceptible clinical isolate was identified as A. baumannii and named SZE. Several common carbapenemases were detected by polymerase chain reaction (PCR). Imipenem-selected mutants were selected from SZE by serial subcultivations on Mueller–Hinton agar, and the minimum inhibitory concentration (MIC) was detected. Gene expressions of four families of efflux pumps, five OMPs, and bla_OXA-51 were determined by reverse transcription quantitative PCR, and comparisons were made between SZE strain and the imipenem-selected mutants. The adeRS system in SZE and its mutant was sequenced and aligned. Results: Under consecutive imipenem-selected stress, the MIC to imipenem increased gradually from 0.125 μg/mL to 8 μg/mL. The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor. The expression of efflux pumps, adeB, adeG, and adeJ, was increased by 6.9-, 4.0-, and 2.1-fold in mutants, respectively, compared to SZE. A single mutation (G to A) at position 58 was detected in the regulatory adeRS system and possibly upregulated the adeB expression, and then affected the carbapenem resistance in A. baumannii strains. Conclusion: In conclusion, under consecutive imipenem-selected stress in vitro, A. baumannii strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps. Especially, the resistance-nodulation-cell division super family and a nucleotide mutant in adeRS regulating system caused the overexpression of adeABC.

The objective of this study was to determine whether Lactobacillus rhamnosus GG culture supernatant (LCS) has a preventive effect against gut-derived systemic neonatal Escherichia coli (E. coli) K1 infection. The preventive effects were evaluated in human colonic carcinoma cell line Caco-2 and neonatal rat models. Our in vitro results showed that LCS could block adhesion, invasion and translocation of E. coli K1 to Caco-2 monolayer via up-regulating mucin production and maintaining intestinal integrity. In vivo experiments revealed that pre-treatment with LCS significantly decrease susceptibility of neonatal rats to oral E. coli K1 infection as reflected by reduced bacterial intestinal colonization, translocation, dissemination and systemic infections. Further, we found that LCS treated neonatal rats have higher intestinal expressions of Ki67, MUC2, ZO-1, IgA, mucin and lower barrier permeability than those in untreated rats. These results indicated that LCS could enhance neonatal resistance to systemic E. coli K1 infection via promoting maturation of neonatal intestinal defense. In conclusions, our findings suggested that LCS has a prophylactic effect against systemic E. coli K1 infection in neonates. Future studies aimed at identifying the specific active ingredients in LCS will be helpful in developing effective pharmacological strategies for preventing neonatal E. coli K1 infection.