Effect of Lactobacillus rhamnosus HN001 and Bifidobacterium longum BB536 on the healthy gut microbiota composition at phyla and species level: a preliminary study

AIM: To evaluate the ability of Lactobacillus rhamnosus HN001 and Bifidobacterium longum BB536 to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. METHODS: Twenty healthy Italian volunteers, eight males and twelve females, participated in the study. Ten subjects took a sachet containing 4 × 109 colony-forming units (CFU) of Bifidobacterium longum BB536 and 109 CFU of Lactobacillus rhamnosus HN001, 30 min before breakfast (pre-prandial administration), while ten subjects took a sachet of probiotic product 30 min after breakfast (post-prandial administration). The ability of Lactobacillus rhamnosus HN001 and Bifidobacterium longum BB536 to colonize human gut microbiota was assessed by means of quantitative real-time PCR, while changes in gut microbiota composition were detected by using Ion Torrent Personal Genome Machine. RESULTS: Immediately after 1-mo of probiotic administration, B. longum BB536 and L. rhamnosus HN001 load was increased in the majority of subjects in both pre-prandial and post-prandial groups. This increase was found also 1 mo after the end of probiotic oral intake in both groups, if compared to samples collected before probiotic consumption. At phyla level a significant decrease in Firmicutes abundance was detected immediately after 1-mo of B. longum BB536 and L. rhamnosus HN001 oral intake. This reduction persisted up to 1 mo after the end of probiotic oral intake together with a significant decrease of Proteobacteria abundance if compared to samples collected before probiotic administration. Whereas, at species level, a higher abundance of Blautia producta, Blautia wexlerae and Haemophilus ducrey was observed, together with a reduction of Holdemania filiformis, Escherichia vulneris, Gemmiger formicilis and Streptococcus sinensis abundance. In addition, during follow-up period we observed a further reduction in Escherichia vulneris and Gemmiger formicilis, together with a decrease in Roseburia faecis and Ruminococcus gnavus abundance. Conversely, the abundance of Akkermansia muciniphila was increased if compared to samples collected at the beginning of the experimental time course. CONCLUSION: B. longum BB536 and L. rhamnosus HN001 showed the ability to modulate the gut microbiota composition, leading to a significant reduction of potentially harmful bacteria and an increase of beneficial ones. Further studies are needed to better understand the specific mechanisms involved in gut microbiota modulation

Role of the Human Breast Milk-Associated Microbiota on the Newborns' Immune System : a Mini Review

The human milk is fundamental for a correct development of newborns, as it is a source not only of vitamins and nutrients, but also of commensal bacteria. The microbiota associated to the human breast milk contributes to create the "initial" intestinal microbiota of infants, having also a pivotal role in modulating and influencing the newborns' immune system. Indeed, the transient gut microbiota is responsible for the initial change from an intrauterine Th2 prevailing response to a Th1/Th2 balanced one. Bacteria located in both colostrum and mature milk can stimulate the anti-inflammatory response, by stimulating the production of specific cytokines, reducing the risk of developing a broad range of inflammatory diseases and preventing the expression of immune-mediated pathologies, such as asthma and atopic dermatitis. The aim of the present Mini Review is to elucidate the specific immunologic role of the human milk-associated microbiota and its impact on the newborn's health and life, highlighting the importance to properly study the biological interactions in a bacterial population and between the microbiota and the host. The Auto Contractive Map, for instance, is a promising analytical methodology based on artificial neural network that can elucidate the specific role of bacteria contained in the breast milk in modulating the infants' immunological response

In vitro activity of telithromycin against Haemophilus influenzae at epithelial lining fluid concentrations

<p>Abstract</p> <p>Background</p> <p><it>Haemophilus influenzae </it>is one of the main aetiological agents of community-acquired respiratory tract infections. The primary aim of this study was to evaluate the antibacterial activity of telithromycin against <it>H. influenzae </it>clinical isolates showing different pattern of resistance in comparison with azithromycin and clarithromycin at 1/4 ×, 1/2 ×, 1 ×, 2 ×, 4 × minimum inhibitory concentration (MIC) and to peak concentrations in epithelial lining fluid (ELF). The secondary aim was to determine the influence of CO₂enriched atmosphere on bacterial susceptibility.</p> <p>Results</p> <p>Telithromycin showed high activity against <it>H. influenzae</it>, including strains susceptible to β-lactams (n = 200), β-lactamase producer (n = 50) and β-lactamase negative ampicillin resistant (BLNAR) (n = 10), with MIC from ≤0.03 to 4 mg/L, and MIC₅₀/MIC₉₀of 1/2 mg/L with susceptibility rate of 100%, and minimum bactericidal concentrations (MBC) from 2 to 4-fold higher than the MIC. Azithromycin was the most active tested macrolide (range: 0.25 – 4 mg/L; MIC₅₀/MIC₉₀: 1/2 mg/L), comparable to telithromycin, while clarithromycin showed the highest MICs and MBCs (range: 0.25 – 8 mg/L; MIC₅₀/MIC₉₀: 2/8 mg/L). In time-kill studies, telithromycin showed a bactericidal activity at the higher concentrations (4 – 2 × MIC and ELF) against all the strains, being complete after 12 – 24 hours from drug exposition. At MIC concentrations, at ambient air, bactericidal activity of telithromycin and azithromycin was quite similar at 12 hours, and better than that of clarithromycin. Besides, telithromycin and clarithromycin at ELF concentrations were bactericidal after 12 hours of incubation for most strains, while 24 hours were needed to azithromycin to be bactericidal. Incubation in CO₂significantly influenced the MICs and MBCs, and only slightly the <it>in vitro </it>killing curves.</p> <p>Conclusion</p> <p>Telithromycin showed an <it>in-vitro </it>potency against <it>H. influenzae </it>comparable to azithromycin, with an <it>in-vitro </it>killing rate more rapid and superior to clarithromycin at 2X-MIC against β-lactamase producers and BLNAR strains, and to azithromycin at ELF concentrations against β-lactamase negative strains. Against all strains, MICs and MBCs were lower in the absence of CO₂for the tested antibiotics, showing an adverse effect of incubation in a CO₂environment. The <it>in-vitro </it>potency together with the tissue concentrations of the antimicrobial, should be considered in predicting efficacy.</p

In vitro selection of resistance in Escherichia coli and Klebsiella spp. at in vivo fluoroquinolone concentrations

<p>Abstract</p> <p>Background</p> <p>Fluoroquinolones are potent antimicrobial agents used for the treatment of a wide variety of community- and nosocomial- infections. However, resistance to fluoroquinolones in Enterobacteriaceae is increasingly reported. Studies assessing the ability of fluoroquinolones to select for resistance have often used antimicrobial concentrations quite different from those actually acquired at the site of infection. The present study compared the ability to select for resistance of levofloxacin, ciprofloxacin and prulifloxacin at concentrations observed <it>in vivo </it>in twenty strains of <it>Escherichia coli </it>and <it>Klebsiella </it>spp. isolated from patients with respiratory and urinary infections. The frequencies of spontaneous single-step mutations at plasma peak and trough antibiotic concentrations were calculated. Multi-step selection of resistance was evaluated by performing 10 serial cultures on agar plates containing a linear gradient from trough to peak antimicrobial concentrations, followed by 10 subcultures on antibiotic-free agar. <it>E. coli </it>resistant strains selected after multi-step selection were characterized for DNA mutations by sequencing <it>gyrA</it>, <it>gyrB</it>, <it>parC </it>and <it>parE </it>genes.</p> <p>Results</p> <p>Frequencies of mutations for levofloxacin and ciprofloxacin were less than 10^-11at peak concentration, while for prulifloxacin they ranged from <10^-11to 10^-5. The lowest number of resistant mutants after multistep selection was selected by levofloxacin followed by ciprofloxacin and prulifloxacin. Both ciprofloxacin- and prulifloxacin-resistant mutants presented mutations in <it>gyrA </it>and <it>parC</it>, while levofloxacin resistance was found associated only to mutations in <it>gyrA</it>.</p> <p>Conclusions</p> <p>Among the tested fluoroquinolones, levofloxacin was the most capable of limiting the occurrence of resistance.</p

In vitro Antimicrobial Activity of Chlorquinaldol against Microorganisms Responsible for Skin and Soft Tissue Infections : Comparative Evaluation with Gentamicin and Fusidic Acid

Skin and soft tissue infections (SSTIs) are a major therapeutic challenge for clinicians. The emergence of pathogens with decreased susceptibility to available therapies has become an emerging problem often associated with treatment failure. Hence, there is an urgent need for novel broad-spectrum antimicrobial agents. The purpose of this study was to assess the feasibility of chlorquinaldol as an alternative approach to currently used topical antibiotics for the treatment of skin and soft tissue infections. The activity of chlorquinaldol was investigated against a collection of bacterial isolates responsible for skin infections, including strains resistant to fusidic acid and gentamicin. After determination of MIC and MBC, time-kill experiments were carried out by counting colonies grown after 0, 3, 6, 9, 24, and 48 h of incubation with concentrations equal to \ubc 7, \ubd 7, 1 7, 2 7, and 4 7 MIC of chlorquinaldol, gentamicin, or fusidic acid. Staphylococci resulted the Gram-positives most sensitive to chlorquinaldol, with MIC-values ranging from 0.016 to 0.5 mg/L. A lower activity was observed against Gram-negative bacteria, with 77% of the isolates being inhibited at concentrations ranging from 128 to 512 mg/L. Generally, in time-kill studies, chlorquinaldol showed a bactericidal activity at the higher concentrations (2 7, 4 7 MIC) after 24-48 h of incubation. In conclusion, chlorquinaldol may represent a valuable alternative to conventional topical antibiotics for the treatment of skin and soft tissue infections

Bifidobacteria and lactobacilli in the gut microbiome of children with non-alcoholic fatty liver disease: which strains act as health players?

Introduction: Non-alcoholic fatty liver disease (NAFLD), considered the leading cause of chronic liver disease in children, can often progress from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). It is clear that obesity is one of the main risk factors involved in NAFLD pathogenesis, even if specific mechanisms have yet to be elucidated. We investigated the distribution of intestinal bifidobacteria and lactobacilli in the stools of four groups of children: obese, obese with NAFL, obese with NASH, and healthy, age-matched controls (CTRLs). Material and methods: Sixty-one obese, NAFL and NASH children and 54 CTRLs were enrolled in the study. Anthropometric and metabolic parameters were measured for all subjects. All children with suspected NASH underwent liver biopsy. Bifidobacteria and lactobacilli were analysed in children’s faecal samples, during a broader, 16S rRNA-based pyrosequencing analysis of the gut microbiome. Results: Three Bifidobacterium spp. (Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium adolescentis) and five Lactobacillus spp. (L. zeae, L. vaginalis, L. brevis, L. ruminis, and L. mucosae) frequently recurred in metagenomic analyses. Lactobacillus spp. increased in NAFL, NASH, or obese children compared to CTRLs. Particularly, L. mucosae was significantly higher in obese (p = 0.02426), NAFLD (p = 0.01313) and NASH (p = 0.01079) than in CTRLs. In contrast, Bifidobacterium spp. were more abundant in CTRLs, suggesting a protective and beneficial role of these microorganisms against the aforementioned diseases. Conclusions: Bifidobacteria seem to have a protective role against the development of NAFLD and obesity, highlighting their possible use in developing novel, targeted and effective probiotics

Strain-dependent release of cytokines modulated by Lactobacillus salivarius human isolates in an in vitro model

<p>Abstract</p> <p>Background</p> <p>Oral administration of probiotics is known to modulate cytokines profile not only locally, but also systemically. Four strains of <it>Lactobacillus salivarius</it>, LDR0723, BNL1059, RGS1746 and CRL1528, were evaluated for their ability to modulate release of pro- and anti-inflammatory cytokines.</p> <p>Findings</p> <p>Strains were assessed for effects on production of Interleukin-12 (IL-12), Interferon-γ (IFN-γ), Interleukin-4 (IL-4) and Interleukin-5 (IL-5) by incubating bacterial suspensions with THP-1 macrophage like cells. Cytokines were determined by means of specific quantitative enzyme-linked immunosorbent assays.</p> <p>LDR0723 and CRL1528 led to a sustained increment in production of IL-12 and IFN-γ and to a decrease in release of IL-4 and IL-5, while BNL1059 and RGS1746 favoured Th2 response, leading to a decrease in Th1/Th2 ratio with respect to unstimulated cells.</p> <p>Conclusions</p> <p>In conclusion, capability of <it>L. salivarius </it>to modulate immune response was strictly strain dependent and strains of the same species might have opposite effects. Therefore, a careful evaluation of anti-inflammatory properties of lactobacilli should be performed on single strain, before any consideration on potential probiotic use.</p

Evaluation of antibiotic and cell-based therapy in preventing S. epidermidis-induced nonunion in rats.

Methicillin-resistant S. epidermidis (MRSE) is responsible for biofilm-related infections (Montanaro,2011; Romanò, 2013) and fracture nonunion, as recently demonstrated by our group (Lovati, 2016).The present study aims to investigate the efficacy of antibiotic or cell-based therapies in preventingbacterial infections and nonunion establishment.Under anesthesia, femoral fractures were performed in 30 rats, then the site of injury was injectedwith a clinical-derived MRSE strain and, finally, synthesized with stainless steel plates. Rats weredifferently treated as follows: MRSE-infected controls (IC); systemically-injected vancomycin (s-VANC);local vancomycin-enriched hydrogel (l-HYD); systemically-injected BMSCs (s-BMSCs); and locallyinjectedBMSCs (l-BMSCs).After 6 weeks, pro-inflammatory cytokines, quantitative micro-CT, histological and microbiologicalanalyses were carried out to investigate the host response to the different treatments.Half of the s-BMSCs rats died closely to the systemic cell injection, thus excluded for further analyses.Our results for the IC group were consistent with previously published data (Lovati, 2016), showingsigns of osteomyelitis and nonunion development. In s-VANC and l-HYD groups, micro-CT detected agood bony bridging and the microbiological counts were significantly lower with respect to the othergroups. Our study suggests that the association of s-VANC and l-HYD is an effective treatment toprevent biofilm-induced nonunions. Differently, we cannot positively support cell therapies for thispurpose due to the high risk related to the systemic cell injection, thus requiring further studies to beeventually proposed in clinics

Ability of Lactobacillus kefiri LKF01 (DSM32079) to colonize the intestinal environment and modify the gut microbiota composition of healthy individuals

Abstract Background Probiotics have been observed to positively influence the host's health, but to date few data about the ability of probiotics to modify the gut microbiota composition exist. Aims To evaluate the ability of Lactobacillus kefiri LKF01 DSM32079 (LKEF) to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. Methods Twenty Italian healthy volunteers were randomized in pre-prandial and post-prandial groups. Changes in the gut microbiota composition were detected by using a Next Generation Sequencing technology (Ion Torrent Personal Genome Machine). Results L. kefiri was recovered in the feces of all volunteers after one month of probiotic administration, while it was detected only in three subjects belonging to the pre-prandial group and in two subjects belonging to the post-prandial group one month after the end of probiotic consumption. After one month of probiotic oral intake we observed a reduction of Bilophila, Butyricicomonas, Flavonifractor, Oscillibacter and Prevotella. Interestingly, after the end of probiotic administration Bacteroides, Barnesiella, Butyricicomonas, Clostridium, Haemophilus, Oscillibacter, Salmonella, Streptococcus, Subdoligranolum, and Veillonella were significantly reduced if compared to baseline samples. Conclusion L. kefiri LKF01 showed a strong ability to modulate the gut microbiota composition, leading to a significant reduction of several bacterial genera directly involved in the onset of pro-inflammatory response and gastrointestinal diseases

Comparison of nested PCR and real time PCR of Herpesvirus infections of central nervous system in HIV patients

BACKGROUND: Molecular detection of herpesviruses DNA is considered as the reference standard assay for diagnosis of central nervous system infections. In this study nested PCR and real time PCR techniques for detection of Herpes simplex virus type 1 (HSV-1), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in cerebrospinal fluid of HIV patients were compared. METHODS: Forty-six, 85 and 145 samples previously resulted positive for HSV-1, CMV and EBV by nested PCR and 150 randomly chosen negative samples among 1181 collected in the period 1996–2003 were retrospectively reassessed in duplicate by real time PCR and nested PCR. RESULTS: Samples giving positive results for CMV, HSV-1 and EBV with nested PCR were positive also with real time PCR. One of the negative samples resulted positive for HSV and one for EBV. Real time PCR showed comparable sensitivity and specificity vs nested PCR. CONCLUSION: Real time PCR proved to be a suitable method for diagnosis of herpesvirus infections in CNS, showing comparable sensitivity and being less time consuming than nested PCR