- Volume 68, Issue 11, 2019

Over the past century, numerous studies have used oral biofilm models to investigate growth kinetics, biofilm formation, structure and composition, antimicrobial susceptibility and host–pathogen interactions. In vivo animal models provide useful models of some oral diseases; however, these are expensive and carry vast ethical implications. Oral biofilms grown or maintained in vitro offer a useful platform for certain studies and have the advantages of being inexpensive to establish and easy to reproduce and manipulate. In addition, a wide range of variables can be monitored and adjusted to mimic the dynamic environmental changes at different sites in the oral cavity, such as pH, temperature, salivary and gingival crevicular fluid flow rates, or microbial composition. This review provides a detailed insight for early-career oral science researchers into how the biofilm models used in oral research have progressed and improved over the years, their advantages and disadvantages, and how such systems have contributed to our current understanding of oral disease pathogenesis and aetiology.

Purpose. Acute bacterial meningitis continues to be a potentially life threatening condition. Hospital-acquired meningitis is rapidly increasing and adding an immense burden to the health system due to the emergence of multidrug resistance isolates. The purpose of this study is to find the antibiotic susceptibility pattern of the bacteria detected from hospital- and community-acquired meningitis.

Methodology. A total of 400 Cerebrospinal fluid (CSF) samples from the suspected meningitis cases were collected and processed for cell count, biochemical examination, Gram staining, latex agglutination and culture. Bacteria grown on blood, chocolate and Mac-conkey agar were identified by matrix-assisted laser desorption/ionization-time of flight. Antibiotic susceptibility tests were performed as per Clinical and Laboratory Standard Institute guidelines.

Results. Of the isolates, most prevalent Gram negative organisms in hospital-acquired bacterial meningitis were Escherichia coli 13 (27.08 %), Acinetobacter baumannii 12 (25 %), Klebsiella pneumoniae 5 (10.42 %), Pseudomonas aeruginosa 4 (8.33 %) and Gram positive organisms were Staphylococcus aureus 4 (8.33 %), Enterococcus faecium 3 (6.25 %) and CONS 2 (4.16 %). Streptococcus pneumoniae 3 (6.25 %) was the predominant organism in community-acquired bacterial meningitis. All the Gram negative isolates were multidrug resistance. Only colistin and imipenem were effective antibiotics against them. Likewise Gram positive organisms were susceptible to most of the antibiotics tested. However, E. faecium was only susceptible to Vanco+Teicoplanin.

Conclusion. In hospital-acquired bacterial meningitis, multidrug resistance Gram negative bacteria are a huge challenge for the treatment of patients. Hence, antimicrobial stewardship should be followed to counteract with the emerging multidrug resistance isolates.

Introduction. Kocuria kristinae is becoming a growing public health challenge, especially for its ability to cause infections in immunocompromised patients. This bacterium is a Gram+coccus, catalase+, coagulase, and it is a common inhabitant of skin and oral mucosa.

Aim. To investigate the spectrum of infections caused by K. K ristinae.

Methodology. Between January–March 2018, we carried out a systematic search in PubMed utilizing the key search term ‘ Kocuria kristinae ’. The selection criteria for studies were studies reporting cases of human infections due to K. kristinae , case-control and cohort studies and studies published in English or Spanish.

Results. The literature search yielded 48 publications: after title, abstract and full-text analysis, 20 papers were consistent with the selection criteria. These studies were carried out in the period 2001–2017 in the USA, Japan, Taiwan, Hong Kong, Ukraine, Egypt, Bahrain, Serbia, India, Italy, Spain, Turkey and Mexico. K. kristinae was involved in 17 cases of central venous catheter-related bacteremia, four infective endocarditis, three acute peritonitis, one abdominal abscess, umbilical sepsis, acute cholecystitis and urinary tract infection. Additionally, K. kristinae was found in 40 % of carious cavities, although it is not clear whether they are directly involved in the development of caries. Antibiotic susceptibility testing has sometimes revealed multi-drug resistance.

Conclusions. The clinical spectrum of K. kristinae infections has recently widened. The increasing spread of this underestimated bacterium and its resistance to antibiotics represent a new challenge for public health, which requires specific actions to limit it.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.

Aim. In the present study attempts were made to characterize ML1899 in detail.

Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.

Results. In silico analysis predicted ML1899 as a member of α/β hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM−1 min−1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min−1 ml−1 and 27 U mg− 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.

Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

Myeloid C-type lectin receptors (CLRs) are innate immune recognition molecules that bind to microorganisms via their carbohydrate recognition domains. In this study, we utilized a library of CLRs that recognize fungal mannans. We used this library to screen against Pneumocystis carinii (Pc) homogenates or purified Pc major surface glycoprotein (Msg) present on Pneumocystis. The results demonstrated that all of the mammalian CLR hFc-fusions tested displayed significant interaction/binding with Pc organisms, and furthermore to isolated Msg. Highest Pc organism and Msg binding activities were with CLR members Mincle, Dectin-2, DC-SIGN and MCL. An immunofluorescence assay with the respective CLR hFc-fusions against whole Pc life forms corroborated these findings. Although some of these CLRs have been implicated previously as important for Pneumocystis pathogenesis (Dectin-1/Dectin-2/Mincle), this is the first analysis of head-to-head comparison of known fungal mannan binding CLR-hFc fusions with Pc. Lastly, heat treatment resulted in reducted CLR binding.

Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis .

Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis .

Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86–0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.

Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis . Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.

Purpose. Listeria monocytogenes is a foodborne pathogen that causes central nervous system (CNS) and maternal-neonatal (MN) infections, bacteremia (BAC), and gastroenteritis in humans and ruminants. Specific clonal complexes (CC) have been associated with severe listeriosis cases, however, less is known about differences among subgroup virulence patterns. This study aimed to assess variation in virulence across different CC and clinical outcomes.

Methodology. Galleria mellonella larvae were used to compare virulence phenotypes of 34 L . monocytogenes strains representing isolates from CC1, CC6 (from lineage I), and CC7, CC9, CC14, CC37 and CC204 (from lineage II) classified by clinical outcome: BAC, CNS and MN infection. Larvae survival, LD50, cytotoxicity, health index scores and bacterial concentrations post-infection were evaluated as quantifiable indicators of virulence.

Results. Isolates belonging to CC14 and MN-associated infections are hypervirulent in G. mellonella as they led to lower G. mellonella survival rates and health index scores, as well as reduced cytotoxic effects when compared to other CC and clinical outcomes included here. CC14 isolates also showed increased bacterial concentrations at 8 and 24 h post-infection, indicating ability to survive the initial immune response and proliferate within G. mellonella larvae.

Conclusion. Subgroups of L. monocytogenes possess different virulence phenotypes that may be associated with niche-specificity. While hypervirulent clones have been identified so far in lineage I, our data demonstrate that hypervirulent clones are not restricted to lineage I, as CC14 belongs to lineage II. Identification of subgroups with a higher ability to cause disease may facilitate surveillance and management of listeriosis.

Introduction. Tunisia is an intermediate hepatitis B virus (HBV) endemic country. The vaccination against hepatitis B was introduced in 1995 including four doses with a first dose administrated at birth. Decreasing the level of antibodies against hepatitis B surface antigen (anti-HBs) over time can be alarming. This study was conducted to explore the anti-HBV immune response among children under 6 years old, vaccinated according to the national vaccination schedule, by evaluating the immunological response to primary vaccination and by exploring the anamnestic immune response to a booster dose.

Methods. We conducted a cross-sectional prospective study from June 2016 to June 2017 (n=180), based on voluntary participation. Children were recruited from the public pediatric ward sectors in Sahloul University Hospital of Sousse in Central Tunisia. An anti-HB titre was determined based on electro-chemiluminescence micro-particle immunoassay (ECLIA), using Elecsys Anti-HBs II kit, Roche.

Results. Mean age at the time of enrollment in the study was 33±14.8 months. The seroprotection rate was 77.2 %. The anti-HB titre differed significantly between the different age groups (P=0.002). The predicting variable for having no seroprotective antibody level was older age. Children with anti-HB levels <10 IU l− 1 were offered an additional dose of HBV vaccine. Anamnestic response 1 month after the challenge dose was observed in 100 % of subjects. The probability of developing a high antibody response, following the booster dose increased in conjunction with an increased pre-booster antibody level.

Conclusion. The response to a booster dose suggests the persistence of immune memory in almost all vaccinated individuals. Although a booster dose increases substantially anti-HB titre, the clinical relevance of such an increase remains unknown.