- Volume 69, Issue 11, 2020

Introduction. The rise of carbapenem-resistant enterobacteriaceae (CRE) is a growing crisis that requires development of novel therapeutics.

Hypothesis. To this end, cationic antimicrobial peptides (CAMPs) represent a possible source of new potential therapeutics to treat difficult pathogens such as carbapenem-resistant Klebsiella pneumoniae (CRKP), which has gained resistance to many if not all currently approved antibiotics, making treatment difficult.

Aim. To examine the anti-CRKP antimicrobial activity of the predicted cathelicidins derived from Varanus komodoensis (Komodo dragon) as well as synthetic antimicrobial peptides that we created.

Methodology. We determined the minimum inhibitory concentrations of the peptides against CRKP. We also characterized the abilities of these peptides to disrupt the hyperpolarization of the bacterial membrane as well as their ability to form pores in the membrane.

Results. We did not observe significant anti-CRKP activity for the predicted native Komodo cathelicidin peptides. We found that the novel peptides DRGN-6,-7 and -8 displayed significant antimicrobial activity against CRKP with MICs of 4–8 µg ml−1. DRGN-6 peptide was the most effective peptide against CRKP. Unfortunately, these peptides showed higher than desired levels of hemolysis, although in vivo testing in the waxworm Galleria mellonella showed no mortality associated with treatment by the peptide; however, CRKP-infected waxworms treated with peptide did not show an improvement in survival.

Conclusion. Given the challenges of treating CRKP, identification of peptides with activity against it represents a promising avenue for further research. Given DRGN-6′s similar level of activity to colistin, DRGN-6 is a promising template for the development of novel antimicrobial peptide-based therapeutics.

Introduction. Totally implanted venous access ports (TIVAPs) are widely used in patients receiving long-term chemotherapy but may lead to serious complications such as catheter-related bloodstream infections (CRBSIs). Diagnosis of CRBSI requires catheter culture, but there is no consensus on microbiological culture methods to be adopted.

Aim. To compare three different procedures to recover bacterial cells from colonized catheters and to determine which section of the TIVAP (i.e. tip, septum, reservoir) is the probable source of infection. To investigate the correlation between blood culture results and TIVAP culture in order to get further evidence about the utility of differential time to positivity (DTP) as a diagnostic tool before TIVAP removal.

Hypothesis/Gap statement. Comparisons of different diagnostic procedures for catheter culture have been rarely reported for TIVAPs. We hypothesized that the optimization of methods to recover micro-organisms from different parts of TIVAPs may help to decrease the number of false-negative results in the diagnosis of TIVAP-related bloodstream infections.

Methodology. A total of 53 TIVAPs removed because of suspected infection (n=36) or end of use (n=17) were evaluated. The reservoir, the septum and the catheter tip were separated and subjected to different treatments for the recovery of adherent micro-organisms: (a) flushing of the catheter lumen, (b) sonication and flushing, (c) treatment with dithiothreitol and flushing. The three methods were also evaluated in an in vitro catheter infection model with Staphylococcus epidermidis . Culture results were compared to those obtained from paired blood cultures drawn from TIVAP and peripheral vein and to the relative DTP.

Results. The results obtained demonstrated that vigorous flushing/vortexing of the catheter lumen/septum, allows the recovery of a number of micro-organisms comparable to that of more complex procedures such as sonication or chemical treatment. Among 24 positive TIVAP-cultures, nine were tip-culture negative, whereas the corresponding reservoirs and septa were culture positive. A good correlation was observed between DTP and TIVAP cultures (P<0.001).

Conclusions. The results support the evidence that sending the port reservoir in addition to the catheter tip to the microbiology laboratory may increase the sensitivity and the accuracy of CRBSI diagnosis. Moreover, when a TIVAP-related infection is suspected, DTP is a useful diagnostic tool to decide between device removal or a more conservative approach.

Introduction. Papiliotrema laurentii, formerly Cryptococcus laurentii, is typically isolated from environmental sources, but also occasionally from clinical specimens. Other close relatives may be misidentified as P. laurentii by phenotypic methods. P. laurentii usually lacks melanin; however, melanin-forming strains have also been isolated.

Hypothesis/Gap Statement. Although melanin production by encapsulated budding yeasts is considered a major virulence factor, the comparative pathogenicity of melanin-forming and non-melanized environmental strains of P. laurentii has rarely been studied.

Aim. We performed phenotypic and molecular identification and determined the genotypic heterogeneity among P. laurentii isolates. We also studied the pathogenicity of melanin-forming and non-melanized strains in normal and immunosuppressed mice.

Methodology. Eleven environmental isolates were tested for their identity by Vitek2 and/or ID32C systems, and by PCR-sequencing of the internal transcribed spacer (ITS) region and D1/D2 domains of ribosomal DNA (rDNA). Genotypic heterogeneity was studied by sequence comparisons. The pathogenicity of melanized and non-melanized P. laurentii strains was studied in intravenously infected normal and immunosuppressed BALB/c mice.

Results. Phenotypic methods identified seven of the environmental isolates, while PCR-sequencing of the ITS region and D1/D2 domains of rDNA detected two and five isolates, respectively, as P. laurentii. Sequence comparisons demonstrated genotypic heterogeneity among P. laurentii. The remaining four environmental isolates yielded expected results. None of the normal mice infected with 105 cells of melanized/non-melanized P. laurentii strains died. Infection of immunosuppressed mice with 107 cells caused higher mortality with non-melanized P. laurentii, while viable counts in brain/lung tissue were higher in mice infected with a melanized strain and were detectable for up to 14 days.

Conclusion. Phenotypic methods lacked specificity, but PCR-sequencing of D1/D2 domains correctly identified P. laurentii and sequence comparisons demonstrated the genotypic heterogeneity of the isolates. Both melanized and non-melanized strains at a higher dose caused mortality in immunosuppressed mice and persisted in brain/lung tissue up to 14 days post-infection.

Introduction. The pathogenesis of atopic dermatitis (AD) is not yet fully understood, but the bacterial composition of AD patients’ skin has been shown to have an increased abundance of Staphylococcus aureus . More recently, coagulase-negative Staphylococcus (CoNS) species were shown to be able to inhibit S. aureus , but further studies are required to determine the effects of Staphylococcus community variation in AD.

Aim. Here we investigated whether analysing metabarcoding data with the more recently developed DADA2 approach improves metabarcoding analyses compared to the previously used operational taxonomic unit (OTU) clustering, and can be used to study Staphylococcus community dynamics.

Methods. The bacterial 16S rRNA region from tape strip samples of the stratum corneum of AD patients (non-lesional skin) and non-AD controls was metabarcoded. We processed metabarcoding data with two different bioinformatic pipelines (an OTU clustering method and DADA2), which were analysed with and without technical replication (sampling strategy).

Results. We found that OTU clustering and DADA2 performed well for community-level studies, as demonstrated by the identification of significant differences in the skin bacterial communities associated with AD. However, the OTU clustering approach inflated bacterial richness, which was worsened by not having technical replication. Data processed with DADA2 likely handled sequencing errors more effectively and thereby did not inflate molecular richness.

Conclusion. We believe that DADA2 represents an improvement over an OTU clustering approach, and that biological replication rather than technical replication is a more effective use of resources. However, neither OTU clustering nor DADA2 gave insights into Staphylococcus community dynamics, and caution should remain in not overinterpreting the taxonomic assignments at lower taxonomic ranks.

Salmonella enterica serotype Enteritidis ( Salmonella Enteritidis) is a major cause of foodborne disease outbreaks worldwide. In 2018, two concurrent outbreaks of Salmonella Enteritidis gastroenteritis in one district of South Africa were investigated. We describe the use of whole-genome sequencing (WGS) analysis of bacterial isolates to assist with the investigation of these outbreaks. Outbreak A affected children (n=27) attending a day-care centre, while outbreak B affected adults (n=16) who ate breakfast at the same restaurant. Salmonella Enteritidis was isolated from stool samples in both outbreaks (four children in outbreak A; 12 restaurant customers and three restaurant food-handlers in outbreak B). In outbreak B, Salmonella Enteritidis was isolated from three food retention samples (raw chicken egg, hollandaise sauce and rocket-herb). Available isolates from both outbreaks (n=13) were investigated using WGS analysis. Sequencing data for isolates were analysed at the EnteroBase web-based platform and included core-genome multi-locus sequence typing (cgMLST). Isolates with epidemiological links to the restaurant (n=10) and day-care centre (n=3), were shown by cgMLST to be highly genetically related, with no more than five allele differences when comparing one isolate against another. On food history, eggs and hollandaise sauce were the common food items consumed by ill restaurant customers. Unfortunately, Salmonella Enteritidis isolated from the egg and hollandaise sauce were not available for WGS analysis. Our investigation concluded that the two concurrent outbreaks were caused by a highly related strain of Salmonella Enteritidis, suggesting the possibility of a common contaminated food source, of which contaminated eggs are strongly implicated.

Introduction. Streptococcus pyogenes is a diverse virulent synthesis pathogen responsible for invasive systemic infections. Establishment of antibiotic resistance in the pathogen has produced a need for new antibiofilm agents to control the biofilm formation and reduce biofilm-associated resistance development.

Aim. The present study investigates the in vitro antibiofilm activity of eucalyptol against S. pyogenes .

Methodology. The antibiofilm potential of eucalyptol was assessed using a microdilution method and their biofilm inhibition efficacy was visualized by microscopic analysis. The biochemical assays were performed to assess the influence of eucalyptol on virulence productions. Real-time PCR analysis was performed to evaluate the expression profile of the virulence genes.

Results. Eucalyptol showed significant antibiofilm potential in a dose-dependent manner without affecting bacterial growth. Eucalyptol at 300 µg ml−1 (biofilm inhibitory concentration) significantly inhibited the initial stage of biofilm formation in S. pyogenes . However, eucalyptol failed to diminish the mature biofilms of S. pyogenes at biofilm inhibitory concentration and it effectively reduced the biofilm formation on stainless steel, titanium, and silicone surfaces. The biochemical assay results revealed that eucalyptol greatly affects the cell-surface hydrophobicity, auto-aggregation, extracellular protease, haemolysis and hyaluronic acid synthesis. Further, the gene-expression analysis results showed significant downregulation of virulence gene expression upon eucalyptol treatment.

Conclusion. The present study suggests that eucalyptol applies its antibiofilm assets by intruding the initial biofilm formation of S. pyogenes . Supplementary studies are needed to understand the mode of action involved in biofilm inhibition.

Introduction. Yersinia enterocolitica is one of the leading food-borne entero-pathogens causing various illnesses ranging from gastroenteritis to systemic infections. Quorum sensing (QS) is one of the prime mechanisms that control the virulence in Y. enterocolitica .

Hypothesis/Gap Statement. Vanillic acid inhibits the quorum sensing and other virulence factors related to Y. enterocolitica . It has been evaluated by transcriptomic and Insilico analysis. Therefore, it can be a prospective agent to develop a therapeutic combination against Y. enterocolitica .

Aim. The present study is focused on screening natural anti-quorum-sensing agents against Y. enterocolitica . The effect of selected active principle on various virulence factors was evaluated.

Methodology. In total, 12 phytochemicals were screened by swarming assay. MATH assay, EPS and surfactant production assay, SEM analysis, antibiotic and blood sensitivity assay were performed to demonstrate the anti-virulence activity. Further, RNA sequencing and molecular docking studies were carried out to substantiate the anti-QS activity.

Results. Vanillic acid (VA) has exhibited significant motility inhibition, thus indicating the anti-QS activity with MQIC of 400 µg ml−1 without altering the cell viability. It has also inhibited the violacein production in Chromobacterium violaceum ATCC 12472, which further confirms the anti-QS activity. VA has inhibited 16 % of cell-surface hydrophobicity (CSH), 52 % of EPS production and 60 % of surfactant production. Moreover, it has increased the sensitivity of Y. enterocolitica towards antibiotics. It has also made the cells upto 91 % more vulnerable towards human immune cells. The transcriptomic analysis by RNA sequencing revealed the down regulation of genes related to motility, virulence, chemotaxis, siderophores and drug resistance. VA treatment has also positively regulated the expression of several stress response genes. In furtherance, the anti-QS potential of VA has been validated with QS regulatory protein YenR by in silico molecular simulation and docking study.

Conclusion. The present study is possibly the first attempt to demonstrate the anti-QS and anti-pathogenic potential of VA against Y. enterocolitica by transcriptomic and in silico analysis. It also deciphers that VA can be a promising lead to develop biopreservative and therapeutic regimens to treat Y. enterocolitica infections.