- Volume 68, Issue 4, 2019

To determine micro-organisms attached to removed urethral catheters and relate this to patient-specific information. Indwelling urethral catheters were collected from patients at a UK teaching hospital. The balloon and lumen were sonicated, and micro-organisms were enumerated. Catheter specimen urine results were retrospectively reviewed. Escherichia coli and Enterococcus faecalis were the most common isolates from 61 catheters. 19.7% of patients received antibiotics and 25 % of those had a multi-drug-resistant (MDR) organism in the lumen. Only 2.04% of catheters from patients not receiving antibiotics had a MDR organism. All lumens were colonized irrespective of antibiotic use. Symptom presentation did not correlate with numbers of colonizing organisms or species. Despite heavy colonization, only 8/61 patients were symptomatic. Indwelling urinary catheters in place for ≥10 days were universally colonized and there was no correlation with symptom presentation. Symptom presentation remains the most important factor for defining catheter-associated urinary tract infection.

Purpose. The public health burden of infections caused by Neisseria gonorrhoeae is magnified due to high rates of resistance to traditional antimicrobials. The aim of this study was to evaluate the in vitro efficacy of an alternative dual therapy comprising gentamicin and azithromycin.

Methodology. The E-test method was used to determine the minimum inhibitory concentrations (MICs) of gentamicin and azithromycin individually prior to testing in combination using the cross or 90o angle formation method. A total of 70 clinical isolates of N. gonorrhoeae displaying varying ceftriaxone MICs along with 2 reference strains (WHO K and P) and 1 ceftriaxone-resistant QA isolate were examined. The fractional inhibitory concentration index (FICI) was calculated and the results were interpreted using the following criteria: synergy, FICI ≤0.5; indifference or additive, FICI >0.5 to ≤4.0; and antagonism, FICI >4.0.

Results. A total of 54 (77.1 %) isolates displayed indifference, while 16 (22.9 %) demonstrated synergy. When azithromycin was tested alone, the MICs ranged from 0.016 to 2 µg ml−1 . However, in combination with gentamicin, the mean MIC value of all isolates decreased from 0.275 µg ml−1 to 0.090 µg ml−1 (P=0.05).When gentamicin was tested alone, the MICs ranged from 0.25 to 8 µg ml−1, with a mean MIC of 4.342 µg ml−1, whereas in combination with azithromycin it decreased significantly to 2.042 µg ml−1 (P=0.04).

Conclusion. No antagonism was observed in this combination, suggesting that it could be a future treatment option as we prepare for a post-cephalosporin era. However, comprehensive in vivo evaluations are warranted and recommendations should be made based on clinical trials.

Purpose. We aimed to investigate the presence of three recently identified point mutations (A2115G, G2141A and A2144T) of the 23 S rRNA gene and compare them with the three most frequently encountered point mutations (A2142G, A2142C and A2143G) in Helicobacter pylori strains in Turkey.

Methodology. A total of 63 patients (mean 47.08±12.27) were included. The E-test method (for clarithromycin) was used for the clarithromycin antimicrobial susceptibility test of isolated H. pylori strains. Real-time PCR was used to detect the point mutations.

Results. A total of 24 out of 63 H. pylori strains (38.1%) were detected as clarithromycin resistant (>0.5 mg l−1 ). The new A2115G (n:6, 25%), A2144T (n:7, 29.1%) and G2141A, 8 (n:8, 33.3%) mutations and the classical A2142G (n:8, 33.3%) and A2143G (n:11, 45.8%) point mutations were detected in the 24 clarithromycin-resistant strains. The A2144T point mutation had the highest median MIC value (3 mg l−1 ) amongst the new mutations, but the classical mutations (A2142G and A2143G) had the highest median MIC values (256 mg l−1 ) overall. The presence of the A2115G (OR:31.66), A2144T (OR:36.92) or G2141A (OR:28.16) mutations increased the likelihood of clarithromycin resistance in H. pylori strains by 31.66-, 36.92- and 28.16-fold (ORs), respectively, according to the binary logistic regression analysis.

Conclusion. We concluded that classical mutations of the 23 S rRNA gene resulted in higher clarithromycin MIC values than new mutations. These new point mutations caused moderate elevations in the MIC values of clarithromycin-resistant H. pylori strains.

Purpose. Otomycosis is a mycotic infection of the external auditory canal and can be caused by a wide range of fungal species. In this study, we aimed to identify fungal isolates from patients suspected of otomycosis.

Methodology. External ear canal samples were taken from patients referred to the outpatient department of Shahid-Mofatteh Clinic in the city of Yasuj, Iran, and examined by direct microscopy and culture. DNA of the isolated fungi was tested by internal transcribed spacer PCR restriction fragment length polymorphism analysis for identification of yeasts and β-tubulin sequencing for identification of Aspergillus species.

Results. Among 275 patients suspected of otomycosis, 144 cases (83 female and 61 male) were confirmed with otomycosis. For 89% (n=128) of positive cultures, microscopy was also positive, while there were no cases with a microscopy-positive and culture-negative result. The predominant predisposing factor was self-cleaning of the external ear using unhygienic tools, and the main risk occupation was ‘housewife’. The most common isolated fungi were typically Aspergillus (n=120), including 73 isolates of Aspergillus section Nigri, 43 of section Flavi, 3 of section Terrei and 1 of section Fumigati. After sequencing, 44 out of 73 strains primarily identified as Aspergillus niger turned out to be Aspergillus tubingensis. Thirty-five isolates were identified as Candida, including Candida parapsilosis (n=22), Candida albicans (n=12) and Candida tropicalis (n=1).

Conclusion. Aspergillus tubingensis was the most common species involved in otomycosis. This work corroborates the difficulty of precise identification of species within the black Aspergilli by morphological characteristics.

Purpose . Trichophyton rubrum is a dermatophyte that causes most human superficial mycoses worldwide. The spliceosome, a large ribonucleoprotein complex responsible for pre-mRNA processing, may confer adaptive advantages to deal with different stresses. Here, we assessed the structural aspects of the Prp4 kinase protein and other pre-mRNA-splicing factors (Prps) in T. rubrum grown in different protein sources and exposed to antifungal drugs.

Methodology. Quantitative Reverse Transcription PCR (RT-PCR) assessed the modulation of prp1, prp31, prp8 and prp4 kinase genes after exposure of T. rubrum to sub-lethal doses of amphotericin B, caspofungin and acriflavine, or after T. rubrum growth on keratin sources for 48 and 72 h. We also performed the in silico analysis of the domain organization of Prps orthologues from filamentous fungi and yeasts.

Results . The prp4 gene was modulated in a time-dependent manner. Transcription levels were mostly up-regulated when T. rubrum was grown on keratin for 72 h, while exposure to amphotericin B promoted prp4 gene down-regulation at the same time point. We also observed co-expression of prp1 and prp31, and their down-regulation after amphotericin B exposure. In silico analysis revealed a conserved domain organization for most Prps orthologues with slight differences, which were mostly related to structural elements such as repetition domains in Prp1 and complexity in motif assembly for the Prp4 kinase. These differences were mainly observed in dermatophyte species and may alter protein interactions and substrate affinity.

Conclusion . Our results improve the understanding of spliceosome proteins in fungi as well as their roles in adaptation to different environmental situations.

Purpose. The oral microbiome is maintained by host- and microbe-derived factors. A shift in microbial composition, as a result of diseases related to the immune system, is the most important step in the development of oral and dental diseases. The aim of this study was to investigate the oral microbial composition of patients with Kostmann syndrome, who have severe neutropenia, compared with healthy children.

Methodology. A group of nine Kostmann syndrome patients and a group of nine healthy controls participated. After clinical investigation, DNA from stimulated saliva specimens was examined by high-throughput sequencing of the V3–V4 hypervariable region of the 16S rRNA gene using Illumina sequencing. The QIIME software package was used for 16 S rRNA amplicon analysis, while the Greengenes database was used for taxonomic classification.

Results. The periodontal pocket depths, plaque indices and bleeding-on-probing percentages and caries status on the deciduous teeth of the patients with Kostmann syndrome were statistically higher than those for the healthy controls. Patients with Kostmann syndrome had significantly lower bacterial diversity as compared to the controls. The presence of Firmicutes was statistically higher in patients with Kostmann syndrome, while that for Proteobacteria was higher in samples from the healthy controls (P<0.05). Streptococcus , Rothia , Granulicatella , Actinomyces , and genera from the family Gemellaceae were present as the core microbiome (abundance >1 % in at least 75  % of samples) in all groups, whereas the genus Porphyromonas was only detected as a member of the core microbiome in Kostmann patients.

Conclusions. The evidence of lower bacterial diversity and differences in microbial profile for patients with Kostmann syndrome not only shows the impact of immune system-related diseases on oral microbiota, but also endorses the ecological plaque hypothesis proposed for the aetiology of oral diseases such as dental caries and periodontitis.

Parvovirus B19 (B19V) transmission may occur through blood transfusion as a result of asymptomatic viral persistence in blood donors. Our study evaluated the prevalence and viral load of B19V in blood donors from Brasilia, Federal District, Central-West Brazil. B19V DNA detection and quantification were performed in 477 blood donors. The positive samples were also tested for anti-B19V IgG and haemoderivative recipients were investigated for adverse effects following transfusion. B19V DNA prevalence was 0.21  % (n=1/477). The positive B19V DNA sample was also anti-B19 IgG-positive (probably persistent infection). The viral load was low and no adverse effects following blood transfusion were registered in the recipients. This study demonstrated that the B19V DNA prevalence in blood donors from Central-West Brazil is low. Nevertheless, the mere presence of B19V DNA in blood donors strengthens the need for viral molecular screening, especially in haemoderivatives that that will go to susceptible recipients.

The liver-expressed pattern recognition receptors mannose-binding lectin (MBL), ficolin-2 and ficolin-3 contribute to the innate immune response by activating complement. Binding of soluble ficolin-2 to viral pathogens can directly neutralize virus entry. We observed that the human hepatoma cell line HuH7.5, which is routinely used for the study of hepatotropic viruses, is deficient in expression of MBL, ficolin-2 and ficolin-3. We generated a cell line that expressed and secreted ficolin-2. This cell line (HuH7.5 [FCN2]) was more resistant to infection with hepatitis C virus (HCV), ebolavirus and vesicular stomatitis virus, but surprisingly was more susceptible to infection with rabies virus. Cell-to-cell spread of HCV was also inhibited in ficolin-2 expressing cells. This illustrates that ficolin-2 expression in hepatocytes contributes to innate resistance to virus infection, but some viruses might utilize ficolin-2 to facilitate entry.

Background. Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. Infection of RSV has been shown to induce Th lymphocyte differentiation. The present study was designed to determine the effects of RSV on the expression of Notch-1 and the related mechanisms on subsequent differentiation of Th lymphocytes.

Methods. A RSV-infected animal model was established and investigated at 7, 28 and 60 days post infection. Real-time qPCR and Western blot were used to observe the expression levels of Notch-1 in CD4+ T cells and its five ligands in lung tissues. The methylation levels of CpG islands in autoimmune regulator (AIRE) and Notch-1 promoters were analysed by time-of-flight mass spectrometry. The differentiation of Th lymphocytes was assayed by real-time qPCR. The distribution of JAG1 and DLL3 in the lung tissues were assayed by immunohistochemistry. The correlation between Th17 and DLL3 was analysed by simple correlation.

Results. The results showed that RSV promoted the expression and de-methylation of Notch-1 promoters in CD4+ T cells. Moreover, RSV infection promoted Th1 differentiation at day 7 and day 28; Th17 differentiation at day 7, day 28 and day 60; Th2 differentiation at day 28 and day 60. At the same time, RSV infection promoted the expression of JAG1 and DLL3. Activation of Notch-1/ DLL 3 in lungs may be associated with the differentiation of Th17 lymphocytes.

Conclusions. Our data showed that activation of RSV stimulated the differentiation of Th17 in airway microenvironment through activation Notch-1/DLL3, which may be associated with the occurrence and development of RSV-induced asthma.

Purpose. To determine the effect of allicin on Staphylococcus aureus cell wall peptidoglycans by the application of MALDI-TOF mass spectrometry on whole cells and to relate this to current knowledge of wall-processing enzymes.

Methodology. Two different S. aureus strains were grown for 48 h after which period each culture was split into two, one part was then treated with sub-inhibitory levels of allicin while the other part left untreated as a control. After a further 24 h whole cells were recovered and analysed by MALDI-TOF mass spectrometry.

Results. Changes in the mass spectra between the treated and untreated cells revealed fragmented peptidoglycans identified by mass calculation only in the treated cells. These peptidoglycan fragments where identified as the products of specific peptidoglycan hydrolases.

Conclusions. Allicin is known to target cysteine thiol groups. These are absent in peptidoglycan hydrolases and we might have expected identical results in both of the treated and untreated cells. Peptidoglycan synthesis enzymes such as the Fem family of enzymes do contain cysteines. Fem enzymes A, B and X all have a conserved conformation of 99 % for over 100 S. aureus strains and are therefore potential targets for allicin. Examination of FemA structure showed that cysteine102 is accessible from the surface. We propose that allicin has an inhibitory mechanism alongside others of targeting FemA and possibly other Fem enzymes by curtailing glycine bridging and leading to fragmentation. This study provided an insight into yet another antimicrobial mechanism of allicin.