- Volume 64, Issue 4, 2015

Biofilms are of great importance in infection control and healthcare-associated infections owing to their inherent tolerance and ‘resistance’ to antimicrobial therapies. Biofilms have been shown to develop on medical device surfaces, and dispersal of single and clustered cells implies a significant risk of microbial dissemination within the host and increased risk of infection. Although routine microbiological testing assists with the diagnosis of a clinical infection, there is no ‘gold standard’ available to reveal the presence of microbial biofilm from samples collected within clinical settings. Furthermore, such limiting factors as viable but non-culturable micro-organisms and small-colony variants often prevent successful detection. In order to increase the chances of detection and provide a more accurate diagnosis, a combination of microbiological culture techniques and molecular methods should be employed. Measures such as antimicrobial coating and surface alterations of medical devices provide promising opportunities in the prevention of biofilm formation on medical devices.

The concept of infertility as a result of asymptomatic microbial colonization of the female reproductive tract has been neglected to date. However, increasing incidence of infertility and advanced research has drawn attention towards this idea. Many of these micro-organisms have been reported to bring about adverse changes in sperm parameters in vitro, but their in vivo potential to cause infertility is still a controversy. The present study was carried out to observe what effect the intravaginal inoculation of sperm-agglutinating Serratia marcescens and sperm-immobilizing Candida albicans had in the reproductive tract and consequently in fertility outcome. When these strains were intravaginally inoculated into female BALB/c mice at 104, 106 and 108 c.f.u. in 20 µl PBS for 10 consecutive days, with mating of mice on day 12, the results showed 100 % decrease in fertility in all groups as compared with control mice receiving PBS alone. Furthermore, no clinical or histopathological changes were observed in the reproductive organs (i.e. ovary, uterus and vagina), suggesting that colonization of the genital tract with sperm-impairing micro-organisms could be a feasible reason for female infertility.

Barrett’s oesophagus (BO) is a complicated condition at the gastroesophageal junction in which normal squamous epithelium is changed to columnar and leads to oesophageal adenocarcinoma (OA). In the past decades, the prevalence of Barrett’s disease and mortality rate of adenocarcinoma has significantly increased throughout the word. Data has shown that molecular pathogenesis of disease has not been clearly identified. However, a wide-range and successful administration of probiotics in cancer and gastrointestinal diseases has lead to the investigation into the possible inhibitory role of probiotics in oesophageal cancer. This study was conducted to evaluate the inhibitory effect of probiotics on the expression of biomarkers in an in vitro model. Two different Barrett’s oesophageal cell lines were selected to co-culture with B. longum and Lactobacillus acidophilus to measure expression of IL-18, TNFα, p53 (tumour suppressor gene), cyclooxygenase 2 and CDX1 (caudal type homeobox 1) genes. In addition, two different aspects of probiotic administration, therapeutic and prophylactic test were also examined. Results showed that micro-organisms could inhibit expression of biomarkers and therapeutic culture conditions were more effective than prophylactic tests. The results obtained suggest that it is possible to incorporate the administration of probiotics in BO and OA prevention.

Electrosyneresis and double diffusion are immunoprecipitation techniques commonly used in the serological diagnosis of Farmer’s lung disease (FLD). These techniques are reliable but lack standardization. The aim of this study was to evaluate Western blotting for the serodiagnosis of FLD. We carried out Western blotting with an antigenic extract of Lichtheimia corymbifera, an important aetiological agent of the disease. The membranes were probed with sera from 21 patients with FLD and 21 healthy exposed controls to examine the IgG antibody responses against purified somatic antigens. Given the low prevalence of the disease, 21 patients could be considered as a relevant series. Four bands were significantly more frequently represented in membranes probed with FLD sera (bands at 27.7, 40.5, 44.0 and 50.5 kDa) than those probed with control sera. We assessed the diagnostic value of different criteria alone or in combination. The diagnostic accuracy of the test was highest with the inclusion of at least two of the following criteria: at least five bands on the strip and the presence of one band at 40.5 or 44.0 kDa. Sensitivity, specificity and positive and negative predictive values were all 81 %, and the odds ratio was 18.06. Inclusion of bands of high intensity diminished rather than improved the diagnostic value of the test. We concluded that Western blotting is a valuable technique for the serodiagnosis of FLD. The industrial production of ready-to-use membranes would enable the routine use of this technique in laboratories, and provide reliable and standardized diagnostic results within a few hours.

Bacteriological examinations were conducted for seven Arcanobacterium haemolyticum strains isolated from elderly patients with skin and soft-tissue infections, such as cellulitis and skin ulcers. Streptococcus dysgalactiae or Gram-positive cocci were isolated together with A. haemolyticum from all patients. The strains were identified as A. haemolyticum based on their being catalase negative, reverse Christie, Atkins and Munch-Petersen (CAMP) positive and phospholipase D gene positive in respective tests. Moreover, API Coryne and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry confirmed the identification of A. haemolyticum. All strains showed good susceptibility to minocycline, vancomycin and β-lactam antibiotics, but several strains were resistant to gentamicin and levofloxacin.

Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix and represent a common mode of microbial growth. Microbes growing as biofilm are highly resistant to commonly used antimicrobial drugs. We aimed to screen and characterize biofilm formation by different isolates of Candida on removed intrauterine devices (IUDs), to perform experimental biofilm formation with isolated strains, and to examine biofilm by the crystal violet and XTT reduction assays and scanning electron microscopy (SEM). A total of 56 IUDs were examined for biofilm formation using Sabouraud's dextrose chloramphenicol agar. Suspected colonies were identified by different methods. Antifungal susceptibility testing with fluconazole (FLU) and amphotericin B for the isolated strains and in vitro experimental biofilm formation was carried out. The biofilm was quantified by crystal violet, XTT reduction assay and SEM. Among the 56 IUDs investigated, 26 were Candida positive (46.4 %). Candida albicans was recovered from 15 isolates. The biofilm MIC of FLU was increased 64 to 1000 times compared to the MIC for planktonic cells. The XTT method results were dependent on the Candida species; biofilm formation was highest in Candida krusei and Candida glabrata strains, followed by C. albicans and Candida tropicalis. SEM of Candida biofilm revealed a heterogeneous thick biofilm with a mixture of micro-organisms. The main conclusion from this study was non-albicans Candida represents more than a half of the Candida biofilm. Better understanding of Candida biofilms may lead to the development of novel therapeutic approaches for the treatment of fungal infections, especially resistant ones among IUD users.

The ability of a non-propagating transport device (test device) to maintain the viability of clinically relevant bacteria was compared with a similar commercial device (predicate device) to establish performance equivalence. Test bacteria, namely Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma hominis, Mycoplasma pneumoniae and Ureaplasma urealyticum, were inoculated into the test [Puritan Medical Products Universal Transport System (UniTranz-RTTM)] and predicate (BD Universal Viral Transport System) devices, and incubated at 4 °C and room temperature for up to 72 h. Bacterial viability was assessed at selected time points post-incubation using shell vial assays followed by immunofluorescence staining (for Chlamydia) or by standard culture techniques (for Mycoplasma and Ureaplasma). Results indicated that the Chlamydia strains were equally stable in both test and predicate devices through 72 h storage, at both test temperatures. Quantifiable levels of Mycoplasma and Ureaplasma were recovered from the test and predicate devices throughout the storage period. Low-temperature storage improved bacterial viability when compared with room temperature storage. In addition, the predicate device demonstrated slightly improved performance versus the test device in the context of Mycoplasma and Ureaplasma following 72 h storage. The overall results of the study confirmed the full performance of UniTranz-RTTM as a microbial transport medium and established equal performance with the predicate device.

It is believed that most microbial infections are caused by pathogens organized in biofilms. Recently, it was shown that the dimorphic fungus Histoplasma capsulatum, estimated to be the most common cause of fungal respiratory diseases, is also able to form biofilm. Although the antifungal therapy commonly used is effective, refractory cases and recurrences have been reported. In the search for new compounds with antimicrobial activity, the sesquiterpene farnesol has gained prominence for its antifungal action. This study aimed to evaluate the in vitro susceptibility of H. capsulatum var. capsulatum to the antifungal agents itraconazole and amphotericin B, and farnesol alone and combined, as well as to determine the in vitro antifungal activity of these compounds against biofilms of this pathogen. The results show that farnesol has antifungal activity against H. capsulatum in the yeast and filamentous phases, with MIC values ranging from 0.0078 to 0.00312 µM. A synergistic effect (fractional inhibitory concentration index ≤0.5) between itraconazole and farnesol was found against 100 and 83.3 % of the isolates in yeast and mycelial forms, respectively, while synergism between amphotericin B and farnesol was only observed against 37.5 and 44.4 % of the isolates in yeast and filamentous forms, respectively. Afterwards, the antifungal drugs, itraconazole and amphotericin B, and farnesol alone, and the combination of itraconazole and farnesol, were tested against mature biofilms of H. capsulatum, through XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) metabolic assay, and the itraconazole and amphotericin B showed lower antibiofilm activity when compared to farnesol alone and farnesol combined with itraconazole. In conclusion, farnesol showed promising results as an antifungal agent against H. capsulatum and also showed adjuvant action, especially when combined with itraconazole, increasing the fungal susceptibility to this drug.

Carbapenem-resistant Acinetobacter baumannii is a major health problem worldwide, especially in intensive care units (ICUs). This study aimed to detect the prevalence of A. baumannii colonization of the gastrointestinal tract of patients admitted to the ICU in two hospitals in Saudi Arabia. In addition, it aimed to characterize the molecular mechanisms of carbapenem resistance in these isolates. From January to June 2014, 565 rectal swab specimens were screened for Acinetobacer strains and carbapenem resistance using CHROMagar Acinetobacter and CHROMagar KPC agar plates, respectively. Organism identification and susceptibility were detected using the Vitek 2 system. A total of 47 Acinetobacter spp. were detected, and 35 were resistant to carbapenem, making the prevalence of Acinetobacter spp. 8.3 % (47/565) and carbapenem resistance (6.2 %, 35/565). The 47 strains showed remarkable clonal diversity as revealed by PFGE. Using PCR, OXA-51, a chromosomal marker for A. baumannii, was detected in 46 strains. OXA-23 β-lactamase was detected in all 35 carbapenem-resistant A. baumannii. No IMP, VIM, SPM, SIM, GIM, KPC or NDM β-lactamases were detected in these isolates. Thus, OXA-23 was the main mechanism of carbapenem resistance in these isolates. To the best of our knowledge, this is the first study to detect the prevalence of Acinetobacter colonization in the digestive tract of ICU patients in Saudi Arabia. This study revealed the importance of having well-established protocols for early identification of these multidrug-resistant organisms, optimizing infection-control strategies and having active surveillance studies to reduce morbidity, mortality and cost.

The antimicrobial activity of cetylpyridinium chloride (CPC) and miramistin (MST) solutions at different concentrations (5×10−5 to 0.4 %) and a dressing, containing 0.15 % CPC, were tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli after 30 (solutions) and 60 min (fleece) incubation, respectively. Furthermore, the cytotoxic effects of CPC and MST were examined in human keratinocyte (HaCaT) and murine fibroblast (L929) cell lines. A dose of 3×10−3 % CPC or MST was sufficient to entirely eradicate S. aureus after 30 min incubation. To achieve the same effect, higher concentrations were required against E. coli (0.025 % CPC; 0.0125 % MST) and P. aeruginosa (0.5 % CPC; 0.05 % MST). The CPC-fleece showed a high antiseptic effect against all three bacterial strains, although it did not completely eliminate P. aeruginosa. Both substances showed a high cytotoxic impact at higher tested concentrations (CPC >3×10−3 %; MST >8×10−4 %). CPC showed high antimicrobial potency at low concentrations against S. aureus, accompanied by low cytotoxic (side) effects at these concentrations, whilst the required minimal concentration to eradicate E. coli and P. aeruginosa was shown to be cytotoxic for keratinocytes and fibroblasts. The necessary antibacterial amounts of MST were lower, but also cytotoxic in direct contact with typical human wound cells. With regard to demographic changes and increasing bacterial resistance, new effective antiseptics, such as CPC and MST, incorporated in wound dressings without releasing an active substance could help to improve the treatment and healing rates of chronic wounds.

Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine – already in clinical use against leishmaniasis – towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values of 1–2 µg ml−1. Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed a decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers and accumulation of an electron-dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine was comparable to those of amphotericin B, but miltefosine showed a higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.

Streptococcus pneumoniae and Staphylococcus aureus are common commensals of the upper respiratory tract in children and adolescents. Understanding the relationship between these two pathogens, including their potential for mutual interference, is needed to evaluate the epidemiology of the diseases they cause, the factors that condition acquisition and carriage, and the impact of related preventative measures. We obtained oropharyngeal and nasal swabs from 497 healthy subjects aged 6–17 years. S. pneumoniae detection and serotyping were performed using a real-time PCR and S. aureus detection was performed using the RIDAGENE MRSA system. We found that 136 (27.3 %) of the children were carriers of both species, 121 (24.3 %) of the children carried S. pneumoniae alone and 128 (25.7 %) of the children carried S. aureus alone. S. aureus carriage was similar between children who carried S. pneumoniae (136/257, 52.9 %, 95 % confidence interval [CI]: 46.8–58.9 %) vs those who did not (128/240, 53.3 %, 95 % CI: 47.0 –59.5 %) and was independent of age and vaccination with 7-valent pneumococcal conjugate vaccine (PCV7). Vaccination with PCV7 did not affect S. aureus carriage [S. pneumoniae: 84/143 (58.7 %, 95 % CI: 50.5 –66.5 %) vaccinated children vs 171/351 (48.7 %, 95 % CI: 43.5 –53.9 %) unvaccinated children; S. aureus: 67/143 (46.9 %, 95 % CI: 38.9–55.0 %) vaccinated children vs 195/351 (55.6 %, 95 % CI: 50.3 –60.7 %) unvaccinated children]. Pneumococcal serotype also did not appear to affect S. aureus carriage. These findings suggested that the carriage of S. pneumoniae did not affect that of S. aureus in older children and adolescents, regardless of age, PCV7 vaccination and pneumococcal serotype.

Diarrhoea is one of leading causes of morbidity and mortality worldwide. Recent estimations suggested the number of deaths is close to 2.5 million. This study examined the causative agents of diarrhoea in children under 5 years of age in suburban areas of Khartoum, Sudan. A total of 437 stool samples obtained from children with diarrhoea were examined by culture and PCR for bacteria, by microscopy and PCR for parasites and by immunoassay for detection of rotavirus A. Of the 437 samples analysed, 211 (48 %) tested positive for diarrhoeagenic Escherichia coli, 96 (22 %) for rotavirus A, 36 (8 %) for Shigella spp., 17 (4 %) for Salmonella spp., 8 (2 %) for Campylobacter spp., 47 (11 %) for Giardia intestinalis and 22 (5 %) for Entamoeba histolytica. All isolates of E. coli (211, 100 %) and Salmonella (17, 100 %), and 30 (83 %) isolates of Shigella were sensitive to chloramphenicol; 17 (100 %) isolates of Salmonella, 200 (94 %) isolates of E. coli and (78 %) 28 isolates of Shigella spp. were sensitive to gentamicin. In contrast, resistance to ampicillin was demonstrated in 100 (47 %) isolates of E. coli and 16 (44 %) isolates of Shigella spp. In conclusion, E. coli proved to be the main cause of diarrhoea in young children in this study, followed by rotavirus A and protozoa. Determination of diarrhoea aetiology and antibiotic susceptibility patterns of diarrhoeal pathogens and improved hygiene are important for clinical management and controlled strategic planning to reduce the burden of infection.

This study aimed to describe the microbiological characteristics of acute septic arthritis (SA) and osteomyelitis (OM) in children. Cases of children (0–15 years) with SA/OM were identified through a retrospective search of hospital discharge codes over a six-year period. In addition, a systematic literature search and meta-analysis of studies reporting culture results of children with SA/OM was performed. In our retrospective chart review, we identified 65 cases of OM and 46 cases of SA. The most frequently cultured organisms in both conditions were Gram-positive cocci, primarily Staphylococcus aureus. On admission, most patients had a normal white blood cell count (WCC) but elevated C-reactive protein (CRP) and/or erythrocyte sedimentation rate (ESR). Bacteraemia was associated with a longer mean length of hospitalization for both infections. Considering our results and the meta-analysis, we found low rates of culture-positivity in cases of clinically confirmed infection. In SA, articular fluid was culture-positive in 42.49 % [95 % confidence interval (CI) 28.39–57.23]. In OM, intra-operative samples were culture-positive in 52.65 % (95 % CI 30.54–74.22). Bacteraemia was detected in 23.91 % (95 % CI 8.40–44.24) of children with SA and 21.48 % (95 % CI 10.89–34.47) with OM. Despite appropriate sampling, a positive microbiological diagnosis is often lacking in paediatric acute osteoarticular infection using standard culture-based methods. This highlights the need for validation and use of more sensitive diagnostic methods, such as PCR.