- Volume 66, Issue 2, 2017

Purpose. Group A rotavirus (RVA) is the leading cause of severe gastroenteritis in children younger than 5 years. The most common human G-types are G1-4 and G9. G12 genotype is currently emerging worldwide, becoming the sixth most prevalent RVA G-genotype. In Tunisia, an emergence of G12 RVA strains was observed. To understand the evolution and origin of these Tunisian G12 strains, phylogenetic analyses were conducted.

Methodology. A total of 1127 faecal samples were collected from Tunisian children under 5 years consulting for gastroenteritis between 2009 and 2014. Samples were screened by ELISA for the presence of RVA antigen. RVA-positive samples were used for the detection of G12 RVA strains by semi-nested RT-PCR. G12-positive specimens were subjected to VP4 genotyping reaction. PCR products of the G12-positive samples were sequenced and characterized by phylogenetic analysis of partial VP7 gene sequence.

Results. Globally, 270 (24 %) stool specimens were RVA-positive. Fourteen presented the G12 genotype (5.2 %) and were found to be in combination with either the P[6] (50.0 %) or the P[8] (50.0 %) genotype. Phylogenetic analysis revealed that all characterized Tunisian G12 strains clustered in the modern G12 lineage III and appear to form three different subclusters.

Conclusion. Thus, the Tunisian G12 strains may have originated from not a single, but at least three distinct ancestral G12 strains. Detailed molecular characterization of the entire genome of these strains remains essential to help determine the extent of genetic variation and the relatedness of Tunisian G12 RVA strains to G12 strains described worldwide.

Purpose. A recently identified colistin resistance gene, mcr-1, has been reported in many countries. In this study, we established a new real-time PCR method to detect it.

Methodology. We used a real-time PCR method to detect the mcr-1 gene in a variety of isolates and faecal samples from 20 provinces and municipal cities in China.

Results. Of the 2330 isolates (from 10 species) screened, 54 (2.3 %) isolates were positive for mcr-1. All of the mcr-1-positive isolates that were identified belonged to Escherichia coli strains, among which 9, 1, and 44 were identified as enteropathogenic E. coli, enteroadherent E. coli, and non-pathogenic E. coli, respectively. The majority of the mcr-1-positive isolates were obtained from farm animals from eight provinces and municipal cities across China. A total of 337 faecal samples, including 229 human and 108 pet animal faecal samples, were also screened for the mcr-1 gene. Of the 337 samples analyzed, six and eight human and pet animal faecal samples were positive for the mcr-1 gene, respectively.

Conclusion. The data demonstrate that the mcr-1 gene is highly prevalent in human and animal populations in China. This occurrence suggests that active surveillance of the mcr-1 gene is imperative in curtailing its spread.

Purpose. Despite the existence of a variety of available yeast-identification strategies, easier and more cost-effective methods are required for routine use in clinical laboratories. The internal transcribed spacer (ITS) regions of fungal rRNA genes exhibit variable sizes depending on the yeast species. In the present study, fragment size polymorphism (FSP) analysis of the ITS1 and ITS2 regions for identification of the clinically most important yeast species was assessed.

Methodology. The ITS1 and ITS2 regions of 190 strains, including isolates of 31 standard strains and 159 clinical isolates, were separately PCR amplified with two primer sets: ITS1–ITS2 and ITS3–ITS4. PCR products were mixed and the two-band electrophoretic pattern of each sample was analysed according to the size of the ITS regions as predicted from the GenBank database.

Results. Using this method and avoiding expensive tools such as sequencing or capillary electrophoresis, we were able to differentiate nearly all pathogenic yeast species, including Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida guilliermondii, Candida kefyr, Candida lusitaniae, Candida rugosa, Cryptococcus neoformans and Saccharomyces cerevisiae. The method showed limited discriminatory power to differentiate species of the Candida parapsilosis complex. Differentiation of Candida albicans and Candida tropicalis needs already identified controls.

Conclusion. FSP method benefits from advantages such as lower cost, higher speed and wider range of species than some commercial yeast-identification methods. We consider this method as one of the easiest molecular approaches for identifying a wide range of human pathogenic yeast species, applicable to both diagnostic and epidemiological purposes.

Purpose. Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are common bacterial causes of sexually transmitted infections. Self-taken meatal swabs are a possible alternative to urine samples in testing for infection; however, the data surrounding their use are limited.

Methodology. We carried out a prospective service review in a large sexual health clinic comparing urine samples and self-taken meatal swabs in men presenting for sexual transmissible infection screening for CT and GC with the BD Viper XTR system.

Results. We found an overall prevalence of 10.5 % for CT infections and 4.2 % for GC infections in our patient population. Meatal swab testing had a sensitivity and specificity of 91 and 99 % with an negative predictive value (NPV) of 99 % and a positive predictive value (PPV) of 96 % for CT testing compared to a sensitivity and specificity of 100 and 99 % with an NPV of 100 % and a PPV of 98 % for urine samples. The sensitivity and specificity of meatal swabs was 100 and 99 %, respectively, for GC detection with an NPV of 100 % and PPV of 89 % compared to urine which had 93 % sensitivity and 99 % specificity with an NPV and PPV of 99 and 93 %, respectively.

Conclusions. Meatal samples were not inferior to urine samples for the detection of CT and GC. Male urethral meatal self-sampling offers an alternative sample type when compared to male urine specimens.

Purpose. Amikacin is one of the most effective antibiotics against Pseudomonas aeruginosa infections, but because of its high toxicity, the use of this antibiotic has been clinically limited. In the present study, amikacin was successfully loaded into a new formulation of nanoparticles (NPs) based on poly(d,l-lactide-co-glycolide) 50 : 50 in order to enhance the treatment efficacy. The synthetized amikacin-loaded PLGA nanoparticles with high drug loading and stability were used to eliminate P. aeruginosa cells in planktonic and biofilm states.

Methodology. P. aeruginosa PAO1 biofilm susceptibility studies were done using the minimum biofilm eradication concentration assay. The association of fluorescently labeled amikacin-loaded nanoparticles (A-NPs) with mouse monocyte macrophage cells (RAW 264.7), and the nanoparticles ability to interact and eradicate the bacterial cells even in the form of biofilms, was investigated using Flow cytometric studies and confocal laser scanning microscopy.

Results. Flow cytometric studies showed that these NPs were able to interact with planktonic and biofilm bacterial cells. Moreover, following 1 h of incubation of A-NPs with 1-day-old biofilm, it was found that particles penetrate through the entire biofilm thickness. Live/dead fluorescent staining followed by CLSM analysis showed that the A-NPs were more effective than free drug in biofilm eradication.

Conclusion. The good antibacterial and antibiofilm activities of A-NPs, in addition to their ability to enter macrophages without any cytotoxicity for these cells, make them a potential candidate to treat P. aeruginosa infections.

Purpose. The molecular epidemiology of Pseudomonas aeruginosa bloodstream infection (BSI) isolates has received limited attention. This study aims to characterize the molecular relationship of P. aeruginosa BSI isolates in the non-outbreak setting at a single tertiary healthcare facility.

Methodology.  P. aeruginosa BSI isolates from patients who were admitted to the Royal Brisbane and Women’s Hospital over a 13 month period from November 2009 were identified retrospectively from the Pathology Queensland Clinical and Scientific Information System. The isolates were typed by the iPLEX MassARRAY matrix assisted lazer desorption/isonisation time of flight (MALDI-TOF) MS genotyping. The DiversiLab automated rapid strain typing platform (bioMérieux) was used to assess the genotypic relationships between study isolates that showed indistinguishable iPLEX20SNP profiles. Clinical data was also collected retrospectively from patient notes.

Results. Fifty-three P. aeruginosa BSI episodes were available for study. Thirty-five different clones or clonal complexes were identified by the iPLEX MassARRAY MALDI-TOF MS genotyping. Seventeen BSI isolates with indistinguishable iPLEX20SNP profiles underwent further DiversiLab genotyping and were found to belong to a further 13 different genotypes. There was no relationship between clonality and acquisition type, source of infection or length of stay in the setting of hospital-acquired infection.

Conclusion. The non-clonal population structure suggests that there is ongoing environmental exposure of inpatients to P. aeruginosa. In clinical areas dealing with at-risk patients, routine attention to mechanism of environmental colonization is important and should be addressed even in the non-outbreak setting.

Purpose. To genetically explore the fusion protein gene (F) in human parainfluenza virus type 1 (HPIV1) and type 3 (HPIV3) strains, we analysed them in patients with acute respiratory infections in Eastern Japan from 2011 to 2015.

Methodology. We constructed phylogenetic trees based on the HPIV and HPIV3 F gene using the maximum likelihood method and conducted P-distance and selective pressure analyses. We also predicted the linear epitopes of the protein in the prototype strains. Furthermore, we mapped the amino acid substitutions of the proteins.

Results. Nineteen strains of HPIV1 and 53 strains of HPIV3 were detected among the clinical acute respiratory infection cases. The phylogenetic trees indicated that the HPIV1 and HPIV3 strains were classified into clusters II and III and cluster C, respectively. The P-distance values of the HPIV1 and HPIV3 F genes were <0.03. Two positive selection sites were inferred in the HPIV1 (aa 8 and aa 10), and one positive selection site was inferred in the HPIV3 (aa 108), but over 10 negative selection sites were inferred. Four epitopes were predicted for the HPIV1 prototype strains, while five epitopes were predicted for the HPIV3 prototype strain. A positive selection site (aa 108) or the HPIV3 F protein was involved in the predicted epitope. Additionally, we found that an amino acid substitution (R73K) in the LC76627 HPIV3 strain presumably may affect the resistance to neutralization by antibodies.

Conclusion. The F gene of HPIV1 and HPIV3 was relatively well conserved in the eastern part of Japan during the investigation period.

Purpose. In this study, we analysed phenotypic resistance profiles and their reflection in the genomic profiles of Enterococcus spp. strains isolated from pigs raised on different farms.

Methodology. Samples were collected from five pig farms (n=90 animals) and tested for Enterococcus. MICs of 12 antimicrobials were determined using the broth microdilution method, and epidemiological molecular analysis of strains belonging to selected species (faecalis, faecium and hirae) was performed using the ADSRRS-fingerprinting (amplification of DNA fragments surrounding rare restriction sites) method with a few modifications.

Results. The highest percentage of strains was resistant to tetracycline (73.4 %), erythromycin and tylosin (42.5 %) and rifampin (25.2 %), and a large number of strains exhibited high-level resistance to both kanamycin (25.2 %) and streptomycin (27.6 %). The strains of E. faecalis, E. faecium and E. hirae (n=184) revealed varied phenotypic resistance profiles, among which as many as seven met the criteria for multidrug resistance (30.4 % of strains tested). ADSRRS-fingerprinting analysis produced 17 genotypic profiles of individual strains which were correlated with their phenotypic resistance profiles. Only E. hirae strains susceptible to all of the chemotherapeutics tested had two different ADSRRS profiles. Moreover, eight animals were carriers of more than one genotype belonging to the same Enterococcus spp., mainly E. faecalis.

Conclusion. Given the possibility of transmission to humans of the high-resistance/multidrug resistance enterococci and the significant role of pigs as food animals in this process, it is necessary to introduce a multilevel control strategy by carrying out research on the resistance and molecular characteristics of indicator bacterial strains isolated from animals on individual farms.

Purpose. Saccharomyces boulardi i may improve the immune response by enhancing the production of anti-inflammatory cytokines, T-cell proliferation and dendritic cell activation. The immunomodulator effect of this probiotic has never been tested with DNA vaccines, which frequently induce low antibody titers. This study evaluated the capacity of Saccharomyces boulardii to improve the humoral and cellular immune responses using DNA vaccines coding for the leptospiral protein fragments LigAni and LigBrep. BALB/c mice were fed with rodent-specific feed containing 108 c.f.u. of Saccharomyces boulardii per gram.

Methodology. Animals were immunized three times intramuscularly with 100 µg of pTARGET plasmids containing the coding sequences for the above mentioned proteins. Antibody titers were measured by indirect ELISA. Expression levels of IL-4, IL-10, IL-12, IL-17, IFN-γ and TGF-β were determined by quantitative real-time PCR from RNA extracted from whole blood, after an intraperitoneal boost with 50 µg of the recombinant proteins.

Results/Key findings. Antibody titers increased significantly after the second and third application when pTARGET/ligAni and pTARGET/ligBrep were used to vaccinate the animals in comparison with the control group (P<0.05). In addition, there was a significant increase in the expression of the IL-10 in mice immunized with pTARGET/ligBrep and fed with Saccharomyces boulardii.

Conclusion. The results suggested that Saccharomyces boulardii has an immunomodulator effect in DNA vaccines, mainly by stimulating the humoral response, which is often limited in this kind of vaccine. Therefore, the use of Saccharomyces boulardii as immunomodulator represents a new alternative strategy for more efficient DNA vaccination.

Purpose. The Miranda donkey (Equus asinus) is an endangeredasinine from Miranda do Douro region, located in the north east of Portugal. We studied the antimicrobial resistance and virulence genes in Escherichia coli and Enterococcus spp. isolates from these animals.

Methodology. In March 2014, a total of 66 faecal samples were recovered from independent animals. Antibiotic resistance was determined by the disc diffusion method. Carriage of genes coding for antibiotic-resistant and virulent factors was analysed by PCR.

Results. A total of 66 E. coli and 41 enterococcal isolates were detected, with Enterococcus faecium (61 %) and Enterococcus hirae (24 %) being the most prevalent species. For enterococcal isolates, high percentages of resistance rates to tetracycline (68.3 %), quinupristin/dalfopristin (51.2 %) and ciprofloxacin (48.8 %) were observed. The genes erm(A) and/or erm(B), tet(M) and/or tet(L), vat(D) and/or vat(E) and aph(3′)-IIIa were also found. The most frequent virulence gene detected was gel(E), followed by ace, cpd and hyl. Escherichia coli isolates were highly resistant to streptomycin (78 %), whereas 39 % of them exhibited resistance to aminoglycosides and tetracycline. Genes sul1 and/or sul2 were detected in 66.7 % of trimethoprim/sulfamethoxazole-resistant isolates. The virulence genes detected were fim(A) (46 %) and cnf1 (27 %).

Conclusion.. To the best of our knowledge, this is the first report showing antibiotic resistance among Escherichia coli and Enterococcus spp. isolates from the Miranda donkey in Portugal, indicating possible antibiotic-resistant bacterial reservoirs. However, the detection of these resistances presents a low risk for other animals and human beings in that rural area.

The antifungal susceptibilities of 598 isolates of Candida spp. (bloodstream and other sterile sites) to liposomal amphotericin B (L-AmB) versus amphotericin B (AmB) were determined. MICs were calculated using the Clinical and Laboratory Standards Institute broth microdilution (M27-A3) method for L-AmB and the Etest method for AmB. The MIC50/MIC90 (µg ml−1) values for L-AmB broth microdilution and AmB Etest were 0.25/1 and 0.19/0.5, respectively. The overall essential agreement (±2 dilutions) was 91.5 %, ranging from 37.5 % (Candida lusitaniae) to 100 % (Candida glabrata and Candida krusei). Categorical agreement between the two methods was categorized based on a previously published breakpoint (susceptible/resistant MIC cut-off of 1 µg ml−1). The overall categorical agreement at the 48 h reading was 97.3 %, ranging from 72.7 % (C. krusei) to 100 % (Candida albicans). Major and very major discrepancies occurred in 2.3 and 0.3 %, respectively. Spearman’s ρ was 0.48 (P<0.0001). These results demonstrate the utility of the AmB Etest as a surrogate marker to predict the sensibility and resistance of Candida spp. to L-AmB and thus to support its use in antifungal treatment.

Purpose. Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen causing diarrhoeal diseases in multiple epidemiological and clinical settings. However, understanding of the pathogenesis of the disease caused by this organism is still suboptimal. Studies have indicated that enteric bacteria induced cell cycle arrest and apoptosis in host intestinal epithelial cells might play a vital role in the pathogenesis caused by these organisms. In this study an attempt was made to assess EAEC-induced apoptosis and cell cycle modulation in human intestinal epithelial cell lines.

Methodology. INT-407 and HCT-15 cells were infected with EAEC-T8 (clinical isolate) as well as plasmid cured variant of EAEC-T8 (EAEC-pT8). Propidium iodide staining was done to select the time of infection and the incubation period of the infected culture. Apoptosis was further assessed in EAEC infected both the cell lines by annexin-V-FLUOS & propidium iodide, cell death detection ELISA, DNA strand breaks and microscopic analysis. Further, the DNA content of the EAEC-infected cells at different phases of cell cycle was also monitored.

Results. We have found that EAEC could induce apoptosis in human small intestinal as well as colonic epithelial cell lines, which was assessed by the expression of phosphatidylserine on host cell surface, internucleosomal cleavage of host cell DNA and microscopic analysis of the characteristic apoptotic features of these cells. EAEC was also found to arrest cells at S phase and G2-M phase of the cell cycle.

Conclusions. EAEC-T8 could induce maximum apoptosis and cell cycle modulation in both small intestinal and colonic epithelial cells. Further, we have observed that the plasmid of this organism had maximum contribution to these processes. The outcome of this study has undoubtedly led to a better understanding of the basic mechanism of pathogenesis caused by EAEC.

Purpose. Multiple Helicobacter pylori strains colonize and coexist in the stomach of one single patient, carrying heterogeneous distributions of cag genotypes. The oesophagus provides a niche for H. pylori colonization; however, little is known about its adaptive role.

Methodology. Using PCR for cagA, cagE and virB11 genes from cag-pathogenicity island (PAI) and Etest for antimicrobial susceptibility test, we determined cag-PAI genotypes associated with H. pylori virulence, when positive cultures were matching in both the stomach and the oesophagus (96 isolates; 8 out of 80 dyspeptic patients).

Results. The stomach showed complete cag-PAI islands in 77 % of the isolates, whereas the oesophagus showed complete cag-PAI islands only in 44 % of the isolates. Expression of CagA and interleukin 8 correlated with inflammatory processes and histopathological changes in the stomach, but not in the oesophagus. Different cag-PAI profiles were found in both mucosae of an individual host, and at least one oesophagus profile corresponded to one profile identified in stomach. The antibiotic resistance profiles showed variability in the colonization by single or mixed H. pylori isolates in the gastric and oesophageal mucosa both intra- and inter-individuals.

Conclusion. These results demonstrate colonization with multiple H. pylori isolates in the oesophageal mucosa, like those found in the stomach of individual hosts. H. pylori was characterized by a dominant partial island, low interleukin 8 induction with lower histopathological damage and lower antibiotic resistance, suggesting that the microenvironmental changes in individual hosts select less virulent isolates in the oesophagus than in the stomach. New approaches to ensure effective eradication therapy in multi-resistant H. pylori strains must be developed.

Purpose. Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas.

Methodology. Blood or spleen samples from 648 koalas where tested for KoRV provirus, and subsequently genotyped, using PCRs to detect the pol and env genes respectively. Clinical data was also recorded where possible and analysed in comparison to infection status.

Results. The prevalence of KoRV was 24.7 % (160/648). KoRV-A was detected in 141/160 cases, but KoRV-B, a genotype associated with neoplasia in captive koalas, was not detected. The genotype in 19 cases could not be determined. Genomic differences between KoRV in Victoria and type strains may have impacted genotyping. Factors associated with KoRV infection, based on multivariable analysis, were low body condition score, region sampled, and ‘wet bottom’ (a staining of the fur around the rump associated with chronic urinary incontinence). Koalas with wet bottom were nearly twice as likely to have KoRV provirus detected than those without wet bottom (odds ratio=1.90, 95 % confidence interval 1.21, 2.98).

Conclusion. Our findings have important implications for the conservation of this iconic species, particularly regarding translocation potential of Victorian koalas.

Purpose. Human bocavirus (HBoV) exsits in four genotypes: 1 to 4, with HBoV-1 being the most prevalent genotype. The aim of the current study was to genetically analyze the full-length genome of the HBoV-1 of recently detected Egyptian strains.

Methodology. Seven overlapping sets of primers were developed to amplify an almost complete HBoV-1 genome from the clinical samples. The primer sets were tested on three recently identified Egyptian HBoV-1 strains with viral loads ≥105 ml−1. Sequencing was conducted using the same sets of primers. HBoV-1 virus strains were genetically analyzed based on the sequences of their complete genomes and the individual ORFs.

Results. The new sets of primers successfully amplified the three tested strains. Sequence analysis of the full-length genome of the HBoV-1 revealed a considerable level of genetic heterogenicity between different strains. Based on the full genome and VP1 ORF, HBoV-1 viruses were clustered into three main lineages, A to C, and lineage A was further subdivided into three sublineages, A1–A3. The Egyptian strains were clustered within two sublineages, A1 and A2. New amino acid substitutions were detected in NS1 and VP1/VP2 proteins. Both inter- and intragenomic recombination events were detected among the Egyptian strains.

Conclusion. The existence of both intragenomic recombination event and multiple amino acid substitutions in the examined Egyptian HBoV-1 strains elucidates considerable level of genetic alterations among bocaviruses. Their possible effects on the virus virulence and multiplication efficiency need to be investigated.