- Volume 64, Issue 11, 2015

The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml− 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml− 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.

Listeria monocytogenes is a dangerous bacterium that causes the food-borne disease listeriosis and accounts for nearly 20 % of food-borne deaths. This organism can survive the body's natural defences within the digestive tract, including acidic conditions and bile. Although the bile response has been analysed, limited information is available concerning the ability of L. monocytogenes to resist bile under anaerobic conditions, especially at acidic pH, which mimics conditions within the duodenum. Additionally, it is not known how the bile response varies between serotypes. In this study, the survival of strains representing six serotypes was analysed under aerobic and anaerobic conditions following exposure to bile. Exposure to bile salts at acidic pH increased toxicity of bile, resulting in a significant reduction in survival for all strains tested. However, following this initial reduction, no significant reduction was observed for an additional 2 h except for strain 10403S (P = 0.002). Anaerobic cultivation increased bile resistance, but a significant increase was only observed in virulent strains when exposed to bile at pH 5.5. Exposure to pH 3.0 prior to bile decreased viability amongst avirulent strains in bile in acidic conditions; oxygen availability did not influence viability. Together, the data suggested that being able to sense and respond to oxygen availability may influence the expression of stress response mechanisms, and this response may correspond to disease outcome. Further research is needed on additional strains to determine how L. monocytogenes senses and responds to oxygen and how this varies between invasive and non-invasive strains.

Clostridium difficile infection (CDI) leads to considerable morbidity and mortality among hospitalized patients. Faecal specimens from 1110 hospitalized patients suspected for CDI were cultured for isolation of C. difficile and characterization of virulence genes. PCR was carried out for toxigenic genes tcdA, tcdB, cdtA and cdtB and PCR-RFLP for fliC and slpA genes. Of 174 (15.7 %) C. difficile isolates, 121 (69.5 %) were toxigenic, amongst which 68 (56.2 %) also had both tcdA and tcdB genes. The remaining 53 (43.8 %) of the isolates also had at least one of the toxin genes. Binary toxin genes (cdtA and cdtB) with only one of the two components were present in 16 (9.2 %) of the 174 isolates. The other virulence genes – fliC and slpA – were present in 100 % of the isolates. The most frequent PCR-RFLP type of fliC gene was type I (n = 101), followed by type VII (n = 49) and type III (n = 24). The slpA gene presented with three combinations of patterns. Characterization of virulence genes in C. difficile isolates is of extreme importance for epidemiological surveillance and control of outbreaks owing to the capacity of this bacterium to adapt to new environmental circumstances, leading to the emergence of new epidemic strains.

This study aimed to investigate the role of quinolone resistance-determining regions (QRDRs) of DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) associated with fluoroquinolone resistance. A total of 114 Ureaplasma spp. strains, isolated from clinical female patients with symptomatic infection, were tested for species distribution and susceptibility to four fluoroquinolones. Moreover, we analysed the QRDRs and compared these with 14 ATCC reference strains of Ureaplasma spp. serovars to identify mutations that caused antimicrobial resistance. Our study indicated that moxifloxacin was the most effective fluoroquinolone against Ureaplasma spp. (MIC range: 0.125–32 μg ml− 1). However, extremely high MICs were estimated for ciprofloxacin (MIC range: 1–256 μg ml− 1) and ofloxacin (MIC range: 0.5–128 μg ml− 1), followed by levofloxacin (MIC range: 0.5–64 μg ml− 1). Seven amino acid substitutions were discovered in GyrB, ParC and ParE, but not in GyrA. Ser-83 → Leu/Trp (C248T/G) in ParC and Arg-448 → Lys (G1343A) in ParE, which were potentially responsible for fluoroquinolone resistance, were observed in 89 (77.2 %) and three (2.6 %) strains, respectively. Pro-462 → Ser (C1384T), Asn-481 → Ser (A1442G) and Ala-493 → Val (C1478T) in GyrB and Met-105 → Ile (G315T) in ParC seemed to be neutral polymorphisms, and were observed and occurred along with the amino acid change of Ser-83 → Leu (C248T) in ParC. Interestingly, two novel mutations of ParC and ParE were independently found in four strains. These observations suggest that amino acid mutation in topoisomerase IV appears to be the leading cause of fluoroquinolone resistance, especially the mutation of Ser-83 → Leu (C248T) in ParC. Moxifloxacin had the best activity against strains with Ser-83 → Leu mutation.

Since 1996 the Meningococcal Reference Unit (MRU) in Manchester has provided a national service for PCR confirmation of meningococcal and pneumococcal disease. Neisseria meningitidis serogroup B is predominant in the UK, accounting for >60 % of cases. In response to this, the MRU has developed a quadruplex quantitative PCR that detects N. meningitidis capsule transporter (ctrA), serogroup B sialyltransferase (siaD), Streptococcus pneumoniae pneumolysin (ply) and an internal control. The assay was prepared in a ready-to-use lyophilized format by Applied Biosystems. Laboratory validation showed excellent performance in a specificity panel of 52 isolates and improved detection in comparison with the routine assay. Testing of 244 patient samples showed sensitivity of 93 % [95 % confidence interval (CI): 88–98 %] for the ctrA assay, 95 % (95 % CI: 91–100 %) for the siaD assay and 100 % (95 % CI: 95–100 %) for the ply assay. Specificity was 100 % (95 % CI: 98–100 %) for both meningococcal targets and 95 % (95 % CI: 92–98 %) for ply. The quadruplex also retained high performance in mixed samples and had acceptable reproducibility. After introduction of the quadruplex into routine use the turnaround time for N. meningitidis group B PCR confirmation reduced from 37 to 29 h and the internal control has proved useful for detecting inhibitory samples. The quadruplex assay provides rapid group B confirmation of meningococcal positive samples, enabling timely public health interventions for the most common disease-causing meningococcal serogroup in the UK.

A loop-mediated isothermal amplification (LAMP) assay was developed to detect Enterocytozoon bieneusi DNA for the first time from human faecal specimens. Four primers specific for Enterocytozoon bieneusi were designed corresponding to small subunit rRNA gene sequences and tested on 100 human faecal specimens. Thirty-nine of the faecal specimens (39 %) were confirmed positive for Enterocytozoon bieneusi by LAMP compared with 33 % by PCR and 32 % by light microscopy. LAMP yielded 94 % sensitivity and 88 % specificity compared with microscopy (sensitivity 48 %, specificity 76 %). No significant differences in positive detection of Enterocytozoon bieneusi were found among the three methods (P>0.05). However, LAMP has shown a substantial agreement with PCR (κ = 0.78) and fair agreement was demonstrated between microscopy and PCR (κ = 0.25). In conclusion, the LAMP assay proved to be useful as a simplified, rapid, sensitive and specific alternative molecular screening tool in the diagnosis of Enterocytozoon bieneusi in faecal specimens.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis complex (MTC), remains one of the leading causes of death in the world. In Korea, the current prevalence of multidrug-resistant TB (MDR-TB) poses a major problem. The most common method for diagnosing TB in developing countries is sputum smear microscopy; however, the sensitivity of this test is relatively low and it usually requires well-trained laboratory staff. Cultures of MTC require up to several weeks in sophisticated facilities, such as Biosafety Level 3. Effective diagnostic techniques are necessary to control TB. In Korea, we evaluated a loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene (TB-hspX-LAMP) of MTC. For clinical evaluation, culture confirmation, smear microscopy and TB-hspX-LAMP were performed on 303 sputum specimens obtained from suspected TB patients in Korea. The sensitivity, specificity, positive predictive value and negative predictive value of TB-hspX-LAMP were 71.1, 98.8, 91.4 and 95.1 %, respectively, compared with TB culture, which is the gold standard for diagnosis of TB. In contrast, the comparable values of smear microscopy were 24.4, 98.1, 68.8 and 88.2 %, respectively. Therefore, we concluded that TB-hspX-LAMP was superior to the use of smear microscopy for the detection of MTC in sputum specimens in clinical settings in Korea.

Using a Clostridium difficile glutamate dehydrogenase (GDH) immunoassay and a sensitive C. difficile toxin A/B immunoassay, human stool specimens from patients with diarrhoea (n = 1085) were classified as either GDH positive/toxin negative, or GDH positive/toxin positive. Overall, 528/725 (73%) of the GDH-positive/toxin-negative specimens contained viable C. difficile, and 433/528 (82%) of these C. difficile isolates were PCR positive for the toxin gene pathogenicity locus. Overall, 867/1078 (80%) of the GDH-positive specimens contained viable C. difficile, and 433/725 (60%) of the GDH-positive/toxin-negative specimens contained a toxigenic C. difficile strain. The diversity of toxigenic C. difficile ribotypes isolated from toxin-negative specimens (n = 433) and toxin-positive specimens (n = 339) was significantly different (P < 0.0001). Specifically, the presence of ribotype 078 strains was very strongly associated (P < 0.0001) with detection of toxin in clinical specimens using a sensitive toxin immunoassay. Specimens positive for ribotype 078 were almost twice as likely to be toxin positive as opposed to toxin negative (risk ratio = 1.90, 95% confidence interval 1.64–2.19). In contrast, other circulating ribotypes were seen with similar frequency in specimens with and without detectable toxin. This supports the view that ribotype 078 strains may be more virulent than other common ribotypes in terms of toxin production.

Rapid identification of bacteria from blood cultures enables early initiation of appropriate antibiotic treatment in patients with bloodstream infections (BSI). The objective of the present study was to evaluate the use of matrix-associated laser desorption ionization-time of flight (MALDI-TOF) MS after a short incubation on solid media for rapid identification of bacteria from positive blood culture bottles. MALDI-TOF MS was performed after 2.5 and 5.5 h plate incubation of samples from positive blood cultures. Identification scores with values ≥ 1.7 were accepted as successful identification if the results were confirmed by conventional methods. Conventional methods included MALDI-TOF MS, Vitek 2, and diverse biochemical and agglutination tests after overnight culture. In total, 515 positive blood cultures with monomicrobial bacterial growth representing one blood culture per patient were included in the study. There were 229/515 (44.5 %) and 286/515 (55.5 %) blood culture bottles with Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB), respectively. MALDI-TOF MS following short-term culture could accurately identify 300/515 (58.3 %) isolates at 2.5 h, GNB being identified in greater proportion (180/229; 78.6 %) than GPB (120/286; 42.0 %). In an additional 124/515 bottles (24.1 %), identification was successful at 5.5 h, leading to accurate identification of bacteria from 424/515 (82.3 %) blood cultures after short-term culture. Interestingly, 11/24 of the isolated anaerobic bacteria could be identified after 5.5 h. The present study demonstrates, in a large number of clinical samples, that MALDI-TOF MS following short-term culture on solid medium is a reliable and rapid method for identification of bacteria from blood culture bottles with monomicrobial bacterial growth.

Identification of bacteria causing lower-airway infections is important to determine appropriate antimicrobial therapy. Flexible bronchoscopy with bronchoalveolar lavage (BAL) is used to obtain lower-airway specimens in young children. The first lavage (lavage-1) is typically used for bacterial culture. However, no studies in children have compared the detection of cultivable bacteria from sequential lavages of the same lobe. BAL fluid was collected from two sequential lavages of the same lobe in 79 children enrolled in our prospective studies of chronic cough. The respiratory bacteria Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus and Haemophilus parainfluenzae were isolated and identified using standard published methods. H. influenzae was differentiated from Haemophilus haemolyticus using PCR assays. Lower-airway infection was defined as ≥ 104 c.f.u. ml− 1 BAL fluid. We compared cultivable bacteria from lavage-1 with those from the second lavage (lavage-2) using the κ statistic. Lower-airway infections by any pathogen were detected in 46 % of first lavages and 39 % of second lavages. Detection was similar in both lavages for all pathogens; the κ statistic was 0.7–0.8 for all bacteria except H. parainfluenzae. Of all infections detected in either lavage, 90 % were detected in lavage-1 and 78 % in lavage-2. However, culture of lavage-2 identified infections that would have been missed in 8 % of children, including infections by additional Streptococcus pneumoniae serotypes. Our findings support the continued use of lavage-1 for bacterial culture; however, culture of lavage-2 may yield additional identifications of bacterial pathogens in lower-airway infections.

In the present study, the adjuvant activity of flagellin was compared, in the conjugated and mixed forms, against a peptide vaccine from human immunodeficiency virus type 1 (HIV-1) p24–Nef vaccine candidate. Mice were immunized with the HIV-1 p24–Nef peptide with flagellin in both conjugated and mixed forms. Lymphocyte proliferation, CTL activity, and IL-4 and IFN-γ cytokines were evaluated by bromodeoxyuridine, carboxyfluorescein succinimidyl ester and ELISA methods, respectively. At the same time, the frequency of IFN-γ-producing T-lymphocytes, as well as total and isotype-specific antibodies, were assessed by ELISPOT and indirect ELISA, respectively. Our experimental results showed that the immunized mice with the HIV-1 p24–Nef conjugated or mixed forms of flagellin led to increases in the proliferative responses and Th1 cytokine pattern. The conjugated form of vaccine led to increased CTL activity and a Th1 cytokine pattern of immune responses, as well as an IgM isotype of humoral responses in comparison with the mixed form. The flagellin-conjugated vaccine seems to be more potent in increasing vaccine immunogenicity.

Limited data exist evaluating outcomes in patients with serious Streptococcus anginosus group infections, particularly bacteraemia. A retrospective, single-centre cohort study was conducted to characterize potential risk factors along with clinical and microbiological outcomes in patients with S. anginosus group bacteraemia (SAGB). Adult inpatients with SAGB identified using the Verigene Gram-positive blood culture assay between March 2013 and April 2014 were included. Patients aged ≤ 18 or >89 years, those with SAGB identified at an outside facility and those who were incarcerated were excluded. Differences between groups were explored using a Wilcoxon rank-sum test, χ2 test, Student's t-test or Fisher's exact test as appropriate and a two-tailed P value of ≤ 0.05 was considered statistically significant. The 34 patients who met the inclusion criteria were 57 ± 14 (mean ± sd) years old and had a median Charlson co-morbidity index of 4 [interquartile range (IQR) 1–6] and 10 (29 %) were immunosuppressed at baseline. Almost half (47 %) had received antibiotics in the previous 90 days. Twelve (35 %) patients had gastrointestinal malignancies and the commonest source of bacteraemia was the gastrointestinal tract (53 %). The primary species responsible for SAGB was S. anginosus (68 %), and overall susceptibility to penicillin was 91 %. Patients were most often treated with a β-lactam/β-lactamase inhibitor combination (36 %) for a duration of 8 (IQR 4–13) days. Length of stay (LOS) and infection-related LOS were 10 (IQR 5–17) and 9 (IQR 4–12) days, respectively. Twenty [59 %] patients achieved a clinical cure, while 29 (85 %) achieved a microbiological cure. Four (12 %) patients died and one patient was readmitted within 30 days. In the largest cohort of patients with SAGB to date, gastrointestinal malignancies may have been an important risk factor for SAGB, while rapid identification via a microarray assay likely contributed to improved disease recognition and timely pharmacological and non-pharmacological therapy.

The cystic fibrosis (CF) airways are colonized by polymicrobial communities with high bacterial load and are influenced by frequent antibiotic exposures. This community includes diverse streptococci, some of which have been directly or indirectly associated with pulmonary exacerbations. As many streptococci are naturally competent, horizontal transfer of antibiotic-resistant determinants coupled with frequent and/or chronic antibiotic exposure may contribute to high resistance rates. In this study, we assessed antibiotic resistance in 413 streptococcal isolates from adult CF patients against nine antibiotics relevant in CF treatment. We observed very low rates of cephalosporin resistance [cefepime and ceftriaxone ( < 2 %)], and higher rates of resistance to tetracycline (∼34 %) and sulfamethoxazole/trimethoprim (∼45 %). The highest rate of antibiotic resistance was to the macrolides [azithromycin (56.4 %) and erythromycin (51.6 %)]. We also investigated the molecular mechanisms of macrolide resistance and found that only half of our macrolide-resistant streptococci isolates contained the mef (efflux pump) or erm (methylation of 23S ribosomal target site) genes. The majority of isolates were, however, found to have point mutations at position 2058 or 2059 of the 23S ribosomal subunit – a molecular mechanism of resistance not commonly reported in the non-pyogenic and non-pneumococcal streptococci, and unique in comparison with previous studies. The high rates of resistance observed here may result in poor outcomes where specific streptococci are contributing to CF airway disease and serve as a reservoir of resistance genes within the CF airway microbiome.

Salmonella enterica subsp. enterica serovar Typhimurium is one of the leading serovars that causes salmonellosis worldwide. However, few studies have molecularly characterized S. Typhimurium strains in Brazil. In this study, we genotyped 92 S. Typhimurium strains isolated from humans (43) and food (49) between 1983 and 2013 in Brazil using PFGE, multiple-locus variable number of tandem repeats analysis (MLVA) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Moreover, we assessed the frequency of 12 virulence markers by PCR and the resistance profile against 12 antimicrobials. More than 85.8 % of the strains studied carried 11 of the virulence markers or more. Thirty-three strains (25 %) were multidrug resistant (MDR). The 92 S. Typhimurium studied were grouped by PFGE as PFGE-A, PFGE-B1 and PFGE-B2; by MLVA as MLVA-A, MLVA-B1 and MLVA-B2; and, finally, by ERIC-PCR as ERIC-A and ERIC-B. The strains isolated from humans before the mid-1990s were allocated to all clusters. The strains isolated from humans after the mid-1990s were distributed in the PFGE-B1, MLVA-B1, MLVA-B2 and ERIC-A clusters. The strains isolated from food were distributed in all clusters, except in PFGE-B2. All typing results suggested that the S. Typhimurium strains of human clinical origin isolated before the mid-1990s were genetically more diverse, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding strains isolated from food, the results suggest the current circulation of more than one subtype. Furthermore, the high frequency of virulence genes and the presence of MDR strains reinforces their potential hazard for humans and the risk of their presence in foods in Brazil.

The resistance of Helicobacter pylori strains to clarithromycin is increasing in several developed countries and their association with a genetic pattern circulation has been variously explained as related to different geographical areas. In this study we have reported: the prevalence of the resistance of H. pylori, isolated in Sicily, to clarithromycin; the principal point of mutation associated with this resistance; and the more frequent association between resistance to clarithromycin and cagA, the EPIYA motif, and the vacA and oipA genes. Resistance to clarithromycin was detected in 25 % of cases, the main genetic mutation involved being A2143G. The cagA gene was present in 48 % of cases and the distribution of the EPIYA motif was: ABC in 35 cases; ABCC in 8 cases; ABCCC in 2 cases; ABC-ABCC in 2 cases; and ABC-ABCC-ABCCC in 1 case. Regarding the vacA allele, an s1i1m1 combination was detected in 35 % of cases, s1i1m2 in 12 %, s1i2m2 in 12 %, s2i2m2 in 40 %, and a double s1m1-m2 mosaic in 1 % of cases. The status of the oipA gene was ‘off’ in 45 % of cases and ‘on’ in 55 %. Resistance to clarithromycin was found to be high in Sicily, but no correlation was found among resistance to clarithromycin, the vacA gene and oipA status; a higher correlation was observed between resistant strains and cagA-negative strains.

Human parechovirus (HPeV) infects humans early in life and typically causes asymptomatic or mild diseases such as gastrointestinal and respiratory illness but sometimes leads to more serious consequences in neonates and young infants. In 2014, we detected HPeV from 38 patients by real-time reverse transcription-PCR in Osaka City, Japan, and 33 HPeV strains were genotyped based on their VP1 sequences. HPeV genotype 3 (HPeV-3) was the most prevalent and accounted for 22 cases (66.7 %) followed by nine HPeV-1 (27.3 %), one HPeV-2 (3.0 %) and one HPeV-4 (3.0 %). Phylogenetic analysis revealed that detected HPeV-3 strains were divided into three genetically distinct groups. One was characterized by a novel single amino acid deletion mutation at the N terminus of the 2A protein as well as the VP1 sequence, whereas the others were closely related to HPeV-3 strains detected in Japan in either 2008 or 2011. These HPeV-3 groups were detected from patients with various symptoms including three myositis cases. Recent papers have demonstrated that HPeV-3 was the aetiological agent for epidemic myalgia exclusively among adults from Yamagata Prefecture in Japan. Here, we provide clinical details and episodes of three myositis patients including an adult and two children in Osaka City, Japan. Our results suggest that HPeV-3 is a causative agent of myositis not only in adults but also in children.