- Volume 60, Issue 9, 2011

Leishmaniasis is a parasitic disease affecting over 12 million individuals worldwide. As current treatments are insufficient, the development of an effective vaccine is a priority. This study generated and assessed the efficacy of Leishmania vaccines engineered from the non-colonizing, non-pathogenic Gram-positive bacterium Lactococcus lactis. A truncated, codon-optimized version of the A2 antigen from Leishmania donovani was engineered for expression in Lactococcus lactis in three different subcellular compartments: in the cytoplasm, secreted outside the cell or anchored to the cell wall. These three A2-expressing Lactococcus lactis strains were tested for their ability to generate A2-specific immune responses and as live vaccines against visceral Leishmania donovani infection in BALB/c mice. Subcutaneous immunization with live Lactococcus lactis expressing A2 anchored to the cell wall effectively induced high levels of antigen-specific serum antibodies. It was demonstrated that Lactococcus lactis-based vaccines are a feasible approach in the generation of live vaccines against leishmaniasis. The Lactococcus lactis strains generated in this study provide an excellent foundation for further studies on live bacterial vaccines against leishmaniasis and other pathogens.

Candida biofilm development can be influenced by diverse factors such as substrate, culture medium, carbohydrate source and pH. We have analysed biofilm formation of Candida albicans SC5314 and Candida glabrata ATCC 2001 wild-type strains in the presence of different media (RPMI 1640 versus YNB) and using different pH values (pH 5.6 or 7.0). We determined adhesion and biofilm formation on polystyrene, changes in the expression of adhesin genes during these processes and the susceptibility of mature biofilms to echinocandins. Biofilms formed on polystyrene by both Candida species proved to be influenced strongly by the composition of the medium rather than pH. C. albicans and C. glabrata formed thicker biofilms in RPMI 1640 medium, whereas in YNB medium, both species manifested adhesion rather than characteristic multilayer biofilm architecture. The stimulated biofilm formation in RPMI 1640 medium at pH 7.0 corroborated positively with increased expression of adhesin genes, essential to biofilm formation in vitro, including ALS3 and EAP1 in C. albicans and EPA6 in C. glabrata. The thicker biofilms grown in RPMI 1640 medium were more tolerant to caspofungin and anidulafungin than YNB-grown biofilms. We also observed that mature C. glabrata biofilms were less susceptible in RPMI 1640 medium to echinocandins than C. albicans biofilms. Environmental conditions, i.e. medium and pH, can significantly affect not only biofilm architecture, but also the expression profile of several genes involved during the different stages of biofilm development. In addition, growth conditions may also influence the antifungal-susceptibility profile of fungal populations within biofilm structures. Therefore, before designing any experimental biofilm set-up, it is important to consider the potential influence of external environmental factors on Candida biofilm development.

The aim of this study was to investigate the interaction of Candida tropicalis with three different human cell lines: TCC-SUP (epithelial cells from urinary bladder), HeLa (epithelial cells from cervical carcinoma) and Caco-2 (epithelial cells from colorectal adenocarcinoma). In particular we sought to assess the degree of cell damage and activity reduction induced by C. tropicalis adhesion and the role of secreted aspartyl proteinase (SAP) gene expression in this process. Two C. tropicalis strains were used: the reference strain ATCC 750 and a clinical isolate from urine (U69). The ability of C. tropicalis to adhere to a confluent layer of human cells was determined using an adaptation of the crystal violet staining method; cell damage and cell activity inhibition induced by the adhesion of C. tropicalis were assessed by measuring lactate dehydrogenase and tetrazolium salt (MTS) reduction, respectively. C. tropicalis SAP gene expression was determined by real-time PCR. Both C. tropicalis strains were able to adhere to the different human cells, although in a strain- and cell-line-dependent manner. Concerning the cellular response to C. tropicalis, the highest inhibition of cell activity was obtained for Caco-2, followed by TCC-SUP and HeLa cells. The highest percentage of cell damage (around 14 %) was observed for TCC-SUP cells in contact with the U69 isolate and for Caco-2 in contact with the reference strain. Real-time PCR analysis revealed a wide range of expression profiles of SAP genes for both C. tropicalis strains in contact with the different types of epithelial cells. SAPT3 was the gene expressed at the highest level for both C. tropicalis strains in contact with the three human epithelial cell lines. The results highlight that the response of human cells to C. tropicalis adhesion, as well as production of SAPs, is dependent on both the strain and the epithelial cell line.

This study provides guidelines on the usefulness of full and 527 bp 16S rRNA gene sequencing and Microseq databases for identifying medically important aerobic Gram-negative bacteria. Overall, full and 527 bp 16S rRNA gene sequencing can identify 26.1 % and 32.6 %, respectively, of medically important aerobic Gram-negative bacteria confidently to the species level, whereas the full-MicroSeq and 500-MicroSeq databases can identify 15.2 % and 26.1 %, respectively, of medically important aerobic Gram-negative bacteria confidently to the species level. Among the major groups of aerobic Gram-negative bacteria, the methods and databases are least useful for identification of Aeromonas, Bordetella and Bartonella species. None of the Aeromonas species can be confidently or doubtfully identified, whereas only 0 % and 0–33.3 % of Bordetella species and 0–10 % and 0–10 % of Bartonella species can be confidently and doubtfully identified, respectively. On the other hand, these methods and databases are most useful for identification of members of the families Pasteurellaceae and Legionellaceae and Campylobacter species: 29.6–59.3 % and 7.4–18.5 % of members of Pasteurellaceae, 36–52 % and 12–24 % of members of Legionellaceae, and 26.7–60 % and 0–13.3 % of Campylobacter species can be confidently and doubtfully identified, respectively. Thirty-nine medically important aerobic Gram-negative bacteria that should be confidently identified by full 16S rRNA gene sequencing are not included in the full-MicroSeq database. Twenty-three medically important aerobic Gram-negative bacteria that should be confidently identified by 527 bp 16S rRNA gene sequencing are not included in the 500-MicroSeq database. Compared with results of our previous studies on anaerobic and Gram-positive bacteria, full and 527 bp 16S rRNA gene sequencing are able to confidently identify significantly more anaerobic Gram-positive and Gram-negative bacteria than aerobic Gram-positive and Gram-negative bacteria.

Epidemiological investigations of Clostridium difficile often focus on differences between separate geographical areas. In this investigation, two populations of C. difficile recovered from separate tertiary referral Trusts within the West Midlands, UK, were characterized using both PCR ribotyping and an optimized RAPD (random amplification of polymorphic DNA) protocol. The PCR ribotyping and RAPD methodologies identified differences between the two C. difficile populations, in both the prevalence and the diversity of types identified. The use of PCR ribotyping in conjunction with RAPD further categorized different types within defined PCR ribotypes, identifying different types within the same PCR ribotype and therefore providing a greater discriminatory power than either of the methods when used alone. The differences observed in this study between the two Trusts in the distribution of both RAPD ‘type’ and PCR ribotype demonstrate the diversity that is present amongst isolates of C. difficile within a relatively small geographical area and warrants a need for further investigation into the local epidemiology of C. difficile.

Breakthrough contamination of tuberculosis (TB) cultures is a problem in that it allows the overgrowth of another bacterium present in the sputum specimen, which can potentially mask the presence of Mycobacterium tuberculosis. The aim of this study was to isolate and characterize the bacterial organisms responsible for such overgrowth and contamination, and to examine their susceptibility to (i) various chemical selective decontamination steps and (ii) antibiotics in liquid culture media, in an attempt to develop a method to help alleviate contamination problems associated with the conventional isolation of M. tuberculosis from routine patient sputum specimens. Bacterial contaminants from 102 routine sputum cultures were identified molecularly by 16S rRNA gene PCR and direct sequencing from contaminated Löwenstein–Jensen (LJ) slopes and BacT/Alert liquid medium. It was found that the contaminants from LJ slopes belonged to 11 different genera and were composed largely of Gram-negative organisms (84.9 %; 45/53), whereas the liquid culture contaminants belonged to 13 different genera, with 37/66 isolates (56.1 %) being Gram-negative. Pseudomonas aeruginosa was the dominant contaminant in both media. The effect of six different selective decontamination protocols was examined. Four of the six methods were effective at eliminating all culturable organisms present; these were 5 % oxalic acid, 5 % oxalic acid/2 % NaOH, 5 % oxalic acid/4 % NaOH and 1 % chlorhexidine. NaOH at a concentration of 2 or 4 % was less effective as it was unable to eliminate all organisms of each species tested, with the exception of P. aeruginosa. In conclusion, breakthrough contamination of TB cultures is due to a diverse range of at least 17 different bacterial genera, with P. aeruginosa and Staphylococcus epidermidis accounting for the dominant contaminating flora. Employment of chemical decontaminating protocols solely involving NaOH may lead to higher rates of contamination. Where such contamination is encountered, TB laboratories should consider the reprocessing of such sputum samples with an alternative decontamination method such as 1 % chlorhexidine.

There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.

The in vitro antifungal susceptibilities of 159 clinical isolates of Candida species from patients with invasive candidiasis in Kuala Lumpur Hospital, Malaysia, were determined against amphotericin B, fluconazole, voriconazole, itraconazole and caspofungin. The most common species were Candida albicans (71 isolates), Candida parapsilosis (42 isolates), Candida tropicalis (27 isolates) and Candida glabrata (12 isolates). The susceptibility tests were carried out using an E-test. The MIC breakpoints were based on Clinical Laboratory Standards Institute criteria. Amphotericin B and voriconazole showed the best activities against all the isolates tested, with MIC90 values of ≤1 µg ml−1 for all major species. Only one Candida lusitaniae isolate was resistant to amphotericin B, and all the isolates were susceptible to voriconazole. In total, six isolates were resistant to fluconazole, comprising two isolates of C. albicans, two of C. parapsilosis, one C. tropicalis and one C. glabrata, and all of these isolates showed cross-resistance to itraconazole. The MIC90 of itraconazole was highest for C. glabrata and C. parapsilosis. Caspofungin was active against most of the isolates except for five isolates of C. parapsilosis. The MIC90 of caspofungin against C. parapsilosis was 3 µg ml−1. In conclusion, amphotericin B remains the most active antifungal agent against most Candida species except for C. lusitaniae. Voriconazole is the best alternative for fluconazole- or itraconizole-resistant isolates. Although five of the C. parapsilosis isolates showed in vitro resistance to caspofungin, more clinical correlation studies need to be carried out to confirm the significance of these findings. Currently, despite the increase in usage of antifungals in our hospitals, especially in the management of febrile neutropenia patients, the antifungal-resistance problem among clinically important Candida isolates in Kuala Lumpur Hospital is not yet worrying. However, continued antifungal-susceptibility surveillance needs to be conducted to monitor the antifungal-susceptibility trends of Candida species and other opportunistic fungal pathogens.

The earliest step in cell division in bacteria is the assembly of FtsZ, an essential cell division protein, into a ring at the division site. FtsZ has GTPase activity and can assemble in vitro to form protein filaments. The present work involved the study of eight phenylpropanoids (cinnamic, p-coumaric, caffeic, chlorogenic, ferulic, 3,4-dimethoxycinnamic and 2,4,5-trimethoxycinnamic acids and eugenol) as inhibitors of Escherichia coli FtsZ. Phenylpropanoids make up the majority of our diet and act as antibacterial agents. Polymerization and GTPase inhibition assays showed that chlorogenic and caffeic acids were the most active amongst these (IC50 of 70 and 106 µM, respectively). Circular dichroism studies indicated that chlorogenic acid perturbed the protein conformation and electron microscopy showed distorted filaments. Bacillus subtilis 168 cells treated with the phenylpropanoids were longer when compared to the control. The highest binding energy was observed between chlorogenic acid and the homology modelled E. coli FtsZ, which was consistent with the experimental results. A strong negative correlation was observed between binding energy and inhibition of the polymerization ability. 3D-Quantitative structure–activity relationship studies using GTPase activity indicated that the presence of more hydrophilic groups around the 3′- and 4′-carbon increased the activity. The effect of stress-induced formation of cell filamentation has to be understood before confirming the role of phenylpropanoids as FtsZ inhibitors.

This study was undertaken to investigate the synergistic interaction between amphotericin B (AmB) and acteoside, isolated from the aerial parts of the shrub Colebrookea oppositifolia (Lamiaceae). Acteoside alone exhibited no intrinsic antifungal activity but showed a potent synergism in combination with AmB against selected pathogenic species, with fractional inhibitory concentration indices in the range of 0.0312–0.1562. The combination of acteoside at 3.12 and 12.5 µg ml–1 with subinhibitory concentrations of AmB resulted in a potent fungicidal effect and also exhibited a significantly extended post-antifungal effect. Furthermore, the combination also reduced the minimum biofilm reduction concentration values of AmB (2–16-fold) in preformed biofilms of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. There was decreased viability of the cells, increased uptake of propidium iodide and enhanced leakage of 260 nm-absorbing material by Candida albicans cells when exposed to AmB in the presence of acteoside. The reason for potentiation is likely to be that the subinhibitory concentrations of AmB facilitated the uptake of acteoside, which resulted in increased killing of the fungal cells. Administration of acteoside in mice at up to 2000 mg (kg body weight)−1 by the intraperitoneal or oral route produced no overt toxicity. The data presented here support synergism between acteoside and AmB, and it is therefore proposed that a prospective new management strategy for therapeutic application of this combination should be explored.

Surgical site infections are the second most common hospital- and community-acquired Gram-positive infections, with the US Centers for Disease Control and Prevention estimating that about 500 000 surgical site infections occur annually in the USA. The aim of this work was to determine the in vitro activity of the saponin diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside hydrochloride (HSM1) and its bactericidal effect for a large number of Gram-positive cocci, as well as to investigate its in vitro interaction with seven clinically used antibiotics. In vivo, a wound model was established through the panniculus carnosus of BALB/c mice and then inoculated with 5×107 c.f.u. Staphylococcus aureus or Enterococcus faecalis. For each bacterial strain, the study included an infected or non-infected group that did not receive any treatment, a group treated with local HSM1, a group treated with intraperitoneal vancomycin, a group treated with intraperitoneal daptomycin and two groups that received HSM1 local treatment plus intraperitoneal vancomycin or daptomycin. All isolates were inhibited by HSM1 at concentrations of 2–32 mg l−1. Synergy was demonstrated when HSM1 was combined with vancomycin and daptomycin. In in vivo studies, all groups treated with single drugs showed a statistically significant result compared with the control group. The two groups treated with drug combinations showed the highest antimicrobial efficacy. The good in vitro activities and the in vivo efficacy suggest HSM1 as a promising therapeutic candidate in Gram-positive wound infections.

Of 803 community Escherichia coli (n = 767) and Klebsiella pneumoniae (n = 36) isolates collected from patients with urinary tract infections in three Moroccan cities, 10 E. coli (1.3%) and 2 K. pneumoniae (5.6 %) isolates were shown to produce extended-spectrum β-lactamases (ESBLs). PFGE revealed that the E. coli isolates comprised seven distinct genotypes. The presence of plasmids in the 12 isolates was revealed by conjugation experiments of plasmids from these Enterobacteriaceae strains with E. coli K12J5, with further isolation of the plasmids in the transconjugants. Subsequent nucleotide sequencing indicated that the plasmids encoded the bla CTX-M, bla OXA, bla TEM and bla SHV genes, including genes for CTX-M-15 (n = 11), OXA-1 (n = 11), TEM-1b (n = 4), SHV-5 (n = 1) and SHV-1 (n = 2). Identification of plasmid-mediated quinolone-resistance genes was performed by PCR. The aac(6′)Ib-cr variant was detected in all strains, and two strains co-expressed qnrS1, bla CTX-M-15 and bla OXA-1 genes. The presence of ESBLs in the Enterobacteriaceae strains studied was probably due to the dissemination of resistance plasmids with the predominant genotype of bla CTX-M-15.

In 2006, an outbreak of aseptic meningitis was noted in Taiwan. From January to October 2006, a total of 3283 specimens collected from patients with viral infection, including 173 cerebrospinal fluid (CSF) samples, were examined for virus isolation and identification. Overall, 339 enterovirus (EV)-positive cases were identified by virus culture: echovirus 18 (E18) formed the majority (27.4 %, 93 cases), followed by coxsackievirus B2 (13.8 %, 47 cases) and coxsackievirus A2 (10.8 %, 37 cases). The manifestations of the 93 E18 cases were aseptic meningitis (44.1 %), viral exanthema (23.6 %), acute tonsillitis (15.1 %), acute pharyngitis (14.0 %), acute gastritis (11.8 %), herpangina (7.5 %) and bronchopneumonia (5.3 %). Of 107 E18 isolates identified, 100, 62.5 and 19 % were obtained following culture in RD, MRC-5 and A549 cells, respectively. E18 was identified most frequently from throat swabs (67.2 %) and less frequently from stool samples (15.9 %) and CSF (16.8 %). The detection rate of E18 was 78.2 % from CSF, 50 % from stool samples and 22.9 % from throat swabs. Phylogenetic relationships among the E18 strains were examined. Analysis of the partial VP1 gene showed 3.7–23.8 % variation in sequence compared with sequences from GenBank and, notably, the amino acid change V152S was detected in a protruding loop within the VP1 protein. These results indicate that a genetic variant of E18 was circulating and caused an outbreak of aseptic meningitis in Taiwan in 2006.