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1.  Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity 
BMC Microbiology  2014;14:174.
Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified.
In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis.
We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis.
PMCID: PMC4087242  PMID: 24975018
Mycobacterium tuberculosis; Cell wall; Rv1096; Peptidoglycan deacetylase; Lysozyme
2.  Perturbation of copper homeostasis is instrumental in early developmental arrest of intraerythrocytic Plasmodium falciparum 
BMC Microbiology  2014;14:167.
Malaria continues to be a devastating disease. The elucidation of factors inducing asexual growth versus arrest of Plasmodium falciparum can provide information about the development of the parasite, and may help in the search for novel malaria medication. Based on information from genome-wide transcriptome profiling of different developmental stages of P. falciparum, we investigated the critical importance of copper homeostasis in the developmental succession of P. falciparum with regard to three aspects of copper function. These were:1) inhibition of copper-binding proteins, 2) copper-ion chelation, and 3) down-regulated expression of genes encoding copper-binding proteins associated with a specific growth-promoting factor.
Inhibition of copper-binding proteins with tetrathiomolybdate (TTM) caused cessation of growth of the parasite. TTM arrested the parasite irreversibly during the trophozoite to schizont stage progression. Target molecules for TTM may be present in P. falciparum. The involvement of copper ions in developmental arrest was also investigated by copper-ion chelating methods, which indicated a critical function of reduced copper ions (Cu1+) in the parasite during the early developmental stage. Copper ions, not only in the parasite but also in host cells, were targets of the chelators. Chelation of Cu1+caused blockage of trophozoite progression from the ring stage. Profound growth arrest was detected in parasites cultured in a chemically defined medium containing hexadecanoic acid alone as a growth-promoting factor. This developmental arrest was associated with down-regulated expression of genes encoding copper-binding proteins. Cis-9-octadecenoic acid completely prevented the down-regulation of gene expression and developmental arrest that were observed with the use of hexadecanoic acid.
The critical importance of copper homeostasis in early developmental stages of P. falciparum was confirmed. Perturbation of copper homeostasis induced profound and early developmental arrest of P. falciparum. These findings should help to elucidate the mechanisms behind the development of P. falciparum, and may be applied in the development of effective antimalarial strategies.
PMCID: PMC4080775  PMID: 24961242
Plasmodium falciparum; Intraerythrocytic growth; Copper homeostasis; Copper-binding protein; Copper ion; Developmental arrest
3.  Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection 
BMC Microbiology  2014;14:166.
Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies.
We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively.
Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization.
PMCID: PMC4081464  PMID: 24961160
H. ducreyi; GUD; Chancroid; Lipoprotein; Human
4.  Functional genomics to identify the factors contributing to successful persistence and global spread of an antibiotic resistance plasmid 
BMC Microbiology  2014;14:168.
The spread of bacterial plasmids is an increasing global problem contributing to the widespread dissemination of antibiotic resistance genes including β-lactamases. Our understanding of the details of the biological mechanisms by which these natural plasmids are able to persist in bacterial populations and are able to establish themselves in new hosts via conjugative transfer is very poor. We recently identified and sequenced a globally successful plasmid, pCT, conferring β-lactam resistance.
Here, we investigated six plasmid encoded factors (tra and pil loci; rci shufflon recombinase, a putative sigma factor, a putative parB partitioning gene and a pndACB toxin-antitoxin system) hypothesised to contribute to the ‘evolutionary success’ of plasmid pCT. Using a functional genomics approach, the role of these loci was investigated by systematically inactivating each region and examining the impact on plasmid persistence, conjugation and bacterial host biology. While the tra locus was found to be essential for all pCT conjugative transfer, the second conjugation (pil) locus was found to increase conjugation frequencies in liquid media to particular bacterial host recipients (determined in part by the rci shufflon recombinase). Inactivation of the pCT pndACB system and parB did not reduce the stability of this plasmid.
Our findings suggest the success of pCT may be due to a combination of factors including plasmid stability within a range of bacterial hosts, a lack of a fitness burden and efficient transfer rates to new bacterial hosts rather than the presence of a particular gene or phenotype transferred to the host. The methodology used in our study could be applied to other ‘successful’ globally distributed plasmids to discover the role of currently unknown plasmid backbone genes or to investigate other factors which allow these elements to persist and spread.
PMCID: PMC4083329  PMID: 24961279
Beta-lactam; ESBL; Mobile genetic element; Plasmid; Recombination
5.  Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/XDR-TB strains isolated in Thailand 
BMC Microbiology  2014;14:165.
The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) makes the treatment and control of tuberculosis difficult. Rapid detection of drug-resistant strains is important for the successful treatment of drug-resistant tuberculosis; however, not all resistance mechanisms to the injectable second-line drugs such as amikacin (AK), kanamycin (KM), and capreomycin (CAP) are well understood. This study aims to validate the mechanisms associated with AK, KM, and CAP resistance in M. tuberculosis clinical strains isolated in Thailand.
A total of 15,124 M. tuberculosis clinical strains were isolated from 23,693 smear-positive sputum samples sent from 288 hospitals in 46 of 77 provinces of Thailand. Phenotypic analysis identified 1,294 strains as MDR-TB and second-line drugs susceptibility was performed in all MDR-TB strains and revealed 58 XDR-TB strains. Twenty-nine KM-resistant strains (26 XDR-TB and 3 MDR-TB) could be retrieved and their genes associated with AK, KM, and CAP resistance were investigated compared with 27 KM-susceptible strains. Mutation of the rrs (A1401G) was found in 21 out of 29 KM-resistant strains whereas mutations of eis either at C-14 T or at G-37 T were found in 5 strains. Three remaining KM-resistant strains did not contain any known mutations. Capreomycin resistance was determined in 28 of 29 KM-resistant strains. Analysis of tlyA revealed that the A33G mutation was found in all CAP-resistant strains and also in susceptible strains. In contrast, the recently identified tlyA mutation T539G and the novel Ins49GC were found in two and one CAP-resistant strains, respectively. In addition, our finding demonstrated the insertion of cytosine at position 581 of the tap, a putative drug efflux encoding gene, in both KM-resistant and KM-susceptible strains.
Our finding demonstrated that the majority of KM resistance mechanism in Thai M. tuberculosis clinical strains was rrs mutation at A1401G. Mutations of the eis promoter region either at C-14 T or G-37 T was found in 5 of 29 strains whereas three strains did not contain any known mutations. For CAP resistance, 3 of 28 CAP-resistant strains contained either T539G or Ins49GC mutations at tlyA that might be associated with the resistant phenotype.
PMCID: PMC4076439  PMID: 24953243
Tuberculosis; Second-line drug; Resistance; Aminoglycoside
6.  The ColRS signal transduction system responds to the excess of external zinc, iron, manganese, and cadmium 
BMC Microbiology  2014;14:162.
The ColRS two-component system has been shown to contribute to the membrane functionality and stress tolerance of Pseudomonas putida as well as to the virulence of Pseudomonas aeruginosa and plant pathogenic Xanthomonas species. However, the conditions activating the ColRS pathway and the signal(s) sensed by ColS have remained unknown. Here we aimed to analyze the role of the ColRS system in metal tolerance of P. putida and to test whether ColS can respond to metal excess.
We show that the ColRS system is necessary for P. putida to tolerate the excess of iron and zinc, and that it also contributes to manganese and cadmium tolerance. Excess of iron, zinc, manganese or cadmium activates ColRS signaling and as a result modifies the expression of ColR-regulated genes. Our data suggest that the genes in the ColR regulon are functionally redundant, as several loci have to be deleted to observe a significant decrease in metal tolerance. Site-directed mutagenesis of ColS revealed that excess of iron and, surprisingly, also zinc are sensed by a conserved ExxE motif in ColS’s periplasmic domain. While ColS is able to sense different metals, it still discriminates between the two oxidation states of iron, specifically responding to ferric and not ferrous iron. We propose a signal perception model involving a dimeric ColS, where each monomer donates one ExxE motif for metal binding.
Several transition metals are essential for living organisms in certain amounts, but toxic in excess. We show that ColRS is a sensor system which detects and responds to the excess of physiologically important metals such as zinc, iron and manganese. Thus, the ColRS system is an important factor for metal homeostasis and tolerance in P. putida.
PMCID: PMC4074579  PMID: 24946800
ColRS two-component system; Metal tolerance; Histidine kinase; Signal perception; Pseudomonas putida
7.  Bdellovibrio bacteriovorus HD100 guards against Pseudomonas tolaasii brown-blotch lesions on the surface of post-harvest Agaricus bisporus supermarket mushrooms 
BMC Microbiology  2014;14:163.
Pseudomonas tolaasii is a problematic pathogen of cultured mushrooms, forming dark brown ‘blotches’ on mushroom surfaces and causing spoilage during crop growth and post-harvest . Treating P. tolaasii infection is difficult, as other, commensal bacterial species such as Pseudomonas putida are necessary for mushroom growth, so treatments must be relatively specific.
We have found that P. tolaasii is susceptible to predation in vitro by the δ-proteobacterium Bdellovibrio bacteriovorus. This effect also occurred in funga, where B. bacteriovorus was administered to post-harvest mushroom caps before and after administration of the P. tolaasii pathogen. A significant, visible improvement in blotch appearance, after incubation, was observed on administration of Bdellovibrio. A significant reduction in viable P. tolaasii cell numbers, recovered from the mushroom tissue, was detected. This was accompanied by a more marked reduction in blotch severity on Bdellovibrio administration. We found that there was in some cases an accompanying overgrowth of presumed-commensal, non-Pseudomonas bacteria on post-harvest mushroom caps after Bdellovibrio-treatment. These bacteria were identified (by 16SrRNA gene sequencing) as Enterobacter species, which were seemingly resistant to predation. We visualised predatory interactions occuring between B. bacteriovorus and P. tolaasii on the post-harvest mushroom cap surface by Scanning Electron Microscopy, seeing predatory invasion of P. tolaasii by B. bacteriovorus in funga. This anti-P. tolaasii effect worked well in post-harvest supermarket mushrooms, thus Bdellovibrio was not affected by any pre-treatment of mushrooms for commercial/consumer purposes.
The soil-dwelling B. bacteriovorus HD100 preys upon and kills P. tolaasii, on mushroom surfaces, and could therefore be applied to prevent spoilage in post-harvest situations where mushrooms are stored and packaged for sale.
PMCID: PMC4077555  PMID: 24946855
8.  Population typing of the causal agent of cassava bacterial blight in the Eastern Plains of Colombia using two types of molecular markers 
BMC Microbiology  2014;14:161.
Molecular typing of pathogen populations is an important tool for the development of effective strategies for disease control. Diverse molecular markers have been used to characterize populations of Xanthomonas axonopodis pv. manihotis (Xam), the main bacterial pathogen of cassava. Recently, diversity and population dynamics of Xam in the Colombian Caribbean coast were estimated using AFLPs, where populations were found to be dynamic, diverse and with haplotypes unstable across time. Aiming to examine the current state of pathogen populations located in the Colombian Eastern Plains, we also used AFLP markers and we evaluated the usefulness of Variable Number Tandem Repeats (VNTRs) as new molecular markers for the study of Xam populations.
The population analyses showed that AFLP and VNTR provide a detailed and congruent description of Xam populations from the Colombian Eastern Plains. These two typing strategies clearly separated strains from the Colombian Eastern Plains into distinct populations probably because of geographical distance. Although the majority of analyses were congruent between typing markers, fewer VNTRs were needed to detect a higher number of genetic populations of the pathogen as well as a higher genetic flow among sampled locations than those detected by AFLPs.
This study shows the advantages of VNTRs over AFLPs in the surveillance of pathogen populations and suggests the implementation of VNTRs in studies that involve large numbers of Xam isolates in order to obtain a more detailed overview of the pathogen to improve the strategies for disease control.
PMCID: PMC4071214  PMID: 24946775
Xanthomonas axonopodis pv. manihotis; Population dynamics; Molecular markers; Plant pathology
9.  The Pseudomonas aeruginosa rhlG and rhlAB genes are inversely regulated and RhlG is not required for rhamnolipid synthesis 
BMC Microbiology  2014;14:160.
Pseudomonas aeruginosa produces rhamnolipid biosurfactants involved in numerous phenomena including virulence. The transcriptional study of the rhlAB operon encoding two key enzymes for rhamnolipid synthesis led to the discovery of the quorum sensing system RhlRI. The latter positively controls the transcription of rhlAB, as well as of rhlC, which is required for di-rhamnolipid synthesis. The rhlG gene encodes an NADPH-dependent β-ketoacyl reductase. Although it was reported to be required for the biosynthesis of the fatty acid part of rhamnolipids, its function in rhamnolipid synthesis was later questioned. The rhlG transcription and its role in rhamnolipid production were investigated here.
Using 5′-RACE PCR, a luxCDABE-based transcriptional fusion, and quantitative reverse transcription-PCR, we confirmed two previously identified σ70- and σ54-dependent promoters and we identified a third promoter recognized by the extra-cytoplasmic function sigma factor AlgU. rhlG was inversely regulated compared to rhlAB and rhlC: the rhlG transcription was down-regulated in response to N-butyryl-l-homoserine lactone, the communication molecule of the RhlRI system, and was induced by hyperosmotic stress in an AlgU-dependent manner. Consistently with this transcriptional pattern, the single or double deletions of rhlG and PA3388, which forms an operon with rhlG, did not dramatically impair rhamnolipid synthesis.
This first detailed study of rhlG transcription reveals a complex regulation involving three sigma factors and N-butyryl-l-homoserine lactone. We furthermore present evidences that RhlG does not play a key role in rhamnolipid synthesis.
PMCID: PMC4074388  PMID: 24943492
RhlG; Rhamnolipid; Pseudomonas aeruginosa; AlgU
10.  Pilus distribution among lineages of group b streptococcus: an evolutionary and clinical perspective 
BMC Microbiology  2014;14:159.
Group B Streptococcus (GBS) is an opportunistic pathogen in both humans and bovines. Epidemiological and phylogenetic analyses have found strains belonging to certain phylogenetic lineages to be more frequently associated with invasive newborn disease, asymptomatic maternal colonization, and subclinical bovine mastitis. Pilus structures in GBS facilitate colonization and invasion of host tissues and play a role in biofilm formation, though few large-scale studies have estimated the frequency and diversity of the three pilus islands (PIs) across diverse genotypes. Here, we examined the distribution of pilus islands (PI) 1, 2a and 2b among 295 GBS strains representing 73 multilocus sequence types (STs) belonging to eight clonal complexes. PCR-based RFLP was also used to evaluate variation in the genes encoding pilus backbone proteins of PI-2a and PI-2b.
All 295 strains harbored one of the PI-2 variants and most human-derived strains contained PI-1. Bovine-derived strains lacked PI-1 and possessed a unique PI-2b backbone protein allele. Neonatal strains more frequently had PI-1 and a PI-2 variant than maternal colonizing strains, and most CC-17 strains had PI-1 and PI-2b with a distinct backbone protein allele. Furthermore, we present evidence for the frequent gain and loss of genes encoding certain pilus types.
These data suggest that pilus combinations impact host specificity and disease presentation and that diversification often involves the loss or acquisition of PIs. Such findings have implications for the development of GBS vaccines that target the three pilus islands.
PMCID: PMC4074840  PMID: 24943359
Streptococcus agalactiae; Pilus; MLST; Molecular epidemiology
11.  OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay 
BMC Microbiology  2014;14:158.
Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks.
The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay.
The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.
PMCID: PMC4078931  PMID: 24943244
12.  Role IL-17A and IL-10 in the antigen induced inflammation model by Mycoplasma pneumoniae 
BMC Microbiology  2014;14:156.
Mycoplasma pneumoniae is one of the causative organisms of community-acquired pneumonia which is found commonly in younger patients. Extrapulmonary complications similar to autoimmune disease are caused by M. pneumoniae following the initial infection. The mechanism and pathology of onset is not clear, but it is considered that excessive host immunoreactions play a part in the onset of mycoplasmal pneumonia and its extrapulmonary complications. In this study, we investigated the participation of the immune response, excluding the participation of Th1 and Th2 which has previously been investigated.
In this study, the host immune response of an antigen induced inflammation model using SPF mice repeatedly sensitized with M. pneumoniae antigens was analyzed. The specificity of M. pneumoniae antigens in the Th17 response of murine lymphocytes in vitro was also examined. Frequent and concentrated sensitization induced exacerbation of lung inflammation immunologically and pathologically, and evoked intrapulmonary IL-17A and IL-10 production. M. pneumoniae antigen stimulation induced proliferation of mouse lymphocytes and caused production of IL-17A and IL-10. In addition, it was shown that IL-17A and IL-10 production was increased in the presence of IL-6 and TGF-β1.
It was shown that M. pneumoniae antigens induced potent immunoreaction and enhanced the Th17 cell response both in vivo and in vitro, and that both Treg and IL-10 are involved in the suppression of IL-17A production. This raises the possibility that breakdown of the immune balance may be part of the process leading to subsequent development of extrapulmonary mycoplasmal pneumonia.
PMCID: PMC4074139  PMID: 24928272
Mycoplasma pneumoniae; Th17; Animal models; Immune response; Cytokines
13.  Development of cross-resistance by Aspergillus fumigatus to clinical azoles following exposure to prochloraz, an agricultural azole 
BMC Microbiology  2014;14:155.
The purpose of this study was to unveil whether azole antifungals used in agriculture, similar to the clinical azoles used in humans, can evoke resistance among relevant human pathogens like Aspergillus fumigatus, an ubiquitous agent in nature. Additionally, cross-resistance with clinical azoles was investigated. Antifungal susceptibility testing of environmental and clinical isolates of A. fumigatus was performed according to the CLSI M38-A2 protocol. In vitro induction assays were conducted involving daily incubation of susceptible A. fumigatus isolates, at 35°C and 180 rpm, in fresh GYEP broth medium supplemented with Prochloraz (PCZ), a potent agricultural antifungal, for a period of 30 days. Minimal inhibitory concentrations (MIC) of PCZ and clinical azoles were monitored every ten days. In order to assess the stability of the developed MIC, the strains were afterwards sub-cultured for an additional 30 days in the absence of antifungal. Along the in vitro induction process, microscopic and macroscopic cultural observations were registered.
MIC of PCZ increased 256 times after the initial exposure; cross-resistance to all tested clinical azoles was observed. The new MIC value of agricultural and of clinical azoles maintained stable in the absence of the selective PCZ pressure. PCZ exposure was also associated to morphological colony changes: macroscopically the colonies became mostly white, losing the typical pigmentation; microscopic examination revealed the absence of conidiation.
PCZ exposure induced Aspergillus fumigatus morphological changes and an evident increase of MIC value to PCZ as well as the development of cross-resistance with posaconazole, itraconazole and voriconazole.
PMCID: PMC4061453  PMID: 24920078
Aspergillus fumigatus; Cross-resistance; Clinical and agricultural azoles
14.  The oxygen sensor MgFnr controls magnetite biomineralization by regulation of denitrification in Magnetospirillum gryphiswaldense 
BMC Microbiology  2014;14:153.
Magnetotactic bacteria are capable of synthesizing magnetosomes only under oxygen-limited conditions. However, the mechanism of the aerobic repression on magnetite biomineralization has remained unknown. In Escherichia coli and other bacteria, Fnr (fumarate and nitrate reduction regulator) proteins are known to be involved in controlling the switch between microaerobic and aerobic metabolism. Here, we report on an Fnr-like protein (MgFnr) and its role in growth metabolism and magnetite biomineralization in the alphaproteobacterium Magnetospirillum gryphiswaldense.
Deletion of Mgfnr not only resulted in decreased N2 production due to reduced N2O reductase activity, but also impaired magnetite biomineralization under microaerobic conditions in the presence of nitrate. Overexpression of MgFnr in the WT also caused the synthesis of smaller magnetite particles under anaerobic and microaerobic conditions in the presence of nitrate. These data suggest that proper expression of MgFnr is required for WT-like magnetosome synthesis, which is regulated by oxygen. Analyses of transcriptional gusA reporter fusions revealed that besides showing similar properties to Fnr proteins reported in other bacteria, MgFnr is involved in the repression of the expression of denitrification genes nor and nosZ under aerobic conditions, possibly owing to several unique amino acid residues specific to MTB-Fnr.
We have identified and thoroughly characterized the first regulatory protein mediating denitrification growth and magnetite biomineralization in response to different oxygen conditions in a magnetotactic bacterium. Our findings reveal that the global oxygen regulator MgFnr is a genuine O2 sensor. It is involved in controlling expression of denitrification genes and thereby plays an indirect role in maintaining proper redox conditions required for magnetite biomineralization.
PMCID: PMC4065386  PMID: 24915802
Magnetospirillum gryphiswaldense; Magnetite biomineralization; Oxygen regulation; Denitrification; Oxygen sensor Fnr
15.  Cell death in amastigote forms of Leishmania amazonensis induced by parthenolide 
BMC Microbiology  2014;14:152.
Leishmania amazonensis infection results in diverse clinical manifestations: cutaneous, mucocutaneous or visceral leishmaniasis. The arsenal of drugs available for treating Leishmania infections is limited. Therefore, new, effective, and less toxic leishmaniasis treatments are still needed. We verified cell death in amastigote forms of Leishmania amazonensis induced by the sesquiterpene lactone parthenolide.
The tested compound was able to concentration-dependently affect axenic and intracellular amastigotes, with IC50 values of 1.3 μM and 2.9 μM, respectively after 72 h incubation. No genotoxic effects were observed in a micronucleus test in mice. Parthenolide induced morphological and ultrastructural changes in axenic amastigotes, including a loss of membrane integrity, swelling of the mitochondrion, cytoplasmic vacuoles, and intense exocytic activity in the region of the flagellar pocket. These results led us to investigate the occurrence of autophagic vacuoles with monodansylcadaverine and the integrity of the plasma membrane and mitochondrial membrane potential using flow cytometry. In all of the tests, parthenolide had positive results.
Our results indicate that the antileishmanial action of parthenolide is associated with autophagic vacuole appearance, a reduction of fluidity, a loss of membrane integrity, and mitochondrial dysfunction. Considering the limited repertoire of existing antileishmanial compounds, the products derived from medicinal plants has been one the greatest advances to help develop new chemotherapeutic approaches.
PMCID: PMC4067685  PMID: 24913205
Leishmania amazonensis; Amastigotes; Parthenolide; Cell death; Autophagy
16.  High prevalence of Cfr-producing Staphylococcus species in retail meat in Guangzhou, China 
BMC Microbiology  2014;14:151.
The emergence and wide distribution of the transferable gene for linezolid resistance, cfr, in staphylococci of human and animal origins is of great concern as it poses a serious threat to the public health. In the present study, we investigated the emergence and presence of the multiresistance gene, cfr, in retail meat sourced from supermarkets and free markets of Guangzhou, China.
A total of 118 pork and chicken samples, collected from Guangzhou markets, were screened by PCR for cfr. Twenty-two Staphylococcus isolates obtained from 12 pork and 10 chicken samples harbored cfr. The 22 cfr-positive staphylococci isolates, including Staphylococcus equorum (n = 8), Staphylococcus simulans (n = 7), Staphylococcus cohnii (n = 4), and Staphylococcus sciuri (n = 3), exhibited 17 major SmaI pulsed-field gel electrophoresis (PFGE) patterns. In 14 isolates, cfr was located on the plasmids. Sequence analysis revealed that the genetic structures (including ΔtnpA of Tn558, IS21-558, ΔtnpB, and tnpC of Tn558, orf138, fexA) of cfr in plasmid pHNTLD18 of a S. sciuri strain and in the plasmid pHNLKJC2 (including rep, Δpre/mob, cfr, pre/mob and partial ermC) of a S. equorum strain were identical or similar to the corresponding regions of some plasmids in staphylococcal species of animal and human origins.
To the best of our knowledge, this is the first study to report the presence of the multiresistance gene, cfr, in animal meat. A high occurrence of cfr was observed in the tested retail meat samples. Thus, it is important to monitor the presence of cfr in animal foods in China.
PMCID: PMC4059476  PMID: 24913069
Plasmids; Linezolid; Staphylococcus spp; Food safety; Resistance epidemiology
17.  A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia 
BMC Microbiology  2014;14:150.
Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China.
Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates.
Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.
PMCID: PMC4063691  PMID: 24912963
18.  The genetic diversity of cereulide biosynthesis gene cluster indicates a composite transposon Tnces in emetic Bacillus weihenstephanensis 
BMC Microbiology  2014;14:149.
Cereulide is a cyclic dodecadepsipeptide ionophore, produced via non-ribosomal peptide synthetases (NRPS), which in rare cases can lead to human death. Early studies had shown that emetic toxin formation belongs to a homogeneous group of Bacillus cereus sensu stricto and the genetic determinants of cereulide (a 24-kb gene cluster of cesHPTABCD) are located on a 270-kb plasmid related to the Bacillus anthracis virulence plasmid pXO1.
The whole genome sequences from seven emetic isolates, including two B. cereus sensu stricto and five Bacillus weihenstephanensis strains, were compared, and their inside and adjacent DNA sequences of the cereulide biosynthesis gene clusters were analyzed. The sequence diversity was observed, which classified the seven emetic isolates into three clades. Different genomic locations of the cereulide biosynthesis gene clusters, plasmid-borne and chromosome-borne, were also found. Potential mobile genetic elements (MGEs) were identified in the flanking sequences of the ces gene cluster in all three types. The most striking observation was the identification of a putative composite transposon, Tnces, consisting of two copies of ISces element (belonging to IS6 family) in opposite orientations flanking the ces gene cluster in emetic B. weihenstephanensis. The mobility of this element was tested by replacing the ces gene cluster by a KmR gene marker and performing mating-out transposition assays in Escherichia coli. The results showed that Tnces::km transposes efficiently (1.04 × 10-3 T/R) and produces 8-bp direct repeat (DR) at the insertion sites.
Cereulide biosynthesis gene clusters display sequence diversity, different genomic locations and association with MGEs, in which the transposition capacity of a resistant derivative of the composite transposon Tnces in E. coli was demonstrated. Further study is needed to look for appropriate genetic tools to analysis the transposition of Tnces in Bacillus spp. and the dynamics of other MGEs flanking the ces gene clusters.
PMCID: PMC4057527  PMID: 24906385
Cereulide; Bacillus cereus; Bacillus weihenstephanensis; Transposon; Plasmid
19.  Activation of JNK1/2 and p38 MAPK signaling pathways promotes enterovirus 71 infection in immature dendritic cells 
BMC Microbiology  2014;14:147.
c-Jun NH2-terminal kinase/stress-activated kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase (p38 MAPK) are important components of cellular signal transduction pathways, which have been reported to be involved in viral replication. However, little is known about JNK1/2 and p38 MAPK signaling pathways in enterovirus 71 (EV71)-infected immature dendritic cells (iDCs). Thus, iDCs were induced from peripheral blood mononuclear cells (PBMC) and performed to explore the expressions and phosphorylation of molecules in the two signaling pathways as well as secretions of inflammatory cytokines and interferons during EV71 replication.
We showed that EV71 infection could activate both JNK1/2 and p38 MAPK in iDCs and phosphorylate their downstream transcription factors c-Fos and c-Jun, which further promoted the production of IL-2, IL-6, IL-10, and TNF-α. Moreover, EV71 infection also increased the release of IFN-β and IL-12 p40. Pretreatment of iDCs with SP600125 and SB203580 (20 μM) could severely impair viral replication and its induced phosphorylation of JNK1/2,p38 MAPK, c-Fos and c-Jun. In addition, treatment of EV71-infected iDCs with SP600125 and SB203580 could inhibit secretions of IL-6, IL-10 and TNF-α.
JNK1/2 and p38 MAPK signaling pathways are beneficial to EV71 infection and positively regulate secretions of inflammatory cytokines in iDCs.
PMCID: PMC4057572  PMID: 24906853
Enterovirus 71; Viral replication; JNK1/2; p38 MAPK; Dendritic cells
20.  Identification of a New Delhi metallo-β-lactamase-4 (NDM-4)-producing Escherichia coli in Italy 
BMC Microbiology  2014;14:148.
During June-July 2012, six imipenem-resistant Escherichia coli isolates were isolated from two patients hospitalized in a ward of one large tertiary-care hospital in Genoa, Italy. Genetic features associated with blaNDM-4 gene were investigated.
The isolates exhibited the same PFGE profile and a multidrug-resistant (MDR) phenotype to aminoglycosides, fluoroquinolones, and β-lactams. The strains produced the NDM-4 carbapenemase and the blaNDM-4 gene was part of the variable region of a class 1 integron. MLST analysis revealed that all isolates belonged to sequence type 405 (ST405).
This is the first report on the emergence of an MDR strain of E.coli producing the NDM-4 MBL in Italy.
PMCID: PMC4060580  PMID: 24906230
Carbapenemases; E.coli; ST405; Class 1 integron
21.  Conservation of streptococcal CRISPRs on human skin and saliva 
BMC Microbiology  2014;14:146.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are utilized by bacteria to resist encounters with their viruses. Human body surfaces have numerous bacteria that harbor CRISPRs, and their content can provide clues as to the types and features of viruses they may have encountered.
We investigated the conservation of CRISPR content from streptococci on skin and saliva of human subjects over 8-weeks to determine whether similarities existed in the CRISPR spacer profiles and whether CRISPR spacers were a stable component of each biogeographic site. Most of the CRISPR sequences identified were unique, but a small proportion of spacers from the skin and saliva of each subject matched spacers derived from previously sequenced loci of S. thermophilus and other streptococci. There were significant proportions of CRISPR spacers conserved over the entire 8-week study period for all subjects, and salivary CRISPR spacers sampled in the mornings showed significantly higher levels of conservation than any other time of day. We also found substantial similarities in the spacer repertoires of the skin and saliva of each subject. Many skin-derived spacers matched salivary viruses, supporting that bacteria of the skin may encounter viruses with similar sequences to those found in the mouth. Despite the similarities between skin and salivary spacer repertoires, the variation present was distinct based on each subject and body site.
The conservation of CRISPR spacers in the saliva and the skin of human subjects over the time period studied suggests a relative conservation of the bacteria harboring them.
PMCID: PMC4063239  PMID: 24903519
CRISPR; Skin microbiome; Saliva microbiome; Virome; Virus
22.  Zinc protects against shiga-toxigenic Escherichia coli by acting on host tissues as well as on bacteria 
BMC Microbiology  2014;14:145.
Zinc supplements can treat or prevent enteric infections and diarrheal disease. Many articles on zinc in bacteria, however, highlight the essential nature of this metal for bacterial growth and virulence, suggesting that zinc should make infections worse, not better. To address this paradox, we tested whether zinc might have protective effects on intestinal epithelium as well as on the pathogen.
Using polarized monolayers of T84 cells we found that zinc protected against damage induced by hydrogen peroxide, as measured by trans-epithelial electrical resistance. Zinc also reduced peroxide-induced translocation of Shiga toxin (Stx) across T84 monolayers from the apical to basolateral side. Zinc was superior to other divalent metals to (iron, manganese, and nickel) in protecting against peroxide-induced epithelial damage, while copper also showed a protective effect.
The SOS bacterial stress response pathway is a powerful regulator of Stx production in STEC. We examined whether zinc’s known inhibitory effects on Stx might be mediated by blocking the SOS response. Zinc reduced expression of recA, a reliable marker of the SOS. Zinc was more potent and more efficacious than other metals tested in inhibiting recA expression induced by hydrogen peroxide, xanthine oxidase, or the antibiotic ciprofloxacin. The close correlation between zinc’s effects on recA/SOS and on Stx suggested that inhibition of the SOS response is one mechanism by which zinc protects against STEC infection.
Zinc’s ability to protect against enteric bacterial pathogens may be the result of its combined effects on host tissues as well as inhibition of virulence in some pathogens. Research focused solely on the effects of zinc on pathogenic microbes may give an incomplete picture by failing to account for protective effects of zinc on host epithelia.
PMCID: PMC4072484  PMID: 24903402
Enterohemorrhagic E. coli; O157:H7; Hemolytic-uremic syndrome; SOS response; Diarrheal diseases; Xanthine oxidase; Manganese; Copper
23.  A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood 
BMC Microbiology  2014;14:144.
The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR .
Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 101 CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi.
Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours.
PMCID: PMC4049433  PMID: 24893651
Nested PCR; Multiplex PCR; Sepsis; Bacteria; Fungi; Gram differentiation
24.  Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers 
BMC Microbiology  2014;14:143.
Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate’s sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles.
The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years.
The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.
PMCID: PMC4057806  PMID: 24889110
Multi-locus sequence typing; MLST; Invasive pneumococcal disease; Molecular epidemiology; Streptococcus pneumoniae; Bacterial typing
25.  Genetic basis for denitrification in Ensifer meliloti 
BMC Microbiology  2014;14:142.
Denitrification is defined as the dissimilatory reduction of nitrate or nitrite to nitric oxide (NO), nitrous oxide (N2O), or dinitrogen gas (N2). N2O is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Legume crops might contribute to N2O production by providing nitrogen-rich residues for decomposition or by associating with rhizobia that are able to denitrify under free-living and symbiotic conditions. However, there are limited direct empirical data concerning N2O production by endosymbiotic bacteria associated with legume crops. Analysis of the Ensifer meliloti 1021 genome sequence revealed the presence of the napEFDABC, nirK, norECBQD and nosRZDFYLX denitrification genes. It was recently reported that this bacterium is able to grow using nitrate respiration when cells are incubated with an initial O2 concentration of 2%; however, these cells were unable to use nitrate respiration when initially incubated anoxically. The involvement of the nap, nirK, nor and nos genes in E. meliloti denitrification has not been reported.
E. meliloti nap, nirK and norC mutant strains exhibited defects in their ability to grow using nitrate as a respiratory substrate. However, E meliloti nosZ was not essential for growth under these conditions. The E. meliloti napA, nirK, norC and nosZ genes encode corresponding nitrate, nitrite, nitric oxide and nitrous oxide reductases, respectively. The NorC component of the E. meliloti nitric oxide reductase has been identified as a c-type cytochrome that is 16 kDa in size. Herein, we also show that maximal expression of the E. meliloti napA, nirK, norC and nosZ genes occurred when cells were initially incubated anoxically with nitrate.
The E. meliloti napA, nirK, norC and nosZ genes are involved in nitrate respiration and in the expression of denitrification enzymes in this bacterium. Our findings expand the short list of rhizobia for which denitrification gene function has been demonstrated. The inability of E. meliloti to grow when cells are initially subjected to anoxic conditions is not attributable to defects in the expression of the napA, nirK, norC and nosZ denitrification genes.
PMCID: PMC4064527  PMID: 24888981
Cu-containing nitrite reductase; Nitrate respiration; Nitric oxide reductase; Nitrous oxide reductase; Periplasmic nitrate reductase

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