Genotypic and Phenotypic Diversity within Species of Purple Nonsulfur Bacteria Isolated from Aquatic Sediments

To assess the extent of genotypic and phenotypic diversity within species of purple nonsulfur bacteria found in aquatic sediments, a total of 128 strains were directly isolated from agar plates that had been inoculated with sediment samples from Haren and De Biesbosch in The Netherlands. All isolates were initially characterized by BOX-PCR genomic DNA fingerprinting, and 60 distinct genotypes were identified. Analyses of 16S rRNA gene sequences of representatives of each genotype showed that five and eight different phylotypes of purple nonsulfur bacteria were obtained from the Haren and De Biesbosch sites, respectively. At the Haren site, 80.5% of the clones were Rhodopseudomonas palustris, whereas Rhodoferax fermentans and Rhodopseudomonas palustris were numerically dominant at the De Biesbosch site and constituted 45.9 and 34.4% of the isolates obtained, respectively. BOX-PCR genomic fingerprints showed that there was a high level of genotypic diversity within each of these species. The genomic fingerprints of Rhodopseudomonas palustris isolates were significantly different for isolates from the two sampling sites, suggesting that certain strains may be endemic to each sampling site. Not all Rhodopseudomonas palustris isolates could degrade benzoate, a feature that has previously been thought to be characteristic of the species. There were differences in the BOX-PCR genomic fingerprints and restriction fragment length polymorphisms of benzoate-coenzyme A ligase genes and form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes between benzoate-degrading and non-benzoate-degrading genotypes. The ability to distinguish these two Rhodopseudomonas palustris groups based on multiple genetic differences may reflect an incipient speciation event resulting from adaptive evolution to local environmental conditions.     

Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates

Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)₅- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)₅ and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)₅ and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)₅ and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection

Background

The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies.

Methodology/Principal Findings

We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA.

Conclusion

This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.     

Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae Isolated from Vegetables Imported from the Dominican Republic,India, Thailand,and Vietnam

To examine to what extent fresh vegetables imported into Switzerland represent carriers of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae, 169 samples of different types of fresh vegetables imported into Switzerland from the Dominican Republic, India, Thailand, and Vietnam were analyzed. Overall, 25.4% of the vegetable samples yielded one or more ESBL-producing Enterobacteriaceae, 78.3% of which were multidrug resistant. Sixty isolates were obtained: Escherichia coli, 26; Klebsiella pneumoniae, 26; Enterobacter cloacae, 6; Enterobacter aerogenes, 1; and Cronobacter sakazakii, 1. We found 29 isolates producing CTX-M-15, 8 producing CTX-M-14, 7 producing CTX-M-55, 3 producing CTX-M-65, 1 each producing CTX-M-1, CTX-M-3, CTX-M-27, and CTX-M-63, 5 producing SHV-2, 3 producing SHV-12, and 1 producing SHV-2a. Four of the E. coli isolates belonged to epidemiologically important clones: CTX-M-15-producing B2:ST131 (1 isolate), D:ST405 (1 isolate), and D:ST38 (2 isolates). One of the D:ST38 isolates belonged to the extraintestinal enteroaggregative E. coli (EAEC) D:ST38 lineage. Two of the K. pneumoniae isolates belonged to the epidemic clones sequence type 15 (ST15) and ST147. The occurrence of antibiotic-resistant pathogenic and commensal Enterobacteriaceae in imported agricultural foodstuffs constitutes a source of ESBL genes and a concern for food safety.     

Biotyping of Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates from France and Algeria Using MALDI-TOF MS

Background

Klebsiella pneumoniae is one of the most important pathogens responsible for nosocomial outbreaks worldwide. Epidemiological analyses are useful in determining the extent of an outbreak and in elucidating the sources and the spread of infections. The aim of this study was to investigate the epidemiological spread of K. pneumoniae strains using a MALDI-TOF MS approach.

Methods

Five hundred and thirty-five strains of K. pneumoniae were collected between January 2008 and March 2011 from hospitals in France and Algeria and were identified using MALDI-TOF. Antibiotic resistance patterns were investigated. Clinical and epidemiological data were recorded in an Excel file, including clustering obtained from the MSP dendrogram, and were analyzed using PASW Statistics software.

Results

Antibiotic susceptibility and phenotypic tests of the 535 isolates showed the presence of six resistance profiles distributed unequally between the two countries. The MSP dendrogram revealed five distinct clusters according to an arbitrary cut-off at the distance level of 500. Data mining analysis of the five clusters showed that K. pneumoniae strains isolated in Algerian hospitals were significantly associated with respiratory infections and the ESBL phenotype, whereas those from French hospitals were significantly associated with urinary tract infections and the wild-type phenotype.

Conclusions

MALDI-TOF was found to be a promising tool to identify and differentiate between K. pneumoniae strains according to their phenotypic properties and their epidemiological distribution. This is the first time that MALDI-TOF has been used as a rapid tool for typing K. pneumoniae clinical isolates.     

Bacteriocin production by members of the genusKlebsiella

Bacteriocin production was tested in 36Klebsiella and 3Enterobacter aerogenes strains. Bacteriocins produced byK. pneumoniae were found to be active on most strains ofK. edwardsi, K. aerogenes, K. rhinoscleromatis andE. aerogenes. The bacteriocin produced byE. aerogenes 37 is also active onK. pneumoniae andK. ozaenae. The bacteriocins produced byK. rhinoscleromatis, K. edwardsi andK. aerogenes are active on only a few strains. The activity spectra of the bacteriocins of a number of strains were similar. The method of classification used for colicins could not be applied to these bacteriocins as mutants resistant to one bacteriocin were nearly always resistant to all other bacteriocins. One mutant, though resistant, still adsorbed the bacteriocin to which it was resistant and it is very likely that the same applies for all other resistant mutants. The hypothesis is made that allKlebsiella bacteriocins have the same biochemical target, or more likely, possess a common transmission mechanism.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003

Background

The high mortality impact of infectious diseases will increase due to accelerated evolution of antibiotic resistance in important human pathogens. Development of antibiotic resistance is a evolutionary process inducing the erosion of the effectiveness of our arsenal of antibiotics. Resistance is not necessarily limited to a single class of antibacterial agents but may affect many unrelated compounds; this is termed ‘multidrug resistance’ (MDR). The major mechanism of MDR is the active expulsion of drugs by bacterial pumps; the treatment of Gram negative bacterial infections is compromised due to resistance mechanisms including the expression of efflux pumps that actively expel various usual antibiotics (ß-lactams, quinolones, …).

Methodology/Principal Findings

Enterobacter aerogenes has emerged among Enterobacteriaceae associated hospital infections during the last twenty years due to its faculty of adaptation to antibiotic stresses. Clinical isolates of E. aerogenes belonging to two strain collections isolated in 1995 and 2003 respectively, were screened to assess the involvement of efflux pumps in antibiotic resistance. Drug susceptibility assays were performed on all bacterial isolates and an efflux pump inhibitor (PAßN) previously characterized allowed to decipher the role of efflux in the resistance. Accumulation of labelled chloramphenicol was monitored in the presence of an energy poison to determine the involvement of active efflux on the antibiotic intracellular concentrations. The presence of the PAßN-susceptible efflux system was also identified in resistant E. aerogenes strains.

Conclusions/Significance

For the first time a noticeable increase in clinical isolates containing an efflux mechanism susceptible to pump inhibitor is report within an 8 year period. After the emergence of extended spectrum ß-lactamases in E. aerogenes and the recent characterisation of porin mutations in clinical isolates, this study describing an increase in inhibitor-susceptible efflux throws light on a new step in the evolution of mechanism in E. aerogenes.     

Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum

Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259–93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum.     

Colistin resistance in <Emphasis Type="Italic">Enterobacter</Emphasis> spp. isolates in Korea

We investigated the colistin resistance rate among 356 Enterobacter spp. clinical isolates from eight hospitals in Korea. Antibiotic susceptibility testing was performed by broth microdilution. While 51 of 213 (23.9%) Enterobacter cloacae isolates were colistin-resistant, only six of 143 (4.2%) E. aerogenes isolates showed resistance. We also identified the skip well phenotype in eight E. cloacae and three E. aerogenes isolates. Multilocus sequence typing for E. cloacae and randomly amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus PCR for E. aerogenes revealed that clonal spreading of colistin-resistant and skip well Enterobacter spp. isolates had not occurred. In vitro time-kill assays were performed with three colistin-resistant, three skip well, and two colistin-susceptible isolates of E. cloacae and E. aerogenes. Inconsistent results were observed among isolates with skip well phenotypes; while some were eradicated by 2 mg/L colistin, others were not. This suggests that skip well isolates have differentiated into different categories. As the high rates of colistin resistance in E. cloacae detected are of clinical concern, continuous monitoring is warranted. In addition, the clinical implications and mechanisms of the skip well phenotype should be investigated to ensure the appropriate use of colistin against Enterobacter infections.     

Isolation and Characteristics of Shiga Toxin 2f-Producing Escherichia coli among Pigeons in Kyushu,Japan

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx _2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx _2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA _O26, irp ₂, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.     

Low-Temperature Isolation of Disease-Suppressive Bacteria and Characterization of a Distinctive Group of Pseudomonads

The influence of environmental factors during isolation on the composition of potential biocontrol isolates is largely unknown. Bacterial isolates that efficiently suppressed wheat seedling blight caused by Fusarium culmorum were found by isolating psychrotrophic, root-associated bacteria and by screening them in a bioassay that mimicked field conditions. The impact of individual isolation factors on the disease-suppressive index (DSI) of almost 600 isolates was analyzed. The bacteria originated from 135 samples from 62 sites in Sweden and Switzerland. The isolation factors that increased the probability of finding isolates with high DSIs were sampling from arable land, Swiss origin of samples, and origination of isolates from plants belonging to the family Brassicaceae. The colony morphology of the isolates was characterized and compared to DSIs, which led to identification of a uniform morphological group containing 57 highly disease-suppressive isolates. Isolates in this group were identified as Pseudomonas sp.; they were fluorescent on King's medium B and had characteristic crystalline structures in their colonies. These isolates were morphologically similar to seven strains that had previously been selected for suppression of barley net blotch caused by Drechslera teres. Members of this morphological group grow at 1.5°C and produce an antifungal polyketide (2,3-deepoxy-2,3-didehydrorhizoxin [DDR]). They have similar two-dimensional polyacrylamide gel electrophoresis protein profiles, phenotypic characteristics, and in vitro inhibition spectra of pathogens. In summary, in this paper we describe some isolation factors that are important for obtaining disease-suppressive bacteria in our system, and we describe a novel group of biocontrol pseudomonads.     

Phenotypic and Genotypic Characterization of Avian Escherichia coli O86:K61 Isolates Possessing a Gamma-Like Intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.     

The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria

In this study, the activity of S-thanatin (an analog of antimicrobial peptide derived from thanatin) against different bacterial pathogens frequently which can cause therapeutic problems was tested. The result showed minimal inhibitory concentrations (MICs) of S-thanatin against all isolates of the Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella ornithinolytica and Klebsiella oxytoca were in the range of 4-16 μg/ml, no matter which antibiotic the bacterial was resistant or susceptible, while almost all MICs to Gram-positive bacterial were >128 μg/ml except Enterococcus faecium. S-thanatin was more effective toward Gram-negative strains, especially for Enterobacter and Klebsiella. The MICs of S-thanatin were no significantly different in the same species regardless of antibiotic sensitive or -resistant isolates to single or multiple antibiotic (P > 0.05). Likewise, no notable difference could be observed between E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica which were sensitive to S-thanatin (P > 0.05). It was implied that the antimicrobial activity of S-thanatin was independent on multi-drug resistance spectrum of bacteria.     

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Karlodinium veneficum

The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) combined with a chromatographic lateral flow dipstick (LFD) assay to rapidly and specifically detect the Karlodinium veneficum ITS gene. Four groups of LAMP primers were specially designed to target the K. veneficum ITS gene. The LAMP-LFD detection limit was 7.4 pg/μL (approximately 6.5 cells/mL) of K. veneficum genomic DNA and was 10 times more sensitive than standard PCR. The LAMP-LFD method exhibited high specificity and accurately identified K. veneficum algal isolates, but not other algal isolates. To test the assay’s accuracy, samples from positive results were further analyzed by sequencing and phylogenetic analysis, all of which were identified as K. veneficum. Over all, the LAMP-LFD assay established in this paper can be used as a reliable and simple method to detect the K. veneficum.     

Phylogenetic Distribution of Phenotypic Traits in Bacillus thuringiensis Determined by Multilocus Sequence Analysis

Diverse isolates from a world-wide collection of Bacillus thuringiensis were classified based on phenotypic profiles resulting from six biochemical tests; production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). Eighty two isolates representing the 15 most common phenotypic profiles were subjected to phylogenetic analysis by multilocus sequence typing; these were found to be distributed among 19 sequence types, 8 of which were novel. Approximately 70% of the isolates belonged to sequence types corresponding to the classical B. thuringiensis varieties kurstaki (20 isolates), finitimus (15 isolates), morrisoni (11 isolates) and israelensis (11 isolates). Generally, there was little apparent correlation between phenotypic traits and phylogenetic position, and phenotypic variation was often substantial within a sequence type. Isolates of the sequence type corresponding to kurstaki displayed the greatest apparent phenotypic variation with 6 of the 15 phenotypic profiles represented. Despite the phenotypic variation often observed within a given sequence type, certain phenotypes appeared highly correlated with particular sequence types. Isolates with the phenotypic profiles TLUAE and LSAE were found to be exclusively associated with sequence types associated with varieties kurstaki and finitimus, respectively, and 7 of 8 TS isolates were found to be associated with the morrisoni sequence type. Our results suggest that the B. thuringiensis varieties israelensis and kurstaki represent the most abundant varieties of Bt in soil.     

Induction by Klebsiella aerogenes of a Melanin-Like Pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro‐intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low‐resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum‐marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.     

Enhanced Utilization of Phosphonate and Phosphite by Klebsiella aerogenes

Klebsiella aerogenes ATCC 9621 was able to utilize phosphonates (P_n), including aminoethylphosphonate, ethylphosphonate, methylphosphonate (MP_n), and phosphonoacetate, and inorganic phosphite (P_t) as sole sources of phosphorus (P). The products of the phn gene cluster were absolutely required for P_n breakdown and P_t oxidation to inorganic phosphate (P_i) in this organism. To determine if K. aerogenes ATCC 9621 could be engineered to enhance the utilization of P_n and P_t, a multicopy plasmid, pBI05, which carried the entire phn gene cluster, was introduced into this strain. Despite the increased dosage of the phn genes, K. aerogenes ATCC 9621(pBI05) could utilize only up to 1.1-fold more P_n and P_t than did the control strain with the parent vector alone. These results suggested that P_i, which was generated from P_n and P_t, might limit further utilization of these P compounds. Consequently, to convert the resulting P_i to polyphosphate (polyP), the plasmid pKP28, which carried the K. aerogenes ppk gene (which encodes polyP kinase), was introduced into K. aerogenes ATCC 9621(pBI05). Overexpression of the ppk gene in K. aerogenes ATCC 9621(pBI05, pKP28) resulted in a 2.5-fold increase in P_t utilization over that of the control strain. This recombinant strain also accumulated approximately sixfold more P than did the control strain when the cells were grown with MP_n as a sole source of P.