Non-pigmented Isolates Research Articles

Antibiotic resistance of P. aeruginosa in the presence and absence of pyocyanin pigment

Purpose: study of antibiotic resistance of Р. aeruginosa strains in the presence and absence of pyocyanin pigment. Materials and methods. Bacteriological studies of pathological material from "asphyxiated embryos", sick chickens and forcibly killed or dead adult birds were carried out according to generally accepted methods. Cultures from bone, brain, heart, liver, spleen, gall bladder, muscles and other organs were carried out on simple, selective and differential diagnostic nutrient media. The sensitivity of P. aeruginosa isolates to antibacterial drugs was determined by diffusion in agar according to the generally accepted method. The results. The obtained results of studies by the diffusion method in agar indicate that the proportion of resistant to the studied antibiotics among non-pigmented isolates was on average 79.00 % (60-100) %, and among strains that formed pyocyanin - 51.00 % (25-100) %. 60.00 % - 90.00 % of resistant non-pigmented isolates were found to fluoroquinolones, 60.00-70.00 % to cephalosporins, 60.00-97.00 % to aminoglycosides. To representatives of semisynthetic penicillins, tetracyclines, and macrolides - 100 % of resistant isolates that did not synthesize pyocyanin were found. Among P. aeruginosa isolates that synthesized pigment, 25.00-40.00 % were resistant to fluoroquinolones, 30.00-35.00 % to cephalosporins, 25.00-50.00 % to aminoglycosides, representatives semi-synthetic penicillins, tetracyclines, macrolides - 100 % of the studied strains. Conclusions. The absence of pigment formation in P. aeruginosa isolates, obtained in association with bacterial pathogens, is not accompanied by the absence of antibiotic resistance. 28 % more antibiotic-resistant isolates were found among non-pigmented isolates compared to isolates that synthesize pyocyanin pigment. The data presented emphasize the need for the use of differential media for the isolation of P. aeruginosa in order to identify non-pigmented strains and prescribe appropriate treatment, which, accordingly, will prevent the spread of latent forms of infection

Pathogenicity of non-pigmented and pigmented isolates of P. aeruginosa during long-term storage

The aim: to study the pathogenicity of non-pigmented P. aeruginosa isolates compared to those that synthesize pigment during long-term storage for broiler chickens. Materials and methods. Bacteriological studies were carried out in accordance with generally accepted methods. Initially, P. aeruginosa and other bacterial microflora were isolated from poultry farms, pathological material from poultry. The type of microorganisms was identified using Bergey's identifier. The obtained isolates were examined by morphological characteristics (according to Gram), tincture, cultural, biochemical, pathogenic properties and sensitivity of selected cultures to antibiotics were studied. After the expiration of 3 years, the main biological properties of the isolates were monitored. To establish pathogenic properties, a bioassay was performed on white mice and one-week-old broiler chickens of the Cobb 500 cross (intraperitoneally by washout from daily agar culture at a dose of 0.2 cm3, which corresponds to the previously established LD50). The results. With parenteral infection by washout from agar daily culture of pigmentless isolates at a dose of LD50, chickens died within the first - second day, in this case 87.50 % of the cultures showed pathogenicity. Experimental parenteral infection with washings from a daily culture of pigmented isolates of P. aeruginosa at a dose of LD50 led to the death of one-week-old chickens within 24-48 hours, in this case 75.00 % of the isolates showed pathogenicity. Clinical and pathological signs of infection with pigmented and non-pigmented P. aeruginosa isolates were similar. Conclusions. A comparative analysis of cases of pathogenicity of non-pigmented and pigmented isolates of P. aeruginosa on one-week-old broiler chickens of the Cobb-500 cross was carried out. In the experiment, we found that among the non-pigmented isolates, compared to the pigmented pathogenic isolates, 12.5 % more were detected in chickens. This emphasizes the importance of differential diagnosis for pseudomonosis, because infection with non-pigmented strains often goes undiagnosed

Genetic and Virulent Difference Between Pigmented and Non-pigmented Staphylococcus aureus.

Staphyloxanthin (STX), a golden carotenoid pigment produced by Staphylococcus aureus, is suggested to act as an important virulence factor due to its antioxidant properties. Restraining biosynthesis of STX was considered as an indicator of virulence decline in pigmented S. aureus isolates. However, it is not clear whether natural non-pigmented S. aureus isolates have less virulence than pigmented ones. In this study, it is aimed to compare the pigmented and non-pigmented S. aureus isolates to clarify the genetic and virulent differences between the two groups. Here, 132 S. aureus isolates were divided into two phenotype groups depending on the absorbance (OD450) of the extracted carotenoids. Then, all isolates were subjected to spa typing and multilocus sequence typing (MLST), and then the detection of presence of 30 virulence factors and the gene integrity of crtN and crtM. Furthermore, 24 typical S. aureus isolates and 4 S. argenteus strains were selected for the murine infection assay of in vivo virulence, in which the histological observation and enumeration of CFUs were carried out. These isolates were distributed in 26 sequence types (STs) and 49 spa types. The pigmented isolates were scattered in 25 STs, while the non-pigmented isolates were more centralized, which mainly belonged to ST20 (59%) and ST25 (13%). Among the 54 non-pigmented isolates, about 20% carried intact crtN and crtM genes. The in vivo assay suggested that comparing with pigmented S. aureus, non-pigmented S. aureus and S. argenteus strains did not show a reduced virulence in murine sepsis models. Therefore, it suggested that there were no significant genetic and virulent differences between pigmented and non-pigmented S. aureus.

Staphylococcal food poisoning caused by Staphylococcus argenteus harboring staphylococcal enterotoxin genes

Staphylococcal food poisoning (SFP) is caused by staphylococcal enterotoxins (SEs) preformed in food materials. SE genes are encoded on mobile genetic elements and are widely found across Staphylococcus species including S. argenteus, although most SFP cases are caused by S. aureus. S. argenteus, recently discriminated from S. aureus as a novel species, are non-pigmented staphylococci phenotypically related to S. aureus. In 2014 and 2015, two independent food poisoning cases occurred in Osaka, Japan, in which non-pigmented staphylococci were predominantly isolated. Several enterotoxin genes (seb, seg, sei, sem, sen, seo, and selu2) were found in their genome and the production of SEB was confirmed by reverse passive agglutination tests. The non-pigmented isolates from patients, food handlers, food, and cooking utensils all produced the same pulsed-field gel electrophoresis pattern. These non-pigmented isolates were coagulase-positive and biochemically identical to S. aureus. We performed further genetic analysis using nucA sequencing and multi-locus sequence typing, and identified these isolates as S. argenteus. We also found that seb was encoded on the Staphylococcus aureus pathogenicity island, while seg, sei, sem, sen, seo, and selu2 were encoded on the enterotoxin gene cluster. From these results, we concluded that the two food poisoning outbreaks were SFP cases caused by S. argenteus harboring SE genes.

Evaluation of the Killing Virulence of Pigmented and Non-Pigmented Clinical Isolates of Pseudomonas Aeruginosa in Mice

Evaluation of the Killing Virulence of Pigmented and Non-Pigmented Clinical Isolates of Pseudomonas Aeruginosa in Mice

Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica.

Respiratory infections are common causes of morbidity and mortality worldwide. We sought to assess the multiple antibiotic resistance (MAR) index, fitness and virulence potential in Pseudomonas aeruginosa from patients with lower respiratory tract infections. Isolates were assessed for antimicrobial susceptibility, in vitro competitive fitness, and pigment, elastase and rhamnolipid production. Oxidative stress tolerance was determined on both planktonic and biofilm cells, and virulence potential was tested in a plant model. Mean MAR index for isolates was 0.34 (range 0.17-0.50). Whilst isolates exhibited good biofilm formation in the presence of ciprofloxacin, there was no significant difference in biofilm production over the concentration range assessed. Several drug-resistant strains were out-competed by a susceptible strain even in the presence of antibiotic. H2O2 exerted a greater oxidative stress than tert-butyl-hydroperoxide and, as expected, biofilms were more resistant than planktonic cells. Whilst most (81 %) isolates were pigmented there was no significant difference between pigmented and non-pigmented isolates when elastolytic activity was compared (P>0.05). More than half of the isolates produced the quorum sensing mediator rhamnolipid and infection of the plant model by bacteria occurred whether elastase or rhamnolipid was present or absent. These data suggest that non-pigmented strains of P. aeruginosa might pose an equally significant microbiological threat as pigmented strains even though pigment production appeared to be strongly associated with elastase expression. Whilst dual expression of elastase and rhamnolipid by these bacteria would cause severe tissue damage (as seen in the plant model), non-production of either does not prevent bacteria from causing serious infection.

Large scale MALDI-TOF MS based taxa identification to identify novel pigment producers in a marine bacterial culture collection

A challenge in the rational exploitation of microbial culture collections is to avoid superfluous testing of replicas. MALDI-TOF MS has been shown to be an efficient dereplication tool as it can be used to discriminate between bacterial isolates at the species level. A bacterial culture collection of more than 10,000 heterotrophic marine bacterial isolates from sea-water surface layers of the Norwegian Trondheimsfjord and neighbouring coastal areas has been established. A sub-collection of pigmented isolates was earlier screened for novel carotenoids with UVA-Blue light absorbing properties. This was a comprehensive analytical task and it was observed that a significant number of extracts with identical pigment profile were recovered. Hence, this study was undertaken to explore the use of MALDI-TOF MS as a dereplication tool to quickly characterize the bacterial collection. Furthermore, LC-DAD-MS analysis of pigment profiles was performed to check if pigment profile diversity was maintained among isolates kept after the potential MALDI-TOF MS selection step. Four hundred isolates comprising both pigmented and non-pigmented isolates were used for this study. The resulting MALDI-TOF MS dendrogram clearly identified a diversity of different taxa and these were supported by the pigment profile clustering, thus linking the pigment production as species-specific properties. Although one exception was found, it can be concluded that MALDI-TOF MS dereplication is a promising pre-screening tool for more efficient screening of microbial culture collection containing pigments with potential novel properties.

Carotenoid Pigmentation in Antarctic Heterotrophic Bacteria as a Strategy to Withstand Environmental Stresses

Bacterial strains isolated from Antarctic environments were used to assess the role of carotenoid pigments as cryo- and solar radiation protectants. Isolates were subjected to one hundred 12-hr freeze-thaw cycles and exposed to ambient simulated solar radiation (300 Wm−2) with growth recovery evaluated after pre-set time intervals. Differences in survival were observed between carotenoid pigmented and non-pigmented strains in response to the different stresses based upon the enumeration of colony forming units. On average carotenoid pigmented strains were more resistant to freeze-thaw cycles as compared to the non-pigmented strains. Survival for non-pigmented strains decreased precipitously from 2 × 107 to 1.5 × 104 cells mL−1, on average, within the first 20 cycles. Similar results were found in the solar radiation experiments. After 2 hrs of solar radiation exposure, 61% of the pigmented organisms survived versus 0.01% for the non-pigmented isolates. We applied an additive mixed model to estimate differences between the carotenoid pigmented and non-pigmented bacterial groups. Modeled results confirmed a positive effect of pigmentation on survivability and provide evidence that carotenoid pigmentation in heterotrophic bacteria isolated from Antarctic environments increases resistance to environmental stressors.

Molecular characterization of pigmented and nonpigmented isolates of Mycobacterium avium subsp. paratuberculosis.

Five pigmented isolates of Mycobacterium avium subsp. paratuberculosis were examined by pulsed-field gel electrophoresis (PFGE), IS900 restriction fragment length polymorphism (IS900-RFLP), and IS1311 polymorphism analysis using PCR. All of the pigmented isolates exhibited one of three distinct PFGE profiles with SnaBI, designated 9, 10, and 11, and with SpeI, designated 7, 8, and 9, which generated three multiplex profiles designated [9-7], [10-8], and [11-9]. All of the pigmented isolates had the same IS900-RFLP BstEII and PvuII profiles. The IS900-RFLP BstEII profile was new, but the IS900-RFLP PvuII profile corresponded to PvuII type 6 of a sheep strain described by Cousins and colleagues (D. V. Cousins, S. N. Williams, A. Hope, and G. J. Eamens, Aust. Vet. J. 78:184-190, 2000). IS1311-PCR analysis typed all of the pigmented isolates as sheep (S) strains. The genetic relationship between pigmented and nonpigmented isolates was investigated by using multiplex PFGE data from the analysis of both the 5 pigmented isolates and 88 nonpigmented isolates of M. avium subsp. paratuberculosis from a variety of host species and geographic locations. It was possible to classify the isolates into two distinct types designated type I, comprising the pigmented isolates, and type II, comprising the nonpigmented isolates, which exhibit a very broad host range.

Effect of solar UV-B radiation on a phyllosphere bacterial community.

The effect of solar UV-B radiation on the population dynamics and composition of the culturable bacterial community from peanut (Arachis hypogeae L.) was examined in field studies using plants grown under UV-B-transmitting (UV-B+) or UV-B-excluding (UV-B-) plastic filters. Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the season. The total bacterial population size was not affected by either UV-B treatment. However, isolates from the UV-B+ plots (n = 368) were significantly more UV tolerant than those from the UV-B- (n = 363) plots. UV sensitivity was determined as the minimal inhibitory dose of UV that resulted in an inhibition of growth compared to the growth of a nonirradiated control. The difference in minimal inhibitory doses among bacterial isolates from UV-B+ and UV-B- treatments was mainly partitioned among nonpigmented isolates, with pigmented isolates as a group being characterized as UV tolerant. A large increase in UV tolerance was observed within isolate groups collected late (89 and 96 days after planting) in the season. Identification of 200 late-season isolates indicated that the predominant UV-tolerant members of this group were Bacillus coagulans, Clavibacter michiganensis, and Curtobacterium flaccumfaciens. We selected C. michiganensis as a model UV-tolerant epiphyte to study if cell survival on UV-irradiated peanut leaves was increased relative to UV survival in vitro. The results showed an enhancement in the survival of C. michiganensis G7.1, especially following high UV-C doses (300 and 375 J m(-2)), that was evident between 24 and 96 h after inoculation. A dramatic increase in the in planta/in vitro survival ratio was observed over the entire 96-h experiment period for C. michiganensis T5.1.

Ultraviolet Radiation (UVR) Sensitivity Analysis and UVR Survival Strategies of a Bacterial Community from the Phyllosphere of Field-Grown Peanut (Arachis hypogeae L.).

> Abstract The short-term population dynamics of the culturable bacterial community from field-grown peanut (Arachis hypogeae L.) was analyzed over three 2-day periods. As in other phyllosphere studies, significant numbers of pigmented organisms were detected, suggesting the importance of pigmentation in the colonization of this habitat. Isolates were grouped according to pigmentation (orange, pink, yellow, nonpigmented), and the sensitivity of each isolate in the collection (n = 617) to ultraviolet radiation (UVR) was determined as the minimal inhibitory dose (MIDC) of UVR that resulted in an inhibition of growth compared to an unirradiated control. The majority of isolates recovered (56.1%) had an MIDC equal to or exceeding that of Pseudomonas syringae 8B48, a known UV-tolerant strain. Among pigmentation groups, the mean MIDC of pink- and orange-pigmented isolates was significantly greater than that of yellow- or nonpigmented isolates at each sampling time of day. Identification of 213 of the isolates using fatty acid methyl ester analysis indicated that a large proportion of the isolates were gram-positive, with Bacillus spp. alone accounting for 35.7% of the total. The genus Curtobacterium contained the largest percentage of highly UVR-tolerant strains. Nonpigmented mutants of four Curtobacterium strains were selected following ethyl methane sulfonate mutagenesis; these nonpigmented mutants were significantly altered in survival following irradiation with UV-A wavelengths. The strategy of avoidance of UVR through colonization of the abaxial leaf surface was evaluated on three separate occasions by leaf imprint sampling. Only 3 of 120 leaves (2.5%) contained larger bacterial populations on the adaxial surface, indicating that colonization of the abaxial leaf surface is important to phyllosphere survival. Our results indicate that tolerance to UVR is a common phenotype among phyllosphere bacteria, suggesting that solar radiation has a strong influence on the microbial ecology of the phyllosphere.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p27.html

Nonpigmented and Bacteriochlorophyll-Containing Bradyrhizobia Isolated from Aeschynomene indica

The legume genus Aeschynomene is unusual, since many species develop stem nodules and the bradyrhizobia isolated from these nodules produce bacteriochlorophyll (Bchl). Evidence is presented that the bradyrhizobia of Aeschynomene indica have wide distribution throughout the world, since A. indica was nodulated when grown in 58 soils collected in 14 different countries. Only 38 of 79 isolates tested synthesized Bchl and carotenoids during heterotrophic growth. Nine isolates produced Bchl constitutively, and cultures were pigmented after growth in the dark. The other isolates required light for Bchl production. The DNA from seven pigmented and three nonpigmented bradyrhizobia hybridized with a DNA probe containing the genes for the photosynthetic apparatus of Rhodobacter capsulatus, but DNA from two other nonpigmented isolates did not hybridize with this probe. A relationship between pigmentation in culture and symbiotic phenotype was not evident, since bradyrhizobia of both Bchl phenotypes nodulated stems of A. indica and formed nitrogen-fixing symbioses. Several isolates, which were ineffective on A. indica, probably do belong to the proposed cross-inoculation group 3 (D. Alazard, Appl. Environ. Microbiol. 50:732-734, 1985), since they did not nodulate Aeschynomene americana or Macroptilium atropurpureum. Since it has been suggested that extant rhizobia arose from photosynthetic ancestors (J. I. Sprent, p. 45-54, in P. M. Gresshoff, L. E. Roth, G. Stacey, and W. E. Newton, ed., Nitrogen Fixation: Achievements and Objectives, 1990), we propose that the nonpigmented isolates may represent an extant lineage of an intermediate evolutionary stage.

DNA:DNA hybridization and chemotaxonomic studies of Thermus scotoductus

The species Thermus scotoductus was recently described as containing several non-pigmented isolates from Selfoss, Iceland, and the X-1 strain from the USA (Kristjansson et al., 1994). In this study, we performed DNA:DNA hybridizations and chemotaxonomic studies on several non-pigmented Thermus isolates from other geographical areas to assess their relationship to the strains originally assigned to this species. The results of DNA:DNA hybridizations showed that strains NH and DI from London and strains VI-7a and VI-13 from Vizela, Portugal, belonged to T. scotoductus. T. scotoductus X-1 (ATCC 27978) was composed of two stable colony types, one of which had a major glycolipid different from the one present in the other colony type and from all other Thermus strains examined as well. The fatty acid composition of the isolates from Selfoss and London were practically identical. However, the fatty acid composition of strain X-1, the individual colony types of this strain and the Vizela strains were different from the Selfoss-London isolates and from each other. Another non-pigmented strain, designated SPS-11, belonged to a different DNA homology group.

Characterisation of non-pigmented species of the genus Prevotella by polyacrylamide gel electrophoresis.

Gram-negative anaerobic bacilli previously known as the melaninogenicus-oralis group of Bacteroides have been assigned to a new genus, Prevotella. The non-pigmented members of this genus share several general characteristics and cannot be readily distinguished by routine tests. A polyacrylamide slab gel electrophoresis procedure, with visual analysis of protein patterns, was used to compare cellular protein patterns from clinical isolates with those from collection (reference) strains. Reference strains of P. oralis, P. veroralis, P. buccalis, P. oris, P. buccae, P. zoogleoformans, P. bivia, P. disiens, P. oulora, B. (P.) capillus and B. (P.) pentosaceus, and 91 non-pigmented isolates from patients with adult periodontal disease were examined by conventional biochemical tests, gas-liquid chromatography (GLC) and enzyme tests, and whole-cell protein profiles were obtained by SDS-PAGE. There was close correlation between patterns of results in biochemical and GLC tests and the SDS-PAGE profiles, and the species were readily distinguished in SDS-PAGE. The periodontal isolates were assigned to 10 groups by conventional test reaction patterns and nine groups by SDS-PAGE; the profiles of 79 isolates corresponded to those of seven species reference strains. By SDS-PAGE, clinical isolates of P. buccae (42 isolates) and P. oralis (eight isolates) showed good similarity with reference strains. However, for P. veroralis (15), P. oris (7), P. bivia (4), P. zoogleoformans (2) and P. buccalis (1), clinical isolates showed some minor variations from reference strains. Twelve isolates remained undesignated in SDS-PAGE analysis. Variant SDS-PAGE profiles divided clinical isolates of P. buccae into two subgroups and those of P. veroralis into five subgroups.

Non-radioactive DNA probe for the rapid identification of Mycobacterium avium complex from clinical specimens

We evaluated an alkaline phosphatase labelled oligonucleotide probe (SNAP(TM), Syngene Co., Molecular Biosystems, Inc., San Diego CA) for the direct culture identification of Mycobacterium avium complex (MAC) isolated from clinical specimens. Mycobacterial species identified by conventional biochemical methods were retested with this DNA probe using the Centri-Dot(TM) format. The probe accurately identified all 69 pigmented M. avium complex and 15 non-pigmented isolates of M. avium complex. There were no false-positives with 45 non-MAC mycobacteria isolates (10 species) and 16 non-mycobacteria organisms (10 species). The sensitivity and specificity of the SNAP(TM) culture identification for M. avium complex were 100%. The alkaline phosphatase labelled DNA probe is stable for at least 9 months. The procedure can be completed within 2 h and is easily adapted in the clinical laboratory. For the strains encountered in our laboratory, we conclude that the SNAP(TM) hybridization is a rapid, specific, and reliable method for culture identification of M. avium complex.

Mating reactions of pigmented and non-pigmented isolates of Arthroderma uncinatum.

Keratinomyces ajelloi the conidial state of Arthroderma uncinatum has 2 colonial forms — one producing dark vinaceous-red pigment and the other devoid of diffusible pigment. Twenty-one isolates of this fungus, including 2 pairs of + and - test strains, were crossed with each other in all possible combinations on soil-hair medium. Of the 13 pigmented isolates 5 were + mating type, 5 were — mating type, 2 were self-fertile, and 1 was incompatible. Of the 8 nonpigmented isolates, 5 were + mating type, and 3 were —. Of 80 crosses between + and - isolates, only 6 were abundantly fertile, and 44 were completely sterile. Two of the + and 3 of the - isolates were strong reactors, being fertile with most of the isolates of the opposite mating type. The rest of the isolates showed a high degree of incompatibility with each other. The compatibility of all the 8 non-pigmented isolates with the pigmented test strains indicates that lack of pigment does not denote a separate taxon.

Bacteriophage studies of isolates from fire blight sources.

Six hundred and sixteen Gram-negative bacterial isolates were obtained from trees and orchards diseased with fire blight. By serological means, 194 Erwinia amylovora isolates were distinguished from 57 nonpigmented isolates of miscellaneous colonial types. One hundred and eighty-nine isolates were distinguished by yellow colony color. In addition, 176 isolates produced a fluorescent pigment. Four other biochemical tests were applied to the isolates.All of the E. amylovora isolates, 26 'miscellaneous white' isolates, and 94 'yellow' isolates were lysed by one or more of seven phages. Two phages, 1507 and 1508, lysed many isolates from all three groups, and in this way relationships were indicated. Phage sensitivity was not correlated with other characteristics, although phage P19 lysed most of the E. amylovora isolates and none of the yellow. Phage P17 lysed a majority of the 'yellow' isolates and none of the E. amylovora. Both phages lysed some 'miscellaneous white' isolates.