Microbial Ecology

Microbial Ecology Bacteria Isolated from the Different Developmental Stages and Larval Organs of the Obligate Parasitic Fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae) E.M. To´th1, E´. Hell1,2, G. Kovaćs1,3, A.K. Borsodi1 and K. Ma´rialigeti1 (1) Department of Microbiology, Faculty of Science, Eo¨tvo¨s Lorańd University, Pa´zmańy P. se´tańy 1/c, H-1117, Budapest, Hungary (2) Department of Parasitology and Zoology, Faculty of Veterinary Science, Szent Istvań University, Budapest, Hungary (3) National Public Health and Health Officer’s Service, Budapest, Hungary Received: 19 April 2004 / Accepted: 17 August 2004 / Online publication: 31 December 2005

Abstract

Wohlfahrtia magnifica (Diptera: Sarcophagidae) is the major myiasis-causing fly species in the whole of Eurasia for most important domestic animals. The aim of the present work was to obtain data on the culturable bacteria isolated under aerobic conditions from this fly: bacteria were isolated from all developmental stages (larvae, pupa, and imago) of Wohlfahrtia magnifica, and the third-stage larval organs were also sampled. To determine the possible antagonistic effects between the dominant bacterial groups, an antibiosis assay was carried out. Plating and isolation of bacteria was performed by classical microbiological methods. Characterization of the isolated strains was carried out via a polyphasic approach; classical phenotypic tests, chemotaxonomical examinations, and 16S rDNA sequence analyses were also applied. In the case of maggot macerate samples, members of the family Enterobacteriaceae were characteristic. Members of a new genus (Schineria) belonging to the g subdivision of proteobacteria were also isolated. According to our data, the shifts in the Schineria and Proteus populations within the larvae are strongly influenced by their interactions with each other and among the members of the family Enterobacteriaceae. The pupa and imago samples contained several other Gram-negative bacteria (Stenotrophomonas, Brevundimonas, etc.). Among Gram-positive bacteria, in all maggot macerate samples, members of the genus Bacillus and the Arthrobacter–Micrococcus group of actinobacteria were dominant (neither of them was a producer or sensitive to the compounds of other microorganisms), and bacteria related to the genus Corynebacterium were also found. From the larvae Aureobacterium liquefaciens Correspondence to: E.M. To´th; E-mail: [email protected] DOI: 10.1007/s00248-005-0090-6

and Enterococcus faecalis were isolated, and from the pupae Dietzia maris and Enterococcus faecalis. In the samples of third-stage larval organs, the dominant groups were the same as in the third-stage larval macerate sample; however, several additional genera/species were observed (Rhodococcus fascians, Streptomyces sp., Rathayibacter sp., Bacillus thuringiensis/cereus).

Introduction

It is generally accepted that the world of insects is a basic source of bacterial species diversity. Insects carry thousands of bacterial symbionts as parasites, mutualistic partners, or protocooperants [1–3]. Flies causing myiasis (infestation of live and/or dead organs and tissues of vertebrates by dipterous larvae) are one of the world’s most devastating insects and are responsible for severe losses in animal husbandry [4]. In the whole of Eurasia, Wohlfahrtia magnifica (Diptera: Sarcophagidae), an obligate larval parasite of live warm-blooded vertebrates, was found to be the major myiasis-causing fly species in the majority of domesticated animals [5–8]. A wealth of data has been published concerning the infestation of animals [7, 9, 10], the differences in the bacterial communities of the healthy skin and myiatic lesions [11, 12], and the bacterial species isolated from the lesions producing fly-attracting volatiles [13]. However, there is no information on the microbe partners of this myiasiscausing fly. The aim of our work is to present data on the natural bacteria isolated from Wohlfahrtia magnifica, tracing changes in the cultured bacterial communities through the developmental stages of the fly, and to obtain information on the organ location of bacteria in the third-stage maggots.

& Volume 51, 13–21 (2006) & * Springer Science+Business Media, Inc. 2005

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Materials and Methods Isolation and Cultivation. First-, second-, and thirdstage maggots of Wohlfahrtia magnifica were collected from vulval lesions of Romney breed sheep at Mezo˜falva State Farm (18-400E, 46-500N, Hungary), in 1997. Maggots were washed three times in sterile PBS solution (physiological saline in 0.025 M phosphate buffer, pH 6.8) and three individuals of each developmental stage were homogenized to obtain composite samples. In parallel, five third-stage maggots were pupated in sterile sawdust. Three-week-old pupae were aseptically opened. In two pupa, imagos were found and used as sample material; in the other three cases earlier stages of metamorphosis were sampled. Thus five composite samples (L1: first-stage maggot; L2: second-stage maggot; L3: third-stage maggot; P: pupa; I: imago) were used to culture bacteria. In 1999, the organs (foregut, midgut, hindgut, cuticule, fat body, salivary glands) of three third-stage maggots collected from an infected sheep at Mezo˜falva were excised under aseptic conditions to investigate the organ location of bacteria. Serial dilutions of the samples were made in PBS. Plating and isolation of bacteria were made on King B [14], nutrient [15], and Endo agar [15] media under aerobic conditions. Later, for maintenance, King B agar medium was used. Isolates were grouped according to their macromorphological properties, and from each sample representative strains were selected for taxonomic investigations (altogether, 159 strains from the developmental stages of the fly and 243 strains from the larval organs). Phenotypic Characterization of the Isolated Colony morphology of the strains was tested Strains.

on King B agar medium by direct and stereomicroscopic observations of single colonies. Cell morphology and motility were studied by phase-contrast microscopy of native preparations. Gram staining was performed according to the procedure described by Claus [16], spore staining according to Smibert and Krieg [17], and capsules were visualized by negative staining after Duguid [18]. Oxidase activity was checked by the method of Tarrand and Gro¨schel [19], and catalase production and Voges–Proskauer reaction were demonstrated via the standard methods of Cowan and Steel [15]. Acid production from different carbohydrates was studied by OF test according to Hugh and Leifson [20]. Temperature (4, 28, 37, and 45-C) and pH tolerance [2, 4, 6, 8–11] values were determined using King B agar and broth _ cultures, respectively. Urease activity, reduction of NO3 _ to NO2 , aesculin and starch hydrolysis, indole production from tryptophane, casease, gelatinase, phosphatase, esterase (Tween 80 hydrolysis) activities, and H2S production from cystein were studied by the

DIFFERENT DEVELOPMENTAL STAGES

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WOHLFAHRTIA MAGNIFICA

methods of Smibert and Krieg [17]. Hemolysin production was tested on ox-blood agar plates [21]. Utilization of citrate was checked on Simmon’s agar [22], and hydrolysis of chitin was studied by the modified method (King B agar was used instead of a nutrient basal medium) of Holding and Collee [23]. Utilization of 95 different carbon sources as sole source of carbon was controlled on Biolog plates (Biolog Inc., Hayward, CA, USA). Chemotaxonomic Investigations. For chemotaxonomic analysis, strains were grown in liquid Rich medium [24] to the middle of their exponential phase on a rotary shaker at 28-C. Isoprenoid quinones were extracted as described by Collins et al. [25], and the profile was analyzed by high performance liquid chromatography [26]. Cellular fatty acids were extracted according to Stead et al. [27] and analyzed by gas chromatography [28]. 16S rDNA Sequence Analysis. As a first step, partial 16S rDNA sequence analysis was carried out; in case of possible new taxa, the full gene was sequenced. Genomic DNA extraction and polymerase chain reaction (PCR)-mediated amplification of the 16S rDNA of the strains were carried out according to the methods described by Rainey et al. [29]. The PCR products were purified by Prep-A-Gene kit (Bio-Rad, Hercules, CA, USA). Cycle sequencing was performed in a Gene-Amp 2400 PCR using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystem, Foster City, CA, USA) according to the manufacturer’s protocol (Applied Biosystems). Base sequences were determined in an Applied Biosystems Model 310 Genetic Analyzer. The 16S rDNA sequence data were aligned against the ARB-formatted RDP Database Release 7.1 [30] using the ARB program package [31]. To check for more recent updates, an additional BLAST search [32] was carried out. Antibiosis Assay. To determine the possible antagonistic effects among the dominant bacterial groups, an agar diffusion assay was carried out following the method of Quener and Lively [33]. Shaken cultures (37-C; at 150 rpm; in a modified Rich [15] medium, which contained 10% glucose and saccharose) of the following possible producing strains were used: Escherichia coli (LE/28), Proteus vulgaris (L2/64), Schineria larvae (L1/68), Bacillus sp. (U60B), Bacillus pumilus (KU31), Providencia stuartii (K14), Arthrobacter luteolus (E53), Acinetobacter johnsonii (E23). The larval Bextract^ (sterile phosphate buffer dilutions from the homogenized material of the third-stage larvae) was also tested. As test microorganisms, the same bacteria were used supplemented with an authentic Staphylococcus aureus strain

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(NCAIMB 0526). All tests were completed in three replicates. Statistical Analysis. Based on phenotypic characteristics, strains were clustered by UPGMA algorithm [34] using SPSS 3.1 statistical software and also with System Release 3.5 for Biolog results. One representative strain of each phenon and ungrouped strains was selected for chemo- and genotaxonomic investigations. To determine the significance levels, the standard deviations as well as analysis of variance (ANOVA) for the results for the antibiosis assay, Statistica 5.1 (StatSoft Inc. Tulsa, OK, USA) and NCSS 2000 statistical software (NCSS Inc., Kaysville, UT, USA) were used.

Results

Out of the 159 strains isolated from whole macerate samples and 243 strains isolated from organ samples, 17 and 13 died out at the beginning of laboratory maintenance, so the total number of examined strains was 142 and 230, respectively. Bacteria detected in the whole maggot, pupa, and imago macerate samples are shown in Table 1, Figs. 1,

Figure 1. Percentage ratio of Gram-negative bacteria originating from the composite samples of the different developmental stages of Wohlfahrtia magnifica.

and 2. In maggot macerate samples, members of the family Enterobacteriaceae were characteristic, and strains belonging to the genus Proteus were isolated in great numbers. Members of a new taxon belonging to the g subdivision of proteobacteria were also found in these

Table 1. Phenon size and closest relatives of phenon representative bacteria isolated from the developmental stages of Wohlfahrtia magnifica

Strain no.

The closest relative of the phenon representative strain

16S rDNA similarity (%)

Phenon size (%)a

L1/68 L1/15 L1/9B L1/54 L2/19 L2/85 L2/64 L3/6 L3/39 L2/22 LE/29 L2/33 L3/36 L3/72 B2/73 LE/24 B2/69 B2/42 B2/74 B2/22 B2/13 LE/66 LE/28 LE/72 LE/65 LE/27

Schineria larvae Aureobacterium liquefaciens Brevibacterium linens Micrococcus lylae Corynebacterium ammoniagenes Providencia stuartii Proteus vulgaris Proteus vulgaris Bacillus subtilis Bacillus sp. Micrococcus luteus Micrococcus luteus Micrococcus luteus Providencia stuartii Pseudomonas fulva Pseudomonas fulva Enterobacter asburiae Comamonas acidovorans Acidovorax delafieldi Alcaligenes xylosoxidans Dietzia maris Delftia acidovorans Escherichia coli Acinetobacter haemolyticus Brevundimonas diminuta Enterococcus faecalis

100.00 100.00 92.79 97.92 91.58 97.63 96.31 97.42 97.87 97.96 95.92 99.78 97.26 99.47 98.37 99.39 98.87 99.30 96.29 98.35 97.89 98.37 99.60 96.59 99.55 99.22

13.38 2.11 0.71 1.41 7.04 3.52 2.82 5.64 6.34 3.52 3.52 4.93 4.23 3.52 4.23 3.52 2.11 1.41 0.71 1.41 6.34 1.41 4.23 1.41 1.41 5.64

a

Percentage ratio of strains belonging to a phenon among strains investigated. L1: first-stage maggot; L2: second-stage maggot; L3: third-stage maggot; P: pupae; I: adult.

b

Origin of the strainb L1 L1 L1 L1 L2 L2 L2 L3 L3 L3 I L2 L3 L3 P I P P P P P I I I I I

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Figure 2. Percentage ratio of Gram-positive bacteria originating from the different developmental stages of Wohlfahrtia magnifica.

samples, described as Schineria larvae [35], which seemed to be codominant with Proteus in the larval stages. So far, the only known habitat of Schineria is Wohlfahrtia magnifica. The proportion of Schineria strains decreased in parallel with the development of the fly maggots, but the proportion of the members of the genus Proteus increased (Fig. 1). In pupae, at the time of metamorphosis members of both genera almost totally disappeared. From the pupa and imago samples, Schineria larvae could not be isolated and the proportion of Proteus strains also decreased, although other aerobic Gramnegative bacteria (Acidovorax delafieldii, Achromobacter xylosoxidans, Delftia acidovorans, Brevundimonas diminuta, etc.) could be detected in great numbers (Table 1, Fig. 1). It is noteworthy that during pupation the number of Gram-negative strains decreased. Among Gram-positive bacteria isolated from all macerate samples, members of the genus Bacillus and the Arthrobacter–Micrococcus group of actinobacteria were characteristic. Among the Bacilli, Bacillus subtilis was present in high numbers and another Bacillus sp. (close to Bacillus firmus) was also observed. As the 16S rDNA similarity of Micrococcus strains to their closest relatives (Table 1) was quite low, some can be considered as members of new taxa within this group (LE/29, L3/36, L1/54, under description). We also isolated Aureobacterium liquefaciens and Enterococcus faecalis from the larvae and Dietzia maris and Enterococcus faecalis from the pupae. Interestingly, the strains of Dietzia maris were present in the pupae only. Among Gram-positive isolates, a group related to the genus Corynebacterium (based on their phenotypic characteristics) was also present in all samples; however, except for one strain (L2/19), they died out before genotaxonomic characterization. The results of different phenotypic tests are depicted in Table 2. Urease activity was found in 39–90% of the


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isolated strains, and the ratio was higher among strains of larval origin. The ratio of gelatinase producing strains was also higher among bacteria originating from larvae (40–76%). Hemolysine was produced by 16.4% of Gramnegative strains and 32.6% of Gram-positive strains. Two groups of strains had strong chitinase activity: strains of Schineria larvae and some members of the Arthrobacter– Micrococcus group of actinobacteria. All of them were isolated from the maggot samples. Results of sequence analysis, phenon size, and genera originating from the third-stage larval organs are depicted in Table 3 and Fig. 3. Determination of the strains from the third-stage maggot organs showed that the main groups of bacteria (members of the family Enterobacteriaceae, the genus Bacillus, and the aerobic actinobacteria) were the same as in the macerate sample of the third-stage larvae, although some additional taxa also appeared (Fig. 3). Among the members of Enterobacteriaceae, Providencia species dominated in all samples (41.9% of all organ origin strains). Rhodococcus, Micrococcus, Gordonia, and Arthrobacter represented the aerobic actinobacterial group. Among Bacillus strains, more species could be detected than before because Bacillus macroides, Bacillus pumilus, and Bacillus thuringiensis/cereus also appeared. Interestingly, the latter species was present in all organ samples. Bacteria related to the genus Pseudomonas (Brevundimonas, Delftia, Acidovorax, and Stenotrophomonas) typical of imago and pupa samples were not present in culturable amounts in the organ samples of the thirdstage larvae. Strains with the broadest generic affiliation could be detected in the cuticle and foregut samples. Variovorax,

Table 2. Percentage ratio (%) of positive reactions in different phenotypical tests among strains originating from all macerate samples

Phenotypical test

L1a

L2

L3

P

I

Glucose oxidative Glucose fermentative Urease Nitrate reduction Casease Gelatinase Amylase H2S production from cysteine Voges–Proskauer reaction Indol production from tryptophane Haemolysis Chitinase

20 20 72 40 32 40 20 38

53 53 90 71 77 76 33 46

58 50 45 55 23 61 27 20

30 24 57 50 10 12 0 8

27 24 39 80 28 32 15 40

0

8

58

0

5

0

18

0

0

42

24 56

12 25

20 16

21 0

51 0

a L1: first-stage maggot; L2: second-stage maggot; L3: third-stage maggot; P: pupae; I: adult.

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Table 3. Phenon size and closest relatives of phenon representative bacteria isolated from the organ samples of third-stage maggots of Wohlfahrtia magnifica

Strain no.

The closest relative of the strain

E65B2 E23 E43 E1 E53 E66 K14 K58B U60B KU29 KU31 KU40 KU18 KU11 KU52 ZS50 ZS35 ZS25 ZS50B NY57 NY18

Acinetobacter sp. Acinetobacter johnsonii Schineria larvae Microbacterium foliorum Arthrobacter luteolus Rathayibacter tritici Providencia stuartii Gordonia sp. Bacillus sp. Bacillus sp. Bacillus pumilus Bacillus thuringiensis/cereus Rhodococcus fascians Variovorax paradoxus Streptomyces sp. Bacillus macroides Bacillus pumilus Bacillus thuringiensis/cereus Micrococcus luteus Acinetobacter johnsonii Providencia stuartii

a b

16S rDNA similarity (%)

Phenon size (%)a

97.63 99.17 94.22 96.27 98.07 99.48 99.67 95.53 99.82 95.32 98.76 99.47 100.00 97.56 99.67 99.77 100.00 98.78 99.30 97.65 98.72

0.87 0.87 1.74 1.74 1.74 0.87 38.15 0.87 13.6 2.61 6.96 6.96 2.61 0.87 0.87 0.87 1.74 0.87 3.48 2.61 8.69

Origin of the strainb F F F F F F M M H C C C C C C FB FB FB FB SG SG

Percentage ratio of strains belonging to a phenon among strains investigated. F: Foregut; M: midgut; H: hindgut; C: cuticule; FB: fat body; SG: salivary gland.

Figure 3. Bacterial genera occurring in the organ samples of third-stage larvae of Wohlfahrtia magnifica.

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Acinetobacter johnsonii (E23)

Staphylococcus aureus (NCAIMB 0526)

Table 4. Size of inhibition zones (mm) among bacteria isolated from larvae of Wohlfahrtia magnifica

Possible antagonistic strain/test microorganism Escherichia coli (LE28) Proteus vulgaris (L2/64) Schineria larvae (L1/68) Bacillus sp. (U60B) Bacillus pumilus (KU31) Providencia stuartii (K14) Arthrobacter luteolus (E53) Acinetobacter johnsonii (E23) Larval extract

Bacillus pumilus (KU31)

Providencia stuartii (K14)

Arthrobacter luteolus (E53)

Proteus vulgaris (L2/64)

Schineria larvae (L1/68)

18a

22

0

0

0

0

16

17

ND

19

0

0

0

0

17

13

0

ND

0

0

0

0

0

0

0

0

ND

0

0

0

0

0

0

0

0

ND

0

0

0

0

15

9

0

0

ND

12

17

18

0

0

0

0

0

ND

0

0

16

20

0

0

0

0

ND

15

0

0

0

0

0

0

0

20

Bacillus sp. (U60B)

a

Results are significant at p G 0.003; standard deviations between the replicates are 0.3–1.2.

Streptomyces, and Rhodococcus strains could be detected from the cuticle, and, similarly, Rathayibacter and Schineria strains were present only in the foregut sample. The ratio of aerobic actinobacteria (Micrococcus, Arthrobacter, Rhodococcus) was also highest in the cuticle and foregut samples. Among the strains isolated from the salivary gland sample, 78.2% belonged to Providencia stuartii and the rest (21.8%) to Acinetobacter johnsonii species. The ratio of facultative anaerobic strains showed shifts among digestive tract samples. It was lowest at hindgut sample. The highest lipase activity (76.3%) could be detected among strains isolated from the fat body of third-stage larvae of Wohlfahrtia magnifica. The ratio of utilization of urea and gelatin was highest in cuticleoriginating strains (76.6% and 67.8%, respectively). Chitinase activity was present in 60% of cuticle, 22% of fat body, 14% of foregut, 2% of midgut, and 18% of hindgut strains. Hemolysis among isolates did not show characteristic shifts among the organ samples, and the ratio of strains having hemolysine-producing activity was always low (9.09–27.27%). Results of the antibiosis assay are shown in Table 4. Schineria larvae was sensitive to the antimicrobial compounds of several other bacteria, but did not produce any antagonistic chemicals. Compounds of Proteus vulgaris were antagonistic against Schineria larvae, Acinetobacter johnsonii, and Staphylococcus aureus, but the L2/64 strain of Proteus vulgaris was sensitive to the filtrate of the members of the family Enterobacteriaceae and Acinetobacter johnsonii. Bacillus and Arthrobacter strains were neither producers nor sensitive to products of any other

tested bacteria. The larval extract was active only against the authentic Staphylococcus aureus strain.

Discussion

The appearance of strains of the family Enterobacteriaceae in all Wohlfahrtia magnifica samples agrees with literature data [36–38], as these bacteria are known to be constant natural partners of flies. From this group, members of the genus Proteus and Providencia stuartii seem to be the most dominant partners of the larvae. Results of the sequence comparisons show that some strains of the genus Proteus isolated from the maggots of Wohlfahrtia magnifica may belong to a new species (Table 1). The habitat of Schineria species is most probably the foregut of the maggots. Besides Schineria larvae, one of its close relatives was detected in the foregut sample of the third-stage maggot. These bacteria were sensitive to antimicrobial compounds produced by other bacteria, among them Proteus vulgaris, isolated from the fly. According to our data, the shifts in the Schineria and Proteus populations within the larvae are strongly influenced by their interactions to each other and among the members of the family Enterobacteriaceae. Schineria larvae has a strong chitinase activity [35]; thus it may contribute to the development of fly larvae. It is known that Pseudomonas species play a role in the pupation of the fly Cochliomyia hominivorax by their chitinase enzyme production [39]. The fact that the highest ratio of strains with this enzyme activity was

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detected among cuticle-originating strains, and the lack of chitinase activity among pupae and imago-originating strains suggests that bacteria may also influence the metamorphosis of Wohlfahrtia magnificaa, although mainly in the development of maggots. The presence of members of the genus Bacillus in all Wohlfahrtia samples is normal as these bacteria (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus) were detected from all previously examined flies [11, 36, 37]. The presence of Bacillus thuringiensis/cereus strains in the organ samples of Wohlfahrtia magnifica is extremely interesting. Unfortunately, Bacillus thuringiensis and Bacillus cereus can only be distinguished by their protoxin production and/or the presence of protoxin genes. As a possible pest control Bacillus thuringiensis strains are of interest, because some are effective against flies [40]. In this case, however, there were no characteristic visible symptoms of disease in the third-stage maggots. Bacteria of the Arthrobacter–Micococcus group are often isolated from the digestive tract of invertebrate animals [3, 41–43]; thus their appearance in our samples is not surprising. All of our strains produced chitinase enzymes, so they may accordingly play a role in the metamorphosis of Wohlfahrtia magnifica. Aureobacterium species have also been isolated from insect cadavers [44] and Enterococcus faecalis is widespread in the digestive tract samples of invertebrate animals [45]. The reason for sheep death due to Wohlfahrtia myiasis is often blood hyperammonaemia caused by Wohlfahrtia magnifica maggots [6]. The relatively high urease activity among the isolated strains suggests that bacteria can also contribute to the disease process. The composition of the bacterial communities of the Wohlfahrtia maggot cuticle sample is similar to that found earlier in myiatic vulval mucosa samples [11, 12]. Streptomyces and Variovorax species most often occur in soils [46]. The soil origin of Streptomyces and Variovorax strains from Wohlfahrtia can not be neglected as the pupation of the fly occurs in soils. The presence of Rhodococcus fascians in cuticle samples agrees with the data of Khoga et al. [13], who isolated bacteria from the healthy skin and myiatic lesions of sheep. When his strain cultures were used as baits for flies in field experiments, cultures of strains identified as Rhodococcus fascians and Mycobacterium aurum proved to be the best fly attractants, producing various volatile sulfur compounds, toluene, and benzene. In the case of Cochliomyia hominivorax (Diptera: Calliphoridae), Proteus mirabilis was found to produce antimicrobial compounds (phenyl acetic acid and phenyl acetaldehyde) active against a series of other bacteria (Staphylococcus sp., Salmonella sp., etc.) [47, 48]. In our experiments, the authentic strain of Staphylococcus aureus was sensitive to the antimicrobials of Proteus vulgaris,

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Providencia stuartii (in cuticule, foregut, and salivary gland samples, the dominance of members of genus Providencia could be demonstrated), which is a codominant bacterial partner of flies [37]), Escherichia coli, and Acinetobacter johnsonii. According to our results, these bacteria play a definite role in the disappearance of pyogenic cocci from Wohlfahrtia-stricken wounds. At the same time, catabolites of the larvae are also responsible for this effect (Table 4). Members of the genus Microbacterium could be detected in Wohlfahrtia third-stage maggot foregut and hindgut samples. Previously, the absence of these bacteria was shown from myiatic lesions, although they were present in healthy sheep skin surfaces [12]. Wohlfahrtia larvae may feed on these bacteria. As can be seen from our data, the microbial communities of different developmental stages are more diverse than in the case of healthy skin surfaces or myiatic regions [12]. At time of their hatching, first-stage larvae mainly carry their autochthonous microbe partners. With the infection of the sheep, they can acquire several bacterium partners also from the host animal and from their surroundings. The shifts in the culturable bacterial communities during the metamorphosis of the parasitic fly are most probably influenced by the developmental processes of Wohlfahrtia magnifica, the connection of the fly to its host animal (sheep), and also by the microbial interactions within the maggots, pupae, and adults. At the same time, until now there have been no data on the unculturable parts of the microbial communities within this fly although, in most cases, only 0.1–10% of bacteria can be cultivated on different media [49, 50]. It is known that with cloning and sequence analysis of ribosomal genes, different habitats are successfully described [51–53]. To describe all the natural bacterial communities of W. magnifica, it would be indispensable to use also culture-independent methods.

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