Laboratory bioassays and field-cage trials of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. isolates with field-collected Mormon crickets (<Emphasis Type="Italic">Anabrus simplex</Emphasis>)

The Mormon cricket (MC) is an important pest in the western United States. This study evaluated the virulence in the laboratory of 32 isolates of Metarhizium spp. towards field-collected MCs. Additionally, four isolates were tested in outdoor field-cage studies. All 32 Metarhizium isolates were pathogenic towards the MC (could induce some mortality in the laboratory), including four isolates of the grasshopper-specialist species M. acridum. Virulence varied considerably among the isolates. Field studies, conducted in 2008–2009, showed a statistically significant effect of fungal treatments in both years. Pairwise comparison of the survival curves, however, revealed that, in 2008, three of the isolates did not differ statistically from the non-fungus control treatment. In 2009, all three of the isolates tested had significantly lower survival rates than the control treatment: MCs exposed to M. robertsii isolate DWR 346 had the lowest survival with a LT₅₀ of 16 days. We hypothesize that the poor field performance resulted from a combination of negative environmental effects and isolate selection, and propose that further field studies with additional isolates are needed to identify an efficacious fungal agent for MC biocontrol.     

Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.)

AIMS: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. METHODS AND RESULTS: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1.9 x 10(5) to 1.8 x 10(6) CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. CONCLUSIONS: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.     

Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

Isolation and characterization of a new subspecies ofMycobacterium chelonei infectious for salmonid fish

Rapidly growing, nonchromogenic mycobacteria were isolated from salmonid fish at five locations in the states of Oregon and Montana, USA. The isolates were characterized by biochemical, physiological, genetic and mycolic acid properties, then subjected to taxonomic analysis. Detection of mycobacterial mycolic acids and a percent guanine plus cytosine value of 63 ± 1.7 mol% confirmed that the isolates belong to the genusMycobacterium. The internal similarity of the isolates was 94.2 ± 3.4 %. None of the isolates grew at 37 °C. A comparison of their properties with those of other rapidly growing, nonchromogenic and photochromogenic mycobacteria was made. The salmonid isolates showed a relationship toM. chelonei subspecieschelonei andM. chelonei subspeciesabscessus, but had biochemical properties which were intermediate to these two subspecies. Acid methanolysates of the salmonid isolates, analyzed by two dimensional thin-layer chromatography, produced lipid patterns identical to those of both subspecies ofM. chelonei. Sufficient differences in biochemical properties and the inability to grow at 37 °C suggest these isolates be regarded as a new subspecies ofM. chelonei. We propose the nameM. chelonei subspeciespiscarium subsp. nov. (L. adj.piscarius of fish). The isolates were not infectious for mice. Experimental infections were produced in juvenile salmonid fish. The occurrence of mycobacterial infections in selected salmonid populations from Oregon hatcheries and the Pacific Ocean ranged from 0 to 26 %.     

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of cultured mycobacteria in a diagnostic laboratory

Background

The development of DNA amplification for the direct detection of M. tuberculosis from clinical samples has been a major goal of clinical microbiology during the last ten years. However, the limited sensitivity of most DNA amplification techniques restricts their use to smear positive samples. On the other hand, the development of automated liquid culture has increased the speed and sensitivity of cultivation of mycobacteria. We have opted to combine automated culture with rapid genotypic identification (ARDRA: amplified rDNA restriction analysis) for the detection resp. identification of all mycobacterial species at once, instead of attempting direct PCR based detection from clinical samples of M. tuberculosis only.

Results

During 1998–2000 a total of approx. 3500 clinical samples was screened for the presence of M. tuberculosis. Of the 151 culture positive samples, 61 were M. tuberculosis culture positive. Of the 30 smear positive samples, 26 were M. tuberculosis positive. All but three of these 151 mycobacterial isolates could be identified with ARDRA within on average 36 hours. The three isolates that could not be identified belonged to rare species not yet included in our ARDRA fingerprint library or were isolates with an aberrant pattern.

Conclusions

In our hands, automated culture in combination with ARDRA provides with accurate, practically applicable, wide range identification of mycobacterial species. The existing identification library covers most species, and can be easily updated when new species are studied or described. The drawback is that ARDRA is culture-dependent, since automated culture of M. tuberculosis takes on average 16.7 days (range 6 to 29 days). However, culture is needed after all to assess the antibiotic susceptibility of the strains.

     

Isolation of extremely halophilic <Emphasis Type="Italic">Archaea</Emphasis> from a saline river in the Lut Desert of Iran,moderately resistant to desiccation and gamma radiation

Admittedly, the Lut Desert of Iran has been remained as an unexplored region from a microbiological standpoint. Domain Archaea contains extremophiles that can live in harsh habitats. Extremely halophilic archaea are exposed to different environmental stresses in the hypersaline environments such as high solar irradiance and periodic desiccation. Haloarchaeal diversity in Shoor River, a saline river in the Lut Desert (a salinity of 134.3 g L^–1 of dissolved salts), was investigated by a culture-dependent method. A large number of extremely halophilic isolates were obtained and a subset of 59 isolates was considered distinct. Firstly, the isolates were screened for their resistance under desiccation stress in 35 days. Eleven of these strains remained viable during the period in a desiccator containing silica gel. Then, three of them were randomly selected and their resistance against desiccation and ionizing radiation were determined. The isolates MS2, MS17, and MS50 were still recovered after 8 weeks in a desiccator and were moderately resistant to gamma radiation with D₁₀ value between 2 and 3 kGy. Strains MS2, MS17, and MS50 were affiliated with three species in the family Halobacteriaceae using 16S rRNA gene sequence analysis as well as morphological and biochemical characteristics—Haloterrigena jeotgali A29^T (99.6% similarity), Natrialba aegyptia 40^T (99.4% similarity) and Natrinema pallidum NCIMB 777^T (99.3% similarity), respectively. Although resistance to desiccation did not follow the sigmoid survival curve pattern of Deinococcus radiodurans, apparently haloarchaea can show a more resistance to desiccation in more long-term periods of time. This is the first report on isolation of extremely halophilic archaea belonged to the family Halobacteriaceae and their radioresistance and desiccation tolerance properties isolated from the Shoor River.     

Pathogenic Escherichia coli found in sewage treatment plants and environmental waters

We previously demonstrated that some Escherichia coli strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264 E. coli isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93 E. coli strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic E. coli (IPEC) or uropathogenic E. coli (UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2(2), B2(3), D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental E. coli in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.     

Multilocus sequence typing for phylogenetic view and <Emphasis Type="Italic">vip</Emphasis> gene diversity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains of the Assam soil of North East India

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes’ crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.     

Phylogenetic and phenotypic analyses of arsenic-reducing bacteria isolated from an old tin mine area in Thailand

An agar plate screening assay was used to determine whether 100 arsenic-resistant bacterial isolates, previously obtained from arsenic-contaminated soils, had the ability to transform arsenite and arsenate. Ninety-five percent of the isolates were capable of reducing arsenate on agar plates. The isolates also grew in the presence of high concentrations of arsenite, but none of the bacterial isolates oxidized arsenite to arsenate under the growth conditions tested. About 14 % (13 of 95) of the tested isolates transformed high levels of arsenate (33–70 μM) when tested using the molybdenum blue method. Partial sequence analysis of 16S rDNA genes indicated that the isolates belonged to two broad taxonomic groups: Firmicutes and Proteobacteria. Ten isolates were assigned to four species in the genus Bacillus, and three isolates belonged to two species in the genera Enterobacter and Ochrobactrum. Taken together these results indicate that phylogenetically diverse bacteria isolated from arsenic-contaminated soils in an old tin mine area in Thailand have the ability to transform arsenate to arsenite.     

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

Background

Tuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.

Results

On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.

Conclusions

Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.     

Variability in virulence of Beauveria spp. soil isolates against Ostrinia nubilalis

European corn borer (Ostrinia nubilalis) is an important maize pest which is treated mainly by synthetic pesticides with many negative effects for ecosystem and human health. Soil borne entomopathogenic fungi of species belonging to Beauveria genus may provide an alternative strategy for biological control of this pest. In this article, we report results of virulence testing of 46 soil isolates of Beauveria spp. against O. nubilalis in relation to isolates origin and haplotype. All strains were collected from soil using Galleria mellonella bait method. Conidia suspension with concentration 10⁷ conidia in ml was used for virulence test against 4th instar larvae of O. nubilalis at temperature 25°C. After 14 days, mortality of larvae was in the range 34%–96%. Median lethal time LT50 was estimated to 5.5–21.3 days. Mortality was not in relation to habitat or any other environmental parameter of sites where isolates came from. Differences among species were insignificant. Isolates were divided into 8 genetic lineages and 14 haplotypes using sequencing of ITS and Bloc nuclear regions. None of the lineages showed higher mortality than others. Despite more or less virulent isolates being found in each lineage, the three most virulent isolates belonged to the same lineage Beauveria bassiana A3. The four most virulent isolates were compared to commercial isolate GHA to assess medium lethal concentration. LC50 were estimated in range 7.5 × 10⁴ to 9.5 × 10⁵. Three of the four isolates showed equal or greater efficiency than strain GHA.     

Pyrolysis mass spectrometry characterisation and numerical taxonomy ofAeromonas spp.

Reference strains (2) and 29 isolates ofAeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of typical isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate betweenA. caviae andA. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis forA. caviae andA. sobria, but the main group ofA. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate betweenA. hydrophila andA. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains ofA. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable nonpathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.Abbreviations CTRP conventional test reaction pattern - PyMS pyrolysis mass spectrometry     

Population analysis of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> by using endogenous repetitive DNA sequences and mating-type alleles in different districts of Karnataka,India

Rice is the staple food crop of more than 60% of the population of the world. This crop suffers from blast disease caused by Magnaporthe oryzae. Information on the mating-type allele distribution and diversity of the pathogen population for the state of Karnataka, India is scanty. With this background, a total of 72 isolates of M. oryzae from rice in different districts of Karnataka were examined for identifying sexual mating alleles MAT1, MAT2 and understanding the genetic diversity based on DNA fingerprint of pot2, an inverted repeat transposon. Among 72 isolates, 44 isolates belonged to MAT1 type (male fertile) and 28 isolates were of MAT2 (female fertile) and there were no hermaphrodite isolates. In a given geographical location, only one mating type was identified. Results revealed that the isolates obtained from these regions are not sexually fertile showing predominant asexual reproduction. Hence, genetic variation observed in the pathogen may be mainly because of high copy number of transposons. A high copy number transposon, namely Pot2, was selected in our study to detect genetic diversity of the pathogen. Pot2 rep-PCR DNA fingerprinting profile showed 27 polymorphic bands with bands ranging in size from 0.65 to 4.0 kb and an average of 10 to 14 bands per isolate. Five distinct clusters were formed with two major, two minor, and one outlier. Clusters 4 and 5 are further subdivided into three sub-clusters. Some of the isolates belonging to clusters 3, 4, and 5 are interlinked as these locations are close to one another sharing common geographical parameters and boundaries. This knowledge on the sexual behavior and genetic diversity of M. oryzae is important with respect to breeding for disease resistance.     

Virulence of entomopathogenic hypocrealean fungi infecting <Emphasis Type="Italic">Anoplophora glabripennis</Emphasis>

Twenty isolates of four species of entomopathogenic hypocrealean fungi (Beauveria bassiana, Beauveria brongniartii, Isaria farinosa, and Metarhizium anisopliae) were found to be pathogenic to adults of the Asian longhorned beetle, Anoplophora glabripennis. Survival times for 50% of the beetles tested (ST₅₀) ranged from 5.0 (M. anisopliae ARSEF 7234 and B. brongniartii ARSEF 6827) to 24.5 (I. farinosa ARSEF 8411) days. Screening studies initially included strains of B. brongniartii, which is registered as a microbial control agent in Europe, Asia and South America but not in North America. At that time, we could not confirm that this fungal species is native to North America which added uncertainty regarding future registration of this species for pest control in the USA. Therefore, subsequent bioassays documented median survival times for three M. anisopliae isolates (5–6 days to death) and two of these isolates are suggested for further development because they are already registered for pest control in the USA. An erratum to this article can be found at     

Characteristics of bacteria isolated by the anaerobic roll-tube method from cheeses and ground beef.

In this study the methods of Hungate were used to quantitate the anaerobic bacteria present in commercially available ground beef, cheddar cheese, and German hand cheese. Of 235 anaerobic roll-tube isolates from ground beef and German hand cheese, all were facultative anaerobes. Of 213 anaerobic roll-tube isolates from cheddar cheese, 91% were facultative anaerobes and 9% were obligate anaerobes. Using results of biochemical tests, 14 or the 17 obligately anaerobic isolates from cheddar cheese were Propionibacterium acnes, two were strains of Propionibacterium that could not be speciated, and one was tentatively identified as a strain of Streptococcus evolutus. Obligate anaerobes were estimated to be present in the cheddar cheese at a level of about 10(6)/g. The possible significance of these levels of P. acnes in nonsterile foods is discussed.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65(PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.     

Biochemical tests for identification of mycelial cultures of basidiomycetes

Twenty-four biochemical tests have been developed to identify basidiomycetes from mycelial cultures. Eleven are designated ‘instant tests’ because results are obtained within 24 h; ten of these test for specific enzymes, the other is based on a reaction between vanillin and unknown fungal metabolites. The remaining thirteen tests require incubation periods ranging from 1 to 4 days and are thus designated ‘incubated tests’. Sixteen of these tests were applied first to identified isolates of Peniophora sacrata, Sclerotium rolfsii, Stereum purpureum and Trametes versicolor. They distinguished these species satisfactorily, so these sixteen tests, and eight others devised subsequently, were applied to unidentified basidiomycetes isolated from diseased roots of fruit crops. The results of these tests enabled the isolates to be placed in seven groups. All isolates in a particular group were morphologically similar which further suggested that each group might correspond to a species and that isolates could be defined on the basis of biochemical tests.     

Wild Animal Mycobacterial Isolates

Thirteen strains of mycobacteria isolated from deer and various species of wild birds were analysed by gas chromatography (GG) for cellular fatty acids and by thin-layer chromatography (TLG) for polar lipids. These strains were compared to reference strains of Mycobacterium avium, M. para tuberculosis and M. mal-moense. All the examined strains exhibited a generally similar fatty acid pattern characterized by relatively large amounts of hexadenca-noate (16:0), octadecenoate (18:1), octadecanoate (18:0) and 10-me-thyl-octadecanoate (tuberculostearic acid, 10-Me-18:0). Several additional acids were also generally present but in smaller amounts. By means of small but distinct differences in fatty acid composition, the wild animal isolates could be distinguished from both M. paratuber-culosis and M. malmoense but not from M. avium. The TLG polar lipid patterns on the other hand separated the wild animal isolates into 2 distinct groups of complex and simple polar lipid composition which corresponded to the morphologically smooth and rough types, respectively. The complex patterns of the smooth strains were comparable to those of the M. avium serovars whereas both the rough wild animal isolates and all the M. paratuber-culosis strains showed a simple pattern of polar lipids. Both fatty acid profiles and TLG polar lipid patterns support allocation of the wild animal isolates to the MAIS complex. Moreover, the 2 chemical techniques, particularly the GC procedure, are very useful for a more rapid and precise identification of the slow-growing wild animal mycobacterial isolates which have hitherto been characterized on basis of vague criteria.     

Extended-Spectrum β-Lactamases and/or Carbapenemases-Producing Enterobacteriaceae Isolated from Retail Chicken Meat in Zagazig,Egypt

Objectives

The aim of the present study was to determine the prevalence and to characterize extended-spectrum β-lactamases- and/or carbapenemases-producing Enterobacteriaceae among Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt.

Methods

One hundred and six Enterobacteriaceae isolates were collected from retail chicken meat samples purchased in Zagazig, Egypt in 2013. Species identification was done by MALDI-TOF MS. Screening for ESBL-E was performed by inoculation of isolates recovered from meat samples onto the EbSA (Cepheid Benelux, Apeldoorn, the Netherlands) selective screening agar. ESBL production was confirmed by combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark). Carbapenemases production was confirmed with double disk synergy tests. Resistance genes were characterized by PCR with specific primers for TEM, SHV, and CTX-M and carbapenemases (KPC, NDM, OXA-48, IMP and VIM). PCR products of CTX-M genes were purified and sequenced. Phylogenetic grouping of E. coli was performed by a PCR-based method.

Results

Of these 106 isolates 69 (65.09%) were ESBL producers. Twelve (11.32%) of these isolates were also phenotypically class B carbapenemases producer. TEM genes were detected in 61 (57.55%) isolates. 49 (46.23%) isolates harbored CTX-M genes, and 25 (23.58%) carried genes of the SHV family. All CPE belonged to the NDM group. The predominant CTX-M sequence type was CTX-M-15 (89.80%). The majority (80%) of the ESBL-EC belonged to low virulence phylogroups A and B1.

Conclusions

This is the first study from Egypt reporting high rates of ESBLs and carbapenemases (65.09% and 11.32%, respectively) in Enterobacteriaceae isolated from retail chicken meat. These results raise serious concerns about public health and food safety as retail meat could serve as a reservoir for these resistant bacteria which could be transferred to humans through the food chain.