Comprehensive phylogeny, biogeography and new classification of the diverse bee tribe Megachilini: Can we use DNA barcodes in phylogenies of large genera?

Classification and evolutionary studies of particularly speciose clades pose important challenges, as phylogenetic analyses typically sample a small proportion of the existing diversity. We examine here one of the largest bee genera, the genus Megachile - the dauber and leafcutting bees. Besides presenting a phylogeny based on five nuclear genes (5480 aligned nucleotide positions), we attempt to use the phylogenetic signal of mitochondrial DNA barcodes, which are rapidly accumulating and already include a substantial proportion of the known species diversity in the genus. We used barcodes in two ways: first, to identify particularly divergent lineages and thus to guide taxon sampling in our nuclear phylogeny; second, to augment taxon sampling by combining nuclear markers (as backbone for ancient divergences) with DNA barcodes. Our results indicate that DNA barcodes bear phylogenetic signal limited to very recent divergences (3-4 my before present). Sampling within clades of very closely related species may be augmented using this technique, but our results also suggest statistically supported, but incongruent placements of some taxa. However, the addition of one single nuclear gene (LW-rhodopsin) to the DNA barcode data was enough to recover meaningful placement with high clade support values for nodes up to 15 million years old. We discuss different proposals for the generic classification of the tribe Megachilini. Finding a classification that is both in agreement with our phylogenetic hypotheses and practical in terms of diagnosability is particularly challenging as our analyses recover several well-supported clades that include morphologically heterogeneous lineages. We favour a classification that recognizes seven morphologically well-delimited genera in Megachilini: Coelioxys, Gronoceras, Heriadopsis, Matangapis, Megachile, Noteriades and Radoszkowskiana. Our results also lead to the following classification changes: the groups known as Dinavis, Neglectella, Eurymella and Phaenosarus are reestablished as valid subgenera of the genus Megachile, while the subgenus Alocanthedon is placed in synonymy with M. (Callomegachile), the subgenera Parachalicodoma and Largella with M. (Pseudomegachile), Anodonteutricharaea with M. (Paracella), Platysta with M. (Eurymella), and Grosapis and Eumegachile with M. (Megachile) (new synonymies). In addition, we use maximum likelihood reconstructions of ancestral geographic ranges to infer the origin of the tribe and reconstruct the main dispersal routes explaining the current, cosmopolitan distribution of this genus.

A study of the relationships between antibiotic resistance phenotypes, phage-typing and biotyping of 117 clinical isolates of Acinetobacter spp.

Two typing systems were used to conduct an epidemiological study of Acinetobacter and to establish their relationship to antibiotic resistance phenotypes. Biotyping was performed with biochemical tests according to the new definition of Acinetobacter baumannii (18 biotypes). Phage typing included two complementary systems: 125 phage-types and 25 subtypes. Resistance phenotype analysis included 11 antibiotics. The results of the study showed that: (1) nine phage-types or subtypes (67%) and two groups of atypical phage-types (5%) or of untypable strains (28%), could be defined; (2) all strains that were resistant to carboxy/ureido-penicillins and cephalosporins (62%) belonged to biotypes 6 or 9; among them 70% belonged to phage-types 17 or 124; (3) imipenem resistance was observed in five isolates of biotype 9 and one of biotype 6; (4) a phenotype including resistance to third generation cephalosporins (but not carboxypenicillins) and to amikacin (but not tobramycin) represented 8.5% of the isolates; 90% of them belonged to biotype 1 and were not phage-typable; (5) 15% of the isolates were not identified as A. baumannii; among them five Acinetobacter haemolyticus strains all had the same resistance phenotype: amikacin-tobramycin-kanamycin-netilmicin resistant; they were however, susceptible to beta-lactams and to gentamicin. There was a clear relationship between biotypes 6 and 9 and phage-types 17 and 124 which were the strains most resistant to beta-lactams and aminoglycosides and were predominant in the survey. The three typing systems were complementary but it seems that antibiotic resistance phenotypes and one of the two other typing systems would be required in parallel to provide suitable information for epidemiological purposes.

Dissemination of amikacin resistance gene aphA6 in Acinetobacter spp.

The distribution of the aphA6 gene, encoding a 3'-aminoglycoside phosphotransferase type VI, was studied by dot blot hybridization with 115 amikacin-resistant Acinetobacter strains from various geographical areas. Nucleotide sequences related to aphA6 were found in 109 strains belonging to seven species. As inferred from results of Southern hybridization, dissemination of amikacin resistance in Acinetobacter spp. is due to a gene rather than a strain or plasmid epidemic.

[Epidemiology of Acinetobacter and resistance to antibiotics at hospitals. A 5-year evaluation]. / Epidémiologie et résistance aux antibiotiques des Acinetobacter en milieu hospitalier. Bilan de 5 années.

The growing number of Acinetobacter strains in hospitals and the rapid increase of their resistance to antibiotics have prompted us to undertake a long-term epidemiological study of this resistance at the Bichat hospital, Paris. Between 1971 and 1984, the resistance of Acinetobacter to antibiotics had already progressed, with only some antibiotics (imipenem, ceftazidime, tobramycin and amikacin) remaining active. During the following 5 years (1984-1988) a study of 1056 strains demonstrated a further increase of resistance and showed how serious the problem was in intensive care units. During the last few years, there has been a considerable increase in the proportion of multiresistant strains, reaching 84 per cent with beta-lactam antibiotics and 64 per cent with aminoglycosides. At present, in most cases the only effective treatment is imipenem, and no antibiotic is active in 5.5 per cent of the cases. Studies of lysotypes, enzymes and phenotypic resistance of bacterial strains completed the epidemiological approach, showing the presence of dominant lysotypes. Two predominant lysotypes are associated with multiresistance of Acinetobacter strains responsible for nosocomial infections.

Species, biotype, and bacteriophage type determinations compared with cell envelope protein profiles for typing Acinetobacter strains.

Species, biotypes, and phage types were determined for 120 Acinetobacter strains from clinical or environmental sources or from culture collections. These characteristics were compared with cell envelope protein profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in previous studies. A considerable heterogeneity of species and types was observed by use of the various methods, in particular among strains from different sources. Acinetobacter baumannii was the most commonly found species in isolates from clinical sources, followed by Acinetobacter species 3. Nine biotypes were observed among A. baumannii strains. Further differentiation within most species and biotypes was achieved by protein profile typing and, to some extent, phage typing. Of 120 strains, 49 (41%) were not typeable by phages. Consistent results for the various classification methods were obtained for strains from common sources. Biotyping seemed an appropriate method for the screening of strains, whereas protein profile and phage typing could serve as additional methods to establish the identity or nonidentity of strains. Results of this study suggest that the combination of the typing methods is useful in epidemiological studies.

A comparative assay of epidemiological markers for Acinetobacter strains isolated in a hospital.

A comparative assay for epidemiological evaluation of three different Acinetobacter typing procedures, i.e. biotyping, phage-typing, and the analysis of the bacterial envelope protein profiles, was carried out using sixty-four multiresistant Acinetobacter strains isolated from clinical specimens. The antibiotic susceptibility of the strains was also considered. After geno-species identification, biotyping allowed the recognition of a relatively large and long-lasting presence, at an Intensive Therapy Unit, of two A. baumannii biotypes. Phage-typing and the analysis of the susceptibility to antibiotics allowed for the differentiation of strains belonging to different geno-species and biotypes, and in some cases also to the same biotypes. On the contrary, the analysis by polyacrylamide gel electrophoresis of the cell-envelope proteins failed to show any diversity not only within, but also between some of the biotypes of A. baumannii, the most prevalent species of the genus in the hospital environment.

Morphology of Pseudomonas aeruginosa typing phages of the Lindberg set.

Eighteen phages were studied by electron microscopy. They belonged to eight morphotypes representing the Myoviridae, Siphoviridae and Podoviridae families of tailed phages. Twelve phages were members of known species and six phages belonged to three new species. Results correlated closely with serological and DNA-DNA hybridization data. Induced and non-induced propagating bacteria were investigated for lysogeny. All strains produced R-type pyocins, phage-like particles or inhibitory reactions. The identity of typing phages should be controlled periodically.

[d-Tartrate-positive Salmonella paratyphi B (var. java): lysotype of 1200 strains isolated in France (1975-1985)]. / Salmonella paratyphi B d-tartrate positif (var. java): lysotypie de 1200 souches isolées en France (1975-1985).

The 1200 d-tartrate positive (dt+) strains of S. paratyphi B, isolated in France during the period 1975-1985, were studied with the aid of the Felix and Callow phage-typing system and the results were compared with those of the study of 1945 d-tartrate negative (dt-) strains of S. paratyphi B examined by means of the same phage-typing method. 2359 dt+ and dt- strains were typable and could be assigned to 25 recognized phage-types. A great variation in the number of strains of each biovar was observed in some phage-types. The proportion of dt+ strains was very high (more than 80%) in four of the most frequent phage-types: 1 var. 3 (87.5%), 1 var. 4 (88.1%), Battersea (90.4%), Worksop (100%); but this proportion was low or very low in eight other frequent phage-types: 1 (29%), BAOR (21.1%), 3aI var. 1 (6.1%), Beccles (5.1%), 3aI var. 4 (4.8%), Dundee (4.7%), 3aI (2.7%), Taunton (2.1%). No recognized phage-type found in France can be considered to be specific of the variety java of S. paratyphi B, with the exception of the phage-type Workshop of all which 76 strains were dt+. On the other hand, 422 dt+ strains were atypical and 93 other were untypable by phages. Among 211 dt+ strains isolated from frozen foods imported from South East Asia, 81.3% were atypical. Six new atypical phage-types were defined and proposed for inclusion into the phage-typing schema which is commonly used in epidemiological investigations on human and animal infections due to either of the two biovars of S. paratyphi B.

[The antigen H:z66 in 1,000 strains of Salmonella typhi from the Antilles, Central America and South America]. / Recherche de l'antigène H:z66 chez 1,000 souches de Salmonella typhi provenant des Antilles, d'Amérique Centrale et d'Amérique du Sud.

The research of the flagellar antigen H:z66 of Salmonella typhi was performed in 1,000 strains from the West Indies, Central America and South America. A method based on the immobilization of motile strains in soft agar with immune serum anti-H: d was used to detect strains carrying this flagellar antigen. No strains had antigen H:z66 irrespective to their biovar, phage-type, colicinogeny, drug susceptibility and geographical origin. Future investigations are needed for a better knowledge of the worldwide distribution of Salmonella typhi strains carrying antigen H:z66.

[Prevalence of lysotype No. 3 of Salmonella paratyphi A in Peru]. / Prédominance du lysotype No 3 de Salmonella paratyphi A au Pérou.

Sixty-nine S. paratyphi A strains isolated from Peru were studied with the aid of the phage-typing system of Banker. 55 strains (79.7%) showed specific lytic reactions of the phage-type no 3 and 12 strains (17.3%) those of the phage-type no 1; one strain was atypical and another untypable. All of these strains were susceptible to ampicillin, chloramphenicol, streptomycin, tetracycline and trimethoprim. These results are compared with the data of the scientific literature on the worldwide distribution of the S. paratyphi A phage-types.

[Epidemiology of Pseudomonas aeruginosa hospital infection in the Ivory Coast]. / Epidémiologie de l'infection hospitalière à Pseudomonas aeruginosa en Côte-d'Ivoire.

210 P. aeruginosa hospital strains isolated at Abidjan from pathological samples or contaminated material were studied by means of serotyping, biotyping, phage-typing and for their resistance to eleven antibiotics. 87% were classified into 14 serogroups: O:1, O:2, O:3, O:4, O:5, O:6, O:8, O:9, O:10, O:11, O:12, O:13, O:15, O:16. No strains were O:7 or O:14. The presence of an ortho-nitrophenyl beta-d-galactopyranoside hydrolase was demonstrated in 42 strains belonging to the serogroup O:11. Sixty-five phage-types were defined but 37% of strains were untypable by phages. The incidence of resistance to each tested antibiotic was as follows: carbenicillin 49%, mezlocillin 44%, ticarcillin 34%, azlocillin 25%, cefamandole 94%, latamoxef 15%, cefotaxime 41%, cefsulodin 32%, tobramycin 35%, amikacin 7%, gentamicin 39%. Thirty-eight strains were multiresistant to carbenicillin, mezlocillin, ticarcillin, azlocillin, and four strains to eleven antibiotics. These results are compared with data of the scientific literature on the epidemiology of hospital Pseudomonas infection.

[Epidemiology of Pseudomonas aeruginosa in Mauritania: study of 239 strains of various origins]. / Epidémiologie de Pseudomonas aeruginosa en Mauritanie: étude de 239 souches d'origines diverses.

239 strains (173 from hospital infections, 20 from carriers, 46 from animal infections and environment) were studied with regard to their serogroups, biovars, phage-types and drug-resistance. 82.2% of all strains were typable by specific immune sera and distributed in 15 serogroups (0:1, 0:2, 0:3, 0:4, 0:5, 0:6, 0:7, 0:8, 0:9, 0:10, 0:11, 0:12, 0:13, 0:15, 0:16). Among them, 8 were ubiquitous (0:1, 0:3, 0:4, 0:5, 0:8, 0:9, 0:11 and 0:16) and found in hospital patients, carriers, animals and environment. Thirty-five 0:11 strains and four no agglutinable strains produced an orthonitrophenyl beta-d-galactopyranoside hydrolase (ONPG +). Among 183 strains typable by phages of the Lindberg phage-typing system, 96 different phage-types were recognized. The proportions of the strains untypable by phages were 8.6% for animal strains, 23.6% for hospital strains and 55% for strains from carriers. The frequency of the resistance to each of eleven tested antibiotics was as follows: carbenicillin: 48%, ticarcillin: 12.2%, azlocillin: 9.1%, cefotaxime: 22.8%, cefsulodin: 27.4%, ceftazidime: 0.5%, latamoxef: 6.2%, gentamycin: 12.2%, amikacin: 15.4%, tobramycin: 12.5%, norfloxacin: 0%. However, 18.7% of hospital strains, 65% from carriers and 92.5% of animal or environmental strains were susceptible to all of these antibiotics. These results are compared with those from a previous report on epidemiology of P. aeruginosa hospital infection in Ivory-Coast (Dosso, Vieu et al., Bull. Soc. Path. exot., 1987, 80, 19-27) and with the data of the scientific literature.

Plasmid-mediated resistance to multiple antibiotics in Salmonella typhi.

Two hundred forty-one strains of Salmonella typhii isolated in Lima, Peru, from October 1981 through February 1983 were tested for susceptibility to antimicrobial agents. Seventy-two strains (29.9%) were resistant to chloramphenicol and other antibiotics, including ampicillin, sulfonamides, and trimethoprim. The minimal inhibitory concentrations of 17 antimicrobial agents were determined for all of these chloramphenicol-resistant strains. Sch 25393, new beta-lactams, new quinolones, and the formulation clavulanic acid-amoxicillin were effective against all the strains. Four different resistance patterns distributed among eight phage types were observed. The 72 resistant S. typhi could transfer the resistance marker into Escherichia coli C1, and all the plasmids belonged to the incompatibility group H1.

[Absence of the antigen H:z66 in 2355 strains of Salmonella typhi from Madagascar and several countries of tropical Africa]. / Absence de l'antigène H:z66 chez 2355 souches de Salmonella typhi provenant de Madagascar et de plusieurs pays d'Afrique tropicale.

The presence of the new flagellar antigen H:z66 (Guinée, 1981) was investigated among 2,355 strains of S. typhi isolated from 1981 to 1985 in Madagascar and some countries of tropical Africa: Burundi, Ivory Coast, Gabon, Mauritania, Central African Republic, Rwanda, Senegal, Zaïre. A method based on the immobilization of motile strains in soft agar with immunserum anti-H:d, was used to detect strains carrying antigen H:z66. No African and malagazy strains had antigen H:z66, irrespective to their biovar, phage-type, drug susceptibility and geographical origin. These results were compared with a study of 2,121 indigenous or imported strains of S. typhi isolated in France during the same period. Except 4 strains from patients contaminated in Indonesia, all of them were also devoid of antigen H:z66. Future research is needed for a complete assessment of the geographical distribution of S. typhi antigen H:z66.

[Is there a geographic epidemiology of serogroups O of Pseudomonas aeruginosa?]. / Existe-t-il une épidémiologie géographique des sérogroupes O de Pseudomonas aeruginosa?

3720 human strains of P. aeruginosa from Egypt, France, Greece, Iracq and Portugal were studied by serotyping, phage typing, and ONPG-test. The comparison of the results with the data of the scientific literature suggests that three classes of P. aeruginosa O groups can be characterized on the basis of their geographical distribution in the world: 1 degree Nine serogroups with worldwide distribution: 0:1, 0:2, 0:3, 0:4, 0:5, 0:6, 0:8, 0:9 and 0:11. 2 degrees Five serogroups absent (or very rare) in a few countries but present in others: 0:7, 0:10, 0:12, 0:13 and 0:16. 3 degrees Two serogroups very rare in all countries: 0:14 and 0:15.

[Biotypes of Salmonella typhimurium in Iraq]. / Biovars de Salmonella typhimurium en Irak.

207 Salmonella typhimurium strains isolated from childrens below three years (190 strains), adult patients (8 strains), health carriers (7 strains) or animals (2 strains) were studied with the biotyping scheme of Cordano, Richard and Vieu (1971). Five biovars were found and 76.8% of the strains were TTR+, Ino+, Tre+, d. Tar- (biovar d). In Irak the epidemiology of the Salmonella typhimurium human infections is associated with an high frequency of strains of Salmonella typhimurium 0:5- (var. Copenhagen), biovar d.

Classification of Vibrio bacteriophages.

85 Vibrio phages, 84 of them tailed and 1 filamentous, were surveyed. The tailed phages belonged to six basic morphotypes and to the Myoviridae, Siphoviridae, or Podoviridae families. 63 phages were classified into 18 species. The filamentous phage is a member of the Inovirus genus of the Inoviridae family. Vibrio phages are very heterogenous and include some morphologically interesting viruses. Several Vibrio phages closely resemble phages of other gram-negative bacteria, possibly indicating phylogenetic relationships between their hosts.

Phage-types and susceptibility to 26 antibiotics of nosocomial strains of Acinetobacter isolated in Portugal.

Sixty-two strains of Acinetobacter calcoaceticus isolated from pathological samples or from the environment in several hospitals in Lisbon, were studied by means of two complementary phage-typing systems. Eighteen phage-types or sub-types, one group of uncommon types (9.6%) and one group of untypable strains (20.9%) were found. A new phage-type (No. 104) and a new sub-type (No. 18) were defined among the Portuguese strains. The in vitro activity of 26 antibiotics against 48 nosocomial strains was studied by the Kirby-Bauer method. The most active antibiotics were, in decreasing order, amikacin, carbenicillin, tobramycin, minocycline, dibekacin, doxycycline, sisomycin, trimethoprim-sulphamethoxacole, ticarcillin and piperacillin. The strains were also tested against 4 new beta-lactam antibiotics, and the MICs were determined by an agar dilution method. N-formimidoyl-thienamycin was found to be the most active against Acinetobacter and ceftazidime was considerably more active than apalcillin and cefotaxime.