Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates.

Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and delta 8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5' side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3' side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus delta 8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5' and 3' proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.     

Antibiotic resistance patterns of gram-negative bacteria isolated from environmental sources.

A total of 2,445 gram-negative bacteria belonging to fecal coliform, Pseudomonas, Moraxella, Acinetobacter, and Flavobacterium-Cytophaga groups were isolated from the rivers and bay of Tillamook, Oregon, and their resistances to chloramphenicol (25 microgram/ml), streptomycin (10 microgram/ml), ampicillin (10 microgram/ml), tetracycline (25 microgram/ml), chlortetracycline (25 microgram/ml), oxytetracycline (25 microgram/ml), neomycin (50 microgram/ml), nitrofurazone (12.5 microgram/ml), nalidixic acid (25 microgram/ml), kanamycin (25 microgram/ml), and penicillin G (10 IU/ml) were determined. Among fecal coliforms the bay isolates showed greater resistance to antibiotics than those from tributaries or surface runoff. No such well-defined difference was found among other bacterial groups. The antibiotic resistance patterns of gram-negative bacteria from different sources correlated well, perhaps indicating their common origin. The antibiotic resistance patterns of gram-negative bacteria of different general also correlated well, perhaps indicating that bacteria which share a common environment also share a common mode for developing antibiotic resistance.     

The hydrophobicity of gram-negative bacteria isolated from patients with inflammatory and suppurative-destructive lung diseases]

Hydrophobic properties of 11 enterobacterial strains and 12 Pseudomonas aeruginosa strains isolated from patient with inflammatory and purulent destructive diseases of the lungs were studied. The hydrophobic properties of the isolated bacteria were determined by the method of salting out with ammonium sulfate. A high correlation (r = 0.56) between the above-mentioned properties of gram-negative bacteria and their isolation rate from patients with destructive diseases was established. The hydrophobic properties of bacteria were supposed to be of importance in the process of the colonization of pulmonary tissues with bacterial flora.     

Antibiotic sensitivity of gram-negative opportunistic bacteria isolated from the feces of newborn infants with intestinal infections

Antibiotic sensitivity of 136 pure cultures of gram negative aerobic potentially pathogenic bacteria was assayed. The bacteria were isolated from feces of newborns with intestinal infections. It was shown that the majority of the strains were resistant to 4-5 antibiotics widely used in treatment of patients. However, they were sensitive to gentamicin and polymyxin M.     

Improved method of lipopolysaccharide isolation from gram-negative bacteria

A modification of the traditional method for lipoplysaccharide isolation from the cells of grammnegative bacteria was elaborated on the basis of extraction by the hot water solution of phenol (the method of Westfahl). To make the method simpler and to raise the yield of the product it was proposed to use the water-phenol extract without its division for plases. The nucleic acids are eliminated by precipitation from dialyzed extract at pH 3,2-3,4 achieved by addition of acetic acid. The comparative isolation of lipopolysaccharides by the classic and modified methods has confirmed the advantages of a new technique.     

A novel glycosphingolipid from gram-negative aquatic bacteria.

The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.     

Rapid method for distinction of gram-negative from gram-positive bacteria

Summary A rapid method for distinction between gram-negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram-positive and 55 gram-negative bacterial strains. The method proved reliable with one exception only, a Bacillus macerans strain. That strain was definately gram-negative on staining. Other Bacillus strains were proved gram-positive by the test, even those being gram-negative on staining.     

Directed isolation of gram-negative asporogenous bacteria from natural substrates

A method for selective isolation of gramnegative nonsporulating bacteria from soil substrates was developed. The method consists of plating out the substrates on a glucose-yeast medium with addition of benzylpenicillin and nalidixic acid. The isolation frequency of gramnegative nonsporulating bacteria increased under such conditions from 9-16 (control) to 80-100 per cent. At the same time the isolation frequency of gram-positive bacteria decreased from 88.5 to 9.6 per cent.     

Anhydrobiotic engineering of gram-negative bacteria

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.     

Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria.

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundstr?m and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.     

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.     

Class 1 and class 2 integrons in multidrug-resistant gram-negative bacteria isolated from the Salmon River, British Columbia

Using an enrichment protocol, we isolated 16 gram-negative, multidrug-resistant strains of known or opportunistic bacterial pathogens from the Salmon River in south-central British Columbia from 2005 to 2009, and investigated the genetic basis of their resistance to a variety of antibiotics. Of the 16 strains, 13 carried class 1 integrons and three carried class 2 integrons. Genes found in cassettes associated with the integrons included those for dihydrofolate reductases (dfrA1, dfrA12, dfrA17, and dfrB7), aminoglycoside adenyltransferases (aadA1, aadA2, aadA5, and aadB), streptothricin acetyltransferase (sat), and hypothetical proteins (orfF and orfC). A new gene cassette of unknown function, orf1, was discovered between dfrA1 and aadA5 in Escherichia sp. Other genes for resistance to tetracycline, chloramphenicol, streptomycin, and kanamycin (tetA, tetB, tetD; catA; strA-strB; and aphA1-Iab, respectively) were outside the integrons. Several of these resistance determinants were transferable by conjugation. The detection of organisms and resistance determinants normally associated with clinical settings attest to their widespread dispersal and suggest that regular monitoring of their presence in aquatic habitats should become a part of the overall effort to understand the epidemiology of antibiotic resistance genes in bacteria.