Morphological and Biochemical Differentiation of Achromobacter and Moraxella (Debord's Tribe Mimeae)

To determine the most useful laboratory tests for the differentiation of Achromobacter anitratus, Achromobacter lwoffii, and Moraxella duplex (DeBord''s tribe Mimeae), 157 strains of these bacteria, isolated from clinical specimens, were examined for their morphological and biochemical characteristics. There were several differences between these nonfermentative, gram-negative diplococci: Moraxella was nonglucolytic in either infusion base or synthetic base, oxidase-positive, and sensitive to penicillin, whereas Achromobacter produced variable carbohydrate activity, and was oxidase-negative and resistant to penicillin. A. anitratus was distinguished from A. lwoffii in that the former utilized infusion media containing either glucose or 10% lactose, whereas the latter did not. Both species utilized the same carbohydrates in a chemically defined medium, although the latter acted more sluggishly.     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Characterization of radiation-resistant vegetative bacteria in beef.

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.     

Studies on the microflora associated with xenic cultures of Entamoeba gingivalis

The microflora associated with xenic stock cultures (ATCC 30927) of Entamoeba gingivalis, the major protozoan of the human oral cavity, were isolated and identified as Citrobacter diversus, Yersinia enterocolitica, Acinetobacter anitratus and Pseudomonas maltophilia. In studies to determine whether the bacterial isolates were able to utilize rice starch as a sole carbon source, Y. enterocolitica exhibited excellent growth in rice starch minimal medium and TYSGM-9 medium (with rice starch), but growth was weak in TYSGM-9 medium (without rice starch). C. diversus, A. anitratus and P. maltophilia exhibited poor growth in rice starch minimal medium, but they produced excellent growth in TYSGM-9 medium with or without rice starch. In order to determine the effect of the rice starch hydrolysis on Entamoeba growth, the filtrate from each isolate grown in rice starch minimal medium was added to an E. gingivalis culture grown in TYSGM-9 medium. The filtrate from a Y. enterocolitica culture grown in rice starch minimal medium enhanced E. gingivalis growth, but the filtrates from cultures of C. diversus, A. anitratus and P. maltophilia suppressed E. gingivalis growth. This supported the concept that Y. enterocolitica is capable of metabolizing rice starch into intermediate products, which in turn can be utilized by the amoeba.     

Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry

The MS/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter lwoffii K24 is an aniline degrading bacterium. In this report, we present the results of a comparison between the proteome profile of A. lwoffii K24 cultured in aniline- or succinate-containing media. Protein analysis was performed using two-dimensional gel electrophoresis (2-DE) with pH 3-10 immobilized pH gradient (IPG) strips followed by ESI-Q-TOF MS. More than 780 protein spots were detected by 2-DE from the soluble proteome. Forty-eight of these proteins were expressed exclusively in aniline cultured bacteria, and 81 proteins increased and 162 proteins decreased in aniline-cultured versus succinate cultured A. lwoffii K24. Internal amino acid sequences of 43 major protein spots were successfully determined by ESI-Q-TOF MS to try to identify the bacterial proteins responding to aniline culture condition. Since the A. lwoffii K24 genome is not yet sequenced, many proteins were found to be hypothetical. Comparative proteome analysis of the insoluble protein fractions showed that one novel protein that was strongly induced by succinate-cultured A. lwoffii K24 was repressed under aniline culture conditions. These results suggest that comprehensive analysis of bacterial proteomes by 2-DE and amino acid sequence analysis by ESI-Q-TOF MS is useful for understanding induced novel proteins of biodegrading bacteria.     

Nonfermentative gram-negative bacteria encountered in clinical specimens

The characteristics of 1192 gram-negative nonfermentative bacteria isolated from human clinical specimens and 32 reference strains representing six genera (Pseudomonas, Alcaligenes, Achromobacter, Acinetobacter, Moraxella, Flavobacterium) encountered in clinical bacteriology are presented. Salient features for their identification in the routine diagnostic laboratory are summarized.     

The incidence of Weeksella- and Bergeyella-like bacteria in the food environment

Eighty-five catalase- and oxidase-positive Gram-negative rods and cocci susceptible to penicillin G were isolated from a variety of food sources. The phenotypic relationships of these isolates with reference cultures of Bergeyella -like, Chryseobacterium, Empedobacter, Myroides , Moraxella , Sphingobacterium and Weeksella -like strains were examined by numerical taxonomy. Seventy-three isolates were recovered in five groups; 80% of the isolates clustered in groups 1, 2 and 3 and produced indole, bearing a strong resemblance to Weeksella and Bergeyella . They could not, however, be regarded as belonging to the known species of W. virosa and B. zoohelcum . It is suggested that three species may be necessary to accommodate the environmental Weeksella - or Bergeyella -like bacteria. The isolates in groups 4 and 5 had white colonies and were unable to produce indole, in this way resembling the Moraxella genus.     

Heat-stable proteases of psychrotrophic bacteria isolated from cooled raw milk

Summary 143 psychrotrophic bacteria were isolated and identified from 53 cooled raw bulk milk samples. The most encountered isolates were Pseudomonas fluorescens and P. fluorescens-like organisms, Achromobacter xylosoxidans, Acinetobacter lwoffii, Aeromonas hydrophila and enterobacteria. The strongest activity was displayed by the pseudomonads, especially the P. fluorescens-like organisms. All the excreted proteases examined were heat-stable: the best inactivation was obtained after a heat-treatment of 1 h at 55°C and 2 min at 140°C. At temperatures of 80, 90 and 100°C often increasing heat-resistance was observed.     

Nosopharyngeal microflora in ambulatory treated children and adults with upper respiratory tract infections

Upper respiratory tract consists resident and transient bacterial microflora, which in appropriate condition can cause infection. Bacteriological study was performed among 201 patients with upper respiratory tract infections treated in ambulatory. From nasal and pharyngeal swabs Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Streptococci group A, B, C, G were isolated. Antibiotic susceptibility testing of isolated strains was performed using CLSI criteria. All isolated strains of streptococci were susceptible to penicillin; some of them demonstrated resistance to macrolides and lincosamides. Few isolated strains of H. influenzae demonstrated resistance to penicillin and cotrimoxazole. Azitromycin resistant strains were not detected. All isolated strains of M. catarrhalis were beta-lactamase positive and demonstrated resistance to penicillin. Strains of methicillin sensitive S. aureus (MSSA) were isolated most frequently from pharyngeal swabs (35.4%) and S. pneumoniae (33.3)--from nasal swabs.     

Metabolism of quaternary carbon compounds: 2,2-dimethylheptane and tertbutylbenzene.

Two Achromobacter strains capable of utilizing 2,2-dimethylheptane or tertbutylbenzene as the sole carbon and energy source were isolated from waste-water. Pivalic acid was found in the cultures of Achromobacter A1 containing 2,2-dimethylheptane. From cultures of Achromobacter A2 in the presence of tertbutylbenzene, a diol was isolated and identified as 2,3-dihydro-2,3-dihydroxytertbutylbenzene. Evidence for meta cleavage of the aromatic ring and for accumulation of pivalic acid in the cultures was also obtained. A metabolic pathway for tertbutylbenzene is suggested.     

Unstable generalized transduction in Achromobacter.

Six auxotrophic markers of a halotolerant collagenolytic strain of Achromobacter were transduced by four alpha hages. Abortive transduction was also demonstrated. The generalized transduction system is unusual as the transductants were unstable, characteristic of transduction by lysogeny. The Achromobacter strain is a cryptic lysogen for alpha and purified transductants were either sensitive or resistant to alpha. Purified clones from four resistant transductants and one sensitive transductant liberated phage spontaneously. The host ranges of these spontaneous phage differed from that of the alpha phage used for the transduction experiment. Some initially resistant transductants became simi-sensitive to alpha (efficiency to plating) e.o.p. (10minus-1 to 10minus-2) after repeated cloning.     

Enhanced detection and characterization of protocatechuate 3,4-dioxygenase in Acinetobacter lwoffii K24 by proteomics using a column separation

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.     

Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant

Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and potentially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequencing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics. Attempts to decontaminate the eukaryotic cell culture used multiple antibiotics at different concentrations. The contaminating Achromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin.     

Comparison of Hup Trait and Intrinsic Antibiotic Resistance for Assessing Rhizobial Competitiveness Axenically and in Soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

Transduction transmission of the extrachromosomal markers of antibiotic resistance in staphylococcal populations]

Transduction of extrachromosomal markers of resistance to penicillin and erythromycin in staphylococcal strains isolated from patients was studied. Two transduction methods were compared, i. e. transduction with a phage filtrate of the donor culture resistant to erythromycin and transduction on mixed cultivation of the donor and recipient. A higher transduction rate was observed with the latter method. Mixed cultivation of the donor cultures resistant to penicillin or erythromycin and the recipient strains of the wild type sensitive to these antibiotic resulted in transduction of the respective markers. Transductants which acquired prophage 6 simultaneously with marker Egg became donors of erythromycin resistance.     

Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids.

Three cryptic plasmids have been discovered in Acinetobacter calcoaceticus BD413. These three plasmids, designated pWM10 (7.4 kb), pWM11 (2.4 kb), and pWM12 (2.2 kb), exhibited extensive homology to one another, as shown by Southern blot hybridization and restriction site analysis data, and also hybridized with three plasmids having slightly different sizes detected in a second strain, A. calcoaceticus BD4. Plasmid pWM11 and a fragment of pWM10 were each subcloned into pUC19, yielding plasmids pWM4 and pWM6, respectively, and were used in a series of inter- and intraspecies transformation experiments. Both plasmids replicated as high-copy-number plasmids in A. calcoaceticus BD413, as well as in strains of Escherichia coli. However, when transformed into the oil-degrading strain Acinetobacter lwoffii RAG-1, both plasmids were maintained at low copy numbers. No modification of the plasmids was detected after repeated transfers between hosts. An analysis of a series of deletions demonstrated that (i) a 185-bp fragment of pWM11 was sufficient to permit replication of the shuttle plasmid in A. calcoaceticus BD413, (ii) the efficiency of transformation of A. calcoaceticus BD413 decreased according to the size of the deletion in the insert by up to 4 orders of magnitude, and (iii) the entire insert was required for transformation and replication in A. lwoffii RAG-1. The sequence of pWM11 contained several small (150- to 300-bp) open reading frames, none of which exhibited any homology to known DNA or protein sequences. In addition, a number of inverted and direct repeats, as well as six copies of the consensus sequence AAAAAAATA previously described for a cryptic plasmid from A. lwoffii (M. Hunger, R. Schmucker, V. Kishan, and W. Hillen, Gene 87:45-51, 1990), were detected. Cloning and expression of the alcohol dehydrogenase regulon from A. lwoffii RAG-1 were accomplished by using the Acinetobacter shuttle plasmid.     

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae

Abstract Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 μg benzylpenicillin ml⁻¹. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.     

Synthesis and antibacterial activities of novel C(3)-aminopyrimidinyl substituted cephalosporins including against respiratory tract pathogens

The variety of cephalosporins 1 and 2 which possessed C(3)-aminopyrimidinyl substituents were prepared and evaluated for their antibacterial activities. They exhibited excellent in vitro activities especially against respiratory tract pathogens such as penicillin resistant Streptococcus pneumonia. Moraxella catarrhalis and Haemophilus influenza.