Antibiotic resistance in coagulase-negative staphylococci isolated from Cope's gray treefrogs (Hyla chrysoscelis).

Organisms belonging to the genus Staphylococcus were isolated on mannitol salt agar from the feces of wild caught Cope's gray treefrogs (Hyla chrysoscelis) from east-central Kansas. All 222 presumptive isolates were confirmed as coagulase-negative staphylococci with Staphylococcus sciuri and Staphylococcus xylosus being most prevalent. Antibiotic susceptibility patterns to five different antibiotics were determined and the results indicated 99% of all isolates were resistant to penicillin G and 59% of the isolates were resistant to oxacillin, a clinical substitute for methicillin. Due to the significance of methicillin resistance in the genus Staphylococcus, 10 randomly chosen oxacillin resistant organisms were analyzed for the presence of the mecA gene, which is known to code for methicillin resistance. The gene was detected in four of the 10 organisms examined. These data indicate that gray treefrogs are harboring inordinately large numbers of methicillin resistant staphylococci as part of their normal flora and that the mechanism of methicillin resistance may be independent of mecA.

Antimicrobial susceptibility of staphylococci isolated from the faeces of wild turkeys (Meleagris gallopavo).

AIMS: The purpose of this study was to investigate the staphylococcal flora associated with wild turkey populations. METHODS AND RESULTS: Faecal samples obtained from 26 wild turkeys over a 16-month period were inoculated onto mannitol salt agar plates to select for staphylococci. Fifty-seven randomly chosen isolates were identified as Staphylococcus lentus and their susceptibility determined against clindamycin, chloramphenicol, ciprofloxacin, erythromycin, oxacillin, penicillin G, rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin. Resistance was minimal as only 3 isolates showed resistance to clindamycin, 3 isolates were resistant to oxacillin, 3 isolates were resistant to penicillin G, and 1 isolate was resistant to erythromycin. Multiple antibiotic resistance was also minimal. CONCLUSIONS: S. lentus is the predominant staphylococcal species associated with wild turkey faeces and antibiotic resistance in these organisms is not problematic. SIGNIFICANCE AND IMPACT OF THE STUDY: S. lentus has been shown as a potential causative agent of inflammatory reactions in the respiratory tract. Due to increased numbers of wild turkeys and more frequent human exposure, surveys to monitor microbial populations are warranted.

Genotyping of clinical Serratia marcescens isolates: a comparison of PCR-based methods.

The polymerase chain reaction (PCR)-based procedures of randomly amplified polymorphic DNA (RAPD) and repetitive element (RE)-based PCR were used to amplify total DNA prepared from each of 62 clinical Serratia marcescens isolates. Three different random primers, designated 1060, 1254 and 1283, were used individually in RAPD-PCR. Primers representing enterobacterial repetitive intergenic consensus (ERIC) sequences, extragenic palindromic (REP) elements, and polymorphic GC-rich repetitive sequences (PGRS) constituted the repetitive element-PCR. We were able to generate 40, 40 and 58 genotypic groupings using the 1060, 1254 and 1283 RAPD primers, respectively. Using the ERIC, REP and PGRS primers, 19, 54 and 60 unique genotypic profiles were yielded, respectively. The PGRS primers, which were developed to amplify GC-rich repetitive sequences in the genome of Mycobacteria, were the most discriminatory. These data indicate that both of these PCR-based approaches are a valid means of discriminating strain differences among isolates of S. marcescens and the amount of differentiation depends on the primer used. These techniques should prove useful for routine surveillance or in examining outbreaks of S. marcescens in clinical settings.

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella.

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.

Avian metallothioneins: structure, regulation and evolution.

This article reviews studies of the molecular biology of the avian metallothionein (MT) genes. Analysis of cloned genes and/or cDNAs from chicken, turkey, pheasant and quail suggests that each of these species possesses a very simple MT gene family. The MT from these birds is a cysteine-rich protein of 63 amino acids that shares extensive structural homology with the mammalian MTs, and, remarkably, the deduced amino acid sequence of the major metallothionein is identical in each of these birds. The chicken MT gene is inducible by dietary or injected metal ions [i.e., zinc (Zn) and copper (Cu)], bacterial lipopolysaccharide and oxidative stress. Furthermore, it is expressed during liver development. The turkey and chicken MT genes are identical in gross structure to other functional MT genes. They consist of three exons separated by two intervening sequences. Comparisons of the nucleotide sequences of the turkey and chicken MT genes revealed regions of exceptionally high sequence conservation, suggesting important functions such as splicing, polyadenylation and transcriptional activation. Structure-function studies of the chicken MT promoter using transient transfection assays and transgenic mice have revealed cis-acting promoter sequences involved in the induction of chicken MT gene expression by metals ions. A metal-responsive enhancer was located in the proximal 107-bp of the chicken MT promoter in a region highly conserved in both the turkey and chicken MT genes. Function of this enhancer element apparently requires cooperation of transcription factors interacting with an Sp 1 binding site and a single palindromic metal-responsive element. In this regard, the structure of the proximal region of the chicken and turkey MT promoters is unique. Our current studies suggest that this enhancer regulates gene expression in a position-independent manner in transgenic mice.

Activation of the complete mouse metallothionein gene locus in the maternal deciduum.

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6-8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation.

Evolution of avian metallothionein: DNA sequence analyses of the turkey metallothionein gene and metallothionein cDNAs from pheasant and quail.

The turkey metallothionein gene (tkMT) was isolated from a phage lambda-turkey genomic DNA library by virtue of high identity with chicken MT cDNA. The nucleotide sequences of the proximal 240 bp of the 5'-flanking region, of each of the three exons, and of the intron/exon boundaries were determined. Comparisons of the nucleotide sequences of the tkMT and cMT genes revealed (1) absolute conservation of intronic DNA immediately flanking each respective intron/exon boundary, (2) high conservation (95.6%) of exonic DNA encoding translated regions of the mRNA, and (3) high conservation (95%) of exonic DNA encompassing the putative transcription start point and polyadenylation signals. Sequence comparisons of the tkMT and cMT promoters regions near the TATA box revealed that both promoters contain a highly conserved proximal metal-responsive enhancer (MRE-enhancer) motif. The deduced amino acid sequence (63 amino acids) of tkMT was identical with that of cMT. In order to further explore the degree of conservation of the protein coding regions of avian MT genes, partial MT cDNAs from turkey, quail (qMT), and pheasant (pMT) were amplified using the reverse transcriptase-polymerase chain reaction (RT-PCR) and primers corresponding to the amino- and carboxyl-terminal coding regions of cMT mRNA. RT-PCR reaction products were cloned and the DNA sequences of multiple cDNA clones from each species were determined. The results suggest the existence of a single MT mRNA in zinc-treated liver from turkey and pheasant and the existence of a major and possibly a minor MT mRNA in quail.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell agglutination by a novel cell surface sialoglycopeptide inhibitor and the relationship between its protease and biological activities.

A bovine sialoglycopeptide, purified to homogeneity and capable of inhibiting cellular protein synthesis and proliferation, was shown to agglutinate a wide variety of nontransformed and transformed cells. The cell agglutination activity was shown to be independent of the biological inhibitory action and most likely related to a protease activity that could not be physically separated during purification of the sialoglycopeptide. Samples that were completely biologically inactivated retained full protease activity and their ability to agglutinate target cells. Balb/c 3T3 cells were not agglutinated by the sialoglycopeptide and they elicited a protein that interfered with the agglutination reaction and even redispursed cells that already had been aggregated by the inhibitor.

Effects of purine amino groups on the development of Drosophila.

The effect of a variety of 6-substituted purines on development of Drosophila melanogaster, eggs and larva, were studied. Purine and 2,6-diaminopurine both were very toxic to egg development. Adenine and 2,6-diaminopurine were moderately and equally toxic to larva development. Substitution on the 6-position of the purine ring was very effective in regulating metamorphosis of Drosophila.

Detection of pigment precursors in white clinical strains of Serratia marcescens.

Filter paper strips containing a pigment precursor extracted from Serratia marcescens strain 933 were used to determine whether white, clinical S. marcescens strains could form pigment syntrophically. In all, 114 strains (113 of clinical origin) were tested, and 99% were found to develop colors ranging from violets to pinks.

Depression of lactose hydrolysis by yeasts.

To investigate the role that the known disaccharidase depression may play in the aetiology of infant gastroenteritis caused by Candida albicans, C. albicans and the rarely pathogenic, Saccharomyces cerevisiae were studied by three different methods. Both types of yeast significantly depressed the lactose-hydrolysis activity of beta-galactosidase, and both depressed lactose hydrolysis in the ligated small intestine of infant rabbits, either in intact animals allowed to survive for 10 h, or in a physiological bath for 20 h. The depression of lactose activity was not temperature dependent; living and inactivated yeast preparations produced comparable degrees of depression of enzyme activity. It is concluded that the depression of lactose hydrolysis is not a virulence factor of C. albicans, but contributes to the often observed disaccharide intolerance associated with candida gastroenteritis in infants.

Toxicity of nalidixic acid on candida albicans, Saccharomyces cerevisiae, and Kluyveromyces lactis.

The antibacterial drug nalidixic acid (Nal) can suppress the growth of Candida albicans at levels of the drug normally found in urine. Growth suppression increases as drug levels are increased, and Nal also causes a similar proportional inhibition of the synthesis of all cellular macromolecules. However, growth temperature (25 versus 37 C) and the divalent cations Mg(2+) and Mn(2+) can increase C. albicans resistance to Nal. Also, nitrogen depletion of Candida shows that Nal-treated and untreated cells exhibit no difference in leucine uptake during readaptation to nitrogen. In Nal-treated, nitrogen-starved cells, ribonucleic acid and deoxyribonucleic acid (DNA) biosynthesis are less affected than in unstarved Nal-treated cells, but of the two nucleic acids DNA synthesis is the most affected. Nal-resistant strains of C. albicans exhibit a slight toxicity for macromolecular synthesis. Nal treatment of a synchronized population of Saccharomyces cerevisiae results in an increase in the culture mean doubling time of, at most, 20%, but Nal causes the loss of synchronous cell division. With a synchronized population of Kluyveromyces lactis, Nal causes an increase in the mean doubling time of upwards of 300%, with synchrony of cell division being maintained. It is known that S. cerevisiae asynchronously synthesizes mitochondrial DNA during the cell cycle, whereas with K. lactis it is synchronous. Thus, with C. albicans Nal toxicity is dependent both on the dose and the physiological state of the cell. Furthermore, Nal inhibits growth of yeast with synchronous mitochondrial DNA synthesis more adversely than yeast with asynchronous mitochondrial DNA synthesis.

Cold-sensitive Pseudomonas RNA polymerase. I. Characterization of the host dependent cold-sensitive restriction of phage CB3.

Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells.

Cold-sensitive Pseudomonas RNA polymerase. II. Cold-promoted restriction of bacteriophage CB3 and the lack of host-dependent bacteriophage-specific RNA transcription.

Cold-sensitive restriction of Pseudomonas phage CB3 by Pseudomonas aeruginosa strain PAT2 involves some aspect of CB3 specific RNA synthesis at 20 C. Experiments using chloramphenicol treatment and RNA-DNA hybridization establish that the amount of CB3 RNA present at 20 C is consistent with the known percentage of phage yielder cells at 20 C. Thus, it appears that nonyielder cells of PAT2 synthesize little or no phage-specific mRNA. Burgess technique extracted PAT2 RNA polymerase (RNAP) is cold sensitive when assayed in vitro with CB3 DNA at 20 C. However, it is not cold sensitive when either calf thymus or PAT2 DNA are the templates for transcription. Low ionic strength assay conditions eliminate the cold sensitivity of PAT2 RNAP. The effect of low ionic environments on transcription initiation along with the in vivo and in vitro suppression of cold sensitivity by host rifampin resistance suggests that the inability of CB3 to reproduce in PAT2 at 20 C is a cold-sensitive step in host RNAP initiation. Our modified RNAP extraction procedure for PAT2 and PAO1C also results in the recovery of cold-sensitive PAT2 RNAP with respect to CB3 DNA templates and points to basic enzymological differences between the two hosts. A model is presented for the unusual influence of temperature on the initiation process of both PAT2 and PAO1C on RNAP transcription.