Silvia A. Pineiro

Evolution of Multiresistance in Nontyphoid Salmonella Serovars from 1984 to 1998 in Argentina

ABSTRACT Molecular evolution of multiresistance in nontyphoid Salmonella spp. was investigated with 155 isolates obtained in Argentina from 1984 to 1998. In 74 isolates obtained from 1984 to 1988 resistance was associated with the presence of Tn3, Tn9, class I (In0) and II (Tn7) integrons, and the aac(3)-IIa gene. Extended-spectrum cephalosporin (ESC) resistance in Salmonella spp. emerged in 1989, and 81 isolates resistant to at least one ESC and one aminoglycoside were collected thereafter. Among these, two patterns of antimicrobial resistance mechanisms were found: from 1989 to 1992, resistance was related to the spreading of Tn1331 and blaCTX-M-2, in addition to the persistence of In0 and Tn7. From 1993 to 1998, several integrons were added to the first pattern and three integron groups (IG), namely, IG1 (38% of the isolates), IG2 (51%), and IG3 (11%), were identified. At least two β-lactamase genes were detected in 65% of the isolates (after 1989) by PCR analysis. Furthermore, five β-lactamase genes, blaCTX-M-2, blaOXA-9, blaOXA-2, blaTEM-1, and blaPER-2, were found in two isolates. The blaCTX-M-2 gene was found in several complex sulI-type integrons with different rearrays within the variable region of class I integrons, suggesting evolution of these integrons in nontyphoid Salmonella. In conclusion, progressive acquisition and accumulation of plasmid-mediated resistance determinants occurred from 1984 to 1998 in nontyphoid Salmonella isolates of the most prevalent serovars from Argentina. It is suggested that antimicrobial resistance mechanisms in these bacteria may have been the consequence of plasmid exchange between Salmonella enterica serovar Typhimurium and Escherichia coli or Shigella flexneri and/or spreading of mobile elements from the nosocomial environment.

Class 1 Integrons Increase Trimethoprim-Sulfamethoxazole MICs against Epidemiologically Unrelated Stenotrophomonas maltophilia Isolates

ABSTRACT Twenty-five plasmid-specified antimicrobial resistance determinants common to gram-negative bacilli from nosocomial infection were investigated from 31 Stenotrophomonas maltophilia isolates. Twenty-four clones were identified by pulsed-field gel electrophoresis, and in three clones that exhibited an increased trimethoprim-sulfamethoxazole MIC, the sul1 determinant was found. These results support not only the higher spread of class 1 integrons compared to other mechanisms but also the potential limitation of using trimethoprim-sulfamethoxazole for therapy of severe S. maltophilia infections.

Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus

ABSTRACT Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.

Predation Pattern and Phylogenetic Analysis of Bdellovibrionaceae from the Great Salt Lake, Utah

The Bdellovibrionaceae are predatory, intraperiplasmic bacteria that prey upon a variety of Gram-negative bacteria. The prey susceptibility pattern is frequently used to characterize new isolates. The objective in this study was to isolate and characterize predators from the Great Salt Lake (GSL) by prey susceptibility testing. To recover the predators, water samples were inoculated into an enrichment medium with Vibrio parahaemolyticus as prey. After several days of incubation, the predators were isolated, pure DNA was extracted, and partial 16S rDNA gene was sequenced. Water samples were also plated for isolation of heterotrophic bacteria. The susceptibility of bacterial isolates from the lake and other sources to each predator isolate was determined. The results revealed that there are predators in the GSL, and they preferentially prey on bacteria from the lake. This is the first report of the isolation of Bdellovibrionaceae from GSL and the predators showing preferences for bacteria from the same habitat.

A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus

Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to Gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500 nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus, the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.

Ecology of the Predatory Bdellovibrio and Like Organisms

This work explores what is known about the ecology of the Bdellovibrio and like-organisms (BALO). Recent studies of these incredibly unique predatory bacteria have revealed new information on some of their genomic features, distribution in the environment, environmental determinants that may select for the predators and their interactions with other bacteria. However, little remains known about the ecology of BALOs and their role in nature. Lack of advances in applications of newer molecular methodologies to study the predators remains a key barrier to their investigation. Nevertheless, a brighter future awaits as molecular techniques aimed at improving their detection and enumeration are being applied. On the basis of culture methods the predators are known to be ubiquitous exhibiting both geographical and seasonal distribution patterns.

Development and evaluation of a quantitative real-time PCR assay for the detection of saltwater Bacteriovorax.

Bdellovibrio-and-like-organisms (BALOs) are small, Gram-negative predatory bacteria with the ability to prey on a wide variety of Gram-negative bacteria, and which may have a significant ecological role. Detection and quantification of BALOs by culture-dependent methods are complicated, as their reproduction is dependent upon the use of appropriate prey. For this reason, a sensitive and specific molecular detection method was developed. This paper describes a SYBR Green-based real-time PCR (quantitative PCR) assay that combines the use of a specific 16S rDNA primer with a universal primer for quantitative detection of halophilic Bacteriovorax. 16S rDNA sequences from 174 BALO strains, including both halophilic and freshwater, were aligned and a consensus region was identified that is unique to the halophilic Bacteriovorax strains. A specific primer was designed and analysed for specificity. The PCR conditions were optimized to obtain high specificity and sensitivity. The specificity was evaluated by testing a series of halophilic Bacteriovorax samples and prey specimens, including both pure cultures and environmental saltwater samples. A linear and reproducible standard curve was obtained over a range of 10(1)-10(6) gene copies and the detection limit was determined to be 10 copies of 16S rRNA gene per reaction. The results presented in this study validate the procedure as a rapid, sensitive and accurate method for the detection and quantification of halophilic Bacteriovorax in environmental saltwater samples. It is anticipated that this culture-independent method will facilitate future investigations of the distribution and population dynamics of these interesting predatory bacteria, leading to a better understanding of their ecological role.

Identification of Bdellovibrio bacteriovorus HD100 Bd0714 as a Nudix dGTPase

Bdellovibrio bacteriovorus bacteria are predatory organisms that attack other gram-negative bacteria. Here, we report that Bd0714 is a Nudix dGTPase from B. bacteriovorus HD100 with a substrate specificity similar to that of Escherichia coli MutT and complements an E. coli mutT-deficient strain. We observed different transcription levels of the gene throughout the predator life cycle.

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from Bdellovibrio bacteriovorus–that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.

Discrepancies in Bacterial Recovery from Dental Unit Water Samples on R2A Medium and a Commercial Sampling Device

Monitoring the number of bacterial colony-forming units is an important step in assuring compliance with the recommendation that water from dental units contain <200 CFU mL−1. Media that have been used for this purpose include R2A, a standard plate counting medium for water samples, and the Millipore HPC Sampler device, designed to facilitate sampling in dental offices. Discrepancies between the two media have been observed. This study tested the hypothesis that differences in counts on the two media were due to the failure of some bacteria to grow on the HPC sampler or to grow at less efficiency than on R2A. Of four different bacterial colony phenotypes tested in three independent experimental trials, one phenotype did not grow on the HPC device, and another grew inconsistently and at lower efficiency. These results confirmed the hypothesis. From these findings, users of the HPC sampler should be aware that microbial undercounts may occur.