Marcelo E. Tolmasky

Aminoglycoside Modifying Enzymes

Aminoglycosides have been an essential component of the armamentarium in the treatment of life-threatening infections. Unfortunately, their efficacy has been reduced by the surge and dissemination of resistance. In some cases the levels of resistance reached the point that rendered them virtually useless. Among many known mechanisms of resistance to aminoglycosides, enzymatic modification is the most prevalent in the clinical setting. Aminoglycoside modifying enzymes catalyze the modification at different -OH or -NH₂ groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be nucleotidyltransferases, phosphotransferases, or acetyltransferases. The number of aminoglycoside modifying enzymes identified to date as well as the genetic environments where the coding genes are located is impressive and there is virtually no bacteria that is unable to support enzymatic resistance to aminoglycosides. Aside from the development of new aminoglycosides refractory to as many as possible modifying enzymes there are currently two main strategies being pursued to overcome the action of aminoglycoside modifying enzymes. Their successful development would extend the useful life of existing antibiotics that have proven effective in the treatment of infections. These strategies consist of the development of inhibitors of the enzymatic action or of the expression of the modifying enzymes.

Whole-genome analyses of speciation events in pathogenic Brucellae.

ABSTRACT Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.

Multiple intensive care unit outbreak of Acinetobacter calcoaceticus subspecies anitratus respiratory infection and colonization associated with contaminated, reusable ventilator circuits and resuscitation bags

PURPOSE Acinetobacter calcoaceticus subspecies anitratus (A. anitratus) can cause nosocomially and community acquired pneumonia. Source identification of the organism is often difficult. An outbreak of respiratory infection and colonization with A. anitratus affecting 93 ventilated patients in all six of a hospitals intensive care units (ICUs) over 10 months is described. PATIENTS AND METHODS In April 1984, the infection control staff started to review positive culture results from all patients in all ICUs. At this point, information on significant isolates was recorded by patient, site, date, genus and species, and antimicrobial susceptibility. During the month of August 1984, an increased number of A. anitratus isolates from sputum began to be detected. Information was expanded to include the date of hospital admission, ICU admission, intubation, and extubation; the dates and types of all surgical procedures; the results and dates of all prior sputum cultures; and the use of nebulized bronchodilator medications. Monthly numbers of cases were compared for four months prior to the outbreak, during the outbreak, and for seven months after the outbreak. Plasmid DNA from isolates was prepared, electrophoresed, and visualized. Isolates were designated according to the molecular weights of visualized plasmids. RESULTS Barrier precautions and improved staff handwashing did not diminish the frequency of new cases. When pasteurized, reusable ventilator circuits and resuscitation bags were cultured for the possibility of low-level contamination, 18 percent were positive for A. anitratus. Terminal ethylene oxide sterilization of these devices was associated with prompt control of the outbreak. Plasmid DNA analysis of isolates from patients involved in the outbreak, contaminated devices, and the hands of personnel responsible for device disinfection revealed two predominant plasmid profiles. After outbreak control, isolates with these profiles were found much less frequently in patient specimens. CONCLUSION Contaminated, reusable ventilator support equipment may be a leading cause for the extent of A. anitratus in the sputum of intubated patients. This problem is potentially correctable by the use of terminal etyhlene oxide sterilization of reusable ventilator circuits and resuscitation bags.

Complete Sequence of Virulence Plasmid pJM1 from the Marine Fish Pathogen Vibrio anguillarum Strain 775

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.

Complex Class 1 Integrons with Diverse Variable Regions, Including aac(6′)-Ib-cr, and a Novel Allele, qnrB10, Associated with ISCR1 in Clinical Enterobacterial Isolates from Argentina

ABSTRACT Transferable quinolone resistance has not previously been reported in Argentina. Here we describe three complex class 1 integrons harboring the novel allele qnrB10 in a unique region downstream of orf513, one of them also containing aac(6′)-Ib-cr within the variable region of integrons. The three arrays differed from blaCTX-M-2-bearing integrons, which are broadly distributed in Argentina.

Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-Negatives: the Klebsiella pneumoniae Paradigm.

Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits, resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about the physiology of plasmids and their role in virulence and antibiotic resistance from the Gram-negative opportunistic pathogen Klebsiella pneumoniae. This bacterium has acquired multidrug resistance and is the causative agent of serious community- and hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most U.S. hospital infections.

Complete Nucleotide Sequence of Klebsiella pneumoniae Multiresistance Plasmid pJHCMW1

ABSTRACT The multiresistance plasmid pJHCMW1, harbored by a clinical Klebsiella pneumoniae strain isolated from a neonate with meningitis, was sequenced. A circular sequence of 11,354 bp was generated, of which 7,993 bp make up Tn1331, a transposon including the antibiotic resistance genes aac(6′)-Ib, aadA1, blaOXA-9, and blaTEM-1. The gene aac(6′)-Ib is included in a gene cassette, and both aadA1 and blaOXA-9 are included in a single-gene cassette that may have arisen as a consequence of a recombination event involving two integrons. The pJHCMW1 plasmid replicates through a ColE1-like RNA-regulated mechanism, includes a functional oriT, and two loci with similarity to XerCD site-specific recombination target sites involved in plasmid stabilization by the resolution of multimers. One of these two loci, mwr, is active and has been the subject of previous studies, and the other, dxs, is not functional but binds the recombinase XerD with low affinity. Two additional open reading frames were identified, one with low similarity to two hypothetical membrane proteins from Mycobacterium tuberculosis and Mycobacterium leprae and the other with low similarity to psiB, a gene encoding a function that facilitates the establishment of the transferring plasmid in the recipient bacterial cell during the process of conjugation.

Sequencing and expression of aadA, bla, and tnpR from the multiresistance transposon Tn1331.

A fragment of Tn1331 including tnpR, aac, aadA, and a bla gene which encodes lower levels of resistance to ampicillin and carbenicillin as compared to those mediated by the TEM beta-lactamase was sequenced. The polypeptide encoded by the bla gene has homology with the OXA-1, PSE-2, and OXA-2 proteins. Genes aac and bla are upstream and downstream respectively of aadA, and are both flanked by recombinational hot spots. Tn1331 has 520-bp direct repeats which include parts of the tnpR and TEM bla genes. Evolutionary models for the genesis of Tn1331 are proposed.

Rise and dissemination of aminoglycoside resistance: the aac(6′)-Ib paradigm

Enzymatic modification is a prevalent mechanism by which bacteria defeat the action of antibiotics. Aminoglycosides are often inactivated by aminoglycoside modifying enzymes encoded by genes present in the chromosome, plasmids, and other genetic elements. The AAC(6′)-Ib (aminoglycoside 6′-N-acetyltransferase type Ib) is an enzyme of clinical importance found in a wide variety of gram-negative pathogens. The AAC(6′)-Ib enzyme is of interest not only because of his ubiquity but also because of other characteristics, it presents significant microheterogeneity at the N-termini and the aac(6′)-Ib gene is often present in integrons, transposons, plasmids, genomic islands, and other genetic structures. Excluding the highly heterogeneous N-termini, there are 45 non-identical AAC(6′)-Ib related entries in the NCBI database, 32 of which have identical name in spite of not having identical amino acid sequence. While some variants conserved similar properties, others show dramatic differences in specificity, including the case of AAC(6′)-Ib-cr that mediates acetylation of ciprofloxacin representing a rare case where a resistance enzyme acquires the ability to utilize an antibiotic of a different class as substrate. Efforts to utilize antisense technologies to turn off expression of the gene or to identify enzymatic inhibitors to induce phenotypic conversion to susceptibility are under way.

A histidine decarboxylase gene encoded by the Vibrio anguillarum plasmid pJM1 is essential for virulence: histamine is a precursor in the biosynthesis of anguibactin.

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJMl which encodes a histidine decarboxlase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44 259.69 Da. The amino acid sequence has extensive homology with the pyridoxal‐P‐dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3‐HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJMI Iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron‐limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore. immunoblot analysis of production of β‐galactosidase by V. anguillarum strains carrying the lacZ fusions demonstrated that expression of histidine decarboxylase is not regulated by the iron concentration of the medium.