Mariana Catalano

Extracellular DNA: A Major Proinflammatory Component of Pseudomonas aeruginosa Biofilms

We previously demonstrated that extracellular bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Biofilms are microbial communities enclosed in a polymeric matrix that play a critical role in the pathogenesis of many infectious diseases. Because extracellular DNA is a key component of biofilms of different bacterial species, the aim of this study was to determine whether it plays a role in the ability of biofilms to induce human neutrophil activation. We found that degradation of matrix extracellular DNA with DNase I markedly reduced the capacity of Pseudomonas aeruginosa biofilms to induce the release of the neutrophil proinflammatory cytokines IL-8 and IL-1β (>75%); reduced the upregulation of neutrophil activation markers CD18, CD11b, and CD66b (p < 0.001); reduced the number of bacteria phagocytosed per neutrophil contacting the biofilm; and reduced the production of neutrophil extracellular traps. Consistent with these findings, we found that biofilms formed by the lasI rhlI P. aeruginosa mutant strain, exhibiting a very low content of matrix extracellular DNA, displayed a lower capacity to stimulate the release of proinflammatory cytokines by neutrophils, which was not decreased further by DNase I treatment. Together, our findings support that matrix extracellular DNA is a major proinflammatory component of P. aeruginosa biofilms.

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.

Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina

Staphylococcus aureus is the most prevalent pathogen causing mastitis of dairy ruminants. This study was developed to ascertain the genotypes and genealogical relationship among strains isolated from milk of bovines with mastitis in Argentina. Molecular epidemiological analysis of S. aureus was performed on 112 isolates from 21 districts. Clonality was assessed by SmaI pulsed-field gel electrophoresis (PFGE) typing, automated EcoRI ribotyping and restriction enzyme analysis of plasmid (REAP) DNA profiles. A total of 22 band patterns distributed in four clusters were found by SmaI PFGE analysis. The similarity of clusters 2, 3 and 4 with cluster 1 was 0.73, 0.69 and 0.33, respectively, and 101 of 112 isolates belonged in cluster 1. PFGE band patterns from 42 isolates within cluster I were indistinguishable from each other (type A). The second largest group of isolates with indistinguishable PFGE band patterns was subtype A11, which was composed of 19 isolates. Automated ribotyping assigned the 112 isolates into 13 ribotypes. Among these, the most prevalent ribotypes I and VI were composed of 49 and 35 isolates respectively. Although there was certain correspondence between PFGE genotypes and ribotypes, further discrimination was achieved by combining both methods. REAP DNA profile analysis was useful to provide even further discrimination between isolates with identical PFGE genotype and ribotype. The most prevalent S. aureus strains A/I and A11/VI were widely distributed in the country and were not restricted to individual nearby locations. Prevalence of these two strains varied consecutively within a period of 8 years. Whether the shift in type prevalence was due to selection of a phenotypic trait remains undisclosed.

Acinetobacter baumannii extensively drug resistant lineages in Buenos Aires hospitals differ from the international clones I-III.

As a way to contribute to the assessment of Acinetobacter baumannii clinical population structure, multi-locus sequence typing (MLST) was performed in a collection of 93 isolates from Buenos Aires (1983-2012) and Rosario (2006-2009) hospitals. Sequence types (STs) were achieved by Bartual (B) and Institut Pasteur (P) schemes. PFGE typing, antimicrobial susceptibility assays, and the amplification of the OXA carbapenemase genes most prevalent in our region, were also performed. e-Burst clustered the 25 STs(B) (15 novels) into 5 clonal complexes (CC) and 5 singletons, and grouped the 18 STs(P) (12 novels) into 3 CC and 4 singletons. Bartual scheme divided the CC79(P) into two groups. CC113(B)/CC79(P) prevailed in Buenos Aires at least in 1992-2009, being responsible for epidemic and for endemic infections and acquiring the XDR (extensively drug-resistant) pattern throughout the years. While, CC119(B)/CC79(P) was apparently present before the CC113(B)/CC79(P)domain. CC103(B)/CC15(P) was the second most prevalent CC. Interestingly, CC110(B)/ST25(P) apparently increased over the last years. Conversely, CC109(B)/CC1(P) (international clone I) predominated in Rosario, although the presence of CC113(B)/CC79(P), CC103(B)/CC15(P) and CC110(B)/ST25(P) was observed. Nineteen novel STs clustered in CC79(P), CC15(P), CC113(B), CC109(B) and CC103(B), suggesting their clonal expansion during persistence. PFGE typing proved transmission of strains intra- and inter-hospitals in each city. Except for one, all the recent isolates (2007-2012) harboured the blaOXA-23-like. All isolates were susceptible to colistin. Tigecycline MIC(90) was 1mg/L and the rifampicin MIC>512mg/l was found among isolates in three hospitals. In conclusion, the international clone II (CC92(B)/CC2(P)) was not found among our isolates. CC113(B)/CC79(P), CC103(B)/CC15(P), and ST25(P), suggested also as major components in the A. baumannii population together with the international clone I, were present in Buenos Aires and Rosario with different prevalence rate. Their recent isolates showed high distribution of the blaOXA-23-like as well as the XDR pattern.

Molecular Epidemiology of orf513-Bearing Class 1 Integrons in Multiresistant Clinical Isolates from Argentinean Hospitals

ABSTRACT The spread of orf513-bearing class 1 integrons is associated with blaCTX-M-2 in gram-negative clinical isolates in Argentina, with In35 being the most frequently found integron (74%). Among 65 isolates without blaCTX-M-2, only one harbored a novel orf513-bearing class 1 integron with the dfrA3b gene. The finding of orf513 not associated with class 1 integrons in two gram-positive strains indicates the widespread occurrence of this putative site-specific recombinase.

Identification of an epidemic carbapenem-resistant Acinetobacter baumannii strain at hospitals in Buenos Aires City

To identify epidemic Acinetobacter baumannii (AB) clones, 38 carbapenem-resistant AB isolates from 5 hospitals were analyzed. Macrorestriction classified 24 isolates as clone IV, susceptibility pattern clustering analysis grouped almost all of them together, and they were uniformly biotype 8. Clone IV was present at all 5 hospitals, so that it represents a carbapenem-resistant AB strain with epidemic behavior.

Molecular epidemiology of Acinetobacter baumannii spread in an adult intensive care unit under an endemic setting

BACKGROUND Acinetobacter baumannii (Ab) clones I, III, and IV were recovered in several Buenos Aires City hospitals. We investigated the prevalence of these clones with epidemic behavior (EB) in our intensive care unit (ICU) under an endemic setting and its spread. METHODS A 10-week prospective cohort study including surveillance cultures of newly admitted patients was conducted. Air, environment, and staff hands were weekly screened. In the seventh week, a new environmental cleaning protocol and a staff hand hygiene reeducation program were implemented. RESULTS Almost 15% of all screening samples (159/1042) were Ab positive. Up to the seventh week, carbapenem-resistant clone If was the main one recovered from patients, environmental frequently touched surfaces (EFTS), and staff hands screening samples. Few air samples were Ab positive. Clone I was also isolated from patients at admission. After the seventh week, a significant reduction of EFTS contamination and of clone If isolation was observed. During the last 3 weeks, clone I was no longer isolated from patients. Instead, the newly identified clone IVb was mainly cross transmitted. It was also recovered from staff hands and from EFTS. In the last week, clone If was again isolated from 1 bed rail. CONCLUSION Patients with EB clones-positive culture at admission provide verification that interhospital patient transfers play a role in these clones spread. However, subtypes such as clone If seem to be endemic in our ICU. EFTS showed to have potential for EB clones transmission via transient staff hand carriage. Transmission did not involve airborne route.

Efficacy of two DNA fingerprinting methods for typing Acinetobacter baumannii isolates

Performance of macrorestriction and repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR) to type Acinetobacter baumannii isolates was quantitatively estimated using a test population of 54 outbreak-related, 29 endemic infection-related and 17 epidemiologically-unrelated isolates. Reproducibility and stability for macrorestriction were 100%, and REP-PCR showed only slightly lower stability. Macrorestriction resolved 18 fingerprints and REP-PCR 10 DNA patterns, forming eight and seven clusters at 75% of similarity level, respectively. Intercluster band variation was > 7 bands for both methods. Although, all endemic isolates, except one, were concordantly grouped by both methods, macrorestriction distinguished a greater number of subtypes over one year study. For outbreaks, the epidemiologic concordance for both methods was 88%. The discriminatory index for macrorestriction and REP-PCR was 0.884 and 0.877, respectively. In conclusion, both methods showed similar efficacy as epidemiological markers, and by concordance, this study demonstrated that for REP-PCR typing, a > or = 7 bands difference seemed an appropriate threshold to identify unrelated strains.

Helicobacter pylori vacA genotypes, cagA status and ureA-B polymorphism in isolates recovered from an Argentine population

Several reports have evidenced geographic differences in the prevalence of vacA (vacuolating cytotoxin gene) alleles and cagA (cytotoxin-associated gene) status among Helicobacter pylori isolates. We investigated the occurrence of these virulence-associated genes status among our isolates, and their relationship with ulcer disease outcome. Besides, ureA-B polymorphism was studied. One hundred isolates, comprising 32 from patients with ulcer disease (UD) and 68 from patients with non-ulcer dyspepsia (NUD), were analyzed. Eighty-four percent of isolates were cagA-positive without statistically significant difference in prevalence between patients with UD or NUD. Genotype vacA-s1m1 was predominant, although unlike other South American regions, subtype s1am1 occurrence was higher than s1b. The multivariate model used to estimate the predictive value of cagA and vacA status for UD development disclosed infection with vacA-s1am1 isolates as the only variable that increased the risk of UD onset. ureAB fingerprinting showed considerable genetic divergence among isolates, however, confirmed that certain DNA banding profiles are conserved worldwide.

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.