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Stenotrophomonas maltophilia intrinsic resistance to β-lactams is mediated by two chromosomal β-lactamases, L1 and L2, whose induction depends on AmpR. Its quorum sensing (QS) signal, the diffusible signal factor (DSF), has a positive role in biofilm production, virulence and induction of β-lactamases. We hypothesized that AmpR has a role in virulence, biofilm production and QS system. Studies were done on S. maltophilia K279a, K279a ampR FS (ampR deficient mutant) and K279aM11 (constitutively active AmpR mutant). K279a ampR FS showed the highest biofilm biomass, thickness and 3D organization. Conversely, K279aM11 was the least efficient biofilm former strain. qRT-PCR showed that spgM, related to biofilm formation and virulence, was upregulated in K279a ampR FS and downregulated in K279aM11. A constitutively active AmpR led to a reduction of DSF production, while K279a ampR FS was the highest producer. Consequently, qRT-PCR showed that AmpR negatively regulated rpfF expression. K279a ampR FS presented the highest oxidative stress resistance, overexpressed sodA gene and showed the highest virulence in the Galleria mellonella killing assay. This is the first evidence of the function of AmpR as a dual regulator in S. maltophilia with a positive role in β-lactam resistance and a negative role in DSF production, biofilm formation, oxidative stress resistance and virulence., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Introduction. Stenotrophomonas maltophilia has emerged as one of the most common multi-drug-resistant pathogens isolated from people with cystic fibrosis (CF). However, its adaptation over time to CF lungs has not been fully established. Hypothesis. Sequential isolates of S. maltophilia from a Brazilian adult patient are clonally related and show a pattern of adaptation by loss of virulence factors. Aim. To investigate antimicrobial susceptibility, clonal relatedness, mutation frequency, quorum sensing (QS) and selected virulence factors in sequential S. maltophilia isolates from a Brazilian adult patient attending a CF referral centre in Buenos Aires, Argentina, between May 2014 and May 2018. Methodology. The antibiotic resistance of 11 S. maltophilia isolates recovered from expectorations of an adult female with CF was determined. Clonal relatedness, mutation frequency, QS variants (RpfC-RpfF), QS autoinducer (DSF) and virulence factors were investigated in eight viable isolates. Results. Seven S. maltophilia isolates were resistant to trimethoprim-sulfamethoxazole and five to levofloxacin. All isolates were susceptible to minocycline. Strong, weak and normomutators were detected, with a tendency to decreased mutation rate over time. XbaI PFGE revealed that seven isolates belong to two related clones. All isolates were RpfC-RpfF1 variants and DSF producers. Only two isolates produced weak biofilms, but none displayed swimming or twitching motility. Four isolates showed proteolytic activity and amplified stmPr1 and stmPr2 genes. Only the first three isolates were siderophore producers. Four isolates showed high resistance to oxidative stress, while the last four showed moderate resistance. Conclusion. The present study shows the long-time persistence of two related S. maltophilia clones in an adult female with CF. During the adaptation of the prevalent clones to the CF lungs over time, we identified a gradual loss of virulence factors that could be associated with the high amounts of DSF produced by the evolved isolates. Further, a decreased mutation rate was observed in the late isolates. The role of all these adaptations over time remains to be elucidated from a clinical perspective, probably focusing on the damage they can cause to CF lungs.

Purpose. The aim of this work was to investigate the presence of selected potential virulence factors, susceptibility and clonal relatedness among 63 Stenotrophomonas maltophilia isolates recovered from patients exposed to invasive devices in a university hospital in Argentina between January 2004 and August 2012. Methodology. Genetic relatedness was assessed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and pulsed-field gel electrophoresis (PFGE). Isolates were characterized by antimicrobial resistance, the presence and/or expression of potential virulence determinants, and virulence in the Galleria mellonella model. Results/Key findings. ERIC-PCR generated 52 fingerprints, and PFGE added another pattern. Resistance to trimethoprim-sulfamethoxazole (6.35 %), levofloxacin (9.52 %) and ciprofloxacin (23.80 %) was detected. All isolates were susceptible to minocycline. All isolates were lipase, protease and siderophore producers, while all but Sm61 formed biofilms. However, 11/63 isolates did not amplify the major extracellular protease-coding gene ( stmPr1 ). Sm61 is an stmPr1- negative isolate, and showed (as did Sm13 and the reference strain K279a) strong proteolysis and siderophore production, and high resistance to hydrogen peroxide. The three isolates were virulent in the G . mellonella model, while Sm10, a low-resistance hydrogen peroxide stmPr1- negative isolate, and weak proteolysis and siderophore producer, was not virulent. Conclusion. This is the first epidemiological study of the clonal relatedness of S. maltophilia clinical isolates in Argentina. Great genomic diversity was observed, and only two small clusters of related S. maltophilia types were found. Minocycline and trimethoprim-sulfamethoxazole were the most active agents. S. maltophilia virulence in the G. mellonella model is multifactorial, and further studies are needed to elucidate the role of each potential virulence factor.

The aim of this study was to compare the in vitro activity of ethanol, EDTA and levofloxacin (Levo), alone or in combination, on biofilms of Stenotrophomonas maltophilia recovered from patients with catheter-related bloodstream infections (CRBSIs) at a university hospital in Argentina. First, 24 and 48 h biofilms were formed in microtitre plates and challenged with 25 or 40 % ethanol for 1 h. Biofilms, of the 14 local isolates and from the reference strain K279a, were eradicated after both treatments as shown by plate counts and the regrowth technique. Second, 24 h biofilms of all isolates were established in silicone catheter segments and challenged with 25 or 40 % ethanol, Levo (2.5 mg ml(-1)), EDTA (30 mg ml(-1)), 25 % ethanol-EDTA or Levo-EDTA for 1, 3 and 24 h. Viable counts of biofilms treated for 1 h with 25 or 40 % ethanol or 25 % ethanol-EDTA were under the limit of detection. Killing of biofilms by Levo or Levo-EDTA was gradual and it was only after 24 h of treatment that no differences could be seen between the effects of these catheter lock solutions (CLSs) and those of ethanol (P>0.05). Levo-EDTA, in combination, did not act synergistically against biofilms. After 24 h of exposure, EDTA did not eradicate biofilms but reduced biofilm survival rates to 1-5 %. The effect of the different CLSs on biomass reduction, estimated by crystal violet staining, was highly dependent on the isolate, and the most effective agents were 25 and 40 % ethanol. Our results suggest that when used as a CLS for short periods, ethanol at low concentrations, alone or in combination with a chelator, can decontaminate the line from S. maltophilia in cases of CRBSI and help, in conjunction with systemic antibiotics, in the retention of precious vascular catheters.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen. Despite the broad spectrum of syndromes associated with S. maltophilia infections, little is known about its virulence factors, including siderophore production. The aims of this work were to detect S. maltophilia siderophores and to determine their chemical nature. We studied 31 S. maltophilia isolates from device-associated infections, recovered over the period 2006-2011 at Hospital de Clínicas José de San Martín, Buenos Aires, Argentina, and the strain K279a, whose genome has been fully sequenced. The production of siderophores was screened by the chrome azurol S (CAS) agar assay, previously modified to detect siderophores in this species. When grown on modified CAS agar plates, all the clinical isolates and K279a were CAS-positive for siderophore production. In order to determine the chemical nature of siderophores, the Csáky (hydroxamate-type) and Arnow (catechol-type) assays were used. All S. maltophilia isolates produced catechol-type siderophores, but hydroxamate-type siderophores were not detected.

Medical devices are often colonized by bacteria which may cause severe infections. The aim of this work was to evaluate biofilm formation by S. maltophilia isolates from device-associated nosocomial infections. The 13 local isolates exhibited different capacities of biofilm formation on hydrophilic and hydrophobic surfaces. All isolates formed strong biofilms in polystyrene microplates, while strong, moderate or weak biofilms were detected in borosilicate (BS) or polypropylene (PP) tubes. The proficiency of biofilm formation was better evaluated by the level of crystal violet staining expressed relative to the final culture density. The microscopic analysis of biofilms formed on glass coverslips revealed the presence of a matrix of exopolysaccharides and microcolonies typical of biofilm architecture. Isolates with increased adhesion to BS showed larger microcolonies. According to our results, twitching correlated well with attachment to the three abiotic surfaces tested, while swimming only showed a slight correlation with biofilm formation on PP. Poor correlation was observed between cell surface hydrophobicity and biofilm formation. One of the highest biofilm-producing isolates adhered to urethral catheters of different materials, and exhibited an increased resistance to oxidative stress, one of the common stresses encountered by bacteria during the infection process due to the immune response.

Aims: The present study shows that Congo red binding and urease activity assays are useful for selection of virulent (Bvg+) Bordetella bronchiseptica cultures., Methods and Results: Congo red binding and urease activity of Bvg+ B. bronchiseptica cultures in different liquid media were compared with the expression of virulence markers such as filamentous haemagglutinin and some outer membrane proteins (OMP). The correlation with the reference virulence markers allowed the establishment of cut-off values for the proposed markers to assure the virulent phenotype (> or = 26 nmol ml-1 of CR and < or = 2.6 U). Using both assays, modulated cultures with avirulent phenotype (Stainer-Scholte broth, with MgSO4 20 mmol l-1 and brain heart infusion broth) and semi-modulated cultures with intermediate phenotypes (tryptose phosphate broth and 83% Stainer-Scholte with MgSO4 5 mmol l-1 cultures) could be distinguished., Conclusion: CR binding assay and urease activity are specific and sensitive enough to detect intermediate phenotypes that could only be detected by subtle changes in OMP profiles., Significance and Impact of the Study: The production of effective veterinary vaccines is hampered by reversible B. bronchiseptica antigenic modulation. The proposed assays are technically suitable for selection of virulent cultures to optimize vaccine production.

Bordetella pertussis virulence-associated 30-, 32-, 90- and 95-kDa outer membrane proteins were purified and their N-terminal amino acid sequences were determined. The 30- and 32-kDa outer membrane proteins showed identity to the C-terminal region of the precursors of the serum resistance protein (BrkA) and the tracheal colonization factor, respectively. We confirmed the cleavage site of these precursors after N731 for BrkA and after N393 for tracheal colonization factor. Associated with the 32-kDa outer membrane protein, we found a new group of 36-kDa virulence-associated peptides. The 95-kDa outer membrane protein showed identity to Vag8. The 90-kDa outer membrane protein did not show homology with the described proteins. We report the N-termini sequence of Vir-90, a novel potential virulence factor.

The bvg or vir locus positively regulates the expression of many Bordetella virulence-associated determinants (encoded by vag genes), including cell envelope proteins, in response to environmental stimuli. On the other hand, several genes named vrg genes are negatively controlled by the bvg regulon (Knapp and Mekalanos, 1988). Flagellin is encoded by a vrg gene, which is expressed when the principal virulence factors are eliminated during antigenic modulation or in phase variants (Akerley et al., 1992). We have previously analyzed SDS-PAGE profiles of Sarkosyl-outer membrane protein (OMP)-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains and reported a major band associated with the avirulent phenotype (Passerini de Rossi et al., 1995). In order to characterize this band we have purified flagellar filaments from Bvg- and modulated Bvg+ strains, and analyzed them by SDS-PAGE. These profiles revealed a single major band of 40 or 45 kDa depending on the strain. The N-terminal amino acid sequence of the putative flagellin expressed by BB7200a was identical over the first 21 residues analyzed to that of the flagellin from the modulated strain BB7865 reported by Akerley et al. (1992). Comparison of the SDS-PAGE profile of flagellar filaments with that of the OMP-enriched fraction of the corresponding strain showed that the flagellum-associated polypeptide had the same electrophoretic mobility as that of the characteristic band of the avirulent phenotype. Furthermore, this band was absent in the OMP-enriched fraction profile from a Bvg- strain subjected to a treatment that removes flagella. Our results indicate that the major protein observed in SDS-PAGE profiles of Sarkosyl-OMP-enriched fractions from B. bronchiseptica Bvg- and modulated Bvg+ strains corresponds to flagellin.

Bordetella pertussis virulent strains (Bvg+) produce a wide array of virulence factors. The production of these factors is coordinately regulated by the locus bvg in response to environmental signals in a process known as antigenic modulation. Mutations in the bvg locus originate avirulent variants (Bvg-) that are unable to express virulence factors regardless of growth conditions. In this paper we have obtained spontaneous variants Bvg- of B. pertussis Tohama I and 10536, which would be useful for studying virulence markers, using the selective media Stainer-Scholte agar supplemented with 1% Casamino-acids (SSA-CAS) and Jones-Kendrick with 0.20 microgram of erythromycin per ml (JK-Ery). We have also studied the efficiency of plating of B. pertussis Tohama I, 10536 and 40103 cells (Bvg+ strains) and of Bvg- strain 347 (control of the avirulent phenotype) on SSA-CAS and Stainer-Scholte agar (SSA), and we have analyzed the phenotype of the cells recovered from these media. The bacterial phenotype was characterized by using the following virulence markers: hemolysis production, hemagglutinin production, and outer membrane protein (OMP) enriched profiles. The three Bvg+ strains showed different behaviour in these selective media. B. pertussis Tohama I and 10536 could not be recovered on SSA, whereas on SSA-CAS the efficiency of plating was poor, less than 0.001%, but nevertheless allowed the selection of stable Bvg- variants of Tohama I since the OMP profile of this stain did not change after subculture in Cyclodextrin liquid medium. By contrast, strain 10536 grown on SSA-CAS suffered the process of antigenic modulation since this strain recovered the virulent phenotype when it was subcultured in Bordet-Gengou. Stable Bvg- variants from Tohama I and 10536 were obtained on JK-Ery. On the other hand, Bvg+ strain 40103 showed a high efficiency of plating on SSA and SSA-CAS and retained the virulent phenotype in all the selective media. Bvg- strain 347, in spite of being an avirulen variant, showed a poor efficiency of plating in SSA and a scant growth in JK-Ery, in agreement with the findings of other investigators which suggest that not all avirulent strains posses the ability to grow on media that inhibit most of virulent strains.