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Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200-900 cm(-1) region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm(-1)) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.

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Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). These opportunistic pathogens are also widely distributed in natural and man-made environments. After a 12-year epidemiological surveillance involving Bcc bacteria from respiratory secretions of Argentinean patients with CF and from hospital settings, we found six isolates of the Bcc with a concatenated species-specific allele sequence that differed by more than 3 % from those of the Bcc with validly published names. According to the multilocus sequence analysis (MLSA), these isolates clustered with the agricultural soil strain, Burkholderia sp. PBP 78, which was already deposited in the PubMLST database. The isolates were examined using a polyphasic approach, which included 16S rRNA, recA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), DNA base composition, average nucleotide identities (ANIs), fatty acid profiles, and biochemical characterizations. The results of the present study demonstrate that the seven isolates represent a single novel species within the Bcc, for which the name Burkholderia puraquae sp. nov. is proposed. Burkholderia puraquae sp. nov. CAMPA 1040 (=LMG 29660=DSM 103137) was designated the type strain of the novel species, which can be differentiated from other species of the Bcc mainly from recA gene sequence analysis, MLSA, ANIb, MALDI-TOF MS analysis, and some biochemical tests, including the ability to grow at 42 °C, aesculin hydrolysis, and lysine decarboxylase and β-galactosidase activities.

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A total of 120 Burkholderia cepacia complex isolates collected during 2004 -2010 from 66 patients in two cystic fibrosis reference centers in Argentina were analyzed. Burkholderia contaminans was the species most frequently recovered (57.6%), followed by Burkholderia cenocepacia (15%), a species distribution not reported so far. The recA-PCR-based techniques applied to the B. contaminans isolates revealed that 85% of the population carried the recA-ST-71 allele. Our results showed the utility of BOX-PCR genotyping in analyzing B. contaminans diversity. This approach allowed us to address clonal transmission during an outbreak and the genetic changes occurring in infecting bacteria over the course of chronic infection. Burkholderia cepacia complex species are capable of causing chronic and often severe respiratory tract infections in cystic fibrosis (CF) patients and other types of infections in immunocompromised patients. Although many CF patients remain infected by these bacteria and yet stay relatively healthy for prolonged periods, others either have a severe decline in their pulmonary status or die shortly after the initial colonization (1). In Argentina, the first reports of B. cepacia complex bacteria infecting CF patients started around 1990. At that time, Burkholderia spp. were recovered sporadically and with a very low prevalence (Ͻ0.1%). The prevalence increased over the last decade from 0.2 to 3.6%, depending on the medical center. In early 2004, an outbreak involving main Argentine CF care centers occurred, and the proportion of patients from whom B. cepacia complex species were recovered ranged from 19 to 36% (2-4). Currently, through strict infection control procedures, the prevalence of Burkholderia spp. in local patients has decreased to approximately 10%. Recent worldwide surveillance studies concerning the distribution of B. cepacia complex species in CF patients have cited B. multivorans and B. cenocepacia as the most frequently recovered species. These species account for approximately 80% of the infected patients, and the prevalence of one or the other is geographically and temporally dependent (5-7). Unlike this worldwide situation, in Argentina a relatively high prevalence of B. contaminans followed by B. cenocepacia has been observed (3, 4). To our knowledge, no other geographical region has been reported in the literature to have such a high occurrence of B. contaminans infecting CF patients. In view of this particular scenario, coupled with the lack of biodiversity information available for B. contaminans species, we decided to study this local population by recA-PCR-based techniques and by repetitive element sequence-based PCR (rep-PCR) approaches. These methods allowed us to address the genetic diversity of B. contaminans isolates recovered during that outbreak along with the genetic changes occurring in the infecting bacteria over the course of a chronic infection.

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