Opposing effects of acellular and whole cell pertussis vaccines on Bordetella pertussis biofilm formation, Siglec-F+ neutrophil recruitment and bacterial clearance in mouse nasal tissues.

Despite global vaccination, pertussis caused by Bordetella pertussis (Bp) is resurging. Pertussis resurgence is correlated with the switch from whole cell vaccines (wPV) that elicit TH1/TH17 polarized immune responses to acellular pertussis vaccines (aPV) that elicit primarily TH2 polarized immune responses. One explanation for the increased incidence in aPV-immunized individuals is the lack of bacterial clearance from the nose. To understand the host and bacterial mechanisms that contribute to Bp persistence, we evaluated bacterial localization and the immune response in the nasal associated tissues (NT) of naïve and immunized mice following Bp challenge. Bp resided in the NT of unimmunized and aPV-immunized mice as biofilms. In contrast, Bp biofilms were not observed in wPV-immunized mice. Following infection, Siglec-F+ neutrophils, critical for eliminating Bp from the nose, were recruited to the nose at higher levels in wPV immunized mice compared to aPV immunized mice. Consistent with this observation, the neutrophil chemokine CXCL1 was only detected in the NT of wPV immunized mice. Importantly, the bacteria and immune cells were primarily localized within the NT and were not recovered by nasal lavage (NL). Together, our data suggest that the TH2 polarized immune response generated by aPV vaccination facilitates persistence in the NT by impeding the infiltration of immune effectors and the eradication of biofilms In contrast, the TH1/TH17 immune phenotype generated by wPV, recruits Siglec-F+ neutrophils that rapidly eliminate the bacterial burden and prevent biofilm establishment. Thus, our work shows that aPV and wPV have opposing effects on Bp biofilm formation in the respiratory tract and provides a mechanistic explanation for the inability of aPV vaccination to control bacterial numbers in the nose and prevent transmission.

Genome Sequences of Bordetella pertussis Isolates from Outbreaks in Northeastern Mexico.

Here, we report the draft genome sequences of 4 Bordetella pertussis isolates which correspond to major clones isolated between 2008 and 2014 from two outbreaks in northeastern Mexico. The B. pertussis clinical isolates belong to the ptxP3 lineage, and they are grouped into two major clusters, defined by the fimH allele.

Architecture and matrix assembly determinants of Bordetella pertussis biofilms on primary human airway epithelium.

Traditionally, whooping cough or pertussis caused by the obligate human pathogen Bordetella pertussis (Bp) is described as an acute disease with severe symptoms. However, many individuals who contract pertussis are either asymptomatic or show very mild symptoms and yet can serve as carriers and sources of bacterial transmission. Biofilms are an important survival mechanism for bacteria in human infections and disease. However, bacterial determinants that drive biofilm formation in humans are ill-defined. In the current study, we show that Bp infection of well-differentiated primary human bronchial epithelial cells leads to formation of bacterial aggregates, clusters, and highly structured biofilms which are colocalized with cilia. These findings mimic observations from pathological analyses of tissues from pertussis patients. Distinct arrangements (mono-, bi-, and tri-partite) of the polysaccharide Bps, extracellular DNA, and bacterial cells were visualized, suggesting complex heterogeneity in bacteria-matrix interactions. Analyses of mutant biofilms revealed positive roles in matrix production, cell cluster formation, and biofilm maturity for three critical Bp virulence factors: Bps, filamentous hemagglutinin, and adenylate cyclase toxin. Adherence assays identified Bps as a new Bp adhesin for primary human airway cells. Taken together, our results demonstrate the multi-factorial nature of the biofilm extracellular matrix and biofilm development process under conditions mimicking the human respiratory tract and highlight the importance of model systems resembling the natural host environment to investigate pathogenesis and potential therapeutic strategies.

Bps polysaccharide of Bordetella pertussis resists antimicrobial peptides by functioning as a dual surface shield and decoy and converts Escherichia coli into a respiratory pathogen.

Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp) are increasing, despite widespread vaccinations. The current acellular pertussis vaccines remain ineffective against nasopharyngeal colonization, carriage, and transmission. In this work, we tested the hypothesis that Bordetella polysaccharide (Bps), a member of the poly-ß-1,6-N-acetyl-D-glucosamine (PNAG/PGA) family of polysaccharides promotes respiratory tract colonization of Bp by resisting killing by antimicrobial peptides (AMPs). Genetic deletion of the bpsA-D locus, as well as treatment with the specific glycoside hydrolase Dispersin B, increased susceptibility to AMP-mediated killing. Bps was found to be both cell surface-associated and released during laboratory growth and mouse infections. Addition of bacterial supernatants containing Bps and purified Bps increased B. pertussis resistance to AMPs. By utilizing ELISA, immunoblot and flow cytometry assays, we show that Bps functions as a dual surface shield and decoy. Co-inoculation of C57BL/6J mice with a Bps-proficient strain enhanced respiratory tract survival of the Bps-deficient strain. In combination, the presented results highlight the critical role of Bps as a central driver of B. pertussis pathogenesis. Heterologous production of Bps in a non-pathogenic E. coli K12 strain increased AMP resistance in vitro, and augmented bacterial survival and pathology in the mouse respiratory tract. These studies can serve as a foundation for other PNAG/PGA polysaccharides and for the development of an effective Bp vaccine that includes Bps.

Generalized and Prolonged Use of Gentamicin-Lock Therapy Reduces Hemodialysis Catheter-Related Infections Due to Gram Negatives.

INTRODUCTION: The incidence of catheter-related bloodstream infections (CRBSI) ranges from 2.2 to 5.5 episodes per 1,000 catheter-days. Our aim was to evaluate the utility of a generalized and prolonged gentamicin-lock therapy in patients undergoing hemodialysis (HD) in a third-level hospital for the reduction in CRBSI. METHODS: A prospective cohort analyzed before and after intervention. During intervention periods after each HD-session, the catheter lumens were locked with gentamicin/heparin for all patients compared to nonintervention periods were the same procedure was performed without gentamicin. Active surveillance was performed for HD CRBSI. Microbiologic assessment and epidemiological data were gathered. Continuous hand hygiene and water quality monitoring were performed. RESULTS: The rates of CRBSI were reduced from 1.28 to 0.2 cases per 1,000 catheter-days when the lock therapy was employed (p = 0.001) The greatest reduction was for CRBSI caused by Pseudomonas aeruginosa were no cases were recorded during the intervention periods (p = 0.001). There was a significant reduction in the total number of isolates; Gram-negative bacterial species (-97.2%) and Gram-positive bacterial species (-61.5%) although only the former reached statistical significance (p = 0.0001). The difference in the absolute risk reduction was 20.56% (95% CI 14.46-26.66%), the calculated Number Needed to Treat was 5 (95% CI 3.8-6.9). No adverse effects were noted. CONCLUSION: In the current study, gentamicin-lock therapy was associated with a significant reduction in CRBSI specially with P. aeruginosa and other Gram-negative bacteria. It proved to be safe and effective intervention when applied to the entire population of HD patients.

Non-typeable Haemophilus influenzae biofilm production and severity in lower respiratory tract infections in a tertiary hospital in Mexico.

Non-typeable Haemophilus influenzae (NTHi) is a common opportunistic bacterial pathogen that primarily infects the respiratory mucosa. This study was conducted to assess clinical and microbiological data related to disease severity in patients with lower respiratory tract infections caused by NTHi in a tertiary care hospital in Mexico. NTHi isolates were subjected to serotyping, antimicrobial susceptibility evaluationand analyses of ß-lactamase production, genetic relatednessand biofilm formation. Clinical and demographic data were retrieved from patients' records. The mean age of the patients was 40.3 years; the majority (n=44, 72.1 %) were male. The main comorbidities were arterial hypertension (n=22, 36.1 %) and diabetes mellitus (n=17, 27.9 %). NTHi isolates (n=98) were recovered from tracheal aspirate (n=57, 58.2 %), sputum (n=26, 26.5 %)and bronchial aspirate (n=15, 15.3 %) specimens. Low resistance to cefotaxime (n=0, 0.0 %), rifampin (n=1, 1.1 %) and chloramphenicol (n=3, 3.2 %) and greater resistance to ampicillin (n=30, 32.3 %) and trimethoprim-sulfamethoxazole (n=49, 52.7 %) were detected. ß-Lactamase production was found in 17 (17.3 %) isolates. Isolates displayed high genetic diversity, and only 10 (10.2 %) were found to be biofilm producers. The antimicrobial susceptibility patterns of biofilm-producing and non-producing isolates did not differ. Biofilm production was associated with prolonged hospital stay (P=0.05). Lower respiratory NTHi isolates from Mexico showed low antimicrobial resistance and weak biofilm production. Younger age was correlated with lower Acute Physiology and Chronic Health Evaluation II score (moderate, P=0.07; severe, P=0.03).

Stenotrophomonas maltophilia in Mexico: antimicrobial resistance, biofilm formation and clonal diversity.

Stenotrophomonas maltophilia is an important multidrug-resistant nosocomial pathogen associated with high mortality. Our aim was to examine antimicrobial susceptibility, biofilm production and clonal relatedness of clinical isolates of S. maltophilia. S. maltophilia isolates were collected between 2006 and 2013 from two tertiary care hospitals in Mexico. Antimicrobial susceptibility was evaluated by the broth microdilution method. PCR was used to determine the presence of ß-lactamase genes L1 and L2. Biofilm formation was assessed with crystal violet staining. Clonal relatedness was determined by PFGE. Among the 119 collected S. maltophilia isolates, 73 (61.3%) were from the respiratory tract. Resistance levels exceeded 75% for imipenem, meropenem, ampicillin, aztreonam, gentamicin and tobramycin. Resistance to trimethoprim-sulfamethoxazole was 32.8%. L1 and L2 genes were detected in 77.1% (91/118) and 66.9% (79/118) of isolates, respectively. All S. maltophilia strains were able to produce biofilms. Strains were classified as weak (47.9%, 57/119), moderate (38.7%, 46/119), or strong (13.4%, 16/119) biofilm producers. A total of 89 distinct PFGE types were identified and 21.6% (22/102) of the isolates were distributed in nine clusters. This is the first study in Mexico to reveal characteristics of clinical isolates of S. maltophilia. Clonal diversity data indicate low cross-transmission of S. maltophilia in a hospital setting. The high antibiotic resistance underscores the need for continuous surveillance of S. maltophilia in hospital settings in Mexico.