N. Ravenni

Use of the gyrB gene to discriminate among species of the Burkholderia cepacia complex

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that can cause serious infections in lungs of cystic fibrosis patients. The Bcc comprises at least nine species that have been discriminated by a polyphasic taxonomic approach. In this study, we focused on the gyrB gene, universally distributed among bacteria, as a new target gene to discriminate among the Bcc species. New PCR primers were designed to amplify a gyrB DNA fragment of about 1900 bp from 76 strains representative of all Bcc species. Nucleotide sequences of PCR products were determined and showed more than 400 polymorphic sites with high sequence similarity values from most isolates of the same species. Phylogenetic tree analysis revealed that most of the 76 gyrB sequences grouped, forming clusters, each corresponding to a given Bcc species.

Application of multiplex single nucleotide primer extension (mSNuPE) to the identification of bacteria: the Burkholderia cepacia complex case.

Burkholderia cepacia complex (BCC) is characterized by a complex taxonomy constituted by seventeen closely related species of both biotechnological and clinical importance. Several molecular methods have been developed to accurately identify BCC species but simpler and effective strategies for BCC classification are still needed. A single nucleotide primer extension (SNuPE) assay using gyrB as a target gene was developed to identify bacteria belonging to the B. cepacia (BCC) complex. This technique allows the successful detection and distinction of single nucleotide polymorphisms (SNPs) and is effectively applied in routine medical diagnosis since it permits to analyze routinely many samples in a few times. Seven SNuPE primers were designed analyzing the conserved regions of the BCC gyrB sequences currently available in databases. The specificity of the assay was evaluated using reference strains of some BCC species. Data obtained enabled to discriminate bacteria belonging to the species B. multivorans, B. cenocepacia (including bacteria belonging to recA lineages III-A, III-C, and III-D), B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina and B. pyrrocinia. Conversely, identification failed for B. cepacia, B. cenocepacia III-B and B. stabilis. This study demonstrates the efficacy of SNuPE technique for the identification of bacteria characterized by a complex taxonomical organization as BCC bacteria.

Antimicrobial Susceptibility and Synergistic Activity of Meropenem Against Gram-Negative Non-Fermentative Bacteria Isolated from Cystic Fibrosis Patients

Abstract The objective of the present study was to evaluate the activity of meropenem (a beta-lactam carbapenem with good bactericidal potency and a very wide spectrum of activity) and of ticarcillin, ceftazidime ciprofloxacin, tobramycin, cefepime, which are the most commonly used antimicrobial agents for treatment of pulmonary infections associated with CF. The effect of these antibiotics was tested on 27 multiresistant strains isolated from 24 CF patients during 2000 and 2001. Furthermore, the in vitro synergistic effect of meropenem in association with the other antibiotics was evaluated. Ciprofloxacin, ticarcillin, meropenem and ceftazidime had the most activity and inhibited 66%, 37%, 36% and 33% of strains respectively. The addition of a second antibiotic to meropenem resulted in a synergistic effect on 5 (18.5%) isolates; on average 2.8 synergistic combinations where determined per strain. Of these 27 isolates, antagonism was observed in 3 (11%) strains (1 antagonistic combination per strain). Our study suggests that selecting effective double antibiotic therapy cannot be made empirically for CF patients infected with Gram-negative multiresistant bacilli. Therefore in vitro methods for testing double antibiotic combinations are mandatory.

73 Impact of community-acquired MRSA and hospital-acquired MRSA on pulmonary function of CF patients

Objective Persistent methicillin-resistant Staphylococcus aureus (MRSA) infection affects both pulmonary function and survival of CF patients. Few reports have investigated the genetic background of MRSA strains involved in persistent lung infection. This study compares lung function decline in CF patients persistently infected by community-acquired MRSA (CA-MRSA) and hospital-acquired MRSA (HA-MRSA). Methods Seventy-five patients attending five Italian CF centers and persistently infected by MRSA were studied. One strain/year/patient was analyzed. SCCmec analysis was performed as previously described in order to characterize CA- and HA-MRSA strains. The mean annual FEV 1 decline was evaluated for each patient. Results Forty-nine out of 75 (65%) patients maintained the same SCCmec type over time and the mean duration of infection was 3.1 years. Twenty-five patients (mean age 20.6 years) were persistently infected by HA-MRSA and 24 (mean age 18.4 years) by CA-MRSA. The prevalence of chronic P. aeruginosa infection was 52% and 29% in the HA- and CA-MRSA group respectively. The yearly mean FEV 1 decline for patients harboring HA-MRSA and CA-MRSA was –1.12±4.7 and –2.2±4 respectively. Conclusion Most of the studied patients maintained the same SCCmec type for several years. Patients infected by CA-MRSA showed more FEV1 decline in comparison with HA-MRSA infected patients. However this difference is not statistically significant. The knowledge of genetic background of MRSA strains should be improved in order to understand the clinical impact of persistent MRSA infection and optimize prevention and treatment strategies. Grant RF-2010-2316176 Bando Ricerca Finalizzata

121 Anti-Pseudomonas aeruginosa antibodies and microbiological outcome in not chronically infected patients

Objectives: The present study explored the possible protective effect of IgY on PA lung infection in vivo. Methods: In vivo model of acute lung infection: Balb/c mice were anaesthetized with isoflurane and PAO1 vaccine strain ± specific (S-IgY) or control (C-IgY) was inoculated intranasally. Mice were sacrificed after 2, 6 and 24h and lungs removed aseptically, weighted and suspended in PBS. A blinded observer engaged a clinical scoring system (0−5) of the mice. Lungs were homogenized, serially diluted and cultured on Conradi-Drigalski medium for estimation of bacterial load. Results: Relative lung weight: Lung weights in the S-IgY treated group were significantly reduced 24h post-infection compared to PBS controls (p< 0.03). No significant difference between C-IgY and PBS groups were observed. Clinical symptom score: The clinical score was significantly lower in the S-IgY group compared to controls after 6h (C-IgY: p< 0.05, PBS: p< 0.05). After 24h the clinical score in the S-IgY was reduced additionally compared to controls (PBS: p< 0.002, C-IgY: p< 0.04). No significant difference between C-IgY and PBS groups were observed. Quantitative bacteriology: The bacterial load of S-IgY treated mice was significantly reduced 2h post-infection compared to PBS group (p< 0.02) and C-IgY (p< 0.03) and further reduced 6h post-infection compared to both control groups (PBS: p< 0.0001, C-IgY: p< 0.03). After 24h the lung bacteriology in S-IgY treated mice was reduced by 2 logs compared to PBS (p< 0.0001) and C-IgY (p< 0.0002) groups. Conclusion: The present results imply that anti-PA IgY antibodies protects against PA lung infection due to readily bacterial clearance in the airways.

46 Delay in meconium emission: should it be considered a risk for cystic fibrosis?

Background: Ten to 15% of CF infants present with meconium ileus (MI), that sometimes can lead to intestinal malformation. Serum immunoreactive trypsin (IRT) levels may be normal and newborn screening (NBS) could be erroneously considered negative. In our CFNBS protocol newborn babies presenting problems with meconium passage (delay in meconium emission − MD − included) undergo sweat test also if IRT is negative Aim: The aim of this study is to investigate the incidence of CF in neonates with MI and MD to determine if infants with this last condition have to be submitted to CF diagnostic test. Patients and Method: A retrospective 5-year study examines neonates screened in our Center that presented problems in meconium passage, with regard to IRT values and CF diagnosis. Results: In a 5-year period (2006–2010) we screened 172,185 newborns and diagnosed 50 CF. Eigthy-two newborns were reported having some problems related to meconium passage: 13 were referred as MI (group 1), 7 as intestinal atresia (IA) (group 2), 62 as MD (group 3). In group 1 8/13 had IRT positive, 6/13 were diagnosed as CF and one of them had IRT normal value; 2 died without diagnosis. In group 2 6/7 had IRT negative, 2/7 had CF and one of them was IRT negative. In group 3 nobody out 62 had IRT positive, nor positive sweat test. Conclusion: In this series 12% of CF neonates presented with MI and/or IA, and 2 of them were IRT negative confirming that CF with MI and IA can have negative NBS. Delay in meconium pass is therefore, asscociated neither with positive screening nor with CF, suggesting that newborns with MD and IRT negative don’t require further analysis for CF.

107 Treatment of multiresistant Gram-negative non-fermentative infections in Cystic Fibrosis patients: more attention required to in vitro studies

Due to the high degree of resistance to antimicrobial agents of Gram-negative nonfermenters bacilli isolated from CF patients, there are limited therapeutic options available. The present study evaluates the activity of the most frequently used antibacterial drugs, and the effect of combination of two antibiotic against multiresistant Gramnegative non-fermentative rods. The bactericidal effect was investigated by E-test on 48 multiresistant strains (16 P aeruginosa and 32 B. cepacia-complex) isolated over a period of 3 years from CF patients attending two Italian CF Centers. Meropenem, ceftazidime, tobramycin, were the most active inhibiting 77, 75, and 71% of strains respectively. Seventy-three out of 342 (21%) tested combinations showed a synergistic effect being meropenem + ciprofloxacin (25%) and tobramycin + ceftazidime/ticarcillin (19%) the most active associations against P aeruginosa. Ceftazidime + tobramycin (41%) and meropenem + tobramycin (34%) were the most active against B. cepacia-complex. The higher percentage of antagonistic effect was demonstrated for meropenem + cefepime/tobramycin (19%), ticarcillin + ciprofloxacin (19%) for P aeruginosa strains and cloramphenicol + cotrimoxazole (26%), meropenem + ciprofloxacin (25%), ceftazidime + tobramycin (22%) for B. cepacia-complex members. This study indicates that a choice of effective double antibiotic therapy cannot be made empirically for Gram-negative multiresistant infections. The addition of a second antibiotic was found to be potentially counterproductive, in fact 16% of tested associations resulted in antagonisms with loss of bactericidal effects. 4. Microbiology