Objetivos. Evaluar la susceptibilidad antimicrobiana in vitro a cloranfenicol (CHL) y ciprofloxacino (CIP) de cepas de Bartonella bacilliformis procedentes de áreas endémicas de la enfermedad de Carrión (EC) en el Perú, mediante tres métodos de laboratorio. Materiales y métodos. Se evaluó la susceptibilidad antimicrobiana a CHL y CIP de 100 cepas de Bartonella bacilliformis, los aislamientos procedieron de pacientes de los departamentos de Ancash, Cusco, Cajamarca, Lima y La Libertad; las cepas se evaluaron mediante: disco difusión, E-Test y dilución en agar. Resultados. El 26% de las cepas de Bartonella bacilliformis evaluadas, presentaron resistencia a CIP y 1% a CHL. Se obtuvieron patrones similares de sensibilidad/resistencia antimicrobiana en los tres métodos utilizados. Conclusiones. Las cepas de Bartonella bacilliformis circulantes en el Perú, presentan elevados niveles de resistencia in vitro a CIP, por lo que se recomienda ampliar la investigación sobre la utilización del fármaco en los esquemas de tratamiento de la EC. Los métodos de E-test y disco difusión resultaron más convenientes para la evaluación de la susceptibilidad antimicrobiana in vitro del microorganismo. Palabras clave: Bartonella bacilliformis; Ciprofloxacino; Cloranfenicol; Pruebas antimicrobianas de difusión por disco; Recuento de dilución agar (fuente: DeCS BIREME). ANTIMICROBIAL RESISTANCE OF Bartonella bacilliformis STRAINS FROM REGIONS ENDEMIC TO BARTONELLOSIS IN PERU ABSTRACT Objectives. To evaluate in vitro antimicrobial susceptibility to chloramphenicol (CHL) and ciprofloxacin (CIP) in strains of Bartonella bacilliformis from areas that are endemic to Bartonellosis in Peru, through three laboratory methods. Materials and methods. Antimicrobial susceptibility to CHL and CIP from 100 strains of Bartonella bacilliformis isolated in patients from the regions of Ancash, Cusco, Cajamarca, Lima and La Libertad were evaluated. Strains were evaluated by: disk diffusion, E-test and agar dilution. Results. 26% of the strains of Bartonella bacilliformis evaluated were resistant to CIP and 1% to CHL. Similar patterns of antimicrobial sensitivity / resistance were obtained in all three methods. Conclusions. Bartonella bacilliformis strains circulating in Peru have high levels of in vitro resistance to CIP, so it is advisable to expand research on the use of drug treatment regimens of the Bartonellosis. The methods of E-test and disk diffusion were the most suitable for assessment in vitro of antimicrobial susceptibility of the microorganism.

The presence of amino acid changes in GyrA, GyrB, ParC, ParE, and in a proposed chromosomal chloramphenicol acetyl transferase (CAT), as well as mutations at 23S rRNA, were established by PCR and sequencing in 38 B. bacilliformis clinical isolates from four different areas in Peru. Eighteen out of 24 (75%) isolates showing ciprofloxacin resistance for both disk-diffusion and e-test presented amino acid substitutions in GyrA (G89C, six isolates, A91V, 1 isolate) GyrB (S474F, 10 isolates) or both (GyrA D95N and GyrB S474F, one isolate). Two out of 14 susceptible isolates presented amino acid substitutions at GyrB (S474F) or a double substitution GyrA D95N and GyrB S474F. Of note, ciprofloxacin-resistant isolates were recovered in the four areas studied. No amino acid change was observed at ParC or ParE. Only one isolate showed chloramphenicol resistance, but no alteration was present in either 23S rRNA or CAT. B. bacilliformis resistant to quinolones are extended throughout Peru, with amino acid substitutions at GyrA or GyrB as the main, albeit not exclusive, cause. B. bacilliformis seems to have an apparent facility to develop mutations on GyrB outside the classical positions 91, 95 of GyrA and 85, 88 of ParC.

The current methods for inferring antigenic, pathogenic or virulence factors in bacteria include in-silico or computational tools. Bartonella bacilliformis, the causal agent of Carrion's disease (CD) is a Gram-negative microorganism transmitted by the sand fly Lutzomya verrucarum mainly in Peruvian Inter-Andean valleys. A better understanding of the pathogenicity of B. bacilliformis, the implementation of serological diagnostic methods and the development of candidate vaccines for the control of CD could be facilitated by the identification of outer membrane exposed proteins such as outer-membrane beta-barrels (OMBB) and outer-membrane lipoproteins (OMLPP). In this study we present the in-silico identification of OMBBs and OMLPPs in B. bacilliformis. The present analysis identified 32 OMBBs and 9 OMLPPs, of which 15 and 4 are reported for the first time as potential antigenic, pathogenic and virulence-related proteins, respectively. Future implications of this study are discussed and compared with proteins validated by experimental assays.

Carrion´s disease is caused by Bartonella bacilliformis, it is a Gram-negative pleomorphic bacterium. B. bacilliformis is transmitted by Lutzomyia verrucarum in endemic areas of the Peruvian Inter-Andean valleys. Additionally, the pathogenicity of B. bacilliformis involves an initial infection of erythrocytes and the further infection of endothelial cells, which mainly affects children and expectant women from extreme poverty rural areas. Therefore, the implementation of serological diagnostic methods and the development of candidate vaccines for the control of CD could be facilitated by the prediction of linear b-cell epitopes in specific proteins of B. bacilliformis by bioinformatics analysis. In this study, We used an in-silico analysis employing six web servers for the identification of epitopes in proteins of B. bacilliformis. The selection of B. bacilliformis-specific proteins and their analysis to identify epitopes allowed the selection of seven protein candidates that are expected to have high antigenic activity.