Intermediate susceptibility to ciprofloxacin among Salmonella Typhi isolates, Lima, Peru

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1 JCM Accepts, published online ahead of print on 26 December 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American ociety for Microbiology. All Rights Reserved. 1 ntermediate susceptibility to ciprofloxacin among almonella Typhi isolates, Lima, Peru Coralith García a#, Veerle Lejon b,c, Gertrudis Horna a, Lizeth Astocondor a, Raymond Vanhoof d, ophie Bertrand d, Jan Jacobs c nstituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru a ; nstitut de Recherche pour le Développement, Montpellier, France b ; nstitute of Tropical Medicine Antwerp, Antwerp, Belgium c ; Belgian National Reference Centre for almonella, nstitute of Public Health, Brussels, Belgium d Running head: usceptibility to ciprofloxacin, almonella Typhi #Address correspondence to Coralith.Garcia@upch.pe

2 15 16 Abstract Thirty-three almonella Typhi blood isolates from Lima, Peru ( ), were fully susceptible to trimethoprim-sulfamethoxazole, chloramphenicol, ceftriaxone, and tetracycline; 8/33 (24.2%) showed intermediate susceptibility to ciprofloxacin carrying mutations in the quinolone resistance-determining region of the gyra gene (er83-phe and Asp87-Asn); and in the gyrb gene (er464-phe). Downloaded from on July 1, 2018 by guest

3 Typhoid fever is a systemic disease mainly caused by almonella enterica serotype Typhi (further referred to as T). Although there is a decreasing incidence, it is still endemic in many countries of Latin America (1). ince the widespread of multidrug resistant isolates (MDR, i.e. resistance to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole), fluoroquinolones (e.g. ciprofloxacin) were the first-line therapy for typhoid fever in adult patients. However, reports of clinical failure in patients treated with ciprofloxacin were described in the outh-east of Asia in the 90s (2). These isolates were resistant to nalidixic acid (NA) showing an increased minimal inhibitory concentration (MC)-value of ciprofloxacin ( µg/ml) but still within the range for susceptibility as defined for Enterobacteriaceae; this phenomenon was named decreased ciprofloxacin susceptibility. The presence of point mutations in the encoding genes of the fluoroquinolone targets, the DNA gyrase (gyra and gyrb) and/or the topoisomerase V (parc and pare) were detected among them (3). everal studies subsequently emphasized the need of changing the breakpoints for fluoroquinolones for almonella (4); as a consequence, the Clinical Laboratories of tandards nstitute changed the breakpoints as follows: (i) susceptible ( 0.06 µg/ml), (ii) intermediate susceptible ( µg/ml) and (iii) resistant ( 1 µg/ml) (5). The emergence of T showing intermediate susceptibility to ciprofloxacin (C) has been almost unstudied in Latin America. Our objective was to assess the susceptibility pattern of T from Peruvian patients, to describe the molecular characteristics of resistance to fluoroquinolones and to analyze their clonal relationship. Blood culture isolates were collected as part of a microbiological surveillance study during through a network of eight public hospitals in Lima, Peru. Blood cultures were drawn during routine patient care. Each hospital followed its own protocol for blood culturing. Of 2897 Gram-

4 negative isolates, 154 (5.3%) belonged to the genus almonella. Only one isolate per patient was considered. Lactose-negative colonies with typical biochemical characteristics of T were further confirmed by agglutination with almonella O9, Vi specific and H:d antisera. usceptibility testing was performed by disk diffusion for ampicillin, ceftriaxone, chloramphenicol, tetracycline and trimethoprim-sulfamethoxazole (6). MC-values for NA and ciprofloxacin were determined by agar dilution (5). Escherichia coli ATCC strain was used as control for susceptibility testing. creening for mutations in the quinolone resistance-determining region (QRDR) was performed by amplification of a fragment of the gyra, gyrb, and parc genes containing the QRDR as previously described (6), the fragments were sequenced on a AB 3130 sequencer. The presence of the plasmid-mediated quinolone resistance qnr genes (qnra, qnrb, and qnr) was determined using PCR (7). The alignment was done using the following strains: almonella Typhimurium LT2, T CT18 and T Ty2. Clonal relationships were assessed by pulse-field gel electrophoresis (PFGE) according to the PulseNet Europe protocol (8). Genomic DNA was digested with the Xba restriction enzymes; almonella Braenderup H9812 was used as a size marker. For cluster analysis, Bionumerics 5.1 was used, with as comparison settings the Dice similarity coefficient and UPMGA dendrogram type (optimization 0.50%, position tolerance 1.50%). A total of 33 isolates were recovered, 11 cases (33.3%) were children (<14 years). All were fully susceptible to trimethoprim-sulfamethoxazole, chloramphenicol, ceftriaxone, ampicillin and tetracycline except two which showed intermediate susceptibility to ampicillin. ix isolates (18.2%) were resistant to NA and eight (24.2%) showed C (Table 1). Most of previous studies in Latin American countries focused on almonella non-typhi

5 (9). Over the last 20 years, only two studies assessing T were found, reporting full susceptibility of 151 T isolates from Colombia and Argentina. Of note, disk diffusion was the method used in both studies. ince MC-values were not determined, it is possible that isolates with C were missed (10,11). A mutation in the gyra, Gly133-Glu, was detected in all C isolates when almonella Typhimurium LT2 was used for alignment. However, it was not detected when T CT18 and T Ty2 were used. This is very likely a serotype specific mutation. t is present in isolates susceptible to NA irrespective of their MC-values for ciprofloxacin. t has been rarely described in Asian countries but was recently found to be the most common mutation in DR Congo (5,13,14). ix isolates had a second mutation in the gyra gene, er83-phe ( n = 3), and Asp87-Asn (n = 3). These mutations are widely spread in T from Asian countries including ndia (4,13). One study which included 12 NA-resistant T isolates from travelers found that seven isolates carried the er83-phe mutation; interestingly six travelers came from ndia and one from Peru (14). n addition, the two remaining isolates had a mutation in the gyrb gene, er464-phe (Table 1). This mutation has been associated to non-classical fluoroquinolone resistance phenotype as both isolates were susceptible to NA; as a consequence C is not predicted by the detection of resistance to NA. uch isolates are typically non-mdr, they have been described from France, UA and England, but they were primarily acquired in ndia (15). No mutations were found in the parc and qnr genes. PFGE revealed a total of 28 different pulsotypes among the 33 isolates, with a maximum of two isolates sharing the same pulsotype (Figure 1). This high diversity has been reported by alve et al. in Argentina and Colombia (11).

6 This study has several limitations. A small number of isolates were included, but they were obtained during a 5-year multicenter laboratory surveillance. Besides, we did not collect clinical information regarding treatment follow-up Among 33 T isolates, none displayed MDR but C was demonstrated in almost a quarter of them. All C isolates showed a mutation in the gyra (Gly133-Glu), combined with either a mutation in gyra at codons 83 and 87 or in the gyrb gene at codon 464. A diverse clonal distribution was demonstrated. ince fluoroquinolones are still the cornerstone of the treatment of typhoid fever in Latin American countries, active surveillance is needed in order to monitor resistance trends to these antimicrobials. Acknowledgements This study was sponsored by the Directorate General for Development Cooperation of the Belgian Government. We thank our collaborators: Aida Palacios, Elba Linares, Rafael Ramírez, Jorge Velásquez, Verónica Medina, Martha Urbina, Nelva Espinoza, ilvia Zevallos, Maria Zamudio, and Ana Meza.

7 References Crump J, Luby P, Mintz ED The global burden of typhoid fever. Bull. World. Health. Org. 82: Parry CM, Hien T, Dougan G, White N, Farrar J Typhoid fever. New. Eng. J. Med. 347: Parry CM, Threlfall EJ Antimicrobial resistance in typhoidal and nontyphoidal salmonellae. Curr. Opin. nfect. Dis. 21: Parry CM, Thuy CT, Dongol, Karkey A, Vinh H, Chinh NT, Thieu Nga TV, Campbell J, Van Minh Hoang N, Arjyal A, Bhutta ZA, Bhattacharya K, Agtini MD, Dong B, Canh do G, Naheed A, Wain J, Tinh Hien T, Basnyat B, Ochiai L, Clemens J, Farrar JJ, Dolecek C, Baker uitable disk antimicrobial susceptibility breakpoints defining almonella enterica serovar Typhi isolates with reduced susceptibility to fluoroquinolones. Antimicrobial. Agents. Chemother. 54: CL Performance standards for antimicrobial susceptibility testing; twentysecond informational supplement. CL document M Clinical and Laboratory tandards nstitute, Wayne, PA. 6. Griggs D, Gensberg K, Piddock L Mutations in gyra gene of quinoloneresistant almonella serotypes isolated from humans and animals. Antimicrob. Agents. Chemother. 40: Cavaco L, Frimodt-Møller N, Hasman H, Guardabassi L, Nielsen L, Aarestrup F Prevalence of quinolone resistance mechanisms and associations to minimum inhibitory concentrations in quinolone-resistant Escherichia coli isolated from humans and swine in Denmark. Microb. Drug. Resist. 14: Ribot E, Fair M, Gautom R, Cameron D, Hunter, waminathan B, Barrett TJ tandardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, almonella, and higella for PulseNet. Foodborne. Pathog. Dis. 3: Cordano a M, Virgilio R Evolution of drug resistance in almonella panama isolates in Chile. Antimicrob. Agents. Chemother. 40: Cardona-Castro N Caracterización de dos brotes de fiebre tifoidea en Apartadó, Antioquia, Biomedica. 27:

8 alve A, Pichel M Molecular subtyping of almonella enterica serovar Typhi isolates from Colombia and Argentina. Foodborne. Pathol. Dis. 3: Lunguya O, Lejon V, Phoba M-F, Bertrand, Vanhoof R, Glupczynski Y, Verhaegen J, Muyembe-Tamfum JJ, Jacobs J Antimicrobial resistance in invasive non-typhoid almonella from the Democratic Republic of the Congo: emergence of decreased fluoroquinolone susceptibility and extended-spectrum beta lactamases. PLo. Negl. Trop. Dis. 7:e Menezes GA, Harish BN, Khan MA, Goessens WHF, Hays JP Antimicrobial resistance trends in blood culture positive almonella Typhi isolates from Pondicherry, ndia, Clin. Microbiol. nfect. 18: Tatavarthy A, anderson R, Peak K, cilabro G, Davenhill P, Cannons A, Amuso P Molecular typing and resistance analysis of travel-associated almonella enterica serotype Typhi. J Clin Microbiol. 50: Accou-Demartin M, Gaborieau V, ong Y, Roumagnac P, Marchou B, Achtman M, Weill FX almonella enterica erotype Typhi with nonclassical quinolone resistance phenotype. Emerg. nfect. Dis.17: Downloaded from on July 1, 2018 by guest

9 Table 1. Cases of typhoid fever caused by almonella Typhi with intermediate susceptibility to ciprofloxacin Code Age Origin ampling date Alignment with MC (µg/dl) almonella Typhi CT18 and Ty2 CP NA gyra gyrb HAMA Pediatric ward 13/05/ er83-phe HAMA Pediatric ward 15/02/ Asp87-Asn HEB Pediatric ward 10/08/ >256 Asp87-Asn HCH Outpatient clinic 19/01/ * er464-phe HGA Emergency room 13/02/ Asp87-Asn HAMA Medicine ward 01/12/ * er464-phe HCH Medicine ward 04/01/ er83-phe HAMA Pediatric ward 28/04/ er83-phe CP: ciprofloxacin, NA: nalidixic acid All isolates tested carried a Glu133-Gly mutation in the gyra gene * solates susceptible to nalidixic acid

10 Graphic 1. Pulsed field gel electrophoresis analysis of 33 almonella Typhi isolates from Lima, Peru. Ciprofloxacin susceptibility Mutations gyra Asp87-Asn Asp87-Asn er83-phe gyrb er464-phe Downloaded from = susceptible, = intermediate susceptibility Asp87-Asn er83-phe er83-phe er464-phe on July 1, 2018 by guest