Canadian Journal of Microbiology. Ciprofloxicin-resistant Neisseria meningitidis in Canada: likely imported strains.

Transcription

1 Ciprofloxicin-resistant Neisseria meningitidis in Canada: likely imported strains. Journal: Manuscript ID cjm r1 Manuscript Type: Note Date Submitted by the Author: 25-Nov-2016 Complete List of Authors: Tsang, Raymond S.W.; CNS Infection Division and Vaccine Preventable Bacterial Diseases Division,, Law, Dennis; Public Health Agency of Canada, Vaccine Preventable Bacterial Diseases Division Deng, Saul; Public Health Agency of Canada, National Microbiology Laboratory Hoang, Linda; British Columbia Center for Disease Control, Keyword: Neisseria meningitidis, Ciprofloxacin resistance

2 Page 1 of 11 Ciprofloxicin-resistant Neisseria meningitidis in Canada: likely imported strains. Formatted Raymond SW Tsang 1*, Dennis KS Law 1, Saul Deng 1, and Linda Hoang 2. Vaccine Preventable Bacterial Diseases, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba 1 ; and BC Public Health Microbiology and Reference Laboratory, Vancouver, British Columbia 2, Canada. Running Title: Ciprofloxacin-resistant N. meningitidis in Canada * Corresponding author: Raymond Tsang, National Microbiology Laboratory, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2. Phone: (204) Fax (204) Raymond.tsang@phac-aspc.gc.ca 1

3 Page 2 of 11 Abstract The prevalence of ciprofloxacin-resistant Neisseria meningitidis in Canada was studied by testing 346 isolates received at the National Microbiology Laboratory during the calendar years 2013 to Of the 277 individual invasive and 69 non-invasive isolates tested, only 2 serogroup C (MenC) isolates were found to be resistant to ciprofloxacin. Both MenC were typed as sequence type (ST)-4821, a unique clone found mainly in China, thus suggesting both isolates might be from travel-related or imported cases. This prompted us to also examine six serogroup A (MenA) isolates in our collection since MenA is not currently endemic in Canada. Three MenA from 2006 were resistant to ciprofloxacin and they were typed as ST A ciprofloxacin-resistant MenA strain of ST-4789 was responsible for a meningococcal disease outbreak in Delhi, India in 2005 to The two MenC and three MenA ciprofloxacin-resistant N. meningitidis were from patients residing in British Columbia. (148 words) Key Words: Neisseria meningitidis, Ciprofloxacin resistance 2

4 Page 3 of 11 Invasive meningococcal disease (IMD) caused by the bacterium Neisseria meningitidis is a serious infection with an average case-fatality rate of approximately 10% (Rosenstein et al. 2001). The choice of antibiotic for treatment of IMD is either penicillin G or a third-generation cephalosporin such as ceftriaxone; or chloramphenicol if patient is allergic to β-lactam antibiotics (Apicella 2009). The currently recommended antibiotics for chemoprophylaxis against meningococci include rifampin, ceftriaxone, or ciprofloxacin. Rifampin treatment of contacts of meningococcal cases has been shown to induce antibiotic resistance (Weidner et al. 1971). Since a single oral dose of ciprofloxacin can eliminate carriage of meningococci (Dworzack et al. 1988), ciprofloxacin has become a common choice for chemoprophylaxis. However, resistance to ciprofloxacin has also been reported in N. meningitidis (Corso et al. 2005; Chu et al. 2007; Skocznska et al. 2008; Enriquez et al. 2008; Strahilevitz et al. 2008; Wu et al. 2009; Lapadua et al. 2009; Donaldson et al. 2010; du Plessis et al. 2010). This study examined the current prevalence of ciprofloxacin resistant N. meningitidis in Canada using strains received at the National Microbiology Laboratory (NML) between the period of January 1, 2013 to December 31, The Public Health Agency of Canada s NML has a laboratory surveillance program on IMD and receives almost all invasive N. meningitidis strains from different provinces and territories. Between January 1, 2013 to December 31, 2015, 277 individual IMD case isolates were received and were included in this study. Identification of isolates as N. meningitidis and serogroup determination were confirmed at the provincial public health laboratories. There were 102, 84, and 91 isolates received in 2013, 2014, and 2015, respectively and their serogroup information was confirmed at the NML. Of the 277 IMD case isolates, 168 were serogroup B, 16 were 3

5 Page 4 of 11 serogroup C, 73 were serogroup Y, 17 were serogroup W, two were serogroup Z and one was a non-encapsulated strain. Sensitivity of N. meningitidis to ciprofloxacin and nalidixic acid were determined by E-test (AB Biodisk Solna, Sweden) and results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guideline (Clinical and Laboratory Standards Institute 2015). Resistant strains were characterized by serotype and serosubtype using a monoclonal antibody kit (Rijksinstitut voor Volksgezondheid en Milieu, National Institute of Public Health, Bethoven, The Netherlands) and whole cell ELISA assay (Abdillahi et al. 1987). PorA genotype and multilocus sequence typing (MLST) were done as previously described (Zhou et al. 2012; Jamieson et al. 2013) using resources described in the Neisseria.org website (www://neisseria.org). Mutations at the quinolone resistance determining regions (QRDRs) of gyra, gyrb, parc, and pare that affects N. meningitidis susceptibility to quinolone antibiotics were determined by DNA sequencing of the respective genes and comparing the sequence results to that obtained from a quinolone sensitive N. meningitidis strain MC58 using published methods. The gyra and parc genes were amplified and sequenced as described by Wu et al.; while gyrb was amplified and sequenced as described by Strahilevitz et al. For amplification and sequencing of the pare gene, an in-house designed forward primer (5 CCCGCAGTTCCAAGGTCS-3 ) was used in combination with the reverse primer described by Hong et al. Of the 277 IMD case isolates tested, only two serogroup C (MenC) strains were found to be resistant to ciprofloxacin with both isolates showing MIC values of 0.38 µg/ml to ciprofloxacin and 258 µg/ml to nalidixic acid. Both MenC isolates were recovered from patients in the province of British Columbia (BC), one in 2013 and the other in Both patients were male aged 84 and 67 years old, respectively. One of the isolates was recovered from blood culture and 4

6 Page 5 of 11 the other was recovered from the joint. Both isolates were typed as C:NT:P1.14 and sequence type (ST)-4821 (ST-4821 clonal complex). MenC of ST-4821 was first described in China causing a MenC outbreak in the province of Anhui (Shao et al. 2006). Of the 19 entries of ST in the Neisseria MLST website ( data accessed on June 14, 2016), thirteen were serogroup C, two were group B, and four were non-groupable (from carriers); and all were reported from China between 2004 and To ascertain that ciprofloxacin-resistant N. meningitidis of ST-4821 was not endemic in Canada, we also examined 69 (37 from BC) non-invasive N. meningitidis isolates received at the NML between 2013 and 2015, including 49 (26 from BC) isolates from the respiratory tract. No ciprofloxacinresistant N. meningitidis nor ST-4821 strain was found in any of the non-invasive isolates. Since our data on ciprofloxacin-resistant N. meningitidis suggests a potential foreign source of such antibiotic-resistant strain and serogroup A N. meningitidis (MenA) is not currently endemic in Canada, we also examined six MenA from our culture collection. These six isolates were recovered from patients in Canada from 2001 to present. Four of these serogroup A isolates were recovered from blood culture, one was isolated from a bronchial washing, and one was of unknown source. Three of the six MenA isolates were resistant to quinolone with ciprofloxacin MIC of 0.19 µg/ml (two isolates) or 0.25µg/ml (one isolate) and all three isolates gave nalidixic acid MIC values of 245 µg/ml. All three ciprofloxacin-resistant MenA were recovered from blood cultures of patients in the province of BC in 2006, involving three male subjects of ages 63, 72, and 88 years old. The three ciprofloxacin-resistant N. meningitidis were typed as A:4:P1.9 and ST MenA is very rarely isolated in Canada and strain of ST-4789 has not been found prior to the isolation of these three strains in Between the period of April 2005 and April 2006, a MenA outbreak was reported in Delhi, India (Mehta et al. 2007; Singhal et al. 5

7 Page 6 of ), and the responsible strain was resistant to ciprofloxacin and subsequently identified as ST-4789, member of the ST-5 clonal complex or subgroup III. Nucleic acid sequence of the QRDRs of gyra, gyrb, parc, and pare were determined in the two MenC and three MenA that showed resistance to ciprofloxacin. In all five ciprofloxacin-resistant case isolates, only the gyra gene was found to contain the characteristic mutation affecting amino acid 91 to change from Threonine to Isoleucine (Enriquez et al. 2008; Hong et al. 2013). In four of these five ciprofloxacin-resistant meningococcal isolates, no mutation was detected in their gyrb, parc, and pare genes when their nucleic acid sequences were compared to the respective genes of the ciprofloxacin-sensitive MenB strain MC58. In the fifth strain, its pare gene shows no mutation while the gyrb and parc genes could not be amplified and therefore, their gene sequences could not be determined. In all five isolates, other than resistance to ciprofloxacin, none showed resistance to penicillin G, chloramphenicol, rifampin, or ceftriaxone. Their antibiotic susceptibility profiles are shown in Table 1. Ciprofloxacin-resistant MenA of ST-4789 was responsible for the outbreak in New Delhi, India in (Mehta et al. 2007; Singhal et al. 2007) and this strain was subsequently reported in Israel (Strahilevitz et al. 2008) and Italy (Lapadua et al. 2009) as imported cases. MenC of ST was first identified in China (Shao et al. 2006), and although it was not reported to be resistant to ciprofloxacin, recent studies of MenW in China indicated a 70.6% resistance rate (Strahilevitz et al. 2008) of 17 isolates studied (Hu et al. 2014). Therefore, our data when interpreted in the current literature suggested that both the ciprofloxacin-resistant MenC and MenA isolates identified in Canada were of potential foreign sources and such antibioticresistant strains did not appear to be endemic in Canada. Furthermore, subsequent surveillance studies did not suggest local spread of these imported antibiotic resistant N. meningitidis strains. 6

8 Page 7 of 11 However, continued surveillance is required to monitor for potential import of exotic antibioticresistant strains or antibiotic resistant strains that arise locally through genetic mutations. (1,312 words) Acknowledgement We thank the Directors and staff of the Provincial Public Health Laboratories across Canada for providing the isolates for the national surveillance of invasive meningococcal disease. We also thank Jianwei Zhou for serotyping and serosubtyping of the strains, and the NML s DNA Core Service for providing assistance in nucleotide sequencing. This publication made use of the Neisseria Multi Locus Sequence Typing website ( neisseria/) developed by Keith Jolley and sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:595). The development of this site has been funded by the Wellcome Trust and European Union. 7

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12 Page 11 of 11 Table 1. The antibiotic susceptibility profiles * of five Canadian Neisseria meningitidis strains showing resistance to ciprofloxacin. Strain 1 Strain 2 Strain 3 Strain 4 Strain 5 # resistant / total # tested # (% resistant) Year of isolation Not applicable Serogroup C C A A A Not applicable Penicillin G S S S S S 0/352 (0%) Ceftriaxone S S S S S 0/352 (0%) Chloramphenicol S S S S S 0/352 (0%) Rifampin S S S S S 0/352 (0%) Ciprofloxacin R R R R R 5/352 ((1.4%) * Antibiotic susceptibility profiles: S = sensitive; R = resistant # Total number tested = 277 invasive isolates from 2013 to 2015; 69 non-invasive isolates from 2013 to 2015; and six serogroup A isolates from our culture collection.