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 ~ Introduction
 ~ Methods
 ~ Results
 ~ Discussion
 ~ Conclusions
 ~  References
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  Table of Contents  
BRIEF COMMUNICATION
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 520-525
 

Six-year susceptibility trends and effect of revised Clinical Laboratory Standards Institute breakpoints on ciprofloxacin susceptibility reporting in typhoidal Salmonellae in a tertiary care paediatric hospital in Northern India


1 Department of Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, New Delhi, India
2 Department of Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya; Department of Microbiology (VM), Maulana Azad Medical College, New Delhi, India

Date of Submission22-Dec-2015
Date of Acceptance05-Oct-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
V Manchanda
Department of Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya; Department of Microbiology (VM), Maulana Azad Medical College, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.195362

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 ~ Abstract 

The antimicrobial trends over 6 years were studied, and the effect of revised Clinical Laboratory Standards Institute (CLSI) breakpoints (2012) for ciprofloxacin susceptibility reporting in typhoidal Salmonellae was determined. A total of 874 (95.4%) isolates were nalidixic acid-resistant (NAR). Using the CLSI 2011 guidelines (M100-S21), 585 (66.9%) isolates were ciprofloxacin susceptible. The susceptibility reduced to 11 (1.25%) isolates when interpreted using 2012 guidelines (M100-S22). Among the forty nalidixic acid susceptible (NAS) Salmonellae, susceptibility to ciprofloxacin decreased from 37 isolates (M100-S21) to 12 isolates (M100-S22). The 25 cases which appeared resistant with newer guidelines had a minimum inhibitory concentration (MIC) range between 0.125 and 0.5 μg/ml. MIC50 for the third generation cephalosporins varied between 0.125 and 0.5 μg/ml over 6 years whereas MIC90 varied with a broader range of 0.19–1 μg/ml. The gap between NAR and ciprofloxacin-resistant strains identified using 2011 guidelines has been reduced; however, it remains to be seen whether additional NAS, ciprofloxacin-resistant isolates are truly resistant to ciprofloxacin by other mechanisms of resistance.


Keywords: Multidrug-resistant organism, revised Clinical Laboratory Standards Institute breakpoints, Salmonella typhi, susceptibility trends, typhoidal Salmonellae


How to cite this article:
Saksena R, Nayyar C, Manchanda V. Six-year susceptibility trends and effect of revised Clinical Laboratory Standards Institute breakpoints on ciprofloxacin susceptibility reporting in typhoidal Salmonellae in a tertiary care paediatric hospital in Northern India. Indian J Med Microbiol 2016;34:520-5

How to cite this URL:
Saksena R, Nayyar C, Manchanda V. Six-year susceptibility trends and effect of revised Clinical Laboratory Standards Institute breakpoints on ciprofloxacin susceptibility reporting in typhoidal Salmonellae in a tertiary care paediatric hospital in Northern India. Indian J Med Microbiol [serial online] 2016 [cited 2017 May 24];34:520-5. Available from: http://www.ijmm.org/text.asp?2016/34/4/520/195362



 ~ Introduction Top


Enteric fever caused by Salmonella typhi and paratyphi A remains an important public health problem in developing countries. In 2000, it was estimated that over 2.16 million episodes of typhoid occurred worldwide, resulting in 216,000 deaths, and that more than 90% of this morbidity and mortality occurred in Asia.[1] India has an annual incidence of 493.5 per 100,000 person-years with 340.1 per 100,000 person-years occurring in children of 2–5 years.[2] Fluoroquinolones have been recommended by the World Health Organization (WHO) as empirical therapy for the management of multidrug-resistant (MDR) enteric fever. Globally, the spread of Salmonella enterica serovar typhi (S. typhi) strains with fluoroquinolone-reduced susceptibility has become a major problem. Earlier, the Clinical Laboratory Standards Institute (CLSI) breakpoints (≥4 mg/mL [resistant] and ≤1 mg/L [susceptible]) for reporting fluoroquinolone susceptibility in Enterobacteriaceae do not detect these strains.[3] Using a disc diffusion method, usually, these strains appear resistant when tested with a nalidixic acid disc (30 µg) and therefore screening with it is a sensitive, specific and cost-effective approach, used by many laboratories in resource-limited countries.[4] Breakpoints for ciprofloxacin minimum inhibitory concentration (MIC) for S. typhi and extraintestinal salmonellosis have been reduced by the CLSI in 2012.[5] This study was conducted to determine the effect of revised CLSI breakpoints for ciprofloxacin susceptibility in typhoidal Salmonellae, and the role of nalidixic acid resistance (NAR) as a marker for ciprofloxacin resistance was reviewed. Third generation cephalosporins (3GC) are now used as the choice of therapy in the management of severe typhoidal infections. However, delayed response to these antimicrobials has emerged. Thus, MIC and susceptibility trends for 3GC were also studied.


 ~ Methods Top


A prospective study was conducted on 916 isolates of S. typhi, paratyphi A and paratyphi B, isolated at our tertiary care paediatric hospital from January 2009 to December 2014. Isolates were identified by VITEK2® compact system (BioMérieux, Marcy-l'Étoile, France) and by serotyping with specific antisera. Antimicrobial susceptibilities were determined by E-test (ciprofloxacin, ceftriaxone, azithromycin, co-trimoxazole and chloramphenicol; AB Biodisk, BioMérieux, France) and disc diffusion method (nalidixic acid 30 µg, co-trimoxazole 1.25/23.75 µg, ampicillin 10 µg; Oxoid, Basingstoke, UK). In 2013 and 2014, a wider panel of antimicrobials was also tested using VITEK2® system. Susceptibility interpretative criteria were taken from the CLSI. Earlier, up to 2011, MIC breakpoints recommended by the CLSI for Enterobacteriaceae (including S. enterica) for ciprofloxacin were ≥4 mg/L (resistant) and ≤1 mg/L (susceptible).[3] In 2012, the CLSI lowered the breakpoints for ciprofloxacin susceptibility for S. typhi and extraintestinal salmonellosis in view of poor response to ciprofloxacin therapy, now categorising isolates with MIC ≥1 mg/L as resistant and MIC ≤0.06 mg/L as susceptible.[5] Zone diameter interpretive criteria of >=19 were taken as susceptible and ≤13 were taken as resistant for nalidixic acid by disc diffusion testing.

For azithromycin, interpretative criteria of the British Society of Antimicrobial Chemotherapy were used (MIC ≤16 mcg/ml was considered susceptible and MIC ≥16 mcg/ml was considered resistant).[6] The antimicrobial susceptibility patterns were analysed using WHONET 5.6 (World Health Organisation, Geneva, Switzerland).


 ~ Results Top


Of the 916 non-repeat isolates studied, 890 (97.1%) were isolated from blood, 15 (1.6%) from urine, 9 (0.98%) from pus and 2 (0.22%) from cerebrospinal fluid. The number of S. typhi isolates was 849 (92.7%) compared to 65 (7.1%) Salmonella paratyphi A isolates and two Salmonella paratyphi B (0.22%) were isolated. A total of 876 (95.6%) isolates out of 916 studied were observed as NAR. Among the 876 NAR strains, using the CLSI 2011 guidelines,[3] 587 (67%) isolates were ciprofloxacin susceptible [Figure 1]. The susceptibility to ciprofloxacin reduced to 11 (1.25%) isolates when interpreted using 2012 guidelines (M100-S22) [Figure 2]. After applying the revised breakpoints, among the resistant strains, MIC value of 218 (23.8%) strains was ≥4 µg/ml whereas 69 strains were between 1 and 4 µg/ml (7.5%). There were 537 (58.6%) strains which had MIC value between 0.12 and 1 µg/ml and thus decreased ciprofloxacin susceptibility (DCS). Among the forty nalidixic acid-susceptible (NAS) Salmonellae, susceptibility to ciprofloxacin decreased from 37 isolates (M100-S21) to 12 isolates (M100-S22) using the newer breakpoints. The 25 NAS cases which appeared resistant to ciprofloxacin with newer guidelines had MIC range between 0.125 and 0.5 µg/ml.
Figure 1: Scatterplot depicting the relative distribution of Salmonella typhi and paratyphi A isolates for ciprofloxacin minimum inhibitory concentrations and nalidixic acid disc diffusion zone diameters with ciprofloxacin breakpoints based on the Clinical Laboratory Standards Institute 2011 guidelines. Clustering of cases in left lower quadrant reveals that using these guidelines large number of isolates that were resistant to nalidixic acid (X-axis), were found as susceptible to ciprofloxacin (Y-axis). Thus, isolates reported susceptible based on only ciprofloxacin minimum inhibitory concentrations may have delayed response or therapeutic failure

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Figure 2: Scatterplot depicting the relative distribution of Salmonella typhi and paratyphi A isolates for ciprofloxacin minimum inhibitory concentrations and nalidixic acid disc diffusion zone diameters with ciprofloxacin breakpoints based on the Clinical Laboratory Standards Institute 2012 guidelines. The number of isolates in left lower quadrant has reduced dramatically when compared with [Figure 1]. This shows that there is now higher concordance between nalidixic acid resistance and ciprofloxacin resistance based on newer breakpoints

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The overall susceptibility to 3GCs was 100%. MIC50 for 3GC varied between 0.125 and 0.5 mg/L over the period of 5 years, whereas MIC90 varied with a broader range of 0.19–2 mg/L [Table 1], [Figure 3] and [Figure 4]. In the case of other antimicrobials tested, the susceptibility to chloramphenicol was 87.5%, to sulfamethoxazole-trimethoprim was 84.9% and 92% to azithromycin.
Table 1: Trends of minimal inhibitory concentration of commonly tested antimicrobials against typhoidal Salmonellae over the years

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Figure 3: Year-on-year trends of minimum inhibitory concentration 50 for different antibiotics against typhoidal Salmonellae

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Figure 4: Year-on-year trends of minimum inhibitory concentration 90 of different antibiotics against typhoidal Salmonellae

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In 2013 and 2014, susceptibility testing was done for the following antimicrobial agents, and the antibiogram for enteric Salmonellae was determined [Table 2].
Table 2: Antibiogram of typhoidal Salmonellae for 2013 (n=161) and 2014 (n=179)

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The rate of MDR Salmonella isolates was 4.34% in 2013 and 2.79% in 2014.


 ~ Discussion Top


The trend of rising MIC and resistance to fluoroquinolones in enteric Salmonellae has been reported since 1990s in various studies from India.[7],[8],[9],[10] Nalidixic acid is the prototype quinolone. Although it has been available for use since 1960s, it is seldom used for the treatment of enteric fever because of the high prevalence of NAR S. typhi. Studies from the United Kingdom, United States and Kuwait reported a higher percentage of discordant nalidixic acid and ciprofloxacin susceptibility results in their Southeast Asian immigrants or travellers (i.e. 7%, 12.5% and 13.8%).[11],[12],[13] In our study, discordance was 69.5%, which was reduced to 0.1% using the modified CLSI guidelines. In the present study, NAR was observed in 95.38% of the strains. Other studies in India have earlier reported resistance to nalidixic acid to be between 47% and 100%.[14],[15]

Earlier MIC breakpoints recommended by the CLSI for Enterobacteriaceae (including S. enterica) for ciprofloxacin were ≥4 mg/L (resistant) and ≤1 mg/L (susceptible). In 2012, the CLSI lowered the breakpoints for ciprofloxacin susceptibility for S. typhi and extraintestinal salmonellosis in view of poor response to ciprofloxacin therapy, now categorising isolates with MIC ≥1 mg/L as resistant and MIC ≤0.06 mg/L as susceptible.

Applying the newer guidelines to the archived data among the NAR strains, the ciprofloxacin susceptible isolates reduced from 585 to 11. Thus, the fluoroquinolone resistance ambiguity in NAR strains has been reduced from 66.9% to 1.25%. Another multicentre study from different settings in India has also reported a sharp decline in the percentage of susceptible strains, from 95% susceptible strains to mere 3%, following application of newer breakpoints. The majority of isolates (88%) fell in the intermediate susceptible zone similar to our study where 69.5% of isolates showed DCS.[16] Studies have shown that this type of resistance is mediated by single mutation in the gyrA gene which encodes a subunit of DNA gyrase and is located in the quinolone resistance-determining region.[17]

It was also observed that among the forty nalidixic acid-susceptible Salmonellae isolates, ciprofloxacin-susceptible isolates reduced from 37 using 2011 CLSI guidelines to 12 after applying the newer breakpoints indicating that there are other mechanisms of resistance also which are not detected by NAR. The MIC range of the additional 25 strains which were reclassified as non-susceptible was between 0.125 and 0.5 mg/L. Although the gap between NAR and ciprofloxacin-resistant strains identified using 2011 guidelines has been considerably reduced with revised breakpoints, we did not determine whether the additional NAS, ciprofloxacin-resistant isolates are resistant to ciprofloxacin by other mechanisms of resistance. It has been reported that mutations outside of gyrA gene like mutation in the gyrB gene or in the plasmid-mediated quinolone resistance determinants result in DCS with nalidixic acid-susceptible strains.[17] Thus, the role of nalidixic acid as a predictor of fluoroquinolone susceptibility and whether there is a need to test for ciprofloxacin susceptibility additionally should be further evaluated.

The recent studies from North India have also reported a high rate of resistance to fluoroquinolones similar to the present findings, but we have detected a higher rate of resistance to chloramphenicol and trimethoprim-sulfamethoxazole at 11.7% and 14%, respectively [Table 3].[18],[19] Among 161 total strains isolated in 2013, only 7 (4.34%) were MDR, i.e., resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. In 2014, this number further decreased to 5 out of a total of 179 strains (2.79%). Thus, the present rate of MDR S. typhi and paratyphi A was <5% as reported from various regions of India.[20] In comparison with a study done at our centre in 2004–2005, a rise in the percentage resistance and MIC50 to fluoroquinolones, 3GCs and chloramphenicol was observed whereas a fall in the resistance to co-trimoxazole from 30.4% in 2004–2005 to 15% in 2009–2014 was seen with only 9.1% resistance in 2013.[21]
Table 3: Comparative susceptibility profile of extraintestinal Salmonellae from various studies in Northern India

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The susceptibility to 3GC remains high and thus can be continued to be treated as the drug of choice for enteric fever. As the MDR rate is <5%, ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol can be considered as alternatives for the treatment of enteric fever.


 ~ Conclusions Top


It is recommended that ciprofloxacin MIC determination in nalidixic acid-susceptible strains should be performed to avoid therapeutic failure. In case of resource-limited settings where ciprofloxacin MIC cannot be determined for all NAS strains, periodic testing for ciprofloxacin MIC can be done to see the trend of ciprofloxacin susceptibility in the community.

Limitations

The genotypic basis for resistance to fluoroquinolones was not determined in the present study. Further investigations are required to ascertain various mutations leading to decreased ciprofloxacin resistance, especially in NAS strains. We did not test for azithromycin susceptibility in 2014 and, thus, the trend for susceptibility to this drug is only available to us till 2013. Furthermore, MICs for 3GCs were determined for only 75 out of 179 isolates in 2014 (disc zones were determined for the rest) and, thus, the lower MIC90 may not be accurate.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Harish BN, Madhulika U, Parija SC. Isolated high-level ciprofloxacin resistance in Salmonella enterica subsp. enterica serotype Paratyphi A. J Med Microbiol 2004;53(Pt 8):819.  Back to cited text no. 8
    
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Dimitrov T, Udo EE, Albaksami O, Al-Shehab S, Kilani A, Shehab M, et al. Clinical and microbiological investigations of typhoid fever in an infectious disease hospital in Kuwait. J Med Microbiol 2007;56(Pt 4):538-44.  Back to cited text no. 13
    
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Renuka K, Kapil A, Kabra SK, Wig N, Das BK, Prasad VV, et al. Reduced susceptibility to ciprofloxacin and gyrA gene mutation in North Indian strains of Salmonella enterica serotype Typhi and serotype Paratyphi A. Microb Drug Resist 2004;10:146-53.  Back to cited text no. 15
    
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Balaji V, Sharma A, Ranjan P, Kapil A. Revised ciprofloxacin breakpoints for Salmonella Typhi: Its implications in India. Indian J Med Microbiol 2014;32:161-3.  Back to cited text no. 16
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Jain S, Das Chugh T. Antimicrobial resistance among blood culture isolates of Salmonella enterica in New Delhi. J Infect Dev Ctries 2013;7:788-95.  Back to cited text no. 19
    
20.
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21.
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[PUBMED]  Medknow Journal  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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