Multidrug-resistant Streptococcus pneumoniae causing invasive pneumococcal disease isolated from a paediatric patient

Journal Pre-proof Multidrug Resistant Streptococcus pneumoniae causing Invasive Pneumococcal Disease (IPD) Isolated from a Pediatric Patient Revathy Arushothy, Hema Ramasamy, Rohaidah Hashim, Santhana Raj A.S., Fairuz Amran, Nazirah Samsuddin, Norazah Ahmad

PII:

S1201-9712(19)30431-X

DOI:

https://doi.org/10.1016/j.ijid.2019.10.037

Reference:

IJID 3813

To appear in:

International Journal of Infectious Diseases

Received Date:

15 August 2019

Revised Date:

22 October 2019

Accepted Date:

27 October 2019

Please cite this article as: Arushothy R, Ramasamy H, Hashim R, Raj A.S. S, Amran F, Samsuddin N, Ahmad N, Multidrug Resistant Streptococcus pneumoniae causing Invasive Pneumococcal Disease (IPD) Isolated from a Pediatric Patient, International Journal of Infectious Diseases (2019), doi: https://doi.org/10.1016/j.ijid.2019.10.037

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Multidrug Resistant Streptococcus pneumoniae causing Invasive Pneumococcal Disease (IPD) Isolated from a Pediatric Patient.

Revathy Arushothy1, Hema Ramasamy3, Rohaidah Hashim1, Santhana Raj A.S2 Fairuz Amran1, Nazirah Samsuddin1, Norazah Ahmad1.

Bacteriology Unit, Infectious Disease Research Center, Institute for Medical Research,

ro of

1

National Institutes of Health, Ministry of Health Malaysia, Selangor, Malaysia. 2

Electron Microscopy Unit, Medical Research Resource Center, Institute for Medical

Hospital Sultanah Aminah, Ministry of Health Malaysia, Johor Bahru, Malaysia.

lP

re

3

-p

Research, National Institutes of Health, Ministry of Health Malaysia, Selangor, Malaysia.

Highlights

Jo



Emergence of non-vaccine pneumococcal serotype is a rising concern in Malaysia Non vaccine serotypes show possibility of horizontal gene transfer from serotype 19A or 23F. Continuous pneumococcal surveillance of serotypes and antimicrobial resistance is crucial for patient management.

ur

 

na

*Corresponding author: Tel : +60146606524: E-mail address : [email protected]

Abstract Emergence of non-vaccine multidrug resistant Streptococcus pneumoniae (MRSP) serotypes are on rise. In this study, we report the investigation of a highly resistant serotype 15A 1

Streptococcus pneumoniae (MRSP) isolated from the blood specimen of a one year and eight months old patient who had succumbed to her infections. The SS40_16 isolate was resistant to erythromycin, co-trimoxazole, tetracycline and chloramphenicol as well as to penicillin, ceftriaxone and cefotaxime (using meningitis cut off points (CLSI)). The isolate belongs to sequence type 1591 (ST1591) and is related to CC81 clonal complex, suggesting a possibility of horizontal gene transfer. SEM comparison between resistant and sensitive pneumococci isolates too indicates similar phenotypic characteristics that confers high resistance. Emergence

ro of

of highly resistant non vaccine pneumococcal are of great concern in public health and clinical setting. Pneumococcal surveillance program is a crucial tool for monitoring selective pressure of serotype replacement besides impact of PCVs for treatment in IPD.

re

-p

Keywords : Emerging non vaccine serotype, Multidrug resistant, serotype 15A

lP

Introduction

Streptococcus pneumoniae is the etiological agent for invasive pneumococcal disease and is

na

responsible for significant morbidity and mortality, particularly in young children (Rosen J.B. et al., 2011). World Health Organization reported that the invasive pneumococcal disease (IPD)

ur

was responsible for increased morbidity and mortality in children worldwide (WHO, 2012). Introduction of conjugate vaccines (PCV7, PCV10 and PCV13), have reduced pneumococcal

Jo

disease in vaccinated children, although this has resulted in the emergence of non-vaccine serotypes such as serotypes 8, 10A, 12F, 15A and 24F. The emergence serotype 15A is of concern due to its multidrug-resistance characteristics which has been reported by several studies (Ho P.L. et al, 2015, Golden A.R. et al, 2015, Strachan J.E. et al, 2013 Satoshi Nakano et al, 2019, Bastiaens G.J.H. et al, 2018, Jin Yang Baek et al, 2018 & Büyükcam A. et al 2017).

2

In this study, we report the investigation of a highly resistant serotype 15A Streptococcus pneumoniae (MRSP) isolated from blood specimen.

Case report A 1 year 8-month-old girl was admitted at the Emergency Department of a hospital in Johor Bahru, Malaysia with respiratory distress and was immediately intubated and transferred

ro of

to the paediatric intensive care unit (PICU). The patient was then treated for severe bronchopneumonia with IV Ceftriaxone and IV Azithromycin. She also required high ventilator setting using pressure control mode (FiO2 70/PEEP 6/PIP 21/ Rate 40). Furthermore,

-p

the patient was noted to have persistent tachycardia with poor pulse volume, hence given IV normal saline bolus 30cc/kg, IV Vitamin K and transfused FFP 15mls/kg. Despite on high

re

ventilator setting, she was still unable to maintain good oxygenation and was started on High Frequency Oscillatory Ventilation-HFOV (FiO2 90/Map 22/rate 10/delta P40) which was

lP

further increased to FiO2 100/delta P 64/rate 8/Map 20). However, despite this the blood gas remained hypoxemic and clinical oxygen saturation showed only 80%, thus treatment was

na

commenced on inhaled nitric oxide (iNO) 20ppm. In view of hypotension, the patient was

ur

started on triple inotropes, namely IV Noradrenaline, IV Dopamine and IV Dobutamine. Echocardiogram, revealed bilateral pleural effusion with normal heart and good function.

Jo

Bilateral chest tubes were inserted and drained brownish fluid 70cc-80cc. Microbiological investigation on both blood and pleural effusion fluids grew S. pneumoniae. The patient was immediately started on two doses of IV vancomycin on day 2 and 3, with continuation of IV Ceftriaxone. IV Immunoglobulin was also given twice on day 2 and day 4. Despite all these efforts, her condition deteriorated and she succumbed to her infections on the 5th day.

3

Laboratory Investigation Bacterial Isolate Streptococcus pneumoniae was isolated from blood culture and pleural effusion of the patient at Hospital Sultanah Aminah, Johor Bahru, Malaysia. The isolate was then sent to Bacteriology Unit, Institute for Medical Research, for further analysis under the National Surveillance for Streptococcus pneumoniae program in Malaysia. The isolate was identified as S. pneumoniae

ro of

based on typical colony morphology, gram staining, β-hemolysis, optochin (Oxoid Company, Britain) susceptibility and positive bile solubility test and was given the lab ID number

-p

SS40_16.

re

Serotyping

The SS40_16 isolate was serotyped by Neufeld’s Quellung reaction method using type-specific

lP

antisera (Staten’s Serum Institute, Copenhagen, Denmark) according to the manufacturer’s

na

protocol. The SS40_16 isolate was serotype 15A.

ur

Antibiotic susceptibility

Susceptibility of the SS40_16 isolate to penicillin, ceftriaxone and cefotaxime was determined

Jo

via the E-test methods (bioMérieux, France) using minimum inhibitory concentrations (MICs). Its susceptibility to erythromycin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin was determined using the disk diffusion method (Oxoid, USA). All tests were performed following the guidelines and cut-offs recommended by the CLSI (2017), and the finding as reported in Table 1. S. pneumoniae ATCC 49619 was used as a quality control strain for the antimicrobial susceptibility testing. 4

As indicated in Table 1, the SS40_16 isolate was resistant to erythromycin, co-trimoxazole, tetracycline and chloramphenicol. Although this isolate originated from blood, if meningitis cut-off points provided by the CLSI (2018) were adopted, it was also shown to be resistant to penicillin, ceftriaxone and cefotaxime. Whole Genome Sequencing The SS40_16 isolate was subjected to whole genome sequencing as previously reported

ro of

(Arushothy R. et al, 2018). The nucleotide sequence of the Streptococcus pneumoniae SS40_16 genome has been deposited in DDBJ/EMBL/GenBank under accession no. PEKA00000000. The strain belongs to serotype 15A and sequence type 1591 (ST1591) (as determined by PubMLST [https://pubmlst.org/]). Moreover, Yun et al. 2018 established that the ST1591 is

-p

related to the CC81 clonal complex, whereby the serotype 15A was proposed to be replacing

na

Scanning Electron Microscopy

lP

successful MDR resistant PMEN-1 clone.

re

serotype 19F and 23F. According to these authors, this genotype is also related to the highly

Four S.pneumoniae isolates SS40_16, drug sensitive 15A isolate, ATCC 49619 and multidrug

ur

resistant ATCC 700673, were subjected to scanning electron microscopy (SEM) analysis. Prior to performing SEM these isolates were fixed with a solution containing 2% glutaraldehyde 0.1

Jo

M phosphate buffer solution (pH 7.4) before being washed with distilled water and dehydrated in increasing ethanol concentrations (50%, 70%, 90% and 100%) and finally dried in Critical Point Dryer. The thus prepared samples were with gold using Sputter Coater to obtain a film of 42nm thickness. The stained samples were examined in a scanning electron microscope (FEI ESEM Quanta 200) at 28 kV accelerating voltage.

5

Based on the SEM findings, both SS40_16 isolate (serotype 15A) and the multidrug-resistant ATCC 700673 (serotype 19A) had very thick lipopolysaccharide matrix, a characteristic that was not shared by the drug sensitive 15A isolate (clinical isolate) and the control strain ATCC 49619 (serotype 19F).

Discussion

ro of

This report pertains to the investigation of a highly resistant serotype 15A Streptococcus pneumoniae (MRSP) isolated from blood in Malaysia. The emergence of serotype 15A S.pneumoniae has been noted following PCV introduction as indicated in several recent

-p

reports. (Satoshi Nakano et al, 2019, Bastiaens G.J.H. et al, 2018, Jin Yang Baek et al, 2018 Büyükcam A. et al 2017). The introduction of PCV7 and PCV13 into the NIP of many countries

re

has resulted in a relative increase in infections rates due to non-vaccine serotypes. Although PCV efficacy is significant, IPD continues to occur as a result of serotype replacement, which

lP

leads to greater circulation of new pneumococcal serotypes such as 6C, 15A, 23A, and 35B

na

(Htar T. T. et al, 2015; Büyükcam A. et al 2017).

In Malaysia, 13.8% of emerging non vaccine serotypes 11C, 15A, 15C, 19B, 23A, 8, and 6C

ur

from IPD were reported to be MDR (Arushothy et al., 2019). The prevalence of 15A isolates in the country is less than 10% (unpublished data), but still unique since PCV is not included

Jo

in the NIP. Natural serotypes switch may occur even without the effect from conjugate vaccine and the clinical features of IPD in children from these infections have shifted towards more a focal infection with increased severity requiring hospitalization (Ricketson et al., 2018). The isolate SS40_16 belongs to ST1591 and is related to the CC81 clonal complex. This clone is linked to serotype 19F and 23F (Yun, K. W. et al, 2018) whereby this may indicate horizontal gene transfer to serotype 15A. Surveillance from Japan (Satoshi Nakano et al, 2019), detected 6

an association between multidrug-resistant serotype 15A-CC63 clonal complex with multidrug resistant ST9084. A similar surveillance and epidemiological study in Malaysia on the emerging non vaccine serotypes especially highly resistant isolates would be able to demonstrate the true dissemination of these isolates in the country. The polysaccharide capsule plays a role in preventing the immune adherence of pneumococci which results in reduction of the phagocytic activity against the bacterium (Andre, G. O. et al., 2017). Phenotypically, the SEM of the SS40_16 suggest a possible horizontal gene transfer

ro of

between serotype 19A (ATCC 700673) and serotype 15A. This would probably be the first evidence to show a possibility and effects of gene transfer between serotypes. The very thick lipopolysaccharide matrix shared between both ATCC 700673 and SS40_16 indicates the

-p

phenotypic characteristic that confers to high resistance.

re

The limitation of this study was the inability to know the true chronology of transmission of the disease and the pneumococcal vaccination status of the patient. It would have provided

lP

better understanding of the actual route of dissemination of 15A serotype pneumococci to the patient. Phylogenetic analysis of the isolate may provide more information on the lineage of

na

the emerging pneumococci and other epidemiological information. In conclusion the emergence of highly resistant non vaccine clones are of great concern for

ur

public health and in clinical setting. The case of this patient indicates importance of

Jo

characterizing emerging serotypes, to understand its virulence and pathogenicity. Surveillance would be the key factor for monitoring the selective pressure of serotype replacement besides impact of PCVs for treatment in IPD.

Ethical considerations

7

This study did not involve any types of intervention in diagnosis and treatment, thus written informed consent was not considered necessary for the study.

Conflict of interest

ro of

The authors declare that they have no conflicts of interest.

Author contributions

Revathy Arushothy, Fairuz Amran, Norazah Ahmad and Rohaidah Hashim conceived and

-p

designed the experiments.

re

Hema Ramasamy prepared the case report. Santhana Raj performed the electron microscopy.

lP

Revathy Arushothy and Nazirah Samsudin performed the experiments.

na

Revathy Arushothy analysed the data.

ur

Revathy Arushothy wrote and reviewed the paper.

Jo

Acknowledgements

This research was funded by Institute Medical Research, Ministry of Health, Malaysia. We would like to thank the Director General of Health Malaysia for his permission to publish this article. The authors would like to thank Hospital Sultanah Aminah, Johor Bahru, Malaysia for providing clinical information and the isolate.

8

9

ro of

-p

re

lP

na

ur

Jo

References 1. Jennifer B. Rosen, Ann R. Thomas, Catherine A. Lexau, Art Reingold, Jim L. Hadler, Lee H. Harrison, Nancy M. Bennett, William Schaffner, Monica M. Farley, Bernard W. Beall, Matt R. Moore, CDC Emerging Infections Program Network, Geographic Variation in Invasive Pneumococcal Disease Following Pneumococcal Conjugate Vaccine Introduction in the United States, Clinical Infectious Diseases, Volume 53, Issue 2, 15 July 2011, Pages 137–143.

ro of

2. World Health Organization. Pneumococcal vaccines for childhood immunization: WHO position paper. Wkly Epidemiol Rec. 2012; 87: 129–144

3. Sheppard C, Fry NK, Mushtaq S, et al. Rise of multidrug-resistant non-vaccine serotype Streptococcus

pneumoniae

in

United

Kingdom,

2001

to

re

2014. Eurosurveillance. 2016;21(50):30423.

the

-p

15A

4. Ho PL, Chiu SS, Law PY, Chan EL, Lai EL, Chow KH. Increase in the nasopharyngeal

lP

carriage of non-vaccine serogroup 15 Streptococcus pneumoniae after introduction of children pneumococcal conjugate vaccination in Hong Kong. Diagn Microbiol Infect

na

Dis. 2015;81(2):145-8.

5. Golden AR, Adam HJ, Gilmour MW, Baxter MR, Martin I, Nichol KA, et al.

ur

Assessment of multidrug resistance, clonality and virulence in non-PCV-13 Streptococcus pneumoniae serotypes in Canada, 2011-13. J Antimicrob Chemother.

Jo

2015;70(7):1960-4

6. Strachan JE, Rowe SL, Dunne EM, Hogg GG. Emergence of Streptococcus pneumoniae serotype 15A after the introduction of the conjugate vaccine in Victoria. Med J Aust. 2013;199(7):461-3.

10

7. Arushothy, R., Ahmad, N., Amran, F., Hashim, R., Samsuddin, N., & Che Azih, C. R. (2018). Draft Genome Sequence of a Highly Resistant Streptococcus pneumoniae Serotype 15A Strain Isolated from Blood. Genome Announcements, 6(16), e00167–18. 8. Yun KW, Choi EH, Lee HJ, Kang JH, Kim KH, Kim DS, Kim YJ, Eun BW, Oh SH, Cho HK, Hong YJ, Kim KN, Kim NH, Kim YK, Lee H, Lee T, Kim HM, Cho EY, Kim CS, Park SE, Oh CE, Jo DS, Choi YY, Lee J. Genetic structures of invasive Streptococcus pneumoniae isolates from Korean children obtained between 1995 and

ro of

2013. BMC Infect Dis. 2018 Jun 8;18(1):268. 9. Büyükcam A, Güdücüoğlu H, Karaman K, et al. Invasive pneumococcal infection due to serotype 15A after the pneumococcal conjugate vaccine implementation in Turkey.

-p

Hum Vaccin Immunother. ;13(8):1892–1894.

10. Jin Yang Baek, So Hyun Kim, Cheol-In Kang, Doo Ryeon Chung, Kyong Ran Peck,

re

Jae-Hoon Song, Kwan Soo Ko,Emergence of an extensively drug-resistant (XDR)

lP

Streptococcus pneumoniae serotype 15A by capsular switching, International Journal of Medical Microbiology, Volume 308, Issue 8,2018,Pages 986-989,ISSN 1438-4221, 11. Satoshi Nakano, Takao Fujisawa, Yutaka Ito, Bin Chang, Yasufumi Matsumura,

na

Masaki Yamamoto, Shigeru Suga, Makoto Ohnishi, Miki Nagao Whole-Genome Sequencing Analysis of Multidrug-Resistant Serotype 15A Streptococcus pneumoniae

ur

in Japan and the Emergence of a Highly Resistant Serotype 15A-ST9084 Clone

Jo

Antimicrobial Agents and Chemotherapy Apr 2019, 63 (5) 12. Tin Tin Htar M, Christopoulou D, Schmitt HJ. Pneumococcal serotype evolution in Western Europe. BMC Infect Dis 2015; 15:419;PMID:2646800

13. Andre, G. O., Converso, T. R., Politano, W. R., Ferraz, L. F., Ribeiro, M. L., Leite, L. C., & Darrieux, M. (2017). Role of Streptococcus pneumoniae Proteins in Evasion of

11

Complement-Mediated

Immunity.

Frontiers

in

microbiology,

8,

224.

doi:10.3389/fmicb.2017.00224 14. Dougherty, K., Smith, B. A., Moore, A. F., Maitland, S., Fanger, C., Murillo, R., & Baltrus, D. A. (2014). Multiple phenotypic changes associated with large-scale

Jo

ur

na

lP

re

-p

ro of

horizontal gene transfer. PloS one, 9(7), e102170. doi:10.1371/journal.pone.0102170

12

ro of -p

re

Figure 1: Scanning Electron Microscopy of (A) Streptococcus pneumoniae SS40_16, (B)

Jo

ur

na

lP

drug-sensitive 15A serotype, (C) ATCC 49619 and (D) multidrug resistant ATCC 700673.

13

Table 1: MICs and antimicrobial susceptibility interpretations of Streptococcus pneumoniae SS40_16 Antibiotic

Values

Intrepretations

>2.0ug/ml

R

>2.0ug/ml

S

1.5ug/ml

R

Penicillin (meningitis) (S:≤0.06ug/ml; R:≥0.12ug/ml) Penicillin (non-meningitis) (S:≤2ug/ml; I:4ug/ml; R:≥8ug/ml) (S:≤0.5ug/ml; I:1ug/ml; R:≥2ug/ml) Ceftriaxone (non-meningitis) (S:≤1ug/ml; I:2ug/ml; R:≥4ug/ml)

1.5ug/ml

Cefotaxime (meningitis) (S:≤1ug/ml; I:2ug/ml; R:≥4ug/ml)

-p

1.5ug/ml

Cefotaxime (non-meningitis) (S:≤1ug/ml; I:2ug/ml; R:≥4ug/ml)

Erythromycin

12mm

lP

(S: ≥19mm; I: 16-18mm; R: ≤15mm)

1.5ug/ml

re

Co-trimoxazole

ro of

Ceftriaxone (meningitis)

na

(S: ≥21mm; I: 16-20mm; R: ≤15mm)

R

I

R R

13mm

20mm

Vancomycin

I

S

(S: ≥17mm )

22mm

ur

Tetracycline

(S: ≥28mm; I: 25-27mm; R: ≤24mm)

Jo

R

18mm

Chloramphenicol

(S: ≥21mm; R: ≤20mm)

14

R