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Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 309-311
Antibiotic pressure mediated selection of non-biofilm forming strain of Elizabethkingia meningosepticum causing fatal nosocomial meningitis in a term infant

Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India

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Date of Web Publication24-Oct-2013


We describe a fatal case of hospital acquired meningitis in a term infant due to the antibiotic pressure mediated selection of Elizabethkingia meningosepticum. The antibiotics were administered for multi-drug resistant Acinetobacter baumannii infection. The strain was also phenotypically characterized for beta lactamase production, biofilm forming capability and resistance to in use disinfectants.

Keywords: Biofilm, carbapenemase, Elizabethkingia meningosepticum, nosocomial meningitis

How to cite this article:
Rai S, Niranjan DK, Mishra P, Singh NP. Antibiotic pressure mediated selection of non-biofilm forming strain of Elizabethkingia meningosepticum causing fatal nosocomial meningitis in a term infant . Indian J Pathol Microbiol 2013;56:309-11

How to cite this URL:
Rai S, Niranjan DK, Mishra P, Singh NP. Antibiotic pressure mediated selection of non-biofilm forming strain of Elizabethkingia meningosepticum causing fatal nosocomial meningitis in a term infant . Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Jun 4];56:309-11. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/309/120408

   Introduction Top

Elizabethkingia meningosepticum is an oxidase positive, non-motile, Gram -negative, aerobic, glucose non-fermenting bacilli from family Flavobacteriaceae. It poses a threat of hospital acquired outbreaks in neonatal intensive care unit (ICU's) and is often difficult to identify by conventional methods. [1] It ubiquitously inhabits hospital environment and survives in chlorinated water, causing colonization of medical devices such as suction machines, ventilator tubings, respirators and injectable fluids. [2] Its inherent resistance against many antimicrobial drugs makes it a potential threat in hospital settings as it may cause outbreaks of neonatal meningitis in premature infants, especially by serotype C. [3]

We describe a fatal case of nosocomial meningitis in a term infant due to E. meningosepticum strain, which got selected as a result of intraventricular polymyxin B therapy for hospital acquired Acinetobacter baumannii infection. The strain was also characterized for beta lactamase activity including extended spectrum beta lactamases and carbapenemase production, biofilm production and resistance to in-use disinfectants.

   Case Report Top

A full-term female neonate was delivered at a tertiary care university hospital of East Delhi with a birth weight of 2.5 kg to a 28-year-old primigravida, by cesarean section. After delivery, the baby developed respiratory distress and was kept on oxygen. At 36 h of life, the baby suffered an episode of generalized tonic clonic seizure. On 7 th day, the infant developed repeated seizures following which a lumbar puncture was performed, which yielded purulent fluid. The investigations were consistent with bacterial meningitis. Ultrasonography of the head demonstrated ventriculitis with obstructive hydrocephalus, which was confirmed on contrast enhanced computed tomography scan. The infant was treated with intravenous phenobarbitone, meropenem and vancomycin. Blood culture isolated Klebsiella pneumonia, which was sensitive to polymyxin B and chloramphenicol but resistant to carbapenems. Repeated lumbar punctures subsequently isolated A. baumannii sensitive only to polymyxin B. The treatment was changed to intravenous polymyxin B and meropenem. An extraventricular drainage (EVD) of Cerebro spinal fluid (CSF) was performed. Intraventricular polymyxin B was also given every alternate day. Fourth CSF culture isolated oxidase positive, lactose non-fermenting bacilli, resistant to polymyxin B and carbapenems, but sensitive to ciprofloxacin and levofloxacin. The organism was identified as E. meningosepticum in the Microscan Walkaway 96 Plus (Siemens, India) automated identification system using the NBPC34 identification and susceptibility cards. In the light of potential E. meningosepticum hospital outbreak in neonatal ICU, the neonatal cubicle was disinfected immediately after notification of the critical alert. [1] The EVD was changed and its proximal tip sent for culture that also isolated the same organism. Patient expired on the 30 th day due to meningitis caused by hospital acquired multi-drug resistant E. meningosepticum.

   Methods Top

Strain characterization

Unlike typical strains of E. meningosepticum, this isolate produced non-hemolytic and non-pigmented colonies on sheep blood agar and nutrient agar. Breakpoint minimum inhibitory concentration (MIC's) of non-Enterobacteriaceae members were used according to the Clinical Laboratory Standards Institute guidelines. [4] The MIC's were evaluated using the HiComb ® MIC strips (HiMedia, India) for ceftazidime, ciprofloxacin, polymyxin B, colistin, amikacin, gentamicin and piperacillin + tazobactam. Escherichia coli American type culture collection (ATCC) 25922 used as a control strain. The MIC's for the above mentioned antimicrobials were: >240, 0.1, >240, >240, 64, 60 and 10 μg/ml respectively, which correlated with MIC's obtained by the automated method.

Evaluation of beta lactamase production

Presence of carbapenemase activity was assessed as previously performed by the authors [Figure 1]a and c. [5] Another template was used for simultaneous detection of Amp C beta lactamase (Amp C) and ESBL's [Figure 1]b. This was used on the template suggested by Yagi et al. with slight modifications. [6] Briefly, pure colonies from an overnight culture of E. meningosepticum were suspended in sterile saline and adjusted to 0.5 McFarland turbidity. A Mueller Hinton Agar (MHA) plate, (Oxoid, Basingstoke, UK) was swabbed with standard inoculum of the isolate. This plate was roughly divided in an upper half where, a sterile blank disc was placed on to which 400 μg of phenyl boronic acid (PBA) (Arcos Organics, New Jersey, USA) was added, which acts as an AmpC enzyme inhibitor. Its solution prepared in dimethyl sulfoxide (HiMedia, India). A disc of cefotaxime (Ctx, 30 μg) and ceftazidime (Caz, 30 μg) (Oxoid, Basingstoke, USA) was placed at a distance of 20 mm edge-to-edge to observe for the presence of synergy with PBA in the presence of AmpC beta lactamase. Two discs of amoxicillin + clavulanic acid (AMC, 20/10 μg) (Oxoid, Basingstoke, UK) were placed in the central part of the plate at a distance of 22 mm edge-to-edge from the Ctx and Caz discs mentioned above to observe synergy in the presence of ESBL enzymes. One another set of discs, each of Ctx and Caz were placed in the lower half of the plate at a distance of 22 mm edge-to-edge to the AMC discs, but to these discs, 400 μg of PBA each was added. This was carried out so as to suppress AmpC enzyme and unmask ESBL activity in case of co-presence of AmpC and ESBL enzymes [Figure 1]b.
Figure 1: (a) Synergy between cefotaxime (Ctx), Ctx + clavulanic acid (Ctx + CA) and boronic acid (BA) disc (left arrows) and ceft azidime (Caz), Caz + clavulanic acid (Caz + CA) and BA disc (right arrows). (b) Demonstrati ng a equal and mild indentati on of Escherichia coli ATCC 25922 indicator strain in both meropenem (Mer) and meropenem + zinc sulfate (Mer + Zn) discs. (c) An increase in zone size on additi on of EDTA to imipenem discs (I + E as well as I + E + BA)

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Carbapenemase activity was assessed by another method as previously described by the authors with slight modifications. In brief, the strain was swabbed on to an MHA plate as described above. This was followed by the placement of six imipenem discs as shown in [Figure 1]c. This was followed by the addition of various inhibitors [ethylene diamine tetra acetate (EDTA), phenyl boronic acid (PBA)] and enhancer (zinc) of carbapenemases in same concentrations as mentioned above in various combinations as depicted in [Figure 1]c. An enhancement of zone by ≥5 mm from the imipenem zone was considered as positive. [5]

Biofilm formation

Biofilm forming capability was assessed using the microtiter plate method. [7]

Disinfectant testing

Basic bactericidal activity of chemical disinfectants and antiseptics was performed by suspension tests as per recommendations. [8] Disinfectant testing was performed on two disinfectants Virkon ® (potassium peroxymonosulfate, sodium dodecylbenzenesulfonate, sulphamic acid and inorganic buffers) and Cidex ® (2% glutaraldehyde) and three antiseptics Savlon ® (Cetrimide and Chlorhexidine Gluconate), Bioclenz ® (Propranolol and Mecetronium ethyl sulfate) and SeptoMed-Scrub ® (Undecylenamidopropyl-trimonium methosulfate and Phenoxyethanol).

   Results Top

The strain was non-pigmented and was found to have zinc dependant carbapenemase activity, which was inhibited by EDTA, but not enhanced by the addition of zinc [Figure 1]a. Addition of PBA alone or with EDTA did not produce any increase in zone size indicating the absence of Amp C beta lactamase [Figure 1]c. [5] Addition of PBA also did not produce any zone of inhibition around the Ctx or Caz discs again indicting absence of AmpC beta lactamases. There was a zone of synergy observed between AMC discs and Ctx, Caz, Ctx + PBA and Caz + PBA discs, indicating presence of ESBL activity [Figure 1]b. Unlike most hospital strains of E. meningosepticum, this strain was not found to produce any biofilm either under different pH conditions or different incubation durations. The strain was susceptible to all five in use disinfectants and antiseptics. Source tracking of the strain could not be performed due to immediate disinfection of the neonatal cubicle.

   Discussion Top

This case differs from previous reported cases as patient was a full-term infant and the causative strain was a non-pigmented and non-hemolytic isolate compared with conventional isolates. Unlike its classical mechanism for survival, this strain did not possess biofilm forming capability. The strain was not the primary pathogen but got selected as a result of selective antibiotic pressure after the commencement of intraventricular polymyxin B therapy. Regions, which are highly endemic for pan drug resistant organisms like carbapenem resistant Enterobacteriaceae harboring the New Delhi metallo-beta-lactamases, polymyxin drugs remain the only plausible choice. [9] Treating physicians must bear in mind the possibility of strain selection when antimicrobials like polymyxin B are being used. Many glucose non-fermenters like Brevundimonas spp and Burkholderia spp are naturally resistant to this drug and may get selected in such patients as a secondary pathogen in hospital settings. [10]

   Acknowledgments Top

The authors would like to thank Dr Archana Kashyap, Dr. Pooja Dewan and Dr. Prerna Batra from Department of Pediatrics, GTB Hospital for providing clinical details for the present case.

   References Top

1.Ceyhan M, Celik M. Elizabethkingia meningosepticum (Chryseobacterium meningosepticum) Infections in Children. Int J Pediatr 2011;2011:215237.  Back to cited text no. 1
2.Chiu CH, Waddingdon M, Greenberg D, Schreckenberger PC, Carnahan AM. Atypical Chryseobacterium meningosepticum and meningitis and sepsis in newborns and the immunocompromised, Taiwan. Emerg Infect Dis 2000;6:481-6.  Back to cited text no. 2
3.Holmes B. Identification and distribution of Flavobacterium meningosepticum in clinical material. J Appl Bacteriol 1987;62:29-41.  Back to cited text no. 3
4.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty First Informational Supplement. Wayne,USA: 2011. M100-S21.  Back to cited text no. 4
5.Rai S, Manchanda V, Singh NP, Kaur IR. Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae. Indian J Med Microbiol 2011;29:275-9.  Back to cited text no. 5
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6.Yagi T, Wachino J, Kurokawa H, Suzuki S, Yamane K, Doi Y, et al. Practical methods using boronic acid compounds for identification of class C beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. J Clin Microbiol 2005;43:2551-8.  Back to cited text no. 6
7.Lin PY, Chen HL, Huang CT, Su LH, Chiu CH. Biofilm production, use of intravascular indwelling catheters and inappropriate antimicrobial therapy as predictors of fatality in Chryseobacterium meningosepticum bacteraemia. Int J Antimicrob Agents 2010;36:436-40.  Back to cited text no. 7
8.Hobson DW, Bolsen K. Methods of testing oral and topical antiseptics and antimicrobials. In: Block SS, editor. Disinfection, Sterilization and Preservation. 5 th ed. Philadelphia: Lippincott Williams and Wilkins; 2001. p. 329-59.  Back to cited text no. 8
9.Manchanda V, Rai S, Gupta S, Rautela RS, Chopra R, Rawat DS, et al. Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniaef Acinetobacter baumannii. Indian J Med Microbiol 2011;29:249-53.  Back to cited text no. 9
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10.Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC Jr. The non fermentative gram negative bacilli. In: Color Atlas and Textbook of Microbiology. 5 th ed., Ch. 5. Philadelphia: Lippincott Williams and Wilkins; 1997. p. 253-320.  Back to cited text no. 10

Correspondence Address:
Sumit Rai
Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, Dilshad Garden, New Delhi - 110 095
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.120408

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