Fakhur Uddin  ( Department of Microbiology, Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, Karachi, Pakistan )
Muhammad Sohail  ( Department of Microbiology, University of Karachi, Karachi, Pakistan )
Qurban Hussain Shaikh  ( Department of Medicine, Jinnah Postgraduate Medical Center, Karachi, Pakistan. )
Mir Tahir Hussain  ( Department of Medicine, Jinnah Postgraduate Medical Center, Karachi, Pakistan. )
Kerry Roulston  ( UCL Centre for Clinical Microbiology, Royal Free Campus, UCL, London, United Kingdom. )
Timothy Daniel McHugh  ( UCL Centre for Clinical Microbiology, Royal Free Campus, UCL, London, United Kingdom. )

Pseudomonas balearica, a saprophyte found in marshy and marine habitats, is not routinely differentiated from P. aeruginosa and P. stutzeri using automated systems and hence has not been reported from clinical samples. This study describes the identification of P. balearica using MALDI-TOF-MS and 16S rDNA sequence from a patient admitted to an intensive care unit (I.C.U.). The isolate was found to be Verona integron-mediated Metallo-b-lactamase (V.I.M.), and Vietnam extended-spectrum b-lactamase (V.E.B.) producer and resistant to Ceftriaxone, Imipenem, and Tobramycin. P. balearica can be a source for horizontal transfer of blaVEB and blaVIM. Its pathogenesis has yet to be understood.

 

Keywords: Emerging pathogen, b-lacamases, Pseudomonas balearica, Pseudomonas stutzeri.

 

DOI: https://doi.org/10.47391/JPMA.3890

 

Introduction

With the advent of new techniques and their implementation in the diagnosis of clinical specimens, many new species have been identified, while previously known species have either been reclassified or have been isolated from unusual habitats. Pseudomonas species other than P. aeruginosa were commonly considered as saprophytes, but recently these have been considered as opportunistic human pathogens, and outbreaks have also been reported from such species.^1,2

P. balearica is mainly found in marine and freshwater, salt marshes, and wastewater.^3,4 It is characteristically tolerant to higher salt concentrations (8.5% NaCl) and was recognized and reclassified as a separate species of Pseudomonas due to its ability of denitrification.^3,5 In this study, P. balearica was isolated from the tracheal aspirate of a patient with ventilator-associated pneumonia (V.A.P.). This is the first report describing the isolation of P. balearica from a clinical specimen.

 

Case Report

 

The specimen was obtained on June 2017 from a patient aged 52 years admitted to the Medical ICU-23 of Jinnah Post Graduate Medical Centre (JPMC), Karachi, Pakistan, diagnosed with Guillain-Barre Syndrome (G.B.S.) and had been referred from a hospital for mechanical ventilation. On the 7th day of admission, the patient developed Ventilator-Associated Pneumonia (V.A.P.) with high fever, leucopenia (2900WBCs/mm2), tachypnoea, increased or purulent secretions, haemoptysis, and bronchospasm. Ceftriaxone was given after sample collection, but no improvement was observed. Considering susceptibility testing results, Ceftriaxone was replaced with Ciprofloxacin. The patient improved and was discharged from the I.C.U. The improved parameters included normalization of the WBC count, temperature, and the absence of other indicators of V.A.P.

The pathogen was isolated from the tracheal aspirate and initially identified as P. stutzeri by the conventional methods using the API 20NE strip (Biomerieux, Marcy I'Etoile France). While identification through BD Phoenix™ Automated Microbiology System (instrument version 5.15A, software version 6.01A/V5.15A) [Becton Dickinson, Oxford, U.K.] presented the pathogen as P. aeruginosa. Consequently, further biochemical tests such as the ability to utilize xylose and to tolerate high salt concentration (Table-1) were carried out which indicated the necessity to investigate the strain through more sophisticated techniques including sequencing 16S rDNA and by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry [MALDI-TOF-MS] (Microflex, Bruker Daltonics, Bremen, Germany) and sequencing. These techniques identified the isolated strain as P. balearica. The cut-off scores (MALDI-TOF-MS) for identification was >1.7 for genus level and >2 for species level, while a score of <1.5 was disregarded. The details of the Biotyper threshold for the log score was interpreted and recorded as recommended by the manufacturer protocols.

 

 

The antimicrobial susceptibility of P. balearica to the most common antipseudomonal drugs was assayed by the disc diffusion and microtitre broth dilution methods, and  automated B.D. Phoenix (version 5.15A) at the UCL Centre for Clinical Microbiology, London,  using CLSI breakpoints for minimum inhibitory concentration (M.I.C.) interpretive standards (µg/mL) for other Non-Enterobacteriaceae.⁷ The antimicrobial susceptibility of imipenem and meropenem was confirmed by the determination of M.I.C.s using Etest strip (BioMérieux, Lyon, France, and M.I.C. Evaluator, Oxoid, Basingstoke, U.K.). The data showed that P. balearica was resistant to imipenem, Ceftriaxone, and tobramycin and sensitive to ceftazidime, cefepime, and antipseudomonal penicillin. Results of automated B.D. Phoenix system for antimicrobial susceptibility was similar to the M.I.C.s by microtitre broth dilution and disc diffusion method (Table-2).

 

 

The detection of carbapenemases, Modified Hodge test (M.H.T.), Rapidec Carba NP (RCNP; Biomerieux, Marcy-l'Etoile, France), and ethylenediaminetetraacetic acid (EDTA) double-disc synergy test with imipenem and meropenem were employed. Rosco kit test for Klebsiella pneumoniae carbapenemase (KPC)/ Metallo-b-lactamase (M.B.L.) for          P. aeruginosa and Acinetobacter species (ROSCO's Diagnostic, Tassstrup, Denmark) was also performed. Positive control strains for phenotypic and genotypic tests included P. aeruginosa NCTC 13437 (for V.I.M. and V.E.B.), Klebsiella pneumoniae NCTC 13443 (for NDM-1), and Escherichia coli NCTC 13476 (for IMP-types). The carbapenem hydrolyzing and other b-lactamases encoding genes were also investigated by the check-MDR CT103XL DNA microarray (Check-Points Health B.V., Wageningen, Netherlands) and by conventional PCR using the primers as listed in Table-3.

 

 

The results of phenotypic tests for ESBL (double disc diffusion), carbapenemases M.H.T. and M.B.L.s (EDTA double-disc synergy and Rosco kit) were negative. In contrast, the Rapedic CARBA NP phenotypic test was positive for carbapenemase production. Genotypic analysis revealed the presence of blaVIM and blaVEB. The results of microarray were also the same as manual PCR, showing that the strain was positive for V.E.B. and V.I.M.

Amplicon and its size were measured with positive and negative controls of blaVIM (Supplementary material Figure-1). The location of blaVIM and blaVEB on the plasmid was determined by extracting plasmid using GeneJET plasmid miniprep kit (Thermo Scientific™ #K0502) according to the manufacturer's instructions. The PCR mixture and conditions were kept the same as for the amplification of V.I.M. and V.E.B. The VIM PCR product was sequenced and submitted to GenBank with an accession number of KY798549, whereas the V.E.B. was not sequenced.

 

 

To study conjugation mediated transfer of blaVIM and blaVEB, the protocol given previously was followed using P. balearica as a donor and azir carrying E. coli J53 as a recipient.⁹ The resultant transconjugants were selected on Mueller- Hinton (M.H.) agar containing sodium azide (100µg mL-1) and imipenem (1µg mL-1). The presence of the resistant markers in transconjugants was further confirmed by PCR as described above.

 

Discussion

 

Previously, P. balearica was included in a diverse group of      P. stutzeri; however, heterogeneity within the group rendered further investigations to update the classification.6 Indeed, P. balearica was separated from this group when sufficient distinguishing features were explored and supported by (a) multilocus sequence with phylogenetic analysis. This species was first separated from P. stutzeri after 16S rRNA gene sequence analysis in 1996 by Bennasar et al.² However, the routine protocols in diagnostic laboratories still report it as P. stutzeri because the semi-automated or API and automated systems cannot differentiate between the two species.

Moreover, the results of antimicrobial susceptibility testing indicate the need for updating surveillance programmes to curtail the spread of this pathogen. Contrary to the present results for susceptibility to ceftazidime, aztreonam, and cefepime, Laudy et al. reported that all the VEB-9 positive isolates were resistant to these antibiotics.¹⁰

The presence of V.I.M. and V.E.B. b-lactamases which are found in P. aeruginosa strains from the same unit needs further analysis, as it suggests P. balearica may be a potential reservoir for outbreaks of highly resistant strains in I.C.U.s and a potential source for the horizontal transfer of antimicrobial resistance genes to other pathogenic bacteria. These findings support the conclusion that the resistance markers are plasmid-borne.

 

Conclusion

 

This case revealed that Pseudomonas species should identified by molecular assays and the prevalence of                P. balearica needs to be determined. P. balearica can be a potential source of V.E.B. and M.B.L. (V.I.M.) by horizontal gene transfer. However, further investigations are required to understand the etiology and pathogenesis of P. balearica.

 

Disclaimer: This case was identified in Ph.D. research work and is part of the thesis.

Conflict of Interest: The authors do not declare any conflict of interest.

Funding Disclosure: Authors are grateful to the Higher Education Commission (H.E.C.) Pakistan for providing travel grant to F.U. for performing experiments at UCL. under IRSIP scheme.

 

References

 

1.       Uddin F, Roulston K, McHugh TD, Khan TA, Sohail M. Bacteremia in a human caused by an XDR strain of Pseudomonas fulva. J Infect Dev Ctries. 2018; 12:597-9.

2.       Liu Y, Liu K, Yu X, Li B, Cao B. Identification and control of a Pseudomonas spp (P. fulva and P. putida) bloodstream infection outbreak in a teaching hospital in Beijing, China. Int J Infect Dis. 2014; 23:105-8.

3.       Bennasar A, Rosselló-Mora R, Lalucat J, Moore ERB. 16S rRNA gene sequence analysis relative to genomovars of Pseudomonas stutzeri and proposal of Pseudomonas balearica sp nov. Int J Syst Bacteriol.1996; 46:200-5.

4.       Mulet M, Gomila M, Gruffaz C, Meyer JM, Palleroni NJ, Lalucat J, et al. Phylogenetic analysis and siderotyping as useful tools in the taxonomy of Pseudomonas stutzeri: Description of a novel genomovar. Int J Syst Evol Microbiol.2008; 58:2309-15.

5.       Bennasar A, Guasp C, Lalucat J. Molecular methods for the detection and identification of Pseudomonas stutzeri in pure culture and environmental samples. Microb Ecol.1998; 35:22-33.

6.       Rossello R, Garcia-Valdes E, Lalucat J, Ursing J. Genotypic and Phenotypic Diversity of Pseudomonas stutzeri. Syst Appl Microbiol.1991; 14:150-7.

7.       CLSI. Performance Standards for Antimicrobial Susceptibility Testing 26th ed. CLSI supplement M100. Wayne, PA, U.S: Clinical and Laboratory Standards Institute, 2016.

8.       Uddin F, McHugh TD, Roulston K, Platt G, Khan ATA, Sohail M. Detection of carbapenemases, AmpC and ESBL genes in Acinetobacter isolates from I.C.U.s by D.N.A. microarray. J Microbiol Methods. 2018; 155:19-23.

9.       Borgia S, Lastovetska O, Richardson D, Eshaghi A, Xiong J, Chung C, et al. Outbreak of carbapenem-resistant Enterobacteriaceae containing blaNDM-1, Ontario, Canada. Clin Infect Dis. 2012; 55:e109-e117.

10.     Laudy AE, Róg P, Smolińska-Król K, Ćmiel M, Słoczyńska A, Patzer J, et al. Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods. PLoS One PLoS One. 2017; 12:e0180121. J Pak