Bacteremia in a human caused by an XDR strain of Pseudomonas fulva

This is the first report from Pakistan of a case of bacteremia in a human due to P. fulva, an opportunistic infection with increased risk of a drug resistant phenotype. P. fulva was isolated from blood of a 45 years male admitted in surgical ICU. Isolate was identified by the MALDI-TOFMS and was extensively drug resistant (XDR) strain. Isolate was found negative for metallo β lactamase (MBL) and extended spectrum β lactamase (ESBL) types by phenotypic and polymerase chain reaction (PCR) assays. It was concluded that P. fulva is an emerging opportunistic pathogen.


Introduction
Pseudomonas fulva is an opportunistic pathogen of the Pseudomonad group that rarely causes infection in man.It has mostly been reported from aquatic environments including rice seed, petroleum fields, oil brine and gills of the molluscs [1].To date, four cases of infections with P. fulva have been reported in humans together with an outbreak of 19 patients in China over 3 years in which the of source the bacteremia was identified as contaminated (insulin, isosorbide dinitrate, and potassium magnesium aspartate with 5% glucose) solutions [2].The case reports regarded isolation of bacterium from sputum of a patient with cystic fibrosis [3], from cerebral drainage of a case of meningitis [1], from the blood of a case of bacteremia [4] and from exudate from a soft tissue wound [5].Infections with P. fulva are characterized by poor clinical outcomes: out of five cases including this case, only 2 patients survived.

Case Report
A 45 years male was admitted to the surgical Intensive Care Unit (ICU) of a tertiary care hospital, in Karachi, Pakistan following a road accident.The case was referred from a public hospital with post-surgical complications, history of head injury and fractured ribs and laceration wounds at various sites of the body together with fever and the need for mechanical ventilation.Total leucocyte count (TLC) was 16.5×10 9   cells/L and septicemia was suspected.Blood was collected for microbiological culture (on the 18 th day after surgery) and empirical treatment with Piperacillin/tazobactam and Vancomycin was initiated.The patient's condition did not improve.Blood culture was processed manually and was positive for Gramnegative bacilli on direct Gram-staining from the incubated blood culture bottle after 24 h of incubation showing slight turbidity.Sub-culturing on MacConkey and Blood agar plates revealed the growth of nonhemolytic colonies on blood agar, with lactose negative colonies on MacConkey agar.Using a routine manual method and API 20NF kit (Biomerieux, Marcy I'Etoile France) the growth was identified as Pseudomonas putida.Before the complete identification, the patient died.However, the identification characteristics in this case, i.e. motile cells, oxidase positive (weak ±) colonies and water-soluble yellow pigments were not observed.Variable results for oxidase have previously been reported from negative, weak positive-to-positive for P. putida [4].To confirm the identity, the isolate was submitted in duplicate to MALDI-TOF-MS (Microflex, Bruker Daltonics, Bremen, Germany) at the UCL Centre for Clinical Microbiology and identified as P. fulva.Antimicrobial susceptibility to the most common antipseudomonal drugs was determined by the disc diffusion method following the Clinical  Diagnostic, Tassstrup, Denmark).It appeared positive for MBL using these phenotypic methods, however, production of MBLs could not be confirmed by the genotypic assay using primers VIM-like F-GGTGTTTGGTCGCATATCGC, R-CCATTCAGCCAGATCGGCATC, IMP-like F-GGAATAGAGTGGCTTAATTC R-CAACCAGTTTTGCCTTACC, and NDM-like F-CACCTCATGTTTGAATTCGCC R-CTCTGTCACATCGAAATCGC. Polymerase chain reaction (PCR) conditions were maintained as previously described [7][8][9].The isolate was also found negative for extended spectrum β lactamase (ESBLs), PER and VEB types) by PCR method [10,11].This suggests that the resistance mechanism was other than these β-lactamases.

Discussion
The members of Pseudomonas putida group are ubiquitously present in variety of environmental niches [12].They are rarely involved in infections in human but few reports of bacteremia and infections of soft tissues have been emerged [13].Studies state higher rate (46%) of VIM positive strains of this group from human specimens [1].Furthermore, these species have been described as reservoirs for the dissemination of the antibiotic resistance genes to the more pathogenic species such as P. aeruginosa [13][14][15].P. fulva is the less characterized member of this group which has scarcely been demonstrated as human pathogen.It is mainly due to its misidentification as Pseudomonas putida (99%) by the Vitek 2 system [1,2] resulting in its under reporting.In all the cases, P. fulva was identified either by 16S rRNA typing or MALDI-TOF-MS.
The MALDI-TOF-MS has been validated by different researchers as an alternate assay for the accurate and reliable tool to distinguish P. fulva, from P. putida group (P.putida, P. fulva, P. monteilii, and P. mosselii) in comparison to sequencing16s rRNA, gyrB, or rpoD genes.It identifies P. fulva with 100% sensitivity and specificity among P. putida group [16][17][18].Hence, cautionary measures should be taken to identify an isolate when unusual biochemical results of Pseudomonas spp.are observed.Correct identification of the isolatesfrom samples may increase the number of cases caused by this species.
History of the patient revealed that source of P. fulva may be traced back to hospital environment or contaminated intravenous solutions as described earlier [2].Susceptibility pattern of our strain was slightly different to the previously isolated stains [1,2,19].Almuzara et al. [1] reported susceptibility of P. fulva to Cefepime, Amikacin, Ciprofloxacin, and Colistin.However, in this case, the isolate was susceptible to Colistin only.The presence of MBL was also detected Contrarily, the presence of blaVIM, MBL type was reported in previously isolated P. fulva strains [1,19].

Conclusion
The increasing isolation of P. fulva foreshadows the increased risk of this organism causing severe infections in critically ill patients.Due to capability of acquiring resistance determinants such as carbapenemase (VIM), it appears as an emerging threat to public health care facilitates.

Table 1 .
Antibiotic susceptibility pattern (MIC) of P. fulva.Resistant by Rebolledo et al. [19] using phenotypic tests, EDTA double disc synergy test and ROSCO test, however, genotypic results did not affirm this finding in our case.