Psychrobacter sanguinis: an Unusual Bacterium for Nosocomial Meningitis

Abstract

We report the first case of postneurosurgical meningitis due to Psychrobacter sanguinis, identified only by 16S rRNA analysis. Psychrobacter spp. usually live in deep sea environments and cold habitats. Despite a strict questioning of the patient and the medical staff, we did not find the source of this bacterium.

CASE REPORT

A 64-year-old woman, living near the English Channel, suddenly experienced severe headaches and vomiting. After hospital admission to an emergency department, clinical examination revealed nuchal rigidity but no focal neurological symptoms. There was no hyperthermia or altered consciousness (Glasgow coma scale evaluated at 14). Computed tomography angiography (CTA) showed a subarachnoid hemorrhage with enlargement of ventricles, due to a basilar artery aneurysm. The patient was immediately transferred to a neurosurgery unit. An external ventricular drain was put in place under local anesthesia to relieve the elevated intracranial pressure. She did not receive any prophylactic antibiotic therapy. Endovascular coiling was performed on day 1 after admission to seal off the basilar aneurysm. A leak of cerebrospinal fluid (CSF) was observed at the drain insertion site, on day 3. The patient kept touching the drain insertion site with her bare hands, despite repeated instructions. On day 8, the external ventricular drain was removed due to the persistence of the CSF leak. There was no local sign of inflammation or infection. However, the patient presented hyperthermia (38.5°C) the same day and a lumbar puncture was performed. The CSF was hemorrhagic, with an increased concentration of proteins (1.64 g/liter) and decreased glucose (0.19 g/liter, with glycemia = 1.32 g/liter). Mild hypoglycorrhachia occurs frequently after subarachnoidal hemorrhage; therefore, it was not considered indicative of meningitis. No bacteria were shown on Gram stain, and the cultures remained sterile after 5 days of incubation (chocolate agar in a CO₂ atmosphere and brain heart infusion [BHI] broth under aerobic conditions at 37°C). On day 15, the patient presented hyperthermia at 39°C, with confusional syndrome and drowsiness. A new lumbar puncture was performed, and the CSF culture allowed a definitive diagnosis of postneurosurgical meningitis. CSF analysis showed hyperleukocytosis (5,900 white blood cells [WBC]/mm³), profound hypoglycorrhachia (0.07 g glucose/liter, with glycemia = 1.78 g/liter), and elevated protein levels (1.53 g/liter). Direct examination revealed few Gram-negative coccobacilli. On the basis of the evolution of CSF parameters (Table 1), empirical antimicrobial therapy was initiated immediately using a combination of meropenem and fosfomycin at 2 g 3 times per day and at 4 g 4 times per day, respectively. A single dose of amikacin (25 mg/kg of body weight) was also administered. No bacterial growth was observed after 48 h of incubation. After 4 days of incubation, the BHI broth became turbid due to the growth of Gram-negative coccobacilli. The BHI broth was subcultured onto blood agar incubated at 37°C, chocolate agar incubated at 37°C and 5% CO₂, and blood agar incubated at 37°C in an anaerobic atmosphere. The Gram-negative coccobacilli grew on the blood and chocolate agar only under aerobic conditions after 48 h. These colonies were catalase, oxidase, and urease positive. Unfortunately, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) (Bruker Daltonik S. A., Wissembourg, France) using FlexControl software (version 3.0) gave no reliable identification (Psychrobacter sp.; score = 1.065). To identify this agent, sequencing of the 16S rRNA gene was performed and a 1,500-bp DNA fragment was amplified using universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1525R (5′-AAAGGAGGTGATCCAGCC-3′). DNA sequence analysis was performed with leBIBI and BLAST systems. There was 100% identity for 1,400 bp with the Psychrobacter sanguinis type strain (GenBank accession no. HM212668). Partial 16S sequences of 19 Psychrobacter species type strains and our isolate were analyzed using the ARB software environment (1). A phylogenetic tree was created using the ARB neighbor-joining algorithm (Fig. 1). Attempts at phenotypical identification using the Vitek2 GN card (bioMérieux) and API 20NE (bioMérieux) were not successful. In vitro antimicrobial susceptibility was determined by Mueller-Hinton agar disc diffusion incubated under aerobic conditions at 37°C, using the critical zone diameters recommended by CA-SFM 2013 (Comité de l'Antibiogramme de la Société Française de Microbiologie; http://www.sfm-microbiologie.org/). Our isolate of P. sanguinis was susceptible to amoxicillin, ticarcillin, cefepime, ceftazidime, imipenem, meropenem, amikacin, ciprofloxacin, and trimethoprim-sulfamethoxazole. Therefore, meropenem and fosfomycin were switched to amoxicillin (4 g 3 times per day), as part of the antimicrobial therapy de-escalation strategy. Amoxicillin treatment was continued until the patient had received 15 days of antimicrobial therapy. The patient experienced a rapid recovery under treatment. The fever subsided 4 days after the initiation of meropenem and fosfomycin treatment. A lumbar puncture performed the same day (on day 19) revealed only 12 WBC/mm³ and a normal level of glucose (0.54 g/liter). The protein level was still slightly elevated (0.92 g/liter). No other complications were observed during the hospitalization.

TABLE 1.

Timeline of CSF parameters

Day	No. of WBCa/mm3	Gram stain result	Culture result	Glycorrhachia (g glucose/liter)	Glycemia (g glucose/liter)	Protein level g/liter
8	680	Negative	Sterile	0.19	1.32	1.64
15	5.900	Gram-negative coccobacilli	P. sanguinis	0.07	1.78	1.53
19	12	Negative	Sterile	0.54	1.43	0.92

^aWBC, white blood cells.

FIG 1.

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences from 19 Psychrobacter type strains, Psychrobacter sanguinis isolate KJ439038 from our study, and an Acinetobacter calcoaceticus type strain as a root. Bootstrap values (>50%) are indicated at branch nodes.

We report the first case of postneurosurgical meningitis due to P. sanguinis. Psychrobacter spp. usually live in deep sea environments and cold habitats, such as Antarctic ice or Siberian permafrost. Members of the Psychrobacter genus are small Gram-negative coccobacilli. Psychrobacter spp. have been observed in marine environments such as deep sea (2) or sea ice (3) and in a wide range of food products, including seafood (4, 5) and cheese (6). P. sanguinis is a new species described in 2012; the type strain was isolated from the blood of an 84-year male in the United States (7). Because P. sanguinis is a recently described species, data are lacking concerning its pathogenicity. P. sanguinis was first isolated from the blood cultures of 4 patients in New York (7). However, P. sanguinis is probably an environmental bacteria and was recovered from seaweed samples in India (GenBank accession no. JX501674 and JX501676). Whereas Psychrobacter spp. usually grow on marine agar, which contains 3.5% NaCl, our P. sanguinis isolate failed to grow on this specific medium, as described by Wirth et al. (7). Moreover, our isolate was able to grow between the temperatures of 4°C and 37°C. Other Psychrobacter species have been found in various environmental sites and are considered only rare opportunistic human pathogens. P. arenosus is a species considered to be closely related to P. sanguinis on the basis of their 16S rRNA sequences and was described in 2004 (8). P. arenosus was isolated from costal sea ice in Japan (9) and was associated with a published case of bacteremia after blood transfusion in France (10). In our case report, investigations were performed to identify the source of P. sanguinis but it is difficult to understand how P. sanguinis was able to cause postneurosurgical meningitis. This was a case of nosocomial meningitis because the bacteria was identified in a CSF analysis performed more than 48 h after the hospitalization, but the nature of the bacteria suggested an extrahospital contamination source. One hypothesis is that the bacteria were present in the patient environment, perhaps before the hospitalization, and entered the central nervous system by colonization of the external ventricular drain. Indeed, the patient frequently touched the drain with her bare hands despite repeated instructions from the hospital staff. It is also possible that the mode of entry was transient bacteremia, but it is less likely. Four sets of blood cultures were drawn during the hospitalization and remained sterile.

This first report of P. sanguinis meningitis emphasizes the need to incubate culture medium for more than 48 h in postneurosurgical patients. Neurosurgery can lead to infection with rare bacteria of low pathogenicity levels, even 1 week after an external ventricular drain is removed.

Nucleotide sequence accession number.

The 16S rRNA gene sequence data of this P. sanguinis strain was deposited in GenBank under accession number KJ439038.