First case of Listeria innocua meningitis in a patient on steroids and eternecept

Received 14 May 2014 Accepted 15 May 2014 Department of Experimental Medicine and Surgery, Tor Vergata University of Rome Via Montpellier 1, 00133 Rome, Italy Clinical Microbiology Laboratories, Polyclinic Tor Vergata, V. le Oxford 81, 00133 Rome, Italy Clinical Infectious Diseases, ‘Tor Vergata’ University, Rome, Italy Neuroscience Department, Polyclinic Tor Vergata, V. le Oxford 81, 00133 Rome, Italy


Introduction
Listeria innocua is considered a non-haemolytic saprophyte that is widely distributed in the natural environment.L. innocua is able to survive in various extreme conditions (high pH, high and low temperatures, and high salt concentrations) and has been isolated from soil, surface water, decaying vegetables, sewage and foodstuffs (Moreno et al., 2012).It is most closely related to Listeria monocytogenes but is generally considered non-pathogenic.In fact, L. innocua does not seem to carry the virulenceassociated genes described in L. monocytogenes and Listeria ivanovii.Nevertheless some atypical L. monocytogenes-like haemolytic L. innocua strains have been described (Johnson et al., 2004;Moreno et al., 2012).Some genetic characteristics of these L. innocua strains, such as possession of the LIPI-1 virulence cluster and part of the inlAB operon, can justify the ability to replicate intracellularly in mammalian cells and so be causative agents of disease, especially in immunocompromised mammals (Buchrieser et al., 2003;Glaser et al, 2001;Johnson et al., 2004;Milillo et al., 2012;Perrin et al, 2003;Volokhov et al, 2007;Walker et al, 1994;Welch, 2007).Perrin et al. (2003) described a fatal bacteraemia caused by a L. innocua strain and this was the first case reported in humans.Corticosteroids increase the risk of listeriosis (Schuchat et al., 1992), and etanercept use is associated with the possible occurrence of L. monocytogenes infection (La Montagna, G., Valentini, G., 2005;Pagliano et al, 2004;Rachapalli & O'Daunt, 2005;Salmon-Ceron et al., 2011;Schett et al., 2005).Here, we describe the first case of L. innocua meningitis, to the best of our knowledge, in a patient treated with etanercept and corticosteroids given for rheumatoid arthritis (Pagliano et al., 2004;Rachapalli & O'Daunt, 2005;Schett et al., 2005;Slifman et al., 2003).

Case report
A 72-year-old woman was admitted to our hospital in June 2010 with a high fever (up to 39.5 u C) and confusion.The patient had a 10-year history of rheumatoid arthritis, and had been treated in the last 3 years with methotrexate 10 mg once a week, and with intravenous infliximab 100 mg every 6 weeks.In the last 5 months, the therapy for rheumatoid arthritis changed to 25 mg prednisone once a day and 25 mg etanercept once a week.It is worth noting that, 5 months prior to the admission, the patient had a 2-week episode of meningomyeloradiculitis with Epstein-Barr virus-positive cerebrospinal fluid (CSF).At the time of admission, on objective examination, the patient appeared stuporous and presented neck stiffness and fever (39.5 u C).A lumbar puncture was performed again.

Investigations
The CSF appeared subclear and the white blood cell count was 3848 mm 23 [the lymphocytes were numerous (60 %) and several were activated, and a discrete number of monocytes were present as well as some red blood cells], protein 499,1 mg dl 21 (reference value 10-45), white blood cell count was 73 mm 23 , CSF glucose was 0 mg dl 21 , serum glucose was 118 mg dl 21 and lactate was 66.6 mg dl 21 .Microscopic examination of the CSF showed Grampositive rods with a coryneform appearance, suggesting a diagnosis of L. monocytogenes.An overnight culture of the CSF on sheep blood agar plates facilitated the growth of small, white, non-haemolytic colonies.The absence of haemolysis was unusual for the expected L. monocytogenes but is otherwise common for other Listeria spp.(such as L. innocua, Listeria welshimeri and Listeria seeligeri) (Rocourt & Grimont, 1983;Seeliger, 1981).Our isolate also exhibited characteristic tumbling motility when viewed under a light microscope (by hanging drop).On Listeria selective agar (PALCAM; bioMe ´rieux Italia), a very faint blackening of the medium (due to the aesculin hydrolysis) was only obtained after 48 h of incubation at 37 u C in aerobic conditions.Catalase production, aesculin hydrolysis (albeit very slow) and the production of acid from glucose, maltose, trehalose and lactose were all positive.Bacterial identification was performed using a GP card on a VITEK 2 system (bioMe ´rieux) and by nucleotide sequencing.In particular, the bacterial DNA was extracted and then amplified as described previously by Turner et al. (1999).The sequencing analysis was performed using an ABI Prism 310 Genetic Analyser (Applera).The sequence obtained was compared with all of the bacterial sequences available in GenBank, using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).The analysis showed 99 % nucleotide identity between the isolate and three published L. innocua sequences (GenBank accession nos HM007562.1,S55473.1 and AL596173.1)and 98 % nucleotide identity between the isolate and L. monocytogenes (GenBank accession no.EU090894.1).Additional auxiliary identification tests were performed, and the absence of haemolytic activity was confirmed by performing a CAMP test (named from Christie, Atkins and Munch-Petersen), which was negative for our isolate (Christie et al., 1944).Detection of phosphatidylinositol phospholipase C (PlcA) activity, which is useful in distinguishing L. innocua from L. monocytogenes, was performed on an agar plate and was also negative (Restaino et al., 1999).Serotyping indicated that our isolate was serotype 4 (Johnson et al., 2004).To better characterize our isolate, L. innocua gene tests were performed.These gene tests are known to be able to identify species of Listeria isolates on the basis of the presence (or absence) of 12 virulence genes, as well as on the detection of some L. innocua-specific genes.The virulence genes were: prfA, hly, plcA, plcB, mpl, actA, inlA, inlB, clpE, iap, daaA and inlC (Johnson et al., 2004;Volokhov et al., 2002), while the L. innocua-specific genes, detectable by PCR assays, were lin2693, lin1074, lin1068, lin0558, lin1073, lin0198, lin2454, lin0372 and lin0419, and for the non-coding intergenic regions were lin0454-lin0455-lin2134 (Glaser et al., 2001;Johnson et al., 2004).We also detected and sequenced the 16S-23S intergenic region (Johnson et al., 2004).The primer sequences and the PCR conditions were the same as those reported by Johnson et al. (2004).The results of the PCR assays are shown in Table 1.Our isolate contained the following virulence genes: prfA, inlA, inlB and the virulence cluster genes hly and plcA (not plcB), which encode, respectively, the L. monocytogenes haemolysin (listeriolysin O) and inositol-specific phospholipase C. The strain had mpl, iap, clpE and daaA but not actA.Moreover, our strain contained some of the L. innocua-specific genes namely: lin0372, lin0454-lin0455, lin0558 and lin1068 (Table 1).The majority of these genes had the expected sizes, particularly when our isolate was compared with the isolate reported by Johnson et al. (2004): L. innocua PRL/ NW 15B95.The sizes of the amplicons are shown in Table 1.Only a few exceptions were observed and concerned the following genes: plcA, mpl, lin0454-lin0455 and lin1068.However, these differences could be explained by the extreme variability of this species regarding gene assortment, as already described by several authors (Glaser et al., 2001;Johnson et al., 2004;Volokhov et al., 2007).The sequence analysis of the 16S-23S intergenic region showed 99 % nucleotide identity with the L. innocua genome deposited in GenBank.The accession number of the sequence is NCBI accession no.HM007562.1.
L. monocytogenes is an invasive opportunistic, foodborne pathogen and is one of the leading causes of mortality from foodborne infections.The complete genome sequence of L. monocytogenes demonstrates that it is closely related to other non-pathogenic species such as L. innocua.The most striking features of the L. monocytogenes genome are an exceptionally large number of surface proteins and an abundance of transport proteins (in particular, proteins dedicated to carbohydrate transport and an extensive regulatory repertoire).Surface proteins have important roles in the interactions of the micro-organism with its environment, in particular during host infection.Thus, the presence/absence of such proteins could be a valuable determinant of virulence.Particularly, among the surface proteins, internalins and InlB, are necessary to enter eukaryotic cells, while ActA plays a key role in the movement of L. monocytogenes in the host cell (Volokhov et al., 2007;Welch, 2007).Another fundamental feature of Listeria spp.characterization is the presence/absence of some genes, particularly those that belong to the LIPI1 pathogenicity island of L. monocytogenes (Volokhov et al., 2002).Data obtained using the PCR assays confirmed the potential pathogenicity of our isolate, which had some genes typical of L. monocytogenes (prfA, hly, plcA, mpl, intA, intB, iap, clpE and daaA).In contrast, the absence of pclB and actA (typical of L. monocytogenes) confirmed the difference of our isolate from L. monocytogenes.The gene asset, plcA + plcB 2 , of our isolate could justify the absence of the phosphatidylinositol phospholipase C activity that was shown on the agar plate.Other features such as the presence of some L. innocuaspecific genes (lin0372, lin0454-lin0455, lin0558 and lin1068) as well as the sequence analysis of the 16S-23S intergenic region confirmed that our isolates was a L. innocua strain and is not a variant of L. monocytogenes.Moreover, our strain appeared to be different from previously reported L. innocua strains.This evidence was particularly clear when we compared our isolate with those reported by Johnson et al. (2004), particularly with the Listeria sp.strain PRL/NW 15B95, which was the main subject of their study (see Table 1).This L. innocua strain was capable of invasive disease in the absence of cell-to-cell spreading, as suggested by the absence of the actA gene product (an important virulence determinant for L. monocytogenes).

Diagnosis
A diagnosis of acute meningitis and late obstructive hydrocephalus was made.

Treatment
At the time of CSF-positive culture, a therapy based on 3 g ampicillin every 6 h and 80 mg gentamicin every 8 h was started.The common dosage for ampicillin is 2 g every 4 h, but some clinical experience has shown that ampicillin given in daily doses of 6 g or more is probably equally effective against L. monocytogenes (Jones et al., 1997).In our case, the dose of ampicillin used was intermediate between the two and this may have negatively influenced the patient's clinical course.

Outcome and follow-up
As the microscopic examination of CSF was positive, the patient was transferred to an Infectious Disease Centre {L.innocua strain described by Johnson et al. (2004).{The gene was not present in the L. innocua PRL/NW 15B95 strain and the amplicon size refers to that of L. monocytogenes.
and, shortly thereafter, to an intensive care unit, continuing treatment with the following specific antibiotic therapy: 3 g ampicillin every 6 h for 1 week and 80 mg gentamicin every 8 h for 2 weeks.After a transient clinical improvement associated with an almost normal CSF examination, the consciousness of the patient worsened.Upon magnetic resonance imaging, the brain showed enlarged lateral ventricles and periventricular oedema, whereas the mesencephalic duct was of a small calibre.The obstructive hydrocephalus was treated with a ventricular shunt.A short improvement of consciousness was interrupted again 2 weeks later by the occurrence of Acinetobacter baumannii pneumonia, which was unresponsive to different cocktails of antibiotics (vancomycin, levofloxacin, linezolid, colistin, tigecycline and fluconazole), and death occurred in approximately 10 days.An autopsy was not performed.

Discussion
L. innocua is widespread in the environment and food.It causes animal infections but was not considered to be a human pathogenic bacterium until the publication by Perrin et al. (2003) who presented a case of fatal sepsis.Due to the strong similarities between L. monocytogenes and L. innocua, the correct identification of L. innocua could be a challenge.Some of the most common biochemical and/or serological tests are often not sufficient to discriminate between the two species.In our case, the automatic identification system correctly identified our strain within 7 h.The subsequent sequence analysis of the DNA extracted from the isolate as well as the PCR assays helped us to confirm the initial identification.This evidence suggests that, to produce a report in a short time, particularly in a case of meningitis, it is not strictly necessary to use a molecular-based test for confirmation.On the contrary, the latter could be considered a secondchoice tool that is just necessary to confirm the pathogen identification.
The present report is the second description of a human invasive infection caused by L. innocua (Perrin et al., 2003) and the first case, to our knowledge, in a patient being treated with etanercept.The rapid identification of the pathogen and the prompt institution of a specific effective antibiotic therapy were effective in treating the acute meningitis.However, the occurrence of obstructive hydrocephalus as a late complication of L. innocua infection should be taken into account and, in our case, could explain the poor outcome.

Table 1 .
Comparison of the gene assortment of L. monocytogenes, L. innocua PRL/NW 15B95 and our isolate *L. monocytogenes from our strain collection.