Detection and DNA quantification of Enterococcus casseliflavus in a foal with septic meningitis

Valentina Stefanetti Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Francesca Beccati Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Fabrizio Passamonti Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Elisa Sgariglia Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Mauro Coletti Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Matteo Vuerich Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Maria Luisa Marenzoni Dipartimento di Medicina Veterinaria, Centro di studio del Cavallo Sportivo, Università degli studi di Perugia, 06124 Perugia, Italy.

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Abstract

CASE DESCRIPTION A 3-month-old 180-kg (396-lb) Hanoverian colt was examined because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture. Initial treatment included empirical antimicrobial treatment and NSAIDs.

CLINICAL FINDINGS Initial findings were consistent with CNS anomalies. Endoscopy revealed hyperemia, ecchymosis, and some mucopurulent exudate in the right guttural pouch. Hematologic findings were consistent with neutrophilic inflammation. On the third day of hospitalization, severe neurologic signs were observed. Computed tomography of the skull revealed a comminuted fracture of the axial aspect of the right mandibular condyle. Examination of CSF revealed turbidity, xanthochromia, and intracellular and extracellular cocci, consistent with septic meningitis. After DNA extraction from blood and CSF, sequenced products from a PCR assay for the bacterial 16S rRNA gene were 99% identical to Enterococcus casseliflavus. Microbial culture of CSF and blood samples yielded bacteria with Enterococcus spp morphology; antimicrobials were selected on the basis of susceptibility testing that identified the isolate as vancomycin resistant. A quantitative PCR assay was used to estimate Enterococcus DNA concentrations in CSF and blood.

TREATMENT AND OUTCOME Treatment for E casseliflavus meningitis, including trimethoprim-sulfadiazine and ampicillin sodium administration, resulted in resolution of clinical signs. Culture of CSF and blood samples after 12 days of the targeted treatment yielded no growth.

CLINICAL RELEVANCE To the authors' knowledge, this was the first report of E casseliflavus meningitis in a horse. Treatment was successful; vancomycin-resistant enterococci can be a clinical problem and may potentially be zoonotic.

Abstract

CASE DESCRIPTION A 3-month-old 180-kg (396-lb) Hanoverian colt was examined because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture. Initial treatment included empirical antimicrobial treatment and NSAIDs.

CLINICAL FINDINGS Initial findings were consistent with CNS anomalies. Endoscopy revealed hyperemia, ecchymosis, and some mucopurulent exudate in the right guttural pouch. Hematologic findings were consistent with neutrophilic inflammation. On the third day of hospitalization, severe neurologic signs were observed. Computed tomography of the skull revealed a comminuted fracture of the axial aspect of the right mandibular condyle. Examination of CSF revealed turbidity, xanthochromia, and intracellular and extracellular cocci, consistent with septic meningitis. After DNA extraction from blood and CSF, sequenced products from a PCR assay for the bacterial 16S rRNA gene were 99% identical to Enterococcus casseliflavus. Microbial culture of CSF and blood samples yielded bacteria with Enterococcus spp morphology; antimicrobials were selected on the basis of susceptibility testing that identified the isolate as vancomycin resistant. A quantitative PCR assay was used to estimate Enterococcus DNA concentrations in CSF and blood.

TREATMENT AND OUTCOME Treatment for E casseliflavus meningitis, including trimethoprim-sulfadiazine and ampicillin sodium administration, resulted in resolution of clinical signs. Culture of CSF and blood samples after 12 days of the targeted treatment yielded no growth.

CLINICAL RELEVANCE To the authors' knowledge, this was the first report of E casseliflavus meningitis in a horse. Treatment was successful; vancomycin-resistant enterococci can be a clinical problem and may potentially be zoonotic.

A 3-month-old 180-kg (396-lb) Hanoverian colt was referred for examination because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture of 2 days' duration. The birth had been assisted, and the foal had reportedly stood without assistance and received colostrum. The dam had been vaccinated against tetanus and influenza and had been treated with ivermectin 1 month prior to foaling. The foal was reportedly healthy before the described clinical signs appeared.

Treatments initiated by the referring veterinarian included antimicrobial (ceftiofur sodium, 4.4 mg/kg [2.0 mg/lb], IM, q 12 h) and anti-inflammatory (meloxicam, 0.6 mg/kg [0.27 mg/lb], IV, q 12 h) drugs. Radiographic evaluation of the neck was performed by the referring veterinarian, and no abnormalities were noted.

Clinical findings on hospital admission were consistent with CNS anomalies: the foal appeared lethargic but responsive and maintained a kyphosis posture with a low head and neck position. Drooping of the left ear, consistent with damage of the auricular branch of the facial nerve, was also evident. Tachycardia (heart rate, 80 beats/min [reference range, 45 to 60 beats/min]), pyrexia (rectal temperature, 39.3°C [102.7°F]), and hyperemic mucous membranes were detected, with all other physical examination variables within expected limits. The foal had apparent difficulties in attempts at nursing but was able to drink mare's milk from a bucket. Results of ultrasonographic examinations of the abdomen and thorax and endoscopy of the upper respiratory tract, including the guttural pouch, were mostly unremarkable. However, an area of hyperemia and ecchymosis was detected on the dorsal aspect of the lateral compartment of the right guttural pouch, and some mucopurulent exudate was identified on its ventral wall.

Hematologic analysis revealed moderate leukocytosis (22.3 × 103 WBCs/μL; reference range, 6.3 × 103 WBCs/μL to 13.6 × 103 WBCs/μL) with neutrophilia (18.3 × 103 neutrophils/μL; reference range, 3.0 × 103 neutrophils/μL to 8.0 × 103 neutrophils/μL). The monocyte count was 100 monocytes/μL (reference range, 0 to 400 monocytes/μL). Hyperfibrinogenemia (8.3 g/L; reference range, 1 to 4 g/L), high serum amyloid A concentration (531.76 mg/L; reference range, 0.5 to 20 mg/L), and mild hyperbilirubinemia (4.1 mg/dL; reference range, 0.4 to 2.0 mg/dL) were present.

Meloxicam administration was discontinued, and treatment with flunixin meglumine (1.1 mg/kg [0.5 mg/lb], IV, q 24 h) was started. Amikacin sulfate (15 mg/kg [6.8 mg/lb], IV, q 24 h) was added to the antimicrobial regimen to increase coverage for gram-negative bacteria. Sucralfate (40 mg/kg [18.2 mg/lb], PO, q 6 h) was administered as a gastric coating agent. The foal received additional supportive care by IV administration of lactated Ringer solution (1 L, q 12 h, as a bolus [5 mL/kg/h {2.3 mL/lb/h}]).

On the third day of hospitalization, 10 mL of blood was collected aseptically from the right jugular vein and submitted for microbial culture. The colt began to show severe neurologic signs, including intermittent episodes of lethargy and somnolence and aimless wandering. Abnormal clinical findings included kyphosis, a stiff and extended neck, muscle tremors, head tilt to the left, drooping of the left ear, dysphagia, lack of coordination, and grade 2 out of 5 ataxia as scored on a published grading scale.1 These signs were consistent with diffuse CNS involvement. No petechial or ecchymotic hemorrhages of the mucous membranes; signs of respiratory distress, enteritis, or diarrhea; lameness or other evidence of joint involvement; or signs of umbilical infection were detected. The clinical and laboratory findings and results of neurologic evaluation were suggestive of diffuse forebrain disease. Differential diagnoses included intracranial abscess, Equine herpesvirus-1 myeloen-cephalitis, West Nile virus encephalitis, and fungal or parasitic meningitis.

On the same day, CT of the skull with and without contrast medium administration was performed to further evaluate the brain. The foal was positioned in dorsal recumbency under general anesthesia. The skull was imaged with a multislice CT scannera (slice thickness, 6 mm; voltage, 120 kV; amperage, 180 mAs; tube rotation time, 0.5 seconds). Images were reformatted as 3-mm thick by use of low-medium and high kernels (H30s, H40s, and H70h) to improve resolution and were displayed in soft tissue and bone tissue windows. The unenhanced CT examination revealed an avulsed and comminuted fracture of the axial aspect of the right mandibular condyle at the insertion of the lateral pterygoid muscle. No swelling of soft tissue surrounding the fracture site was detected (Figure 1). Postcontrast CT images were acquired immediately after administration of iopamidol (250 mL; 350 mg of iodine/mL) through a jugular vein; mild contrast enhancement of the meningeal sulci was detected on the postcontrast images. During the same anesthetic episode, CSF was collected from the atlanto-occipital subarachnoid space. A venous blood sample was collected on the same day and both samples were stored at 4°C until analysis.

Figure 1—
Figure 1—

Pre- and postcontrast CT images (A and B, respectively) of the right temporomandibular joint of a 3-month-old Hanoverian colt that was evaluated because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture of 2 days' duration. Images were obtained at the level of the zygomatic process of the temporal bone. A—Image displayed in a bone window highlights a bone fragment (arrow) at the medial margin of the mandibular condyle (MC). B—Image displayed in a soft tissue window highlights the blood vessels around the fracture site: the right external carotid artery (arrow) and maxillary artery (arrowhead).

Citation: Journal of the American Veterinary Medical Association 249, 1; 10.2460/javma.249.1.96

The CSF was turbid and xanthochromic. Biochemical examination results revealed a high total protein concentration (200 mg/dL; reference range, 20 to 80 mg/dL), a high nucleated cell count (9.29 × 103 WBCs/μL; reference range, 0 to 8 WBCs/μL), and a high RBC count (10.0 × 103 RBCs/μL; reference range, 0 to 100 RBCs/μL).2 Examination of a direct smear of CSF with Romanowsky stain revealed intracellular and extracellular cocci, and the cytologic findings were consistent with septic neutrophilic inflammation. A PCR assay for equine herpesvirus-1 in CSF and an ELISA for West Nile virus in a blood sample had negative results.

Blood and CSF samples were cultured for aerobic bacteria and fungi; DNA was also extracted from both sample types for biomolecular assay with a commercial kitb in accordance with the manufacturer's instructions. The extracted DNA was subjected to PCR assay to amplify the 466-bp fragment of the bacterial 16S rRNA (16S ribosomal RNA) gene, following the procedure described by Nadkarni et al3 with modifications. The PCR assay products were subjected to electrophoresis on 1.5% agarose gel stained with ethidium bromide (0.5 μg/mL) and visualized under UV light. The products were then purified in accordance with the manufacturer's recommended protocolc and subjected to direct sequencing. All gel-purified PCR assay products were sequencedd by use of the previously described gene-specific primers. The final sequences were submitted to a basic local alignment search tool analysise to verify specific amplification. Alignments in the GenBank-European nucleotide archive database revealed that the sequences were 99% identical to the 16S rRNA gene of Enterococcus casseliflavus (GenBank accession number, NR_102793.1).

After 48 hours of incubation, the CSF and blood sample cultures yielded a pure growth of bacteria with the morphology of Enterococcus spp. The colonies were identified by assessment of morphological features and results of a Gram stain, coagulase test, and catalase test. A biochemical test result profile was obtained by use of a commercial kit in accordance with the manufacturer's instructions.f This biochemical identification method gave a probability score of 90% for E casseliflavus. Susceptibility to a panel of 10 antimicrobial agents was determined by the disk diffusion method on Mueller-Hinton agar plates according to Clinical Laboratory Standards Institute guidelines.g Disks containing ceftiofur (30 μg), cefquinome (30 μg), penicillin (6 μg), ampicillin (10 μg), gentamicin (10 μg), amikacin (30 μg), enrofloxacin (5 μg), marbofloxacin (5 μg), trimethoprim-sulfadiazine (25 μg), and vancomycin (30 μg) were tested. The E casseliflavus isolate was identified as susceptible to ceftiofur, cefquinome, ampicillin, amikacin, and trimethoprim-sulfadiazine by measurement of inhibition zone diameters; it was found to be resistant to all other tested antimicrobial agents.

To estimate Enterococcus DNA concentrations in the CSF and blood, a real-time quantitative PCR assay was performed as described by Ryu et al,4 with slight modifications. A reference curve, relating the quantification cycle as a function of the bacterial DNA concentration in the samples, was constructed by use of 6 tenfold serial dilutions of the Enterococcus DNA in a scalar manner starting with 5.35 × 105 bacterial GE and ending with a final dilution of 5.35 × 10°. The efficiency of the standard curve was 107.2% (R2, 0.98; slope, −3.160). The quantitative PCR assay was performed with 20 μL of total volume, comprising 10 μL of a commercial master mix and 100 ng of DNA template in sterile water. The amplification was carried out under the following conditions: 98°C for 3 minutes, 40 cycles of 98°C for 5 seconds, then 61°C for 1 minute. Each reaction was run in triplicate with the appropriate negative controls (reaction mix and water).

The E casseliflavus GE value (GE/mL of template) was quantified by use of the standard curve; the detection limit was approximately 5.35 × 103 GE/mL of template. Both blood and CSF samples tested positive by quantitative PCR assay, with GE values well above the detection limit. The bacterial load was 1.88 × 104 GE/mL of template in the CSF and 3.74 × 104 GE/mL of template in blood.

A diagnosis of E casseliflavus meningitis was made on the basis of clinical and laboratory findings. On day 6 of hospitalization, the previous antimicrobial treatments were discontinued and trimethoprim-sulfadiazine (30 mg/kg [13.6 mg/lb], IV, q 12 h) and ampicillin sodium (20 mg/kg [9.1 mg/lb], IV, q 6 h) were administered on the basis of results of the antimicrobial susceptibility testing and because these were expected to have good blood-brain barrier penetration. Total duration of the antimicrobial treatments was 6 days for ceftiofur, 4 days for amikacin, and 12 days for trimethoprim-sulfadiazine and ampicillin sodium. Total duration of NSAID administration was 15 days.

The targeted treatments resulted in rapid clinical improvement. The fever and neurologic deficits were resolved, and normal suckling behavior was observed, beginning on the fourth day of the revised treatment protocol. Eleven days after the initiation of targeted antimicrobial treatment, acute-phase protein measurement showed a partial response (serum amyloid A concentration was within the reference range at 12.58 mg/L; fibrinogen concentration was still high at 7.32 g/L), but the clinical improvement was strongly suggestive of recovery. Moreover, after 12 days of the targeted treatment, examination of a direct smear of the CSF revealed no bacteria and the culture of CSF and blood samples yielded no growth. All hematologic values were within the reference ranges, and after 15 days of targeted treatment, the patient was discharged from the hospital.

Discussion

In human medicine, bacterial meningitis is still an unresolved problem and can have a high mortality rate and prolonged recovery time.5 The outcome critically depends on the rapid initiation of an effective antimicrobial treatment.6 In human medicine, meningitis caused by E casseliflavus is extremely rare: it was reported as a potential pathogen of postneurosurgical meningitis in immunocompromised hosts,7 and hospital-acquired enterococcal meningitis has emerged as an important problem because vancomycin resistance has been increasingly identified among these organisms.8

Bacterial meningitis in horses has been associated with high mortality rates. Toth et al9 reported death in 27 of 28 horses (96% mortality rate) with meningitis or meningoencephalomyelitis over a 25-year period. In a study10 of horses with a history of bacterial meningitis secondary to infectious disease processes involving the head, 7 of 7 horses died spontaneously or were euthanized because of clinical deterioration. Because of the closed-space localization of the infection and the proximity to CNS control centers, bacterial meningitis is associated with a high risk of a fatal outcome.11 There are only a few reports12–14 of successfully treated bacterial meningitis cases in horses in the literature. Meningitis in horses is typically caused by infectious agents9; however, to the authors' knowledge, meningitis caused by E casseliflavus has not previously been reported in horses.

A study by Graves et al15 that characterized populations of enterococci in livestock manure revealed that E casseliflavus and Enterococcus mundtii are the most predominant Enterococcus spp in fresh or dry manure from horses. This suggests that environmental spread of these Enterococcus spp may be common.

Nine distinct gene clusters conferring vancomycin resistance have been described in enterococci, and some of these can be transferred among the organisms by conjugation.8 Interestingly, in vivo transfer of a resistance gene (vanA) from a vancomycin-resistant Enterococcus faecium isolate of chicken origin to an E faecium isolate of human origin has been shown to occur in the intestines of humans.16 Thus, livestock may be a reservoir for vancomycin-resistant enterococci and a source of zoonotic infection.17,18 The isolate from the horse of this report showed a vancomycin-resistant phenotype that was not investigated further. Although vancomycin is generally not used in equine medicine, it can represent a therapeutic alternative to other drugs and thus the presence of vancomycin-resistant enterococci can also be a clinical problem.

There are 4 likely main pathways for infections involving the head in horses: pathogens may invade through erosion of thin bones, by hematogenous spread, or by spreading along cranial nerves, or direct inoculation can result from head trauma.19 In the horse of this report, the mandibular condyle fracture might have been the initial site of systemic entry of E casseliflavus. The external communication induced by trauma could have permitted the entry of E casseliflavus, and the consequent disruption of the periosteal blood supply could have provided an environment suitable for bacterial settling and proliferation. The authors speculate that spread of bacteria to the maxillary artery could have led to subsequent hematogenous dissemination to the meninges.

A mechanism for cell-to-cell spread has been postulated for encephalitic listeriosis in ruminants, involving migration of bacteria from a site of head injury along the trigeminal nerve to the brainstem, with bacteria observed within neurons and axons.20 In horses, the possibility of ascending infections along the olfactory nerve exists.10 Much of the CNS injury associated with bacterial meningitis results from the host response rather than damage directly caused by infectious organisms.12 The presence of bacteria in CSF induces production of prostaglandins, leukotrienes, and cytokines and migration of leukocytes. The resultant inflammation leads to many of the observed neurologic sequelae.9,12 Previous investigations found bacterial meningitis in 6 of 74 (8%)21 to 4 of 38 (11%)22 neonatal foals with septicemia. In the case described here, the foal was 3 months old at the time of onset of clinical signs, and we could not presume that the meningitis was secondary to hematogenous dissemination of the bacteria from an umbilical infection.

Computed tomography is used commonly to obtain images of the skull and brain of equine patients with neurologic conditions, and it can be a useful tool to aid in diagnosis of intracranial disorders23,24; however, this imaging technique has some limitations in the evaluation of soft tissues. Although a useful tool for identification of intracranial space-occupying masses and acute intracranial, particularly subarachnoid, hemorrhages, CT and contrast-enhanced CT were found to have limited sensitivity to detect multifocal, diffuse, inflammatory lesions of the brain parenchyma, such as meningoencephalitis or meningitis, in horses.24 Magnetic resonance imaging allows a better evaluation of demyelinating and other white matter diseases, cerebral neoplasm, degenerative diseases, cerebral infarction, and inflammatory lesions, because better contrast can be achieved between soft tissues with this method.24,25 For the foal of this report, CT was performed because intracranial abscess was included in the differential diagnoses on the basis of the clinical and laboratory findings, and the method is very useful for detection of brain abscesses.26

Interestingly, the results of quantitative PCR assays for the foal of this report indicated the bacterial load in CSF (1.88 × 104 GE/mL of template) was approximately half of that in the blood (3.74 × 104 GE/mL of template). Hackett et al27 showed that the bacterial load of Neisseria meningitidis in blood samples was positively associated with a clinical severity score in children and could be as high as 1.6 × 108 DNA copies/mL (representing the bacterial load) of blood in patients with severe septicemia. Bacterial load in the order of 104 and 105 colony forming units/mL of CSF or blood, respectively, was found for human patients with meningococcal disease by use of a quantitative direct plating procedure.28 Unfortunately, limited information is available in the veterinary literature concerning bacterial load quantification in CSF and blood in horses with septicemia or meningitis, so future research is needed to better understand our results.

The prognosis has been described as poor to grave in horses with bacterial or fungal meningitis despite treatment.11 In the foal of this report, the authors believe that the prompt diagnosis and consequent appropriate antimicrobial treatment were the primary factors in achieving a favorable outcome. The optimal approach to a critically ill patient with sepsis involves the initiation of aggressive, broad-spectrum empirical treatment.29 In our patient, a combination of ceftiofur and amikacin was initially used to provide coverage for gram-positive and gram-negative pathogens. Ampicillin and trimethoprim-sulfadiazine replaced the earlier empirical treatment, in accordance with the results of culture and susceptibility testing. However, ampicillin is one of the drugs to which Enterococcus spp have the highest degree of susceptibility in vitro,8 and its use alone would likely have been effective in this case.

The advantages of the biomolecular assay used for assessment of bacteria in samples from our patient included rapid identification of the organism; compared with a conventional blood culture, the time saved by a biomolecular analysis can range from 20 to 44 hours. This technique is extremely sensitive, rapid, capable of high throughput,30 and relatively easy to perform. In general, a PCR assay and amplified PCR product detection can be completed in 2 hours, which is also considerably faster than conventional detection methods.31 However, the PCR procedure also has limitations. The detection of degraded bacterial DNA from dead organisms can lead to false-positive results,32 and antimicrobial susceptibility data would be lacking. On the other hand, although bacterial culture of CSF is considered the gold standard in the diagnosis of bacterial meningitis33 and allows antimicrobial susceptibility testing of isolates, it may require days to develop a positive result. A challenge for future research on bacterial meningitis in horses could be the use of an algorithmic approach linking clinical findings with results of biochemical testing, hematologic analysis, bacterial culture, quantitative PCR assay of CSF, and tests for specific biomarkers to develop a useful scoring system for prognostication in this potentially fatal neurological emergency.

Acknowledgments

The authors declare that there was no conflict of interest.

ABBREVIATIONS

GE

Genome equivalents

Footnotes

a.

Somatom Volume Zoom, Siemens, Forchheim, Germany.

b.

QIAamp DNA Mini kit, Qiagen, Hilden, Germany.

c.

Wizard SV Gel and PCR Clean-up System, Promega Corp, Madison, Wisc.

d.

Primm srl, Milan, Italy.

e.

Basic Local Alignment Search Tool (BLAST), National Center for Biotechnology Information, Bethesda, Md.

f.

API-20 Strep Microbial Identification Strip, bioMérieux Sa, Marcy-l'Etole, France.

g.

Clinical Laboratory Standards Institute, 2009. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Information Supplement CLSI document M100-S19. Wayne, Pa. 2009.

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  • 33. Sanchez LC. Equine neonatal sepsis. Vet Clin North Am Equine Pract 2005; 21:273293.

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