A very rare case of bacteraemia in a 4-year-old girl with osteopetrosis with probable Leuconostoc lactis infection

Leuconostoc lactis (LLac) is a Gram-positive coccus of the family Leuconostocaceae . It can be found in a variety of vegetables and dairy products. LLac is an opportunistic pathogen with intrinsic resistance to vancomycin and teicoplanin. In this case report, we discuss a rare case of LLac-associated bacteraemia in a patient with osteopetrosis. A 4-year-old girl was admitted to the paediatric emergency department with acute fever without other signs. Blood culture revealed an infection with LLac. Using the streptococcus antibiogram, the isolate was resistant to vancomycin, teicoplanin, rifampicin and sulfamethoxazole-trimethoprim but sensitive to β-lactams, gentamicin, streptomycin, azithromycin, clarithromycin, lincomycin, clindamycin and erythromycin. The patient was treated with intravenous ceftriaxone and gentamicin, and subsequently with oral amoxicillin. After a favourable course, she was discharged from the hospital on the 10th day. The modes of transmission and physiopathology of LLac remain unknown. Factors associated with this infection include compromised immunity, previous antibiotic therapy especially with vancomycin, and application of a central venous catheter. In our patient, the risk factors for infection were pancytopenia and multiple transfusions used to treat bone marrow failure. The source of the bacteraemia could have been the cutaneous route, but it could also have been digestive due to the reservoir of the bacteria. LLac is known as an opportunistic bacterium. Further studies on its pathogenesis and other risk factors are needed to understand the true prevalence of this potentially fatal bacterium in compromised individuals, such as the case of our patient.


INTRODUCTION
Leuconostoc lactis (LLac) belongs to Gram-positive cocci of the family Leuconostocaceae [1].This bacterium grows easily on columbia blood agar, but it is difficult to identify because of its rarity [2].LLac can be morphologically confused with Enterococcus sp. or Streptococcus sp. in clinical microbiology laboratories where bacterial identification is limited to classical biochemical techniques [2].It is intrinsically resistant to vancomycin and teicoplanin [3].LLac can be found in various vegetables and dairy products, and also milk and wine [3].In humans, it has been isolated from vaginal and stool samples [4,5].The biological agents committee classifies LLac in risk group 1 [6].This group defines agents that are unlikely to cause infectious disease in humans [6].However, published cases of infection with this agent in immunocompromised individuals have confirmed that it can be an opportunistic pathogen in humans [7][8][9][10][11][12].Most of the reported cases were bloodstream infections, ventriculitis, osteomyelitis, meningitis, pneumonia, endocarditis, pleural empyema or urinary tract infections [7][8][9][10][11][12].Several factors may be associated with these infections such as immunosuppression, catheter use and prior antibiotic therapy with vancomycin [7][8][9][10][11][12].In this report, we discuss a rare case of a child diagnosed with osteopetrosis who had LLac bacteraemia.To the best of our knowledge, this association is the first to be documented worldwide.

REPORT
Our patient was a 4-year-old girl treated in the paediatric department of the Mohammed VI University Hospital (Oujda, Morocco) since 2015 for osteopetrosis with bone marrow aplasia.Her treatment was mainly based on blood transfusion and an oral iron chelator (deferiprone, 30 mg kg -1 day -1 in three divided doses).She was admitted to the emergency department (13 March 2018; day 1) with 4 days of acute fever without other symptoms.Clinical examination found a conscious, pale and febrile child (39.4 °C).She had a psychomotor delay, and growth failure with craniofacial dysmorphism.Abdominal examination found a distension with splenomegaly reaching the umbilicus, and hepatomegaly with discrete abdominal collateral venous circulation.
Two blood culture aerobic bottles (BD Aerobic, BACTEC; Becton Dickinson), taken at a 7 h interval, were incubated in the BD BACTEC FX-400 (Becton Dickinson Microbiology Systems) automated blood culture system according to the manufacturer's instructions (day 1).Immediately after the second blood sampling, a probabilistic antibiotic therapy combining ceftriaxone (50 mg kg -1 24 h -1 ) and gentamicin (5 mg kg -1 24 h -1 ) was started (day 1).No further sampling for a possible entry point to the bloodstream was performed.In addition, the time to positivity of both blood culture bottles was 2 h.Microscopic examination after Gram staining found abundant Gram-positive cocci in both bottles.Subculture of both bottles in blood agar for 24 h at 37 °C allowed the isolation of monomorphic colonies with alpha haemolysis.Microscopic examination after Gram staining showed monomorphic Gram-positive cocci.Isolated bacteria were catalase-negative and oxidase-negative.We used the BD Phoenix 100 system and BD Phoenix Gram Positive Panel (both Becton Dickinson Microbiology Systems) for phenotypic identification of the isolated colonies.The identified bacterium was LLac with 99.99 % confidence.The main reactions that distinguished our isolated bacterium from all other catalase-negative Gram-positive cocci were negative pyrrolidonyl-arylamidase (pyrrolidonyl aminopeptidase) and negative leucine aminopeptidase (leucine-arylamidase) [13].
As there are no standard methods or interpretation criteria of antimicrobial susceptibilities established for LLac, we adopted the breakpoints of the streptococci based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [14].The method used was Muller-Hinton agar diffusion.Inhibition diameters were based on the streptococcal antibiogram.Our strain was resistant to vancomycin, teicoplanin, rifampin and sulfamethoxazole-trimethoprim.LLac was susceptible to β-lactams, gentamicin, streptomycin, azithromycin, clarithromycin, lincomycin, clindamycin and erythromycin.After bacteriological examination and based on the results, initial antibiotic therapy was maintained for 10 days, including 6 days of ceftriaxone (50 mg kg -1 24 h -1 ), followed by 4 days of oral amoxicillin (50 mg kg -1 24 h -1 ).In addition, this regimen was combined with gentamicin (5 mg kg -1 24 h -1 ) for 3 days.Her course was marked by apyrexia at day 2, and decreased C-reactive protein to 26 mg l −1 .Our patient was discharged on the sixth day and died 1 year later from a haemorrhagic syndrome related to her disease.

DISCUSSION
LLac is a Gram-positive bacterium known by several features including spherical or lenticular form [15].It is non-sporulated, capsulated, immobile, and with negative oxidase and catalase activities.It is also a facultative aero-anaerobic and heterofermentative lactic acid bacterium [15].LLac is used in the food industry, especially in dairies and sugar factories, which explains its carriage in the oral cavity and the digestive tract of humans and animals [16][17][18].LLac grows easily on columbia blood agar, but it is difficult to identify because of its rarity, and it can be morphologically confused with Enterococcus sp. or Streptococcus sp. in clinical microbiology laboratories where bacterial identification is limited to classical biochemical techniques [15].It shares common characteristics with alpha-haemolytic or non-haemolytic Streptococcus sp. or Enterococcus sp.[19].In addition, the increasing existence of vancomycin-resistant strains of enterococci may lead to confusion with other bacteria that are naturally resistant to these glycopeptides [19][20][21][22].
The difference between different genera of the family Leuconostocacae, essentially between Leuconostoc sp. and Weissella sp., can only be determined with molecular biology techniques, particularly metagenomic sequencing using the 16S rRNA marker [23,24].In fact, Weissella sp.belongs to the family Leuconostocacae and it shares the same biochemical characteristics with Leuconostoc spp., and it is also known as an opportunistic pathogen [23,24].In our case report, infection by Weissella sp. could not be excluded.
Since the 1990s, various types of infections have been described confirming the potential human pathogenicity of LLac [25].However, its modes of transmission and pathophysiology remain unknown [26].Described factors associated with infection include compromised immunity, previous antibiotic therapy (particularly vancomycin), parenteral nutrition, central venous catheters, surgery, liver failure, chronic renal failure, haemodialysis and extensive burns [12,19,27,28].Yet, LLac-associated infections have also been documented in healthy patients [29].To our knowledge, we report the first case of LLac bacteraemia associated with osteopetrosis (Table 2).Osteopetrosis ('marble bone disease') encompasses a group of rare inherited autosomal metabolic bone diseases.One of the consequences of this fatal disease is pancytopenia [30].There is currently no curative medical treatment for osteopetrosis [30].Management of this disease is essentially supportive and aims to ensure multidisciplinary monitoring and symptomatic management of complications, and a supportive management of associated infections [31].In our patient, pancytopenia, as a state of immunodeficiency, increased the risk of LLac bacteraemia.Another factor that might be associated with this bacteraemia is the multiple transfusion therapy protocol used to treat bone marrow insufficiency.The starting point could be the cutaneous route, either in the patient or the paramedical staff, but it could also be digestive as this is the reservoir of the bacterium [3].The lack of clinical data on our proposal did not allow us to confirm with certainty the portal of entry of the bacteria in the blood nor the pathogenic mechanism of bacteraemia.
After treating our patient, her course was marked by clinical and biological improvements and the patient was discharged 10 days after admission.To date, there are no recommendations for the treatment of patients infected with LLac.Published cases of LLac infection showed variability in the antibiotics used (Table 2).For example, penicillin, clindamycin, linezolid, macrolides, aminoglycosides, cephalosporins and tetracyclines were all tested [7,8,11].Susceptibility to these or other antibiotics was not specified.The reported cases of death showed that this bacterial infection may be fatal [9,32].However, this was not the case for our patient.In fact, the combination of ceftriaxone and aminoglycosides led to a successful cure.
In summary, we discuss a case of a fragile patient who developed bacteraemia associated with low-pathogenicity or nonpathogenic but opportunistic bacteria.LLac is naturally resistant to vancomycin.This is an option for treating infections, but new drugs against Gram-positive bacteria are available (e.g.linezolid).Study of the sensitivity of LLac to antibiotics and the therapeutic protocol has not yet been standardized.Particularly noteworthy is the management of Gram-positive bacterial infections resistant to vancomycin, where clinicians must consider the presence of naturally vancomycin-resistant bacteria to avert severe consequences, especially with rare bacteria such as LLac.Additionally, the microbiology laboratory should assess susceptibility to alternative antibiotics to avoid therapeutic failures.The formulation of specific recommendations for such cases is eagerly anticipated to ensure effective treatment.

CONCLUSION
We report a case of a compromised patient who developed bacteraemia with LLac.This species is known as a low-pathogenicity or non-pathogenic but opportunistic bacterium.The species must be determined to provide rapid effective therapy and improve clinical outcomes for these individuals.
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Table 1 .
Complete blood count parameters

Table 2 .
Cases of Leuconostoc lactis infection reported in the literature