Keywords
lactobacillus - probiotics - infection - sepsis - infant
It is well known that necrotizing enterocolitis (NEC)[1] and nosocomial infections[2] increase morbidity and mortality in preterm infants and, therefore, their prevention
is of crucial importance for improving outcome in these patients. Probiotic supplementation
has been widely studied as one of the proposed interventions for the prophylaxis of
NEC and nosocomial infections. Probiotics can enhance the enteric microbiota composition
and counteract the loss of gut commensals such as Bifidobacterium and Lactobacillus species, as occurs in preterm infants undergoing prolonged antibiotic treatment,
delayed enteral feeding, and lack of human milk, which can favor the proliferation
of pathogenic microflora and abnormal gut colonization.[3] Thus, probiotics may help to decrease translocations of pathogens from the gut and
ultimately the development of NEC and nosocomial infections.[3]
A recent meta-analysis of 24 randomized controlled studies showed that probiotics
are effective in significantly decreasing NEC occurrence and mortality, but not nosocomial
infections, and concludes that these findings support a change in the current practice
and they should be widely used.[4] It is noteworthy that this review specifies that none of the included studies report
systemic infections due to administered probiotic organisms, thus supporting the safety
of probiotic supplementations in preterm infants.[4]
On this basis, every day thousands of extremely and very preterm infants have been
and are supplemented with probiotics. However, some cases of sepsis attributable to
Lactobacillus species have been documented in patients supplemented with probiotics, such as two
preterm infants with short-gut syndrome,[5] one child with short-gut syndrome,[5] one infant with congenital heart disease,[6] one child with cerebral palsy,[6] and one term infant with intrauterine growth restriction.[7] Moreover, these reports confirm previous concerns regarding the risk of infections
due to Lactobacillus species previously documented in adult human beings.[8]
[9]
Thus, the purpose of this report is to document two further cases of sepsis caused
by Lactobacillus rhamnosus that occurred in our neonatal intensive care in a term infant affected by multiple
chromosomal disorders and in an extremely preterm infant, respectively, and to discuss
the emerging issue of probiotic supplementation safety in neonates.
Data Sources
The National Library of Medicine (MEDLINE) database was searched from 1995 to 2014.
Search criteria included the following MESH: (1) Lactobacillus or probiotic; (2) sepsis, bacteremia, or short-gut syndrome; and (3) infant, newborn,
preterm, or premature.
Case 1
A Caucasian female was born at 39 weeks of gestation by vaginal delivery and was affected
by trisomy 18 and triple-X syndromes. Her birthweight was 1,660 g and Apgar scores
were 5 and 8 at 1 and 5 minutes, respectively. Heart ultrasound demonstrated atrial
and ventricular septal defects, bicuspid aortic valve, and patent ductus arteriosus
(PDA). Furthermore, her postnatal course was complicated by status epilepticus, relapsing
systemic infections (sepsis caused by Staphylococcus aureus, pneumonia caused by Stenotrophomonas maltophilia and Staphylococcus aureus), respiratory failure requiring mechanical ventilation (MV), need of pulmonary arterial
banding, and surgical closure of the PDA for hemodynamic worsening. On day 97 of life,
during MV and with central venous catheter (CVC) in place, the patient had a temperature
of 38.7°C and pulse of 120 beats/min, without other signs and symptoms. Blood sample
for culture was drawn from CVC and a peripheral vein, bronchoalveolar lavage for culture
was performed, and empiric antibiotic treatment with daptomycin (6 mg/kg, dose every
24 hours) and ceftazidime (30 mg/kg, dose every 6 hours) was started. Laboratory analyses
evidenced a white blood cell count (WBC) of 8,040 cells/mL, platelet count of 80,000
cells/mL, serum C-reactive protein (CRP) level of 135.3 mg/L, and serum procalcitonin
(PCT) level of 10.12 ng/mL. Blood culture from the peripheral vein was positive for
L. rhamnosus species. Since the 9th day of life our patient was given oral drop supplementation
with 5 × 109 colony-forming unit (CFU) of L. rhamnosus GG (Dicoflor, Dicofarm, Rome, Italy) twice daily, through the orogastric tube, for
the prevention antibiotic-associated diarrhea. After the results of a positive blood
culture the probiotic supplementation was discontinued. The isolate Lactobacillus isolate was susceptible to penicillin G, erythromycin, ampicillin, gentamicin, clindamycin,
linezolid, and was resistant to vancomycin. Therefore, we discontinued ceftazidime
and started clindamycin (5 mg/kg, dose every 6 hours). Our patient's clinical conditions
remained stable and after 10 days of therapy with clindamycin, her WBC, CRP, and PCT
normalized and antibiotic therapy was discontinued. Ultimately, our patient was discharged
at 300 days of life with gastrostomy for enteral nutrition and tracheotomy for respiratory
support due to her syndromes.
Case 2
A white male, was born at 23 weeks of gestation by vaginal delivery. His birth weight
was 660 g and Apgar scores were 4 and 7 at 1 and 5 minutes, respectively. The postnatal
course was complicated by the development of respiratory distress syndrome, pharmacological
closure of PDA, and sepsis caused by Staphylococcus haemolyticus. On the 18th day of life, during noninvasive respiratory support (nasal intermittent
MV) and with CVC in place, the patient developed episodes of mixed apnea associated
with metabolic acidosis without other signs and symptoms of infection. Blood samples
for culture were made from the CVC and a peripheral vein, and empiric antibiotic treatment
with linezolid (10 mg/kg, dose every 8 hours) and gentamicin (4 mg/kg, dose every
36 hours) was started. Laboratory analyses evidenced a WBC of 20,500 cells/mL, CRP
of 25.7 mg/L, and PCT of 2.90 ng/mL. The blood culture from the peripheral vein was
positive for L. rhamnosus. Since the 2nd day of life our patient was given daily oral drop supplementation
with 5 × 109 CFU of L. rhamnosus GG (Dicoflor), through the orogastric tube, to prevent NEC. After positive blood
culture appeared, probiotic supplementation was discontinued. The isolate had the
same antibiotic susceptibility and resistance of the previous case. Therefore, we
discontinued gentamicin and started clindamycin (5 mg/kg, dose every 6 hours) that
was given for 10 days.
On the 26th day of life, the infant developed severe respiratory failure requiring
MV and 100% oxygen, caused by a chest X-ray confirmed pneumonia. Laboratory analyses
evidenced a WBC of 6,950 cells/mL, platelet count of 119,000 cells/mL, CRP of 84.7
mg/L, and PCT of 3.80 ng/mL. Blood samples for culture were made from the CVC and
a peripheral vein, and daptomycin (6 mg/kg, dose every 24 hours) and meropenem (20
mg/kg, dose every 8 hours) were administered empirically. Also, the second peripheral
blood culture was positive for L. rhamnosus with the same antimicrobial susceptibility profile as the first positive blood culture
and therefore meropenem was stopped and gentamicin (4 mg/kg, dose every 24 hours)
was given. Persistence of L. rhamnosus bacteremia was documented in a third blood sample, obtained from a peripheral vein
after 6 days (34th day of life). However, after 10 days of therapy with gentamicin,
WBC, CRP, PCT, and chest X-ray normalized, and his clinical condition progressively
improved. Ultimately, our patient was discharged at 117 days of life in good health.
Characterization of the Lactobacillus Isolates
Characterization of the Lactobacillus Isolates
The Lactobacillus isolates from the two blood cultures were identified by MALDI-TOF mass spectrometry
(VITEKMS, bioMérieux, Marcy L'Etoil, France) as L. rhamnosus, suggesting a correlation with the probiotic preparation given to the infants. To
compare the two cultures with the probiotic strain, genotyping by pulsed field gel
electrophoresis (PFGE) profiling of the genomic DNA digested with the NotI and SfiI
restriction endonucleases was performed.
The genomic DNA in agarose blocks was prepared by the method of Tynkkynen et al.[10] Restriction enzyme digestion was performed overnight at 37°C. Electrophoresis was
performed with a CHEF-DRIII apparatus (Bio-Rad, Hemel Hempstead, United Kingdom) in
1% PFGE certified agarose (Bio-Rad) with 0.5× TBE buffer. The pulse time was 1 to
15 seconds the current was 5 V/cm, the temperature was 14°C, and running time was
22 hours. After electrophoresis, the agarose gel was stained with ethidium bromide
(0.5 μg/mL), visualized under ultraviolet light, and the PFGE profiles were compared.
The isolates from both the patients exhibited an identical PFGE profile to that of
the probiotic strain L. rhamnosus GG (ATCC 53103) (data not shown). Antimicrobial susceptibility testing of the L. rhamnosus GG strain from the probiotic formulation revealed a profile identical to that of
the two clinical isolates, with minimal inhibitory concentrations of penicillin G,
erythromycin, ampicillin, gentamicin, clindamycin, linezolid, daptomycin, and vancomycin
of 0.5, ≤ 0.12, 1, 4, ≤ 0.12, 1, 1, and > 256 mg/L, respectively ([Table 1]).
Table 1
Comparison of MIC values (determined by broth microdilution method) of penicillin,
erythromycin, ampicillin, gentamycin, clindamycin, linezolid, and vancomycin of the
two Lactobacillus rhamnosus clinical isolates and L. rhamnosus GG. Results were interpreted according to CLSI M45-A2 document
|
Isolate case 1
|
Interpretation
|
Isolate case 2
|
Interpretation
|
Lactobacillus rhamnosus GG
|
Interpretation
|
|
Penicillin
|
0.5
|
S
|
1
|
S
|
0.5
|
S
|
|
Erythromycin
|
≤ 0.12
|
S
|
≤ 0.12
|
S
|
≤ 0.12
|
S
|
|
Ampicillin
|
2
|
S
|
1
|
S
|
1
|
S
|
|
Gentamicin
|
4
|
S
|
2
|
S
|
4
|
S
|
|
Clindamycin
|
≤ 0.12
|
S
|
≤ 0.12
|
S
|
≤ 0.12
|
S
|
|
Linezolid
|
2
|
S
|
2
|
S
|
1
|
S
|
|
Vancomycin
|
> 256
|
R
|
> 256
|
R
|
> 256
|
R
|
Abbreviations: MIC, minimum inhibitory concentration, expressed in µg/mL; R, resistant;
S, susceptible.
Discussion
In this study, we report two cases of sepsis caused by L. rhamnosus GG that developed during the patients' probiotic supplementation with the same strain,
thus supporting a cause–effect relationship between supplementation and the development
of sepsis. By reviewing the international literature we identified six other cases
of sepsis due to L. rhamnosus GG occurring during probiotic supplementation with the same strain in infants[5]
[7]
[8] and children[6]
[7] ([Table 2]). All these patients were supplemented with L. rhamnosus GG with the purpose of preventing or treating gastrointestinal complications, such
as antibiotic-associated diarrhea or NEC.
Table 2
Summary of reported cases of sepsis by Lactobacillus rhamnosus GG during its supplementation in infants and children
|
Age
|
Main risk factors
|
Dose (CFU)
|
Exposure before sepsis (d)
|
Outcome
|
Effective antibiotic therapy
|
Typing methods
|
|
Kunz et al[5]
|
3 mo
|
Prematurity, short-gut syndrome
|
Unknown
|
23
|
Unknown
|
Ampicillin
|
No confirmatory typing
|
|
10 wk
|
Prematurity, gastroschisis, short-gut syndrome
|
Unknown
|
169
|
Unknown
|
Ceftriaxone, ampicillin
|
PFGE
|
|
De Groote et al[6]
|
11 mo
|
Prematurity, gastrostomy, short-gut syndrome, CVC
|
Not reported
|
35
|
Unknown
|
Ampicillin, gentamycin
|
rRNA sequencing
|
|
Land et al[7]
|
6 wk
|
CHD, antibiotic related diarrhea
|
10 × 109
|
20
|
Alive after 6 wk
|
Penicillin G, gentamycin
|
PCR DNA fingerprinting
|
|
6 y
|
Cerebral palsy, jejunostomy feeding, CVC, antibiotic- associated diarrhea
|
10 × 109
|
44
|
Discharged after 86 d
|
Ampicillin
|
PCR DNA fingerprinting
|
|
Sadowska-Krawczenko et al[8]
|
6 d
|
IUGR
|
3 × 109
|
4
|
Discharged after 86 d
|
Ticarcillin plus clavulanic acid
|
PCR DNA fingerprinting
|
|
Present cases
|
3 mo
|
Trisomy 18, triple X syndrome, CHD, CVC
|
5 × 109
|
88
|
Discharged after 300 d
|
Clindamycin
|
PFGE
|
|
18 d
|
Prematurity, PDA, CVC
|
5 × 109
|
16
|
Discharged after 117 d
|
Clindamycin, gentamycin
|
PFGE
|
Abbreviations: CHD, congenital heart disease; CVC, central venous catheter; IUGR,
intrauterine growth restriction; PDA, patent ductus arteriosus; PFGE, pulsed field
gel electrophoresis.
These cases are in agreement with previous studies reporting the development of systemic
infections caused by Lactobacillus species in infants and children who were not supplemented with probiotics.[11]
[12]
[13]
[14]
[15]
[16]
[17] Both supplemented and nonsupplemented patients had similar risk factors, such as
immune-deficiency (including that associated with prematurity[18]), previous gastrointestinal or cardiac surgery, previous antibiotic therapy, particularly
with vancomycin, NEC, ileostomy, malabsorption, and placement of CVC, but it is probable
that supplementation may further enhance the risk of developing L. rhamnosus GG sepsis through the daily prolonged overload of microorganisms.
Thus, L. rhamnosus GG is considered a commensal microbe in human beings and part of the normal gut microbial
flora,[6] is safe and nonpathogenic in most patients,[19] but can induce serious infections, including sepsis,[5]
[6]
[7]
[8] pneumonia, and meningitis[14]
[15] in compromised newborns and children. It is likely that similar considerations may
be extended to other probiotics commonly given to preterm infants, such as Bifidobacterium species, since five cases of bacteremia/sepsis have already been documented in newborns.[20]
[21]
[22] However, it must be underlined that only a few cases of severe infection by probiotics
have been reported in comparison to the thousands of preterm infants who have been
or are supplemented for preventing NEC.
The pathogenesis of Lactobacillus infection is not well known, but its adhesion to the intestinal mucosa and subsequent
colonization are considered important steps because they can prolong persistence in
the intestine.[23] This consideration seems to support our speculation that prolonged daily probiotic
supplementation, as occurred in our and previous patients,[5]
[6]
[7]
[8] may represent a relevant risk factor for the development of related infections.
When supplemented patients develop L. rhamnosus GG sepsis its only plausible portal of entry is through enteral administration that
is probably followed by Lactobacillus access to the bloodstream through translocation across the epithelium. This event
might be favored by local gut injuries, such as those potentially caused by decreased
blood perfusion able to injure the gastrointestinal mucosa (i.e., systemic hypotension,
gastrointestinal surgery, congenital heart disease, intrauterine growth restriction,
treatment with nonsteroidal anti-inflammatory drugs for PDA closure, treatment with
corticosteroids, etc.). Another uncommon possibility might be CVC contamination, either
during the opening of the probiotic bottle or through hand-related transmission.[6]
We evaluated the possible role of dose and duration of exposure to L. rhamnosus GG in our cases in comparison with previous reports,[4]
[5]
[6]
[7] and we observed a great heterogeneity. In fact, while some reports did not detail
the supplementation dose,[4]
[5] we administered 10 × 109 CFU in the first case and 5 × 109 CFU in the second, Land et al[7] gave 10 × 109 CFU, and Sadowska-Krawczenko et al[7] gave 3 × 109 CFU. Moreover, the duration of supplementation with L. rhamnosus GG ranged from 4 to 95 days.[5]
[8] Such heterogeneity precludes the possibility of drawing conclusions regarding the
possible effect of probiotic dose and exposure duration on the risk of developing
related sepsis. However, after these two cases and due to the lack of evidence-based
recommendations on these points,[24] we have decided to decrease the daily dose of L. rhamnosus GG in our patients to 3 × 109 CFU.
Antimicrobial susceptibility of the infecting L. rhamnosus GG strains has been reported in only some of the L. rhamnosus case reports reviewed in this article.[5]
[7]
[8] These data showed some variability, but these discrepancies could also be attributed
to differences among susceptibility testing techniques and interpretative criteria
adopted by different laboratories. However, a consistent finding among all the reports
was the resistance of L. rhamnosus strains to vancomycin and their susceptibility to ampicillin.[5]
[7]
[8]
In summary, we report two cases of sepsis in neonates caused by L. rhamnosus GG during enteral supplementation in addition to the six cases previously reported.[5]
[6]
[7]
[8] Probiotic supplementation most likely caused the sepsis in these patients, although
all of them had further documented risk factors for sepsis. In these few cases, the
dose and duration of probiotic supplementation do not seem to be positively related
to the risk of developing sepsis and the antibiotic susceptibility of isolated strains
varied between patients. We conclude that, although none of the thousands of patients
enrolled in previous studies[4] developed systemic infections due to administered probiotics, neonatologists must
be aware that supplementation with L. rhamnosus GG can cause sepsis in high-risk patients on rare occasions. Further studies evaluating
the most effective and safe dose and duration of each probiotic supplementation should
be performed.
