Members of the Lactobacillus Genus Complex (LGC) as Opportunistic Pathogens: A Review

Microorganisms belonging to the Lactobacillus genus complex (LGC) are naturally associated or deliberately added to fermented food products and are widely used as probiotic food supplements. Moreover, these bacteria normally colonize the mouth, gastrointestinal (GI) tract, and female genitourinary tract of humans. They exert multiple beneficial effects and are regarded as safe microorganisms. However, infections caused by lactobacilli, mainly endocarditis, bacteremia, and pleuropneumonia, occasionally occur. The relevance of Lactobacillus spp. and other members of the LGC as opportunistic pathogens in humans and related risk factors and predisposing conditions are illustrated in this review article with more emphasis on the species L. rhamnosus that has been more often involved in infection cases. The methods used to identify this species in clinical samples, to distinguish strains and to evaluate traits that can be associated to pathogenicity, as well as future perspectives for improving the identification of potentially pathogenic strains, are outlined.


Introduction
Bacteria currently classified in the genus Lactobacillus are a paraphyletic group of gram-positive, non-spore forming, mostly non-respiratory, but aerotolerant, lactic acid bacteria (LAB), comprising at this time more than 237 species and 29 subspecies (http://www.bacterio.net/lactobacillus.html) [1]. Morphologically, they can be elongated or short non-motile rods, frequently found in chains and sometimes bent. They produce lactic acid as a major end-product of carbohydrate fermentation.
Lactobacilli are part of the normal human microbiota that colonizes the mouth, gastrointestinal (GI) tract, and female genitourinary tract. Moreover, these bacteria have been used for centuries for food and feed fermentation processes aimed at the transformation of perishable raw materials of animal or plant origin into more preservable products. Their activities are relevant to the production of dairy products, bread, sausages, fermented vegetables, wine, and silage.
According to the type of sugar fermentation pathway, lactobacilli fall into the following three groups, all including species that are industrially exploited: (i) obligately homofermentative, that produce only lactic acid as an end product of carbohydrate metabolism through the glycolysis pathway; (ii) facultatively heterofermentative, that produce a mixture of lactic and acetic acid as end products of carbohydrate metabolism through the glycolysis or the phosphoketolase pathway, and; (iii) obligately heterofermentative, that produce lactic and acetic acid, or ethanol, and CO 2 as end products of carbohydrate metabolism through the phosphoketolase pathway [2].
A metagenomic analysis on a human subject showed that over a period of two years, more than 50 Lactobacillus species, and individual Lactobacillus genotypes, were repeatedly detected in numbers of up to 10 8 cells/g in the stool [25], suggesting that a persistent population of lactobacilli could inhabit the gastrointestinal tract (GIT) of individuals.
The efficacy of lactobacilli as probiotics derives from their ability to tolerate very low pH values, which allows them to survive transit through the stomach, and adhere to the mucus layer by surface structures, such as pili and cell-wall anchored proteins [30]. Some of their beneficial activities are favoring GIT health by inhibiting the growth of pathogenic organisms with the production of lactic acid and other metabolites. Some Lactobacillus strains are able to immunomodulate human cells and elicit an anti-inflammatory response [31]. In addition, some strains produce antioxidants [32].
As other probiotics, they are sold as constituents of food, food additives, or food supplements, but control on their use to safeguard consumer's health needs to be improved [33].
Lactobacillus organisms are rarely associated with pathology in immunocompetent people, but in the presence of risk factors and underlying conditions, they can cause infections such as endocarditis, bacteremia, neonatal meningitis, dental caries, and intra-abdominal abscesses including liver abscess, pancreatic necrosis infection, pulmonary infections, pyelonephritis, meningitis, postpartum endometritis, and chorioamnionitis [34,35].
In a retrospective analysis carried out in Argentina between January 2012 and July 2017, Lactobacillus spp. were isolated from patients with bacteremia (67%), meningitis, empyema, urinary infection, vaginosis, and hepatic abscess and underlying conditions such as cancer, surgery interventions, diabetes, and intestinal malformation. However, it is not clear from the report if it was ascertained that these organisms were the primary cause of infection. L. rhamnosus was most commonly isolated, followed by L. fermentum, L. paracasei, L. oris, L. gasseri, L. iners, and L. salivarius [36].
A recent systematic review of case reports and case series of infection complications after probiotic treatments found that both Lactobacillus spp. and Pediococcus spp. were involved as causative agents, among other probiotic organisms of common use [37].
Finally, members of the genus Weissella, that can be isolated from a variety of habitats including raw milk, feces, saliva, breast milk, urine, and fermented food, have been implicated in cases of bacteremia, abscesses, prosthetic joint infections, and infective endocarditis as a possible consequence of translocation after disruption of the mucosal barrier caused by surgery or therapies [38].
This review presents the evidences for the behavior of LGC organisms as opportunistic pathogens able to cause different types of infection and the related risk factors and predisposing health conditions or medical treatments. The scope is increasing awareness that, for immunocompromised individuals, or those affected by particular medical conditions, these bacteria can represent a hazard and must be used more cautiously as health promoting microorganisms than it is currently done.
Moreover, perinatal infections caused by lactobacilli indicate preterm neonates as a population category at risk. Though a meta-analysis indicated that probiotics reduce the incidence of necrotising enterocolitis and all-cause mortality in preterm infants, excluding infants with a birth weight of <1000 g, cases of infections in premature infants have been reported. These include late-onset sepsis due to L. rhamnosus following a laparotomy, amnionitis, and neonatal meningitis, cases of bacteremia, lactobacillemia of amniotic fluid origin, L. rhamnosus GG bacteremia associated with probiotic use in a child with short gut syndrome, and L. rhamnosus infection in a child following bone marrow transplantation [45][46][47].
Experiments with athymic mice have shown the potential for probiotics to cause sepsis in immune deficient neonates. This possibility was supported by case reports of probiotic sepsis in humans [48].
The most common predisposing events for Lactobacillus infections are dental manipulation, poor dental hygiene, intravenous drug abuse, abdominal surgery, colonoscopy, probiotic use, and heavy dairy product consumption [49].
Recent opinion articles invite safety assessments to be conducted for Lactobacillus probiotics, since they represent a risk for individuals with underlying medical conditions [33,50]. In particular, Cohen (2019) [33] stated that the ability of these strains to infect humans is not controversial and that live bacteria sold as commercial probiotics are capable of infecting immunocompromised hosts and have well-established "inherent infective qualities".
Theoretically, the potential pathogenicity of probiotics may be enhanced in strains selected on the basis of the capacity to adhere to the intestinal mucosa, a trait that is considered important for their mechanism of action. Indeed, adherence can favor translocation across the intestinal barrier and ability to cause infections. The finding that Lactobacillus spp. isolated from blood adhere to intestinal mucus in greater numbers than isolates from human feces or dairy products supports the relationship between mucosal adhesion and pathogenicity [34].

Endocarditis
Among infections caused by lactobacilli, endocarditis, with or without bacteremia, is the most common. It occurred in patients who had dental extractions or gingival bleeding after toothbrushing [51,52], suggesting that these could be considered risk factors, especially in the presence of underlying immunosuppression and valvular heart disease [53].
An L. rhamnosus endocarditis case was reported in an 80 year old man who frequently consumed yogurt containing the organism following an upper endoscopy. This patient required aortic and mitral valve replacement for a cure. Cases of Lactobacillus endocarditis have also been described following colonoscopy [54]. Patients with hereditary hemorrhagic telangiectasia (HHT) are also exposed to this infection because of telangiectasias and arteriovenous malformations (AVMs). In a habitual consumer of fermented dairy products with this pathological condition, the portal of entry was intestine following a colonoscopy [55].
In a middle-aged man, L. acidophilus endocarditis led to an aneurysmal rupture of the sinus of Valsalva into the right ventricular outflow tract with fistula formation from the right coronary sinus to the right ventricular outflow tract that required surgical repair with an aortic valve replacement [56]. A case of mitral valve endocarditis due to Lactobacillus was recently reported in an 81 year old woman [57].
P. pentosaceus caused endocarditis in a 66 year old male in association with Lactococcus lactis subsp. lactis [58].
The species L. rhamnosus and L. casei have been most frequently involved in endocarditis, presumably for their ability to induce platelet aggregation and generate fibrin by producing a factor Xa-like enzyme that catalyzes steps of the coagulation process favoring clot formation. It is supposed that these bacteria colonize thrombotic vegetations where they grow, evading host defenses [59].

Bacteremia
Lactobacillus bacteremia has been associated with the consumption of probiotics in special medical conditions, including hematopoietic stem cell transplantation [60] and HIV-infection [61].
Bacteremia caused by Veillonella and Lactobacillus spp., secondary to occult dentoalveolar abscess, was reported in a pediatric patient [62].
In a patient with chronic lymphocytic leukemia and recurrent bacteremia caused by L. casei/paracasei and L. rhamnosus, the source of infection was unknown, since probiotics had not been assumed and entry from dental infections or the gastrointestinal and urinary tract was excluded [63].
Bacteremia caused by isolates indistinguishable from the L. rhamnosus probiotic strain GG based on pulsed field gel electrophoresis (PFGE) typing was associated with a higher mortality rate than bacteremia caused by other Lactobacillus species [40].
Lactobacillus sepsis was normally resolved with antimicrobial therapy, but in some cases, patients developed septic shock. In other cases, the outcome has been fatal, but due mostly to underlying diseases rather than probiotic sepsis. On the basis of the characteristics of the cases reported, a list of major and minor risk factors for probiotic sepsis was proposed and caution in using probiotics in the presence of a single major risk factor or more than one minor risk factor was suggested. Major risk factors are being immune-compromised and preterm births, while minor risk factors are presence of central venous catheters (CVCs), impaired intestinal epithelial barrier caused by intestinal infections or inflammation, administration of probiotic by jejunostomy, concomitant administration of antibiotics to which the probiotic is resistant, probiotics with properties of high mucosal adhesion or known pathogenicity, and cardiac valvular disease (Lactobacillus probiotics only) [34].

Pleuropneumonia
Lactobacillus species were a primary cause of pleuropneumonia without bacteremia, especially in immunocompromised patients. From 1982 to 2016, 15 cases of pleuropneumonia caused by Lactobacillus spp. were reported, and involved L. rhamnosus, L. fermentum, L. acidophilus, L. paracasei, and L. coryneformis. All the patients had severe associated co-morbidities comprising immunosuppression, caused in most cases by AIDS, carcinoma, chronic diseases, and neutropenia. One patient had Lactobacillus pneumonia linked to consumption of a probiotic supplement. The route of entry was probably GIT in some patients, the transplanted lung in one patient, ventilator in an immunocompetent patient with thoracic trauma. In one patient, diagnosed with trachea-esophageal fistula, the route of Lactobacillus pneumonia was aspiration of a probiotic strain. Only one patient had concurrent lactobacillemia. The authors of the study suspected that infections due to Lactobacillus species are under-reported because appropriate growth conditions, such as microaerophily or anaerobiosis, are not applied in clinical microbiology laboratories for their isolation [64].

Meningitis
The first reported case of meningitis in which Lactobacillus was isolated from blood and cerebrospinal fluid was in an early-term neonate (38 weeks gestation) within the first day of life. Transmission from the mother's genital tract to the neonate's oral mucosa at the time of delivery was identified as the probable route of infection, since no immunological abnormalities, structural defects, or peripartum complications were observed. Another case involved a 10 year old neutropenic child affected by acute leukemia with four successive episodes of L. rhamnosus bacteriemia and unknown origin of infection.
A lethal case of meningitis due to L. rhamnosus was reported in an 80 year old woman not immunocompromised but with a fistula between the esophagus and the meningeal space, caused by dislodged and eroded plates and screws used several years earlier for cervical spine surgery, that facilitated bacterial translocation.
Meningoencephalitis caused by L. plantarum was reported in a 63 year old man with metastatic planoepithelial lung cancer.
Bacteremia and endocarditis, which are the two main manifestations of Lactobacillus infection, can lead to the onset of neurological sequelae through mechanisms mediated by embolic material.
This was not the case of the latter patient, who had no signs of endocarditis. Therefore, direct bacterial dissemination from the gastrointestinal tract was hypothesized [65].

Urinary Tract Infections
Cases of urinary tract infections caused by lactobacilli in women have been reported, with symptoms such as chronic pyuria and pyelonephritis with bacteremia, in which L. delbrueckii or L. jensenii were the causative microorganisms [66][67][68]. A case of urinary tract infection caused by Lactobacillus spp. was reported in a newborn [69].

Virulence of LGC Members
Studies on Lactobacillus virulence have regarded mainly the species L. rhamnosus and L. paracasei, that comprise the most widely used Lactobacillus probiotics. These possess potential virulence factors such as production of enzymes which break down human glycoproteins, and proteins that bind extracellular proteins such as fibronectin, fibrinogen, and collagen, which may be important in early stage colonization and adherence. Moreover, some strains have the ability to aggregate human platelets, a trait that has a role in the pathogenesis of various infections [70,71]. The ability to bind fibrinogen is known to help gram-positive pathogens in escaping the immune system and can be sufficient to induce platelet aggregation and lead to infections, such as endocarditis [72]. Recently, a L. salivarius isolated from a case of sepsis was found to aggregate human platelets by binding human fibrinogen through a newly described fibrinogen-binding protein [73]. In some species of the genus Weissella, genome analysis revealed the presence of potential virulence determinants, such as collagen, adhesins, and hemolysins [74].
Virulence aspects were better studied in the species L. rhamnosus that comprises highly effective probiotic strains of wide use. The species L. rhamnosus comprises strains able to exert many proven beneficial effects on health, with L. rhamnosus GG as the best studied and most recommended probiotic for the prevention and treatment of conditions like antibiotic associated diarrhea (AAD) caused by Clostridium difficile, Crohn's disease, atopic dermatitis [75,76], and pathological states of the respiratory tract and the vaginal tract [77]. Its use in pediatric patients is justified by its ability to survive to amoxicillin-clavulanate treatment, with relevance for the frequent use of this antibiotic treatment in children [78].
L. rhamnosus strain GG has been applied successfully to treat infections caused by vancomycin-resistant Enterococcus faecium (VRE). A mechanism explaining the efficacy of this probiotic against VRE intestinal colonization is the prevention of their binding to mucus by competition exerted by the SpaC pilus protein of L. rhamnosus, very similar to its counterpart in the clinical E. faecium strain E1165 [79].
L. rhamnosus GG inhibits biofilm formation by various pathogens, including Salmonella spp. and uropathogenic E. coli, by the production of lectin-like proteins Llp1 and Llp2. These proteins are also involved in the adhesion capacity of L. rhamnosus GG to gastrointestinal and vaginal epithelial cells and could improve the prophylaxis of urogenital and gastrointestinal infections [80].
L. rhamnosus strains are endowed with a catalase gene, and are therefore more resistant to oxidative stress, with possible anti-oxidant applications that were recently described [81].
Beneficial effects of L. rhamnosus strains proven in vivo in human trials are synthetized in Table 2.

GG
Decrease of total and LDL cholesterol and increase in natural killer activity in elderly persons [82] prevention and relief of various types of diarrhea, and treatment of relapsing Clostridium difficile colitis [83,84] Anti-inflammatory effect by interleukin-10 generation in atopic children and alleviation of atopic eczema-dermatitis symptoms [85,86] reduced duration of respiratory tract infections in in children [87] SD11 Decrease of oral mutans streptococci [88] PL60 Production of c9,t11-and t10,c12-conjugated linoleic acids with anticarcinogenic and antiatherogenic activities, reduction of the catabolic effects of immune stimulation, and reduction of body fat [89] Not specified modulation of dendritic cells function to induce a novel form of T cell hyporesponsiveness; this mechanism might be an explanation for the observed beneficial effects of probiotic treatment in clinical disease [90] HN001 increased tumoricidal activity of circulating natural killer (NK) cells significantly correlated with age [91] 3.

Implication of L. rhamnosus in Infection Cases
L. rhamnosus has caused infections more frequently than other Lactobacillus species. It was implicated in 68 of 85 cases examined, among which 22 were attributable to L. rhamnosus GG [92]. Among 60 strains of Lactobacillus spp. from blood cultures identified in a retrospective study, L. rhamnosus was the most commonly isolated species and was found in blood cultures from 16 patients. Of patients with L. rhamnosus bacteremia, 66% were immunosuppressed and 83% had catheters [49]. A case of bacteremia caused by L. rhamnosus GG in an adult patient affected by severe active ulcerative colitis under treatment with corticosteroids and mesalazine was associated with candidemia and occurred while the patient was receiving a probiotic formulation containing the same strain (as determined by PFGE typing), and was concomitantly treated with vancomycin, to which the Lactobacillus strain was intrinsically resistant [93]. L. rhamnosus GG bacteremia was apparently a consequence of the translocation of bacteria from the intestinal lumen to the blood in an immunocompetent 58 year old male suffering from ischemic colitis. The authors of the study underlined that the Lactobacillus infection can represent a clue for a serious underlying pathological state [94].
Probiotics are commonly administered to infants to prevent adverse effects of antibiotic treatment and necrotizing enterocolitis. However, the supplementation with L. rhamnosus GG has been associated with the development of sepsis with a cause-effect relationship in eight newborns and children. Therefore, physicians must be made aware that supplementation with L. rhamnosus GG can cause sepsis in high-risk patients on rare occasions [95].
Other infections caused by L. rhamnosus GG were empyema in a human HIV-infected lung transplant recipient receiving a probiotic containing this strain [96], aspiration pneumonia in an eleven month old child with trisomy 21 affected by respiratory syncytial virus (RSV) bronchiolitis who had assumed a probiotic culture containing L. rhamnosus GG for 3 months prior to her illness [97], disseminated infection in a 6 day old newborn with intrauterine growth restriction to whom L. rhamnosus GG was administered to prevent gastrointestinal complications [98], septic shock caused by yogurt derived L. rhamnosus GG in a 54 year old male patient with acute promyelocytic leukemia in second complete remission, and who received high doses of chemotherapy and autologous peripheral blood stem cell transplantation [99], and endocarditis in a patient who regularly ate a yogurt brand labeled as containing Lactobacillus bulgaricus, Lactobacillus acidophilus, and L. casei. In the latter case, though not declared in the label, a L. rhamnosus strain identical to blood L. rhamnosus isolates based on PFGE and with 2-band difference with the valve isolate was isolated from the product [54].

Methodologies Used for L. rhamnosus Identification and Strain Discrimination
Correct species identification and strain discrimination is of utmost importance for the recognition of infection etiological agents. In the case of L. rhamnosus, species identification can be carried out by species-specific PCR as described by Alander et al. (1999) [100] or MALDI-TOF MS [97].
Identification can be accomplished by 16S rRNA or tuf gene sequencing [49,98]. PFGE with four restriction enzymes, NotI, SfiI, AscI, and FseI, used separately, is the gold standard typing technique applied for the comparison of clinical and probiotic L. rhamnosus strains [101].
Another typing method adopted for L. rhamnosus strain distinction is repetitive-sequence PCR (rep-PCR) with the primer RW3A. PCR products can be resolved on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA), and the relatedness of the strains can be evaluated using the Diversilab software (bioMérieux, Durham, NC). Identical strains have a similarity index of >99% [97].
Moreover, amplified fragment length polymorphism (AFLP) can be applied as genetic fingerprinting method for L. rhamnosus strain distinction [102].
Finally, methods of whole genome comparison have been applied in different occasions for this bacterial species [71,81,103].

Recent Advances in the Study of L. rhamnosus Capacity to Behave as Opportunistic Pathogen
Recent developments in the study of L. rhamnosus pathogenic potential consist of the analysis of virulence characters at phenotypic and genotypic level.
Comparison of isolates from dental pulp infection with L. rhamnosus GG indicated as possible biomarkers for pathogenicity the presence of a modified exopolysaccharide cluster, altered transcriptional regulators of families RpoN, NtrC, MutR, ArsR, and zinc-binding Cro/CI, and changes in the two-component sensor kinase response regulator and ABC transporters for ferric iron. Clinical strains appeared to be segregated on the basis of genomic distance analysis and SNP divergence from L. rhamnosus GG and were found to possess only the SpaFED pilus gene cluster instead of SpaCBA and SpaFED, as in the latter strain [103]. Nissilä et al. (2017) [71] studied virulence related characters, i.e. surface exposed structures, complement evasion, platelet aggregation, and biofilm formation in 4 newly sequenced and 12 already described L. rhamnosus strains from blood cultures collected from bacteremic patients between 2005 and 2011.
L. rhamnosus isolates were clearly different from L. rhamnosus GG and from each other at sequence level. The blood isolates showed no common phenotypic trait possibly involved in the persistence in the host, like biofilm formation, platelet aggregation, and pilus production.
Two strain clusters were defined: cluster A, with sequence similarity at nucleic acid level to L. rhamnosus GG between 99.942 and 99.984%, and cluster B, with a similarity to L. rhamnosus GG between 97.0 and 98.5%. All strains that were found to contain plasmids fell in the genome cluster B. All strains possessed a unique set of LPXTG proteins that are recognized by sortases and are involved in interactions with the environment and in vivo.
All the L. rhamnosus strains were able to activate the complement system, measured as C3a and terminal pathway complement complex (TCC) formation in serum. However, the strains expressing pili showed a borderline increase in TCC formation compared to the group without pili.
None of the strains bound complement inhibitors C4bp or FH, indicating that L. rhamnosus, differently from some pathogens, have not the ability to escape the complement system. Four of the sixteen strains induced platelet aggregation and four strains in cluster B formed stronger biofilm. One strain had both characteristics. Most of these strains belonged to cluster B. There was a significant association between biofilm formation and the presence of the SpaCBA pilus. Similar features are not found in L. rhamnosus GG and were observed in pathogenic strains, as reported in earlier studies with strains isolated from infectious endocarditis (5/5 tested strains), laboratory strains (8/16 strains), and strains from infection of aortic aneurysm graft and carcinoma with liver metastasis [70].
Distinctive characters of cluster B compared to strains in cluster A, similar to L. rhamnosus GG also in exopolysaccharide (EPS) gene cluster composition, were the presence of only some of the genes in one EPS gene cluster and a different type EPS/CPS cluster comprising 19 genes. This could influence tissue adherence capacity, biofilm formation, and evasion of host defense.
It was concluded that L. rhamnosus strains isolated from blood cultures are distinct from L. rhamnosus GG, suggesting that use of this probiotic is safe in healthy subjects with a functional immune system.
On the other hand, in silico analysis of regulatory motifs in L. rhamnosus GG has indicated that some sortases, as well as a fibronectin binding protein, could be upregulated during exposure to stress factors that induce the heat shock response (HSR) [104]. This suggests that the expression of those characters in stress conditions could influence the ability of L. rhamnosus GG to be virulent and should be experimentally investigated.
Though the studies on genetic determinants of the capacity to express pathogenicity of lactobacilli or other LGC genera are still limited, these can be synthetized to date as in Table 3 according to the observations in L. rhamnosus.

Conclusions
The capacity of L. rhamnosus and lactobacilli in general to behave as opportunistic pathogens has been linked to characters such as platelet aggregation capacity and biofilm formation. Still, little is known on the cell wall structures involved in these activities, so this aspect should be investigated by correlating the cell surface protein profile, including the sortase-recognized LPXTG proteins, with the virulence phenotype.
Moreover, the expression of structures and proteins involved in adherence in different growth conditions should be investigated.
EPS production, which influences biofilm structure and strength, is highly variable among strains and even genetically unstable, being determined by genome regions prone to rearrangements and loss. The implication of type of EPS and production conditions in virulence needs to be better defined by elucidating the link between presence and expression of specific genes and biofilm formation and tenacity on materials used for CVCs or prosthetic heart valve manufacturing.
A better definition of the relationships between expression of specific characters and virulence could lead to the selection of Lactobacillus probiotic strains with no intrinsic capacity to pose health risks.
On the other hand, it was shown that belonging to a specific intra-species cluster of plasmid endowed strains from bacteremic patients is per se an indication of potential pathogenicity, so that genome regions specific for those strains could be used to design PCR tests that enable to exclude the membership of probiotic candidates to those clusters.
Since infections due to Lactobacillus species are probably under-reported because appropriate growth conditions, such as microaerophily or anaerobiosis, are not applied in clinical microbiology laboratories for their isolation, improved isolation methods should be implemented to correctly estimate the involvement of lactobacilli in infection cases.
Author Contributions: F.R., C.A. and G.C. equally contributed to writing, review and editing of the article.