The Role of the Bacteroides spp. in Bacteraemia

Anaerobic bacteria are clinically significant pathogens in blood stream infections and septicaemia [15]. It is estimated that the consequence of bacteraemia of this aetiology has a high mortality rate of 16–27% [9, 28, 37, 41, 57, 62, 68]. Mortality is even higher when it comes to patients with antimicrobial empirical treatment not covering anaerobes – then the mortality rate increases to 45% [7, 37] or even to 63% [70]. Bacteroides/Parabacteroides anaerobic Gram-negative bacilli are the most commonly identified anaerobic bacteria causing bacteraemia and account for 1,2–13,7% of all positive blood samples from hospitalized patients [11, 13, 18, 28, 29, 62].

Anaerobic infections including bacteraemia nearly always arise from contamination by endogenous bacteria into contiguous or other sites. The most common sources of bacteraemia caused by anaerobes are: gastrointestinal tract, female genitourinary tract, abscesses and wound-/skin- and soft tissues and lower respiratory tract infections [14]. Postoperative gastrointestinal tract and genitourinary system patients and those with malignant tumours are at higher risk of bacteraemia caused by anaerobes [9, 57, 68]. Clinical significance of Bacteroides spp. bacilli is caused by the occurrence of numerous factors of virulence and increasing antibiotic resistance [69]. In the past decade the number of multidrug resistant isolates from the Bacteroides/Parabacteroides spp. has increased [11, 18, 36].

Among all species of genus Bacteroides, phenotypically similar species were identified to the Bacteroides fragilis group (BFG). This group consists of species of significant clinical importance: Bacteroides caccae, B. eggerthii, B. fragilis, B. nordii, B. ovatus, B. salyersiae, B. stercoris, B. thetaiotaomicron, B. uniformis, B. vulgatus. Within BFG, the number of newly-recognized species is increasing and several members have been renamed to Parabacteroides group. They are the dominant component of the flora of the gastrointestinal tract and are also present on the genital tract and in the upper respiratory tract [20, 54, 66].

BFG is found in approximately 50% of all bacteraemia caused by anaerobes. One study shows that BFG was detected in 46.6% of all episodes, among which B. fragilis were responsible for 54.4% and B. thetaiotaomicron – 22% of the infections [11]. Similar results, with a significant dominance of B. fragilis, are presented by other researchers [24, 28, 34, 57, 60].

Bacteria of the genus Bacteroides have numerous virulence factors, with the adhesins among them, which are responsible for adhering to tissues [65]. Polysaccharide envelope protects the bacteria from the immune response of the host, both cellular and humoral. It is also responsible for initiating the formation of abscesses, which are an essential element of BFG-induced infections. Untreated abscesses can grow and cause intestinal obstruction, erosion of blood vessels or fistula formation, depending on location. Abscesses can break and cause scattered infections and bacteraemia. The envelope also performs an important function in the adhesion of bacteria to the host cells and tissues. Additionally these bacteria produce a variety of enzymes that mediate tissue destruction: neuraminidase, protease, collagenase, hyaluronidase or chondroitinsulfatase. As a result of metabolic processes, Bacteroides strains produce short-chain fatty acids, mainly succinic acid, which protect them from phagocytosis by inhibiting granulocyte chemotaxis. Like all Gram-negative bacteria, Bacteroides contain lipopolysaccharide (LPS) a component of outer membrane cell. But the biologic activity of this endotoxin is 100 to 1000 times lower than that of LPS from Enterobacterales. The LPS of B. fragilis contains a lipid A moiety, but there are structural and chemical composition differences that render this LPS less potent than the LPS of Escherichia coli. The inability of B. fragilis LPS to activate TLR2 may be responsible for this difference. A large number of anaerobic bacteria, including B. fragilis can tolerate exposure to oxygen but do not replicate in atmospheric oxygen. The ability to survive exposure to oxygen is a different type of factor that facilitates the survival and thus pathogenicity of the organism [27, 39, 43, 65, 69].

Factors leading to anaerobic bacteraemia are mainly surgical procedures, crush injuries, the presence of foreign bodies, tissue necrosis, tumours, diabetes. In some cases, such as gastrointestinal perforation or aspiration pneumonia, sterile sites are exposed to large bacterial inoculum, which increases the chances of infection [62]. Finegold et al. [14] analysed 855 episodes of bacteraemia involving anaerobic microorganisms and identified abdominal cavity (52%), female genital tract (20%), lower respiratory tract (6%), upper respiratory tract (5%), and soft tissue infections (8%) as the main sources of disease [23]. The data presented by Tan et al. [57] are similar: abdominal infections (43%), followed by soft tissue infections (36%) and respiratory infections (5.5%) as a source of bacteraemia.

Other factors predisposing to anaerobic bacteraemia are: malignant tumours, haematological disorders, organ transplantation, alcoholism, drug addiction, immunosuppression, cytostatic therapy and corticosteroids [5, 11, 61]. Dumont et al. [11] analysed blood samples over a period of several months. Anaerobic aetiology has been shown in 5.8% of all cases of bacteraemia, most commonly in patients in abdominal and haematological surgery departments. In transplant departments in one of clinical hospitals in Warsaw, anaerobic bacteria were isolated from blood samples in 6.2% of cases of bacteraemia during the three-year follow-up period [25].

The elderly with comorbidities are definitely at higher risk of infection. Tan et al. [57] reported that 84% of anaerobic bacteraemia cases occurred in people over 60 years of age and the average age of affected patients was 73 years. Anaerobic bacteraemia mainly affects adults, with elderly patients over 65 years having high risk for developing bacteraemia. In contrast the prevalence of anaerobes in bloodstream infections in children is extremely rare with children between 2 and 6 years of age having the least risk ranging 0–0.5% overall [15]. An important aspect of anaerobe bacteraemia in children is that anaerobes frequently are present in cases of polymicrobial bacteraemia reflecting the fact that localized anaerobic infections are usually poly-microbial [5]. The importance of anaerobic bacteria in neonatal bacteraemia and sepsis is a relatively new phenomenon. The incidence of recovery of anaerobes in neonatal bacteraemia varies between 1.8% and 12.5%. The majority of cases reported in the literature were due to Bacteroides spp. (41%) other cultured anaerobes belonged to Clostridium spp. (32%), Peptostreptococcus spp. (20%) [5]. Risk factors of anaerobic blood infections in new-borns are as following: prolonged birth, premature rupture of foetal membranes, premature birth and respiratory failure [4, 15]. Aerobic-anaerobic blood culture pairs are suggested as a routine in neonatal practice [32].

Infections caused by anaerobic bacteria may be facilitated by the use of antibacterial agents to which those organisms are naturally resistant, such as aminoglycosides, aztreonam, phosphomycin, trimethoprim and 1^st and 2^nd generation fluoroquinolones (ciprofloxacin, levofloxacin) [6]. Nguyen et al. [37] demonstrated that the onset of highly resistant anaerobes infections correlates significantly with the previous treatment with beta-lactam antibiotics such as piperacillin, cefoxitin and cefotetan.

Clinical characteristics of anaerobic bloodstream infections do not differ from bacteraemia caused by aerobic pathogens, but due to their longer generation time and rigorous growth requirements, it usually takes longer to establish the aetiology of infection [15]. In addition to identifying the pathogen present in the blood, the diagnosis should include the detection of the primary source of infection. Anaerobic bacteraemia carries a risk of developing systemic inflammatory response syndrome (SIRS), which presents with fever, tachycardia, tachypnoea, leukocytosis or leukopenia with neutrophilia [14, 62].

Blood cultures remain the gold standard for detection of the etiologic both anaerobes and aerobes agent of bloodstream infection. Blood culture for anaerobic bacteria is routinely carried out in all adult patients and in paediatric patients who have or are suspected of having such infection. Some authors suggest that anaerobic blood cultures should only be used selectively, if anamnestic data or clinical signs and symptom are suggestive of anaerobic bacteraemia. The opposite argument for this proceeding is the fact that routine use of anaerobic blood cultures gives opportunity of quick and effective culture of facultative anaerobes [15].

Blood samples collected to blood culture medium is cultivated on continuously monitored automated blood culture systems. Advances in contemporary blood culture media include use of resin-based media that absorb antibiotics and other inhibitory substances in the specimen to increase the detection rate. Additional advances to promote faster time to positivity include automation of workflow steps including loading bottles and measurement of blood volume, optimalization of temperature stabilization within the instrument. Contemporary systems of blood culture are as follows: BacT/Alert Virtuo (bioMérieux, France), BD Bactec FX (Becton, Dickinson and Company, Franklin Lakes, NJ), Versa Trek (Thermo Fisher Scientific, Wltham, MA). Routinely blood is collected to two bottles: an aerobic one allowing preferential growth of aerobic and facultative anaerobic microorganisms, and an anaerobic one allowing preferential growth of strict and facultative anaerobic bacteria [18, 67].

Previously, the identification of strict anaerobes in positive blood culture and other clinical samples mainly relied on in-house, classical biochemical testing, biochemical strips e.g. API ID 32A Kit, Rapid ID ANA II Systems or automated systems e.g. VITEK ANC Card and gas-liquid chromatography. These methods were available in only a few diagnostics laboratories and provided identification results only 48–72 hours later, mostly on the genus level. Introduction of novel technological modalities, most importantly MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and 16S rRNA sequencing, into the routine diagnostics workflow sped up and modernized diagnostics of anaerobes. Continuous developments in improving and complementing databases of bacterial spectra for MALDI-TOF MS analysis enables detection of rarely occurring or taxonomically close microorganisms. As a result of these developments several “new” or so far unknown anaerobic species have been described as causative agents in bacteraemia e.g. Solobacterium moorei, Actinotigum schaalii [15].

In vitro susceptibility tests are usually not done by clinical laboratories for anaerobes because of technically difficulties and the length of examination time to have impact on antibiotic decisions. However, resistance patterns of many anaerobes have changed significantly over the last decades. It forced some clinical laboratories to perform anaerobic susceptibility testing. International guidelines suggest that susceptibility testing of anaerobes is indicated for isolates from blood and other normally sterile body sites for e.g.: brain abscess, endocarditis, osteomyelitis, join infection and vascular graft. Susceptibility testing is obligatory in case of isolation of highly virulent strains or strains which have unpredictable susceptibility patterns. By 2021 routinely used method of determination antibiotic susceptibility of anaerobes was minimal inhibitory concentration (MIC) performed by standardized gradient strips (E-test) or agar and broth microdilution methods used by reference laboratories [20, 35]. Testing by gradient strips is relatively simple but costly. Recently, a standardized disc diffusion method for the susceptibility testing of Bacteroides spp. and other 4 important anaerobic species and genera has been compiled and published by EUCAST in EUCAST Clinical Breakpoint Tables v. 12.0. The present recommendation do not split anaerobes on Gram positive and Gram negative but determines clinical breakpoints in species-specific way. The most critical factor for this method is the time of incubation which cannot exceed 18+/−2 h [12].

What is worth noticing, anaerobic bacteria from blood samples do not always grow in monoculture. Tan et al. [57] reports that 57% of anaerobic bacteraemia were caused by a combination of microbes. Most frequently, other anaerobes (29%) and Enterobacterales bacilli (25%) were isolated. Other researchers report that in about 13–38% of cases, aerobic microorganisms were also present [11, 24, 28, 36, 57, 62].

Carbenicillin, piperacillin, and ticarcillin are generally active against anaerobes but are considered suboptimal for infections involving B. fragilis. The β-lactam/β-lactamase inhibitor combination class of antibiotics still remains very active against B. fragilis. An exception is P. distasonis which is resistant to ampicillin/sulbactam [56]. Carbapenems have very good activity against BFG and other anaerobes. Tigecycline and tetracycline antibiotics are slightly less active than carbapenems and the β-lactam/β-lactamase inhibitor agents against B. fragilis [46].

Clindamycin was once a preferred antimicrobial agent for anaerobic infections including B. fragilis bacteraemia, but resistance has emerged with some B. fragilis strains. According to Sanford Guide clindamycin is a non-recommended agent, as resistance is likely to be present [46]. The same situation refers to moxifloxacin which was previously the preferred agent in the fluoroquinolone class for infections involving BFG. Recently resistance rates of 57 % to B. fragilis have been reported [55]. As for the fluoroquinolone only delafloxacin is active against BFG [46]. Metronidazole continue to be the most active agents against BFG [56].

Therefore, metronidazole, carbapenems and β-lactam/β-lactamase inhibitor combinations are still recommended to empirical therapy of anaerobic infections [55]

Antibiotic selection in anaerobic infections including bacteraemia is generally made empirically based on susceptibility test results from sentinel laboratories or literature reports. Empirical therapy in these infections depends on the clinical condition of the patient and the location of potential primary infection. When the source of bacteraemia is an extravascular site, surgical intervention and drainage are necessary to prevent the continuance of bacteraemia and to reduce the time of therapy. The location also depends on the duration of treatment, ranging from 10 days to 3 months. Knowledge of the antibiotic sensitivity profiles of anaerobic infections in individual hospitals/wards may be crucial in the choice of empirical therapy. Tan et al. [57] describe that the most commonly used empirical antibiotic therapy in Bacteroides bacteraemia were β-lactam with β-lactam inhibitor (amoxicillin/clavulanic acid or piperacillin/tazobactam) (44%), metronidazole (10%) and carbapenems (8.8%). Treatment of the majority of patients (72.65%) started with an appropriate initial antibiotic therapy. Sixteen percent of patients received antibiotics without anti-anaerobic activity.

Nguyen et al. [37] conducted a prospective observational study of 128 cases of bacteraemia involving BFG and presented that, in view of the increasing antibiotic resistance in these microbes. The conclusion of the study points that antibiotics traditionally used in empirical treatment, such as piperacillin with tazobactam or metronidazole, may be proven ineffective. They presented that clinical failure and mortality were more common in patients who did not receive a properly selected antibacterial agent.

The problem of antibiotic resistance, which is on the rise also among anaerobes, is particularly pronounced in BFG and makes it difficult to choose a reliable empirical therapy. Although ineffectiveness of metronidazole against Bacteroides spp. bacilli is rare, cases of resistance are reported increasingly. Essentially, non-prudent use of metronidazole can be held responsible for this phenomenon. One of the mechanisms of resistance is the production of 5-nitroimidazole nitroreductases, encoded by genes nim, which can be present in a “silent genes” form, which may not undergo expression [1, 3, 10, 31].

Clindamycin resistance of Bacteroides spp. is mainly associated with the production of adenyl-N-methyltransferase 23S rRNA, encoded by erm genes. The proportion of clindamycin-resistant strains has steadily increased over the past few decades [26, 33, 45, 47, 65].

As regards resistance to β-lactam antibiotics, the production of different classes of β-lactamases (cephalosporinase, carbapenemase) is of the greatest importance. The β-lactamase most commonly found in Bacteroides spp. is CepA (Cephalosporinase of class A), encoded by the chromosomal cep gene, and CfxA cephamycinase (Cefoxitin resistance class A), a product of the cfxA gene [40, 52]. Sometimes the bacilli produce CfiA carbapenemase encoded by the cfiA gene [16, 44, 52, 58], but not all strains with the cfiA gene are resistant to carbapenems. Similarly to the nim genes, those are also “silent” and are not always expressed. Resistance to β-lactam antibiotics may also be associated with a decrease in the permeability of these drugs through the outer membrane, as well as changes in the qualitative and quantitative composition of penicillin-binding proteins (PBPs) [47, 53]. Some authors suggest that when choosing carbapenems for empirical treatment, imipenem should be preferred, as its MICs were lower than MIC of doripenem and meropenem [22, 50].

Infections caused by multidrug-resistant (MDR) strains of Bacteroides spp. are still rare, but can cause serious therapeutic problems and are often fatal. The definition of MDR strains refers to aerobic bacteria resistant to at least three antibiotics from different groups. This condition may lead to overuse of this term when referring to anaerobes because Bacteroides isolates are often resistant to antibiotics from several groups, e.g. moxifloxacin, clindamycin and various beta-lactams. As Dumont et al. [11] suggest, the criteria for MDR for bacteria of the genus Bacteroides should be established with a distinction between less (e.g. to moxifloxacin, clindamycin) and greater resistance (to metronidazole, carbapenems). The literature describes cases of MDR infections [21]. The first publication on the MDR strain of B. fragilis dates back to 1995 from the United Kingdom (a patient with complications after gynaecological surgery) [59]. Since then, many researchers have described cases of bacteraemia caused by MDR-Bacteroides with different phenotypes and genotypes of antibiotic resistance. Most of the cases involved primary infections in the abdominal cavity, e.g. pancreatitis [64], a condition after gastric resection [23] or colorectal cancer [2, 8, 19, 49].

Ogane et al. [38] presented the antimicrobial susceptibility of 50 isolates of B. fragilis originated from blood samples from patients hospitalized in two hospitals in Japan between 2014 and 2019. Isolates were more sensitive to piperacillin with tazobactam (94% susceptible) than ampicillin/sulbactam (70% susceptible). Ninety six percent of isolates were sensitive to imipenem, while 90% were sensitive to meropenem and doripenem.

In Dumont et al. [11], BFG isolates were sensitive to piperacillin with tazobactam (97%), amoxicillin with clavulanic acid (92.5%) and imipenem (98.5%). According to the published studies sensitivity to clindamycin and moxifloxacin is significantly rare and occurs in 68% and 64% of isolates [38] and in 50.8% and 58.2% [11] respectively. Similar results are presented by other researchers [30, 57, 63]. Increasing resistance to clindamycin is observed in Europe [17, 42] including Poland [26]. The results were confirmed in study covering 8 medical centres. In the study 1957 isolates collected for 4 years (2008–9) were analysed. Resistance rates ranging 60% have been found for clindamycin and even higher above 80% to moxifloxacin, resistance for tigecycline was on the level 5.4%. For carbapenems, resistance of B. fragilis was 1.1–2.5%. B. fragilis isolate resistant to all antibiotics, with the exception of metronidazole, was also identified [57]. It was shown in one study, that among 67 Bacteroides spp. isolates only one, B. fragilis was resistant to metronidazole (1.5%) [11] and in a pool of 116 isolates also one, Parabacteroides distasonis, was resistant to metronidazole (1%). Alarming reports come from Pakistan including metronidazole resistance rate of 17.5% d [48].

To sum up, carbapenems and metronidazole should be considered the most active drugs to be used in the empirical therapy of anaerobic bacteraemia. The importance of the problem of strain resistance into these two antibiotics is even greater since the occurrence of such resistance is particularly related to the outcome of treatment and mortality in anaerobic bacteraemia [51]. Therefore, the collection of epidemiological data at local and global level, before treating patients with bacteraemia, can play an important role not only in public health but also in improving treatment outcomes [37].

Anaerobic bacteria remain an important cause of blood infections, mainly when it comes to elderly people with comorbidities. Most of them are caused by Gram-negative bacilli of the Bacteroides genus, which are a part of the natural human microflora. The increasing resistance to antibiotics among anaerobic bacteria prompts monitoring of the drug sensitivity profile in individual hospitals and wards. The development of MDR is worrying as it also affects broad-spectrum antibiotics. Therefore, in the case of bacteraemia, the determination of drug sensitivity should be a necessity. Rapid microbial diagnosis, targeted therapy and surgical treatment of a possible source of infection are crucial for prognosis improvement.